We are pleased that the following section authors have continued as members of the seventh edition team: Drs. Kalman Rubinson and Eric Lang (nervous system), Dr. James Watras (muscle), Dr. Achilles Pappano (cardiovascular system), Drs. Michelle Cloutier and Roger Thrall (respiratory system), Drs. Kim Barrett and Helen Raybould (gastrointestinal system), and Dr. Bruce White (endocrine and reproductive systems). We also welcome the following authors: Dr. Withrow Gil Wier (cardiovascular system), and Dr. John Harrison (endocrine and reproduction systems).

As in the previous editions of this textbook, we have attempted to emphasize broad concepts and to minimize the compilation of isolated facts. Each chapter has been written to make the text as lucid, accurate, and current as possible. We have included both clinical and molecular information in each section, as feedback on these features has indicated that this information serves to provide clinical context and new insights into physiologic phenomena at the cellular and molecular levels. New to this edition is a list of sources that the reader can consult for further information on the topics covered in each chapter. We hope that you find this a valuable addition to the book.

The human body consists of billions of cells that are organized into tissues (e.g., muscle, epithelia, and nervous tissue) and organ systems (e.g., nervous, cardiovascular, respiratory, renal, gastrointestinal, endocrine, and reproductive). For these tissues and organ systems to function properly and thus allow humans to live and carry out daily activities, several general conditions must be met. First and foremost, the cells within the body must survive. Survival requires adequate cellular energy supplies, maintenance of an appropriate intracellular milieu, and defense against a hostile external environment. Once cell survival is ensured, the cell can then perform its designated or specialized function (e.g., contraction by skeletal muscle cells). Ultimately, the function of cells, tissues, and organs must be coordinated and regulated. All of these functions are the essence of the discipline of physiology and are presented throughout this book. What follows is a brief introduction to these general concepts.

Cells need a constant supply of energy. This energy is derived from the hydrolysis of adenosine triphosphate (ATP). If not replenished, the cellular ATP supply would be depleted in most cells in less than 1 minute. Thus, ATP must be continuously synthesized. This in turn requires a steady supply of cellular fuels. However, the cellular fuels (e.g., glucose, fatty acids, and ketoacids) are present in the blood at levels that can support cellular metabolism only for a few minutes. The blood levels of these cellular fuels are maintained through the ingestion of precursors (i.e., carbohydrates, proteins, and fats). In addition, these fuels can be stored and then mobilized when ingestion of the precursors is not possible. The storage forms of these fuels are triglycerides (stored in adipose tissue), glycogen (stored in the liver and skeletal muscle), and protein. The maintenance of adequate levels of cellular fuels in the blood is a complex process involving the following tissues, organs, and organ systems:

Liver: Converts precursors into fuel storage forms (e.g., glucose → glycogen) when food is ingested, and converts storage forms to cellular fuels during fasting (e.g., glycogen → glucose and amino acids → glucose).

Skeletal muscle: Like the liver, stores fuel (glycogen and protein) and converts glycogen and protein to fuels (e.g., glucose) or fuel intermediates (e.g., protein → amino acids) during fasting.

Gastrointestinal tract: Digests and absorbs fuel precursors.

Adipose tissue: Stores fuel during feeding (e.g., fatty acids → triglycerides) and releases the fuels during fasting.

Cardiovascular system: Delivers the fuels to the cells and to and from their storage sites.

Endocrine system: Maintains the blood levels of the cellular fuels by controlling and regulating their storage and their release from storage (e.g., insulin and glucagons).

Nervous system: Monitors oxygen levels and nutrient content in the blood and, in response, modulates the cardiovascular, pulmonary, and endocrine systems and induces feeding and drinking behaviors. In addition to energy metabolism, the cells of the body must maintain a relatively constant intracellular environment to survive. This includes the uptake of fuels needed to produce ATP, the export from the cell of cellular wastes, the maintenance of an appropriate intracellular ionic environment, the establishment of a resting membrane potential, and the maintenance of a constant cellular volume. All of these functions are carried out by specific membrane transport proteins.

The composition of the extracellular fluid (ECF) that bathes the cells must also be maintained relatively constant. In addition, the volume and temperature of the ECF must be regulated. Epithelial cells in the lungs, gastrointestinal tract, and kidneys are responsible for maintaining the volume and composition of the ECF, while the skin plays a major role in temperature regulation. On a daily basis, H2O and food are ingested, and essential components are absorbed across the epithelial cells of the gastrointestinal tract. This daily intake of solutes and water must be matched by excretion from the body, thus maintaining steady-state balance. The kidneys are critically involved in the maintenance of steady-state balance for water and many components of the ECF (e.g., Na+ , K+ , HCO3 − , pH, Ca++ , organic solutes). The lungs ensure an adequate supply of O2 to “burn” the cellular fuels for the production of ATP and excrete the major waste product of this process (i.e., CO2). Because CO2 can affect the pH of the ECF, the lungs work with the kidneys to maintain ECF pH.

Because humans inhabit many different environments and often move between environments, the body must be able to rapidly adapt to the challenges imposed by changes in ambient temperature and availability of food and water. Such adaptation requires coordination of the function of cells in different tissues and organs as well as their regulation. The nervous and endocrine systems coordinate and regulate cell, tissue, and organ function. The regulation of function can occur rapidly (seconds to minutes), as is the case for levels of cellular fuels in the blood, or over much longer periods of time (days to weeks), as is the case for acclimatization when an individual moves from a cool to a hot environment or changes from a high-salt to a low-salt diet.

The function of the human body represents complex processes at multiple levels. This book explains what is currently known about these processes. Although the emphasis is on the normal function of the human body, discussion of disease and abnormal function is also appropriate, as these often illustrate physiologic processes and principles at the extremes.

The authors for each section have presented what they believe to be the most likely mechanisms responsible for the phenomena under consideration. We have adopted this compromise to achieve brevity, clarity, and simplicity.

Bruce M. Koeppen, MD, PhD Bruce A. Stanton, PhD 29TheGastricPhaseoftheIntegratedResponsetoaMeal,529 30TheSmallIntestinalPhaseoftheIntegratedResponsetoaMeal,541 31TheColonicPhaseoftheIntegratedResponsetoaMeal,559 32TransportandMetabolicFunctionsoftheLiver,568

Section 7: The Renal System, 580 37RoleoftheKidneysintheRegulationofAcid-BaseBalance,670

Section 8: The Endocrine and Reproductive Systems, 685

SECTION 1Cellular Physiology BRUCE M. KOEPPEN AND BRUCE A. STANTON

Upon completion of this chapter, the student should be able to answer the following questions: 1.

What organelles are found in a typical eukaryotic cell, and what is their function?

2.

What is the composition of the plasma membrane?

3.

What are the major classes of membrane transport proteins, and how do they transport biologically important molecules and ions across the plasma membrane?

4.

What is the electrochemical gradient, and how it is used to determine whether the transport of a molecule or ion across the plasma membrane is active or passive?

5.

What are the driving forces for movement of water across cell membrane and the capillary wall?

In addition, the student should be able to define and understand the following properties of physiologically important solutions and fluids: he human body is composed of billions of cells. Although cells can perform different functions, they share certain common elements. This chapter provides an overview of these common elements and focuses on the important function of the transport of molecules and water into and out of the cell across its plasma membrane.

Overview of Eukaryotic Cells

Eukaryotic cells are distinguished from prokaryotic cells by the presence of a membrane-delimited nucleus. With the exception of mature human red blood cells and cells within the lens of the eye, all cells within the human body contain a nucleus. The cell is therefore effectively divided into two compartments: the nucleus and the cytoplasm. The cytoplasm is an aqueous solution containing numerous organic molecules, ions, cytoskeletal elements, and a number of organelles. Many of the organelles are membrane-enclosed compartments that carry out specific cellular function. An idealized eukaryotic cell is depicted in

Fig. 1.1 , and the primary function of some components and compartments of the cell are summarized in . Readers who desire a more in-depth presentation of this material are encouraged to consult one of the many textbooks on cell and molecular biology that are currently available.

The Plasma Membrane

The cells within the body are surrounded by a plasma membrane that separates the intracellular contents from the extracellular environment. Because of the properties of this membrane and, in particular, the presence of specific membrane proteins, the plasma membrane is involved in a number of important cellular functions, including the following:

Selective transport of molecules into and out of the cell. A function carried out by membrane transport proteins.

Cell recognition through the use of cell surface antigens.

Cell communication through neurotransmitter and hormone receptors and through signal transduction pathways.

Tissue organization, such as temporary and permanent cell junctions, and interaction with the extracellular matrix, with the use of a variety of cell adhesion molecules.

Membrane-dependent enzymatic activity.

Determination of cell shape by linkage of the cytoskeleton to the plasma membrane. In this chapter, the structure and function of the plasma membrane of eukaryotic cells are considered. More specifically, the chapter focuses on the transport of molecules and water across the plasma membrane. Only the principles of membrane transport are presented here. Additional details that relate to specific cells are presented in the various sections and chapters of this book.

The plasma membrane of eukaryotic cells consists of a 5-nm-thick lipid bilayer with associated proteins (

Fig. 1.2 ). Some of the membrane-associated proteins are integrated into the lipid bilayer; others are more loosely attached to the

CHAPTER 1 Principles of Cell and Membrane Function 3 • Fig. 1.1 Schematic drawing of a eukaryotic cell. The top portion of the cell is omitted to illustrate the nucleus and various intracellular organelles. See text for details.

Primary Functions of Some Eukaryotic Cellular Components and Compartments 1.1

CytosolMetabolism, protein synthesis (free ribosomes)CytoskeletonCell shape and movement, intracellular transportNucleusGenome (22 autosomes and 2 sex chromosomes), DNA and RNA synthesisMitochondriaATP synthesis by oxidative phosphorylation, Ca2+ storageSmooth endoplasmic reticulumSynthesis of lipids, Ca2+ storageFree ribosomesTranslation of mRNA into cytosolic proteinsRough endoplasmic reticulumTranslation of mRNA into membrane associated proteins or for secretion out of the cellLysosomeIntracellular degradationEndosomeCellular uptake of cholesterol, removal of receptors from the plasma membrane, uptake of small molecules and water into the cell, internalization of large particles (e.g., bacteria, cell debris)Golgi apparatusModification, sorting, and packaging of proteins and lipids for delivery to other organelles within

ATP, adenosine triphosphate; mRNA, messenger RNA.

inner or outer surfaces of the membrane, often by binding molecules that contain a charged (or polar) hydrophilic to the integral membrane proteins. head and two (nonpolar) hydrophobic fatty acyl chains (

Fig.

1.3 ). The amphipathic nature of the phospholipid molecule Membrane Lipids is critical for the formation of the bilayer: The hydrophobic The major lipids of the plasma membrane are phospholipids fatty acyl chains form the core of the bilayer, and the polar and phosphoglycerides. Phospholipids are amphipathic head groups are exposed on the surface.

• Fig. 1.2 Schematic diagram of the cell plasma membrane. Not shown are lipid rafts. See text for details. (Modified from Cooper GM. The Cell—A Molecular Approach. 2nd ed. Washington, DC: Sinauer; 2000, Fig. 12.3.)

Phospholipid Glycolipid (e.g., phosphatidylcholine) (e.g., galactosylceramide) Cholesterol

Sugar (e.g., galactose) • Fig. 1.3 Models of the major classes of plasma membrane lipids, depicting the hydrophilic and hydrophobic regions of the molecules. The molecules are arranged as they exist in one leaflet of the bilayer. The opposing leaflet is not shown. One of the fatty acyl chains in the phospholipid molecule is unsaturated. The presence of this double bond produces a “kink” in the fatty acyl chain, which prevents tight packing of membrane lipids and increases membrane fluidity. (Modified from Hansen JT, Koeppen BM: Netter’s Atlas of Human Physiology. Teterboro, NJ: Icon Learning Systems; 2002.)

The majority of membrane phospholipids have a glycerol carbons in length and may be saturated or unsaturated (i.e., “backbone” to which are attached the fatty acyl chains, contain one or more double bonds). and an alcohol is linked to glycerol via a phosphate group. The phospholipid composition of the membrane The common alcohols are choline, ethanolamine, serine, varies among different cell types and even between the inositol, and glycerol. Another important phospholipid, bilayer leaflets. For example, in the erythrocyte plasma sphingomyelin, has the amino alcohol sphingosine as its membrane, phosphatidylcholine and sphingomyelin are “backbone” instead of glycerol.

lists these common found predominantly in the outer leaflet of the membrane, phospholipids. The fatty acyl chains are usually 14 to 20 whereas phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol are found in the inner leaflet.

, phosphatidylinositol plays an important role in signal transduction, and its location in the inner leaflet of the membrane facilitates this signaling role.

The sterol molecule cholesterol is also a critical component of the bilayer (see

Fig. 1.3 ). It is found in both leaflets and serves to stabilize the membrane at normal body temperature (37°C). As much as 50% of the lipids found in the membrane can be cholesterol. A minor lipid component of the plasma membrane is glycolipids. These lipids, as their name indicates, consist of two fatty acyl chains linked to polar head groups that consist of carbohydrates (see

Fig.

1.3 ). As discussed in the section on , one glycolipid, glycosylphosphatylinositol (GPI), plays an important role in anchoring proteins to the outer leaflet of the membrane. Both cholesterol and glycolipids, like the phospholipids, are amphipathic, and they are oriented with their polar groups on the outer surface of the leaflet in which they are located. Their hydrophobic portion is thus located within the interior of the bilayer.

The lipid bilayer is not a static structure. The lipids and associated proteins can diffuse within the plane of the membrane. The fluidity of the membrane is determined by temperature and by its lipid composition. As temperature increases, the fluidity of the membrane increases. The presence of unsaturated fatty acyl chains in the phospholipids and glycolipids also increases membrane fluidity. If a fatty acyl chain is unsaturated, the presence of a double bond introduces a “kink” in the molecule (see

Fig. 1.3).

This kink prevents the molecule from associating closely with surrounding lipids, and, as a result, membrane fluidity is increased. Although the lipid bilayer is “fluid,” movement of proteins in the membrane can be constrained or limited. For example, membrane proteins can be anchored to components of the intracellular cytoskeleton, which limits their movement. Membrane domains can also be isolated from one another. An important example of this can be found in epithelial tissues. Junctional complexes (e.g., tight junctions) separate the plasma membrane of epithelial cells into two domains: apical and basolateral (see ). The targeted localization of membrane proteins into one or other of these domains allows epithelial cells to carry

CHAPTER 1 Principles of Cell and Membrane Function out vectorial transport of substances from one side of the epithelium to the opposite side. The ability to carry out vectorial transport is crucial for the functioning of several organ systems (e.g., the gastrointestinal tract and kidneys). In addition, some regions of the membrane contain lipids (e.g., sphingomyelin and cholesterol) that aggregate into what are called lipid rafts. These lipid rafts often have an association with specific proteins, which diffuse in the plane of the membrane as a discrete unit. Lipid rafts appear to serve a number of functions. One important function of these rafts is to segregate signaling molecules.

As much as 50% of the plasma membrane is composed of proteins. These membrane proteins are classified as integral, lipid-anchored, or peripheral.

Integral membrane proteins are imbedded in the lipid bilayer, where hydrophobic amino acid residues are associated with the hydrophobic fatty acyl chains of the membrane lipids. Many integral membrane proteins span the bilayer; such proteins are termed transmembrane proteins. Transmembrane proteins have both hydrophobic and hydrophilic regions. The hydrophobic region, often in the form of an α helix, spans the membrane. Hydrophilic amino acid residues are then exposed to the aqueous environment on either side of the membrane. Transmembrane proteins may pass through the membrane multiple times.

Thereisasuperfamilyofmembraneproteinsthatserveasreceptorsformanyhormones,neurotransmitters,andnumerousdrugs.ThesereceptorsarecoupledtoheterotrimericGproteinsandaretermedG protein–coupled receptors (see ).Theseproteinsspanthemembranewithsevenα-helicaldomains.Thebindingsiteofeachligandiseitherontheextracellularportionoftheprotein(largeligands)orinthemembrane-spanningportion(smallligands),whereasthecytoplasmicportionbindstotheGprotein.Thissuperfamilyofmembraneproteinsmakesupthethirdlargestfamilyofgenesinhumans.NearlyhalfofallnonantibioticprescriptiondrugsaretargetedtowardGprotein–coupledreceptors.

A protein can also be attached to the membrane via lipid anchors. The protein is covalently attached to a lipid molecule, which is then embedded in one leaflet of the bilayer. Glycosylphosphatidylinositol (GPI) anchors proteins to the outer leaflet of the membrane. Proteins can be attached to the inner leaflet via their amino-terminus by fatty acids (e.g., myristate or palmitate) or via their carboxyl-terminus by prenyl anchors (e.g., farnesyl or geranylgeranyl).

Peripheral proteins may be associated with the polar head groups of the membrane lipids, but they more commonly bind to integral or lipid-anchored proteins.

In many cells, some of the outer leaflet lipids, as well as many of the proteins exposed on the outer surface of the membrane, are glycosylated (i.e., have short chains of sugars, called oligosaccharides, attached to them). Collectively, these glycolipids and glycoproteins form what is called the glycocalyx. Depending on the cell these glycolipids and glycoproteins may be involved in cell recognition (e.g., cell surface antigens) and formation of cell-cell interactions (e.g., attachment of neutrophils to vascular endothelial cells).

Although plasma membrane proteins perform many important cellular functions, as noted previously, the remainder of this chapter focuses on one group of plasma membrane proteins: the membrane transport proteins, or transporters. It has been estimated that approximately 10% of human genes (≈2000) code for transporters. They are also targets for numerous drugs.

The normal function of cells requires the continuous movement of water and solutes into and out of the cell. The intracellular and extracellular fluids are composed primarily of H2O, in which solutes (e.g., ions, glucose, amino acids) are dissolved. The plasma membrane, with its hydrophobic core, is an effective barrier to the movement of virtually all of these biologically important solutes. It also restricts the movement of water across the membrane. The presence of specific membrane transporters in the membrane is responsible for the movement of these solutes and water across the membrane.

Membrane transporters have been classified in several different ways. In this chapter, the transporters are divided into four general groups: water channels, ion channels, solute carriers, and adenosine triphosphate (ATP)–dependent transporters.

lists these groups of membrane transporters, their modes of transport, and estimates of the rates at which they transport molecules or ions across the membrane.

Water channels, or aquaporins (AQPs), are the main routes for water movement into and out of the cell. They are widely distributed throughout the body (e.g., the brain, lungs, kidneys, salivary glands, gastrointestinal tract, and liver). Cells express different AQP isoforms, and some cells even express multiple isoforms. For example, cells in the collecting ducts of the kidneys express AQP3 and AQP4 in their basolateral membrane and AQP2 in their apical membrane. Moreover, the abundance of AQP2 in the apical membrane is regulated by antidiuretic hormone (also called arginine vasopressin), which is crucial for the ability of the kidneys to concentrate the urine (see

Although all AQP isoforms allow the passive movement of H2O across the membrane, some isoforms also provide a pathway for other molecules such as glycerol, urea, mannitol, purines, pyrimidines, CO2, and NH3 to cross the membrane. Because glycerol was one of the first molecules identified as crossing the membrane via some AQPs, this group of AQPs is collectively called aquaglyceroporins (see also ). Regulation of the amount of H2O that can enter or leave the cell via AQPs occurs primarily by altering the number of AQPs in the membrane.

EachAQPmoleculeconsistsofsixmembrane-spanningdomainsandacentralwater-transportingpore.FourAQPmonomersassembletoformahomotetramerintheplasmamembrane,witheachmonomerfunctioningasawaterchannel.

Ion channels are found in all cells, and are especially important for the function of excitable cells (e.g., neurons and muscle cells). Ion channels are classified by their selectivity, conductance and mechanism of channel gating (i.e., opening and closing). Selectivity is defined as the nature of the ions that pass through the channel. At one extreme, ion channels can be highly selective, in that they allow only a specific ion through. At the other extreme, they may be nonselective, allowing all or a group of cations or anions through. Channel conductance refers to the number of ions that pass through the channel and is typically expressed in picosiemens (pS). The range of conductance is considerable: Some channels have a conductance of only 1 to 2 pS, whereas others have a conductance of more than 100 pS. For some channels, the conductance varies, depending on the direction in which the ion is moving. For example, if the channel has a larger conductance when ions are moving into the cell than when they are moving out of the cell, the channel is said to be an inward rectifier. Moreover, ion channels fluctuate between an open state or a closed state, a process called gating (

Fig. 1.4 ). Factors that can control gating include membrane voltage, extracellular agonists or antagonists (e.g., acetylcholine is an extracellular agonist mitochondria,andwaterchannels(i.e.,aquaporins)thatfunctionas that controls the gating of a cation-selective channel in the apore.

ATP,adenosinetriphosphate.motor end plate of skeletal muscle cells; see

Chapter 6), intracellular messengers (e.g., Ca++ , ATP, cyclic guanosine

CHAPTER 1 Principles of Cell and Membrane Function •Fig. 1.4 Recordingofcurrentflowthroughasingleionchannel.Thechannelspontaneouslyfluctuatesbetweenanopenstateandaclosedstate.Theamplitudeofthecurrentisapproximately2pA(2× 10−12amps);thatis,12.5millionions/secondcrossthemembrane.

monophosphate), and mechanical stretch of the plasma membrane. Ion channels can be regulated by a change in the number of channels in the membrane or by gating of the channels.

Solute carriers (denoted SLCs by the HUGO Gene Nomenclature Committee) represent a large group of membrane transporters categorized into more than 50 families; almost 400 specific transporters have been identified to date. These carriers can be divided into three groups according to their mode of transport. One group, uniporters (or facilitated transporters), transports a single molecule across the membrane. The transporter that brings glucose into the cell (glucose transporter 1 [GLUT-1], or SLC2A1) is an important member of this group. The second group, symporters (or cotransporters), couples the movement of two or more molecules/ions across the membrane. As the name implies, the molecules/ions are transported in the same direction. The Na+,K+,2Cl− (NKCC) symporter found in the kidney (NKCC2, or SLC12A1), which is crucial for diluting and concentrating the urine (see ), is a member of this group. The third group, antiporters (or exchange transporters), also couples the movement of two or more molecules/ions across the membrane; in this case, however, the molecules/ions are transported in opposite directions. The Na+-H+ antiporter is a member of this group of solute carriers. One isoform of this antiporter (NHE-1, or SLC9A1) is found in all cells and plays an important role in regulating intracellular pH.

The ATP-dependent transporters, as their name implies, use the energy in ATP to drive the movement of molecules/ ions across the membrane. There are two groups of ATP-dependent transporters: the ATPase ion transporters and the ATP-binding cassette (ABC) transporters. The ATPase ion transporters are subdivided into P-type ATPases and V-type ATPases.

The P-type ATPases are phosphorylated during the transport cycle. Na+,K+-ATPase is an important example of a P-type ATPase. With the hydrolysis of each ATP molecule, it transports three Na+ ions out of the cell and two K+ ions into the cell. Na+,K+-ATPase is present in all cells and plays a critical role in establishing cellular ion and electrical gradients, as well as maintaining cell volume (see

V-type H+-ATPases are found in the membranes of several intracellular organelles (e.g., endosomes, lysosomes); as a result, they are also referred to as vacuolar H+-ATPases. The aAnother type of ATPases, F-type ATPases, is found in the mitochondria, and they are responsible for ATP synthesis. They are not considered in this chapter.

Na+,K+-ATPase(alsocalledtheNa+,K+-pump orjusttheNa+pump)isfoundinallcellsandisresponsibleforestablishingthegradientsofNa+ andK+ acrosstheplasmamembrane.Thesegradientsinturnprovideenergyforseveralessentialcellfunctions(see ).Na+,K+-ATPaseiscomposedofthreesubunits(α,β,andγ),andtheproteinexistsinthemembranewithastoichiometriccompositionof1α,1β,1γ.Theα subunitcontainsbindingsitesforNa+,K+ andATP.Itisalsothesubunitthatbindscardiacglycosides(e.g.,ouabain),whichspecificallyinhibittheenzyme.Ithasatransmembranedomainandthreeintracellulardomains:phosphorylation(P-domain),nucleotidebinding(N-domain),andactuator(A-domain).Althoughtheα subunitisthefunctionalsubunitoftheenzyme(i.e.,ithydrolyzesATP,bindsNa+ andK+ ,andtranslocatesthemacrossthemembrane),itcannotfunctionwithouttheβ subunit.Theβ subunitisresponsiblefortargetingtheα subunittothemembraneandalsoappearstomodulatetheaffinityoftheNa+,K+-ATPaseforNa+ andK+ .Theα andβ subunitscancarryoutNa+ andK+ transportintheabsenceoftheγ subunit.However,theγ subunitappearstoplayaregulatoryrole.Theγ subunitisamemberofafamilyofproteinscalledFXYD proteins (sonamedfortheFXYDaminoacidsequencefoundintheprotein).

H+-ATPase in the plasma membrane plays an important role in urinary acidification (see

ABC transporters represent a large group of membrane transporters. They are found in both prokaryotic and eukaryotic cells, and they have amino acid domains that bind ATP (i.e., ABC domains). Seven subgroups of ABC transporters in humans and more than 40 specific transporters have been identified to date. They transport a diverse group of molecules/ions, including Cl−, cholesterol, bile acids, drugs, iron, and organic anions.

Because biologically important molecules enter and leave cells through membrane transporters, membrane transport is specific and regulated. Although some membrane transporters are ubiquitously expressed in all cells (e.g., Na+,K+-ATPase), the expression of many other transporters is limited to specific cell types. This specificity of expression tailors the function of the cell to the organ system in which it is located (e.g., the sodium-glucose–linked transporters SGLT-1 and SGLT-2 in the epithelial cells of the intestines and renal proximal tubules). In addition, the amount of a molecule being transported across the membrane can be regulated. Such regulation can take place through altering the number of transporters in the membrane or altering the rate or kinetics of individual transporters (e.g., the time an ion channel stays in the open versus closed state), or both.

Solute and water can be brought into the cell through a process of endocytosis and released from the cell through the process of exocytosis. Endocytosis is the process whereby a piece of the plasma membrane pinches off and

Cystic fibrosis isanautosomalrecessivediseasecharacterizedbychroniclunginfections,pancreaticinsufficiency,andinfertilityinboysandmen.Deathusuallyoccursbecauseofrespiratoryfailure.Itismostprevalentinwhitepeopleandisthemostcommonlethalgeneticdiseaseinthispopulation,occurringin1per3000livebirths.Itisaresultofmutationsinageneonchromosome7thatcodesforanABCtransporter.Todate,morethan1000mutationsinthegenehavebeenidentified.Themostcommonmutationisadeletionofaphenylalanineatposition508(F508del).Becauseofthisdeletion,degradationoftheproteinbytheendoplasmicreticuluminenhanced,and,asaresult,thetransporterdoesnotreachtheplasmamembrane.Thistransporter,calledcystic fibrosis transmembrane conductance regulator (CFTR), normallyfunctionsasaCl− channelandalsoregulatesothermembranetransporters(e.g.,theepithelialNa+ channel[ENaC]).Thusinindividualswithcysticfibrosis,epithelialtransportisdefective,whichisresponsibleforthepathophysiologicprocess.Forexample,inpatientsnotaffectedbycysticfibrosis,theepithelialcellsthatlinetheairwayofthelungarecoveredwithalayerofmucusthatentrapsinhaledparticulatesandbacteria.Ciliaontheepithelialcellsthentransporttheentrappedmaterialoutofthelung,aprocesstermedmucociliary transport (see formoredetails).Inpatientswithcysticfibrosis,theinabilitytosecreteCl− ,Na+ ,andH2Oresultsinanincreaseintheviscosityoftheairwaysurfacemucus;thustheciliacannottransporttheentrappedbacteriaandotherpathogensoutofthelung.Thisinturnleadstorecurrentandchroniclunginfections.Theinflammatoryprocessthataccompaniestheseinfectionsultimatelydestroysthelungtissue,causingrespiratoryfailureanddeath.In2015,the

U.S.FoodandDrugAdministrationapprovedlumacaftor/ivacaftor(Orkambi),adrugthatincreasestheamountofF508delCFTRintheplasmamembraneoflungepithelialcells.

is internalized into the cell interior, and exocytosis is the process whereby vesicles inside the cell fuse with the plasma membrane. In both of these processes, the integrity of the plasma membrane is maintained, and the vesicles allow for the transfer of the contents among cellular compartments. In some cells (e.g., the epithelial cells lining the gastrointestinal tract), endocytosis across one membrane of the cell is followed by exocytosis across the opposite membrane. This allows the transport of substances inside the vesicles across the epithelium, a process termed transcytosis.

Endocytosis occurs in three mechanisms. The first is pinocytosis, which consists of the nonspecific uptake of small molecules and water into the cell. Pinocytosis is a prominent feature of the endothelial cells that line capillaries and is responsible for a portion of the fluid exchange that occurs across these vessels. The second form of endocytosis, phagocytosis, allows for the cellular internalization of large particles (e.g., bacteria, cell debris). This process is an important characteristic of cells in the immune system (e.g., neutrophils and macrophages). Often, but not always, phagocytosis is a receptor-mediated process. For example,

CHAPTER 1 Principles of Cell and Membrane Function 9

Proteins within the plasma membrane of cells are constantly being removed and replaced with newly synthesized proteins. As a result, membrane proteins are constantly being replaced. One mechanism by which membrane proteins are “tagged” for replacement is by the attachment of ubiquitin to the cytoplasmic portion of the protein. Ubiquitin is a 76–amino acid protein that is covalently attached to the membrane protein (usually to lysine) by a class of enzymes called ubiquitin protein ligases. One important group of these ligases is the developmentally downregulated protein 4 (Nedd4)/Nedd4-like family. Once a membrane protein is ubiquitinated, it undergoes endocytosis and is degraded either by lysosomes or by the proteosome. Cells also contain deubiquitinating enzymes (DUBs). Thus the amount of time a protein stays in the plasma membrane depends on the rate that ubiquitin groups are added by the ligases versus the rate that they are removed by the DUBs. For example, Na+ reabsorption by the collecting ducts of the kidneys is stimulated by the adrenal hormone aldosterone (see ). One of the actions of aldosterone is to inhibit Nedd4-2. This prevents ubiquitination of ENaC in the apical membrane of epithelial cells. Thus the channels are retained for a longer period of time in the membrane, and as a result, more Na+ enters the cell and is thereby reabsorbed.

macrophages have receptors on their surface that bind the Fc portion of immunoglobulins. When bacteria invade the body they are often coated with antibody, a process called opsonization. These bacteria then attach to the membrane of macrophages via the fragment crystallizable (Fc) portion of the immunoglobulin, undergo phagocytosis, and are destroyed inside the cell. The third mechanism of endocytosis is receptor-mediated endocytosis, which allows the uptake of specific molecules into the cell. In this form of endocytosis, molecules bind to receptors on the surface of the cell. Endocytosis involves a number of accessory proteins, including adaptin, clathrin, and the GTPase dynamin (

Fig. 1.5

Exocytosis can be either constitutive or regulated. Constitutive secretion occurs, for example, in plasma cells that are secreting immunoglobulin or in fibroblasts secreting collagen. Regulated secretion occurs in endocrine cells, neurons, and exocrine glandular cells (e.g., pancreatic acinar cells). In these cells, the secretory product (e.g., hormone, neurotransmitter, or digestive enzyme), after synthesis and processing in the rough endoplasmic reticulum and Golgi apparatus, is stored in the cytoplasm in secretory granules until an appropriate signal for secretion is received. These signals may be hormonal or neural. Once the cell receives the appropriate stimulus, the secretory vesicle fuses with the plasma membrane and releases its contents into the extracellular fluid. Fusion of the vesicle with the membrane is mediated by a number of accessory proteins. One important group is the SNARE (soluble N-ethylmaleimide sensitive fusion protein [NSF] attachment protein receptors) proteins. These membrane proteins help target the secretory

Receptor Formation of Formation of coated pit coated vesicle Dynamin

Uncoated vesicle ready to fuse Vesicle (e.g., lysosome) uncoating • Fig. 1.5 Receptor-mediated endocytosis. Receptors on the surface of the cell bind the ligand. A clathrin-coated pit is formed with adaptin linking the receptor molecules to clathrin. Dynamin, a guanosine triphosphatase (GTPase), assists in separation of the endocytic vesicle from the membrane. Once inside the cell, the clathrin and adaptin molecules dissociate and are recycled. The uncoated vesicle is then ready to fuse with other organelles in the cell (e.g., lysosomes). (Adapted from Ross MH, Pawlina W: Histology. 5th ed. Baltimore: Lippincott Williams & Wilkins; 2006.)

Cholesterol is an important component of cells (e.g., it is a key component of membranes). However, most cells are unable to synthesize cholesterol and therefore must obtain it from the blood. Normally, cholesterol is ingested in the diet, and it is transported through the blood in association with lipoproteins. Low-density lipoproteins (LDLs) in the blood carry cholesterol to cells, where they bind to LDL receptors in the plasma membrane. After the receptors bind LDL, they collect into “coated pits” and undergo endocytosis as clathrin-coated vesicles. Once inside the cell, the endosomes release LDL and then recycle the LDL receptors back to the cell surface. Inside the cell, LDL is then degraded in lysosomes, and the cholesterol is made available to the cell. Defects in the LDL receptor prevent cellular uptake of LDL. Individuals with this defect have elevated levels of blood LDL, often called “bad cholesterol,” because it is associated with the development of cholesterol-containing plaques in the smooth muscle layer of arteries. This process, atherosclerosis, is associated with an increased risk for heart attacks as a result of occlusion of the coronary arteries.

vesicle to the plasma membrane. The process of secretion is usually triggered by an increase in the concentration of intracellular Ca++ ([Ca++]). However, two notable exceptions to this general rule exist: (1) Renin secretion by the juxtaglomerular cells of the kidney occurs with a decrease in intracellular Ca++ (see ), as does (2) the secretion of parathyroid hormone by the parathyroid gland (see

Basic Principles of Solute and Water Transport

As already noted, the plasma membrane, with its hydrophobic core, is an effective barrier to the movement of virtually all biologically important molecules into or out of the cell. Thus membrane transport proteins provide the pathway that allows transport to occur into and out of cells. However, the presence of a pathway is not sufficient for transport to occur; an appropriate driving force is also required. In this section, the basic principles of diffusion, active and passive transport, and osmosis are presented. These topics are discussed in greater depth, as appropriate, in the other sections of the book.

Diffusion is the process by which molecules move spontaneously from an area of high concentration to one of low concentration. Thus wherever a concentration gradient exists, diffusion of molecules from the region of high concentration to the region of low concentration dissipates the gradient (as discussed later, the establishment of concentration gradients for molecules requires the expenditure of energy). Diffusion is a random process driven by the thermal motion of the molecules. Fick’s first law of diffusion quantifies the rate at which a molecule diffuses from point A to point B:

Equation 1.1 where J = the flux or rate of diffusion per unit time D = the diffusion coefficient A = area across which the diffusion is occurring ΔC = the concentration difference between point A and B ΔX = the distance along which diffusion is occurring

The diffusion coefficient takes into account the thermal energy of the molecule, its size, and the viscosity of the medium through which diffusion is taking place. For spherical molecules, D is approximated by the Stokes-Einstein equation:

Equation 1.2 where k = Boltzmann’s constant T = temperature in degrees Kelvin r = radius of the molecule η= viscosity of the medium

According to eqs. 1.1 and 1.2 , the rate of diffusion will be faster for small molecules than for large molecules. In addition, diffusion rates are high at elevated temperatures, in the presence of large concentration gradients, and when diffusion occurs in a low-viscosity medium. With all other variables held constant, the rate of diffusion is linearly related to the concentration gradient.

Fick’s equation can also be applied to the diffusion of molecules across a barrier, such as a lipid bilayer. When applied to the diffusion of a molecule across a bilayer, the diffusion coefficient (D) incorporates the properties of the bilayer and especially the ability of the molecule to diffuse through the bilayer. To quantify the interaction of the molecule with the bilayer, the term partition coefficient (β) is used. For a molecule that “dissolves” equally in the fluid bathing the lipid bilayer (e.g., water) and in the lipid bilayer, β= 1. If the molecule dissolves more easily in the lipid bilayer, β> 1; and if it dissolves less easily in the lipid bilayer, β< 1. For a simple lipid bilayer, the more lipid soluble the molecule is, the larger the partition coefficient is, and thus the diffusion coefficient—therefore the rate of diffusion of the molecule across the bilayer—is greater. In this situation, ΔC represents the concentration difference across the membrane, A is the membrane area, and ΔX is the thickness of the membrane.

Another useful equation for quantitating the diffusion of molecules across the plasma membrane (or any membrane) is as follows:

Equation 1.3

J =−PC( i − Co), where J = the flux or rate of diffusion across the membrane P = is the permeability coefficient Ci = concentration of the molecule inside the cell Co = the concentration of the molecule outside the cell

This equation is derived from Fick’s equation ( eq. 1.1 ). P incorporates D, ΔX, A, and the partition coefficient (β). P is expressed in units of velocity (e.g., centimeters per second), and C the units of moles/cm3. Thus the units of flux are moles per square centimeter per second (mol/cm2/ sec). Values for P can be obtained experimentally for any molecule and bilayer.

As noted, the phospholipid portion of the plasma membrane represents an effective barrier to many biologically important molecules. Consequently, diffusion through the lipid phase of the plasma membrane is not an efficient process for movement of these molecules across the membrane. It has been estimated that for a cell 20 µm in diameter, with a plasma membrane composed only of phospholipids, dissipation of a urea gradient imposed across the membrane would take approximately 8 minutes. Similar gradients for glucose and amino acids would take approximately 14 hours to dissipate, whereas ion gradients would take years to dissipate.

As noted previously, the vast majority of biologically important molecules cross cell membranes via specific membrane transporters, rather than by diffusing through the lipid portion of the membrane. Nevertheless, eq. 1.3 can be and has been used to quantitate the diffusion of molecules across many biological membranes. When this is done, the value of the permeability coefficient (P) reflects the properties of the pathway (e.g., membrane transporter or, in some cases, multiple transporters) that the molecule uses to cross the membrane.

Despite the limitations of using diffusion to describe and understand the transport of molecules across cell membranes, it is also important for understanding gas exchange in the lungs (see ), the movement of molecules through the cytoplasm of the cell, and the movement of molecules between cells in the extracellular fluid. For example, one of the physiological responses of skeletal muscle to exercise is the recruitment or opening of capillaries that are not perfused at rest. This opening of previously closed capillaries increases capillary density and thereby reduces the diffusion distance between the capillary and the muscle fiber so that oxygen and cellular fuels (e.g., fatty acids and glucose) can be delivered more quickly to the contracting muscle fiber. In resting muscle, the average distance of a muscle fiber from a capillary is estimated to be 40 µm. However, with exercise, this distance decreases to 20 µm or less.

The electrochemical gradient (also called the electrochemical potential difference) is used to quantitate the driving force acting on a molecule to cause it to move across a membrane. The electrochemical gradient for any molecule (Δµx) is calculated as follows:

Equation 1.4 ˜µx = RTln + zFV ,[X]o xm where R = the gas constant T = temperature in degrees Kelvin Ln = natural logarithm [X]i = the concentration of X inside the cell [X]o = the concentration of X outside the cell zx = the valence of charged molecules F = the Faraday constant Vm = the membrane potential (Vm = Vi − Vo)

The electrochemical gradient is a measure of the free energy available to carry out the useful work of transporting the molecule across the membrane. It has two components: One component represents the energy in the concentration gradient for X across the membrane (chemical potential bBy convention, membrane voltages are determined and reported with regard to the exterior of the cell. In a typical cell, the resting membrane potential (Vm) is negative. Positive Vm values can be observed in some excitable cells at the peak of an action potential.

CHAPTER 1 Principles of Cell and Membrane Function difference). The second component (electrical potential difference) represents the energy associated with moving charged molecules (e.g., ions) across the membrane when a membrane potential exits (i.e., Vm ≠ 0 mV). Thus for the movement of glucose across a membrane, only the concentrations of glucose inside and outside of the cell need to be considered (Fig. 1.6A ). However, the movement of K+ across the membrane, for example, would be determined both from the K+ concentrations inside and outside of the cell and from the membrane voltage (see

Fig. 1.6B

Eq. 1.4 can be used to derive the Nernst equation for the situation in which a molecule is at equilibrium across the membrane (i.e., Δµ = 0):

Equation 1.5a

Equation 1.5b

The value of Vm calculated with the Nernst equation represents the equilibrium condition and is referred to as the Nernst equilibrium potential (Ex, the Vm at which there is no net transport of the molecule across the membrane). It should be apparent that the Nernst equilibrium potential quantitates the energy in a concentration gradient and expresses that energy in millivolts. For example, for the cell depicted in

Fig. 1.6B , the energy in the K+ gradient (derived from the Nernst equilibrium potential for K+ [EK+]) is proportional to 90.8 mV (causing K+ to move out of the cell). This is opposite to, and of greater magnitude than, the energy in the membrane voltage (Vm =−60 mV), which causes K+ to enter the cell. As a result, the electrochemical gradient is such that the net movement of K+ across the membrane will be out of the cell. Another way to state this is that the net driving force for K+ (Vm − EK+) is 30.8 mV (driving K+ out of the cell). This is described in more detail in

The Nernst equation, at 37° C, can be written as follows by replacing the natural logarithm function with the base 10 logarithm function:

Equation 1.6a 61 5. mV [X]i

Equation 1.6b 615. mV [X]E = log o x zx [X]i •Fig. 1.6 Electrochemicalgradientsandcellulartransportofmolecules.A, Becauseglucoseisuncharged,theelectrochemicalgradientisdeterminedsolelybytheconcentrationgradientforglucoseacrossthecellmembrane.Asshown,theglucoseconcentrationgradientwouldbeexpectedtodriveglucoseintothecell.B, BecauseK+ ischarged,theelectrochemicalgradientisdeterminedbyboththeconcentrationgradientandthemembranevoltage(Vm).TheNernstequilibriumpotentialforK+ (EK+ ),calculatedwith eq.1.5a ,is−90.8mV(EK+= Vm atequilibrium).Theenergyintheconcentrationgradient,whichdrivesK+ outofthecell,isthusproportionalto+90.8mV.Themembranevoltageof−60mVdrivesK+ intothecell.Thustheelectrochemicalgradient,ornetdrivingforce,is2.97kJ/mol(equivalentto30.8mV),whichdrivesK+ outofthecell.

These are the most common forms of the Nernst equation in use. In these equations, it is apparent that for a univalent ion (e.g., Na+ , K+ , Cl−), a 10-fold concentration gradient across the membrane is equivalent in energy to an electrical potential difference of 61.5 mV (at 37° C), and a 100-fold gradient is equivalent to an electrical potential difference of 123 mV. Similarly, for a divalent ion (e.g., Ca++), a 10-fold concentration gradient is equivalent to a 30.7-mV electrical potential difference, because z in eqs. 1.6a and 1.6b is equal to 2.

When the net movement of a molecule across a membrane occurs in the direction predicted by the electrochemical gradient, that movement is termed passive transport. Thus for the examples given in

Fig. 1.6 , the movement of glucose into the cell and the movement of K+ out of the cell would be considered passive transport. Transport that is passive is sometimes referred to as either “downhill transport” or “transport with the electrochemical gradient.” In contrast, if the net movement of a molecule across the membrane is opposite to that predicted by the electrochemical gradient, that movement is termed active transport, a process that requires the input of energy (e.g., ATP). Active transport is sometimes referred to as either “uphill transport” or “transport against the electrochemical gradient.”

In the various classes of plasma membrane transport proteins, the movement of H2O through water channels is a passive process (see later discussion), as is the movement of ions through ion channels and the transport of molecules via uniporters (e.g., transport of glucose via GLUT-1). The ATPase-dependent transporters can use the energy in ATP to drive active transport of molecules (e.g., Na+,K+-ATPase, H+-ATPase, or ABC transporters). Because the transport is directly coupled to the hydrolysis of ATP, it is referred to as primary active transport. Solute carriers that couple movement of two or more molecules (e.g., 3Na+,Ca++ antiporter) often transport one or more molecules (one Ca++ molecule in this example) against their respective electrochemical gradient through the use of the energy in the electrochemical gradient of the other molecule or molecules (three Na+ in this example). When this occurs, the molecule or molecules transported against their electrochemical gradient are said to be transported by secondary active transport mechanisms (

Fig. 1.7

The movement of water across cell membranes occurs by the process of osmosis. The movement of water is passive, with the driving force for this movement being the osmotic pressure difference across the cell membrane.

Fig. 1.8 illustrates the concept of osmosis and the measurement of the osmotic pressure of a solution.

Osmotic pressure is determined by the number of solute molecules dissolved in the solution. It is not dependent on such factors as the size of the molecules, their mass, or their chemical nature (e.g., valence). Osmotic pressure (π), measured in atmospheres (atm), is calculated by van’t Hoff’s law as follows:

Equation 1.7 ˜= nCRT,

CHAPTER 1 Principles of Cell and Membrane Function

K+ Na+ 3Na+ Na+, K+ -ATPase 3Na+-Ca++ antiporter IntracellularconcentrationsExtracellularconcentrations3Na+ Na+: 145 mEq/L K+: 4 mEq/L Glucose: 5 mmol/L Ca++: 2.5 mEq/L (ionized) Na+: 12 mEq/L K+: 120 mEq/L Glucose: 2 mmol/L Ca++: 0.001 mEq/L (ionized) Ca++ Secondary active transport of Ca++ Primary active transport of Na+ and K+ Passive transport Na+ channel K+ channel Glucose uniporter Glucose 2K+ ATP •Fig. 1.7 Examplesofseveralmembranetransporters,illustratingprimaryactive,passive,andsecondaryactivetransport.Seetextfordetails.ATP,adenosinetriphosphate.

Glucoseistransportedbytheepithelialcellsthatlinethegastrointestinaltract(smallintestine),andbycellsthatformtheproximaltubulesofthekidneys.Inthegastrointestinaltract,theglucoseisabsorbedfromingestedfood.Inthekidney,theproximaltubulereabsorbstheglucosethatwasfilteredacrosstheglomerularcapillariesandtherebypreventsitfrombeinglostintheurine.Theuptakeofglucoseintotheepithelialcellfromthelumenofthesmallintestineandfromthelumenoftheproximaltubuleisasecondaryactiveprocessinvolvingthesodium-glucose–linkedtransportersSGLT-1andSGLT-2.SGLT-2transportsoneglucosemoleculewithoneNa+ ion,andtheenergyintheelectrochemicalgradientforNa+ (intothecell)drivesthesecondaryactiveuptakeofglucose.Accordingtothefollowingequation,forcalculatingtheelectrochemicalgradient,andifthemembranepotential(Vm)is−60mVandthereisa10-fold[Na+]gradientacrossthemembrane,anapproximate100-foldglucosegradientcouldbegeneratedbySGLT-2:[Glucose]i [Na+ ]o V 615. mV

Thus,iftheintracellularglucoseconcentrationwas2mmol/L,thecellcouldlowertheextracellularglucoseconcentrationtoapproximately0.02mmol/L.However,byincreasingthenumberofNa+ ionstransportedwithglucosefromonetotwo,SGLT-1cangenerateanearly10,000-foldglucosegradient: 615. mV

Again,iftheintracellularglucoseconcentrationis2mmol/L,SGLT-1couldremovevirtuallyallglucosefromeitherthelumenofthesmallintestineorthelumenoftheproximaltubule(i.e.,theluminalglucoseconcentration≅ 0.0002mmol/L).

where molecules at 37°C can exert an osmotic pressure of 2.54 n = number of dissociable particles per molecule × 10−2 atm, as calculated with eq. 1.7 and the following C = total solute concentration values: R = gas constant n = 1 T = temperature in degrees Kelvin C = 0.001 mol/L

For a molecule that does not dissociate in water, such as R = 0.082 atm L/mol K glucose or urea, a solution containing 1 mmol/L of these T = 310 °K •Fig. 1.8 Schematicrepresentationofosmoticwatermovementandthegenerationofanosmoticpressure.CompartmentAandcompartmentBareseparatedbyasemipermeablemembrane(i.e.,themembraneishighlypermeablebywaterbutimpermeablebysolute).CompartmentAcontainsasolute,whereascompartmentBcontainsonlydistilledwater.Overtime,watermovesbyosmosisfromcompartmentBtocompartmentA.(Note:Thiswatermovementisdrivenbytheconcentrationgradientforwater.BecauseofthepresenceofsoluteparticlesincompartmentA,theconcentrationofwaterincompartmentAislessthanthatincompartmentB.Consequently,watermovesacrossthesemipermeablemembranefromcompartmentBtocompartmentAdownitsconcentrationgradient.)ThiscausestheleveloffluidtoberaisedincompartmentAandloweredincompartmentB.Atequilibrium,thehydrostaticpressureexertedbythecolumnofwater(h)stopsthenetmovementofwaterfromcompartmentBtoA.Thusatequilibrium,thehydrostaticpressureisequalandoppositetotheosmoticpressureexertedbythesoluteparticlesincompartmentA.(RedrawnfromKoeppenBM,StantonBA.Renal Physiology. 4thed.St.Louis:Mosby;2006.)

Because 1 atm equals 760 mm Hg at sea level, π for this solution can also be expressed as 19.3 mm Hg. Alternatively, osmotic pressure is expressed in terms of osmolarity (see the following section). Regardless of the molecule, a solution containing 1 mmol/L of the molecule therefore exerts an osmotic pressure proportional to 1 mOsm/L.

For molecules that dissociate in a solution, n of eq. 1.7 will have a value other than 1. For example, a 150-mmol/L solution of NaCl has an osmolarity of approximately 300 mOsm/L because each molecule of NaCl dissociates into a Na+ and a Cl− ion (i.e., n = 2).

If dissociation of a molecule into its component ions is not complete, n will not be an integer. Accordingly, osmolarity for any solution can be calculated as follows:

Equation 1.8

Osmolarity = concentration × number of dissociable particles mOsm/L = mmol/L × number of particles

The terms osmolarity and osmolality are frequently confused and incorrectly interchanged. Osmolarity refers to the osmotic pressure generated by the dissolved solute molecules in 1 L of solvent, whereas osmolality is the number of molecules dissolved in 1 kg of solvent. For a dilute solution, the difference between osmolarity and osmolality is cNaCl does not completely dissociate in water. The value for n is 1.88 rather than 2. However, for simplicity, the value of 2 is most often used.

insignificant. Measurements of osmolarity are temperature dependent because the volume of the solvent varies with temperature (i.e., the volume is larger at higher temperatures). In contrast, osmolality, which is based on the mass of the solvent, is temperature independent. For this reason, osmolality is the preferred term for biologic systems and is used throughout this book. Because the solvent in biological solutions and bodily fluids is water, and because of the dilute nature of biological solutions and bodily solutions, osmolalities are expressed as milliosmoles per kilogram of water (mOsm/kg H2O).

The tonicity of a solution is related to the effect of the solution on the volume of a cell. Solutions that do not change the volume of a cell are said to be isotonic. A hypotonic solution causes a cell to swell, whereas a hypertonic solution causes a cell to shrink. Although related to osmolality, tonicity also accounts for the ability of the molecules in solution to cross the cell membrane.

Consider two solutions: a 300-mmol/L solution of sucrose and a 300-mmol/L solution of urea. Both solutions have an osmolality of 300 mOsm/kg H2O and therefore are said to be isosmotic (i.e., they have the same osmolality). When red blood cells—which for the purpose of this illustration also have an intracellular fluid osmolality of 300 mOsm/kg H2O—are placed in the two solutions, those in the sucrose solution maintain their normal volume, whereas those placed in urea swell and eventually burst. Thus the sucrose solution is isotonic and the urea solution is hypotonic. The differential effect of these solutions on

CHAPTER 1 Principles of Cell and Membrane Function red blood cell volume is related to the permeability of the red blood cell plasma membrane to sucrose and urea. The red blood cell membrane contains uniporters for urea. Thus urea easily crosses the cell membrane (i.e., the cell is permeable by urea), driven by the concentration gradient (i.e., extracellular urea concentration > intracellular urea concentration). In contrast, the red blood cell membrane does not contain sucrose transporters, and sucrose cannot enter the cell (i.e., the cell is impermeable by sucrose).

To exert an osmotic pressure across a membrane, a molecule must not cross the membrane. Because the red blood cell membrane is impermeable by sucrose, it exerts an osmotic pressure equal and opposite to the osmotic pressure generated by the contents within the red blood cell (in this case, 300 mOsm/kg H2O). In contrast, urea is readily able to cross the red blood cell membrane, and it cannot exert an osmotic pressure to balance that generated by the intracellular solutes of the red blood cell. Consequently, sucrose is termed an effective osmole, whereas urea is an ineffective osmole.

To take into account the effect of a molecule’s ability to permeate the membrane on osmotic pressure, it is necessary to rewrite eq. 1.7 as follows:

Equation 1.9 ˜e = ˙(nCRT), where σ is the reflection coefficient (or osmotic coefficient) and is a measure of the relative ability of the molecule to cross the cell membrane, and Πe is the “effective osmotic pressure.”

For a molecule that can freely cross the cell membrane, such as urea in the preceding example, σ= 0, and no effective osmotic pressure is exerted (e.g., urea is an ineffective osmole for red blood cells). In contrast, σ= 1 for a solute that cannot cross the cell membrane (in the preceding example, sucrose). Such a substance is said to be an effective osmole. Many molecules are neither completely able nor completely unable to cross cell membranes (i.e., 0 <σ< 1) and generate an osmotic pressure that is only a fraction of what is expected from the molecules’ concentration in solution.

Oncotic pressure is the osmotic pressure generated by large molecules (especially proteins) in solution. As illustrated in

Fig. 1.9 , the magnitude of the osmotic pressure generated by a solution of protein does not conform to van’t Hoff’s law. The cause of this anomalous relationship between protein concentration and osmotic pressure is not completely understood, but it appears to be related to the size and shape of the protein molecule. For example, the correlation to van’t Hoff’s law is more precise with small, globular proteins than with larger protein molecules.

The oncotic pressure exerted by proteins in human plasma has a normal value of approximately 26 to 28 mm Hg. Although this pressure appears to be small in relation to osmotic pressure (28 mm Hg ≅ 1.4 mOsm/kg H2O), it is an important force involved in fluid movement across capillaries (see •Fig. 1.9 Relationshipbetweentheconcentrationofplasmapro-teinsinsolutionandtheosmoticpressure(oncoticpressure)theygenerate.Proteinconcentrationisexpressedingramsperdeciliter.Normalplasmaproteinconcentrationisindicated.Notehowtheactualpressuregeneratedexceedsthatpredictedbyvan’tHoff’slaw.80 60 40 20 0 2 6 10 14 Protein (g/dl) Predicted by van’t Hoff’s law Normal plasma ActualOsmotic pressure (g/dL)

The total concentration of all molecules in a solution can also be measured as specific gravity. Specific gravity is defined as the weight of a volume of solution divided by the weight of an equal volume of distilled water. Thus the specific gravity of distilled water is 1. Because biological fluids contain a number of different molecules, their specific gravities are greater than 1. For example, normal human plasma has a specific gravity in the range of 1.008 to 1.010.

Thespecificgravityofurineissometimesmeasuredinclinicalsettingsandusedtoassesstheurineconcentratingabilityofthekidneys.Thespecificgravityofurinevariesinproportiontoitsosmolality.However,becausespecificgravitydependsbothonthenumberofmoleculesandontheirweight,therelationshipbetweenspecificgravityandosmolalityisnotalwayspredictable.Forexample,inpatientswhohavereceivedaninjectionofradiocontrastdye(molecularweight> 500g/mole)forx-raystudies,valuesofurinespecificgravitycanbehigh(1.040to1.050),eventhoughtheurineosmolalityissimilartothatofplasma(e.g.,300mOsm/kgH2O).

The plasma membrane is a lipid bilayer composed of phospholipids and cholesterol, into which are embedded a wide range of proteins. One class of these membrane proteins (membrane transport proteins or transporters) is involved in the selective and regulated transport of molecules into and out of the cell. These transporters include water channels (aquaporins), ion channels, solute carriers, and ATP-dependent transporters.

The movement of molecules across the plasma membrane through ion channels and solute carriers is driven by chemical concentration gradients and, chemical concentration gradients and electrical potential differences (charged molecules only). The electrochemical gradient is used to quantitate this driving force. ATP-dependent transporters use the energy in ATP to transport molecules across the membrane and often establish the chemical

Alberts B, et al. Essential Cell Biology. 4th ed. New York: Garland Science; 2014.

Altenberg GA, Ruess L. Mechanisms of water transport across cell membranes and epithelia. In: Alpern R, Moe O, Kaplan M, eds. Seldin and Giebisch’s The Kidney—Physiology and Pathophysiology. 5th ed. New York: Academic Press; 2013.

Hediger MA, et al. The ABCs of membrane transporters in health and disease (SLC series): introduction. Mol Aspects Med. 2013; 34:95-107.

and electrical gradients that then drive the transport of other molecules through channels and by the solute carriers. Water movement through aquaporins is driven by an osmotic pressure difference across the membrane.

• Transport across the membrane is classified as passive or active. Passive transport is the movement of molecules as expected from the electrochemical gradient for that molecule. Active transport represents transport against the electrochemical gradient. Active transport is further divided into primary active and secondary active transport. Primary active transport is directly coupled to the hydrolysis of ATP (e.g., ATP-dependent transporters). Secondary active transport occurs with coupled solute carriers, for which passive movement of one or more molecules drives the active transport of other molecules (e.g., Na+-glucose symporter, Na+-H+ antiporter).

Pawlina W. Histology: A Text and Atlas with Correlated Cell and Molecular Biology. 7th ed. Alphen aan den Rijn, The Netherlands: Wolters Kluwer; 2016.

Rojeck A, et al. A current view of mammalian aquaglyceroporins. Annu Rev Physiol. 2008;70:301-327.

Ruess L, Altenberg GA. Mechanisms of ion transport across cell membranes. In: Alpern R, Moe O, Kaplan M, eds. Seldin and Giebisch’s The Kidney—Physiology and Pathophysiology. 5th ed. New York: Academic Press; 2013.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

What is steady-state balance, and, with water balance as an example, what are the elements needed to achieve steady-state balance?

2.

What are the volumes of the body fluid compartments, and how do they change under various conditions?

3.

How do the body fluid compartments differ with regard to their composition?

4.

What determines the resting membrane potential of cells?

5.

How do cells regulate their volume in isotonic, hypotonic, and hypertonic solutions?

6.

What are the structural features of epithelial cells, how do they carry out vectorial transport, and what are the general mechanisms by which transport is regulated?

ormal cellular function requires that the intracellular composition—with regard to ions, small molecules, water, pH, and a host of other substances—be maintained within a narrow range. This is accomplished by the transport of many substances and water into and out of the cell via membrane transport proteins, as described in . In addition, each day, food and water are ingested, and waste products are excreted from the body. In a healthy individual, these processes occur without significant changes in either the volume of the body fluid compartments or their composition. The maintenance of constant volume and composition of the body fluid compartments (and their temperature in warm-blooded animals and humans) is termed homeostasis. The human body has multiple systems designed to achieve homeostasis, the details of which are explained in the various chapters of this book. In this chapter, the basic principles that underlie the maintenance of homeostasis are outlined. In addition, the volume and composition of the various body fluid compartments are defined.

Concept of Steady-State Balance

The human body is an “open system,” which means that substances are added to the body each day and, similarly, substances are lost from the body each day. The amounts added to or lost from the body can vary widely, depending on the environment, access to food and water, disease processes, and even cultural norms. In such an open system, homeostasis occurs through the process of steady-state balance.

To illustrate the concept of steady-state balance, consider a river on which a dam is built to create a synthetic lake. Each day, water enters the lake from the various streams and rivers that feed it. In addition, water is added by underground springs, rain, and snow. At the same time, water is lost through the spillways of the dam and by the process of evaporation. For the level of the lake to remain constant (i.e., steady-state balance), the rate at which water is added, regardless of source, must be exactly matched by the amount of water lost, again regardless of route. Because the addition of water is not easily controlled and the loss by evaporation cannot be controlled, the only way to maintain a constant level of the lake is to regulate the amount that is lost through the spillways.

To understand steady-state balance as it applies to the human body, the following key concepts are important.

1.

There must be a “set point” so that deviations from this baseline can be monitored (e.g., the level of the lake in the preceding example, or setting the temperature in a room by adjusting the thermostat).

2.

The sensor or sensors that monitor deviations from the set point must generate “effector signals” that can lead to changes in either input or output, or both, to maintain the desired set point (e.g., electrical signals to adjust the spillway in the dam analogy, or electrical signals sent to either the furnace or air conditioner to maintain the proper room temperature).

3.

“Effector organs” must respond in an appropriate way to the effector signals generated by the set point monitor (i.e., the spillway gates must operate, and the furnace or air conditioner must turn on).

4.

The sensitivity of the system (i.e., how much of a deviation from the set point is tolerated) depends on several factors, including the nature of the sensor (i.e., how much of a deviation from the set point is needed for the sensor to detect the deviation), the time necessary for generation of the effector signals, and how rapidly the effector organs respond to the effector signals.

1.

2.

1.

2.

3.

4.

•Fig. 2.1 Whole-Body Steady-State Water Balance. Seetextfordetails.ADH,antidiuretichormone(alsocalledargininevasopressin);CNS,centralnervoussystem;GI,gastrointestinal.

It is important to recognize that deviations from steady-state balance do occur. When input is greater than output, a state of positive balance exists. When input is less than output, a state of negative balance exists. Although transient periods of imbalance can be tolerated, prolonged states of positive or negative balance are generally incompatible with life.

Fig. 2.1 illustrates several important concepts for the maintenance of steady-state water balance (details related to the maintenance of steady-state water balance are presented in ). As depicted in

Fig. 2.1 , there are multiple inputs and outputs of water, many of which can vary but nevertheless cannot be regulated. For example, the amount of water lost through the lungs depends on the humidity of the air and the rate of respiration (e.g., low humidity and rapid breathing increase water loss from the lungs). Similarly, the amount of water lost as sweat varies according to ambient temperature and physical activity. Finally, water loss via the gastrointestinal tract can increase from a normal level of 100 to 200 mL/day to many liters with acute diarrhea. Of these inputs and outputs, the only two that can be regulated are increased ingestion of water in response to thirst and alterations in urine output by the kidneys (see

Chapter 35).

Water balance determines the osmolality of the body fluids. Cells within the hypothalamus of the brain monitor body fluid osmolality for deviations from the set point (normal range: 280-295 mOsm/kg H2O). When deviations are sensed, two effector signals are generated. One is neural and relates to the individual’s sensation of thirst. The other is hormonal (antidiuretic hormone, also called arginine vasopressin), which regulates the amount of water excreted by the kidneys. With appropriate responses to these two signals, water input, water output, or both are adjusted to maintain balance and thereby keep body fluid osmolality at the set point.

Volumes and Composition of Body Fluid Compartments

Unicellular organisms maintain their volume and composition through exchanges with the environment they inhabit (e.g., sea water). The billions of cells that constitute the human body must maintain their volume and composition as well, but their task is much more difficult. This challenge, as well as its solution, was first articulated by the French physiologist Claude Bernard (1813-1878). He recognized that although cells within the body cannot maintain their volume and composition through exchanges with the environment, they can do so through exchanges with the fluid environment that surrounds them (i.e., the extracellular fluid). Bernard referred to the extracellular fluid as the milieu intérieur (“the environment within”). He also recognized that the organ systems of the body are designed and function to maintain a constant milieu interieur or a “constant internal environment.” This in turn allows all cells to maintain their volume and composition through exchanges with the extracellular fluid as a result of membrane transport (see

Transport by the epithelial cells of the gastrointestinal tract, kidneys, and lungs are the body’s interface with the

CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments external environment and control both the intake and excretion of numerous substances, as well as water. The cardiovascular system delivers nutrients to and removes waste products from the cells and tissues and keeps the extracellular fluid well mixed. Finally, the nervous and endocrine systems provide regulation and integration of these important functions.

To provide background for the study of all organ systems, this chapter presents an overview of the normal volume and composition of the body fluid compartments and describes how cells maintain their intracellular composition and volume. Included is a presentation on how cells generate and maintain a membrane potential, which is fundamental for understanding the function of excitable cells (e.g., neurons and muscle cells). Finally, because epithelial cells are so central to the process of regulating the volume and composition of the body fluids, the principles of solute and water transport by epithelial cells are also reviewed.

Definition and Volumes of Body Fluid Compartments

Water makes up approximately 60% of the body’s weight; variability among individuals is a function of the amount of adipose tissue. Because the water content of adipose tissue is lower than that of other tissue, increased amounts of adipose tissue reduce the fraction of water in the total body weight. The percentage of body weight attributed to water also varies with age. In newborns, it is approximately 75%. This decreases to the adult value of 60% by the age of 1 year.

Fig. 2.2, total body water is distributed between two major compartments, which are divided by the cell membrane.

The intracellular fluid (ICF) compartment is the larger compartment, and contains approximately two thirds of the total body water. The remaining third is contained in the extracellular fluid (ECF) compartment. Expressed as percentages of body weight, the volumes of total body water, ICF, and ECF are as follows:

Total body water = 0 6. × (body weight) ICF = 0 4. × (body weight) ECF = 0 2. × (body weight)

The ECF compartment is further subdivided into interstitial fluid and plasma. The ECF also includes fluid contained within bone and dense connective tissue, as well as the cerebrospinal fluid. The interstitial fluid surrounds the cells in the various tissues of the body and makes up three fourths of the ECF volume. Plasma is contained within the vascular compartment and represents the remaining fourth of the ECF. In some pathological conditions, additional fluid may accumulate in what is referred to as a third space. Third-space collections of fluid are part of the ECF; an aIn these and all subsequent calculations, it is assumed that 1 L of fluid (e.g., ICF and ECF) has a mass of 1 kg. Although, 1 L of the ICF and ECF has a mass of slightly more than 1 kg, this simplification allows conversion from measurements of body weight to volume of body fluids.

Total body water (TBW) 0.6 x body weight 42 L Extracellular fluid (ECF) 0.2 x body weight 14 L Intracellular fluid (ICF) 0.4 x body weight 28 L Cell membrane

Interstitial fluid 3/4 of ECF 10.5 L

Plasma 1/4 of ECF 3.5 L •Fig. 2.2 Relationship Between the Volumes of the Various Body Fluid Compartments. Theactualvaluesshownareforanindividualweighing70kg.(ModifiedfromLevyMN,KoeppenBM,StantonBA.Berne & Levy’s Principles of Physiology.4thed.St.Louis:Mosby;2006.) example is the accumulation of fluid in the peritoneal cavity (ascites) of individuals with liver disease.

Movement of Water Between Body Fluid Compartments

Fig. 2.2 , water moves between the ICF and ECF compartments across the plasma membranes of cells, and it moves between the vascular (plasma) and interstitial compartments across capillary walls. The pathways and driving forces for this water movement are different across cell membranes, in comparison to the capillary walls.

Movement of water between the ICF and ECF compartments, across cell membranes, occurs through aquaporins expressed in the plasma membrane (see

Chapter 1).

The driving force for this water movement is an osmotic pressure difference. The osmotic pressure of both the ICF and ECF is determined by the molecules/ions present in these fluids. For simplicity, these can be divided into (1) molecules of low molecular weight (e.g., glucose) and ions (e.g., Na+) and (2) macromolecules (e.g., proteins). The osmotic pressures of both the ICF and ECF are in the range of 280 to 295 mOsm/kg H2O. For the ECF, the low-molecular-weight molecules and ions account for nearly all of this pressure because the osmotic pressure contributed by proteins is only 1 to 2 mOsm/kg H2O. The molecules/ions contributing to the osmotic pressure within the cell are less well understood, but they also include low-molecular-weight molecules (e.g., glucose), ions (e.g., Na+), and macromolecules (e.g., proteins). The fact that cell volume remains constant when ECF osmolality is constant means that the osmotic pressure inside the cells is equal to that of the ECF. If an osmotic pressure difference did exist, the cells would either swell or shrink, as described in the section “Nonisotonic Cell Volume Regulation.”

Movement of water between the vascular (plasma) compartment and the interstitial fluid compartment occurs across the capillary wall. The amount of water that moves across the capillary wall, and the mechanism of the water movement varies depending on the capillary. For example, in the capillary sinusoids of the liver, endothelial cells are often separated by large gaps (discontinuous capillary). As a result, water and all components of the plasma (and some cellular elements) can pass easily across the wall. Other capillaries are lined by endothelial cells that contain fenestrations that are up to 80 to 100 nm in diameter (e.g., in the kidneys). These fenestrations allow all components of the plasma (only cellular elements of blood cannot pass through the fenestrations) to move across the capillary wall. Some capillaries (e.g., in the brain) form a relatively tight barrier to water and small molecules and ions, and water movement occurs through small pores on the endothelial cell surface or through clefts between adjacent endothelial cells. These pores and clefts allow water and molecules smaller than 4 nm to pass. In addition, a small amount of water traverses the capillary wall via pinocytosis by endothelial cells.

The driving forces for fluid (water) movement across the capillary wall are hydrostatic pressure and oncotic pressure (i.e., osmotic pressure generated by proteins). Collectively, these are called the Starling forces. Capillary fluid movement is discussed in detail in in brief, hydrostatic pressure within the capillary (as a result of the pumping of the heart and the effect of gravity on the column of blood in the vessels feeding a capillary) is a force that causes fluid to move out of the capillary. Hydrostatic pressure in the surrounding interstitial tissue opposes the effect of the capillary hydrostatic pressure. The oncotic pressure of the plasma in the capillary tends to draw fluid from the interstitium into the capillary. The oncotic pressure of the interstitial fluid opposes this. Thus the amount of fluid moving across the wall of the capillary is determined as follows:

Equation 2.1

Kf = filtration constant (measure of surface area + in πi = interstitial fluid oncotic pressure.

Depending on the magnitude of these forces, fluid may move out of the capillary or into the capillary.

The compositions of the various body fluid compartments differ; however, as described later, the osmolalities of the fluid within these compartments are essentially identical.

Thus the compartments are in “osmotic equilibrium.” In addition, any change in the osmolality of one compartment quickly causes water to redistribute across all compartments, which brings them back into osmotic equilibrium. Because of this rapid redistribution of water, measuring the osmolality of plasma or serum, which is easy to do, reveals the osmolality of the other body fluid compartments (i.e., interstitial fluid and intracellular fluid).

As described later, Na+ is a major constituent of the ECF. Because of its high concentration in comparison with other molecules and ions, Na+ (and its attendant anions, primarily Cl− and HCO3 −) is the major determinant of the osmolality of this compartment. Accordingly, it is possible to obtain an approximate estimate of the ECF osmolality by simply doubling the sodium concentration [Na+]. For example, if a blood sample is obtained from an individual, and the [Na+] of the serum is 145 mEq/L, its osmolality can be estimated as follows:

Equation 2.2

In contrast to water, the movement of ions across cell membranes is more variable from cell to cell and depends on the presence of specific membrane transport proteins (see the section “

Composition of Body Fluid Compart ”). Consequently, in trying to understand the physiology of fluid shifts between body fluid compartments, it can be assumed that while water moves freely between the compartments, there is little net movement of solutes. For most situations, this is a reasonable assumption.

To illustrate the physiologic characteristics of fluid shifts, consider what happens when solutions containing various amounts of NaCl are added to the ECF.

Example 1: Addition of Isotonic Sodium Chloride to the Extracellular Fluid

Addition of an isotonic NaCl solution (e.g., intravenous infusion of 0.9% NaCl: osmolality ≈ 290 mOsm/kg H2O) bSome exceptions do exist. The cerebrospinal fluid is part of the ECF, but its osmolality is slightly higher than that of the ECF elsewhere in the body. Also, regions within the kidney can have osmolalities that are either less than or greater than that of the ECF. However, these volumes are small (≈150 mL) in comparison with the total volume of the ECF (≥12 L). cSerum is derived from clotted blood. Thus serum differs from plasma by the absence of clotting factors. With regard to osmolality and the concentrations of other molecules and ions, the osmolality and concentrations in plasma and serum are virtually identical. dFluids are usually administered intravenously. When electrolyte solutions are infused by this route, equilibration between plasma and interstitial fluid is rapid (i.e., minutes) because of the high permeability of many capillary walls for water and electrolytes. Thus these fluids are essentially added to the entire ECF. eA 0.9% NaCl solution (0.9 g NaCl/100 mL) contains 154 mmol/L of NaCl. Because NaCl does not dissociate completely in solution (i.e., 1.88 Osm/mol), the osmolality of this solution is 290 mOsm/kg H2O, which is very similar to that of normal ECF.

CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments

Insomeclinicalsituations,itispossibletoobtainamoreaccurateestimateoftheserumosmolality,andthustheosmolalitiesoftheECFandICF,byalsoconsideringtheosmolescontributedbyglucoseandurea,asthesearethenextmostabundantsolutesintheECF(theothercomponentsoftheECFcontributeonlyafewadditionalmilliosmoles).Accordingly,serumosmolalitycanbeestimatedasfollows: 18 28.

Theglucoseandureaconcentrationsareexpressedinunitsofmilligramsperdeciliter(dividingby18forglucoseand2.8for allowsconversionfromtheunitsofmilligramsperdecilitertomillimolesperliterandthustomilliosmolesperkilogramofH2O).Thisestimationofserumosmolalityisespeciallyusefulintreatingpatientswhohaveanelevatedserumglucoseconcentrationsecondarytodiabetesmellitus,andinpatientswithchronicrenalfailure,whoseserumureaconcentrationiselevatedbecauseofreducedrenalexcretion.

,theabilityofasubstancetocausewatertomoveacrosstheplasmamembraneofacelldependsonwhetherthesubstanceitselfcrossesthemembrane.Recall

Eq.1.9 whereΠe= theeffectiveosmoticpressureandσ= thereflectioncoefficientforthesubstance.Formanycells,glucoseandureacrossthecellmembrane.Althoughtheycontributetoserumosmolality,asmeasuredbyalaboratoryosmometerwhereallmoleculesare“effectiveosmoles,”theyareineffectiveosmolesforwatermovementacrossmany,butnotall,cellmembranes.Incontrast,Na+ isan“effectiveosmole”forwatermovementacrosstheplasmamembraneofvirtuallyallcells.

Eq.2.2 givesthebestestimateoftheeffectiveosmolalityoftheserum.

*Theureaconcentrationinplasmaismeasuredasthenitrogenintheureamolecule,orbloodureanitrogen(BUN).

to the ECF increases the volume of this compartment by the volume of fluid administered. Because this fluid has the same osmolality as does the ECF, and therefore the ICF, there is no driving force for fluid movement between these compartments, and the volume of the ICF remains unchanged. Although Na+ can cross cell membranes, it is effectively restricted to the ECF by the activity of the Na+,K+-ATPase, which is present in the plasma membrane of all cells (see the section “

Ionic Composition of Cells”).

Therefore, there is no net movement of the infused isotonic NaCl solution into cells.

Example 2: Addition of Hypotonic Sodium Chloride to the Extracellular Fluid

Addition of a hypotonic NaCl solution to the ECF (e.g., intravenous infusion of 0.45% NaCl; osmolality ≅ 145 mOsm/kg H2O) decreases the osmolality of this fluid compartment, which results in the movement of water into

Neurosurgicalproceduresandcerebrovascularaccidents(strokes)oftenresultintheaccumulationofinterstitialfluidinthebrain(i.e.,edema)andswellingoftheneurons.Becausethebrainisenclosedwithintheskull,edemacanraiseintracranialpressureandtherebydisruptneuronalfunction,whichleadstocomaanddeath.Theblood-brainbarrier,whichseparatesthecerebrospinalfluidandbraininterstitialfluidfromblood,canbepermeatedfreelybywaterbutnotbymostothersubstances.Asaresult,excessfluidinbraintissuecanberemovedbyimposinganosmoticgradientacrosstheblood-brainbarrier.Mannitolcanbeusedforthispurpose.Mannitolisasugar(molecularweight,182g/mol)thatdoesnotreadilycrosstheblood-brainbarrierandmembranesofcells(neuronsandothercellsinthebody).Therefore,mannitolisaneffectiveosmole,andintravenousinfusionresultsinthemovementofinterstitialfluidoutofthebrainbyosmosis.

the ICF. After osmotic equilibration, the osmolalities of the ICF and ECF are again equal but lower than before the infusion, and the volume of each compartment is increased. The increase in ECF volume is greater than the increase in ICF volume.

Example 3: Addition of Hypertonic Sodium Chloride to the Extracellular Fluid

Addition of a hypertonic NaCl solution to the ECF (e.g., intravenous infusion of 3% NaCl: osmolality ≅ 1000 mOsm/kg H2O) increases the osmolality of this compartment, which results in the movement of water out of cells. After osmotic equilibration, the osmolalities of the ECF and ICF are again equal but higher than before the infusion. The volume of the ECF is increased, whereas that of the ICF is decreased.

Composition of Body Fluid Compartments

The compositions of the ECF and ICF differ considerably. The ICF has significantly more proteins and macromolecules than the ECF. There are also differences in the concentrations of many ions. The composition of the ICF is maintained by the action of a number of specific cell membrane transport proteins. Principal among these transporters is the Na+,K+adenosine triphosphatase (Na+,K+-ATPase), which converts the energy in ATP into ion and electrical gradients, which can in turn be used to drive the transport of other ions and molecules by means of ion channels and solute carriers (e.g., symporters and antiporters).

The compositions of the plasma and interstitial fluid compartments of the ECF are similar because those compartments are separated only by the capillary endothelium, a barrier that ions and small molecules can permeate. The major difference between the interstitial fluid and plasma is that the latter contains significantly more protein. Although this differential concentration of protein can affect the

Fluidandelectrolytedisordersareobservedcommonlyinclinicalpractice(e.g.,inpatientswithvomitingordiarrhea,orboth).Inmostinstances,thesedisordersareself-limited,andcorrectionofthedisorderoccurswithoutneedforintervention.However,moresevereorprolongeddisordersmaynecessitatefluidreplacementtherapy.Suchtherapymaybeadministeredorally,withspecialelectrolytesolutions,orintravenously,withfluid.

Intravenoussolutionsareavailableinmanyformulations.Thetypeoffluidadministeredtoaparticularpatientisdictatedbythepatient’sneed.Forexample,ifanincreaseinthepatient’svascularvolumeisnecessary,asolutioncontainingsubstancesthatdonotreadilycrossthecapillarywallisinfused(e.g.,5%proteinordextransolutions).Theoncoticpressuregeneratedbythealbuminmoleculescausesfluidtoberetainedinthevascularcompartment,whichexpandsitsvolume.ExpansionoftheECFisaccomplishedmostoftenwithisotonicsalinesolutions(e.g.,0.9%NaClorlactatedRingersolution).Asalreadynoted,administrationofanisotonicNaClsolutiondoesnotresultinthedevelopmentofanosmoticpressuregradientacrosstheplasmamembraneofcells.Therefore,theentirevolumeoftheinfusedsolutionremainsintheECF.

Patientswhosebodyfluidsarehyperosmoticneedhypotonicsolutions.ThesesolutionsmaybehypotonicNaCl(e.g.,0.45%NaCl)or5%dextroseinwater(D5W).AdministrationoftheD5WsolutionisequivalenttotheinfusionofdistilledwaterbecausethedextroseismetabolizedtoCO2andwater.AdministrationofthesefluidsincreasesthevolumesofboththeICFandECF.Inaddition,patientswhosebodyfluidsarehypotonicneedhypertonicsolutions.ThesearetypicallyNaCl-containingsolutions(e.g.,3%or5%NaCl).ThesesolutionsexpandthevolumeoftheECFbutdecreasethevolumeoftheICF.Otherconstituents,suchaselectrolytes(e.g.,K+)ordrugs,canbeaddedtointravenoussolutionstotailorthetherapytothepatient’sfluid,electrolyte,andmetabolicneeds.

distribution of cations and anions between these two compartments by the Gibbs-Donnan effect (see the section “ ” for details), this effect is small, and the ionic compositions of the interstitial fluid and plasma can be considered to be identical.

Maintenance of Cellular Homeostasis

Normal cellular function requires that the ionic composition of the ICF be tightly controlled. For example, the activity of some enzymes is pH dependent; therefore, intracellular pH must be regulated. In addition, the intracellular composition of other electrolytes is similarly held within a narrow range. This is necessary for the establishment of the membrane potential, a cell property especially important for the normal function of excitable cells (e.g., neurons and muscle cells) and for intracellular signaling (e.g., intracellular [Ca++]; see for details). Finally, the volume of cells must be maintained because shrinking or swelling of cells can lead to cell damage or death. The regulation of intracellular

K+ 3.5-5.0mEq/L120-150mEq/L

Ca++ 2.1-2.8(total)mmol/L 1.1-1.4(ionized)≈10−7M(ionized)

Pi1.0-1.4(total)mmol/L 0.5-0.7(ionized)0.5-0.7(ionized) *Ca++ andPi(H2PO4−/HPO4−2)areboundtoproteinsandotherorganicmolecules.Inaddition,largeamountsofCa++ canbesequesteredwithincells.LargeamountsofPiarepresentincellsaspartoforganicmolecules,suchasadenosinetriphosphate(ATP).

composition and cell volume is accomplished through the activity of specific transporters in the plasma membrane of the cells. This section is a review of the mechanisms by which cells maintain their intracellular ionic environment and their membrane potential and by which they control their volume.

Ionic Composition of Cells

The intracellular ionic composition of cells varies from tissue to tissue. For example, the intracellular composition of neurons is different from that of muscle cells, both of which differ from that of blood cells. Nevertheless, there are similar patterns, and these are presented in . In comparison with the ECF, the ICF is characterized by a low [Na+] and a high [K+]. This is the result of the activity of the Na+,K+-ATPase, which transports 3 Na+ ions out of the cell and 2 K+ ions into the cell for each ATP molecule hydrolyzed. As discussed later in this chapter, the activity of the Na+,K+-ATPase not only is important for establishing the cellular Na+ and K+ gradients but also is involved in determining, indirectly, the cellular gradients for many other ions and molecules. Of importance is that the cellular K+ gradient generated by the activity of the Na+,K+-ATPase is a major determinant of the membrane voltage because of the leak of K+ out of the cell through K+selective channels (see the section “

Membrane Potential”).

Thus the Na+,K+-ATPase converts the energy in ATP into ion gradients (i.e., Na+ and K+), and a voltage gradient (i.e., membrane voltage).

The Na+,K+-ATPase–generated ion and electrical gradients are used to drive the transport of other ions and molecules into or out of the cell (

Fig. 2.3 ). For example, as described in , a number of solute carriers couple the transport of Na+ to that of other ions or molecules. The Na+-glucose and Na+–amino acid symporters use the energy in the Na+ electrochemical gradient, directed to bring Na+

CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments •Fig. 2.3 Cell Model Depicting How Cellular Gradients and the Membrane Potential (Vm) Are Established. (1)TheNa+,K+-ATPasedecreasestheintracellular[Na+]andincreasestheintracellular[K+].SomeK+ exitsthecellviaK+-selectivechannelsandgeneratestheVm(cell’sinterioriselectricallynegative).(2)TheenergyintheNa+ electrochemicalgradientdrivesthetransportofotherionsandmoleculesthroughtheuseofvarioussolutecarriers.(3)TheVmdrivesCl− outofthecellviaCl−-selectivechannels.(4)TheCa++-ATPaseandthe3Na+-Ca++ antiportersmaintainthelowintracellular[Ca++].

into the cell, to drive the secondary active cellular uptake of glucose and amino acids. Similarly, the inwardly directed Na+ gradient drives the secondary active extrusion of H+ from the cell and thus contributes to the maintenance of intracellular pH. The 3Na+-Ca++ antiporter, along with the plasma membrane Ca++-ATPase, extrudes Ca++ from the cell and thus contributes to the maintenance of a low intracellular [Ca++].

In addition, the membrane voltage drives Cl− out of the cell through Cl−-selective channels, thus lowering the intracellular concentration below that of the ECF.

As described previously, the Na+,K+-ATPase and K+-selective channels in the plasma membrane are important determinants of the membrane potential (Vm) of the cell. For all cells within the body, the resting Vm is oriented with the interior of the cell electrically negative in relation to the ECF. However, the magnitude of the Vm can vary widely.

To understand what determines the magnitude of the Vm, it is important to recognize that any transporter that transfers charge across the membrane has the potential to influence the Vm. Such transporters are said to be fIn muscle cells, in which contraction is regulated by the intracellular [Ca++], the maintenance of a low intracellular [Ca++] during the relaxed state involves not only the activity of the plasma membrane 3Na+-Ca++ antiporter and the Ca++-ATPase but also a Ca++-ATPase molecule located in the smooth endoplasmic reticulum (see

Chapters 12 to 14 electrogenic. As might be expected, the contribution of various electrogenic transporters to the Vm is highly variable from cell to cell. For example, the Na+,K+-ATPase channel transports three Na+ and two K+ ions and thus transfers one net positive charge across the membrane. However, the direct contribution of the Na+,K+-ATPase to the Vm of most cells is only a few millivolts at the most. Similarly, the contribution of other electrogenic transporters, such as the 3Na+-Ca++ antiporter and the Na+-glucose symporter is minimal. The major determinants of the Vm are ion channels. The type (e.g., selectivity), number, and activity (e.g., gating) of these channels determine the magnitude of the Vm. As described in , rapid changes in ion channel activity underlies the action potential in neurons and other excitable cells, such as those of skeletal and cardiac muscle (see

As ions move across the membrane through a channel, they generate a current. As described in , this current can be measured, even at the level of a single channel. By convention, the current generated by the movement of cations into the cell, or the movement of anions out of the cell, is defined as negative current. Conversely, the movement of cations out of the cell, or the movement of anions into the cell, is defined as positive current. Also by convention, the magnitude of the Vm is expressed in relation to the outside of the cell; thus for a cell with a Vm of −80 mV, the interior of the cell is electrically negative in relation to the outside of the cell.

The current carried by ions moving through a channel depends on the driving force for that ion and on the conductance of the channel. As described in , the driving force is determined by the energy in the concentration gradient for the ion across the membrane (Ei), as calculated by the Nernst equation (Eq. 1.5a ) and the Vm:

Equation 2.3

Driving force = Vm − Ei.

Thus as defined by Ohm’s law, the ion current through the channel (Ii) is determined as follows:

Equation 2.4 where gi is the conductance of the channel. For a cell, the conductance of the membrane to a particular ion (Gi) is determined by the number of ion channels in the membrane and by the amount of time each channel is in the open state.

Fig. 2.4 , the Vm is the voltage at which there is no net ion flow into or out of the cell. Thus for a cell that has ion channels selective for Na+ , K+ , and Cl− ,

Equation 2.5

Equation 2.6 + [(Vm − E − )× G − ]= 0.

•Fig. 2.4 Current-Voltage Relationship of a Hypothetical Cell Containing Na+-, K+-, and Cl−-Selective Channels. Membranecurrentsareplottedoverarangeofmembranevoltages(i.e.,current-voltagerelationships).EachioncurrentiscalculatedwiththeuseofOhm’slaw,theNernstequilibriumpotentialfortheion(ECl,EK,andENa),andthemembraneconductancefortheion.Thecurrent-voltagerelationshipforthewholecellisalsoshown.Totalcellcurrent(Icell)wascalculatedwiththechordconductanceequation(see

Eq.2.7).Because80%of cellconductanceisduetoK+ ,therestingmembranevoltage(Vm)of−64.4mVisneartothatoftheNernstequilibriumpotentialforK+ .

Equation 2.7 where ˜G = G ++ G ++ G −.

Inspection of

Eq. 2.7 , which is often called the chord conductance equation, reveals that the Vm will be near to the Nernst equilibrium potential of the ion to which the membrane has the highest conductance. In

Fig. 2.4 , 80% of the membrane conductance is attributable to K+; as a result, Vm is near to the Nernst equilibrium potential for K+ (EK+). For most cells at rest, the membrane has a high conductance to K+ , and thus the Vm approximates EK+. Moreover, the Vm is greatly influenced by the magnitude of EK+ , which in turn is greatly influenced by changes in the [K+] of the ECF. For example, if the intracellular [K+] is 120 mEq/L and the extracellular [K+] is 4 mEq/L, EK+ has a value of −90.8 mV. If the extracellular [K+] is increased to 7 mEq/L, EK+ would be −79.9 mV. This change in EK+ depolarizes the Vm (i.e., Vm is less negative). Conversely, if the extracellular [K+] is decreased to 2 mEq/L, EK+ becomes −109.4 mV, and the Vm hyperpolarizes (i.e., Vm is more negative).

•Fig. 2.5 Nerve Action Potential Showing the Changes in Na+ and K+ Conductances ( G and GK+ , Respectively) and the Membrane

Potential (Vm). Atrest,themembranehasahighK+ conductance,andVmisneartheNernstequilibriumpotentialforK+ (EK+ ).Withtheinitiationoftheactionpotential,thereisalargeincreaseintheNa+ conductanceofthemembrane,andtheVmapproachestheNernstequilibriumpotentialforNa+ (ENa+ ).TheincreaseinNa+ conductanceistransient,andtheK+ conductancethenincreasesaboveitsvaluebeforetheactionpotential.ThishyperpolarizesthecellasVmapproachesEK+.AstheK+ conductancereturnstoitsbaselinevalue,Vmreturnstoitsrestingvalueof−70mV.(ModifiedfromLevyMN,KoeppenBM,StantonBA.Berne & Levy’s Principles of Physiology. 4thed.St.Louis:Mosby;2006.)

Changesintheextracellular[K+]canhaveimportanteffectsonexcitablecells,especiallythoseoftheheart.Adecreaseinextracellular[K+](hypokalemia) hyperpolarizestheVmofcardiacmyocytesand,insodoing,makesinitiatinganactionpotentialmoredifficult,becausealargerdepolarizingcurrentisneededtoreachthreshold(see ).Ifsevere,hypokalemiacanleadtocardiacarrhythmias,andeventuallytheheartcanstopcontracting(asystole). Anincreaseintheextracellular[K+](hyperkalemia) canbeequallydeleterioustocardiacfunction.Withhyperkalemia,theVmisdepolarized,anditiseasiertoinitiateanactionpotential.However,oncetheactionpotentialfiresthechannelsbecomeinactivated,andareunabletoinitiateanotheractionpotential,untiltheyarereactivatedbynormalrepolarizationoftheVm.BecausetheVmisdepolarizedinhyperkalemia,thechannelsstayinaninactivatedstate.ThusdepolarizationoftheVmwithhyperkalemiacanleadtocardiacarrhythmiasandlossofcardiacmusclecontraction.

Eq. 2.7 also defines the limits for the membrane potential. In the example depicted in

Fig. 2.4, it is apparent that the Vm cannot be more negative than EK+ (−90.8 mV), as would be the case if the membrane were only conductive to K+ . Conversely, the Vm could not be more positive than ENa+ (66.6 mV); such a condition would be met if the membrane were conductive only to Na+ . The dependence of the Vm on the conductance of the membrane to specific ions is the basis by which action potentials in excitable cells are generated (

Fig. 2.5). As noted previously, in all excitable cells, the

CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments membrane at rest is conductive predominantly to K+ , and thus Vm is near EK+. When an action potential is initiated, Na+-channels open and the membrane is now conductive predominantly to Na+ . As a result, Vm now approaches ENa+. The generation of action potentials is discussed in more detail in

TheestablishmentoftheVmrequirestheseparationofchargeacrosstheplasmamembrane.However,thenumberofionsthatmustmoveacrossthemembraneisatinyfractionofthetotalnumberofionsinthecell.Forexample,considerasphericalcellwithadiameterof20µmandaVmof−80mV.Furthermore,assumethatthisVmof−80mVistheresultofthediffusionofK+ outofthecellandthattheintracellular[K+]is120mmol/L.TheamountofK+ thatwouldhavetodiffuseoutofthecelltoestablishtheVmof−80mVisthencalculatedasfollows.

Firstthechargeseparationacrossthemembraneneedstobecalculated.Thisisdonewiththeknowledgethattheplasmamembranebehaveselectricallylikeacapacitor,thecapacitance(C)ofwhichisapproximately1µF/cm2,and whereQ= chargeandisexpressedinunitsofcoulombs.Ifthesurfaceareaofthecellis4πr2or1.26× 10−5cm2,thecapacitanceofthecelliscalculatedasfollows: 1× 10 F/cm × . × 10 cm = . × 10 126 126 F.

Thusthechargeseparationacrossthemembraneiscalculatedasfollows: 1 01× 10−12 coulombs.

Because1moleofK+ contains96,480coulombs,theamountofK+ thathadtodiffuseacrossthemembranetoestablishtheVmof−80mViscalculatedasfollows: . coul = 1 05. × 10 mole ofK , ombs/mol

Withacellvolumeof4.19× 10−12L(volume= 4πr3/3)andanintracellular[K+]of120mmol/L,thetotalintracellularK+ contentis 4 19 × 10 × . mol/L = 5 03 . 012 . × 10 moles

Therefore,thediffusionof1.05× 10−17molesofK+ outofthecellrepresentsonlya0.002%changeintheintracellularK+ content: . mol −13 ° 0 002. % 5 03 × 10 es . mol

Thustheintracellular[K+]ofthecellisnotappreciablyalteredbythediffusionofK+ outofthecell.

Regulation of Cell Volume

As already noted, changes in cell volume can lead to cell damage and death. Cells have developed mechanisms to regulate their volume. Most cells are highly permeable by water because of the presence of aquaporins in their plasma membranes. As discussed in

Chapter 1, osmotic pressure gradients across the cell membrane that are generated by effective osmoles cause water to move either into or out of the cell, which result in changes in cell volume. Thus cells swell when placed in hypotonic solutions and shrink when placed in hypertonic solutions (see the section ”). However, even when a cell is placed in an isotonic solution, the maintenance of cell volume is an active process requiring the expenditure of ATP and specifically the activity of the Na+,K+-ATPase.

The importance of the Na+,K+-ATPase in isotonic cell volume regulation can be appreciated by the observation that red blood cells swell when chilled (i.e., reduced ATP synthesis) or when the Na+,K+-ATPase is inhibited by cardiac glycosides (e.g., ouabain, digoxin [Lanoxin]). The necessity for energy expenditure to maintain cell volume in an isotonic solution is the result of the effect of intracellular proteins on the distribution of ions across the plasma membrane: the so-called Gibbs-Donnan effect (

Fig. 2.6

The Gibbs-Donnan effect occurs when a membrane separating two solutions can be permeated by some but not all of the molecules in solution. As noted previously, this effect accounts for the small differences in the ionic compositions of the plasma and the interstitial fluid. In this case, the capillary endothelium represents the membrane, and the plasma proteins are the molecules whose ability to permeate across the capillary is restricted. For cells, the membrane is the plasma membrane, and the impermeant molecules are the intracellular proteins and organic molecules.

Fig. 2.6 , the presence of impermeant molecules (e.g., protein) in one compartment results over time in the accumulation of permeant molecules/ions in the same compartment. This increases the number of osmotically active particles in the compartment containing the impermeant anions, which in turn increases the osmotic pressure, and water thereby enters that compartment. For cells, the Gibbs-Donnan effect would increase the number of osmotically active particles in the cell, and result in cell swelling. However, the activity of the Na+,K+-ATPase counteracts the Gibbs-Donnan effect by actively extruding cations (three Na+ ions are extruded, whereas two K+ ions are brought into the cell). In addition, the K+ gradient established by the Na+,K+-ATPase allows for the development of the Vm (in which the cell’s interior is electrically negative), that in turn drives Cl− and other anions out of the cell. Thus through the activity of the Na+,K+-ATPase, the number of intracellular osmotically active particles is

B the intracellular osmotic pressure and thereby restoring cell volume to normal. Conversely with cell shrinking a regulatory volume increase response transports osmolytes into the cell, raising the intracellular osmotic pressure and thereby restoring cell volume to normal. These osmolytes include ions and organic molecules such as polyols (sorbitol and myo-inositol), methylamines (glycerophosphorylcholine and betaine), and some amino acids (taurine, glutamate, and β-alanine). If the cell is exposed to the nonisotonic ECF for an extended period of time, the cell alters the intracellular levels of the organic osmolytes through metabolic processes.

•Fig. 2.6 The Gibbs-Donnan Effect. Top, TwosolutionsareseparatedbyamembranethatispermeablebyNa+ ,Cl− ,andH2Obutnotpermeablebyprotein(P−).TheosmolalityofsolutionAisidenticaltothatofsolutionB.Bottom, Cl− diffusesfromcompartmentBtocompartmentAdownitsconcentrationgradient.ThiscausescompartmentAtobecomeelectricallynegativewithregardtocompartmentB.ThemembranevoltagethendrivesthediffusionofNa+ fromcompartmentBtocompartmentA.TheaccumulationofadditionalNa+ andCl− incompartmentAincreasesitsosmolalityandcauseswatertoflowfromcompartmentBtocompartmentA(Note:theincreasevolumeofcompartmentAresultsinalower[P−]).IfthecontainercontainingthetwosolutionsweresealedatthetopsothatwatercouldnotmovefromcompartmentBtocompartmentA,thepressureincompartmentAwouldincreaseasthenumberofosmoticallyactiveparticlesincreasesinthatcompartment.

reduced from what would be caused by the Gibbs-Donnan effect, and cell volume is maintained in isotonic solutions.

Most cells throughout the body are bathed with isotonic ECF, the composition of which is tightly regulated (see ). However, certain regions within the body are not isotonic (e.g., the medulla of the kidney), and with disorders of water balance, the ECF can become either hypotonic or hypertonic. When this occurs, cells either swell or shrink. Cell swelling or shrinkage can result in cell damage or death, but many cells have mechanisms that limit the degree to which the cell volume changes. These mechanisms are particularly important for neurons, in which swelling within the confined space of the skull can lead to serious neurological damage.

In general, when a cell is exposed to nonisotonic ECF, volume-regulatory responses are activated within seconds to minutes to restore cell volume (

Fig. 2.7 ). With cell swelling, a regulatory volume decrease response transports osmotically active particles (osmolytes) out of the cell, reducing

TheECFofindividualswithdisordersinwaterbalancemaybeeitherhypotonic(positivewaterbalance)orhypertonic(negativewaterbalance).WithadecreaseinECFosmolality,neuronsandglialcellsswellaswaterentersthecell.Tominimizethisswelling,theneuronsandglialcellsreduceintracellularosmolytes.IftheECFosmolalityiscorrected(i.e.,increased)tooquickly,theneuronsandglialcellsthenshrinkbecauseofthereducednumberofintracellularosmolytes.ThisresponsetoarapidcorrectionofECFosmolalitycanleadtocelldamage.Damagetotheglialcellsthatsynthesizemyelinwithinthebraincanresultindemyelinization.Thisdemyelinizationresponse,termedosmotic demyelinization syndrome, canaffectanyofthewhitematterofthebrain,butespeciallyregionsofthepons.Theseeffectsareoftenirreversible.Therefore,correctionofdisordersofwaterbalanceisusuallyaccomplishedslowlytoavoidthisseriousneurologicalcomplication.

The regulatory volume increase response results in the rapid uptake of NaCl and a number of organic osmolytes. To increase cell volume there is an activation of the Na+-H+ antiporter (NHE-1), the 1Na+,1K+,2Cl− symporter (NKCC1), and a number of cation-selective channels, which together bring NaCl into the cell. The Na+,K+-ATPase then extrudes the Na+ in exchange for K+ , so that ultimately the KCl content of the cell is increased. Several organic osmolyte transporters are also activated to increase cell volume. These include a 3Na+,1Cl−-taurine symporter, a 3Na+,2Cl−-betaine symporter, a 2Na+–myo-inositol symporter, and a Na+– amino acid symporter. These transporters use the energy in the Na+ and Cl− gradients to drive the secondary active uptake of these organic osmolytes into cells.

The regulatory volume decrease response results in the loss of KCl and organic osmolytes from the cell. The loss of KCl occurs through the activation of a wide range of K+selective, Cl−-selective, and anion-selective channels (the specific channels involved vary depending on the cell), as well as through activation of K+-Cl− symporters. Some of the organic osmolytes appear to leave the cell via anion channels (e.g., volume-sensitive organic osmolyte-anion channels).

Several mechanisms are involved in activation of these various transporters during the volume regulatory

CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments 2Cl Na+, Cl– Na+ + •Fig. 2.7 Volume Regulation of Cells in Hypotonic and Hypertonic Media. Top, Whencellsareexposedtoahypotonicmedium,theyswellandthenundergoavolume-regulatorydecrease(RVD).TheRVDinvolveslossofKClandorganicosmolytesfromthecell.ThedecreaseincellularKClandorganicosmolytescausesintracellularosmoticpressuretodecrease,waterleavesthecell,andthecellreturnstonearlyitsoriginalvolume.Bottom, Whencellsareexposedtoahypertonicmedium,theyshrinkandthenundergoavolume-regulatoryincrease(RVI).DuringtheRVI,NaClandorganicosmolytesenterthecell.TheincreaseintheactivityofNa+,K+-ATPase(notdepicted)enhancestheexchangeNa+ forK+ sothattheK+ (andCl−)contentofthecellisincreased.TheincreaseincellularKCl,alongwithariseinintracellularorganicosmolytes,increasesintracellularosmoticpressure,whichbringswaterbackintothecell,andthecellvolumereturnstonearlyitsoriginalvolume.π,theoncoticpressureinsidethecell.

responses. Changes in cell volume appear to monitored by the cytoskeleton, by changes in macromolecular crowding and ionic strength of the cytoplasm, and by channels whose gating is influenced, either directly or indirectly, by stretch of the plasma membrane (e.g., stretch-activated cation channels). A number of second messenger systems may also be involved in these responses (e.g., intracellular [Ca++], calmodulin, protein kinase A, and protein kinase C), but the precise mechanisms have not been defined completely.

Principles of Epithelial Transport

Epithelial cells are arranged in sheets and provide the interface between the external world and the internal environment (i.e., ECF) of the body. Depending on their location, epithelial cells serve many important functions, such as establishing a barrier to microorganisms (lungs, gastrointestinal tract, and skin), prevention of the loss of water from the body (skin), and maintenance of a constant internal environment (lungs, gastrointestinal tract, and kidneys). This latter function is a result of the ability of epithelial cells to carry out regulated vectorial transport (i.e., transport from one side of the epithelial cell sheet to the opposite side). In this section, the principles of epithelial transport are reviewed. The transport functions of specific epithelial cells are discussed in the appropriate chapters throughout this book.

Fig. 2.8 shows a schematic representation of an epithelial cell. The free surface of the epithelial layer is referred to as the apical membrane. It is in contact with the external environment (e.g., air within the alveoli and larger airways of the lungs and the contents of the gastrointestinal tract) or with extracellular fluids (e.g., glomerular filtrate in the nephrons of the kidneys and the secretions of the ducts of the pancreas or sweat glands). The basal side of the epithelium rests on a basal lamina, which is secreted by the epithelial cells, and this in turn is attached to the underlying connective tissue.

Epithelial cells are connected to one another and to the underlying connective tissue by a number of specialized junctions (see

Fig. 2.8 ). The adhering junction, desmosomes, and hemidesmosomes provide mechanical adhesion by linking together the cytoskeleton of adjacent cells (adhering junction and desmosome) or to the underlying connective tissue (hemidesmosome). The gap junction and tight junction play important physiological roles.

Gap junctions provide low-resistance connections between cells.

The functional unit of the gap junction is the connexon. The connexon is composed of six integral gGap junctions are not limited to epithelial cells. A number of other cells also have gap junctions (e.g., cardiac myocytes and smooth muscle cells).

•Fig. 2.8 Schematic of an Epithelial Cell, Illustrating the Various Adhering Junctions. Thetightjunctionseparatestheapicalmembranefrombasolateralmembrane(seetextfordetails).

membrane protein subunits called connexins. A connexon in one cell is aligned with the connexon in the adjacent cell, forming a channel. The channel may be gated, and when it is open, it allows the movement of ions and small molecules between cells. Because of their low electrical resistance, they effectively couple electrically one cell to the adjacent cell.

The tight junction serves two main functions. It divides the cell into two membrane domains (apical and basolateral) and, in so doing, restricts the movement of membrane lipids and proteins between these two domains. This so-called fence function allows epithelial cells to carry out vectorial transport from one surface of the cell to the opposite surface by segregating membrane transporters to one or other of the membrane domains. They also serve as a pathway for the movement of water, ions, and small molecules across the epithelium. This pathway between the cells is referred to as the paracellular pathway, as opposed to the transcellular pathway through the cells.

The apical surface of epithelial cells may have specific structural features. One such feature is microvilli (Fig.

2.9A ). Microvilli are small (typically 1 to 3 µm in length), nonmotile projections of the apical plasma membrane that serve to increase surface area. They are commonly located on cells that must transport large quantities of ions, water, and molecules (e.g., epithelial cells lining the small intestine and cells of the renal proximal tubule). The core of the microvilli is composed of actin filaments and a number of accessory proteins. This actin core is connected to the cytoskeleton of the cell via the terminal web (a network of actin fibers at

Epithelialcelltightjunctions(alsocalledzonula occludens)arecomposedofseveralintegralmembraneproteins,includingoccludins, claudins, andseveralmembersoftheimmunoglobulinsuperfamily(e.g.,thejunctional adhesion molecule [JAM]).Occludinsandclaudinsaretransmembraneproteinsthatspanthemembraneofonecellandlinktotheextracellularportionofthesamemoleculeintheadjacentcell.Cytoplasmiclinkerproteins(e.g.,tightjunctionprotein[ZO-1,ZO-2,andZO-3])thenlinkthemembranespanningproteinstothecytoskeletonofthecell.

Ofthesejunctionalproteins,claudinsappeartobeimportantindeterminingthepermeabilitycharacteristicsofthetightjunction,especiallywithregardtocationsandanions.Todate,27mammalianclaudingeneshavebeenidentified,and26arefoundinthehumangenome(thegeneforclaudin13isnotfoundinhumans).Certainclaudinsserveasbarrierproteinsthatrestrictthemovementofionsthroughthetightjunction,whereasothersforma“pore”thatfacilitatesthemovementofionsthroughthejunction.Thusthepermeabilitycharacteristicsofthetightjunctionofanepitheliumaredeterminedbythecomplementofclaudinsexpressedbythecell.Forexample,theproximaltubuleofthekidneyistermeda“leaky”epithelium,inwhichwaterandsolutes(e.g.,Na+)movethroughthejunction.Claudin4andclaudin10areexpressedinthetightjunctionofproximaltubulecells.Incontrast,thecollectingductofthekidneyisconsidereda“tight”epithelium,withrestrictedmovementofionsthroughthetightjunction.Collectingductcellsexpressclaudins3,4,7,8,10,and18.

Thefunctionofclaudinscanberegulatedatseverallevels,includinggeneexpression,posttranslationalmodification,interactionswithcytoplasmicscaffoldingproteins,andinteractionswithotherclaudinsinthesamemembrane(cis-interaction),aswellaswithclaudinsofadjacentcells(trans-interaction).ThemineralocorticoidhormonealdosteronestimulatesNa+ reabsorptionbydistalsegmentsoftherenalnephron(see ).Inadditiontothehormone’seffectonNa+ transportersinthecell,aldosteronealsoupregulatesexpressionofclaudin8inthetightjunction.Theincreasedexpressionofclaudin8reducestheabilityofNa+ topermeatethetightjunction,whichthenreducesthebackwardsleakofNa+ fromtheinterstitiumintothetubulelumen,therebyallowingmoreefficientNa+ reabsorptionbytheepithelium.

Mutationsinthegenethatcodesforclaudin16resultintheautosomalrecessiveconditionknowasfamilial hypomagnesemia, hypercalcuria, and nephrocalcinosis (FHHNC).Claudin16isfoundinthetightjunctionofthethickascendingportionofHenle’sloopinthekidneysandservesasaroutefortheparacellularreabsorptionofCa++ andMg++ fromthetubularfluid.IndividualswithFHHNClackfunctionalcopiesofclaudin16,andreabsorptionofthesedivalentionsisthusreduced,whichleadstohypomagnesemia,hypercalcuria,andnephrocalcinosis.

CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments 29 • Fig. 2.9 Illustration of Apical Membrane Specializations of Epithelial Cells (Not Drawn to Scale). A, Microvilli 1 to 3 µm in length serve to increase the surface area of the apical membrane (e.g., those of the epithelial cells of the small intestine). B, Stereocilia can be up to 120 µm in length (e.g., those of the epididymis of the male reproductive tract). Both microvilli and stereocilia have a core structure composed primarily of actin, with a number of associated proteins. Both are nonmotile. (Redrawn from Pawlina, W. Histology: A Text and Atlas, with Correlated Cell and Molecular Biology. 7th ed. Philadelphia: Wolters Kluwer Health, 2016.) the base of the microvilli) and provides structural support for the microvilli. Another surface feature is stereocilia (see

Fig. 2.9B ). Stereocilia are long (up to 120 µm), nonmotile membrane projections that, like microvilli, increase the surface area of the apical membrane. They are found in the epididymis of the testis and in the “hair cells” of the inner ear. Their core also contains actin filaments and accessory proteins.

A third apical membrane feature is cilia (

Fig. 2.10 ). Cilia may be either motile (called secondary cilia) or non-motile (called primary cilia). The motile cilia contain a microtubule core arranged in a characteristic “9+2” pattern (nine pairs of microtubules around the circumference of the cilium, and one pair of microtubules in the center). Dynein is the molecular motor that drives the movement of the cilium. Motile cilia are characteristic features of the epithelial cells that line the respiratory tract. They pulsate in a synchronized manner and serve to transport mucus and inhaled particulates out of the lung, a process termed mucociliary transport (see

Chapter 26).

Nonmotile cilia serve as mechanoreceptors and are involved in determining left-right asymmetry of organs during embryological development, as well as sensing the flow rate of fluid in the nephron of the kidneys (see ). Only a single nonmotile cilium is found in the apical membrane of cells. Nonmotile cilia have a microtubule core (“9+0” arrangement) and lack a motor protein.

As noted previously, the tight junction effectively divides the plasma membrane of an epithelial cell into two domains: an apical surface and a basolateral surface. The basolateral membrane of many epithelial cells is folded or invaginated. This is especially so for epithelial cells that have high transport rates. These invaginations serve to increase the membrane surface area to accommodate the large number of membrane transporters (e.g., Na+,K+-ATPase) needed in the membrane.

Because the tight junction divides the plasma membrane into two domains (i.e., apical and basolateral), epithelial cells are capable of vectorial transport, whereby an ion or molecule can be transported from one side of the epithelial sheet to the opposite side (

Fig. 2.11 ). The accomplishment of vectorial transport requires that specific membrane transport proteins be targeted to and remain in one or the other of the membrane domains. In the example shown in

Fig. 2.11 , the Na+ channel is present only in the apical •Fig. 2.10 Ciliaareapicalmembranespecializationsofsomeepithelialcells.Ciliaare5to10µminlengthandcontainarraysofmicrotubules,asdepictedinthesecross-sectiondiagrams.Left, Theprimaryciliumhasnineperipheralmicrotubulearrays.Itisnonmotileandservesasamechanoreceptor(e.g.,cellsoftherenalcollectingduct).Cellsthathaveaprimaryciliumhaveonlyasinglecilium.Right, Thesecondaryciliumhasacentralpairofmicrotubulesinadditiontothenineperipheralmicrotubulearrays.Alsointhesecondarycilium,themotorproteindyneinisassociatedwiththemicrotubulearraysandthereforeismotile.Asinglecellcanhavethousandsofsecondaryciliaonitsapicalsurface(e.g.,epithelialcellsoftherespiratorytract).(RedrawnfromRodat-DespoixL,DelmasP.Ciliaryfunctionsinthe nephron.Pflugers Archiv.2009;458:179.) membrane, whereas the Na+,K+-ATPase and the K+ channels are confined to the basolateral membrane. The operation of the Na+,K+-ATPase channel and the leakage of K+ out of the cell across the basolateral membrane sets up a large electrochemical gradient for Na+ to enter the cell across the apical membrane through the Na+ channel (intracellular [Na+] < extracellular [Na+], and Vm which is oriented with the cell’s interior electrically negative with respect to the cell’s exterior). The Na+ is then pumped out of the cell by the Na+,K+-ATPase, and vectorial transport from the apical side of the epithelium to the basolateral side of the epithelium occurs. Transport from the apical side to the basolateral side of an epithelium is termed either absorption or reabsorption: For example, the uptake of nutrients from the lumen of the gastrointestinal tract is termed absorption, whereas the transport of NaCl and water from the lumen of the renal nephrons is termed reabsorption. Transport from the basolateral side of the epithelium to the apical side is termed secretion.

As noted previously, the Na+,K+-ATPase and K+-selective channels play an important role in establishing cellular ion gradients for Na+ and K+ and in generating the Vm. In all epithelial cells except the choroid plexus and retinal pigment epithelium, the Na+,K+-ATPase channel is located in the basolateral membrane of the cell. Numerous K+selective channels are in epithelial cells and may be located in either membrane domain. Through the establishment of these chemical and voltage gradients, the transport of other ions and solutes can be driven (e.g., Na+-glucose symporter, Na+-H+ antiporter, 1Na+,1K+,2Cl− symporter, 1Na+-3HCO3 − symporter). The direction of transepithelial transport (reabsorption or secretion) depends simply on which membrane domain the transporters are located. Because of the dependence on the Na+,K+-ATPase, epithelial transport requires the expenditure of energy. Other ATP-dependent transporters, such as the H+-ATPase, H+,K+-ATPase, and a host of ABC transporters—such as P-glycoprotein (PGP) and multidrug resistance-associated protein 2 (MRP2), which transport xenobiotics (drugs), and cystic fibrosis transmembrane conductance regulator (CFTR), which transports Cl−—are involved in epithelial transport.

hThe choroid plexus is located in the ventricles of the brain and secretes the cerebrospinal fluid. The Na+,K+-ATPase channel is located in the apical membrane of these cells.

CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments •Fig. 2.11 Insymmetricalcells(A; e.g.,redbloodcells),membranetransportproteinsaredistributedovertheentiresurfaceofthecell.Epithelialcells(B), incontrast,areasymmetricalandtargetvariousmembranetransportproteinstoeithertheapicalorthebasolateralmembrane.Whenthetransportersareconfinedtoamembranedomain,vectorialtransportcanoccur.Inthecelldepicted,Na+ istransportedfromtheapicalsurfacetothebasolateralsurface.ATP,adenosinetriphosphate.

Solutes and water can be transported across an epithelium by traversing both the apical and basolateral membranes (transcellular transport) or by moving between the cells across the tight junction (paracellular transport). Solute transport via the transcellular route is a two-step process, in which the solute molecule is transported across both the apical and basolateral membrane. Uptake into the cell, or transport out of the cell, may be either a passive or an active process. Typically, one of the steps is passive, and the other is active. For the example shown in

Fig. 2.11B , the uptake of Na+ into the cell across the apical membrane through the Na+-selective channel is passive and driven by the electrochemical gradient for Na+ . The exit of Na+ from the cell across the basolateral membrane is primary active transport via the Na+,K+-ATPase channel. Because a transepithelial gradient for Na+ can be generated by this process (i.e., the [Na+] in the apical compartment can be reduced below that of the basolateral compartment, the overall process of transepithelial Na+ transport is said to be active). Any solute that is actively transported across an epithelium must be transported via the transcellular pathway.

Depending on the epithelium, the paracellular pathway is an important route for transepithelial transport of solute and water. As noted, the permeability characteristics of the paracellular pathway are determined, in large part, by the specific claudins that are expressed by the cell. Thus the tight junction can have low permeability for solutes, water, or both, or it can have a high permeability. For epithelia in which there are high rates of transepithelial transport, •Fig. 2.12 The Electrical Profile Across an Epithelial Cell. Themagnitudeofthemembranevoltages,andthetransepithelialvoltagearedeterminedbythevariousmembranetransportproteinsintheapicalandbasolateralmembranes.Thetransepithelialvoltageisequaltothesumoftheapicalandbasolateralmembranevoltages(seetextfordetails).

the tight junctions typically have a high permeability (i.e., are leaky). Examples of such epithelia include the proximal tubule of the renal nephron and the early segments of the small intestine (e.g., duodenum and jejunum). If the epithelium must establish large transepithelial gradients for solutes, water, or both, the tight junctions typically have low permeability (i.e., are tight). Examples of this type of epithelium include the collecting duct of the renal nephron, the urinary bladder, and the terminal portion of the colon. In addition, the tight junction may be selective for certain solutes (e.g., cation versus anion selective).

All solute transport that occurs through the paracellular pathway is passive in nature. The two driving forces for this transport are the transepithelial concentration gradient for the solute and, if the solute is charged, the transepithelial voltage (Fig. 2.12 ). The transepithelial voltage may be oriented with the apical surface electrically negative in relation to the basolateral surface as shown in

Fig. 2.12 , or it may be oriented with the apical surface electrically positive in relation to the basolateral surface. The polarity and magnitude of the transepithelial voltage is determined by the specific membrane transporters in the apical and basolateral membranes, as well as by the permeability characteristics of the tight junction.

It is important to recognize that transcellular transport processes set up the transepithelial chemical and voltage gradients, which in turn can drive paracellular transport. This is illustrated in

Fig. 2.13 for an epithelium that reabsorbs NaCl and for an epithelium that secretes NaCl. In both epithelia, the transepithelial voltage is oriented with the apical surface electrically negative in relation to the basolateral surface. For the NaCl-reabsorbing epithelium, the transepithelial voltage is generated by the active, transcellular reabsorption of Na+ . This voltage in turn drives Cl− reabsorption through the paracellular pathway. In contrast, for the NaCl-secreting epithelium, the transepithelial voltage is generated by the active transcellular secretion of Cl− . Na+ is then secreted passively via the paracellular pathway, driven by the negative transepithelial voltage.

Water movement across epithelia is passive and driven by transepithelial osmotic pressure gradients. Water movement can occur by a transcellular route involving aquaporins in both the apical and basolateral membranes.

In addition, water may also move through the paracellular pathway. In the NaCl-reabsorbing epithelium depicted in

Fig. 2.13A , the reabsorption of NaCl from the apical compartment lowers the osmotic pressure in that compartment, whereas the addition of NaCl to the basolateral compartment raises the osmotic pressure in that compartment. As a result, a transepithelial osmotic pressure gradient is established that drives the movement of water from the apical to the basolateral compartment (i.e., reabsorption). The opposite occurs with NaCl-secreting epithelia (see

Fig. 2.13B ), in which the transepithelial secretion of NaCl establishes a transepithelial osmotic pressure gradient that drives water secretion.

In some epithelia (e.g., proximal tubule of the renal nephron), the movement of water across the epithelium via the paracellular pathway can drive the movement of additional solute. This process is termed solvent drag and reflects the fact that solutes dissolved in the water will traverse the tight junction with the water.

As is the case with the establishment of transepithelial concentration and voltage gradients, the establishment of transepithelial osmotic pressure gradients requires transcellular transport of solutes by the epithelial cells.

iDifferent aquaporin isoforms are often expressed in the apical and basolateral membrane. In addition, multiple isoforms may be expressed in one or more of the membrane domains.

CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments

NaCl reabsorption •Fig. 2.13 The Role of the Paracellular Pathway in Epithelial

Transport. A, Na+ transportthroughthecellgeneratesatransepithelial voltagethatthendrivesthepassivemovementofCl− throughthetightjunction.NaClreabsorptionresults.B, Cl− transportthroughthecellgeneratesatransepithelialvoltagethatthendrivesthepassivetransportofNa+ throughthetightjunction.NaClsecretionresults.

Regulation of Epithelial Transport

Epithelial transport must be regulated to meet the homeostatic needs of the individual. Depending on the epithelium, this regulation involves neural or hormonal mechanisms, or both. For example, the enteric nervous system of the gastrointestinal tract regulates solute and water transport by the epithelial cells that line the intestine and colon. Similarly, the sympathetic nervous system regulates transport by the epithelial cells of the renal nephron. Aldosterone, a steroid hormone produced by the adrenal cortex (see ), is an example of a hormone that stimulates NaCl transport by the epithelial cells of the colon, renal nephron, and sweat ducts. Epithelial cell transport can also be regulated by locally produced and locally acting substances, a process termed paracrine regulation. The stimulation of HCl secretion in the stomach by histamine is an example of this process. Cells that are located near the epithelial cells of the stomach release histamine, which acts on the HCl-secreting cells of the stomach (parietal cells) and stimulates them to secrete HCl.

When acted upon by a regulatory signal, the epithelial cell may respond in several different ways, including: • Retrieval of transporters from the membrane, by endocytosis, or insertion of transporters into the membrane from an intracellular vesicular pool, by a process called

Change in activity of membrane transporters (e.g., channel gating)

Synthesis of specific transporters, and their insertion into the membrane

Na+ The first two mechanisms can occur quite rapidly (seconds 2Cl– to minutes), but the synthesis of transporters takes addi-K+ tional time (minutes to days).

The body maintains steady-state balance for water and a number of important solutes. This occurs when input into the body equals output from the body. For each solute and water, there is a normal set point. Deviations from this set point are monitored (i.e., when input ≠ output), and effector mechanisms are activated that restore balance. This balance is achieved by adjustment of either intake or excretion of water and solutes. Thereafter, input and output are again equal to maintain balance.

The Na+,K+-ATPase and K+-selective channels are critically important in establishing and maintaining the intracellular composition, the membrane potential (Vm), and cell volume. Na+,K+-ATPase converts the energy in

Altenberg GA, Ruess L. Mechanisms of water transport across cell membranes and epithelia. In: Alpern R, Moe O, Kaplan M, eds. Seldin and Giebisch’s The Kidney—Physiology and Pathophysiology. 5th ed. New York: Academic Press; 2013.

Günzel D, Yu ASL. Claudins and the modulation of tight junction permeability. Physiol Rev. 2013;93:525-569.

Hoffman EK, et al. Physiology of cell volume regulation in vertebrates. Physiol Rev. 2009;89:193-277.

Lang F. Cell volume control. In: Alpern R, Moe O, Kaplan M, eds. Seldin and Giebisch’s The Kidney—Physiology and Pathophysiology. 5th ed. New York: Academic Press; 2013.

ATP into potential energy of ion gradients and the membrane potential. The ion and electrical gradients created by this process are then used to drive the transport of other ions and other molecules, especially by solute carriers (i.e., symporters and antiporters).

• Epithelial cells constitute the interface between the external world and the internal environment of the body. Vectorial transport of solutes and water across epithelia helps maintain steady-state balance for water and a number of important solutes. Because the external environment constantly changes, and because dietary intake of food and water is highly variable, transport by epithelia is regulated to meet the homeostatic needs of the individual.

Pawlina W. Histology: A Text and Atlas, with Correlated Cell and Molecular Biology. 7th ed. Philadelphia: Wolters Kluwer Health; 2016.

Pedersen SF, Kapus A, Hoffmann EK. Osmosensory mechanisms in cellular and systemic volume regulation. J Am Soc Nephrol. 2011;22:1587-1597.

Sackin H, Palmer LG. Electrophysiological analysis of transepithelial transport. In: Alpern R, Moe O, Kaplan M, eds. Seldin and Giebisch’s The Kidney—Physiology and Pathophysiology. 5th ed. New York: Academic Press; 2013.

3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression

Upon completion of this chapter, the student should be able to answer the following questions: 1.

How do cells communicate with each other?

2.

What are the four classes of receptors, and what signal transduction pathways are associated with each class of receptors?

3.

How do steroid and thyroid hormones, cyclic adenosine monophosphate, and receptor tyrosine kinases regulate gene expression?

he human body is composed of billions of cells, each with a distinct function. However, the function of cells is tightly coordinated and integrated by external chemical signals, including hormones, neurotransmitters, growth factors, odorants, and products of cellular metabolism that serve as chemical messengers and provide cell-to-cell communication. Mechanical and thermal stimuli and light are physical external signals that also coordinate cellular function. Chemical and physical messengers interact with receptors located in the plasma membrane, cytoplasm, and nucleus. Interaction of these messengers with receptors initiates a cascade of signaling events that mediate the response to each stimulus. These signaling pathways ensure that the cellular response to external messengers is specific, amplified, tightly regulated, and coordinated. This chapter provides an overview of how cells communicate via external messengers and a discussion of the signaling pathways that process external information into a highly coordinated cellular response. In subsequent chapters, details on signaling pathways in the nervous system, muscular system, cardiovascular system, respiratory system, gastrointestinal system, renal system, and endocrine system are discussed in greater detail.

The significance of signaling pathways in medicine is illustrated by the following short list of popular drugs that act by regulating signaling pathways. Details on these pathways are presented later in this and other chapters.

Aspirin, the first pharmaceutical (1899), inhibits cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2) and therefore is antithrombotic (i.e., reduces the formation of blood clots).

β-Adrenergic receptor agonists and antagonists are used to treat a variety of medical conditions. β1-Agonists increase cardiac contractility and heart rate in patients with low blood pressure. β2-Agonists dilate bronchi and are used to treat asthma and chronic obstructive lung disease. In contrast, β-adrenergic antagonists are used to treat hypertension, angina, cardiac arrhythmias, and congestive heart failure (see

Chapter 18).

Fluoxetine (Prozac) is an antidepressant medication that inhibits reuptake of the neurotransmitter serotonin into the presynaptic cell, which results in enhanced activation of serotonin receptors (see

Several monoclonal antibodies are used to treat cancer caused by the activation of growth factor receptors in cancer cells. For example, trastuzumab (Herceptin) is a monoclonal antibody used to treat metastatic breast cancer in women who overexpress HER2/neu, a member of the family of epidermal growth factor (EGF) receptors, which stimulate cell growth and differentiation. Cetuximab (Erbitux) and bevacizumab (Avastin) are monoclonal antibodies that are used to treat metastatic colorectal cancer and cancers of the head and neck. These antibodies bind to and inhibit the EGF receptor and thereby inhibit EGF-induced cell growth in cancer cells.

Drugs that inhibit cyclic guanosine monophosphate (cGMP)–specific phosphodiesterase type 5, such as sildenafil (Viagra), tadalafil (Cialis), and vardenafil (Levitra), prolong the vasodilatory effects of nitric oxide and are used to treat erectile dysfunction and pulmonary arterial hypertension (see

Chapter 17).

An overview of how cells communicate with each other is presented in

Fig. 3.1 . Cells communicate by releasing extracellular signaling molecules (e.g., hormones and neurotransmitters) that bind to receptor proteins located in the plasma membrane, cytoplasm, or nucleus. This signal is transduced into the activation, or inactivation, of one or more intracellular messengers by interacting with receptors. Receptors interact with a variety of intracellular signaling proteins, including kinases, phosphatases, and guanosine triphosphate (GTP)–binding proteins (G proteins). These signaling proteins interact with and regulate the activity of target proteins and thereby modulate cellular function. Target proteins include, but are not limited to, ion channels and other transport proteins, metabolic enzymes, cytoskeletal proteins, gene regulatory proteins, and cell cycle proteins that regulate cell growth and division. Signaling pathways are characterized by (1) multiple, hierarchical steps; (2) amplification of the signal-receptor binding event, which magnifies the response; (3) activation of multiple pathways and regulation of multiple cellular functions; mechanisms, which minimize the response and provide tight regulatory control over these signaling pathways. A brief description of how cells communicate follows. Readers who desire a more in-depth presentation of this material are encouraged to consult one of the many cellular and molecular biology textbooks currently available.

Cells in higher animals release into the extracellular space hundreds of chemicals, including (1) peptides and proteins (e.g., insulin); (2) amines (e.g., epinephrine and norepinephrine); (3) steroid hormones (e.g., aldosterone, estrogen); and (4) small molecules, including amino acids, nucleotides, ions (e.g., Ca++), and gases, such as nitric oxide and carbon dioxide. Secretion of signaling molecules is • Fig. 3.1 An Overview of How Cells Communicate. A signaling molecule (i.e., hormone or neurotransmitter) binds to a receptor, which may be in the plasma membrane, cytosol, or nucleus. Binding of ligand to a receptor activates intracellular signaling proteins, which interact with and regulate the activity of one or more target proteins to change cellular function. Signaling molecules regulate cell growth, division, and differentiation and influence cellular metabolism. In addition, they modulate the intracellular ionic composition by regulating the activity of ion channels and transport proteins. Signaling molecules also control cytoskeleton-associated events, including cell shape, division, and migration and cell-to-cell and cell-to-matrix adhesion. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.)

CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression 37 cell-type specific. For example, beta cells in the pancreas release insulin, which stimulates glucose uptake into cells. The ability of a cell to respond to a specific signaling molecule depends on the expression of receptors that bind the signaling molecule with high affinity and specificity. Receptors are located in the plasma membrane, the cytosol, and the nucleus (

Fig. 3.2

Fig. 3.3).

Contact-dependent signaling, in which a membrane-bound signaling molecule of one cell binds directly to a plasma membrane receptor of another cell, is important during development, in immune responses, and in cancer (see

Fig. 3.3A ). Molecules that are released and act locally are called paracrine (see

Fig. 3.3B

Fig. 3.3C ) hormones. Paracrine signals are released by one type of cell and act on another type; they are usually taken up by target cells or rapidly degraded (within • Fig. 3.2 Signaling molecules, especially ones that are hydrophilic and cannot cross the plasma membrane, bind directly to their cognate receptors in the plasma membrane (A). Other signaling molecules— including steroid hormones, triiodothyronines, retinoic acids, and vitamin D—bind to carrier proteins in blood and readily diffuse across the plasma membrane, where they bind to cognate nuclear receptors in the cytosol or nucleus (B). Still other signaling molecules, including nitric oxide, can diffuse without carrier proteins and cross the membrane to act on intracellular protein targets (B). Both classes of receptors, when ligand bound, regulate gene transcription. mRNA, messenger RNA. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.) minutes) by enzymes. For example, enterochromaffin-like cells in the stomach secrete histamine, which stimulates the production of acid by neighboring parietal cells (see for details). Autocrine signaling involves the release of a molecule that affects the same cell or other cells of the same type (e.g., cancer cells). In synaptic signaling (see

Fig. 3.3D ), neurons transmit electrical signals along their axons and release neurotransmitters at synapses that affect the function of other neurons or cells that are distant from the neuron cell body. The close physical relationship between the nerve terminal and the target cell ensures that the neurotransmitter is delivered to a specific cell. Details on synaptic signaling are discussed in

Chapter 6.

Endocrine signals are hormones that are secreted into the blood and are widely dispersed in the body (see

Fig. 3.3E ). Details on endocrine signaling are discussed in

In addition to paracrine, autocrine, endocrine, and synaptic signaling, cell-to-cell communication also occurs via gap junctions that form between adjacent cells (see ). Gap junctions are specialized junctions that allow intracellular signaling molecules, generally less than 1200 D in size, to diffuse from the cytoplasm of one cell to an adjacent cell. The permeability of gap junctions is regulated by cytosolic [Ca++], [H+], and cyclic adenosine monophosphate (cAMP) and by the membrane potential. Gap junctions also allow cells to be electrically coupled, which is vitally important for the coordinated activity of cardiac and smooth muscle cells (see

The speed of a response to an extracellular signal depends on the mechanism of delivery. Endocrine signals are relatively slow (seconds to minutes) because time is required for diffusion and blood flow to the target cell, whereas synaptic signaling is extremely fast (milliseconds). If the response involves changes in the activity of proteins in the cell, the response may occur in milliseconds to seconds. However, if the response involves changes in gene expression and the de novo synthesis of proteins, the response may take hours to occur, and a maximal response may take days. For example, the stimulatory effect of aldosterone on sodium transport by the kidneys requires days to develop fully (see

The response to a particular signaling molecule also depends on the ability of the molecule to reach a particular cell, on expression of the cognate receptor (i.e., receptors that recognize a particular signaling molecule or ligand with a high degree of specificity), and on the cytoplasmic signaling molecules that interact with the receptor. Thus signaling molecules frequently have many different effects that are dependent on the cell type. For example, the neurotransmitter acetylcholine stimulates contraction of skeletal muscle but decreases the force of contraction in heart muscle. This is because skeletal muscle and heart cells express different acetylcholine receptors.

aThe acetylcholine receptor in skeletal muscle is termed nicotinic because nicotine can mimic this action of the neurotransmitter. In contrast, the acetylcholine receptor in cardiac muscle is termed muscarinic because this effect is mimicked by muscarine, an alkaloid derived from the mushroom

Amanita muscaria.

• Fig. 3.3 Cell-to-cell communication is mediated by five basic mechanisms: contact-dependent (A), paracrine (B), autocrine (C), synaptic (D), and endocrine signaling (E). These mechanisms are described in detail in the text. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.)

CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression

Nuclear receptors Extracellular ligand:

GABA ACh (muscle) ATP Glutamate: NMDA

Intracellular ligand:

Neurotransmitters (ACh) Peptides (PTH, oxytocin) Odorants Cytokines, lipids

ANP TGF-β Insulin, EGF Interleukin-6, erythropoietin Steroid hormones:

Miscellaneous hormones:

Membrane currents: Cl− Na+ , K+ , Ca++ Ca++ , Na+ , K+ Na+ , K+ , Ca++

K+ Na+ , K+ Ca++ βγ Subunits activate ion channels α Subunit activates enzymes:

Cyclases that generate cAMP, cGMP, phospholipases that generate InsP3 and diacylglycerol, and phospholipases that generate arachidonic acid and its metabolites.

Monomeric G proteins Receptor guanylyl cyclase Receptor serine/threonine kinase Receptor tyrosine kinase Tyrosine kinase–associated receptor Bind to regulatory sequences in DNA and increase or

Bind to regulatory sequences in DNA and increase or decrease gene transcription

ACh, acetylcholine; ANP, atrial natriuretic peptide; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; EGF, epidermal growth factor; GABA, gamma-aminobutyric acid; InsP3, inositol 1,4,5-triphosphate; NMDA, N-methyl-D-aspartate; PTH, parathyroid hormone; TGF, transforming growth factor.

All signaling molecules bind to specific receptors that act as signal transducers, thereby converting a ligand-receptor binding event into intracellular signals that affect cellular function. Receptors can be divided into four basic classes on the basis of their structure and mechanism of action: (1) ligand-gated ion channels, (2) G protein–coupled receptors (GPCRs), (3) enzyme-linked receptors, and (4) nuclear receptors (

Table 3.1

Figs. 3.4 3.5).

Fig. 3.4A). Neurotransmitters bind to receptors and either open or close ion channels, thereby changing the ionic permeability of the plasma membrane and altering the membrane potential. For examples and more details, see

GPCRs regulate the activity of other proteins, such as enzymes and ion channels (see

Fig. 3.4B ). In the example in

Fig. 3.4B , the interaction between the receptor and the target protein is mediated by heterotrimeric G proteins, which are composed of α, β, and γ subunits. Stimulation of G proteins by ligand-bound receptors activates or inhibits downstream target proteins that regulate signaling pathways if the target protein is an enzyme or changes membrane ion permeability if the target protein is an ion channel.

Enzyme-linked receptors either function as enzymes or are associated with and regulate enzymes (see

Fig. 3.4C ). Most enzyme-linked receptors are protein kinases or are associated with protein kinases, and ligand binding causes the kinases to phosphorylate a specific subset of proteins on specific amino acids, which in turn activates or inhibits protein activity.

Nuclear receptors are small hydrophobic molecules, including steroid hormones, thyroid hormones, retinoids, and vitamin D, that have a long biological half-life (hours to days), diffuse across the plasma membrane, and bind to nuclear receptors or to cytoplasmic receptors that, once bound to their ligand, translocate to the nucleus (see

Fig.

3.5 ). Some nuclear receptors, such as those that bind cortisol

Signal molecule as a dimer Signal molecule • Fig. 3.4 Three of the Four Classes of Plasma Membrane Receptors. See text for details. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.) and aldosterone, are located in the cytosol and enter the nucleus after binding to hormone, whereas other receptors, including the thyroid hormone receptor, are located in the nucleus. In both cases, inactive receptors are bound to inhibitory proteins, and binding of hormone results in dissociation of the inhibitory complex. Hormone binding causes the receptor to bind coactivator proteins that activate gene transcription. Once activated, the hormone-receptor complex regulates the transcription of specific genes. Activation of specific genes usually occurs in two steps: an early primary response (≈30 minutes), which activates genes that stimulate other genes to produce a delayed (hours to days) secondary response (see

Fig. 3.5).

Each hormone elicits a specific response that is based on cellular expression of the cognate receptor, as well as on cell type–specific expression of gene regulatory proteins that interact with the activated receptor to regulate the transcription of a specific set of genes (see for more details). In addition to steroid receptors that regulate gene expression, evidence also suggests the existence of membrane and juxtamembrane steroid receptors that mediate the rapid, nongenomic effects of steroid hormones.

Some membrane proteins do not fit the classic definition of receptors, but they subserve a receptor-like function in that they recognize extracellular signals and transduce the signals into an intracellular second messenger that has a biological effect. For example, on activation by a ligand, some membrane proteins undergo regulated intramembrane proteolysis (RIP), which elaborates a cytosolic peptide fragment that enters the nucleus and regulates gene expression (

Fig. 3.6 ). In this signaling pathway, binding of ligand to a plasma membrane receptor leads to ectodomain shedding, facilitated by members of the metalloproteinase-disintegrin family, and produces a carboxy-terminal fragment that is the substrate for γ-secretase. γ-Secretase induces RIP, thereby causing the release of an intracellular domain of

CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression 41 • Fig. 3.5 Steroid Hormones Stimulate the Transcription of Early-Response Genes and Late-Response Genes. See text for details. (Redrawn from Alberts B, et al: Molecular Biology of Cell. 6th ed. New York:

Garland Science; 2015.)

Translocation to the nucleus • Fig. 3.6 Regulated Intramembrane Proteolysis. See text for details. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.) the protein that enters the nucleus and regulates transcription (see

Fig. 3.6 ). The best characterized example of RIP is the sterol regulatory element–binding protein (SREBP), a transmembrane protein expressed in the membrane of the endoplasmic reticulum. When cellular cholesterol levels are low, SREBP undergoes RIP, and the proteolytically cleaved fragment is translocated into the nucleus, where it transcriptionally activates genes that promote cholesterol biosynthesis.

When hormones bind to plasma membrane receptors, signals are relayed to effector proteins via intracellular signaling pathways. When hormones bind to nuclear or cytosolic receptors, they relay signals primarily through regulation of gene expression. Signaling pathways can amplify and integrate signals but can also downregulate and desensitize signals, reducing or terminating the response, even in the continued presence of hormone.

Alzheimer’s disease, a progressive neurodegenerative brain disease characterized by the formation of amyloid plaques, affects approximately 44 million people worldwide. In Alzheimer’s disease, regulated intramembrane proteolysis of amyloid β-protein precursor (APP) causes the accumulation of amyloid β-protein (Aβ), which forms amyloid plaques that contribute to the pathogenesis of Alzheimer’s disease. APP is a type I transmembrane protein (i.e., its spans the membrane only once). After ectodomain shedding, its sequential proteolysis by β-secretase and γ-secretase produces the Aβ40 and Aβ42 peptides that are normally produced throughout life but accumulate in individuals with Alzheimer’s disease. Missense mutations in presenilins, proteins that regulate γ-secretase protease activity, enhance the production of Aβ42, which is more hydrophobic and prone to aggregation into amyloid fibrils than is the more abundant Aβ40 protein.

Intracellular signaling molecules—so-called second messengers (the first messenger of the signal is the ligand that binds to the receptor)—include small molecules such as cAMP, cGMP, Ca++, and diacylglycerol. Signaling pathways often include dozens of small molecules that form complicated networks within the cell (Fig. 3.7 ). Some proteins in the intracellular signaling pathways relay the signal by passing the message directly to another protein (e.g., by phosphorylating a target, or by binding and causing an allosteric change). Such intracellular signaling proteins act as reversible molecular switches: When a signal is received, they switch from an inactive to an active form or vice versa, until another signaling molecule reverses the process. This principle of reversibility is central to many signaling pathways. In many cases, activation is achieved by reversing inhibition: For example, the thyroid hormone receptor is bound to an inhibitory protein in the absence of signal.

Signaling complexes, composed of multiple proteins that interact physically, enhance the speed, efficiency, and specificity of signaling. Many proteins, usually enzymes or ion channels, transduce the signal into a different chemical form and simultaneously amplify the signal either by producing large amounts of additional signaling molecules or by activating a large number of downstream signaling proteins. For example, adenylyl cyclase, the enzyme that makes cAMP, transduces a signal (receptor activation of G proteins) and amplifies the signal by generating large amounts of cAMP. Other types of signaling proteins include those that integrate multiple signals. Other proteins carry the signal from one region of the cell to another: for example, by translocating from the cytosol to the nucleus.

Cells can adjust rapidly to changing signals. Cells can respond quickly and in a graded manner to increasing concentrations of hormone, and the effect of a signaling molecule can be either long-or short-lived. Cells can also adjust their sensitivity to a signal by desensitization, whereby prolonged exposure to a hormone decreases the cell’s response over time. Desensitization is a reversible process that can involve a reduction in the number of receptors expressed in the plasma membrane, inactivation of receptors, or changes in signaling proteins that mediate the downstream effect of the receptors. Homologous desensitization involves a reduction in the response only to the signaling molecule that caused the response (e.g., opioid dependence and tolerance), whereas heterologous desensitization is when one ligand desensitizes the response to another ligand.

Table 3.1 summarizes the four general classes of receptors and provides a few examples of the signal transduction pathways associated with each class of receptors.

This class of receptors transduces a chemical signal into an electrical signal, which elicits a response. For example, the ryanodine receptor, located in the membrane of the sarcoplasmic reticulum of skeletal muscle, is activated by Ca++ , caffeine, adenosine triphosphate (ATP), or metabolites of arachidonic acid to release Ca++ into the cytosol, which facilitates muscle contraction (see for details). In glutamergic synapses in which high levels of prior synaptic activity have led to partial membrane depolarization, activation of the N-methyl-D-aspartate receptor by glutamate stimulates Ca++ influx important for synaptic plasticity.

There are two classes of GTP-binding proteins (i.e., GTPases, which are named for their ability to hydrolyze GTP to guanosine diphosphate [GDP] and an inorganic phosphate): low-molecular-weight, monomeric G proteins and heterotrimeric G proteins composed of α, β, and γ subunits. GTP binding activates, whereas hydrolysis of GTP to GDP inactivates, GTP-binding proteins (Fig. 3.8A ). All GTPases are controlled by regulatory proteins, including GTPase-activating proteins, which induce the hydrolysis of GTP to GDP and thus inactivate the GTPase, and guanine nucleotide exchange factors (GEFs) that causes the GTPase to release GDP, which is rapidly replaced by GTP, thereby activating the GTPase (see

Fig. 3.8B

Monomeric G proteins are composed of a single 20to 40-kDa protein and can be membrane bound because of the addition of lipids posttranslationally. Monomeric G proteins have been classified into five families (Ras, Rho, Rab, Ran, and Arf), play a central role in many enzyme-linked receptor pathways, and regulate gene expression and cell proliferation, differentiation, and survival. Rho GTPases regulate actin cytoskeletal organization, cell cycle progression, and gene expression. The Rab GTPases regulate intravesicular transport and trafficking of proteins between organelles in the secretory and endocytic pathways. Ran GTPases regulate nucleocytoplasmic transport of RNA and

CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression 43 • Fig. 3.7 Illustration of How Intracellular Signals Are Amplified and Integrated. Signaling pathways often include dozens of proteins and small molecules that form complicated networks within the cell. Some signaling proteins relay the signal by passing the message to another protein. Many proteins amplify the signal either by producing large amounts of additional signaling molecules or by activating a large number of downstream signaling proteins. Other proteins carry the signal from one region of the cell to another. See text for more details. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.).

• Fig. 3.8 GTP-Binding Proteins. GTP binding activates whereas hydrolysis of GTP to GDP inactivates GTP-binding proteins (A). All GTPase are controlled by regulatory proteins, including GTPase-activating proteins (GAP), which induce the hydrolysis of GTP to GDP, thus inactivating the GTPase, and guanine nucleotide exchange factors, (GEF) that which cause the GTPase to release GDP, which is rapidly replaced by GTP, thereby activating the GTPase (B). (Redrawn from Kantrowitx ER, Lipscomb WN. Escherichia coli aspartate transcarbamoylase: the molecular basis for a concerted allosteric transition. Trends Biochem

Sci. 1990;15:53-59.) proteins. Ras GTPases are involved in many signaling pathways that control cell division, proliferation and death. Arf GTPases, like Rab GTPases, regulate vesicular transport.

Heterotrimeric G proteins couple to more than 1000 different receptors and thereby mediate the cellular response to an incredibly diverse set of signaling molecules, including hormones, neurotransmitters, peptides, and odorants. Like monomeric G proteins, they can be membrane bound because of the addition of lipids posttranslationally. Heterotrimeric complexes are composed of three subunits: α, β, and γ. There exist 16 α subunits, 5 β subunits, and 11 γ subunits, which can assemble into hundreds of different combinations and thereby interact with a diverse number of receptors and effectors. The assembly of subunits and the association with receptors and effectors depend on the cell type.

An overview of heterotrimeric G protein activation is illustrated in

Fig. 3.9 . In the absence of ligand, these G proteins are inactive and form a heterotrimeric complex in which GDP binds to the α subunit. Binding of a signal molecule to an inactive GPCR induces a conformational change in the G protein that promotes the release of GDP and the subsequent binding of GTP to the α subunit. Binding of GTP to the α subunit stimulates dissociation of the α subunit from the heterotrimeric complex and results in release of the α subunit from the βγ dimer, each of which can interact with and regulate downstream effectors such as adenylyl cyclase and phospholipases (see

Fig. 3.9 ). Activation of downstream effectors by the α subunit and βγ dimer is terminated when the α subunit hydrolyzes the bound GTP to GDP and inorganic phosphate (Pi). The α subunit bound to GDP associates with the βγ dimer and terminates the activation of effectors.

Another way to attenuate or terminate signaling through a GPCR involves desensitization and endocytic removal of receptors from the plasma membrane. Binding of hormone to a GPCR increases the ability of GPCR kinases to phosphorylate the intracellular domain of GPCRs, which recruits proteins called β-arrestins to bind to the GPCR. β-Arrestins inactivate the receptor and promote endocytic

CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression 45 • Fig. 3.9 Activation of a G Protein–Coupled Receptor (GPCR) and Effector Activation. In the absence of ligand, heterotrimeric G proteins are in an inactive state because GDP binds to the α subunit. Binding of a signal molecule to an inactive G protein–coupled receptor (GPCR) induces a conformational change in the G protein that promotes the release of GDP and the subsequent binding of GTP to the α subunit. Binding of GTP to the α subunit stimulates dissociation of the α subunit from the heterotrimeric complex and results in release of the α subunit from the βγ dimer, each of which can interact with and regulate downstream effectors. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York:

Garland Science; 2015.) removal of the GPCR from the plasma membrane. GPCR kinase/β-arrestin inactivation with endocytosis of GPCRs is an important mechanism whereby cells downregulate (desensitize) a response during prolonged exposure to elevated hormone levels. One of the major benefits of β blockers, given for congestive heart failure, is that they reverse chronic desensitization and the associated recovery of adrenergic responsiveness.

Activated G protein α subunits couple to a variety of effector proteins, including adenylyl cyclase, phosphodiesterases, and phospholipases (A2, C, and D). A very common downstream effector of heterotrimeric G proteins is adenylyl cyclase, which facilitates the conversion of ATP to cAMP (

Fig. 3.10 ). When a signal molecule binds to a GPCR that interacts with a G protein composed of an α subunit of the αs class, adenylyl cyclase is activated, which causes an increase in cAMP levels and, as a result, activation of protein kinase A (PKA). By phosphorylating specific serine and threonine residues on downstream effector proteins, PKA regulates effector protein activity. In contrast, when a ligand binds to a receptor that interacts with a G protein composed of a α subunit of the αi class, adenylyl cyclase is inhibited, which causes reductions in cAMP levels and, consequently, in PKA activity.

Some effector proteins, such as ion-gated channels, are also regulated directly by cAMP. cAMP is degraded to AMP activated, phosphorylated CREB •Fig. 3.10 GPCR Stimulation of Adenylyl Cyclase, cAMP, and Protein Kinase A.Binding of a signal molecule to a GPCR mediates Gs stimulation of adenylyl cyclase, which increases cytosolic cAMP, which in turn activates protein kinase A (PKA). Activated PKA phosphorylates a number of target proteins to elicit many effects. PKA also enters the nucleus where it phosphorylates CREB (cyclic adenosine mono-phosphate [cyclic AMP] response element–binding protein). Phosphorylated CREB recruits coactivator CBP, which stimulates gene transcription. (Redrawn from Alberts B, etal: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.) by cAMP phosphodiesterases, which are inhibited by caf-and phosphorylate and activate the transcription factor feine and other methylxanthines. Thus by interfering with cAMP response element–binding (CREB) protein (see a constitutive “off” signal, caffeine can prolong a cellular

Fig. 3.10 ). Phospho-CREB protein increases the transcripresponse mediated by cAMP and PKA. Because these effects tion of many genes, which can in turn produce a distinct set target existing proteins, they can be extremely rapid (e.g., of responses with much slower kinetics. Hence, cAMP has adrenaline response). In addition to cytoplasmic signaling, many cellular effects, including direct and indirect effects the catalytic subunit of PKA can enter the nucleus of cells mediated by PKA.

CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression

Cholera toxin, secreted by Vibrio cholera, catalyzes the ADP ribosylation of the G-protein αs subunit, which inhibits the GTPase activity of αs. Thus αs remains in its activated, GTP-bound state, which in turn causes activation of adenylyl cyclase and an increase in cAMP/PKA levels. In the intestines, elevated levels of PKA increase cystic fibrosis transmembrane conductance regulator (CFTR)–mediated chloride secretion, which leads to secretory diarrhea and extensive loss of fluids, characteristic of cholera. Bordetella pertussis, the bacterium that causes whooping cough, secretes pertussis toxin, which ADP ribosylates the αi subunit. In this case, the ribosylation inactivates αi, reducing the inhibition of adenylyl cyclase and thus also leading to increased levels of cAMP/PKA.

Heterotrimeric G proteins also regulate phototransduction. In rod cells in the eye, absorption of light by rhodopsin activates the G protein transducin, which via the αt subunit activates cGMP phosphodiesterase. Activation of this phosphodiesterase lowers the concentration of cGMP and thereby closes a cGMP-activated cation channel. The ensuing change in cation channel activity alters the membrane voltage. The exquisite sensitivity of rods to light—rods can detect a single photon of light—is due to the abundance of rhodopsin in rods and amplification of the signal (a photon of light) by the G protein–cGMP phosphodiesterase–cGMP channel signaling pathway (see for more details).

Heterotrimeric G proteins also regulate phospholipases, a family of enzymes that modulate a variety of signaling pathways. Ligands that activate receptors that are coupled to the αq subunit stimulate phospholipase C, an enzyme that converts phosphatidylinositol 4,5-biphosphate to inositol 1,4,5-triphosphate (InsP3) and diacylglycerol. InsP3 is a second messenger that diffuses to the endoplasmic reticulum, where it activates a ligand-activated Ca++ channel to release Ca++ into the cytosol, whereas diacylglycerol activates protein kinase C, which phosphorylates effector proteins. As noted earlier, both Ca++ and protein kinase C influence effector proteins, as well as other signaling pathways, to elicit responses.

Ligand binding to GPCRs can also activate phospholipase A2, an enzyme that releases arachidonic acid from membrane phospholipids Arachidonic acid, which can also be released from diacylglycerol via an indirect pathway, can be released from cells and thereby regulate neighboring cells or stimulate inflammation. It can also be retained within cells, where it is incorporated into the plasma membrane or is metabolized in the cytosol to form intracellular second messengers that affect the activity of enzymes and ion channels. In one pathway, cytosolic cyclooxygenases facilitate the metabolism of arachidonic acid to prostaglandins, thromboxanes, and prostacyclins. Prostaglandins mediate aggregation of platelets, cause constriction of the airways, and induce inflammation. Thromboxanes also induce platelet aggregation and constrict blood vessels, whereas prostacyclin inhibits platelet aggregation and causes dilation of blood vessels. In a second pathway of arachidonic acid metabolism, the enzyme 5-lipoxygenase initiates the conversion of arachidonic acid to leukotrienes, which participate in allergic and inflammatory responses, including those causing asthma, rheumatoid arthritis, and inflammatory bowel disease. The third pathway of arachidonic acid metabolism is initiated by epoxygenase, an enzyme that facilitates the generation of hydroxyeicosatetraenoic acid (HETE) and cis-epoxyeicosatrienoic acid (cis-EET). HETE and cis-EET and their metabolites increase release of Ca++ from the endoplasmic reticulum, stimulate cell proliferation, and regulate inflammatory responses.

Ca++ is also an intracellular messenger that elicits cellular effects via Ca++-binding proteins, most notably calmodulin (CaM). When Ca++ binds to CaM, its conformation is altered, and the structural change in CaM allows it to bind to and regulate other signaling proteins, including cAMP phosphodiesterase, an enzyme that degrades cAMP to AMP, which is inactive and unable to activate PKA. By binding to CaM-dependent kinases, CaM also phosphorylates specific serine and threonine residues in many proteins, including myosin light-chain kinase, which facilitates smooth muscle contraction (see

Protein Phosphatases and Phosphodiesterases Counteract the Activation of Cyclic Nucleotide Kinases

There are two ways to terminate a signal initiated by cAMP and cGMP: enhancing degradation of these cyclic nucleotides by phosphodiesterases and dephosphorylation of effectors by protein phosphatases. Phosphodiesterases facilitate the breakdown of cAMP and cGMP to AMP and GMP, respectively, and are activated by ligand activation of GPCRs. Phosphatases dephosphorylate effector proteins that were phosphorylated by kinases such as PKA. The balance between kinase-mediated phosphorylation and phosphatase-mediated dephosphorylation allows rapid and exquisite regulation of the phosphorylated state and thus the activity of signaling proteins.

There are several classes of receptors that have enzymatic activity or are intimately associated with proteins that have enzymatic activity. Four of these classes are discussed next, including receptors that mediate the cellular responses to atrial natriuretic peptide (ANP) and nitric oxide (guanylyl cyclase receptors); transforming growth factor-β (TGF-β; threonine/serine kinase receptors); EGF, platelet-derived growth factor (PDGF), and insulin (tyrosine kinase receptors); and interleukins (tyrosine kinase–associated receptors).

There are two isoforms of cyclooxygenase: COX1 and COX2. When activated in endothelial cells, COX1 facilitates the production of prostacyclins, which inhibit blood clots (thrombin). In vascular smooth muscle cells and platelets, COX1 facilitates the production of thromboxane A2, which is prothrombotic. Thus cardiovascular health depends in part on the balance between prostacyclins and thromboxane A2, generated by distinct cell types. Low doses of aspirin, a nonsteroidal antiinflammatory drug (NSAID), reduce thromboxane A2 production by platelets with little effect on endothelial prostacyclin production. Thus low-dose aspirin is antithrombotic (i.e., reduces blood clots). COX2 is activated by inflammatory stimuli. Thus the ability of NSAIDs (e.g., aspirin, ibuprofen, naproxen, acetaminophen, indomethacin) to suppress the inflammatory response is due to inhibition of COX2. Both COX1 and COX2 facilitate the production of prostanoids that protect the stomach. Several lines of evidence suggest that both COX1 and COX2 must be inhibited to elicit damage to the gastrointestinal tract. Consequently, the negative effects of NSAIDs on the gastric mucosa (e.g., increased incidence of gastrointestinal bleeding) are most likely due to inhibition of COX1 and COX2 by these nonselective COX inhibitors.

Selective COX2 inhibitors (e.g., celecoxib, rofecoxib) are very effective in selectively inhibiting COX2 and are used extensively to reduce the inflammatory response. Because COX2 inhibitors are thought to lack the negative effects elicited by NSAIDs on the gastrointestinal tract, their use has increased dramatically. However, in 2005, the U.S. Food and Drug Administration (FDA) announced that selective COX2 inhibitors, as well as nonselective NSAIDs, increase the risk for heart attacks and strokes and required that COX2selective and nonselective NSAIDs carry a warning label on product packaging that highlighted the potential for the increased risk for adverse cardiovascular events and stroke. In addition, although much evidence suggests that COX2selective inhibitors do not cause gastrointestinal bleeding, in 2005 the FDA also required the pharmaceutical industry to add to the warning label on COX2-selective drugs a caution about the potential for increased risk for gastrointestinal bleeding. In 2015, the FDA strengthened warnings that both COX2-selective and COX2-nonselective NSAIDs increase the risk of heart attacks and strokes.

bSee U.S. Food and Drug Administration. FDA Drug Safety Communication: FDA Strengthens Warning That Non-aspirin Nonsteroidal Anti-inflammatory Drugs (NSAIDs) Can Cause Heart Attacks or Strokes. < http://www.fda.gov/

Drugs/DrugSafety/ucm451800.htm >; 2015 Accessed 25.07.16.

ANP binds to the extracellular domain of the plasma membrane receptor guanylyl cyclase and induces a conformational change in the receptor that causes receptor dimerization and activation of guanylyl cyclase, which metabolizes GTP to cGMP. cGMP activates cGMP-dependent protein kinase, which phosphorylates proteins on specific serine and threonine residues. In the kidney, ANP inhibits reabsorption of sodium and water by the collecting duct (see

Nitric oxide activates a soluble receptor guanylyl cyclase that converts GTP to cGMP, which relaxes smooth muscle. Because nitroglycerin increases blood concentrations of

Ras GTPases, monomeric G proteins, are involved in many signaling pathways that control cell division, proliferation, and death. Many mutations of proteins in the Ras signaling pathway are oncogenic (cancer causing) or inactivate tumor suppressors. Mutations in Ras genes that inhibit GTPase activity, as well as overexpression of Ras proteins as a result of transcriptional activation, lead to continuous cell proliferation, a major step in the development of cancer in many organs, including the pancreas, colon, and lungs. In addition, mutations in and overexpression of GEFs, which facilitate exchange of GTP for GDP, and GTPase-activating proteins, which accelerate GTP hydrolysis, may also be oncogenic (see

Fig. 3.8B nitric oxide, which increases cGMP and thereby relaxes smooth muscle in coronary arteries, it has long been used to treat angina pectoris (i.e., chest pain caused by inadequate blood flow to heart muscle; see

The TGF-β receptor is a threonine/serine kinase that has two subunits. Binding of TGF-β to the type II subunit induces it to phosphorylate the type I subunit on specific serine and threonine residues, which in turn, phosphorylates other downstream effector proteins on serine and threonine residues and thereby elicits cellular responses, including cell growth, cell differentiation, and apoptosis.

There are two classes of tyrosine kinase receptors. Nerve growth factor (NGF) receptors are typical examples of one class. Ligand binding to two NGF receptors facilitates their dimerization and thus enables the cytoplasmic tyrosine kinase domain of each monomer to phosphorylate and activate the other monomer. Once the other monomer is phosphorylated, the cytoplasmic domains can recruit GEFs such as growth factor-receptor bound protein 2 to the plasma membrane, which in turn activates Ras and downstream kinases that regulate gene transcription programs important for cell survival and proliferation.

Activation of the insulin receptor (which is tetrameric and composed of two α and two β subunits) by insulin is an example of the other type of tyrosine kinase receptor. Binding of insulin to the α subunits produces a conformational change that facilitates interaction between the two α and β pairs. Binding of insulin to its receptor causes autophosphorylation of tyrosine residues in the catalytic domains of the β subunits, and the activated receptor then phosphorylates cytoplasmic proteins to initiate its cellular effects, including stimulating the absorption of glucose from the blood into skeletal muscle and fat tissue.

The tyrosine kinase–associated receptors have no intrinsic kinase activity but associate with proteins that do have

CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression tyrosine kinase activity, including tyrosine kinases of the Src family and Janus family. Receptors in this class bind several cytokines, including interleukin-6, a proinflammatory cytokine that is necessary for resistance to bacterial infections, and erythropoietin, which stimulates the production of red blood cells. Tyrosine kinase–associated receptor subunits assemble into homodimers (αα), heterodimers (αβ), or heterotrimers (αβγ) when ligands bind. Subunit assembly enhances the binding of tyrosine kinases, which induces kinase activity and thereby phosphorylates tyrosine residues on the kinases, as well as on the receptor. Most polypeptide growth factors bind to tyrosine-kinase-associated receptors.

Regulation of Gene Expression by Signal Transduction Pathways

Steroid and thyroid hormones, cAMP, and receptor tyrosine kinases are transcription factors that regulate gene expression and thereby participate in signal transduction pathways. This section discusses the regulation of gene expression by steroid and thyroid hormones, cAMP, and receptor tyrosine kinases.

The family of nuclear receptors includes more than 30 genes and has been divided into two subfamilies on the basis of structure and mechanism of action: (1) steroid hormone receptors and (2) receptors that bind retinoic acid, thyroid hormones (iodothyronines), and vitamin D. When ligands bind to these receptors, the ligand-receptor complex activates transcription factors that bind to DNA and regulate the expression of genes (see

Figs. 3.2B, 3.5, and 3.7

The location of nuclear receptors varies. Glucocorticoid and mineralocorticoid receptors are located in the cytoplasm, where they interact with chaperones (i.e., heat shock proteins; see

Fig. 3.2B ). Binding of hormone to these receptors results in a conformational change that causes chaperones to dissociate from the receptor, thereby revealing a nuclear localization motif that facilitates translocation of the hormone-bound receptor complex to the nucleus. Estrogen and progesterone receptors are located primarily in the nucleus, and thyroid hormone and retinoic acid receptors are located in the nucleus bound to DNA.

When activated by hormone binding, nuclear receptors bind to specific DNA sequences in the regulatory regions of responsive genes called hormone response elements. Ligand-receptor binding to DNA causes a conformational change in DNA that initiates transcription. Nuclear receptors also regulate gene expression by acting as transcriptional repressors. For example, glucocorticoids suppress the transcription activator protein-1 (AP-1) and nuclear factor κB, which stimulate the expression of genes that cause inflammation. By this mechanism glucocorticoids reduce inflammation.

As noted previously, cAMP is an important second messenger. In addition to its importance in activating PKA, which phosphorylates specific serine and threonine residues on proteins, cAMP stimulates the transcription of many genes, including those that code for hormones, including somatostatin, glucagon, and vasoactive intestinal polypeptide (see

Fig. 3.10 ). Many genes activated by cAMP have a cAMP response element (CRE) in their DNA. Increases in cAMP stimulate PKA, which not only acts in the cytoplasm but also can translocate to the nucleus, where it phosphorylates CREB and thereby increases its affinity for CREB-binding protein (CBP). The CREB-CBP complex activates transcription. The response is terminated when PKA phosphorylates a phosphatase that dephosphorylates CREB (see

Fig. 3.10).

Many growth factors, including EGF, PDGF, NGF, and insulin, bind to and activate enzyme-linked receptors that have tyrosine kinase activity. Activation of tyrosine kinases initiates a cascade of events that enhance the activity of the small GTP-binding protein Ras, which in a series of steps and intermediary proteins phosphorylates the mitogenactivated protein kinase, which then translocates to the nucleus and stimulates transcription of genes that stimulate cell growth.

Tyrosine kinase–associated receptors, as noted earlier, are activated by a variety of hormones, including cytokines, growth hormone, and interferon. Although these receptors do not have tyrosine kinase activity, they are associated with Janus family proteins, which do have tyrosine kinase activity. Once activated, hormone tyrosine kinase– associated receptors activate Janus family protein, which phosphorylates latent transcription factors called signal transducers and activators of transcription (STATs). When phosphorylated on tyrosine residues, STATs dimerize and then enter the nucleus and regulate transcription.

1. The function of cells is tightly coordinated and integrated by external chemical signals, including hormones, neurotransmitters, growth factors, odorants, and products of cellular metabolism that serve as chemical messengers and provide cell-to-cell communication. Chemical and physical signals interact with receptors located in the plasma membrane, cytoplasm, and nucleus. Interaction of these signals with receptors initiates a cascade of events that mediate the response to each stimulus.

These pathways ensure that the cellular response to external signals is specific, amplified, tightly regulated, and coordinated.

2. There are two classes of GTP-binding proteins: monomeric G proteins and heterotrimeric G proteins composed of α, β, and γ subunits. Monomeric G proteins regulate actin cytoskeleton organization, cell cycle progression, intracellular vesicular transport, and gene expression. Heterotrimeric G proteins regulate ion channels, adenylyl cyclase and the cAMP-PKA signaling pathway, phosphodiesterases (which also regulate cAMP and cGMP signaling pathways), and

Cheung E, Kraus WL. Genomic analyses of hormone signaling and gene regulation. Annu Rev Physiol. 2010;72:191-218.

Huang P, Chandra V, Rastinejad F. Structural overview of the nuclear receptor superfamily: insights into physiology and therapeutics. Annu Rev Physiol. 2010;72:247-272.

Levin ER. Extranuclear steroid receptors are essential for steroid hormone actions. Annu Rev Med. 2015;66:271-280.

Riccardi D, Kemp P. The calcium-sensing receptor beyond extracellular calcium homeostasis: conception, development, adult physiology, and disease. Annu Rev Physiol. 2012;74:271-297.

Wu H. Higher-order assemblies in a new paradigm of signal transduction. Cell. 2013;153:287-292.

phospholipases, which regulate the production of prostaglandins, prostacyclins, and thromboxanes.

3.

There are four subtypes of enzyme-linked receptors that mediate the cellular response to a wide variety of signals, including ANP, nitric oxide, TGF-β, PDGF, insulin, and interleukins.

4.

There are two types of nuclear receptors: (1) one type that in the absence of ligand is located in the cytoplasm and when bound to ligand translocates to the nucleus and (2) another type that permanently resides in the nucleus. Both classes of receptors regulate gene transcription.

Cantley L. Signal transduction. In: Boron WF, Boulpaep EL, eds. Medical Physiology. 3rd ed. Philadelphia: Elsevier; 2016 [Chapter 3].

Caplan MJ. Functional organization of the cell. In: Boron WF, Boulpaep EL, eds. Medical Physiology. 3rd ed. Philadelphia: Elsevier; 2016 [Chapter 2].

Heald R. Cell signaling. In: Alberts B, Johnson A, Lewis J, et al., eds. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015 [Chapter 15].

Igarashi P. Regulation of gene expression. In: Boron WF, Boulpaep EL, eds. Medical Physiology. 3rd ed. Philadelphia: Elsevier; 2016 [Chapter 4].

SECTION 2The Nervous System ERIC J. LANG AND KALMAN RUBINSON

Chapter 4 The Nervous System: Introduction to Cells and Systems

Chapter 5 Generation and Conduction of Action Potentials

The Somatosensory System

The Special Senses

Organization of Motor Function

Chapter 10 Integrative Functions of the Nervous System

Chapter 11 The Autonomic Nervous System and Its Central Control

Upon completion of this chapter the student should be able to answer the following questions: 1.

What are the major cell types of the central and peripheral nervous systems?

2.

What are the major components of a neuron, and what are their functional roles?

3.

What are the functional roles of the major glial cell types?

4.

What are the main divisions of the central nervous system?

5.

How and where is the cerebrospinal fluid formed, and how does it circulate and exit the ventricular system?

6.

How is axon transport related to the response of the axon to transection?

he nervous system is a communications and control network that allows an organism to interact rapidly and adaptively with its environment, where environment includes both the external environment (the world outside the body) and the internal environment (the components and cavities of the body). To carry out its function the nervous system takes in sensory information from a variety of sources using specialized sensors (receptors), integrates this information with previously obtained information stored as memories and with the intrinsic goals and drives of the organism that have been embedded in its nervous system through evolution, decides on a course of action, and then issues commands to the effector organs (muscles and glands) to execute the chosen behavioral response.

Moreover, almost all behavioral responses require the coordination of many body parts. For example, even a simple reaching movement of the arm may require coactivation of axial muscles and possibly muscles in the lower extremity to maintain posture and balance, which themselves may be monitored by up to three different sensory systems (vision, vestibular, and proprioceptive) whose information has to be integrated. Furthermore, movements can alter the internal environment and thus can require compensatory changes in heart and breathing rates, blood vessel diameters, and other internal processes. All these variables are monitored and controlled by various specialized subsystems of the nervous system, all of which must work together for the organism to perform movements and more generally to survive. The succeeding chapters will describe these major subsystems individually; however, it should be remembered that in reality their activity is integrated to generate normal behavior.

To begin, it is useful to divide the nervous system into central and peripheral parts. The central nervous system (CNS) consists of the brain and spinal cord. The peripheral nervous system (PNS) consists of nerves and ganglia (small groups of neurons) that innervate all parts of the body and provide an interface between the environment and the CNS. The transition between the CNS and PNS occurs on the dorsal and ventral rootlets near to where they emerge from the spinal cord and on the cranial nerve fibers near to where they arise from the brain.

Cellular Components of the Nervous System

The nervous system is made up of cells, connective tissue, and blood vessels. The major cell types are neurons (nerve cells) and glia (neuroglia = “nerve glue”). In its most general form a neuron’s function can be defined as generation of signals (to be sent to other neurons or effector cells [e.g., muscle cells]) based on an integration of its own electrical properties with electrochemical signals from other neurons. The points where specific neuron-to-neuron communication occurs are known as synapses, and the process of synaptic transmission is critical to neuronal function (see ). Neuroglia, or just glia, traditionally have been characterized as supportive cells that sustain neurons both metabolically and physically, isolate individual neurons from each other, and help maintain the internal milieu of the nervous system; however, it is now known that they also have important roles in shaping the flow of activity through the nervous system.

The typical neuron consists of three main cellular compartments: a cell body (also referred to as a perikaryon or

CHAPTER 4 The Nervous System: Introduction to Cells and Systems •Fig. 4.1 Schematicdiagramofanidealizedneuronanditsmajorcomponentsandconnections.A,Afferentinputfromaxonsofothercellsterminatesinsynapsesonthedendritesandcellbody.Theinitialsegmentoftheaxonattachesattheaxonhillock.Thisaxonismyelinated,asindicatedbythebluestructuresthatencapsulatesegmentsoftheaxon.Theaxonterminatesontwopostsynapticneuronsbyformingsynapticterminals.B,NodesofRanvierarethegapsbetweenthemyelinsegmentswheretheaxonmembraneisexposedtotheextracellularspace.C,Higher-magnificationviewofsynapse.(RedrawnfromBlumenfeldH.Neuroanatomy Through Clinical Cases. 2nded.Sunderland,MA:SinauerAssociates;2010.) soma), a variable number of processes that extend from the soma called dendrites, and an axon (

Fig. 4.1 ). A tremendous number of morphological variants of this basic template exist, including cases where dendrites or an axon may be absent (

Fig. 4.2 ). These variations do not occur randomly but rather relate to the distinct functional properties of each neuronal class. Indeed, neurons with similar morphologies often characterize specific regions of the CNS and reflect the distinct neuronal processing performed in each CNS region.

The cell body is the main genetic and metabolic center of the neuron. Correspondingly it contains the nucleus and nucleolus of the cell and also possesses a well-developed biosynthetic apparatus for manufacturing membrane constituents, synthetic enzymes, and other chemical substances needed for the specialized functions of nerve cells. The neuronal biosynthetic apparatus includes Nissl bodies, which are stacks of rough endoplasmic reticulum, and a prominent Golgi apparatus. The soma also contains numerous mitochondria and cytoskeletal elements, including neurofilaments and microtubules.

The cell body is also a region in which the neuron receives synaptic input (i.e., electrical and chemical signals from other neurons). Although quantitatively the synaptic input to the soma is usually much less than that to dendrites, it often differs qualitatively from dendritic inputs, and by virtue of the closeness of the soma to the axon, inputs to the soma can override those to the dendrites (see

Dendrites are tapering and branching extensions of the soma and are the main direct recipients of signals from other neurons. They can be thought of as a way to expand and specialize the surface area of a neuron, and indeed, they may account for more than 90% of the surface area available for synaptic contact (soma plus dendrites). Dendrites can be divided into primary dendrites (those that extend directly from the soma) and higher-order dendrites (daughter branches extending from a more proximal branch, in •Fig. 4.2 A,Purkinjecell.B,Pyramidalcell.C,Golgicell.D,Granulecell.E,Inferiorolivecells.F,Bipolarcells.(A,CourtesyofBorisBarbour.B,CourtesyofT.F.Fletcher,from http://vanat.cvm.umn.edu/ neurHistAtls/pages/neuron3.html .C,FigurewasprovidedbyCourtHullandWadeRegehr,DepartmentofNeurobiology,HarvardMedicalSchool.D,FromDelvendahlIetal.Front Cell Neurosci 2015;9:93,Fig.1A.E,FromMathyA,ClarkBA.In:MantoMetal[eds].Handbook of the Cerebellum and Cerebellar Disorders. Dordrecht,Netherlands:SpringerScience+BusinessMediaDordrecht;2013.F,FromLiW,DeVriesSH.Nat Neurosci 2006;9:669-675,Fig.2.) which proximal refers to closeness to the soma). The main cytoplasmic organelles in dendrites are microtubules and neurofilaments; however, the primary dendrites can also contain Nissl bodies and parts of the Golgi apparatus.

A neuron’s set of dendrites is termed its dendritic tree. Dendritic trees differ tremendously between different types of neurons in terms of the size, number, and spatial organization of the dendrites. A dendritic tree can consist of just a few unbranched dendrites or of many highly ramified dendrites. Individual dendrites can be longer than 1 mm or only 10 to 20 µm in length. Another major morphological variation is whether or not a dendrite has spines, which are small mushroomor lollipop-shaped protrusions from the main dendrite. Spines are sites specialized for synaptic contact (usually, but not always) from excitatory inputs. The shape and size of the dendritic tree, as well as the population and distribution of channels in the dendritic membrane, are all important determinants of how the synaptic input will affect the neuron (see

Chapter 6).

The axon is an extension of the cell that conveys the output of the cell to other neurons or, in the case of a motor neuron, to muscle cells as well. In general, each neuron has only one axon, and it is usually of uniform diameter. The length and diameter of axons vary with the neuronal type. Some axons do not extend much beyond the length of the dendrites, whereas others may be a meter or more long. Axons may have orthogonal branches en passant, but they often end in a spray of branches called a terminal arborization (represented by the four terminal branches and their synaptic terminals in

Fig. 4.1A ). The size, shape, and organization of the terminal arborization determine which other cells it will contact. The first part of the axon is known as the initial segment and arises from the soma (or sometimes from a proximal dendrite) in a specialized region called the axon hillock. The axon differs from the soma and proximal dendrites in that it lacks rough endoplasmic reticulum, free ribosomes, and a Golgi apparatus. The initial segment is usually the site where action potentials (spikes) that are propagated down the axon are initiated (see ). An axon may terminate in a synapse and/or it may make synapses along its length. Synapses will be described in detail in

CHAPTER 4 The Nervous System: Introduction to Cells and Systems •Fig. 4.3 Axonaltransport.Schematicofneuronandenlargementofaxonaltransportmechanism.Axonaltransportdependsonmovementofmaterialalongtransportfilamentssuchasmicrotubules.Transportedcomponentsattachtotransportfilamentsbymeansofcross-bridges.Differentobjectsaretransportedanterogradely(fromcellbodytoaxonterminal)andothersretrogradely(towardthecellbody).Thedirectionoftransport—retrogradeandanterograde—isdeterminedbyspecificproteinssuch asdyneinandkinesin,respectively.

Neurons are special because of their ability to control and respond to electricity. Moreover, the response and control mechanisms of each part of a neuron are distinct from those in other parts. This intraneuronal specialization is a consequence of the particular morphology and the ion channel composition of each part of the neuron. For example, dendrites have ligand gated ion channels that allow neurons to respond to chemicals released by other neurons, and their characteristic branching pattern allows for integration of multiple input signals. In contrast the axon typically has a long length and high concentration of voltage-gated channels that allows it to convey electrical signals (action potentials) rapidly over long distances without alteration.

Because the soma is the metabolic engine of the neuron, substances needed to support axonal and synaptic function are synthesized there. These substances must be distributed to replenish secreted or inactivated materials along the axon and especially to the presynaptic terminals. Most axons are too long to allow efficient movement of substances from the soma to the synaptic endings by simple diffusion. Thus special axonal transport mechanisms have evolved to accomplish this task (

Fig. 4.3).

A consequence of this metabolic dependency is that axons degenerate when disconnected from the cell body, a fact that has been used by scientists tracing out neuronal pathways; they would cut an axonal pathway and then determine where the degenerating axons distal to the cut projected to.

Several types of axonal transport exist. Membrane-bound organelles and mitochondria are transported relatively rapidly by fast axonal transport. Substances that are dissolved in cytoplasm (e.g., proteins) are moved by slow axonal transport. In mammals, fast axonal transport proceeds as rapidly as 400 mm/day, whereas slow axonal transport occurs at about 1 mm/day. Synaptic vesicles, which travel by fast axonal transport, can travel from the soma of a motor neuron in the spinal cord to a neuromuscular junction in a person’s foot in about 2.5 days. In comparison the movement of some soluble proteins over the same distance can take nearly 3 years.

Axonal transport requires metabolic energy and involves calcium ions. Microtubules provide a system of guidewires along which membrane-bound organelles move (see

Fig.

4.3 ). Organelles attach to microtubules through a linkage similar to that between the thick and thin filaments of skeletal muscle fibers. Ca++ triggers movement of the organelles along the microtubules. Special microtubule-associated motor proteins called kinesin and dynein are required for axonal transport.

Axonal transport occurs in both directions. Transport from the soma toward the axonal terminals is called anterograde axonal transport. This process involves kinesin, and it allows replenishment of synaptic vesicles and enzymes responsible for the synthesis of neurotransmitters in synaptic terminals. Transport in the opposite direction, which is driven by dynein, is called retrograde axonal transport. This process returns recycled synaptic vesicle membrane to the soma for lysosomal degradation.

Certainvirusesandtoxinscanbeconveyedbyaxonaltransportalongperipheralnerves.Forexample,herpeszoster,thevirusofchickenpox,invadesdorsalrootganglioncells.Thevirusmaybeharboredbytheseneuronsformanyyears.However,eventuallythevirusmaybecomeactivebecauseofachangeinimmunestatus.Thevirusmaythenbetransportedalongthesensoryaxonstotheskin,causingshingles,averypainfuldisease.Anotherexampleistheaxonaltransportoftetanustoxin.Clostridium tetani bacteriamaygrowinadirtywound,andifthepersonhadnotbeenvaccinatedagainsttetanustoxin,thetoxincanbetransportedretrogradelyintheaxonsofmotorneurons.Thetoxincanescapeintotheextracellularspaceofthespinalcordventralhornandblockthesynapticreceptorsforinhibitoryaminoacids.Thisprocesscanresultintetanicconvulsions.

The major nonneuronal cellular elements of the nervous system are the glia (

Fig. 4.4 ). Glial cells in the human CNS outnumber neurons by an order of magnitude: there are about 1013 glia and 1012 neurons. Glial cells in the CNS include astrocytes, oligodendrocytes, microglia, and ependymal cells (see

Fig. 4.4 ); in the PNS the glial cells are Schwann cells and satellite cells. Traditionally, glial cells were thought of as supportive cells, and consistent with that conception, their functions include regulation of the microenvironment and myelination of axons. Glial cells are now also recognized to be important determinants of the flow of signals through neuronal circuits, and related to that they act to modulate synaptic and nonsynaptic transmission and have important roles in synaptogenesis and maintenance.

Astrocytes (named for their star shape) help regulate the microenvironment of the CNS, both under normal conditions and in response to damage to the nervous system. Astrocytes have a cell body from which several main branches arise. Through repeated branching these main processes give rise to hundreds to thousands of branchlets. Astrocyte processes contact neurons and surround synaptic endings, isolating them from adjacent synapses and the general extracellular space. Astrocytes also have foot •Fig. 4.4 SchematicrepresentationofcellularelementsintheCNS.Twoastrocytesareshownendingonasomaanddendritesofaneuron.Astrocytesalsocontactthepialsurfaceorcapillariesorboth.Anoligodendrocyteprovidesthemyelinsheathsforaxons.Alsoshownaremicrogliaandependymalcells. N,neuron.(RedrawnfromWilliamsPL,WarwickR.Functional Neuroanatomy of Man. Edinburgh:ChurchillLivingstone;1975.) processes that contact the capillaries and connective tissue at the surface of the CNS, the pia mater (see

Fig. 4.4 ). These foot processes may help mediate the entry of substances into the CNS. Astrocytes can actively take up K+ ions and neurotransmitter substances, which they metabolize, biodegrade, or recycle. Thus astrocytes serve to buffer the extracellular environment of neurons with respect to both ions and neurotransmitters. The cytoplasm of astrocytes contains glial filaments that provide mechanical support for CNS tissue. After injury the astrocytes undergo a variety of changes to become reactive astrocytes. One example is a class of reactive astrocytes that act to form a glial scar around an area of focal damage, which segregates the damaged tissue and thereby allows inflammatory processes to act selectively at the site of damage, minimizing the impact on surrounding normal tissue. Astrocytes can also affect the properties of synaptic transmission, which is discussed in

Oligodendrocytes and Schwann cells are critical for the function of axons. Many axons are surrounded by a myelin sheath, which is a spiral multilayered wrapping of glial cell membrane (

Fig. 4.5A–B ). In the CNS, myelin is formed by the oligodendrocytes, whereas in the PNS Schwann cells form myelin. Myelin increases the speed of action potential conduction, in part by restricting the

CHAPTER 4 The Nervous System: Introduction to Cells and Systems •Fig. 4.5 Axonal/glialassociations.A,MyelinatedaxonsintheCNS.Asingleoligodendrocyte(G)emitsseveralprocesses,eachofwhichwindsinaspiralfashionaroundanaxontoformamyelinsegment.Theaxonisshownincutaway.Themyelinfromasingleoligodendrocyteendsbeforethenextwrappingfromanotheroligodendrocyte.ThebareaxonbetweenthemyelinatedsegmentsisthenodeofRanvier(N).B,ElectronmicrographofmyelinatedaxoninthePNSshownincrosssection.Theaxon(Ax)isseenatthecenterwithinasheathconsistingofmultiplewrappingsoftheSchwanncell’scytoplasmicmembrane.TheSchwanncellsoma(SC)isattheupperright.mes,mesaxon,internalandexternal.C,Electronmicro-graphofunmyelinatedaxonsinPNS.Nineaxons(asterisks) cutincrosssectionareseenembeddedinaSchwanncellwhosenucleusisatthecenter(N).Atlowerrightaportionofamyelinatedaxonisvisible.(B,FromPetersAetal.The Fine Structure of the Nervous System. NewYork:OxfordUniversityPress;1991,Fig.6.5.C,FromPanneseE.Neurocytology. 2nded.Basel,Switzerland:SpringerInternational;2015.)

Astrocytesarecoupledtoeachotherbygapjunctionssuchthattheyformasyncytiumthroughwhichsmallmoleculesandionscanredistributealongtheirconcentrationgradientsorbycurrentflow.Whennormalneuralactivitygivesrisetoalocalincreaseinextracellular[K+],thiscouplednetworkcanenablespatialredistributionofK+ overawideareaviacurrentflowinmanyastrocytes.

Underconditionsofhypoxia,suchasmightbeassociatedwithischemiasecondarytoblockageofanartery(i.e.,astroke),[K+]intheextracellularspaceofabrainregioncanincreasebyafactorofasmuchas20.Thiswilldepolarizeneuronsandsynapticterminalsandresultinreleaseoftransmitterssuchasglutamate,whichwillcausefurtherreleaseofK+ fromneurons.Theadditionalreleaseonlyexacerbatestheproblemandcanleadtoneuronaldeath.Undersuchconditions,localastrogliawillprobablytakeuptheexcessK+ byK+-Cl− symportratherthanbyspatialbuffering,becausetheelevationinextracellular[K+]tendstobewidespreadratherthanlocal.

flow of ionic current to small unmyelinated portions of the axon between adjacent glial cells, the nodes of Ranvier (see ). Although both act to increase the speed of conduction, there are several important differences in the relationship between axons and either oligodendrocytes or Schwann cells. One major difference is that a single oligodendrocyte typically helps myelinate multiple axons in the CNS, whereas each Schwann cell helps myelinate only a single axon in the PNS. A second difference is that in the CNS, unmyelinated axons are bare, whereas in the PNS, unmyelinated axons are not. Rather, they are surrounded by Schwann cell processes; the Schwann cell, however, does not form a multilayered covering (i.e., myelin), but instead extends processes that surround parts of several axons (the Schwann cell with its set of unymyelinated axons is called a Remak bundle) (see

Fig. 4.5C

Satellite cells encapsulate dorsal root and cranial nerve ganglion cells and regulate their microenvironment in a fashion similar to that of astrocytes.

Microglia are derived from erythromyeloid stem cells that migrate into the CNS early in development. They play an important role in immune responses within the CNS. When the CNS is damaged, microglia help remove the cellular products of the damage by phagocytosis. They are assisted by other glia and by other phagocytes that invade the CNS from the circulation. In addition to their role in immune responses, recent evidence suggests they are also active in healthy brain tissue and may have important roles in normal brain development and function, including pruning of excess synapses that are formed during development and synaptic plasticity.

Ependymal cells form the epithelium lining the ventricular spaces of the brain, which contain (cerebrospinal fluid) CSF. CSF is secreted in large part by specialized ependymal cells of the choroid plexuses located in the ventricular system. Many substances diffuse readily across the ependyma, which lies between the extracellular space of the brain and the CSF.

Mostneuronsintheadultnervoussystemarepostmitoticcells(althoughsomestemcellsmayalsoremainincertainsitesinthebrain).Manyglialprecursorcellsarepresentintheadultbrain,andtheycanstilldivideanddifferentiate.Thusthecellularelementsthatgiverisetomostintrinsicbraintumorsintheadultbrainaretheglialcells.Forexample,braintumorscanbederivedfromastrocytes(whichvaryinmalignancyfromtheslowlygrowingastrocytomatotherapidlyfatalglioblastomamultiforme),fromoligodendroglia(oligodendroglioma),orfromependymalcells(ependymoma).Meningealcellscanalsogiverisetoslowlygrowingtumors(meningiomas)thatcompressbraintissue,ascanSchwanncells(e.g.,acousticschwannomas,whicharetumorsformedbySchwanncellsoftheeighthcranialnerve).Inthebrainofinfants,neuronsthatarestilldividingcansometimesgiverisetoneuroblastomas(e.g.,oftheroofofthefourthventricle)orretinoblastomas(intheeye).

The Peripheral Nervous System

The PNS provides an interface between the environment and the CNS, both for sensory information flowing to the CNS and for motor commands issued from the CNS. It includes sensory (or primary afferent) neurons, somatic motor neurons, and autonomic motor neurons.

Sensory pathways into the nervous system start with a receptor, which may simply be a specialized part of an axon in the PNS or may include additional cells. Each sensory receptor is organized so that it transduces a specific type of energy into an electrical signal. Thus they can be classified in terms of the type of energy they transduce (e.g., photoreceptors transduce light, mechanoreceptors transduce displacement and force). They may also be classified according to the source of the input (e.g., exteroceptors signal external events, proprioceptors signal the state of a body part such as the angle of elbow, and interoceptors signal the distension of the gut).

The transduction process leads to an electrical response in the primary afferent called a receptor potential, which triggers action potentials in the primary afferent fibers innervating the receptor. These action potentials contain information about the sensory stimulus that is conveyed to the CNS via the primary afferent.

Somatic and autonomic motor neurons convey signals from the CNS to their respective effector targets. The somatic motor neurons innervate the skeletal muscles throughout the body. Their cell bodies lie in the ventral horn (or equivalent brainstem nuclei) and project out of the CNS via a ventral root or cranial nerve. The details of their relationship to muscles is covered in . The autonomic motor pathway is responsible for controlling the functioning of organs, smooth muscle, and glands. It is actually a two-neuron pathway, and its properties are covered in

Chapter 11.

The Central Nervous System

The CNS is built from the cellular elements just described and includes the spinal cord and brain (

Fig. 4.6A ). These cellular elements are connected in a variety of complex ways to form the subsystems that underlie the multitude of functions performed by the CNS. The physiology of these systems is covered in ; however, a basic knowledge of CNS anatomy is needed to understand systems physiology and will be briefly discussed here.

Regions of the CNS containing high concentrations of axon pathways (and very few neurons) are called white matter because the axonal myelin sheaths of the axons are highly refractive to light. Regions containing high concentrations of neurons and dendrites are by contrast called gray matter. Note that axons are also present in gray matter. These axons may be related to local processing (i.e., either originating from local neurons or terminating on them) or may be fibers of passage. Thus effects of damage to an area may reflect either loss of local function or disconnection

CHAPTER 4 The Nervous System: Introduction to Cells and Systems pallidus Fimbria of Temporal (fornix) Caudate Inferior horn nucleus of lateral (tail) Mammillary body ventricle

OpticB Cerebral peduncle of hypothalamus tract Hippocampus •Fig. 4.6 A,SchematicofthemajorcomponentsoftheCNSasshowninalongitudinalmidlineview.B–F,Representativesectionsthroughthebrainandspinalcord,withthemajorlandmarkslabeled.B,Cerebrumandthalamus;C,midbrain;D,pons;E,medulla;F,cervicalspinalcord.Notethatmanypathways(e.g.,corticospinalfibers)crosssides(decussate)astheytravelthroughtheCNS,butthesedescussationsarenotindicatedinthefigure(seeChapters7and9fordetailsonthemotorandsensorypathwaycrossings).(A,FromHainesDE[ed].Fundamental Neuroscience for Basic and Clinical Applications. 3rded.Philadelphia:ChurchillLivingstone;2006.) of remote regions that had been linked by fibers of passage through the area that was damaged.

In the CNS, axons often travel in bundles or tracts. The names applied to tracts usually describe their origin and termination. For example, the spinocerebellar tracts convey information from the spinal cord to the cerebellum. The term pathway is similar to tract but is generally used to suggest a particular function (e.g., the auditory pathway: a series of neuron-to-neuron links across several synapses that convey and process auditory information).

Gray matter exists in two main configurations in the CNS. A nucleus is a group of neurons in the CNS (in the PNS such a grouping is called a ganglion). Examples include the thalamic, cerebellar, and cranial nerve nuclei. A cortex is neurons that are organized into layers and usually found on the surface of the CNS. The most prominent are the cerebral and cerebellar cortices, which cover the surface of the cerebral hemispheres and the cerebellum, respectively (

Fig. 4.7

In most nuclei and cortices, one can classify neurons into two broad categories: projection cells and local interneurons. Projection cells are neurons that send their axon to another region and thus are the origins of the various tracts of the nervous system. In contrast, local interneurons have axons that terminate in the same neural structure as their cell of origin and are involved with local computations rather than conveying signals from one region to another. These categories are not exclusive; many neurons have axons that both give off local branches and project to one or more distant regions.

• Fig. 4.7 Lateral view of the human brain showing the left cerebral hemisphere, cerebellum, pons, and medulla. Note the division of the lobes of the cerebrum (frontal, parietal, occipital, and temporal) and the two major fissures (lateral and central). (From Nolte J, Angevine J. The Human Brain in Photographs and Diagrams. 2nd ed. St Louis: Mosby; 2000.)

Regional Anatomy of the CNS

The spinal cord can be subdivided into a series of regions (see

Fig. 4.6A ), each composed of a number of segments named for the vertebrae where their nerve roots enter or leave: 8 cervical, 12 thoracic, 5 lumbar, 5 sacral, and 1 coccygeal. Each portion maintains its tubular appearance, although its lumen, the spinal canal, may not remain patent. Within the gray matter the dorsal horn receives and processes sensory information from the dorsal roots, whereas the ventral horn is primarily a motor structure and contains the motor neurons whose axons project out via the ventral roots (

Fig. 4.8

The surrounding white matter consists of many tracts interconnecting spinal cord levels and for communication with the brain. Three major ones are the lateral corticospinal tract (motor), spinothalamic tract/anterolateral system (sensory), and dorsal column-medial lemniscus pathway (sensory) (see

Fig. 4.6F

The brainstem consists of the medulla, pons, and midbrain (

Fig. 4.9

Fig. 4.6 ). In addition to the longitudinal pathways interconnecting with the spinal cord, the brainstem contains nuclei and many additional pathways that vary by level. These structures have many functions, some of which are analogous to those of the spinal cord (e.g., conveying basic sensory information and motor commands) and others related to a variety of other brain functions, such as cardiac control and state of consciousness. The brainstem also receives input and sends motor output via cranial nerves (

Table 4.1

The cerebellum sits dorsal to the pons and medulla. It receives inputs from spinal cord, brainstem, and cerebral cortex and projects back to many of these same structures. The cerebellum is critical for motor coordination but is increasingly recognized as having key roles in other cognitive functions.

• Fig. 4.8 Diagram of the spinal cord, spinal roots, and spinal nerve. The spinal nerve begins where the dorsal and ventral roots fuse, and has multiple branches (rami), the first few of which are represented. A primary afferent neuron is shown with its cell body in the dorsal root ganglion and its central and peripheral processes distributed, respectively, to the spinal cord gray matter and to a sensory receptor in the skin. An α motor neuron is shown to have its cell body in the spinal cord gray matter and to project its axon out the ventral root to innervate a skeletal muscle fiber.

CHAPTER 4 The Nervous System: Introduction to Cells and Systems

Massa intermedia of Thalamus Interventricular foramen Fornix Anterior commissure Infundibular recess Lamina terminalis

Choroid plexus of third ventricle •Fig. 4.9 Midsagittalviewofthebrainshowingthethirdandfourthventricles,thecerebralaqueductofthemidbrain,andthechoroidplexus.TheCSFformedbythechoroidplexusinthelateralventriclesentersthiscirculationviatheinterventricularforamen.Notealsothelocationofthecorpuscallosumandothercommissures.(FromHainesDE[ed]:Fundamental Neuroscience for Basic and Clinical Applications. 3rded.Philadelphia:ChurchillLivingstone;2006.)

The thalamus sits at the upper end of the brainstem and is enclosed by the cerebrum with which it is highly interconnected (see

Fig. 4.6B ). With a few exceptions, ascending information first reaches the thalamus, which conveys it to the cerebral cortex. These structures play a major role in many functions, including conscious awareness, volition, memory, and language. In addition to the cortex, the cerebrum contains a group of deep nuclei, the basal ganglia, that are interconnected with the cortex and thalamus and whose function will be described in

The major functions of the different parts of the CNS are listed in

Table 4.1

CSF fills the ventricular system, a series of interconnected spaces within the brain, and the subarachnoid space directly surrounding the brain. The intraventricular CSF reflects the composition of the brain’s extracellular space via free exchange across the ependyma, and the brain “floats” in the subarachnoid CSF to minimize the effect of external mechanical forces. The volume of CSF within the cerebral ventricles is approximately 30 mL, and that in the sub-arachnoid space is about 125 mL. Because about 0.35 mL of CSF is produced each minute, CSF is turned over more than three times daily.

CSF is a filtrate of capillary blood formed largely by the choroid plexuses, which comprise pia mater, invaginating capillaries, and ependymal cells specialized for transport. The choroid plexuses are located in the lateral, third, and fourth ventricles (see

Fig. 4.9 ). The lateral ventricles are situated within the two cerebral hemispheres. They each connect with the third ventricle through one of the inter-ventricular foramina (of Monro). The third ventricle lies in the midline between the diencephalon on the two sides. The cerebral aqueduct (of Sylvius) traverses the midbrain and connects the third ventricle with the fourth ventricle. The fourth ventricle is a space defined by the pons and medulla below and the cerebellum above. The central canal of the spinal cord continues caudally from the fourth ventricle, although in adult humans the canal is not fully patent and continues to close with age.

CSF escapes from the ventricular system through three apertures or foramina (a medial foramen of Magendie and two lateral foramina of Luschka) located in the roof of the fourth ventricle. After leaving the ventricular system, CSF circulates through the subarachnoid space that surrounds the brain and spinal cord. Regions where these spaces are expanded are called subarachnoid cisterns. An example is the lumbar cistern, which surrounds the lumbar and sacral spinal roots below the level of termination of the spinal cord. The lumbar cistern is the target for lumbar puncture, a clinical procedure to sample CSF. A large part of CSF is removed by bulk flow through the valvular arachnoid granulations into the dural venous sinuses in the cranium.

Because the extracellular fluid within the CNS communicates with the CSF, the composition of the CSF is a useful indicator of the composition of the extracellular environment of neurons in the brain and spinal cord. The main constituents of CSF in the lumbar cistern are listed in

Table 4.2.

For comparison the concentrations of the same constituents in blood are also given. CSF has a lower concentration of K+ , glucose, and protein but a greater concentration of Na+ and Cl− than blood does. Furthermore, CSF contains practically no blood cells. The increased concentration of Na+ and Cl− enables CSF to be isotonic to blood.

The pressure in the CSF column is about 120 to 180 mm H2O when a person is recumbent. The rate at which CSF is formed is relatively independent of the pressure in the

Na+ (mEq/L)148136–145K+ (mEq/L)2.93.5–5Cl− (mEq/L)120–130100–106Glucose(mg/dL)50–7570–100Protein(mg/dL)15–456.8× 103pH7.3

FromWillisWD,GrossmanRG.Medical Neurobiology. 3rded.StLouis:Mosby;1981.

ventricles and subarachnoid space, as well as systemic blood pressure. However, the absorption rate of CSF is a direct function of CSF pressure.

ObstructionofthecirculationofCSFleadstoincreasedCSFpressureandhydrocephalus,anabnormalaccumulationoffluidinthecranium.Inhydrocephalustheventriclesbecomedistended,andiftheincreaseinpressureissustained,brainsubstanceislost.Whentheobstructioniswithintheventricularsystemorintheforaminaofthefourthventricle,theconditioniscalledanoncommunicating hydrocephalus. Iftheobstructionisinthesubarachnoidspaceorthearachnoidvilli,itisknownasacommunicating hydrocephalus.

The Blood-Brain Barrier

The local environment of most CNS neurons is controlled such that neurons are normally protected from extreme variations in the composition of the extracellular fluid that bathes them. Part of this control is provided by the presence of a blood-brain barrier (other mechanisms are the buffering functions of glia, regulation of CNS circulation, and exchange of substances between the CSF and extracellular fluid of the CNS). Movement of large molecules and highly charged ions from blood into the brain and spinal cord is severely restricted. The restriction is at least partly due to the barrier action of the capillary endothelial cells of the CNS and the tight junctions between them. Astrocytes may also help limit the movement of certain substances. For example, astrocytes can take up potassium ions and thus regulate [K+] in the extracellular space. Some pharmaceutical agents, such as penicillin, are removed from the CNS by transport mechanisms.

Nervous Tissue Reactions to Injury

Injury to nervous tissue elicits responses by neurons and glia. Severe injury causes cell death. Except in specific instances, once a neuron is lost it cannot be replaced because, in general,

CHAPTER 4 The Nervous System: Introduction to Cells and Systems 63

The blood-brain barrier can be disrupted by pathology of the brain. For example, brain tumors may allow substances that are otherwise excluded to enter the brain from the circulation. Radiologists can exploit this by introducing a substance into the circulation that normally cannot penetrate the blood-brain barrier. If the substance can be imaged, its leakage into the region occupied by the brain tumor can be used to demonstrate the distribution of the tumor.

neurons are postmitotic cells. In animals, two exceptions are olfactory bulb and hippocampal neurons; however, in humans, only for the hippocampus has evidence been found for significant levels of neurogenesis in the adult CNS.

When an axon is transected, the soma of the neuron may show chromatolysis, or “axonal reaction.” Normally, Nissl bodies stain well with basic aniline dyes, which attach to the RNA of ribosomes (

Fig. 4.10A ). After injury to the

Fig. 4.10B), the neuron attempts to repair the axon by making new structural proteins, and the cisterns of the rough endoplasmic reticulum become distended with the products of protein synthesis. The ribosomes appear to be disorganized, and the Nissl bodies are stained weakly by basic aniline dyes. This process, called chromatolysis, alters the staining pattern (see

Fig. 4.10C ). In addition, the soma may swell and become rounded, and the nucleus may assume an eccentric position. These morphological changes reflect the cytological processes that accompany increased protein synthesis.

Because it cannot synthesize new protein, the axon distal to the transection dies (see

Fig. 4.10C ). Within a few days the axon and all the associated synaptic endings disintegrate. If the axon had been a myelinated axon in the CNS, the myelin sheath would also fragment and eventually be removed by phagocytosis. However, in the PNS the Schwann cells that had formed the myelin sheath remain viable, and in fact they undergo cell division. This sequence of events was originally described by Waller and is called wallerian degeneration.

If the axons that provide the sole or predominant synaptic input to a neuron or to an effector cell are interrupted, the • Fig. 4.10 A, Normal motor neuron innervating a skeletal muscle fiber. B, A motor axon has been severed, and the motor neuron is undergoing chromatolysis. C, This is associated in time with sprouting and, in D, with regeneration of the axon. The excess sprouts degenerate. E, When the target cell is reinnervated, chromatolysis is no longer present.

and even death. The best known example of this is atrophy of skeletal muscle fibers after their innervation by motor neurons has been interrupted. However, if only one or a few of the innervating axons are removed, the other surviving axons may sprout additional terminals, thereby taking up the synaptic space of the damaged axons and increasing their influence on the postsynaptic cell.

In the PNS, after an axon is lost through injury, many neurons can regenerate a new axon. The proximal stump of the damaged axon develops sprouts (see

Fig. 4.10C ), these sprouts elongate, and they grow along the path of the original nerve if this route is available (see

Fig. 4.10D ). The Schwann cells in the distal stump of the nerve not only survive the wallerian degeneration but also proliferate and form rows along the course previously taken by the axons. Growth cones of the sprouting axons find their way along these rows of Schwann cells, and they may eventually reinnervate the original peripheral target structures (see

Fig.

4.10E ). The Schwann cells then remyelinate the axons. The rate of regeneration is limited by the rate of slow axonal transport to about 1 mm/day.

In the CNS, transected axons also sprout. However, proper guidance for the sprouts is lacking, in part because the oligodendroglia do not form a path along which the sprouts can grow. This limitation may be a consequence of the fact that a single oligodendroglial cell myelinates many central axons, whereas a single Schwann cell provides myelin for only a single axon in the periphery. In addition, different chemical signals may affect peripheral and central attempts at regeneration differently. Other obstacles to successful CNS regeneration include formation of a glial scar by astrocytes and lack of trophic influences that guided axonal trajectories during development.

1.

The functions of the nervous system include excitability, sensory detection, information processing, and behavior.

2.

The CNS includes the spinal cord and brain. The brain includes the medulla, pons, cerebellum, midbrain, thalamus, hypothalamus, basal ganglia, and cerebral cortex.

3.

The neuron is the functional unit of the nervous system. Neurons have three major compartments: the dendrites, cell body, and axon. The first two receive and integrate signals, and the axon conveys the output signals of the neuron to other cells.

4.

The PNS includes primary afferent neurons and the sensory receptors they innervate, the axons of somatic motor neurons, and autonomic neurons.

5.

Information is conveyed through neural circuits by action potentials in the axons of neurons and by synaptic transmission between axons and the dendrites and somas of other neurons or between axons and effector cells.

6.

Different types of neurons are specialized as a consequence of their individual morphology and the ion channel distribution in the cell membrane of their soma, dendrites, and axons.

7.

Sensory receptors include exteroceptors, interoceptors, and proprioceptors. Stimuli are environmental events that excite sensory receptors, responses are the effects of stimuli, and sensory transduction is the process by which stimuli are detected by transforming their energy into electrical signals.

8.

Sensory receptors can be classified in terms of the type of energy they transduce or by the source of the input. Central pathways are usually named by their origin and termination or for the type of information conveyed.

9.

Chemical substances are distributed along the axons by fast or slow axonal transport. The direction of axonal transport may be anterograde or retrograde.

10.

Glial cells include astrocytes (regulate the CNS microenvironment), oligodendroglia (form CNS myelin), Schwann cells (form PNS myelin), ependymal cells (line the ventricles), and microglia (CNS macrophages). Myelin sheaths increase the conduction velocity of axons.

11.

The choroid plexus forms CSF. CSF differs from blood in having a lower concentration of K+ , glucose, and protein and a higher concentration of Na+ and Cl−; CSF normally lacks blood cells.

12.

The extracellular fluid composition of the CNS is regulated by CSF, the blood-brain barrier, and astrocytes.

13.

Damage to the axon of a neuron causes an axonal reaction (chromatolysis) in the cell body and wallerian degeneration of the axon distal to the injury. Regeneration of PNS axons is more likely than regeneration of CNS axons.

Squire L, Berg D. Fundamental Neuroscience. 4th ed. Waltham, MA:

Kiernan JA, Rajakumar N. Barr’s The Human Nervous System: An Academic Press; 2012. Anatomical Viewpoint. 10th ed. Philadelphia: Lippincott Williams Vanderah T, Gould D. Nolte’s The Human Brain: An Introduction to its & Wilkins; 2013. Functional Anatomy. 7th ed. St. Louis: Mosby; 2015.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

How is a nerve membrane’s response to small-amplitude stimuli like a passive electric circuit comprising batteries, resistors, and capacitors?

2.

What factors determine the time and length constants of a nerve membrane? How do these constants shape the electric responses of the nerve membrane?

3.

How does an action potential differ from the subthreshold responses of a membrane (i.e., the passive and local responses)?

4.

What is the sequence of conductances that underlies the action potential?

5.

How are the responses of Na+ and K+ channels to membrane depolarization similar? How does the presence of the Na+ channel inactivation gate cause the responses to differ?

6.

How do the gating properties of Na+ and K+ channels relate to the absolute and relative refractory periods of the action potential?

7.

How is the action potential propagated without decrement? What are the factors that determine its propagation velocity?

8.

What are the structural properties of myelin that underlie its ability to increase conduction velocity?

9.

Given the all-or-none nature of action potentials, how are the characteristics of different stimuli distinguished by the central nervous system?

n action potential is a rapid, all-or-none change in the membrane potential, followed by a return to the resting membrane potential. This chapter describes how action potentials are generated by voltage-dependent ion channels in the plasma membrane and propagated with the same shape and size along the length of an axon. The influences of axon geometry, ion channel distribution, and myelin are discussed and explained. The ways in which information is encoded by the frequency and pattern of action potentials in individual cells and in groups of nerve cells are also described. Finally, because the nervous system provides important information about the external world through specific sensory receptors, general principles of sensory transduction and coding are introduced. More detailed information about these sensory mechanisms and systems is provided in other chapters.

When a microelectrode (tip diameter <0.5 µm) is inserted through the plasma membrane of a neuron, a difference in potential is observed between the tip of the microelectrode inside the cell and an electrode placed outside the cell. The internal electrode is approximately 70 mV negative with regard to the external electrode, and this difference is referred to as the resting membrane potential or, simply, the resting potential (see for details on the basis of the resting potential). (By convention, membrane potentials are expressed as the intracellular potential minus the extracellular potential.) Neurons have a resting potential that typically is around −70 mV.

One of the signature features of neurons is their ability to change their membrane potential rapidly from rest in response to an appropriate stimulus. Two such classes of responses are action potentials and synaptic potentials, which are described in this chapter and the next, respectively. Current knowledge about the ionic mechanisms of action potentials comes from experiments with many species. One of the most studied is the squid because the large diameter (up to 0.5 mm) of the squid giant axon makes it an excellent model for electrophysiological research with intracellular electrodes.

The Passive Response

To understand how an action potential is generated and why it is needed, it is necessary to understand the passive electrical properties of the nerve cell membrane. The term passive properties refers to the fact that components of the cell membrane behave very similarly to some of the passive elements of electric circuits, including batteries, resistors, and capacitors. This is very useful because the properties of these elements are well understood. In particular, a piece of membrane containing ion channels responds to changes in voltage across it much as a circuit containing a resistor and capacitor in parallel (parallel RC circuit) would: The ion channels correspond to the resistor, and the lipid bilayer acts as a capacitor. When a battery is first connected across the two terminals of a parallel RC circuit, all of the current flows through the branch of the circuit with the capacitor, causing the voltage across it to begin changing (recall that for a capacitor, I ˜ dV/dt). Over time, however, the current flow through the capacitor decreases, whereas that through the resistor increases. As this happens, the rate of voltage change across the capacitor (and resistor) slows, and the voltage approaches a steady-state value. This change in voltage has an exponential time course whose specific characteristics depend on the resistance (R) and capacitance (C) of the resistor and capacitor. Moreover, a time constant, τ, for this circuit can be defined by the equation τ= R * C, and it equals the time it takes for the voltage to rise (or fall) exponentially by approximately 63% of the difference between its initial and final values.

With regard to how an axon actually responds to electrical stimulation,

Fig. 5.1 illustrates the results of an experiment in which the membrane potential of an axon is altered by passing rectangular pulses of depolarizing (upward-going 1.0 0.89 0.44 0.22 0.11 –0.11 –0.22 –0.44 –0.89 –1.0 0 5 10 15 20 25 Change in Voltage from baseline (mV)

Slope ˜ 4.6 MegaOhms Linear range Upward deviation due to activation of Na conductance 1.0 0.0 –1.0 –1.0 –0.5 0.0 0.5 1.0 •Fig. 5.1 (A) Voltageresponsesofanaxontorectangularpulsesofhyperpolarizingcurrent(negativenumbers)ordepolarizingcurrent(positivenumbers)asinjectedandrecordedfromanintracellularelectrode.Thechangesintransmembranepotentialaremirrorimagesofthesmallamplitudepulses.Atthethresholdlevel(current= 1.0),thereisa50:50chanceofreturningtorestingpotentialorofgeneratinganactionpotential.Forclarity,onlytherisingphaseoftheactionpotentialisshown.B, Current-voltage(I-V)plotderivedfromdatainA.Currentpulseamplitudeisplottedonthex-axis,andvoltageresponse(measuredatdottedline)isplottedonthey-axis.Notethedeviationfromlinearitywithlargedepolarizations,whichisduetoactivationofvoltage-gatedconductances.(RedrawnfromHodgkinAL,RushtonWAH:Theelectricalconstantsofacrustaceannervefibre.Proc R Soc Lond B Biol Sci. 1946;133:444-479.) pulses) or hyperpolarizing (downward-going pulses) current across its cell membrane. The injection of positive charge is depolarizing because it makes the cell less negative (i.e., decreases the potential difference across the cell membrane). Conversely, the injection of negative charge makes the membrane potential more negative, and this change in potential is called hyperpolarization. The larger the current that is injected, the greater the change in the membrane potential will be. The responses to hyperpolarizing and small-amplitude depolarizing current pulses (see

Fig. 5.1A ) all have the same fundamental shape because of the passive properties of the membrane. In contrast, the shapes of the responses to the larger depolarizing stimulus pulses differ from those to hyperpolarizing and small-amplitude depolarizing current pulses because the larger stimuli activate nonpassive elements in the membrane.

For the responses to hyperpolarizing current pulses, once a long enough time has elapsed from the start of the current pulse to allow the membrane voltage to plateau (essentially several times τ), virtually all of the injected current is flowing through the membrane resistance. If the difference between the initial and steady-state voltages is plotted against the amplitude of the current pulse (see

Fig. 5.1B ), a linear relationship is observed for the hyperpolarizing pulses, which is exactly what is expected from Ohm’s law (V = I *

R) for current flowing through a resistor. The slope of this line (ΔV/ΔI) is referred to as the input resistance of the cell (Rin) and is determined experimentally, exactly as just described. Rin is related to the membrane resistance (rm) of the cell, but the exact relationship depends on the geometry of the cell and is complex in most cases.

Next, note that although the current is injected as rectangular pulses, with vertical rising and falling edges, the shape of the membrane voltage responses just after the starts and ends of the pulses have slower rises and falls. Moreover, with regard to only the responses to hyperpolarizing and small-amplitude depolarizing current pulses (see

Fig. 5.1A ), the fall and rise in the membrane voltage have exponential shapes. This shows that the membrane is responding to these current pulses as a parallel RC circuit would; that is, the stimulus causes no change in membrane resistance or capacitance (cm), and thus the time course of the rise and fall in voltage is the same in all cases because it is governed by the same membrane time constant (τ).

The relationships between voltage and current just described show that within a certain range of stimulation, the cell membrane in one region of the axon can be modeled by a passive RC circuit. However, this model circuit, with only a single resistor and capacitor, takes no account of the fact that axons are spatially extended structures and that because of this, the resistance of the intracellular space is a significant factor in how electrical events in one region affect other regions. That is, if axons had no intracellular resistance, their intracellular space would be isoelectric, and voltage changes, like those just described, across one part of the axonal membrane would occur across all regions instantaneously. In this case, there would be no need for a

CHAPTER 5 Generation and Conduction of Action Potentials special mechanism (i.e., the action potential) to propagate signals actively down the axon. In actuality, axons (and neurons in general) are spatially extended structures with significant resistance to current flow between different regions (this is one reason the relationship of Rin and rm is complicated). Therefore, it is important to understand how current injected at one point along the axon affects the membrane potential at other points because this both helps explain why action potentials are needed and helps explain some of their characteristics.

When current pulses that elicit only passive responses are passed across the plasma membrane, the size of the change in potential recorded depends on the distance of the recording electrode from the point of passage of the current (

Fig. 5.2).

The closer the recording electrode is to the site of current passage, the larger and steeper the change in potential is. The magnitude of the change in potential decreases exponentially with distance from the 0.0 mm 0.5 mm

Change of membrane potential 1.0 mm (mV) 1.5 2.0 2.5 mm 4.5mV •Fig. 5.2 Responsesofanaxonofashorecrabtoasubthresholdrectangularcurrentpulsebyanextracellularelectrodeappliedcloselytoitssurfaceandlocatedatdifferentdistancesfromthecurrent-passingelectrode.Astherecordingelectrodeismovedfartherfromthepointofstimulation,theresponseofthemembranepotentialisslowerandsmaller.(RedrawnfromHodgkinAL,RushtonWAH:Theelectricalconstantsofacrustaceannervefibre.Proc R Soc Lond B Biol Sci. 1946;133:444-479.) site of passage of the current, and the change in potential is said to reflect passive or electrotonic conduction. Such passively conducted changes in potential do not spread very far along the membrane before they become insignificant. As shown in

Fig. 5.2 , an electrotonically conducted signal dies away over a distance of a few millimeters. The distance over which the change in potential decreases to 1/e (37%) of its maximal value is called the length constant or space constant (where e is the base of natural logarithms and is equal to 2.7182). A length constant of 1 to 3 mm is typical for mammalian axons, which can be more than a meter long, which makes obvious the need for a mechanism to propagate information about electrical events generated at the soma to the far end of the axon.

The length constant can be related to the electrical properties of the axon according to cable theory because nerve fibers have many of the properties of an electrical cable. In a perfect cable, the insulation surrounding the core conductor prevents all loss of current to the surrounding medium, so that a signal is transmitted along the cable with undiminished strength. If an unmyelinated nerve fiber (discussed later) is compared to an electrical cable, the plasma membrane equates to the insulation and the cytoplasm as the core conductor, but the plasma membrane is not a perfect insulator. Thus the spread of signals depends on the ratio of the membrane resistance to the axial resistance of the axonal cytoplasm (ra). When the ratio of rm to ra is high, less current is lost across the plasma membrane per unit of axonal length, the axon can function better as a cable, and the distance that a signal can be conveyed electrotonically without significant decrement is longer. A useful analogy is to think of the axon as a garden hose with holes poked in it. The more holes there are in the hose, the more water leaks out along its length (analogous to more loss of current when rm is low) and the less water is delivered to its nozzle. According to cable theory, the length constant can be related to axonal resistance and is equal to rm/ra . This relationship can be used to determine how changes in axonal diameter affect the length constant and, hence, how the decay of electrotonic potentials varies. An increase in the diameter of the axon reduces both ra and rm. However, rm is inversely proportional to diameter (because it is related to the circumference of the axon), whereas ra varies inversely to the diameter squared (because it is related to the cross-sectional area of the axon). Thus ra decreases more rapidly than rm does as axonal diameter increases, and the length constant therefore increases (

Fig. 5.3 ˜ = Distance over which response decays to 1 or ~ 37% of original size (Vo)

Vo/e Vo/e Vo/e Vo/e Large-diameter axon ra is lowra is high˜Vo ˜˜˜˜ = 3 mm ˜ = 1 mm 1. 2.

•Fig. 5.3 Comparison of the Length Constant to Axon Diameter. Notethattheincreaseindiameterisassociatedwithadecreaseinaxialresistanceoftheaxonalcytoplasm(ra)andanincreaseinthelengthconstant(λ).(RedrawnfromBlankenshipJ.Neurophysiology. Philadelphia:Mosby;2002.)

CHAPTER 5 Generation and Conduction of Action Potentials

In sum, in the passive domain, the membrane response to electrical stimuli is essentially identical to that of a circuit composed of passive electrical elements, and it can thus be characterized by the length and time constants of the membrane, which will determine how far and how rapidly electrical signals at one point in the cell spread to other parts.

The Local Response

With regard to the experiment shown in

Fig. 5.1, if larger depolarizing current pulses are injected, the voltage response of the membrane no longer resembles that of a passive RC circuit. This is most easily observed with pulses that elicit depolarizations either just below or to the threshold membrane potential for an action potential but fail to evoke an action potential (tracings 0.89 and 1.0; the threshold membrane potential can be defined as the voltage at which the probability of evoking an action potential is 50%). In these cases, the voltage response shape is altered from that of the passive responses because the stimulus has changed the membrane potential sufficiently to cause the opening of significant numbers of voltage-sensitive Na+ channels (described later).

Also, note the upward deviation from linearity for the corresponding points in the I-V curve (see

Fig. 5.1B ). Opening of these voltage-sensitive channels changes the membrane’s resistance and allows Na+ to enter more easily, driven by its electrochemical gradient. This entry of positive charge (Na+ current) enhances the depolarization by adding to the current pulse delivered by the electrode. The resulting depolarization is called a local response. The local response results from active changes in membrane properties (specifically, its Na+ conductance), whereas in a passive electrotonic response, the conductance to various ions remains constant. Nevertheless, the local response is not self-regenerating but, again, decreases in amplitude with distance. The change in membrane properties is insufficient for what is needed to generate an action potential.

Suprathreshold Response: The Action Potential

Local responses will increase in size as the amplitude of the depolarizing current pulse is increased, until the threshold membrane potential is reached, at which point a different sort of response, the action potential (or spike), can occur. The threshold value is typically near −55 mV. Normally, when the membrane potential exceeds this value, an action potential is always triggered.

Fig. 5.4 shows the typical shape of an action potential. When the membrane is depolarized past threshold, the depolarization becomes explosive and overshoots in such a way that the membrane potential reverses from negative to positive and approaches, but does not reach, the Nernst equilibrium potential for Na+ (ENa; see ). The membrane potential then returns toward the resting membrane potential (repolarizes) almost as rapidly as it was depolarized, and in general, it hyperpolarizes beyond its resting potential (the afterhyperpolarization). The main phase of the action potential (from the onset to the return to the resting potential) typically has a duration of 1 to 2 msec, but the afterhyperpolarization, it can persist •Fig. 5.4 Components of the Action Potential With Regard to Time and Voltage. Markersindicatetheabsoluteandrelativerefractoryperiods.Notethatthetimescaleforthefirstfewmillisecondshasbeenexpandedforclarity.RMP,restingmembranepotential.(RedrawnfromBlankenshipJ.Neurophysiology. Philadelphia:Mosby;2002.) from a few to 100 msec, depending on the particular type of neuron.

The action potential differs from the subthreshold and passive responses in three important ways: (1) It is a response of much larger amplitude, in which the polarity of the membrane potential actually overshoots 0 mV (the cell interior becomes positive in relation to the exterior).

(2) The action potential is generally propagated down the entire length of the axon without decrement (i.e., it maintains its size and shape because it is regenerated as it travels along the axon). (3) It is an all-or-none response, which means that a stimulus normally either produces a full-sized action potential or fails to produce one. This all-or-none nature is in contrast both to the graded nature of the passive and local responses described previously and to synaptic responses (see

Ionic Basis of Action Potentials

Recall that the resting membrane potential is determined primarily by the weighted average of the Nernst potentials for Na+ (ENa) and K+ (EK), as defined by the chord conductance equation (see ). The weighting factors are the conductance (g = 1/resistance) to each ion. At rest the conductance to K+ (gK) is high in relation to that for Na+ (gNa), and so the resting membrane potential (Vr) is closer to EK (Vr ≅−70 mV). If, however, the relative conductances to these ions were to change, this would cause a corresponding change in the membrane potential. For example, an increase in gK would hyperpolarize the membrane, whereas a decrease in gK would depolarize the membrane because EK is approximately −100 mV. Conversely, an increase in gNa would depolarize the membrane and, if of sufficient magnitude, even a lead to reversal in membrane polarity because ENa is approximately +65 mV.

An axonic action potential is, in fact, the result of a rapid sequence of transient changes in gNa or gK, or both. In all axons there is a brief rise in gNa, followed by a decline back to baseline levels. In some axons, this change in gNa occurs against a fixed resting gK (because of leak channels, which are not voltage-gated; discussed later). In many other cases, however, both gNa and gK change. Thus as with the resting membrane potential, the action potential depends on the opposing tendencies of (1) the Na+ gradient to bring the resting membrane potential toward the Nernst potential for Na+ and (2) the K+ gradient to bring the resting membrane potential toward the Nernst potential for K+; but in contrast to when the neuron is at rest, the gK/gNa ratio is not constant but is changing continuously. One additional difference is that because the membrane potential is changing, a capacitative current also exists, and this must also be taken into account to describe the membrane potential quantitatively during an action potential (as a corollary, note that the chord conductance equation is valid only when the membrane potential is constant because then there is no capacitative current).

The early phase of the action potential (the positive deflection of the membrane potential toward ENa) is a result of a rapid increase in gNa and thus of the Na+ current (INa). These changes cause the membrane potential to move toward the equilibrium potential for Na+ . The peak of the action potential does not reach ENa because the rise in gNa is not infinite (i.e., the gK/gNa ratio does not fall to zero).

Because of the nature of the underlying Na+ channels (described later), the rise in gNa with depolarization is transient. Moreover, in many cases, the depolarization leads to a rise in gK. These two factors cause the gK/gNa ratio to stop falling and start increasing; as a result, the membrane potential is driven back toward EK and thus repolarizes toward its resting value. In cases in which the repolarization involves a rise in gK, the membrane potential hyperpolarizes temporarily beyond its normal rest value (if gK does not change, the drop in gNa causes the membrane simply to return to its resting potential). This afterhyperpolarization occurs because gK remains elevated for a period of time after the action potential. As gK returns to its baseline level, the membrane potential returns to its rest value.

These changes in conductance can be explained by the properties of Na+ and K+ ion channels, which are described next.

Early studies of the mechanism underlying action potentials indicated that ion currents pass through separate Na+ and K+ channels, each with distinct characteristics, in the cell membrane. Subsequent research has supported this interpretation. The amino acid sequences of the channel proteins and many of the functional and structural characteristics of the channels are now known in detail.

The structure of a voltage-gated Na+ channel (

Fig. 5.5 ) consists of four α subunits and two β subunits. The α subunit has four repeated motifs each of six transmembrane helices that surround a central ion pore. The pore walls are partly formed by the six helices in each motif. Most voltage-gated K+ channels are composed of four separate subunits, each consisting of a polypeptide with six membrane-spanning segments, similar to the motifs that make up the α subunit of the Na+ channel.

An important characteristic of some channels, such as those that underlie the action potential, is that they are gated by the membrane voltage. These voltage-gated channels sense the potential across the membrane and then act to either open or close the pore according to the membrane potential. The gates are formed by groups of charged amino acid residues, and the voltage dependence of the Na+ and K+ channel gates can account for the complex changes in gNa and gK that occur during an action potential.

The Characteristics of the Na+ and K+ Channels Explain the Conductance Changes During the Action Potential

Use of standard intracellular recording along with voltage-clamp techniques enabled investigators to characterize the underlying ionic currents and conductance changes

CHAPTER 5 Generation and Conduction of Action Potentials 71 •Fig. 5.5 A Model of the Voltage-Gated Na+ Channel. A, Thelargeredelementsrepresentthefourα subunitsandthetwoyellowelementsareβ subunitswiththereceptorsitesforα scorpiontoxin(ScTX)andtetrodotoxin(TTX)indicated.B, β1andβ2subunitsflankinganα subunitareshownwiththeirtransmembranehelices.(RedrawnfromCatterallWA:Structureandfunctionofvoltage-gatedsodiumchannelsatatomicresolution.Exp Physiol. 2014;99:35-51.) associated with the action potential. Detailed statistical analyses of these recordings also allowed remarkable inferences to be made about the nature of the channels that passed these Na+ and K+ currents. The development of the patch recording, however, enabled direct observation of the behavior of individual channels. In this technique, a specially shaped microelectrode is placed against the surface of a cell, and suction is applied to the microelectrode. As a result, a high-resistance seal is formed between the mem brane and the tip of the microelectrode (Fig. 5.6A ), which allows recording of the activity of whatever channels happen to be in the patch of membrane that is inside the seal. Under ideal conditions only one or a few ion channels of a single type are present in the membrane patch.

Patch recordings show that many ion channels flip spontaneously between open and closed conductance states as if they have gates that open and close the entrance to their pore. In the case of voltage-gated channels, the gate is sensitive to the voltage across the membrane, and thus the time a gate spends in each state is a probabilistic function of the membrane potential. A patch recording of a K+ channel demonstrates this probabilistic behavior (see

Fig. 5.6B ). As the membrane potential is clamped to more depolarized levels, the channel spends more time in its open state, which reflects the voltage dependence of the probability that the channel will open (see

Fig. 5.6C ). Also, the amplitude of the current in the open state increases with the level of depolarization; this is because the driving force for K+ is greater at more depolarized levels (i.e., the membrane potential is farther from the K+ Nernst potential).

The behavior of a Na+ channel is more complex than that of the K+ channel. Like the K+ channel, it has a 72 SECTION2Berne & Levy Physiology X˜X˜X˜X˜X˜Cytosol Pipette solution Patch pipette Ground Operational amplifier Feedback resistor Micropipette Cell membrane Ion channel A 1.0 0.8 0.6 0.4 0.2 C Po °80 °60 °40 °20 200 mV D Time (1ms/division)B ˜°°50 0 50 Membrane potential (mV) °1.0 0 Na+ current density (mA/cm2)

CHAPTER 5 Generation and Conduction of Action Potentials •Fig. 5.6 A, Amicropipetteisappliedtothecellmembraneandsufficientsuctionisappliedtoelectricallyisolateasinglechannelatthetip.Anamplifierrecordsthecurrentthatpassesthroughthechannel.B, EachlineshowsthecurrentpassedthroughaK+ channelasitopensspontaneously.Notethatasthetransmembranevoltageisprogressivelydepolarized(fromtoptobottom),boththeprobabilitythatthechannelwillopenandtheamplitudeofthecurrentareincreased.C, Agraphoftheprobabilitythatthechannelwillopenversusmembranedepolarization.D, Agraphoftransmembranevoltage(lowertracing,right-sidedscale)andthecurrentdensityfromapopulationofNa+ channels(uppertracing,left-sidedscale)isolatedinapatchsimilartothatinA (exceptthatitcontainsseveralNa+ channels).Initially,atrestingpotential,thereisnocurrentflow.Withdepolarizationto0mV,thereisaninwardNa+ currentthatiscurtailedevenwhilethedepolarizationcontinues.Thisisduetotheclosingofthechannels’inactivationgates.Afterabriefreturntorestingpotential,anotherdepolarizationto0mVevokesinwardcurrentflow,butitissmallerandbrieferbecausetherehasnotbeenenoughtimeformostoftheslowinactivationgatestoreopen.(B, C, andD areredrawnfrom http://www.physiologymodels.info/electrophysiology voltage-sensitive gate (activation gate) whose probability of being open increases with depolarization. However, unlike K+ channels, with maintained depolarization, Na+ channels open only at the onset of the depolarization and then remain closed. This suggests that Na+ channels have a second gate (inactivation gate) whose probability of being open goes down as the membrane is depolarized. Thus any Na+ current conducted by these channels will be transient (see

Fig. 5.6D ), because the same stimulus (depolarization) increases both the probabilities that the activation gate will open and that the inactivation gate will close. Note that the Na+ channel thus has two closed states, one in which the activation gate is closed, and the channel is said to be “closed,” and one in which the inactivation gate is closed, and the channel is referred to as “inactivated.” The reason the channel is called “inactivated” when the second gate closes is that once this gate closes, it will remain so until the membrane is repolarized. The activation gate, in contrast, can open and close at all membrane potentials, just with differing probabilities.

With the knowledge of the Na+ and K+ channel gating behavior just discussed, we can understand how the action potential is generated by the interaction of these channels (in the following, we assume that both gNa and gK change during the action potential). As stated previously, the action potential starts with a rapid increase in Na+ conductance (gNa;

Fig. 5.7 ). This increase in Na+ conductance reflects the opening of many Na+ channels in response to the depolarization. The open channels allow the influx of Na+ ions, and the effect of this current is to depolarize the membrane further. Note that this is a positive feedback loop, which accounts for the explosive nature of the action potential: the Na+ current depolarizes the membrane, which causes more Na+ channels to open, which in turn increases the Na+ current. In sum, the voltage-dependent opening of Na+ channels and the depolarizing action of the Na+ current account for the rising phase of the action potential.

The end of the rising phase and the subsequent falling (repolarization) phase of the action potential is the result of two processes: a reduction in gNa and an increase in gK. The rise in gK is simply a consequence of membrane depolarization, which increases the probability that the K+ channel will be open. The decrease in gNa results from two •Fig. 5.7 The Action Potential and the Conductance and Currents That Underlie the Action Potential With Regard to Time. NotethattheincreasedconductanceforNa+ (gNa),aswellasitsinwardflow,isassociatedwiththerisingphaseoftheactionpotential,whereastheslowerincreaseinconductanceforK+ (gK),aswellasitsoutwardflow,isassociatedwithrepolarizationofthemembraneandwithafterhyperpolarization.ThereductionintheNa+ current(INa)beforethepeakoftheactionpotential(eventhoughgNaisstillhigh)isduetoinactivationoftheNa+ channels.(RedrawnfromSquiresLR,etal:Fundamental Neuroscience. 2nded.SanDiego,CA:AcademicPress;2002.) factors. First, Na+ channels are inactivated as a result of the closing of the inactivation gate with depolarization. Unlike the activation gate, which can flip between states even when the membrane is depolarized, the inactivation gate, once closed, remains closed until significant repolarization occurs. Second, as the gK/gNa ratio increases (as a result of both inactivation of Na+ channels and opening of K+ channels), the membrane begins to repolarize, and this repolarization acts to shut the activation gate of the Na+ channel. The closure of both voltage-gated Na+ and K+ channels during the falling phase brings the membrane back to its resting state. If only Na+ channels had opened during the action potential (as is the case for some axons), the membrane would simply return to its rest potential. If voltage-gated K+ channels also had opened during the action potential, an afterhyperpolarization would be present because these K+ channels close slowly in response to hyperpolarization.

Knowledgeofthemolecularstructureofchannelshasincreasedtheunderstandingofthebasisoftheirproperties.Forexample,mostchannelsarehighlyselectiveforaparticularion.First,ifthechannelwallsarelinedwitheitherpositiveornegativecharges,theneithercationsoranionscanbeexcluded;however,mostchannelsarealsodifferentiallypermeablebydifferentionsofthesamecharge.Thisfurtherselectivityappearstobetheresultofrequiringionstobecomedehydratedastheypassthroughthenarrowestpartofachannel,knownastheselectivity filter. Ionsinsolutionarehydrated(aresurroundedbyashellofH2Omolecules),andtheradiusofthishydrationshellisdifferentforeachtypeofion.InNa+ andK+ channels,tomakedehydrationenergeticallypossible,theporeofthechannelislinedwithnegativelypolarizedaminoacidsubstituentsofaparticulargeometry,andthesesubstituentssubstituteforthewatermolecules.Suchsubstitution,however,requiresclosematchingofthefilter’ssizetotheion’shydrationshell.Becauseeachionhasadifferent-sizedshell,aparticularchannelwillbestallowpassageofoneparticularionicspecies.

Tetrodotoxin (TTX), oneofthemostpotentpoisonsknown,specificallyblockstheNa+ channel.TTXbindstotheextracellularsideofthesodiumchannel(see

Fig.5.5A ).Tetraethylammonium (TEA+), anotherpoison,blocksK+ channels.TEA+ enterstheK+ channelfromthecytoplasmicsideandblocksthechannelbecauseTEA+ isunabletopassthroughit.Theovariesofcertainspeciesofpufferfish,alsoknownasblowfish,containTTX.RawpufferfishisahighlyprizeddelicacyinJapan.ConnoisseursofpufferfishenjoythetinglingnumbnessofthelipscausedbytheminusculequantitiesofTTXpresentintheflesh.Sushichefswhoaretrainedtoremovetheovariessafelyarelicensedbythegovernmenttopreparepufferfish.Despitetheseprecautions,severalpeopledieeachyearasaresultofeatingimproperlypreparedpufferfish.

SaxitoxinisanotherblockerofNa+ channelsthatisproducedbythereddishdinoflagellatesthatareresponsibleforso-calledredtides.Shellfisheatthedinoflagellatesandconcentratesaxitoxinintheirtissues.Apersonwhoeatstheseshellfishmayexperiencelife-threateningparalysiswithin30minutesafterthemeal.

When a nerve is depolarized very slowly, the normal threshold may be passed without the firing of an action potential; this phenomenon is called accommodation. Both Na+ and K+ channels are involved in accommodation. In response to membrane depolarization, gNa first increases and then, a short time later, decreases. This is due to the opening of the activation gates and closing of the inactivation gates of the Na+ channels. Normally, membrane depolarization to threshold or beyond triggers an action potential; however, the explosive depolarization of the action potential can occur only if a critical number of Na+ channels are recruited. Thus if a cell is slowly depolarized, Na+ channels can become inactivated even without the occurrence of an action potential, and the pool of available noninactivated Na+ channels (i.e., channels in the closed state) can be reduced to the point at which a stimulus to may not be able to recruit a sufficient number of Na+ channels to generate an action potential. An additional factor in causing accommodation is that K+ channels open slowly in response to the depolarization. The increased gK tends to oppose depolarization of the membrane, which makes it even less likely to fire an action potential.

When a cell is refractory, it is either completely unable to fire an action potential or it requires a much stronger stimulation than usual. During much of the action potential, the cell is completely refractory because it will not fire another action potential no matter how strongly it is stimulated. This absolute refractory period (see

Fig. 5.4 ) occurs when a large fraction of the Na+ channels are inactivated and therefore cannot be reopened until the membrane is repolarized. During this period, the critical number of Na+ channels required to produce an action potential cannot be recruited.

Inaninheriteddisordercalledprimary hyperkalemic paralysis, patientshaveepisodesofpainfulspontaneousmusclecontractionsfollowedbyperiodsofparalysisoftheaffectedmuscles.Thesesymptomsareaccompaniedbyelevated[K+]inplasmaandextracellularfluid.Somepatientswiththisdisorderhavemutationsofvoltage-gatedNa+ channelsthatresultinadecreasedrateofvoltageinactivation.Thisresultsinlonger-lastingactionpotentialsinskeletalmusclecellsandincreasedeffluxofK+ duringeachactionpotential,whichcanraiseextracellular[K+].

Theelevationinextracellular[K+]causesdepolarizationofskeletalmusclecells.Initially,thedepolarizationbringsmusclecellsclosertothreshold,andsospontaneousactionpotentialsandcontractionsaremorelikelytooccur.Asdepolarizationofthecellsbecomesmoremarked,thecellsbecomerefractorybecauseincreasingnumbersofNa+ channelsbecomeinactivated.Consequently,thecellsbecomeunabletofireactionpotentialsandarenotabletocontractinresponsetoactionpotentialsintheirmotoraxons.

During the latter part of the action potential, and during the afterhyperpolarization period, the cell is able to fire a second action potential, but a stimulus stronger than normal is required. This period is called the relative refractory period. Early in the relative refractory period, before the

CHAPTER 5 Generation and Conduction of Action Potentials membrane potential has returned to the resting potential level, some Na+ channels are still voltage inactivated, but there are enough in the closed state (and therefore have the potential to open when the membrane is depolarized) to support the generation of an action potential if they are stimulated to open. However, a stimulus stronger than normal is necessary to recruit the critical number of Na+ channels needed to trigger an action potential (i.e., the reduction in the total number of available Na+ channels is countered by increasing the probability of opening). Throughout the relative refractory period, conductance to K+ is elevated, which opposes depolarization of the membrane. This increase in K+ conductance continues throughout the afterhyperpolarization and accounts for most of the duration of the relative refractory period.

Conduction of Action Potentials

Fundamental to nervous system function is the transmission of information along neuronal pathways. To accomplish this, neurons generate action potentials that propagate down the length of their axon without decrement in size in order to trigger neurotransmitter release from the presynaptic terminals. How action potentials propagate down an axon and how the characteristics of the axon affect this propagation are discussed in this section. How they trigger transmitter release is covered in

Action Potential as a Self-Reinforcing Signal

Passive conduction will not transport a signal from one end of an axon to the other unless the axon is very short (i.e., on the order of its length constant) because passively conducted signals decrease in size rapidly with distance from their origin. Neurons with such short axons exist; for example, in the retina of the eye, the distance from one neuron to the next is so small that electrotonic (passive) conduction is sufficient. However, in most cases, axons are many times longer than their length constant. In fact, they can be up to 1 m or more in length (e.g., those of motor neurons) and thus hundreds of times their length constant. Nonetheless, if researchers were to record from points along a typical axon, they would find that as the action potential arrives at successive points traveling along the axon, its shape and size remain constant. This is because the action potential regenerates itself as it is conducted along the fiber and thus is said to be actively propagated.

Fig. 5.8 shows how in a local response the current that flows in through one part of the membrane acts to depolarize the neighboring membrane. The same thing happens when the Na+ channels are opened by an action potential at one site along the axon, except that in this case, the current will be large enough to depolarize the areas on either side past threshold and thus generate action potentials in these neighboring areas. The inward Na+ current in these areas can then provide the current to depolarize their neighbors past threshold so that they in turn generate action potentials, •Fig. 5.8 Mechanism of Electrotonic Spread of Depolarization. A, Thereversalofmembranepolaritythatoccurswithlocaldepolariza-tion.B, Thelocalcurrentsthatflowtodepolarizeadjacentareasofthemembraneandallowconductionofthedepolarization.B + – + – + – + – + – + – + – + – + – + – + – + – + – + – – + – + – + + – + – + – – + – + – + – + – + – + – + – + – + – + – + – + – + – + and so on. In short, action potential propagation along an axon involves recurring cycles of depolarization to provide sufficient local current flow for generation of an action potential in an adjacent region of the cell membrane. Thus the action potential is said to be propagated down the axon, with “new” action potentials being generated along its length. In this way, the action potential can propagate down the entire length of the axon while retaining the same size and shape.

Normally, action potentials are first generated at the axon’s initial segment (i.e., where the axon is attached to the neuron cell body or proximal dendrite) and then conducted to the terminal end. The reason for this is that the initial segment has a very high density of voltage-gated Na+ channels, and thus it has a lower threshold for spiking than does the soma or dendrites. However, axons are not inherently unidirectional conductors. For example, as implied by the local circuits shown in

Fig. 5.8 , an action potential generated by a depolarization in the middle of an axon is conducted in both directions from its initiation site simultaneously.

Why does a spike that starts at the initial segment not propagate in both directions? In fact, it does. In addition to propagating down the axon, the current flowing from the initial segment back to the soma can cause a spike to be generated in the soma because the soma also has voltage-gated Na+ channels. Additional “backpropagating” spikes from the axon do not occur, however, nor does the somatic spike cause the initial segment of the axon to fire a second time (and thereby send another spike down the axon and start a repeating cycle). This does not happen because the refractory period of the membrane makes any area that has already spiked unable to fire a second spike for a short time. Thus for a spike that started at the initial segment and has begun traveling down the axon, current flowing in at the site of the spike depolarizes the membrane on both sides of that site. However, the side closer to the cell body, which has recently fired a spike, cannot respond to this depolarization because its Na+ channels are still inactivated. By the time Na+ channels are de-inactivated (have returned to their closed state and would be able to open) the depolarization of membrane at that site has ended (because the action potential lasts only for ≈1 msec). Thus the inactivation gate of the Na+ channel not only helps determine the duration of the action potential but it also is responsible for its singular and unidirectional propagation from its origin at the initial segment.

The speed of conduction in a nerve fiber is determined by the electrical properties of the cytoplasm and the plasma membrane that surrounds the fiber, as well as by its geometry. In nonmyelinated fibers, conduction velocity is proportional to the square root of the cross-sectional diameter (Fig. 5.9 ). This effect is related to the changes in ra and rm with diameter. As the diameter of a fiber increases, ra decreases with the square of the diameter, and rm increases only linearly with diameter; as a result, resistance to current •Fig.

5.9 Conductionvelocitiesofunmyelinated(blue) andmyelinated(red) felineaxonsasfunctionsofaxondiameter.Solid lines representmeasureddata.Dotted lines representextrapolationsthatshowtheadvantageofmyelinationoversimplyincreasingaxondiameterasamechanismforincreasedconductionvelocity.(FromSchmidt-Nielsen

K.

Animal Physiology: Adaptation and Environment. 5thed.Cambridge,UK:CambridgeUniversityPress;1997.) flow down the axon decreases more than it does to current flow across the membrane. This increases the length constant (see

Fig. 5.3 ), which means that a greater amount of the current entering at one site is delivered to neighboring regions of the axon, which brings those regions to threshold more quickly, and thus the action potential is conducted faster along fibers with large diameters.

However, increasing the diameter also increases the surface area of the plasma membrane over which inner negative and outer positive charges are held to each other. Discharging this increased capacitance tends to slow conduction and mitigate the increase in conduction velocity gained by increasing diameter.

In vertebrates, many nerve fibers are coated with myelin, and such fibers are said to be myelinated. Myelin consists of the plasma membranes of Schwann cells (in the peripheral nervous system) or oligodendroglia (in the central nervous system [CNS]), which wrap around and insulate the nerve fiber (

Fig. 5.10A and B ). The myelin sheath consists of several to more than 100 layers of glial cell plasma membrane. Gaps about 1-2 µm wide, known as nodes of Ranvier, separate the contribution of one Schwann cell (or oligodendrocyte) from that of another. For all but the axons of smallest diameter, a myelinated axon has much greater conduction velocity than does an unmyelinated fiber of the same caliber because the myelin sheath increases the effective membrane resistance of the axon, decreases the capacitance of the axon membrane, and limits the generation of action potentials to the nodes of Ranvier. In short, myelination greatly alters the electrical properties of the axon.

Because the many wrappings of membrane around the axon increase the effective membrane resistance rm/ra and the length constant are much greater. The increased membrane resistance means that less current is lost through the membrane per length of axon, and thus the amplitude of a conducted signal decreases less with distance along the axon and needs to be regenerated (by opening of Na+ channels) less often.

In addition, the thicker myelin-wrapped membrane results in a much larger separation of charges across it than exists across the bare membrane of an axon, so that the charges across it are much less tightly bound to each other. This is analogous to when the plates of a capacitor are moved apart and reduce its capacitance. Because the effect of membrane capacitance is to slow the rate at which the membrane potential can be changed, the reduced capacitance of myelinated axons means that the depolarization occurs more rapidly. For all these reasons, conduction velocity is greatly increased by myelination, and the current generated at one node of Ranvier is conducted at great speed to the next (see

Fig. 5.10

In myelinated axons, the Na+ channels that bring about generation of an action potential are highly concentrated

CHAPTER 5 Generation and Conduction of Action Potentials •Fig. 5.10 A, Schematicillustrations,incrosssectionandlongitudinalsectionthroughanodeofRanvier,ofaSchwanncellwrappedaroundanaxontoformmyelin.NotethattheaxonisexposedtotheextracellularspaceonlyatthenodeofRanvier.B, Viewoftwonodesandtheinterveninginternodeofmyelin.C, Saltatoryconductioninamyelinatedaxonwithaplotoftheactionpotentiallocationalongtheaxon(x-axis)versustime(y-axis).Notetheshorttimetakenfortheactionpotentialtotraversethelargedistancebetweennodes(shallow sloped lines ontheplot)becauseofthehighresistanceandlowcapacitanceoftheinternodalregion.Incontrast,theactionpotentialslowsasitcrosseseachnode(steep sloped line segments). (B, RedrawnfromSquiresLRetal:Fundamental Neuroscience. 2nded.SanDiego,CA:AcademicPress;2002.C, RedrawnfromBlankenshipJ:Neurophysiology. Philadelphia:

Mosby;2002.) at the nodes of Ranvier and are not found between them. Thus the action potential is regenerated only at the nodes of Ranvier (0.3-2 mm apart) rather than being regenerated continuously along the fiber, as is the case in an unmyelinated fiber. Resistance to the flow of ions across the many layers that make up the myelin sheath is so high that transmembrane currents are largely restricted to the short stretches of naked plasma membrane that are present at the nodes of Ranvier (see

Fig. 5.10C ). Therefore, the action potential is regenerated at each successive node. The local currents entering the node are almost entirely conducted from one node to the next node, bringing each node to threshold in about 20 µsec. Thus the action potential appears to “jump” from one node of Ranvier to the next, and the process is called saltatory (from the Latin word saltare, “to leap”) conduction (

Fig. 5.11

Functional Consequences of Myelination

The functional consequences of myelination can be highlighted by a comparison of squid and mammalian axons. Although human nerve fibers are much smaller in diameter than squid giant axons, human axons conduct at comparable or even faster speeds because of myelination. The unmyelinated squid giant axon has a 500-µm diameter and a conduction velocity of about 20 m/sec. In mammals, axon diameters range from about 0.2 to 20 µm, and all fibers with diameters larger than 1-2 µm are myelinated. An unmyelinated mammalian nerve fiber, which has a diameter of less than 1 to 2 µm, has a conduction velocity of less than 2 m/sec (see

Fig. 5.9 ), as expected because of its smaller diameter in comparison to the squid giant axon. In contrast, a 10-µm myelinated mammalian fiber has a conduction velocity in the range of 50 m/sec, more than twice that of the 500-µm squid giant axon, despite being 1/50 of its diameter. Thus the high conduction velocity with far narrower axons achieved by myelination allows a tremendous increase in neuronal connectivity without enormously expanding the volume of the CNS. This is certainly one factor that enabled the evolution of mammalian nervous systems with their huge numbers of neurons that are able to generate everything from fast reflexes to efficient and complex mental processing.

To receive information about the world, the CNS contains a wide variety of sensory receptors, each of which is specialized •Fig. 5.11 Comparison of Action Potential Conduction in an Unmyelinated Axon and in a Myelinated Axon. Attheinitialtime(A andC),anactionpotentialisbeinggeneratedattheleftsideofeachaxon.Notethattheinwardcurrentintheunmyelinatedaxon(A) isdepolarizinganadjacentportion,whereastheinwardcurrentinthemyelinatedaxon(C) isdepolarizingallofthemembranetothenextnode.Atthesecondinstantintime(B andD),theactionpotentialintheunmyelinatedaxon(B) hasbeengeneratedintheadjacentportion,whereastheactionpotentialinthemyelinatedaxon(D) hasbeengeneratedatsubsequentnodesandisalreadydepolarizingthelastnodetotheright.(RedrawnfromCastroA,etal:Neuroscience: An Outline Approach. Philadelphia:Mosby;2002.)

Insomediseasesknownasdemyelinating disorders, themyelinsheathdeteriorates.Inmultiple sclerosis, scatteredprogressivedemyelinationofaxonsintheCNSresultsinlossofmotorcontrolandsensorydeficits.Theneuropathycommoninseverecasesofdiabetesmellitusiscausedbythedemyelinationofperipheralaxons.Whenmyelinislost,thelengthconstantbecomesmuchshorter.Hence,theactionpotentiallosesamplitudeasitiselectrotonicallyconductedfromonenodeofRanviertothenext.Ifdemyelinationissufficientlysevere,theactionpotentialmayarriveatthenextnodeofRanvierwithinsufficientstrengthtofireanactionpotentialatthatnode,leadingtopropagationfailure.

to detect a particular type of energy (stimulus). When a stimulus activates a sensory receptor, it initiates a process called sensory transduction by which information about the stimulus (e.g., its intensity and duration) is converted into local electrical signals. These local signals are called

TheactionpotentialsofmyelinatedaxonsmaynothaveahyperpolarizingafterpotentialoranextendedrelativerefractoryperiodbecausetheirK+ channelsaredisplacedfromthenodesintothepartlyexposedflankingparanodes.Thatincreasestherateatwhichthesefast-conductingaxonscanfire.Myelinatedaxonsarealsomoremetabolicallyefficientthanunmyelinatedaxons.Na+,K+-ATPaseextrudestheNa+ thatentersthecellandcausestheK+ thatleavesthecelltoreaccumulateduringactionpotentials.Inmyelinatedaxons,ioniccurrentsarerestrictedtothesmallfractionofthemembranesurfaceatthenodesofRanvier.Forthisreason,farfewerionstraverseaunitlengthoffibermembrane,andmuchlessionpumping—andenergyexpenditure—isnecessarytomaintainthegradients.

Investigatorscanrecordanactionpotentialwithamicroelectrodewithoutpenetratingtheaxonbyplacingtwospacedelectrodesonitssurfaceandcomparingtheelectricalchargeateachpoint.Anelectrodelocatedwherethereisanactionpotentialwouldyieldasomewhatnegativesignalincomparisontoanelectrodewherethereisnoactionpotential.Astheactionpotentialisconductedtothesecondelectrode,thepolarityoftherecordingreverses.Thistechniqueisusedclinicallytoassessnervefunction.Peripheralnervesandmanycentralpathwaysconsistofapopulationofaxons ofvariousdiameters(Fig.5.12 );someoftheaxonsaremyelinated,andsomearenot.Consequently,actionpotentialstravelatdifferentvelocitiesintheindividualaxons.Asaresult,arecordingfromsuchanervewithexternalelectrodesdoesnotshowasinglesynchronouspeakbutaseriesofpeaksthatvaryintime(whichreflectstheconductionvelocityofgroupsofaxons)andinmagnitude(whichreflectsthenumberofaxonsineachvelocitygroup).Thisiscalledacompound action potential.Theclinicalvalueofsucharecordingisitsability,incertaindiseasestates,torevealthedysfunctionofaparticulargroupofaxonsassociatedwithspecificfunctions,aswellasthenoninvasivenatureofthetechniquebecauseitcanbeperformedwithskinsurfaceelectrodes( receptor or generator potentials. The receptor potentials can then be transformed into patterns of action potentials that are conducted over one or more axons into the CNS. In order for this to happen, the stimulus must produce receptor potentials that are large enough to change the spiking levels of one or more primary afferent fibers that are connected to the receptor. Weaker intensities of stimulation can produce subthreshold receptor potentials, but such stimuli do not change the activity of central sensory neurons and thus are not detected. Thus stimulus threshold is defined as the weakest stimulus that can be reliably detected.

Environmental events that evoke sensory transduction can be mechanical, thermal, chemical, or other forms of energy. However, the types of information used by a

CHAPTER 5 Generation and Conduction of Action Potentials particular organism depend on its set of sensory receptors.

For example, humans cannot sense electrical or magnetic fields, but other animals can sense such stimuli. In particular, many fish have electroreceptors, and various fish and birds use the earth’s magnetic field to orient themselves during migration.

The transduction process varies with the type of environmental stimulus being detected.

Fig. 5.13 shows three examples of how stimuli can alter the membrane properties of the specific sensory receptors that transduce such stimuli 0246 (further details for each of these examples are given in other 36 72 108 m/s chapters).

Fig. 5.13A illustrates how a chemoreceptor, such A as that used for taste and smell, might respond when a chemical stimulant reacts with receptor molecules on the plasma membrane of the sensory receptor. Binding of the •Fig. 5.12 Thedistributionofaxons,bysizeandconductionvelocity,inamixed(muscle)nerve(A) andacutaneousnerve(B). Notetheincreasednumberofsmall-diameterfibersandtheabsenceofAα fibersinthecutaneousnerve.(FromHainesDE[ed].Fundamental Neuroscience for Basic and Clinical Applications. 3rded.Philadelphia:ChurchillLivingstone;2006.) chemical stimulant to the receptor molecule opens an ion channel, which enables the influx of an ionic current that depolarizes the sensory receptor cell. (This is similar to what is described for ligand-gated channels in

Chapter 6.)

Fig. 5.13B, the ion channel of a mechanoreceptor, such as those in the skin, opens in response to the application of a mechanical force along the membrane, and this allows an influx of current to depolarize the sensory receptor. In

Fig. 5.13C, the ion channel of a retinal photoreceptor cell (so-called because it responds to light) is open in the dark and closed when a photon is absorbed by pigment on an internal disc membrane. In this case, an influx of current occurs in the dark; the current ceases when light is applied. When the current stops, the photoreceptor hyperpolarizes. (Because capture of the photon is distant from the ion channel that it influences, this process must involve an intracellular “second messenger” mechanism.)

The nature of the receptor also can vary. In the simplest situation, a receptor is just a specialized portion of an axon,

Aα N/A8-1344-78ExtrafusalskeletalmusclefibersAγ N/A2-812-48IntrafusalmusclefibersBN/A1-36-18PreganglionicautonomicfibersCN/A0.2-20.5-2Postganglionicautonomicfibers

FromHainesDE(ed).Fundamental Neuroscience for Basic and Clinical Applications. 3rded.Philadelphia:ChurchillLivingstone;2006.N/A,notapplicable.

•Fig. 5.13 Models of Transducer Mechanisms in Three Types of Receptors. A, Chemoreceptor.B, Mechanoreceptor.C, Photoreceptor.

in which case the transduction of a stimulus into receptor potential and the translation of this potential into a spike train all take place in the same cell. For example, a mechanical stimulus, such as pressure on the skin of a finger, can distort the membrane of an axon that forms part of a mechanoreceptor, as shown in

Fig. 5.14A . This distortion causes inward current flow at the end of the axon and longitudinal and outward current flow along the neighboring parts of the axon. The outward current produces a depolarization (the receptor potential) that might exceed the threshold for an action potential (see

Fig. 5.14B ). If so, one or more action potentials are evoked and then travel along this primary afferent fiber to the CNS and thereby convey information about the mechanical stimulus.

In many other cases, the receptor is composed of more than one cell. In this situation, transduction occurs in one cell, but spikes are generated in other cells that are •Fig. 5.14 A, Currentflow(thin arrows) producedbystimulation(thick arrow) ofamechanoreceptoratthetipofanaxon.AnintracellularrecordingelectrodeisplacedatthefirstnodeofRanvier.B, ThereceptorpotentialproducedbythecurrentandanactionpotentialthatwouldbesuperimposedonthereceptorpotentialifitweretoexceedthresholdatthefirstnodeofRanvier.

synaptically connected to it (see ). For example, in the cochlea, the primary afferent fibers get synaptic input from mechanoreceptive hair cells. Sensory transduction in such sense organs can be more complex in this arrangement. In photoreceptors, moreover, the receptor potential is hyperpolarizing, as mentioned earlier, and interruption of the dark current is the signal event. Information about each of these mechanisms is discussed in

Chapter 8.

Although the mechanisms of sensory transduction vary between stimulus types, the end result is typically a receptor potential in either the receptor cell or the primary afferent neuron (i.e., the first neuron in a sensory pathway) that has a synapse with the receptor cell.

The relationship between the location of a stimulus and activation of particular sensory neurons is a major theme in the field of sensory physiology. The receptive field of a sensory neuron is the region that, when stimulated, affects the activity of that neuron. For example, a sensory receptor might be activated by indentation of only a small area of skin. That area is the excitatory receptive field of the sensory receptor. Moreover, a neuron in the CNS might have a receptive field several times as large as that of a sensory receptor because it may receive information from many sensory receptors, each with a slightly different receptive field. The receptive field of that CNS neuron is thus the sum of the receptive fields of the sensory receptors that influence it. The location of the receptive field is determined by the location of the sensory transduction apparatus responsible for signaling information about the stimulus to the sensory neuron.

In general, sensory receptive fields are excitatory. However, a central sensory neuron can have either an excitatory or an inhibitory receptive field or, indeed, a complex receptive field that includes areas that excite it and areas that inhibit it. Examples of such complex receptive fields are discussed in

Coding of Information by Action Potentials

Central to CNS function is the transmission of information between neurons. This is accomplished primarily through action potentials, which propagate down the axon to the presynaptic terminals and cause neurotransmitter release, signaling the postsynaptic cells. As already explained, the regenerative nature of action potentials allows them to carry signals regardless of the length of the axon, whereas local signals, such as receptor or synaptic potentials (see ), decay with distance and are therefore not suitable for this purpose. The tradeoff, however, is that the all-or-none nature of action potentials means that their shape and size do not generally convey information in the way gradations of local potentials do. Instead, the variations in the rate or timing of action potentials appear to be used primarily as the “codes” for transmission of information between neurons.

Rate coding refers to information being coded in the firing rate of a neuron, where firing rate is defined as the number of spikes fired per unit time, usually expressed as spikes/second, also called hertz (Hz). For example, the force of a mechanical stimulus to the skin can be encoded in the firing rate of the primary afferent neuron that innervates the skin; the greater the force applied to the skin, the larger the resulting receptor potential in the primary afferent neuron will be and, as a consequence, the faster the rate of action potentials triggered by the receptor potential will be. Research has shown many neurons employ rate coding in the sense that the firing rate of a neuron shows a consistent relationship to particular parameters of sensory stimuli, upcoming movements, or other aspects of behavior.

The amount of information in such rate codes is constrained by several factors. One factor is a neuron’s range of firing rates. The upper limit of this range is set by the maximal frequency that a neuron can fire action potentials, which is determined by the duration of the absolute and relative refractory periods (see

Fig. 5.4 ) and rarely exceeds 1000 Hz. The lower limit of the firing range is, of course, 0 Hz, as neurons cannot fire at negative rates. To avoid this problem, many neurons have spontaneous activity levels. These can be quite high (e.g., some Purkinje cells fire spontaneously at 100 Hz) and let a cell either increase or decrease its activity over a similar range in response to inputs. A second constraining factor is the variability of neuron’s firing rate, which determines the resolution of the neuron’s information coding.

CHAPTER 5 Generation and Conduction of Action Potentials

Timing, or temporal coding, refers to spike codes in which the specific timing of spikes rather than the overall firing rate encodes information. One often-studied version of temporal coding is the synchronization of spikes across neurons. Synchronization of neuronal spiking has been shown to occur in a number of brain regions and has been related to function in a number of instances. An advantage of temporal coding is that it can convey information more quickly than can rate coding, inasmuch as it does not require averaging, which takes time. Moreover, rate coding and temporal coding are not mutually exclusive, inasmuch as overall firing rates can be varied while synchronous events are superimposed. Such multiplexing of codes may increase the information transmission capacity of neuronal pathways.

Sensory neurons encode information about stimuli. In the process of sensory transduction, one or more aspects of the stimulus must be encoded in a way that can be interpreted by the CNS. The encoded information is an abstraction based on (1) which sensory receptors are activated, (2) the responses of sensory receptors to the stimulus, and (3) information processing in the sensory pathway. Some stimulus parameters that can be encoded include sensory modality, location, intensity, frequency, and duration. Other aspects of stimuli that are encoded are described in relation to particular sensory systems in later chapters.

A sensory modality is a class of sensation. For example, sustained mechanical stimuli applied to the skin result in sensations of touch or pressure, and transient mechanical stimuli may evoke sensations of flutter or vibration. Other cutaneous modalities include cold, warmth, and pain. Vision, audition, taste, and smell are examples of noncutaneous sensory modalities. The specific sensory receptors define the normal energy associated with the modality of a sensory pathway. For example, the visual pathway includes photoreceptors, neurons in the retina, the lateral geniculate nucleus of the thalamus, and the visual areas of the cerebral cortex (see ). The normal means of activating the visual pathway is light striking the retina. However, mechanical stimulation (e.g., pressure on the eyeball) or electrical stimulation of neurons in the visual pathway also produce a visual sensation. Thus neurons of the visual system can be regarded as a labeled line, which, when activated by whatever means, results in a visual sensation.

The location of a stimulus is signaled by activation of the particular population of sensory neurons whose receptive fields are affected by the stimulus. The information may be encoded in the CNS as a neural map. For example, a somatotopic map is formed by arrays of neurons in the somatosensory cortex that receive information from corresponding locations on the body surface (see ). In the visual system, points on the retina are represented by neuronal arrays that form retinotopic maps (see

Intensity may be encoded in a number of ways. Because action potentials have a uniform magnitude, some sensory neurons encode intensity by their frequency of discharge (rate coding). The relationship between stimulus intensity and response can be plotted as a stimulus-response function. For many sensory neurons, the stimulus-response function approximates an exponential curve with an exponent that can be less than, equal to, or greater than 1. Stimulus-response functions with fractional exponents characterize many mechanoreceptors. Thermoreceptors, which detect changes in temperature, have linear stimulus-response curves (exponent of 1). Nociceptors, which detect painful stimuli, may have linear or positively accelerating stimulus-response functions (i.e., the exponent for these curves is 1 or greater). The positively accelerating stimulus-response functions of nociceptors help explain the urgency that is experienced as the pain sensation increases.

Another way in which stimulus intensity is encoded is according to the number of sensory receptors that are activated. A stimulus at the threshold for perception may activate only one or only a few primary afferent neurons of an appropriate class, whereas a strong stimulus of the same type may recruit many similar receptors. Central sensory neurons that receive input from sensory receptors of this particular class would be more powerfully affected as more primary afferent neurons discharge. Greater activity in central sensory neurons may be perceived as a stronger stimulus.

Stimuli of different intensities may also activate different sets of sensory receptors. The limit of a neuron’s firing rate of action potentials can also limit its range of response to a stimulus. However, mechanoreceptors with different thresholds can overcome this problem: Those with low thresholds can signal over a range of low input intensities, whereas others with higher thresholds can signal higher input intensities. Together they allow fine resolution over an extended range of intensities. In addition, still higher intensities might recruit nociceptors, and that will also change the perceived quality of the stimulus.

Stimulus frequency can sometimes be encoded by action potentials whose interspike intervals correspond exactly to the intervals between stimuli (e.g., at intervals corresponding •Fig. 5.15 Responses of Slowly and Rapidly Adapting Mechanoreceptors to Displacement of the Skin. A toC arethedischargesofprimaryafferentfibersduringaramp-and-holdstimulusshowninD. A, Theresponseofaslowlyadaptingreceptorthatsignalsthemagnitudeanddurationofdisplacement.B, Theresponseofarapidlyadaptingreceptorwhoseoutputsignalsthevelocityofdisplacement.C, Theresponseofadifferentrapidlyadaptingreceptorthatrespondstoacceleration.p,displacement;R,response;t,time.

to that of a low-frequency vibration). However, this mechanism is limited by the firing rate limits of neurons as discussed earlier. When higher frequencies need to be encoded (e.g., the auditory system, which in humans is capable of detecting frequencies up to 20,000 Hz; see ), other strategies are needed. Other candidate codes depend on the spatiotemporal patterns of firing across populations of neurons.

The duration and the onset and offset of events are encoded by different populations of sensory neurons. For example, slowly adapting receptors in the skin produce a repetitive discharge throughout a prolonged stimulus. However, rapidly adapting receptors produce spikes at the onset (or offset) of the same stimulus.

Fig. 5.15 shows the responses of three types of receptors to the slow deflection of the skin, which is depicted in the graph at the bottom of the figure. The functional implication is that different temporal features of a stimulus can be signaled by receptors with different adaptation rates.

1.

Ion channels are integral membrane proteins that have ion-selective pores. An ion channel typically has two states: high conductance (open) and zero conductance (closed). Different regions of an ion channel protein act as gates to open and close the channel. The channel flips spontaneously between the open and closed states.

2.

For a voltage-dependent channel, the fraction of time that the channel spends in the open state is a function of the transmembrane potential difference.

3.

The action potential is generated by the rapid opening and subsequent voltage inactivation of voltage-dependent Na+ channels and by the delayed opening and closing of voltage-dependent K+ channels.

4.

The absolute and relative refractory periods result from voltage inactivation of Na+ channels and the delayed closure of K+ channels in response to membrane repolarization. These refractory periods limit the firing rate of action potentials.

5.

Subthreshold signals and action potentials are conducted along the length of a cell by local circuit currents. Subthreshold signals are conducted only electrotonically, and thus decrease with distance.

6.

The action potential is propagated rather than merely conducted; it is regenerated as it moves along the axon. In this way, an action potential retains the same size and shape as it travels along the axon.

7.

A large-diameter axon has greater propagation velocity because increased axon diameter lowers axial resistance and allows greater amounts of current to flow farther down the axon.

8.

Myelination dramatically increases the conduction velocity of a nerve axon because myelin increases membrane resistance and lowers membrane capacitance. Myelination allows an action potential to

Fain GL. Molecular and Cellular Physiology of Neurons. 2nd ed. Cambridge, MA: Harvard University Press; 2014.

Hille B. Ion Channels of Excitable Membranes. 3rd ed. Sunderland, MA: Sinauer Associates; 2001.

Hodgkin AL, Huxley AF. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol. 1952;117:500-544.

CHAPTER 5 Generation and Conduction of Action Potentials be conducted very rapidly from one node of Ranvier to the next. This makes the action potential appear to jump from node to node in a form of conduction called saltatory conduction.

9.

A receptor responds preferentially to a particular form of stimulus energy. Its receptive field is that part of a sensory domain in which energy can affect the receptor.

10.

Receptor potentials are the result of transduction of sensory stimuli. These potentials reflect the specific parameters of the stimulus and, if they exceed threshold, alter the action potential firing patterns of the afferent neurons.

Johnston D, Wu SM-S. Foundations of Cellular Neurophysiology. Cambridge, MA: MIT Press; 1994.

Sakmann B, Neher E. Single-Channel Recording. 2nd ed. Philadelphia: Springer; 1995.

Upon completion of this chapter the student should be able to answer the following questions: 1.

What are the characteristics of electrical synapses?

2.

What are the specializations found in the presynaptic and postsynaptic elements of a chemical synapse?

3.

What sequence of events connect the arrival of the action potential at the presynaptic terminal to the entry of calcium?

4.

What sequence of events connect the entry of calcium at the presynaptic terminal to release of neurotransmitter?

5.

What is the quantal hypothesis of synaptic transmission, and how does the presence of miniature end plate potentials support this hypothesis?

6.

Why is the reversal potential of a typical EPSP near 0 mV?

7.

What distinguishes EPSPs and IPSPs in terms of underlying ionic conductances, effect on membrane potential, and neuronal firing probability?

8.

How does an IPSP still inhibit a neuron when its reversal potential is equal to or more positive than the neuron’s resting potential?

9.

What are the mechanisms by which synaptic effects can change over time?

10.

What are the criteria for determining a substance is a neurotransmitter, and what are the major excitatory and inhibitory neurotransmitters?

11.

What are the major classes of neurotransmitter receptors?

ynaptic transmission is the major process by which electrical signals are transferred between cells within the nervous system (or between neurons and muscle cells or sensory receptors). Within the nervous system, synaptic transmission is usually conceived of as an interaction between two neurons that occurs in a point-to-point manner at specialized junctions called synapses. Two main classes of synapses are distinguished: electrical and chemical. However, as the list of chemical neurotransmitters has grown and as understanding of their mechanisms of action has increased, the definition and conception of what constitutes synaptic transmission has had to be refined and expanded. We no longer think of synaptic transmission as a process that involves only neurons, but now realize that glia form an important element of the synapse and that signaling occurs between neurons and glia. Moreover, in some cases, neurotransmitter released at a synapse will act over a widespread territory rather than just at the synapse from which it is released. Thus, we must either generalize the definition of synaptic transmission or consider classically defined synaptic transmission as but one of several mechanisms by which cells in the nervous system communicate with each other. In this chapter we first describe the classic conception of synaptic transmission (electrical and chemical) and then introduce some of the nontraditional neurotransmitters and discuss how they have forced modifications in our conception of chemical communication between cells in the nervous system.

Although their existence in the mammalian central nervous system (CNS) has been known for a long time, electrical synapses, or gap junctions, between neurons were thought to be of relatively little importance for the functioning of the adult mammalian CNS. Only recently has it become apparent that these synapses are quite common and that they may underlie important neuronal functions.

An electrical synapse is effectively a low-resistance pathway between cells that allows current to flow directly from one cell to another and, more generally, allows the exchange of small molecules between cells. Electrical synapses are present in the CNS of animals from invertebrates to mammals. They are present between glial cells as well as between neurons. Electrical coupling of neurons has been demonstrated for most brain regions, including the inferior olive, cerebellum, spinal cord, neocortex, thalamus, hippocampus, olfactory bulb, retina, and striatum.

A gap junction is the morphological correlate of an electrical synapse (see also ). These junctions are plaque-like structures in which the plasma membranes of the coupled cells become closely apposed (the intercellular space narrows to ≈ 3 nm) and filled with electron-dense material (Fig. 6.1 ). Freeze-fracture electron micrographs of gap junctions display regular arrays of intramembrane particles that correspond to proteins that form the intercellular channels connecting the cells. The typical channel diameter is large (1 to 2 nm), thus making it permeable not only to ions but also other small molecules up to approximately 1 kDa in size.

.28 µm • Fig. 6.1 Gap junction structure. A, Schematic view of the gap junction showing narrowing of the intercellularspaceto3.5nmatthejunction.Thegapjunctionhasmultiplechannels,witheachchannel formed by two connexon hemichannels. Each connexon in turn comprises six connexin subunits. B,Electronmicrographofpartofacomplexsynapticarrangementcalledaglomerulusthatisfoundin theinferioroliveandsomeotherCNSregions.Twodendriticspinesarecoupledbyagapjunction(small black arrows).Anaxonterminalpackedwithsynapticvesiclesfillstheupperrightpartofthepanel.Large arrowheadspointtotheelectron-densematerialthatmarkstheactivezones.Blackdotsareimmunogold labelingforGABA,thusidentifyingthisterminalasGABAergic.Redarrowheadspointtosynapticvesicles. (FromDeZeeuwCIetal.J Neurosci1996;16:3420.Copyright1996bytheSocietyforNeuroscience.)

Eachgapjunctionchannelisformedbytwohemichannels(calledconnexons),onecontributedbyeachcell.Eachconnexon,inturn,isahexamerofconnexinproteinsubunits,whichareencodedforbyagenefamilyofatleast21differentmembersinmammals.(Asecondfamilyofproteinsthatformgapjunctions,thepannexins,hasalsobeenidentified.)Gapjunctionsformedbydifferentconnexinshavedistinctbiophysicalproperties(gatingandconductance)andcellulardistributions.Althoughatleast10connexintypesareexpressedintheCNS,connexin36(connexinsarenamedaccordingtotheirmolecularweight;thus,thenumberreferstotheapproximatemolecularweightoftheconnexininkilodaltons)isthemajorneuronalconnexinintheadultCNS.OtherconnexintypesfoundintheCNSformgapjunctionsbetweenglialcellsorareprimarilyexpressedtransientlyduringdevelopment.

Electrical synapses are fast (essentially no synaptic delay) and bidirectional (i.e., current generated in either cell can flow across the gap junction to influence the other cell). In addition they act as low-pass filters. That is, slow electrical events are much more readily transmitted than are fast signals such as action potentials. One important role for neuronal gap junctions appears to be synchronization of network activity. For example, the activity of inferior olivary neurons is normally synchronized but becomes uncorrelated when pharmacological blockers of gap junctions are injected into the inferior olive. It also appears that the patterns of electrical coupling by gap junctions may be highly specific. For example, neocortical interneurons almost exclusively couple to interneurons of the same type. This specific gap junction–coupling pattern suggests that multiple, independent, electrically coupled networks of interneurons may coexist across the neocortex.

Finally, although electrical synapses are generally regarded as relatively simple and static in comparison to chemical synapses, they may actually be fairly dynamic entities. For example, the properties of electrical synapses can be modulated by several factors, including voltage, intracellular pH, and [Ca++]. Moreover, they are subject to regulation by G protein–coupled receptors, and connexins (the protein subunits that form a gap junction, see

At The Cellular ) contain sites for phosphorylation. These factors can change the coupling between cells by causing changes in single-channel conductance, formation of new gap junctions, or removal of existing ones.

Chemical synaptic transmission was first demonstrated between the vagus nerve and the heart by a simple experiment by Otto Loewi. The vagus nerve of a frog was stimulated to slow the heart rate down while the solution perfusing the heart was collected. This solution was then used to perfuse a second heart, whose beating then also slowed, demonstrating that the vagal nerve stimulation had caused a chemical to be released into the solution. The chemical responsible was found to be acetylcholine, which we now know is also a neurotransmitter at the neuromuscular junction and at other synapses in the peripheral and central nervous systems.

Unlike the situation at electrical synapses, at chemical synapses there is no direct communication between the cytoplasm of the two cells. Instead the cell membranes are separated by a synaptic cleft of some 20 nm, and interaction between the cells occurs via chemical intermediaries known as neurotransmitters. Chemical synapses are generally unidirectional, and thus one can refer to the presynaptic and postsynaptic elements that are diagramed in

Fig. 6.2.

The presynaptic element is often the terminal portion of an axon and is packed with small vesicles whose exact shape and size vary with the neurotransmitter they contain. In addition, the presynaptic membrane apposed to the postsynaptic element has regions, known as active zones, of electron-dense material that corresponds to the proteins involved in transmitter release (see

Fig. 6.1B). Moreover, mitochondria and rough endoplasmic reticulum are typically found in the presynaptic terminal. The postsynaptic membrane is also characterized by electron-dense material, which in this case corresponds to the receptors for the neurotransmitter.

Chemical synapses occur between different parts of neurons. Traditionally, focus has been placed on synapses formed by an axon onto the dendrites or soma of a second cell (axodendritic or axosomatic synapses), and our description will be based primarily on such synapses. However, there are many additional types of chemical synapses, such as axoaxonic (axon to axon), dendrodendritic (dendrite to dendrite), and dendrosomatic (dendrite to soma). Furthermore, complex synaptic arrangements are possible, such as mixed synapses, in which cells form both electrical and chemical synapses with each other; serial synapses, in which an axoaxonic synapse is made onto the axon terminal and influences the efficacy of that terminal’s synapse with yet a third element; and reciprocal synapses, in which both cells can release transmitter to influence the other.

Fig. 6.1B shows a complex synaptic arrangement called a glomerulus that involves both chemical and electrical synapses among the participating elements.

Much of what we know about chemical synapses comes from the study of two classic preparations, the frog neuromuscular junction (the synapse from a motor neuron onto a muscle fiber) and the squid giant synapse (the synapse from a second-order neuron onto third-order neurons that innervate the muscle of the squid’s mantle; i.e., the motor neurons whose axons were used to characterize the conductances underlying the action potential [see ]). The principles governing transmission at these synapses mostly apply to synapses within the mammalian CNS as well, at least with regard to synapses using what are called the “classic” neurotransmitters (see the section mitters). Thus, much of the following discussion will be based on results from these two preparations; however, some differences in CNS synapses will also be pointed out.

3 Gaseous transmitters Arginine NO synthase Citrulline Mitochondria Peptides diffuse in extracellular space and bind to synaptic and extrasynaptic G protein–complex receptors Gaseous transmitters diffuse out of cell of origin and directly into other cells. They can act inside cell of origin or in cells distant from point of release G protein– coupled receptors NO Activates variety of enzymes Ionotropic and G protein– coupled receptors Acetyl CoA + Choline Reuptake by transporter 1 Small-molecule neurotransmitters AChAcetyl cholinesterase Other small-molecule neurotransmittersSmall-molecule neurotransmitters diffuse across synaptic cleft and bind to postsynaptic receptors Vesicle transporter concentrates neurotransmitter into vesicles Postsynaptic cell Presynaptic terminal Ca++ channels Large electron-dense vesicles NO O2 Nitrogen oxide (inactive) 2 Neuropeptides •Fig. 6.2 Schematicofachemicalsynapticterminalreleasingallthreemainclassesofneurotransmitter.Foreach,themechanismsofrelease,sitesofaction,andmechanismsforterminationofactivityareshown.Realsynapsesreleasetransmitterfromoneormoreclasses.

Synaptic transmission at a chemical synapse may be summarized as follows. Synaptic transmission is initiated by arrival of the action potential at the presynaptic terminal. The action potential depolarizes the terminal, which causes Ca++ channels to open. The subsequent rise in [Ca++] within the terminal triggers the fusion of vesicles containing neurotransmitter with the plasma membrane. The transmitter is then expelled into the synaptic cleft, diffuses across it, and binds to specific receptors on the postsynaptic membrane. Binding of transmitter to receptors then causes the opening (or less often, the closing) of ion channels in the postsynaptic membrane, which in turn results in changes in the potential and resistance of the postsynaptic membrane that alter the excitability of the cell. The changes in membrane potential of the postsynaptic cell are termed excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) (

Fig. 6.3 ), depending on whether they increase or decrease, respectively, the cell’s excitability, which can be defined as its probability of firing action potentials. The transmitter acts for only a very short time (milliseconds) because reuptake and degradation mechanisms rapidly clear the transmitter from the synaptic cleft.

The succeeding sections will amplify specific points of this summary. However, it is worth mentioning at this point that some of the nonclassic types of neurotransmitters (e.g., neuropeptides and gaseous neurotransmitters such as nitric oxide) and the discovery of metabotropic receptors have required modifications of several aspects of this basic conception. (Whereas an ionotropic receptor usually contains the ion channel as an integral part of itself, a metabotropic receptor does not contain an ion channel but instead is coupled to a G protein that initiates second messenger cascades that can ultimately affect ion channels.) Some of the differences between classic and peptide transmitters are listed in

Table 6.1 . More details on the properties of peptide and gaseous transmitters are provided in the relevant parts of the section of this chapter, and metabotropic receptors are covered in the Receptors section.

Calcium Entry Is the Signal for Transmitter Release

Depolarization of the presynaptic membrane by the action potential causes voltage-gated Ca++ channels to open, which makes it possible for Ca++ to flow into the terminal and trigger the release of transmitter. However, Ca++ will enter the terminal only if there is a favorable electrochemical gradient to do so. Recall that it is the combination of the concentration and voltage gradients that determines the direction of ion flow through open channels. Extracellular [Ca++] is high relative to intracellular [Ca++], which favors entry into the terminal; however, during the peak of the action potential, the membrane potential is positive, and the voltage gradient opposes the entry of Ca++ because of its positive charge. Thus, at the peak of the action potential, relatively little Ca++ enters the terminal because although the membrane is highly permeable to Ca++ , the overall driving force is small. In fact, by using a voltage clamp, one can experimentally make the membrane potential positive and equal to the Nernst equilibrium potential for Ca++ . If this is done, no Ca++ will enter the terminal despite Ca++ channels being open, and as a result no transmitter is released and no postsynaptic response is observed. This voltage is known as the suppression potential. If the membrane potential is rapidly made negative again (because of either the end of the action potential or by adjusting the voltage clamp), Ca++ rushes into the terminal as a result of the large driving force (which arises instantaneously on repolarization) and the high membrane permeability to Ca++ (which remains high because it takes the Ca++ channels several milliseconds to close in response to the new membrane potential), thereby resulting in release of transmitter and a postsynaptic response (

Fig. 6.4 •Fig. 6.3 IPSPsandEPSPsrecordedwithamicroelectrodeinacatspinalmotorneuroninresponsetostimulationofappropriateperipheralafferentfibers.Fortytracesaresuperimposed.NotethattheseIPSPsarehyperpolarizing,butinsomecasesIPSPscanbedepolarizing—seetextforanexplanation.(RedrawnfromCurtisDR,EcclesJC.J Physiol 1959;145:529.)

Synaptic Vesicles and the Quantal Nature of Transmitter Release

How neurotransmitter is stored and how it is released are questions fundamental to synaptic transmission. Answering these questions began with two observations. The first was the discovery of small round or irregularly shaped organelles known as synaptic vesicles in presynaptic terminals by electron microscopy (see

Figs. 6.1B 6.2 ). The second observation came from recordings of postsynaptic responses at the neuromuscular junction. Normally an action potential in a motor neuron causes a large depolarization in the postsynaptic muscle, termed an end plate potential (EPP), which is equivalent to an EPSP in a neuron. However, under conditions of low extracellular [Ca++], the EPP amplitude is reduced (because the presynaptic Ca++ current is reduced, leading to a smaller rise in intracellular [Ca++], and transmitter release is proportional to [Ca++]). In this condition, the EPP is seen to fluctuate among discrete values (

Fig. 6.5 ). Moreover, small spontaneous depolarizations of the postsynaptic membrane, termed miniature end plate potentials (mEPPs), are observable. The amplitude of the mEPP (≤1 mV) corresponds to that of the smallest EPP evoked under low [Ca++], and the amplitudes of other EPPs were shown to be integral multiples of the mEPP amplitude; thus, it was proposed that each mEPP corresponded

SynthesizedandpackagedinthenerveterminalSynthesizedandpackagedinthecellbody;transportedtothenerveterminalbyfastaxonaltransportSynthesizedinactiveformActivepeptideformedwhenitiscleavedfromamuchlargerpolypeptidethatcontainsseveralneuropeptidesUsuallypresentinsmallclearvesiclesUsuallypresentinlargeelectron-densevesiclesReleasedintoasynapticcleftMaybereleasedsomedistancefromthepostsynapticcellTheremaybenowell-definedsynapticstructureActionofmanyterminatedbecauseofuptakebypresynapticActionterminatedbyproteolysisorbythepeptidediffusingawayterminalsviaNa+-poweredactivetransportTypically,actionhasshortlatencyandshortduration(msec)Actionmayhavelonglatencyandmaypersistformanyseconds •Fig. 6.4 PresynapticCa++ currentanditsrelationshiptothepostsynapticresponse.A, Schematicofasquidgiantsynapsepreparation.Electrodes1and2areusedtovoltage-clampthepresynapticterminalandrecorditsvoltageandcurrent.(NotethattetrodotoxinandtetraethylammoniumwerepresenttoblockNa+ andK+ conductancetoisolatetheCa++ conductance.)Electrode3recordsthemembranepotentialofthepostsynapticaxon.Thepresynapticterminalwasvoltage-clampedtoincreasinglymoredepolarizedlevels(blue traces). Withasmalldepolarization(B), asmallCa++ currentstartsshortlyafterthevoltagestep,continuestogrowforthedurationofthestep(oncurrent),andthendecaysexponentiallyafteritstermination(offortailcurrent).Alargervoltagestep(C) increasesboththeonandtheoffcomponentsoftheCa++ current,andnowdistinctonandoffresponsesareobservedinthepostsynapticresponse.D, ThevoltagestepistotheNernstpotentialforCa++ ,sothereisnoCa++ currentduringthestep,butalargetailcurrentandoffresponseareobserved.(BasedondataofLlinasRetal.Biophys J 1981;33:323.) to the release of transmitter from a single vesicle and that EPPs represented the combined simultaneous release of transmitter from many vesicles.

This linking of mEPPs and vesicles implies that each mEPP is caused by the action of many molecules of neurotransmitter binding to postsynaptic receptors. The alternative that each mEPP could be caused by a single transmitter molecule binding to and opening a single postsynaptic receptor was rejected, in part because responses smaller in amplitude than mEPPs could be generated experimentally by directly applying dilute solutions of acetylcholine to the muscle. In fact, mEPPs were calculated to be caused by the action of approximately 10,000 molecules, which corresponds well to estimates of the number of neurotransmitter molecules contained within a single vesicle.

Many additional studies have confirmed the vesicle hypothesis of neurotransmitter release. For example, biochemical studies have shown that neurotransmitter is concentrated in vesicles, and fusion of vesicles to the plasma membrane and their depletion in the terminal cytoplasm after action potentials have been shown with electron microscopic techniques.

3.6 mV 47 msec A 1 mV 20 msec B •Fig. 6.5 A, SpontaneousmEPPsrecordedataneuromuscularjunctioninafiberoffrogextensordigitorumlongus.B, EPPsevokedbynervestimulationunderlow-[Ca++]conditions,whichreducetheprobabilityoftransmitterrelease.Thesmall-amplitudeEPPsevokedundertheseconditionsvaryinamplitudeinastep-likemanner,wherethesizeofthestepisequaltothesmallestEPP,whichinturnequalsthesizeofthemEPPs.(Notethatintheseconditionsthestimulusoftenfailstoevokeanyresponse,asindicatedbyaflatresponse.)(A, DatafromFattP,KatzB.Nature 1950;166:597;B, datafromFattP,KatzB.J Physiol 1952;117:109.)

The small vesicles that contain nonpeptide neurotransmitters can fuse with the presynaptic membrane only at specific sites called active zones. To become competent to fuse with the presynaptic membrane at an active zone, a small vesicle must first dock at the active zone and then undergo a priming process. Once primed the vesicle can fuse and release its transmitter into the synaptic cleft in response to an increase in local cytoplasmic [Ca++]. On the order of 25 proteins may play roles in docking, priming, and fusion. Some of these proteins are cytosolic, whereas others are proteins associated with the vesicle membrane or the presynaptic plasma membrane. The functions of most of these proteins are incompletely understood; however, knowledge of the molecular details of transmitter release has increased dramatically in recent years.

As with other exocytotic processes, neurotransmitter release involves SM (sec1/Munc18-like) and SNARE (soluble N-ethyl maleimide-sensitive factor attachment protein receptor) proteins: v-SNARES in the vesicle membrane and t-SNARES in the (target) presynaptic plasma membrane. Zipper-like interactions between synaptobrevin (a v-SNARE) and syntaxin and SNAP-25 (both are t-SNARES) with the assistance of SM proteins bring the vesicle membrane and the presynaptic plasma membrane close together before fusion. The SNARE proteins are targets for various botulinum toxins, which disrupt synaptic transmission, thus demonstrating their critical role in this process. Nevertheless, they do not bind Ca++ , so another protein must be the Ca++ sensor that triggers the actual fusion event. Evidence indicates that a synaptotagmin protein is almost certainly the Ca++ sensor and, even more specifically, that the second of its two cytoplasmic domains contains the Ca++ binding site. Interestingly, synaptotagmins differ in their kinetics, and brain regions vary as to which synaptotagmin family member acts as the Ca++ sensor for vesicular fusion. Thus, differential expression of synaptotagmin genes in neurons may be a mechanism to adapt the kinetics of vesicle release and thereby tailor the specific characteristics of synaptic transmission to the functional needs of each CNS region.

Calcium channels are located in the active zone membrane at sites adjacent to the docked vesicles. When they open, a small area of high [Ca++], a microdomain is created at the active zone. This local high concentration (which lasts for less than a millisecond), allows the rapid binding of Ca++ to synaptotagmin, triggering the fusion of a docked vesicle and allowing release of its neurotransmitter. Despite the multiple steps involved, the process of vesicular release at a synapse is extremely rapid because of the close proximity of the molecular apparatuses involved to each other. Indeed, the time from Ca++ influx to vesicle fusion is about 0.2 msec.

“Reserve” pool Competence maturation Fusion and collapse Coated-vesicle retrieval “Recycling” pool “Docked” “Primed” (?) (2) (2') (3') ? ? (1) (3) (4) (5) “Cocked” (?) Armed Fire! ? ? “Readily releasable” pool (RRP) Kiss... Run! Kiss-and-run fusion Phosphorylations, Ca++ fusion machine assembly ? ?? Ca++ •Fig. 6.6 Vesiclerecyclingpathways.Synapticvesicleshavebeenthoughttofusewiththemembranewhileemptyingtheircontentsandthenberecycledbyformingclathrin-coatedpitsthatareendocytosedtoformcoatedvesicles(1→ [2or2′]→ 3′→ 1).Analternativepathwaythatmayallowmorerapidrecyclingofvesicleshasbeenproposed.Thispathway,called“kissandrun,”involvesonlytransientfusionofthevesicletothepresynapticmembranetoformaporethroughwhichthevesiclecontentsmaybeemptied,followedbydetachmentofthevesiclefromthemembrane(1→ 2→ 3→ 4→ 5→ 1).(RedrawnfromValtortaF,MeldolesiJ,FesceR.Trends Cell Biol 2001;11:324.)

During synaptic transmission, vesicles must fuse with the plasma membrane to release their contents into the synaptic cleft. However, there must be a reverse process; otherwise, not only would it be hard to sustain the vesicle population, but the presynaptic membrane’s surface area would also grow with each bout of synaptic transmission, and its molecular content and functionality would likewise change (because, as just discussed, the protein content of the vesicle membrane is distinct from that of the terminal membrane).

There appear to be two distinct mechanisms by which vesicles are retrieved after release of their neurotransmitter content (Fig. 6.6 ). One mechanism is the endocytotic pathway commonly found in most cell types. Coated pits are formed in the plasma membrane, which then pinch off to form coated vesicles within the cytoplasm of the presynaptic terminal. These vesicles then lose their coat and undergo further transformations (i.e., acquire the correct complement of membrane proteins and be refilled with neurotransmitter) to become once again synaptic vesicles ready for release.

Evidence for a second, more rapid recycling mechanism has been obtained (see

Fig. 6.6 ). It involves transient fusion of the vesicle to the synaptic membrane and has been called “kiss and run.” In this case, fusion of the vesicle with the synaptic membrane leads to the formation of a pore through which the transmitter is expelled, but there is no wholesale collapse of the vesicle into the membrane. Instead, the duration of the fusion is very brief, after which the vesicle detaches from the plasma membrane and reseals itself. Thus, the vesicle membrane retains its molecular identity. Its contents can then simply be replenished, thereby making the vesicle ready for use again.

The relative importance of these two mechanisms is still being debated. However, at central synapses, which tend to be small and contain relatively few vesicles in comparison to the neuromuscular junction, the rapid time course of the kiss-and-run mechanism may help avoid the problem of vesicle depletion and the consequent failure of synaptic transmission during periods of high activity (many neurons in the CNS can show firing rates of several hundred hertz, and a few types of neuron can fire at rates of ≈ 1000 Hz).

Following vesicle fusion the neurotransmitter molecules are released and diffuse across the synaptic cleft (a very rapid process) and bind to receptors on the postsynaptic membrane. This binding leads to the opening (or less often the closing) of ion channels. These channels are termed ligand-gated because their opening and closing are primarily controlled by the binding of neurotransmitter. This mechanism can be contrasted with that of the voltage-gated channels underlying the action potential, whose opening and closing are determined by the membrane potential. However, there are some channels, most notably the NMDA (N-methyl-Daspartate) channel, that are both ligand and voltage gated.

It is also worth noting here that what follows in this section refers to what happens when neurotransmitter binds to receptors in which the ion channel is part of the receptor itself. These receptors are referred to as ionotropic receptors and underlie what is now called “fast” synaptic transmission. There is also “slow” synaptic transmission, mediated by what are called metabotropic receptors, in which the receptor and ion channel are not part of the same molecule, and binding of neurotransmitter to the receptor initiates biochemical cascades that lead to postsynaptic potentials with slow onsets (see the section Receptors for details). Despite the differing time courses, many of the same basic principles apply to both types of postsynaptic potential.

EPSPs. As stated earlier, the binding of neurotransmitter generally changes the membrane potential of the postsynaptic cell, and these changes are referred to as EPSPs when they increase the excitability of the neuron and IPSPs when they inhibit the neuron from firing action potentials. EPSPs are always depolarizing potentials, and IPSPs are usually hyperpolarizing.

Once a ligand-gated channel is open, the direction of current flow through it is determined by the electrochemical driving force for the permeant ion(s). It turns out that the pores of most channels that underlie EPSPs are relatively large and therefore allow passage of most cations with similar ease. As an example, consider the acetylcholinegated channel that is opened at the neuromuscular junction. Na+ and K+ are the major cations present (Na+ extracellularly and K+ intracellularly); therefore, the net current through the channel is approximately the sum of the Na+ and K+ currents (Inet = INa + IK). Recall that the current through a channel from a particular ion is dependent on two factors: the conductance of the channel to the ion and the driving force on the ion. This relationship is expressed by the equation

Equation 6.1 where gx is the conductance of the channel to ion x, Vm is the membrane potential, and Ex is the Nernst equilibrium potential for ion x. In this case gx is similar for Na+ and K+ , so the main determinant of net current is the relative driving forces (Vm − Ex). If the membrane is at its resting potential (typically around −70 mV), there is a strong driving force (Vm − ENa) for Na+ to enter the cell because this potential is far from the Na+ Nernst potential (about +55 mV), whereas there is only a small driving force for K+ to leave the cell because Vm is close to the K+ Nernst potential (about −90 mV). Thus, if acetylcholine-gated channels open when the membrane is at its resting potential, a large inward Na+ current and a small outward K+ current will flow through the acetylcholine channel, thereby resulting in a net inward current, which acts to depolarize the membrane.

The net inward current that results from opening such channels is called the excitatory postsynaptic current (EPSC).

Fig. 6.7A contrasts the time course of the EPSC and the resulting EPSP for fast synaptic transmission. The EPSC is much shorter (≈1 to 2 msec in duration) and corresponds to the time the channels are actually open. The short duration of the EPSC is due to the fact that the released neurotransmitter remains in the synaptic cleft for only a short while before being either enzymatically degraded or taken up by either glia or neurons. Binding and unbinding of a neurotransmitter to its receptor take place rapidly, so once its concentration falls in the cleft, the postsynaptic receptor channels rapidly close as well and terminate the EPSC. Note how the end of the EPSC corresponds to the peak of the EPSP, which is followed by a long tail. The duration of the tail and the rate of the decay in EPSP amplitude reflect the passive membrane properties of the cell (i.e., its RC properties) (see ). In slow synaptic transmission, the duration of the EPSP reflects the activation and deactivation of biochemical processes more than the membrane properties. The long duration of even fast EPSPs (relative to EPSCs and action potentials) is functionally important because it allows EPSPs to overlap and thereby summate. Such summation is central to the integrative properties of neurons (see the next section,

Normally an EPSP depolarizes the membrane, and if this depolarization reaches threshold, an action potential is generated. However, consider what happens if the channels underlying the action potential are blocked and the membrane of the postsynaptic cell is experimentally depolarized by injecting current through an intracellular electrode. Because the membrane potential is now more positive, the driving force for Na+ is decreased and that for K+ increased. If the synapse is activated at this point, the net current through the receptor channel (the EPSC) will be smaller because of changes in the relative driving force. This implies that if the membrane potential is depolarized enough, there will be a point at which the Na+ and K+ currents through the channel are equal and opposite, and thus there is no net current and no EPSP. If the membrane is depolarized beyond this point, there is a net outward current through the receptor channels and the membrane will hyperpolarize (i.e., the EPSP will be negative). Thus, the potential at which there is no EPSP (or EPSC) is known as the reversal potential. For excitatory synapses, the reversal potential is usually around 0 mV (±10 mV), depending on the synapse (see

Fig. 6.7B–C

It is worth noting that a reversal potential is a key criterion for demonstrating the chemical-gated as opposed to the voltage-gated nature of a synaptic response because currents through voltage-gated channels do not reverse, except at the Nernst potential of the ion for which they are selective (and then only if the channel is open at that potential). Consequently, beyond a certain membrane potential, no current will flow through voltage-gated channels because they will be closed. In contrast, ligand-gated channels can be opened at any membrane potential and thus can always have a net current flow through them, except at one specific voltage, the reversal potential.

0.1 msec/div Action potentials triggered by EPSP 5 mV ˜V •Fig. 6.7 PropertiesofEPSPs.A, TimecourseofafastEPSPcomparedwiththatoftheunderlyingEPSC.Inmanycases,suchasthisone,theEPSCismuchshorterthantheEPSP;however,sometimestheEPSCcanhaveafairlyextensivetail.B, IntracellularlyrecordedEPSPsatdifferentlevelsofdepolarization.EPSPswereevokedinmotorneuronsbystimulationofIaafferents.Thenumbertotheleftofeachtraceindicatesthemembranepotentialinducedbyinjectionofcurrentthroughtheelectrode.Atinitialmembranepotentialsof−42and−60mV,theEPSPtriggeredanactionpotential.Atmoredepolarizedlevels,Na+ channelsareinactivated,sonospikeoccurs.C, TodeterminetheEPSPreversalpotential,theinitialmembranepotentialisplottedagainstthesizeoftheEPSP(ΔV).ThisEPSPreversedat−7mV.(A, DatafromCurtisDR,EcclesJC.J Physiol 1959;145:529;B, datafromCoombsJSetal.J Physiol 1955;130:374.)

IPSPs. Like EPSPs, IPSPs are triggered by the binding an action potential (otherwise it would increase the prob-of neurotransmitter to receptors on the postsynaptic ability of the cell spiking and by definition be an EPSP). It membrane and typically involve an increase in membrane may seem counterintuitive that something that depolarizes permeability as a result of the opening of ligand-gated the membrane can still be considered inhibitory, but if channels. They differ in that IPSP channels are permeable it decreases the probability of spiking, then it is indeed to only a single ionic species, either Cl− or K+ . Thus, IPSPs inhibitory (a further explanation is given in the Synaptic will have a reversal potential equal to the Nernst potential Integration section). of the ion carrying the underlying current. Typically the In sum, starting from the resting membrane potential, Nernst potential for these ions is somewhat negative relative EPSPs are always depolarizing, IPSPs can be either depolarto the resting potential, so when IPSP channels open, there izing or hyperpolarizing, and a hyperpolarizing potential is is an outward flow of current through them that results in always an IPSP. Thus, the key distinction between inhibitory hyperpolarization of the membrane (see

Fig. 6.3 ). and excitatory synapses (and IPSPs and EPSPs) is how they

However, in some cells, activation of an inhibitory affect the probability of the cell firing an action potential: synapse may produce no change in potential (if the mem-EPSPs increase the probability, whereas IPSPs decrease the brane potential equals the Nernst potential for Cl− or K+) or probability. may actually result in a small depolarization. Nevertheless, Safety factor. Synapses between cells vary in strength and in both these cases, the reversal potential for the IPSP thus in the size of the PSP generated in the postsynaptic cell. is still negative with regard to the threshold for eliciting Many factors determine synaptic strength, including the size and number of synaptic contacts between two cells, its activity level and past history, and the probability of vesicle fusion for the synapse. For excitatory synapses, the strength of the synapse may be quantified by what is known as its safety factor (the ratio of EPSP amplitude to the amplitude needed to reach the threshold to trigger an action potential). Most synapses have low safety factors (<1), and thus it takes the summed EPSPs of multiple active synapses to trigger an action potential in the postsynaptic neuron. This summation process is at the core of synaptic integration, which is taken up in the next section. Synapses with high (>1) safety factors exist, however, and the neuromuscular junction is one prominent example. When a motor neuron action potential triggers release of neurotransmitter at the neuromuscular junction, an end plate potential (EPP; the equivalent of an EPSP in a neuron) is generated in the muscle fiber. The EPP is so large that under normal circumstances it depolarizes the sarcolemma well above the action potential threshold and thus always triggers a spike, leading to contraction of the muscle cell. A high safety factor makes sense for the neuromuscular junction because each muscle cell is contacted by only a single motor neuron, and if that motor neuron is firing, the nervous system has basically made the decision to contract that muscle. In certain diseases of the neuromuscular junction, such as myasthenia gravis and Lambert-Eaton syndrome, the EPPs are reduced such that the safety factor can fall below 1, and thus the EPPs sometimes fail to trigger action potentials in the muscle fibers, leading to weakness.

The overall effect of a particular synapse is dependent on its location. To understand this concept fully, we must first recall that action potentials are typically generated at the initial segment of the cell because it has the highest density of voltage-gated Na+ channels and therefore the lowest threshold for initiation of a spike. Thus, it is the summed amplitudes of the synaptic potentials at this point, the initial segment, that is critical for the decision to spike. EPSPs generated by synapses close to the initial segment (i.e., synapses onto the soma or proximal dendrites) will result in a larger depolarization at the initial segment than will EPSPs generated by synapses on distal dendrites (

Fig.

6.8A single action potential in axon 2 versus 1). This is because the cell membrane is leaky and synaptic currents are generated locally at the synapse, so even if two synapses generate a local EPSC of the same size, less of the initial current will arrive at the initial segment from the more distal synapse than from the more proximal one, thereby resulting in the generation of a smaller EPSP at the initial segment by the distal synapse (see discussion of length constant in ). Thus, the synapse’s spatial location in the dendritic tree is an important determinant of its efficacy. However, as already mentioned, EPSPs generated by most CNS synapses, even those in favorable positions (i.e., close to the initial segment), are too small by themselves to reach •Fig. 6.8 SynapticintegrationofEPSPsrecordedattheaxonhillockadjacenttotheinitialsegment.A, ComparisonofEPSPsevokedbyproximalversusdistalsynapses(2versus1).B, Temporalsummation.EPSPsinresponsetotwospikesinthesameaxonoccurringinrapidsuccession(axon2).C, Spatialsummation.Responsesevokedbysynapsesthatareelectricallydistantfromeachother(1and3).D, Sublinearsummationoftwosynapseslocatedneareachotherbecauseofshunting(2and4).

the spiking threshold in the postsynaptic cell, as illustrated in

Fig. 6.8A , where an action potential in either axon 1 (distal) or 2 (proximal) both produce EPSPs that are too small to trigger a spike. Thus, generally the summed EPSPs from multiple synapses are required to reach threshold and trigger a spike.

The requirement for multiple EPSPs to summate in order to trigger a spike is what makes the relatively long duration of EPSPs so important. Temporal summation refers to the fact that EPSPs that are separated by a latency less than their duration can sum. This is illustrated in

Fig.

6.8B , where the same synapse is activated multiple times in rapid succession (axons can fire action potentials at rates well over 100 Hz); in this situation, successive EPSPs will be less than 10 msec apart and therefore overlap and sum. Note the higher amplitude of the second peak.

Spatial summation refers to the fact that synaptic potentials generated by different synapses can interact. For example, in

Fig. 6.8 , suppose axon 1 and 3 each fire an action potential but at widely separated times. Each produces an EPSP that depolarizes the cell but is too small to reach threshold (see

Fig. 6.8C , EPSP1, EPSP3). Instead, if both axons fire within a short enough time of each other, their effect can be additive, as shown in

Fig. 6.8C (EPSP 1+3). The combined EPSP amplitude may then reach threshold and lead to spiking of the cell. If the EPSPs generated by axons 1 and 3 were simultaneous, then we would have an example of pure spatial summation. In the example shown, however, the times of the two EPSPs were slightly separated, thus we have both spatial and temporal summation present. The fact that EPSPs have a long time course (when compared with action potentials or the underlying EPSCs) facilitates both types of synaptic integration.

In the foregoing example, the combined EPSP was approximately the linear summation of the two individual EPSPs evoked by action potentials in axons 1 and 3. This is the case when two synapses are far apart. If the two synapses are close together, such as for axons 2 and 4 (see

Fig. 6.8D ), the summation becomes less than linear because of what is known as a shunting effect. That is, when synapse 2 is active, channels are opened in the cell membrane, which means that it is more leaky. Therefore, when synapse 4 is also active, more of its EPSC will be lost (shunted) through the dendritic membrane, and less current will be left to travel down the dendrite to the initial segment. The result is that synapse 4 causes a smaller EPSP at the initial segment than it would have generated in isolation. Nevertheless, the combined EPSP is still larger than an EPSP caused by either synapse 2 or 4 alone.

Where do IPSPs fit into synaptic integration? In many cases one can think of them as negative EPSPs. Thus, whereas EPSPs add together to help bring the membrane potential up to and beyond the spiking threshold, IPSPs subtract from the membrane potential to make it more negative and therefore further from threshold. In deciding whether to spike, a cell adds up the ongoing EPSPs and subtracts the IPSPS to determine whether the sum reaches threshold. As with an EPSP, the efficacy of an IPSP varies with its location.

In addition to subtracting algebraically from the membrane potential, IPSPs exert an inhibitory action via the shunting mechanism, just as was described earlier for EPSPs. That is, while the IPSP channels are open, they make the membrane more leaky (i.e., lower its resistance) and thereby reduce the size of EPSPs, thus making them less effective. This shunting mechanism explains how IPSPs that do not change the membrane potential—or even those that slightly depolarize it—can still decrease the excitability of the cell. An alternative way to look at this effect is to view each synapse as a device that tries to bring the membrane potential to its own equilibrium potential. Because this potential is below the action potential threshold in the case of IPSPs, IPSPs make it harder for the cell to spike.

Thus far the interaction of synaptic potentials has been presented under the assumption that the postsynaptic cell membrane is passive (i.e., it acts as though it were simply resistors and capacitors in parallel with each other). However, it is clear that the dendrites and somas of most, if not all, neurons contain active elements (i.e., gated channels) that can amplify and alter EPSPs and IPSPs. For example, a distal EPSP can have a larger-than-expected effect if the EPSP activates dendritically-located voltage-gated Na+ or Ca++ channels that boost its amplitude or even generate propagated dendritic action potentials. Another example is Ca++-activated K+ channels that are present in the dendrites of some neurons. These channels are activated by the influx of Ca++ either through synaptic channels or via dendritic voltage-gated Ca++ channels opened by EPSPs and can cause long-lasting hyperpolarizations that effectively make the cell inexcitable for tens to hundreds of milliseconds. As a final example, there are some Ca++ channels that underlie a low-threshold Ca++ spike. These channels are normally inactive at resting membrane potentials, but the hyperpolarization that results from a large IPSP can de-inactivate them and allow them to open (and produce a spike) after termination of the IPSP. In this case “inhibition” actually increases the cell’s excitability. In sum, synaptic integration is a highly complex, nonlinear process. Nevertheless, the basic principles just described remain at its core.

Modulation of Synaptic Activity

Integration of synaptic input by a postsynaptic neuron, as described in the previous section, represents one aspect of the dynamic nature of synaptic transmission. A second aspect is that the strength of individual synapses can vary as a function of their use or activity. That is, a synapse’s current functional state reflects, to some extent, its history.

Activation of a synapse typically produces a response in the postsynaptic cell (i.e., a postsynaptic potential) that will be roughly the same each time, assuming the postsynaptic cell is in a similar state. Certain patterns of synaptic activation, however, result in changes in the response to subsequent activation of the synapse. Such use-related changes may remain for short (milliseconds) or long (minutes to days) durations and may be either a potentiation or suppression of the synapse’s strength. These changes probably underlie cognitive abilities such as learning and memory. Thus, the processes by which activity results in changes in a synapse’s efficacy are a critical feature of synaptic transmission.

When a presynaptic axon is stimulated twice in rapid succession, it is often found that the postsynaptic potential evoked by the second stimulus are larger in amplitude than the one evoked by the first (Fig. 6.9 ). This increase

A Facilitation B Facilitation C Posttetanic potentiation 3 msec 10 msec 1/sec 5/sec 10/sec 12.5/sec 31.2/sec 25/sec 1 mV 4 sec tetanic stimulation 8 sec 14 sec 27 sec Time after tetanic stimulation •Fig. 6.9 A, Facilitationataneuromuscularjunction.EPPsataneuromuscularjunctionintoadsartoriusmusclewereelicitedbysuccessiveactionpotentialsinthemotoraxon.Neuromusculartransmissionwasdepressedby5mMMg++ and2.1mMcuraresothatactionpotentialsdidnotoccur.B, EPPsatafrogneuromuscularjunctionelicitedbyrepetitivelystimulatingthemotoraxonatdifferentfrequencies.Notethatfacilitationfailedtooccuratthelowestfrequencyofstimulation(1/sec)andthatthedegreeoffacilitationincreasedwithincreasingfrequencyofstimulationintherangeoffrequencyused.Neuromusculartransmissionwasinhibitedbybathingthepreparationin12to20mMMg++ .C, Posttetanicpotentiationatafrogneuromuscularjunction.ThetoptwotracesindicatecontrolEPPsinresponsetosingleactionpotentialsinthemotoraxon.SubsequenttracesindicateEPPsinresponsetosingleactionpotentialsaftertetanicstimulation(50impulses/secfor20seconds)ofthemotorneuron.Thetimeintervalbetweentheendoftetanicstimulationandthesingleactionpotentialisshownoneachtrace.Themusclewastreatedwithtetrodotoxintopreventgenerationofactionpotentials.(A, RedrawnfromBelnaveRJ,GagePW.J Physiol 1977;266:435;B, redrawnfromMagelbyKL.J Physiol 1973;234:327;C, redrawnfromWeinrichD.J Physiol 1971;212:431.) is known as paired-pulse facilitation (PPF). Note PPF is distinct from temporal summation, in which two EPSPs overlap and sum to a larger response; with PPF the second EPSP itself is greater in size. If one plots the relative size of the two postsynaptic potentials (PSPs) as a function of the time between two stimuli, the amount of increase in the second PSP will be seen to depend on the time interval. Maximal facilitation occurs at around 20 msec, followed by a gradual reduction in facilitation as the interstimulus interval continues to increase; with intervals of several hundred milliseconds, the two PSPs are equal in amplitude and no facilitation is observed. Thus, PPF is a relatively rapid and short-lasting change in synaptic efficacy.

Posttetanic potentiation (PTP) is similar to PPF; however, in this case the responses are compared before and after stimulation of the presynaptic neuron tetanically (tens to hundreds of stimuli at a high frequency). Such a tetanic stimulus train causes an increase in synaptic efficacy (see

Fig. 6.9C ). PTP, like PPF, is an enhancement of the postsynaptic response, but it lasts longer: tens of seconds to several minutes after the cessation of tetanic stimulation.

Numerous experiments have shown that PPF and PTP are the result of changes in the presynaptic terminal and do not generally involve a change in the sensitivity of the postsynaptic cell to transmitter. Rather, the repeated stimulation leads to an increased number of quanta of transmitter being released. This increase is thought to be due to residual amounts of Ca++ that remain in the presynaptic terminal after each stimulus and help potentiate subsequent release of transmitter. However, the exact mechanism or mechanisms by which this residual Ca++ enhances release is not yet clear. The residual Ca++ does not, however, appear to act simply by binding to the same sites as the Ca++ that enters at the active zone and directly triggers vesicle fusion in response to the action potential.

Use of a synapse can also lead to a short-term depression in its efficacy. Most commonly, the postsynaptic cell at such a fatigued or depressed synapse responds normally to transmitter applied from a micropipette; hence, as was the case for PPF and PTP, the change is presynaptic. In general, the depression is thought to reflect depletion of the number of releasable presynaptic vesicles. Thus, short-term depression of synaptic transmission is most often and most easily seen at synapses in which the probability of release after a single stimulus is high and under conditions that favor release (i.e., high [Ca++]). A postsynaptically related cause of synaptic depression can be desensitization of the receptors in the postsynaptic membrane.

Both potentiation and depressive processes can occur at the same synapse. So in general, the type of modulation observed will depend on which process dominates. This in turn can reflect stimulus parameters, local ionic conditions, and the properties of the synapse. In particular, synapses have different baseline probabilities for releasing vesicles. Synapses with a high release probability will be more likely to show poststimulus depression, whereas those with low release probability are less likely to deplete their vesicle store and thus can be facilitated more easily. Sometimes mixed responses can occur. For example, during a tetanic stimulus train a synapse may show a depressed response, but after the train the synapse can show posttetanic facilitation once the vesicles are recycled.

Just as the postsynaptic membrane contains receptors for neurotransmitters, so does the presynaptic membrane. When these presynaptic receptors bind neurotransmitter, they cause events that can modulate subsequent release of transmitter by the terminal. There are several sources of transmitter that bind to presynaptic receptors: it can be the transmitter released by the terminal itself (i.e., self-modulation, in which case the receptors are referred to as autoreceptors), it can be released by another presynaptic terminal that synapses onto the terminal (a serial synapse), or it can be a nonsynaptically acting neurotransmitter (see the section

Presynaptic receptors can be either ionotropic or metabotropic. In the latter case, recall that their action will be relatively slow in onset and long in duration and the effect will depend on the specific second messenger cascades that are activated. Such cascades can ultimately regulate presynaptic voltage-gated Ca++ and K+ channels and other presynaptic proteins and thereby alter the probability of vesicle release.

In contrast, activation of presynaptic ionotropic receptors will directly alter the electrical properties of the presynaptic terminal and cause rapid transient (millisecond time scale) changes in the probability of vesicle release (although they too can have much longer lasting effects). Binding of an ionotropic receptor will open channels in the presynaptic terminal and thereby alter the amount of transmitter released by an action potential.

Presynaptic inhibition refers to occasions when binding of presynaptic receptors leads to a decrease in release of transmitter, and it can be the result of one or more mechanisms (Fig. 6.10 ). First, opening of channels decreases membrane resistance and creates a current shunt. The shunt acts to divert the current associated with the action potential from the active zone membrane and thereby lessens the depolarization of the active zone, which results in less activation of Ca++ channels, less Ca++ entry, and less release of transmitter. A second mechanism is the change in membrane potential caused by the opening of presynaptic •Fig. 6.10 Presynapticinhibition.Activeregenerationofactionpotentialsinaxon2endsatthelastnode.Theactionpotentialisthenpassivelyconductedintotheterminal.Axon1makesanaxoaxonicsynapsewithaxon2.Activationofthissynapsereducesconductionoftheactionpotentialinaxon2totheactivezoneofitssynapticterminalbymechanismsdescribedinthetext.Thisreducestheopeningofvoltage-gatedCa++ channelsandthereforereleaseofneurotransmitter.

ionotropic channels. If a small depolarization is the result, there will be inactivation of voltage-gated Na+ channels and thereby lessening of the action potential–associated current and transmitter release. Presynaptic γ-aminobutyric acid A receptors (GABAA) occur in the spinal cord and mediate presynaptic inhibition by these mechanisms. They control Cl− channels. Generally, opening of Cl− channels generates a hyperpolarization. However, in the presynaptic terminal, the [Cl−] gradient is such that Cl− flows out of the cell and generates a small depolarization. This depolarization is small enough that it does not cause significant opening of voltage-gated Ca++ channels; otherwise, it would increase release of transmitter (presynaptic facilitation). In fact, there are other receptors that control cation channels and create large depolarizations, thereby increasing the release of transmitter. In addition, presynaptic nicotinic acetylcholine receptors control a cation channel that is permeable to Ca++ . By allowing additional entry of Ca++ , these receptors increase the release of transmitter from the terminal.

Repetitive stimulation of certain synapses in the brain can also produce more persistent changes in the efficacy of transmission at these synapses, a process called long-term potentiation or long-term depression. Such changes can persist for days to weeks and are believed to be involved in the storage of memories.

The increased synaptic efficacy that occurs in long-term potentiation probably involves both presynaptic (greater transmitter release) and postsynaptic (greater sensitivity to transmitter) changes, in contrast to the short-term changes that involve changes only in presynaptic function. Entry of calcium into the postsynaptic region is an early step required for initiating the changes that result in long-term enhancement of the response of the postsynaptic cell to neurotransmitter. Entry of calcium occurs through NMDA and some AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (classes of glutamate receptors; see the section Receptors). Entry of Ca++ is believed to activate Ca++-calmodulin kinase II, a multifunctional protein kinase that is present in very high concentrations in postsynaptic densities. In the presence of high [Ca++], this kinase can phosphorylate itself and thereby become active. Calciumcalmodulin kinase II is believed to phosphorylate proteins that are essential for the induction of long-term potentiation. Long-term potentiation may also have an anatomic component. After appropriate stimulation of a presynaptic pathway, the number of dendritic spines and the number of synapses on the dendrites of postsynaptic neurons may increase rapidly. Changes in the presynaptic nerve terminal may also contribute to long-term potentiation. The postsynaptic neuron may release a signal (nitric oxide has been suggested) that enhances release of transmitter by the presynaptic nerve terminal.

Neurotransmitters are the substances that mediate chemical signaling between neurons. For a substance to be considered a neurotransmitter, it must meet several generally recognized criteria. First, the substance must be demonstrated to be present in the presynaptic terminal, and the cell must be able to synthesize the substance. It should be released on depolarization of the terminal. Finally, there should be specific receptors for it on the postsynaptic membrane. This last criterion is certainly true for substances that act as synaptic transmitters, but if we want to be inclusive and include substances that act over widespread territories rather than just at a single synapse, the last criterion needs to be relaxed to include situations in which receptors are located at sites outside the synapse. Neurotransmission has been suggested as a general term to describe both synaptic and nonsynaptic signaling between cells.

More than 100 substances have been identified as potential neurotransmitters because they have met some (hence the “potential” qualifier) or all of these criteria. These substances can be subdivided into three major categories: small-molecule transmitters, peptides, and gaseous transmitters. The small-molecule neurotransmitters may be further subdivided into acetylcholine, amino acids, biogenic amines, and purines. The first three groups of small-molecule transmitters contain what are considered the classic neurotransmitters. Remaining transmitters are substances that are more recent additions to the list of neurotransmitters, although many of them have been known as biologically important molecules in other contexts for a long time.

In the peripheral nervous system, acetylcholine is the transmitter at neuromuscular junctions, at sympathetic and parasympathetic ganglia, and of the postganglionic fibers from all parasympathetic ganglia and a few sympathetic ganglia. It is also a transmitter within the CNS, most prominently of neurons in some brainstem nuclei, in several parts of the basal forebrain (septal nuclei and nucleus basalis) and basal ganglia, and in the spinal cord (e.g., motor neuron axon collaterals). Cholinergic neurons from the basal forebrain areas project diffusely throughout the neocortex and to the hippocampus and amygdala, and they have been implicated in memory functions. Indeed, degeneration of these cells occurs in Alzheimer’s disease, a form of dementia in which memory function is gradually and progressively lost.

Anumberofdrugsknownasanticholinesterases interferewithacetylcholinesteraseandtherebyenhancetheactionofacetylcholinebyprolongingitspresenceatitssynapses.Suchdrugsincludeinsecticidesandchemicalwarfareagents,aswellassometherapeuticdrugs,suchasthoseusedtotreatmyasthenia gravis. Myastheniagravisisanautoimmunediseaseinwhichantibodiesbindtoacetylcholinereceptorsattheneuromuscularjunction,therebydisruptingtheirfunctionalityandcausingthemtobemorerapidlydegraded.Thisreductioninreceptorsleadstosevereweaknessandultimatelyparalysis.Theweaknessischaracterizedbyrapidtiringofthemusclewithrepeateduse.Rapidtiringoccursbecausethenumberofpresynapticvesiclesavailableforreleasedropsduringthehigh-frequencytrainofmotorneuronactionpotentialsthatgeneratessuchcontractions.Normally,becauseofthehighsafetyfactoroftheneuromuscularjunction,smallerbutstillsuprathresholdEPPswouldstillbegeneratedandmaintainmusclecontractionduringrepeateduse.Inpeoplewithmyastheniagravis,thesafetyfactorissoreducedbythelossofacetylcholinereceptorsthatthedecreaseinreleaseofacetylcholinewithrepeatedactivityleadstoEPPsthatfailtotriggerspikes,andthusmuscularcontractionfails.Standardtreatmentsincludeanticholinesterases,whichallowagreaterconcentrationofacetylcholinetopartiallyovercomethedeficitcausedbythereducednumberoffunctionalpostsynapticreceptors,andimmunosuppressivetherapiesandplasmaexchange,whichreducelevelsofautoantibodiesagainsttheacetylcholinereceptor.Thesetherapiesareallrelativelynonspecificandcanthereforehavemanysideeffects.PotentialfuturetherapiesarebeingdevelopedandincludeinducingtolerancetotheacetylcholinereceptorandselectivedestructionoftheBcellsthatmakeantibodiesagainstthereceptor.

Acetylcholine is synthesized from acetyl coenzyme A and choline by the enzyme choline acetyltransferase, which is located in the cytoplasm of cholinergic presynaptic terminals. After synthesis, acetylcholine is concentrated in vesicles. After release, the action of acetylcholine is terminated by the enzyme acetylcholinesterase, which is highly concentrated in the synaptic cleft. Acetylcholinesterase hydrolyzes acetylcholine into acetate and choline. The choline is then taken up by an Na+ symporter in the presynaptic membrane for the resynthesis of acetylcholine. The extracellular enzymatic degradation of acetylcholine is unusual for a neurotransmitter inasmuch as the synaptic action of other classic neurotransmitters is terminated via reuptake by a series of specialized transporter proteins.

•Fig. 6.11 Glutamatetransportcycle.A, Schematicshowsthefateofglutamatereleasedfromapresynapticterminal.Distinctglutamatetransportersexistonthepresynapticandpostsynapticcellmembranesforreuptake.Inaddition,glialcellstakeupglutamateandconvertittoglutamine.Theglutamineisthenreleasedandtakenintothepresynapticterminal,whereitisconvertedbacktoglutamatebeforebeingrepackagedintosynapticvesicles.B, Schematicoftransportershowingdirectionofionflowassociatedwiththemovementofglutamateacrossthemembrane.

A variety of amino acids function as neurotransmitters. The three most important are glutamate, glycine, and GABA.

Glutamate is the neurotransmitter at the overwhelming majority of excitatory synapses throughout the CNS. Despite its ubiquity, it was initially difficult to identify specific neurons as glutamatergic because glutamate is present in all cells; it has a key role in multiple metabolic pathways, and it is a precursor to GABA, the major inhibitory neurotransmitter. Nevertheless, experimental results have now clearly established glutamate as the major excitatory CNS neurotransmitter. When applied to cells, it causes depolarization and is released from neurons, and specific receptors and transporters for it have been identified.

In addition to being the main excitatory neurotransmitter, glutamate is a potent neurotoxin at high concentrations. Thus, strict limitation of glutamate’s activity after its release from the presynaptic terminal is necessary, not only to allow normal synaptic transmission but also to prevent cell death. This task is accomplished by specialized membrane transporter proteins (

Fig. 6.11

At the Cellular Level box).

GABA and glycine act as inhibitory neurotransmitters. GABA is the major inhibitory transmitter throughout the nervous system. GABA is produced from glutamate by a specific enzyme (glutamic acid decarboxylase) that is present only in neurons that use GABA as a transmitter. Thus, experimentally, it is possible to identify cells as inhibitory GABAergic neurons by using antibodies to this enzyme to mark them (immunolabeling, see

Fig. 6.1B ). Many local interneurons are GABAergic. In addition, several brain regions contain large numbers of GABAergic projection neurons. The most notable are the spiny neurons of the striatum and the Purkinje cells of the cerebellar cortex. The inhibitory nature of Purkinje cells was especially surprising because they represent the entire output of the cerebellar cortex, and thus cerebellar cortical activity basically functions to suppress the activity of its downstream targets (cerebellar and vestibular nuclei).

Glycine functions as an inhibitory neurotransmitter in a much more restricted territory. Glycinergic synapses are predominantly found in the spinal cord, where they represent approximately half of the inhibitory synapses. They are likewise present in the lower brainstem, cerebellum, and retina in significant numbers. Interestingly, glycine also has another synaptic function. At excitatory NMDA-type glutamate receptors, glycine must also be bound for the ion channel to open. Thus, it acts as a cotransmitter at these synapses. It was generally thought that under physiological conditions the extracellular glycine concentration was high enough that the glycine binding sites of the NMDA channel were always saturated, but recent results suggest that this may not always be true, which implies that fluctuations in glycine levels may also be an important modulator of NMDA-mediated synaptic transmission.

After GABA and glycine are released from the presynaptic terminal, they are taken back up into the nerve terminal and neighboring glia by high-affinity Na+-Cl−–coupled membrane transporters. These Na+-Cl− transporters are part of what is called the solute carrier 6 (SLC6) family of transporters that also includes those for the biogenic amine neurotransmitters, but it is distinct from those for glutamate. Transport of the neurotransmitter into the cell is accomplished by symport with two Na+ and one Cl− ion. Four GABA transporter (GAT1, GAT2, GAT3, and BGT1) genes have been identified; however, GAT1 and GAT3 are the ones that are highly expressed in the CNS. Depending on region and species, they may be expressed in neurons and/or in glia. There are two main glycine transporters, GlyT1 and GlyT2. GlyT1 is found predominantly on astrocytes and is present throughout the CNS. In contrast, GlyT2 is located on glycinergic nerve terminals and is largely restricted to the spinal cord, brainstem, and cerebellum.

Many of the neurotransmitters in this category may be familiar because they have roles outside the nervous system, often as hormones. Among the amines known to act as neurotransmitters are dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), serotonin (5-hydroxytryptamine [5-HT]), and histamine. Dopamine, norepinephrine, and epinephrine are catecholamines, and they share a common biosynthetic pathway that starts with the amino acid tyrosine. Tyrosine is converted to L-dopa by the enzyme tyrosine hydroxylase. L-Dopa is then converted to dopamine by dopa-decarboxylase. In dopaminergic neurons, the pathway stops here. In noradrenergic neurons, another enzyme, dopamine β-hydroxylase, converts dopamine to norepinephrine. Epinephrine is obtained by adding a methyl group to norepinephrine via phenylethanolamineN-methyl transferase. In serotoninergic neurons, serotonin is synthesized from the essential amino acid tryptophan. Tryptophan is first converted to 5-hydroxytryptophan by tryptophan 5-hydroxylase, which is then converted to serotonin by aromatic L-amino acid decarboxylase. Finally, in histaminergic neurons the conversion of histidine to histamine is catalyzed by histidine decarboxylase.

Removal of synaptically released biogenic amines is generally accomplished by reuptake into glia and neurons via transporters belonging to the Na+-Cl−–dependent transporter family. The catecholamines are then degraded by two enzymes, monoamine oxidase and catechol Omethyltransferase.

Atleastfivetransporters(EAAT1toEAAT5,inwhichEAAT standsforexcitatory amino acid transporter)thatcarryglutamateacrosstheplasmamembranehavebeenidentified.TheyareallpartoftheNa+-K+–dependentfamilyoftransporters.InwardmovementofeachglutamatemoleculeisdrivenbythecotransportofthreeNa+ ionsandoneH+ ionandthecountertransportofoneK+ ionoutofthecell(see

Fig.6.11B ).Inaddition,thetransporterhasCl− conductance,althoughpassageofCl− ionsisnotstoichiometricallylinkedtoglutamatetransport.Glutamatetransportersarefoundonbothneuronsandglia.However,thetransportersdifferintheirregionalandcellulardistributionandintheirpharmacologicalandbiophysicalproperties.Forexample,EAAT2isfoundongliaandisgenerallyresponsibleformorethan90%ofglutamateuptakefromtheextracellularspace.TheglutamatetakenupintoglialcellsbyEAAT2iseventuallyreturnedtothepresynapticterminalbytheglutamateglutaminecycle(see

Fig.6.11 ).Insideglialcells,glutamateisconvertedtoglutamine.Glutamineisthentransportedoutoftheglialcellandbackintothepresynapticterminal,whereitissubsequentlyconvertedbacktoglutamate.GlutamateinsidethepresynapticterminalispackagedintosynapticvesiclesbyasecondsetofglutamatetransportersknownasvGLUTs (vesicular glutamate transporters),whicharepresentinthemembraneofglutamatergicvesicles.TransportofglutamateintosynapticvesiclesbyvGLUTisdrivenbythecountertransportofH+ ions,theelectrochemicalgradientforwhichhavingbeenestablishedbyanH+-ATPaseinthevesiclemembrane.

Within the CNS, nerve cells that use biogenic amines as neurotransmitters are primarily found within one of a few brainstem nuclei, most of which project rather diffusely throughout large areas of the brain. Noradrenergic neurons are primarily found in the locus coeruleus and nucleus subcoeruleus, which are located near each other in the dorsal part of the rostral pons. The neurons of the locus coeruleus project throughout the entire brain. Targets of the nucleus subcoeruleus are more limited but still widespread and include the pons, medulla, and spinal cord. (Norepinephrine is also important in the peripheral nervous system because it is used by postganglionic sympathetic cells.) Serotoninergic fibers arise from a series of nuclei located at the midline of the brainstem, known as the raphe nuclei. Similar to the noradrenergic fibers, serotoninergic fibers are distributed throughout most of the brain and spinal cord. Dopaminergic fibers arise from two main brainstem regions: the substantia nigra pars compacta, which projects to the striatum, and the ventral tegmental area, which projects more widely to the neocortex and subcortical areas, including the nucleus accumbens. Histaminergic neurons are located within the tuberomammillary nucleus of the hypothalamus and project diffusely throughout the CNS. Finally, adrenergic neurons are relatively few in number when compared with the other biogenic amine transmitters. They have cell bodies localized to small cell groups in the rostral medulla. The largest group, termed C1, has projections to the locus coeruleus and down to the thoracic and lumbar levels of the spinal cord, where they terminate in the autonomic nuclei of the intermediolateral and intermediomedial cell columns. Thus, these neurons are important for autonomic functions, particularly vasomotor ones, such as control of arterial pressure.

The diffuse nature of the projection pattern of most of the amine systems is mirrored in their proposed functions. Activity in the different aminergic systems is believed to be important in setting global brain states. For example, these systems are involved in setting the level of arousal (sleep, waking), attention, and mood. Their involvement in pathways connected with the hypothalamus and other autonomic centers also indicates that they have important homeostatic functions. The role of dopamine in balancing the flow of activity through the basal ganglia pathways and how its loss leads to the motor symptoms observed in Parkinson’s disease are described in

ATP has the potential to act as a transmitter or cotransmitter at synapses in the peripheral and central nervous systems. ATP is found in all synaptic vesicles and thus is co-released during synaptic transmission. ATP has its own receptors, which like standard neurotransmitters, are coupled to ion channels, but it can also modify the action of other neurotransmitters with which it is co-released, including norepinephrine, serotonin, glutamate, dopamine, and GABA. Glial cells may also release ATP after certain types of stimulation. Once released, ATP is broken down by ATPases and 5-nucleotidase to adenosine, which can be taken up again by the presynaptic terminal.

Peptide neurotransmitters consist of chains of between 3 and about 40 amino acids. Studies of neuropeptides

Hyperactivityofdopaminergicsynapsesmaybeinvolvedinsomeformsofpsychosis.Chlorpromazine andrelatedantipsychoticdrugsinhibitdopaminereceptorsonpostsynapticmembranesandthusdiminishtheeffectsofdopaminereleasedfrompresynapticnerveterminals.Overdosesofsuchantipsychoticdrugscanproduceatemporaryparkinsonian-likestate.

focused on the hypothalamus for many years. However, it is now clear that neuropeptides are released by neurons and act on receptors throughout the CNS and thus are a fundamental mechanism of neurotransmission throughout the CNS. To date, more than 100 neuropeptides have been identified. They can be classified into several functional groups, as shown in

Box 6.1 , which lists some of the known neuropeptides. It is now clear that many neurons that release classic neurotransmitters also release neuropeptides. As detailed later, understanding the interaction between coexisting classic and peptide transmitters has become an important area of research. In addition to being co-released with another transmitter, neuropeptides can also function as the sole or primary neurotransmitter at a synapse.

In some ways neuropeptides are like the classic neurotransmitters: they are packaged into synaptic vesicles, their release is dependent on Ca++ , and they bind to specific receptors on target neurons. However, there are also significant differences, ones that have led to alternative names for the intercellular communication mediated by neuropeptides, such as nonsynaptic, parasynaptic, and volume transmission.

Table 6.1 summarizes some of these differences between classic and peptide neurotransmitters.

Unlike classic neurotransmitters, which are synthesized at the presynaptic terminal, neuropeptides are synthesized at the cell body and then transported to the terminal (see

Fig. 6.2 ). Neuropeptides are packaged into large electron-dense vesicles that are scattered throughout the presynaptic terminal rather than in small electron-lucent vesicles docked at the active zone, where small-molecule transmitters are stored. (In neurons that make multiple neuropeptides, the various peptides are co-stored in the same vesicles.) Neuropeptide receptors are not confined to the synaptic region, and in general, peptide action is not limited by reuptake mechanisms.

Each of these differences has functional implications. For example, the separate storage of peptide and nonpeptide transmitters immediately raises the question of whether the two transmitters are co-released or differentially released in response to particular stimulation patterns.

In fact, differential release of peptide and classic transmitters from the same cell has been demonstrated for several types of neurons and is probably a result of the differences in vesicle storage described earlier. Because of their proximity to the active zones, nonpeptide vesicles can be released rapidly (<1 msec) in response to single action potentials • BOX 6.1 Some Neuroactive Peptides as a result of localized influx of Ca++ . Thus, low-frequency stimulation of the cell causes just the release of nonpeptide transmitter. In contrast, with higher-frequency stimulation of the presynaptic neuron, there is a more global increase in [Ca++] throughout the nerve terminal that leads to release of neuropeptide as well as neurotransmitter.

When neuropeptides are co-released with other transmitters, they may act synergistically or antagonistically. For example, in the spinal cord, tachykinins and calcitonin gene–related peptide (CGRP) act synergistically with glutamate and with substance P to enhance the action of serotonin. Conversely, tachykinins and CGRP antagonize norepinephrine’s action at other synapses. The interactions, however, are not simply a one-to-one synergism or antagonism at a particular synapse because of the differing temporal and spatial profiles of the action of peptides versus classic transmitters. In particular, the slower release and lack of rapid reuptake mean that neuropeptides can act for long durations, diffuse over a region of brain tissue, and affect all cells in that region (that have the appropriate receptors) rather than just acting at the specific synapse at which it was released. In fact, studies have shown that there is often a spatial mismatch between the presynaptic terminals that contain a particular neuropeptide and the sites of the receptors for that peptide. In sum, peptides released from a particular synapse probably affect the local neuronal population as a whole, whereas the co-released classic transmitters act in more of a point-to-point manner.

Opiates are drugs derived from the juice of the opium poppy. Compounds that are not derived from the opium poppy but that exert direct effects by binding to opiate receptors are called opioids and form a clinically and functionally important class of neuropeptides. Operationally, opioids are defined as compounds whose effects are stereospecifically antagonized by a morphine derivative called naloxone.

The three major classes of endogenous opioid peptides in mammals are enkephalins, endorphins, and dynorphins. Enkephalins are the simplest opioids; they are pentapeptides. Dynorphins and endorphins are somewhat longer peptides that contain one or the other of the enkephalin sequences at their N-terminal ends.

Opioid peptides are widely distributed in neurons of the CNS and intrinsic neurons of the gastrointestinal tract. The endorphins are discretely localized in particular structures of the CNS, whereas the enkephalins and dynorphins are more widely distributed. Opioids inhibit neurons in the brain involved in the perception of pain. Indeed, opioid peptides are among the most potent analgesic (pain-relieving) compounds known, and opiates are used therapeutically as powerful analgesics. They exert their analgesic effect by binding to specific opiate receptors.

Substance P is a peptide consisting of 11 amino acids. It is present in specific neurons in the brain, in primary sensory neurons, and in plexus neurons in the wall of the gastrointestinal tract. The wall of the gastrointestinal tract is richly innervated with neurons that form networks or plexuses (see also ). The intrinsic plexuses of the gastrointestinal tract exert primary control over its motor and secretory activities. These enteric neurons contain many of the neuropeptides, including substance P, that are found in the brain and spinal column. Substance P is involved in pain transmission and has a powerful effect on smooth muscle.

Substance P is probably the transmitter used at synapses made by primary sensory neurons (their cell bodies are in the dorsal root ganglia) with spinal interneurons in the dorsal horn of the spinal column, and thus it is an example of a peptide acting as a primary transmitter at a synapse.

Enkephalins act to decrease the release of substance P at these synapses and thereby inhibit the pathway for pain sensation at the first synapse in the pathway.

This is the newest category of neurotransmitter to be defined and stretches the usual definition of synaptic transmission even further than neuropeptides do. Gas neurotransmitters are neither packaged into synaptic vesicles nor released by exocytosis. Instead, gas neurotransmitters are highly permeant and simply diffuse from synaptic terminals to neighboring cells after synthesis, their synthesis being triggered by depolarization of the nerve terminal (the influx of Ca++ activates synthetic enzymes). Moreover, there are no specific reuptake mechanisms, nor do they undergo enzymatic destruction, so their action appears to be ended by diffusion or binding to superoxide anions or various scavenger proteins. Both nitric oxide (NO) and carbon monoxide (CO) are examples of gaseous neurotransmitters. NO is a transmitter at synapses between inhibitory motor neurons of the enteric nervous system and gastrointestinal smooth muscle cells (see ). NO also functions as a neurotransmitter in the CNS. The enzyme NO synthase catalyzes the production of NO as a product of the oxidation of arginine to citrulline. This enzyme is stimulated by an increase in cytosolic [Ca++].

In addition to serving as a neurotransmitter, NO functions as a cellular signal transduction molecule both in neurons and in nonneuronal cells (e.g., vascular smooth muscle; see

Chapter 14).

One way that NO functions as a signal transduction molecule is by regulating guanylyl cyclase, the enzyme that produces cGMP from GTP. NO binds to a heme group in soluble guanylyl cyclase and potently stimulates the enzyme. Stimulation of this enzyme leads to an elevation in cGMP in the target cell. The cGMP can then influence multiple cellular processes.

The multitude of neurotransmitters used in the nervous system provides it with a specific and flexible interneuronal communications system. These characteristics are even further enhanced by the variety of receptors for each neurotransmitter. Receptors for a particular neurotransmitter were traditionally distinguished primarily by pharmacological differences in their sensitivity to particular agonists and antagonists. For example, acetylcholine receptors were split into muscarinic and nicotinic classes, depending on whether they bind muscarine or nicotine. Similarly, glutamate receptors were split into three main groups according to their sensitivity to the agonists NMDA, kainic acid, or AMPA. Though useful, this classification scheme has several limitations: some receptors fail to be activated by agonists, and it fails to disclose all the various receptor subtypes for a particular transmitter. Over the past 15 years or so, molecular biological approaches have been used to identify and sequence the receptor genes for many of the known neurotransmitters. It is thought that we now have a relatively complete catalog of the genes for these receptors. What this work has revealed is that there is a tremendous diversity of actual and potential receptor subtypes that are or could be used by the nervous system. Moreover, knowledge of the gene sequences has enabled an understanding of the relationship of different receptor proteins to each other and to other important proteins. This knowledge, combined with the results of biochemical, crystallographic, and other types of studies, has led to a much deeper understanding of the structural and functional workings of receptor proteins. In particular, various receptors can be grouped into families based on gene sequences, and members of each family share various structural and functional features.

Neurotransmitter receptors are members of one of two large groups or families of proteins: ligand-gated ion channels, also known as ionotropic receptors, and G protein– coupled receptors, also referred to as metabotropic receptors (

Fig. 6.12A–B ). Almost all classic neurotransmitters and neuropeptides have at least one metabotropic-type receptor. Many of the classic neurotransmitters also have at least one ionotropic receptor. Ionotropic receptors are protein complexes that both have an extracellular binding site for the transmitter and form an ion channel (pore) through the cell membrane. The receptor is made up of several protein subunits, usually three to five, each of which typically has a series of membrane-spanning domains, some of which contribute to the wall of the ion channel. Binding of the neurotransmitter alters (usually increases) the probability of the ion channel being in the open state and thus typically results in postsynaptic events that are rapid in both onset and decay, with a duration of several milliseconds. Ionotropic receptors underlie fast synaptic EPSPs and IPSPs, as described earlier.

Theionotropicreceptorscanbedividedintoseveralsuperfamilies.Membersofthecys-loopsuperfamilyhavepeptidesubunitsthathaveanN-terminalextracellulardomainthatcontainsaloopdelimitedbycysteineresidues.Thisfamilyincludestheionotropicreceptorsforacetylcholine,serotonin,GABA,andglycine.Inadditiontotheirfamily-definingcysteineloop,thesereceptorssharethefollowingcommonfeatures:theyarepentamers,witheachpeptidesubunithavingfourtransmembranedomains;theneurotransmitterbindstotheN-terminaldomain;andthesecondtransmembranedomainsarethoughttoformthewalloftheionpore.

IonotropicglutamateandATPreceptorsformtwootherionotropicreceptorsuperfamilies;thedetailsforeacharegiveninthelatercorrespondingsections.Transientreceptorpotential(TRP)channels,whichareimportantfortransductionofpainandthermalsensations,formyetanotherfamily(see 1. Neurotransmitter Neurotransmitter Ions binds 1. Neurotransmitter binds 2. Channel opens 5. Ions flow across membrane 4. Ion 3. Ions flow Ions across membrane 2. G protein is 3. G protein subunits or • Fig. 6.12 Neurotransmitter receptors. The basic structure and mechanism of action are shown for ligand-gated ion channels (ionotropic receptors) (A) and G protein–coupled (metabotropic) receptors (B). Detailed structures of cys-loop and glutamate ionotropic receptors are shown in C and D, respectively. Cys-loop receptors include ionotropic receptors for GABA, glycine, serotonin, and acetylcholine. Note the differing membrane topologies of the individual subunits of these two classes of receptors: four transmembrane domains for cys-loop receptors and three plus a pore loop for glutamate receptors. Pore loops form the internal wall of the glutamate channel, whereas transmembrane domain 2 forms the internal wall of cys-loop receptors. (A and B, From Purves D, Augustine GJ, Fitzpatrick D. Neuroscience. 2nd ed. Sunderland, MA: Sinauer Associates; 2001.)

Metabotropic receptors are not ion channels. Instead, they are protein monomers that have an extracellular binding site for a particular transmitter and an intracellular site for binding a G protein. Binding of the receptor leads to activation of a G protein, which is the first step in a signal transduction cascade that alters the function of an ion channel in the postsynaptic membrane. In contrast to ionotropic receptors, metabotropic receptors mediate post-synaptic phenomena that have a slow onset and that may persist from hundreds of milliseconds to minutes. Because of the various biochemical cascades they initiate, they have great potential to cause changes in the neuron beyond just generating a postsynaptic potential.

Acetylcholine receptors were originally classified on a pharmacological basis (being sensitive to nicotine or muscarine) into two major groups. This grouping corresponds to groupings based on structural and molecular biological studies. Nicotinic receptors are members of the ionotropic cys-loop family, and muscarinic receptors are part of the metabotropic family of receptor proteins.

The nicotinic receptors mediate synaptic transmission at the neuromuscular junction as described earlier; however, nicotinic receptors are also present within the CNS. The nicotinic receptor contains a relatively nonselective cationic channel, so binding of acetylcholine produces an EPSP. Being members of the cys-loop family, acetylcholine receptors are pentamers constructed from a series of subunit types called α, β, γ, δ, ε, some of which contain multiple members. At the neuromuscular junction the channel is constructed from 2α, β, δ, ε, whereas in the CNS the composition is typically 3α, 2β. Furthermore, the junctional receptors all use the α1 subunit, whereas centrally located receptors use one of the α subunits between α2 and α10. As noted, the differing subunits result in receptors with differing pharmacological sensitivities and channel kinetics and selectivity.

There are five known muscarinic subtypes of acetylcholine receptors (M1 to M5). All are metabotropic receptors; however, they are coupled to different G proteins and can thus have distinct effects on the cell. M1, M3, and M5 are coupled to pertussis toxin–insensitive G proteins, whereas M2 and M4 are coupled to pertussis toxin–sensitive G proteins. Each set of G proteins is coupled to different enzymes and second messenger pathways (see for details of these pathways).

Inhibitory Amino Acid Receptors: GABA and Glycine

As noted, the most common inhibitory synapses in the CNS use either glycine or GABA as their transmitter. Glycine-mediated inhibitory synapses are common in the spinal cord, whereas GABAergic synapses make up the majority of inhibitory synapses in the brain.

Both glycine and GABA have ionotropic receptors that are members of the cys-loop family, thus sharing a number of characteristics as already described. In addition, each of these receptors has a Cl− channel, which opens while the receptor portion is bound. Therefore, the probability of these channels opening and the average time a channel stays open are controlled by the concentration of the neurotransmitter for which the receptor is specific.

Glycine receptors are pentamers and may be heteromers of α and β subunits (3:2 ratio) or homomers. Interestingly, the molecular composition appears to be related to its cellular location, with heteromers located postsynaptically and homomers located extrasynaptically. The β subunit seems to bind to an intracellular scaffold protein called gephyrin that appears to help localize receptors to the postsynaptic site. The α subunit contains the glycine binding site, and there are four genes coding for distinct α subunits (and splice variants of each). Each variant results in a receptor having distinct conductance, kinetics, agonist and antagonist affinity, and modulatory sites. Intriguingly, subunit variants are differentially expressed during development and in different brain regions.

GABA has two separate ionotropic receptors (GABAA and GABAC) coded for by distinct sets of genes. Like glycine receptors, both control a Cl− channel. GABAA receptors are heteromers generated from seven classes of subunits, three of which have multiple members. The most common configuration is α1, β2, γ2 in a 2:2:1 stoichiometry, which may account for 80% of the receptors; however, many other heteromers are found in the brain. As with glycine, different subunits confer distinct properties on the receptor. For example, GABAA receptors are the targets of two major classes of drugs: benzodiazepines and barbiturates. Benzodiazepines (e.g., diazepam) are widely used antianxiety and relaxant drugs. Barbiturates are used as sedatives and anticonvulsants. Both classes of drugs bind to distinct sites on the α subunits of GABAA receptors and enhance opening of the receptors’ Cl− channels in response to GABA. The sedative and anticonvulsant actions of benzodiazepines appear to be mediated by receptors with the α1 subunit, whereas the anxiolytic effects reflect binding to receptors with the α2 subunit. GABAC receptors are structurally similar to GABAA receptors but have a distinct pharmacological profile (e.g., they are not affected by benzodiazepines) and are coded for by a separate set of genes (ρ1, ρ2, and ρ3).

The GABAB receptor is a metabotropic receptor. Binding of GABA to this receptor activates a heterotrimeric GTP-binding protein (G protein; see ), which leads to activation of K+ channels and hence hyperpolarization of the postsynaptic cell, as well as inhibition of Ca++ channels (when located presynaptically) and thus a reduction in release of transmitter.

Excitatory Amino Acid Receptors: Glutamate

Glutamate has both ionotropic and metabotropic receptors. Based on pharmacological properties and subunit composition, several distinct ionotropic receptor subtypes are recognized: AMPA, kainate, and NMDA. Overall, there are 18 known genes that code for glutamate subunits for the ionotropic glutamate receptors. The genes are divided into several families (AMPA, kainate, NMDA, and δ) that essentially correspond to the pharmacological subtypes of receptors. Each glutamate receptor is a tetramer. Thus, there is a certain correspondence between the genes and the receptor types that are formed. For example, AMPA receptors are formed from GluR1 to GluR4 subunits, kainate receptors require either KA1 or KA2 and GluR5 to GluR7 subunits, and NMDA receptors all have NR1 subunits plus some combination of NR2 and NR3 subunits. As was mentioned for the other receptors, the receptor properties vary with subunit composition. Ionotropic glutamate receptors are excitatory and contain a cationic-selective channel. Thus, all the channels are permeable to Na+ and K+ , but only a subset allow Ca++ to pass.

AMPA and kainate receptors behave as classic ligand-gated channels as already discussed; on binding of glutamate to the receptor, the channel opens and allows current to flow, thereby generating an EPSP. NMDA channels are different. First, they require binding of both glutamate and glycine to open. Second, they display voltage sensitivity as a result of Mg++ blockade of the channel. That is, at resting (or more negative) membrane potentials, a Mg++ ion blocks the entrance to the channel so that even when glutamate and glycine are bound, no current flows through the channel. However, if the cell is depolarized (either experimentally by injection of current through an electrode or by other EPSPs), the Mg++ block is relieved and current can flow through the channel. A further interesting feature of NMDA channels is that they are generally permeable to Ca++, which can act as a second messenger. The combination of voltage sensitivity and Ca++ permeability of the NMDA channels has led to hypotheses concerning their role in learning and memory-related functions (see

Eight genes coding for metabotropic glutamate receptors have been identified and classified into three groups. Group I receptors are found postsynaptically, whereas groups II and III are found presynaptically. These receptors generate slow EPSPs, but probably at least as importantly, they trigger second messenger cascades (see

Purines have two receptor families: an ionotropic (P2X) and a metabotropic (P2Y) family. There are seven identified P2X subunit types that form channels, and they represent their own superfamily of ligand-gated channels. Each subunit has only two transmembrane domains, with the loop between these two domains located extracellularly and containing the ATP binding site. The receptors are heterotrimers or homotrimers or hexamers. In general, these receptors form a cationic channel that is permeable to Na+ , K+ , and Ca++ . The distribution of subunits in the brain varies significantly, with some subunits having a widespread distribution (P2X2) and others being quite limited (P2X3 is present mostly on cells involved in pain-related pathways).

Metabotropic purine receptors are coded for by 10 genes, but only 6 are expressed in the human CNS. They have the typical features of G protein–coupled receptors and are known to activate K+ currents and modulate both NMDA and voltage-gated Ca++ currents. An interesting localization distinction between P2X and P2Y receptors is that although both are present on neurons, the latter dominates on astrocytes.

Finally, in addition to the P2X and P2Y receptors, which respond to ATP, there are adenosine receptors that respond to the adenosine that is released after the enzymatic breakdown of ATP. These receptors are located presynaptically and act to inhibit synaptic transmission by inhibiting influx of Ca++ .

Biogenic Amine Receptors: Serotonin, Dopamine, Noradrenaline, Adrenaline, Histamine

With the exception of one class of serotonin receptors (5-HT3) that are part of the cys-loop ionotropic family, receptors for the various biogenic amines are all metabotropic-type receptors. Thus, these neurotransmitters tend to act on relatively long time scales by generating slow synaptic potentials and by initiating second messenger cascades. Agonists and blockers of many of these receptors are important clinical tools for treating various neurological and psychiatric disorders. The role of different dopamine receptors in basal ganglia disorders will be covered in the motor systems (see

As is the case with the biogenic amines, receptors for the various peptides are essentially all of the metabotropic type and are coupled to G proteins that mediate effects via second messenger cascades. It is worth mentioning again that studies consistently show a mismatch between the locations of terminals containing a particular peptide and the receptors for it. Thus, these receptors are often activated by neurotransmitter diffusing through the extracellular space rather than at synapses. This implies that these receptors will experience much lower concentrations of agonist, and indeed, they are very sensitive to their agonists.

Unlike the other neurotransmitters that were covered, NO and CO do not bind to receptors. One way they do affect cell activity is to activate enzymes involved in second messenger cascades, such as guanylyl cyclase. In addition, NO has been shown to modify the activity of other proteins, such as NMDA receptors and the Na+,K+-ATPase pump, by nitrosylating them.

1.

Both electrical and chemical synapses are important means of cellular communication in the mammalian nervous system.

2.

Electrical synapses directly connect the cytosol of two neurons and allow rapid bidirectional current flow between neurons. They act as low-pass filters.

3.

Gap junctions are the morphological correlate of electrical synapses. Gap junctions contain channels formed by hemichannels called connexons. Connexons are formed by proteins called connexins.

4.

Standard chemical synaptic transmission involves the release of transmitter from a presynaptic terminal, diffusion of transmitter across a synaptic cleft, and binding of the transmitter to receptors on the apposed postsynaptic membrane.

5.

Entry of calcium into the presynaptic terminal triggers the release of neurotransmitter. Release of neurotransmitter is quantal, as first demonstrated by the recording of mEPPs at the frog neuromuscular junction.

6.

Transmitter is packaged into synaptic vesicles in the presynaptic terminal. The vesicles are the quantal elements. That is, the release of transmitter from one vesicle causes a mEPP at the neuromuscular junction or, equivalently, one mPSP at a central synapse.

7.

Many proteins are involved in priming, docking, and fusion of synaptic vesicles. Synaptotagmin is the Ca++ sensor for triggering vesicle fusion.

8.

Excitatory and inhibitory synapses increase or decrease, respectively, the probability that the postsynaptic neuron will spike.

9.

The reversal potential is the membrane potential at which net current flow through a ligand-gated channel reverses. Excitatory synapses generate depolarizing potentials (EPSPs) that have reversal potentials positive to the spike threshold, most often as a result of the opening of nonselective cation channels.

10.

Inhibitory synapses generate IPSPs that have reversal potentials more negative than the spike threshold but not necessarily negative to the resting potential. Inhibitory synapses can decrease spike probability by two mechanisms: hyperpolarization of the membrane and a decrease in the input resistance of the neuron, leading to a shunt of synaptic currents.

Alabi AA, Tsien RW. Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission. Annu Rev Physiol. 2013;75:393-422.

11.

The process by which a neuron decides to fire an action potential as a result of its inputs is referred to as synaptic integration. The summation of EPSPs and IPSPs can be highly nonlinear and depends on many factors, including the geometry of the dendritic tree, location of the synaptic inputs relative to the initial segment, and the passive (RC) and active membrane properties of the cell.

12.

The efficacy of synaptic transmission depends on the timing and frequency of action potentials in the presynaptic neuron. Facilitation, posttetanic potentiation, and long-term potentiation are examples of increased efficacy of synaptic transmission in response to previous multiple stimulations of a synapse. Long-term depression is an example of reduced efficacy resulting from previous activation of the synapse.

13.

The nervous system uses hundreds of neurotransmitters. Neurotransmitters can be subdivided into a few broad functional classes: small-molecule transmitters (acetylcholine, amino acids, biogenic amines, and purines), peptides, and gases (CO, NO). The action of a neurotransmitter depends on its postsynaptic receptors. Most nongaseous transmitters have both ionotropic and metabotropic receptors.

14.

Small-molecule transmitters act locally, mainly across a single synapse, and their duration of action is limited by reuptake and enzymatic degradation. Peptides can diffuse from their presynaptic release site and thus have the potential to affect all cells within a local region. Gaseous transmitters are free to diffuse from their release site.

15.

Ionotropic receptors contain an ion channel whose state (open versus closed) is gated by the binding of neurotransmitter to the receptor. Metabotropic receptors activate second messengers on binding neurotransmitter.

16.

Many synapses can release multiple types of transmitters, and which ones they release depends on the activity pattern of the terminal. Co-released transmitters may function independently or act synergistically or antagonistically.

Fain GL. Molecular and Cellular Physiology of Neurons. 2nd ed. Cambridge, MA: Harvard University Press; 2014.

Hille B. Ion Channels of Excitable Membranes. 3rd ed. Sunderland, MA: Sinauer Associates; 2001.

Upon completion of this chapter the student should be able to answer the following questions: 1.

What are the major modalities of somatosensory information, and what are the corresponding pathways that convey each from the periphery to the primary somatosensory cortex?

2.

What body regions and categories of information are the exteroceptive, proprioceptive, and enteroceptive divisions of the somatosensory system associated with?

3.

What are the main receptors for fine/discriminatory touch sensations?

4.

What types of somatosensory information does the cerebellum receive?

5.

What are the main receptors for pain and temperature touch sensations?

6.

What is the phenomenon of referred pain?

7.

What proteins are involved in transducing different categories of somatosensory information?

8.

How do descending pathways act to regulate the flow of activity in ascending somatosensory pathways?

he somatosensory system provides information to the central nervous system (CNS) about the state of the body and its contact with the world. It does so by using a variety of sensory receptors that transduce mechanical (pressure, stretch, and vibrations) and thermal energies into electrical signals. These electrical signals are called generator or receptor potentials and occur in the distal ends of axons of first-order somatosensory neurons, where they trigger action potential trains that reflect information about the characteristics of the stimulus. The cell bodies of these neurons are located in dorsal root (

Fig. 7.1A

Fig.

4.8 ) and cranial nerve ganglia.

Each ganglion cell gives off an axon that after a short distance divides into a peripheral process and a central process. The peripheral processes of the ganglion cells coalesce to form peripheral nerves. A purely sensory nerve will have only axons from such ganglion cells; however, mixed nerves, which innervate muscles, will contain both afferent (sensory) fibers and efferent (motor) fibers. At the target organ the peripheral process of an afferent axon divides repeatedly, with each terminal branch ending as a sensory receptor. In most cases the free nerve ending by itself forms a functional receptor, but in some the nerve ending is encapsulated by accessory cells and the entire structure (axon terminal plus accessory cells) forms the receptor.

The central axonal process of the ganglion cell either enters the spinal cord via a dorsal root or enters the brainstem via a cranial nerve. A central process typically gives rise to numerous branches that may synapse with a variety of cell types, including second-order neurons of the somatosensory pathways. The terminal location of these central branches varies depending on the type of information being transmitted. Some terminate at or near the segmental level of entry, whereas others project to brainstem nuclei.

Second-order neurons that are part of the pathway for the perception of somatosensory information project to specific thalamic nuclei where the third-order neurons reside. These neurons in turn project to the primary somatosensory cortex (S-I). Within the cortex, somatosensory information is processed in S-I and in numerous higher-order cortical areas. Somatosensory information is also transmitted by other second-order neurons to the cerebellum for use in its motor coordination function.

The organization of the somatosensory system is quite distinct from that of the other senses, which has both experimental and clinical implications. In particular, other sensory systems have their receptors localized to a single organ, where they are present at high density (e.g., the eye for the visual system). In contrast, somatosensory receptors are distributed throughout the body (and head).

Subdivisions of the Somatosensory System

The somatosensory system receives three broad categories of information based on the distribution of its receptors. Its exteroceptive division is responsible for providing information about contact of the skin with objects in the external world, and a variety of cutaneous mechanoceptive, nociceptive (pain), and thermal receptors are used for this purpose. Understanding this division will be the main focus of this chapter. The proprioceptive component provides information about body and limb position and movement and relies primarily on receptors found in joints, muscles, and tendons. The ascending central pathways that originate

CHAPTER 7  The Somatosensory System 109

Pain, temperature, crude touch 7 • Fig. 7.1 Ascending somatosensory pathways from the body. A, First-, second-, and third-order neurons are shown for the two main pathways conveying cutaneous information from the body to the cerebral cortex: the dorsal column/medial lemniscal and the spinothalamic pathways. Note that the axon of the second-order neuron crosses the midline in both cases, so sensory information from one side of the body is transmitted to the opposite side of the brain, but the levels in the neuraxis at which this takes place are distinct for each pathway. Homologous central pathways for the head originate in the trigeminal nucleus and are described in text, but they are not illustrated for clarity. B, Major spinocerebellar pathways carrying tactile and proprioceptive information to the cerebellum from the upper and lower parts of the body. Again, pathways from the head originate in the trigeminal nuclei but are not shown for clarity. A midsagittal view of the nervous system shows the levels of the spinal and brainstem cross sections in panels A and B.

with them and that underlie conscious and unconscious proprioceptive functions will be covered in this chapter. However, because these receptors also initiate pathways that are intimately involved in the control of movement, they will be discussed again in . Finally, the enteroceptive division has receptors for monitoring the internal state of the body and includes mechanoreceptors that detect distention of the gut or fullness of the bladder. Aspects of enteroceptive division are also covered in as they are related to autonomic functions.

The somatosensory pathways can also be classified by the type of information they carry. Two broad functional categories are recognized, each of which subsumes several somatosensory submodalities. Fine discriminatory touch sensations include light touch, pressure, vibration, flutter (low-frequency vibration), and stretch or tension. The second major functional group of sensations is that of pain and temperature. Submodalities here include both noxious and innocuous cold and warm sensations and mechanical and chemical pain. Itch is also closely related to pain and appears to be carried by particular fibers associated with the pain system.

Thesensoryfunctionsofvariouscutaneoussensoryreceptorshavebeenstudiedinhumansubjectswithatechniqueknownasmicroneurography, inwhichafinemetalmicroelectrodeisinsertedintoanervetrunkinthearmorlegtorecordtheactionpotentialsfromsinglesensoryaxons.Whenarecordingcanbemadefromasinglesensoryaxon,thereceptivefieldofthefiberismapped.Mostofthevarioustypesofsensoryreceptorsthathavebeenstudiedinexperimentalanimalshavealsobeenfoundinhumanswiththistechnique.

Afterthereceptivefieldofasensoryaxonhasbeencharacterized,theelectrodecanbeusedtostimulatethesamesensoryaxon.Intheseexperimentsthesubjectisaskedtolocatetheperceivedreceptivefieldofthesensoryaxon,whichturnsouttobeidenticaltothemappedreceptivefield.

Of great importance experimentally, the afferent fibers that convey these somatosensory submodalities to the CNS are different sizes. Recall that the compound action potential recorded from a peripheral nerve (see 5.1 ) consists of a series of peaks, thus implying that the diameters of axons in a nerve are grouped rather than being uniformly distributed. Information about tactile sensations is carried primarily by large-diameter myelinated fibers in the Aβ class, whereas pain and temperature information travels via small-diameter, lightly myelinated (Aδ) and unmyelinated (C) fibers. It is possible to block or stimulate selectively a class of axons of particular size, thereby allowing study of the different somatosensory submodalities in isolation.

Innervation of the Skin

The skin is an important sensory organ and not surprisingly is richly innervated with a variety of afferents. We first consider the afferent types related to fine or discriminatory touch sensations. These afferents are related to what are called low-threshold mechanoreceptors. Nociceptor and thermoceptor innervation will be considered separately in a later section of this chapter.

To study the responsiveness of tactile receptors, a small-diameter rod or wire is used to press on a localized region of skin. With this technique, two basic types of responses may be seen when recording sensory afferent fibers: fast-adapting (FA) and slow-adapting (SA) responses (

Fig. 7.2). They are present in similar quantities. FA fibers will show a short burst of action potentials when the rod first pushes down on the skin, but then they will cease firing despite continued application of the rod. They may also burst at the cessation of the stimulus (i.e., when the rod is lifted off). In contrast, SA units will start firing action potentials (or increase their firing rate) at the onset of the stimulus and continue to fire until the stimulus ends.

Both the FA and SA afferent classes can be subdivided on the basis of other aspects of their receptive fields, where receptive field is defined as the region of skin from which stimuli can evoke a response (i.e., change the firing of the afferent axon). Type 1 units have small receptive fields with well-defined borders. Particularly for glabrous skin (i.e., hairless skin, such as on the palms of the hands and soles of the feet), the receptive field has a circular or ovoid shape within which there is relatively uniform and high sensitivity to stimuli that decreases sharply at the border (

Fig. 7.3).

Type 1 units, particularly SA1 units, respond best to edges. That is, a larger response is elicited from them when the edge of a stimulus cuts through their receptive field than when the entire receptive field is indented by the stimulus.

Type 2 units have wider receptive fields with poorly defined borders and only a single point of maximal sensitivity, from which there is a gradual reduction in sensitivity with distance (see

Fig. 7.3 ). For comparison a type 1 unit’s receptive field typically will cover approximately four papillary ridges in the fingertip, whereas a type 2 unit will have a receptive field that covers most or all of a finger.

Thus, four main classes of low-threshold mechanosensitive afferents have been identified physiologically (FA1, FA2, SA1, and SA2). Peripherally these axons may terminate either as free nerve endings, associated with a hair follicle, or within a specialized receptor structure made up of supporting cells.

For glabrous skin the four afferent classes have been associated with four specific types of histologically identified receptor structures whose locations and physical

CHAPTER 7 The Somatosensory System •Fig. 7.2 Cutaneousmechanoreceptorsandtheresponsepatternsofassociatedafferentfibers.A,Schematicviewsofglabrous(hairless)andhairyskinshowingthearrangementofthevariousmajormechanoreceptors.B,Firingpatternsofthedifferentcutaneouslow-thresholdmechanosensitiveafferentfibersthatinnervatethevariousencapsulatedreceptorsoftheskin.(TracesinBarebasedondatafromJohanssonRS,VallboÅB.Trends Neurosci 1983;6:27.) •Fig. 7.3 Receptivefieldcharacteristicsfortype1andtype2sensoryafferents.Plotsinthetoprowshowthethresholdlevelofforceneededtoevokearesponseasafunctionofthedistanceacrossthereceptivefield.Receptivefieldsizeisshownonthehandbeloweachplot.(DatafromJohanssonRS,VallboÅB.Trends Neurosci 1983;6:27.) characteristics help explain the firing properties of these sensory afferents. FA1 afferents terminate in Meissner’s corpuscles, whereas SA1 afferents terminate in Merkel’s disks. In both cases the receptor is located relatively superficially, either in the basal epidermis (Merkel) or just below the epidermis (Meissner) (see

Fig. 7.2 ). These receptors are small and oriented to detect stimuli pressing down on the skin surface just above them, thus allowing SA1 and FA1 afferents to have small receptive fields. For glabrous skin, SA2 afferents terminate in Ruffini endings and FA2 afferents end in Pacinian corpuscles. Both of these receptors lie deeper in the dermis and connective tissue and therefore are sensitive to stimuli applied over much larger territory. The capsules of both Pacinian and Meissner receptors act to filter out slowly changing or steady stimuli, thus making these afferents selectively sensitive to changing stimuli.

For hairy skin, the relationship between receptors and afferent classes is similar to that of glabrous skin. SA1 and SA2 fibers connect to Merkel and Ruffini endings, the same as for glabrous skin. Pacinian corpuscles also underlie the properties of FA2 afferents; however, they are not found in hairy skin but instead are located in deep tissues surrounding muscles and blood vessels. There is not an exact analogue to the FA1 afferents. Rather there are hair units, which are afferents whose free endings wrap around hair follicles (see

Fig. 7.2 ). Each such hair unit will connect with about 20 hairs to produce a large ovoid or irregularly shaped receptive field. These units are extremely sensitive to movement of even a single hair. There are also field units that respond to touch of the skin, but unlike FA1 units, they have large receptive fields.

Several psychophysical and neural coding questions can be related to the receptive field properties and sensitivities of the various categories of afferents. For example, is the threshold of perception of tactile stimuli due to the sensitivity of the peripheral receptors or to central processes? In fact, by using microneurography it is possible to show that a single spike in an FA1 afferent from the finger can be perceived, thus indicating that the receptors limit the sensitivity; however, for other skin regions, perception is more dependent on central factors such as attention.

An important behavioral and clinical measure of somatosensory function is spatial acuity or two-point discrimination. Clinically a doctor will apply two needle-like points simultaneously to the skin of a patient. The patient will generally perceive the points as two distinct stimuli as long as they are farther apart than some threshold distance, which varies across the body. The best discrimination (shortest threshold distance) is at the fingertips. Type 1 units underlie spatial acuity, which is not surprising given the smaller receptive fields of type 1 units than type 2 units. Moreover, the threshold distance for a region of skin is most closely related to its density of type 1 units because these units have similarly sized receptive fields throughout the glabrous skin, but their density falls off from fingertip to palm to forearm, and this fall-off correlates with the rise in threshold distance. Note that this variation in innervation density also matches the overall sensitivity of different skin regions to cutaneous stimuli.

The relationship of the firing rates in the various afferent classes to perceived stimulus quality is another important issue that has been addressed with microneurographic techniques. When a single SA fiber is stimulated with brief current pulses such that each pulse triggers a spike, a sensation of steady pressure is felt at the receptive field area of that fiber. As pulse frequency is increased a concurrent increase in pressure is perceived. Thus the firing rate in SA fibers codes for the force of the tactile stimulus. As another example, when an FA fiber is repetitively stimulated, a sensation of tapping results first, and as the frequency of the stimulus is increased, the sensation turns to one of vibration. Interestingly, in neither case does the stimulus change its qualitative character—for example, to a feeling of pain—so long as the stimulus activates only a particular fiber class. This is evidence that pain is a distinct submodality that uses a set of fibers distinct from those used by low-threshold mechanoreceptors.

These findings illustrate an important principle of sensory systems called labeled line. The idea is that the quality (i.e., modality) of a particular sensation results from the fact that it is conveyed to the CNS by a specific set of afferents that have a distinct set of targets in the nervous system. Alterations in activity in these afferents will therefore change only quantitative aspects of the sensation. As will be seen in more detail later, the various somatosensory submodalities (i.e., information arising from FA and SA mechanoreceptors, proprioceptors, and nociceptors) appear to use relatively separate dedicated cell populations even at relatively high levels of the CNS such as the thalamus and primary somatosensory cortex.

Innervation of the Body

Axons of the peripheral nervous system (PNS) enter or leave the CNS through the spinal roots (or through cranial nerves). The dorsal root of a given spinal segment is composed entirely of the central processes of its associated dorsal root ganglion cells. The ventral root consists chiefly of motor axons, including α and γ motor neuron axons (see ), and at certain segmental levels, autonomic preganglionic axons (see

CHAPTER 7 The Somatosensory System

The pattern of innervation is determined during embryo-logical development. In adults a given dorsal root ganglion supplies a specific cutaneous region called a dermatome. Many dermatomes become distorted during development, chiefly because of rotation of the upper and lower extremities as they are formed, but also because humans maintain an upright posture. However, the sequence of dermatomes can readily be understood if depicted on the body of a person in a quadrupedal position (

Fig. 7.4).

Although a dermatome receives its densest innervation from the corresponding spinal cord segment, collaterals of afferent fibers from the adjacent spinal segments also supply the dermatome. Thus transection of a single dorsal root causes little sensory loss in the corresponding dermatome. Anesthesia of any given dermatome requires interruption of several adjacent dorsal roots.

Acommondiseasethatillustratesthedermatomalorganizationofthedorsalrootsisshingles. Shinglesistheresultofreactivationoftheherpeszostervirus,whichtypicallycauseschickenpoxduringtheinitialinfection.Duringtheinitialinfectionthevirusinfectsdorsalroot(andcranialnerve)ganglioncells,whereitcanremainlatentforyearstodecades.Whenthevirusreactivates,thecellsofthatparticulardorsalrootganglionbecomeinfected,andthevirustravelsalongtheperipheralaxonbranchesandgivesrisetoapainfuloritchyrashthatisconfinedtoonesideofthebody(endsatthemidline)inadermatomal,belt-likedistributionortothedistributionofacranialnerve.

Within the dorsal roots, fibers are not randomly distributed. Rather the large myelinated primary afferent fibers assume a medial position in the dorsal root, whereas the small myelinated and unmyelinated fibers are more lateral. The large medially placed afferent fibers enter the dorsal column, where they bifurcate into rostrally and caudally directed branches. These branches give off collaterals that terminate in several neighboring segments. The rostral branch also ascends to the medulla as part of the dorsal column–medial lemniscus pathway. The axonal branches that terminate locally in the spinal cord gray matter transmit sensory information to neurons in the dorsal horn and also provide the afferent limb of reflex pathways (see

Chapter 9).

Innervation of the Face

The arrangement of primary afferent fibers that supply the face is comparable to that of fibers that supply the body and is provided for primarily by fibers of the trigeminal nerve. Peripheral processes of neurons in the trigeminal ganglion (also called the gasserian or semilunar ganglion) pass through the ophthalmic, maxillary, and mandibular divisions of the trigeminal nerve to innervate dermatome-like regions of the face. These fibers carry both tactile information and pain and temperature information. The trigeminal nerve •Fig. 7.4 A,Dermatomesrepresentedonadrawingofapersonassumingaquadrupedalposition.NotenerveC1generallyhaslittleornosensorycomponent,andtheunlabeledportionoftheheadandthefaceareinnervatedbysensoryfibersofthecranialnerves,primarilythetrigeminalnerve.B,SagittalviewofthespinalcordshowingtheoriginofnervescorrespondingtoeachofthedermatomesshowninA.

Thetrigeminalnuclearcomplexconsistsoffourmaindivisions,threeofwhicharesensory.Thethreesensorydivisions(fromrostraltocaudal)arethemesencephalic, chief (ormain)sensory, anddescending (orspinal)trigeminal nuclei. Thelattertwoaretypicalsensorynucleiinthatthecellbodiescontainedinthemaresecond-orderneurons.Themesencephalicnucleusactuallycontainsfirst-orderneuronsandthusisanalogoustoadorsalrootganglion.Thefourthdivisionofthetrigeminalcomplexisthemotornucleusofthetrigeminalnerve,whosemotorneuronsprojecttoskeletalmusclesoftheheadviathetrigeminalnerve(see

Fig.4.6D–E also innervates the teeth, the oral and nasal cavities, and the cranial dura mater.

The central processes of trigeminal ganglion cells enter the brainstem at the midpontine level, which also corresponds to the level of the chief sensory trigeminal nucleus (nucleus of cranial nerve V). Some axons terminate in this nucleus (primarily large-caliber axons carrying the information needed for fine discriminative touch), whereas others (intermediateand small-caliber axons that carry information about touch as well as pain and temperature) form the descending trigeminal tract, which descends through the medulla just lateral to the descending trigeminal nucleus. As the tract descends, axons peel off and synapse in this nucleus.

Proprioceptive information is also conveyed via the trigeminal nerve; however, in this unique case the cell bodies of the first-order fibers are located within the CNS in the mesencephalic portion of the trigeminal nucleus. The central processes of these neurons terminate in the motor trigeminal nucleus (to subserve segmental reflexes equivalent to the segmental spinal cord reflexes [see ]), the reticular formation, and the chief sensory trigeminal nucleus.

As may already be clear, information related to the different somatosensory submodalities travels to a large extent into the CNS via distinct sets of axons and targets different structures in the spinal cord and brainstem. Within the CNS this segregation continues as the information travels via separate pathways up the spinal cord and brainstem. For example, from the body, fine discriminatory touch information is conveyed by the dorsal column–medial lemniscus pathway, whereas pain, temperature, and crude touch information is conveyed by the anterolateral system.

Proprioceptive information is transmitted by yet another route that partially overlaps with the dorsal column–medial lemniscal pathway. Note, however, that this functional segregation is not absolute, so, for example, there can be some recovery of discriminative touch ability after a lesion of the dorsal columns. The anterolateral system will be discussed in the section on pain because it is the critical pathway for that information. Here, the central pathways

CHAPTER 7 The Somatosensory System for discriminatory touch and proprioception are considered in detail.

This pathway is shown in its entirety in

Fig. 7.1A.

The dorsal columns are formed by ascending branches of the large myelinated axons of dorsal root ganglion cells (the first-order neurons). These axons enter at each spinal segmental level and travel rostrally up to the caudal medulla to synapse in one of the dorsal column nuclei: the nucleus gracilis, which receives information from the lower part of the body and leg, and the nucleus cuneatus, which receives information from the upper part of the body and arm. Note that across the dorsal columns and across the dorsal column nuclei there is a somatotopic representation of the body, with the legs represented most medially, followed by the trunk and then the upper limb. This somatotopy is a consequence of newly entering afferents being added to the lateral border of the dorsal funiculus as the spinal cord is ascended. Such somatotopic maps are present at all levels in the somatosensory system, at least through the primary sensory cortices.

The dorsal column nuclei are located in the medulla and contain the second-order neurons of the pathway for discriminatory touch sensation. These cells respond similarly to the primary afferent fibers that synapse on them (see the earlier description of afferent types). The main differences between the responses of dorsal column neurons and primary afferent neurons are as follows: (1) dorsal column neurons have larger receptive fields because multiple primary afferent fibers synapse on a given dorsal column neuron, (2) dorsal column neurons sometimes respond to more than one class of sensory receptor because of the convergence of several different types of primary afferent fibers on the second-order neurons, and (3) dorsal column neurons often have inhibitory receptive fields that are mediated through local interneurons.

The axons of dorsal column nuclear projection neurons exit the nuclei and are referred to as the internal arcuate fibers as they sweep ventrally and then medially to cross the midline at the same medullary level as the nuclei. Immediately after crossing the midline, these fibers form the medial lemniscus (see

Fig. 4.6C–E ), which projects rostrally to the thalamus. Knowledge of the level of this decussation is clinically important because damage to the dorsal column–medial lemniscal pathway below this level, which includes all of the spinal cord, will produce loss of fine somatosensory discriminatory abilities on the same, or ipsilateral, side of the lesion, whereas lesions above this level will produce contralateral deficits. Moreover, because there is a clear somatotopic arrangement of fibers in the medial lemniscus, localized lesions cause selective loss of fine-touch sensations limited to specific body regions.

The third-order neurons of the pathway are located in the ventral posterior lateral (VPL) nucleus of the thalamus and project to somatosensory areas of the cerebral cortex (

Fig. 7.5 •Fig. 7.5 Diagramofconnectionsfromthesomatosensoryreceivingnucleiofthethalamustothesomatosensorycortexoftheparietallobe.Notetheparallelflowofdifferenttypesofsomatosensoryinformationthroughthethalamusandontothecortex.CS,centralsulcus;S1andS2areprimaryandsecondarysomatosensoryareas,respectively.Note:collectively,areas3a,3b,1,and2arereferredtoasS1.

The dorsal column–medial lemniscus pathway conveys information about fine-touch and vibratory sensations. This information is critical for many of the discriminatory tactile abilities we have. For example, spatial acuity is lowered by damage to this pathway, and the ability to identify objects by their shape and texture can be lost by damage to this pathway. Clinically, one may test for impaired graphesthesia, or the ability to recognize letters or numbers traced on the skin, or for loss of the ability to tell the direction of a line drawn across the skin. Importantly, some tactile function remains even after complete loss of the dorsal columns, and awareness and localization of nonnoxious tactile stimuli can still occur. Thus at least some of the information carried by the dorsal column pathway is also conveyed by additional ascending pathways. In contrast to the severe deficits in discriminatory touch sensation, cutaneous pain and temperature sensations are unaffected by lesions of the dorsal columns. However, visceral pain is substantially diminished by damage to the dorsal columns.

Trigeminal Pathway for Fine-Touch Sensation From the Face

Primary afferent fibers that supply the face, teeth, oral and nasal cavities, and cranial meninges synapse in several brainstem nuclei, including the main sensory nucleus and the descending nucleus of the trigeminal nerve.

The pathway through the main sensory nucleus resembles the dorsal column–medial lemniscus pathway. This sensory nucleus relays tactile information to the contralateral ventral posterior medial (VPM) thalamic nucleus by way of the trigeminothalamic tract. Third-order neurons in the VPM nucleus project to the facial area of the somatosensory cortex.

Proprioceptors provide information about the positions and movement of parts of the body. In addition to being used for local reflexes (see

Chapter 9), this information has two main targets, the cerebellum and the cerebral cortex. The cerebellum uses this information for its motor coordination functions. The information sent to the cerebral cortex is the basis for conscious awareness of our body parts (e.g., position of our hand), which is referred to as kinesthesia.

The major pathways by which somatosensory information is brought to the cerebellum are shown in

Fig. 7.1B . These pathways carry both cutaneous and proprioceptive information to the cerebellum. For the trunk and lower limb, the pathway starts with dorsal root ganglion cells whose axons synapse in Clarke’s column (nucleus dorsalis). The cells of Clarke’s column send their axons into the ipsilateral lateral funiculus to form the dorsal spinocerebellar tract, which enters the cerebellum via the inferior cerebellar peduncle. The ventral spinocerebellar tract also provides somatosensory input from the lower limb to the cerebellum. Note the double decussation of the ventral spinocerebellar pathway (one decussation at the spinal cord levels and a second one in the cerebellar white matter). This double crossing highlights the general rule that each half of the cerebellum is functionally related to the ipsilateral side of the body.

To provide proprioceptive information from the lower limb to the cerebral cortex, the main axons of the dorsal spinocerebellar tract give off a branch in the medulla that terminates in nucleus z, which is just rostral to the nucleus gracilis. The axons of cells from nucleus z then form part of the internal arcuate fibers and medial lemniscus and ascend to the VPL nucleus of the thalamus.

The ascending somatosensory pathways to the cerebellum for the upper limb are simpler than those from the lower limb (see

Fig. 7.1B ). The route to the cerebellum starts with dorsal root ganglion fibers from the cervical spinal levels that ascend in the cuneate fasciculus to the external cuneate nucleus. The axons of the external cuneate nucleus then form the cuneocerebellar tract, which enters the cerebellum via its inferior peduncle.

The route to the cerebral cortex for proprioceptive information from the upper limb is identical to that for discriminative touch: the dorsal column–medial lemniscal pathway, with a synapse in the cuneate nucleus and then in the VPL nucleus of the thalamus.

For the head, proprioceptive input is carried by cells of the mesencephalic nucleus of the trigeminal nerve. Recall that the neurons in this nucleus are actually the cell bodies of the primary afferents that innervate stretch receptors in the muscles of mastication (muscles that move the jaw) and in other muscles of the head. The central processes of these neurons project to the trigeminal motor nucleus for local reflexes or to the nearby reticular formation. Axons from these reticular formation neurons join the trigeminothalamic tract, which terminates in the VPM of the thalamus. There are also trigeminocerebellar pathways for conveying somatosensory (tactile and proprioceptive) information from the head to the cerebellum.

The ventroposterior nuclear complex of the thalamus represents the main termination site for ascending somatosensory information in the diencephalon. It consists of two major nuclei, the VPL and VPM, and a smaller nucleus called the ventral posterior inferior (VPI) (see

Fig. 7.5 ). The medial lemniscus forms the main input to the VPL nucleus, and the equivalent trigeminothalamic tract from the main sensory nucleus of the trigeminal nerve forms the main input to the VPM nucleus. These nuclei also receive input conveying pain and temperature information from the spinothalamic or equivalent trigeminothalamic tracts, respectively. The VPI nucleus receives input from the spinothalamic tract. In addition, the spinothalamic tract terminates in parts of the posterior nuclear complex and several other thalamic nuclei.

Single-unit recordings from the ventroposterior complex of nuclei have shown that the responses of many of the neurons in these nuclei to stimuli resemble those of firstand second-order neurons in the ascending tracts. The receptive fields of thalamic cells are small but somewhat larger than those of primary afferent fibers. Moreover, the responses may be dominated by a particular type of sensory receptor. For example, VPL and VPM nuclei have cells whose receptive fields typically reflect input either from one type of cutaneous receptor (FA or SA) or from proprioceptive receptors, as expected from their medial lemniscal input. In contrast, cells of the VPI and posterior nuclei show responses to activation of nociceptors, the main input to the spinothalamic pathway.

Thalamic neurons often have inhibitory as well as excitatory receptive fields. The inhibition may actually take place in the dorsal column nuclei or in the dorsal horn of the spinal cord. However, inhibitory circuits are also situated within the thalamus. For example, the VPL and VPM nuclei contain GABAergic inhibitory interneurons (in primates but not in rodents), and GABAergic inhibitory interneurons in the reticular nucleus of the thalamus project into the VPL and VPM nuclei. (The reticular nucleus of the thalamus is a thin shell of neurons that surrounds much of the thalamus. Thalamocortical and corticothalamic neurons both send axon collaterals to the reticular nucleus, whose neurons then respectively complete feedback and/ or feed-forward inhibitory circuits with neurons in other thalamic nuclei.)

One difference between neurons in the VPL and VPM nuclei and sensory neurons at lower levels of the somatosensory system is that thalamic neuron excitability depends on the stage of the sleep/wake cycle and on the presence or absence of anesthesia. During a state of drowsiness or during barbiturate anesthesia, thalamic neurons tend to undergo an alternating sequence of excitatory and inhibitory postsynaptic potentials. The alternating bursts of discharges in turn intermittently excite neurons in the cerebral cortex. Such patterns of excitation and inhibition result in an α rhythm or in spindling on the electroencephalogram. This alternation of excitatory and inhibitory postsynaptic potentials during these two states may reflect the level of excitation of thalamic neurons by excitatory amino acids that act at NMDA and non-NMDA receptors. It may also reflect inhibition of the thalamic neurons by recurrent pathways through the reticular nucleus.

Thalamic neuron receptive fields are on the side of the body contralateral to the neuron, and the receptive field locations vary systematically across the ventroposterior nuclear complex. That is, the VPL and VPM nuclei are somatotopically organized such that the lower limb is represented most laterally and the upper limb most medially in the VPL nucleus, and the head is represented even more medially in the VPM nucleus. Moreover, the fact that thalamic neurons often receive input from only one class of receptor suggests that there are multiple somatotopic maps laid out across the ventroposterior nuclear complex. That is, there appear to be separate somatotopic maps for SA, FA, and proprioceptive and pain sensations laid out across the ventroposterior nuclear complex.

These maps are not randomly interspersed. As already mentioned, pain sensation is largely mapped across the VPI nucleus. In addition, the cutaneous receptors appear to drive cells located in a central “core” region of the VPL-VPM complex, whereas proprioceptive information is directed to cells that form a “shell” (VPS) around this core. This parallel flow of information into thalamus and then onto the cortex is diagramed in

Fig. 7.5

The spinothalamic tract also projects to other thalamic regions, including the posterior nucleus and the central lateral nucleus of the intralaminar complex of the thalamus. The intralaminar nuclei of the thalamus are not somatotopically organized, and they project diffusely to the cerebral cortex as well as to the basal ganglia (see ). The projection of the central lateral nucleus to the S-I cortex may be involved in arousal of this part of the cortex and in selective attention.

Third-order sensory neurons in the thalamus project to the somatosensory cortex. The details of this projection pattern are shown in

Fig. 7.5 . The main somatosensory receiving

CHAPTER 7 The Somatosensory System areas of the cortex are called the S-I and S-II areas. The S-I cortex (or primary somatosensory cortex) is located on the postcentral gyrus, and the S-II cortex (secondary somatosensory cortex) is in the superior bank of the lateral fissure (see

Fig. 7.5

As previously discussed, the S-I cortex, like the somatosensory thalamus, has a somatotopic organization. The S-II cortex also contains a somatotopic map, as do several other less understood areas of the cortex. In the S-I cortex the face is represented in the lateral part of the postcentral gyrus, above the lateral fissure. The hand and the rest of the upper extremity are represented in the dorsolateral part of the postcentral gyrus, and the lower extremity on the medial surface of the hemisphere. A map of the surface of the body and face of a human on the postcentral gyrus is called a sensory homunculus. The map is distorted because the volume of neural tissue devoted to a body region is proportional to the density of its innervation. Thus in humans, the perioral area, the thumb, and other digits take up a disproportionately large expanse of cortex relative to their size.

The sensory homunculus is an expression of place coding of somatosensory information. A locus in the S-I cortex encodes the location of a somatosensory stimulus on the surface of the body or face. For example, the brain knows that a certain part of the body has been stimulated because certain neurons in the postcentral gyrus are activated.

The S-I cortex has several morphological and functional subdivisions, and each subdivision has a somatotopic map. These subdivisions were originally described by Brodmann, and they were based on the arrangements of neurons in the various layers of the cortex, as seen in Nissl-stained preparations. The subdivisions are therefore known as Brodmann areas 3a, 3b, 1, and 2 (see ). Cutaneous input dominates in areas 3b and 1, whereas muscle and joint input (proprioceptive) dominates in areas 3a and 2. Thus separate cortical zones are specialized for processing tactile and proprioceptive information (see

Fig. 7.5

Within any particular area of the S-I cortex, all the neurons along a line perpendicular to the cortical surface have similar response properties and receptive fields. The S-I cortex is thus said to have a columnar organization. A comparable columnar organization has also been demonstrated for other primary sensory receiving areas, including the primary visual and auditory cortices (see ). Nearby cortical columns in the S-I cortex may process information for different sensory modalities. For example, the cutaneous information that reaches one cortical column in area 3b may come from FA mechanoreceptors, whereas the information that reaches a neighboring column might originate from SA mechanoreceptors.

Besides being responsible for the initial processing of somatosensory information, the S-I cortex also begins higher-order processing such as feature extraction. For example, certain neurons in area 1 respond preferentially to a stimulus that moves in one direction across the receptive field but not in the opposite direction (Fig. 7.6 ). Such neurons presumably contribute to the perceptual ability to recognize the direction of an applied stimulus and could help detect slippage of an object being grasped by the hand.

118 SECTION2Berne & Levy Physiology •Fig. 7.6 Featureextractionbycorticalneurons.Theresponseswererecordedfromaneuroninthesomatosensorycortexofamonkey.Thedirectionofastimuluswasvaried,asshownbythearrowsinthedrawing.NotethattheresponsesweregreatestwhenthestimulusmovedinthedirectionfromUWtoRFandleastfromRWtoUF.F,fingers;R,radialside;U,ulnarside;W,wrist.(FromCostanzoRM,GardnerEP.J Neurophysiol 1980;43:1319.)R W F UW RF RW UF U R U R F U W UF RW F W RF UW 1 second

Effects of Lesions of the Somatosensory Cortex

A lesion of the S-I cortex in humans produces sensory changes similar to those produced by a lesion of the somatosensory thalamus. However, usually only a part of the cortex is involved, and thus the sensory loss may be confined, for example, to the face or to the leg, depending on the location of the lesion with respect to the sensory homunculus. The sensory modalities most affected are discriminative touch and position sense. Graphesthesia and stereognosis (i.e., the ability to recognize objects such as coins and keys as they are handled) are particularly disturbed. Pain and thermal sensation may be relatively unaffected, although loss of pain sensation may follow cortical lesions. Conversely, cortical lesions can result in a central pain state that resembles thalamic pain (see “

Effects of Interruption of the Spinotha lamic Tract and Lesions of the Thalamus on Somatosensory

The sensations of pain and temperature are related and often grouped together because they are mediated by overlapping sets of receptors and are conveyed by the same types of fibers in the PNS and the same pathways in the CNS. One consequence of these labeled lines is that pain sensations in particular are not due to stronger activation of touch pathways, as might naively be thought. This difference is borne out experimentally because if SA afferents, for example, are stimulated more and more frequently, the sensation of tactile pressure becomes stronger but not painful.

The axons that carry painful and thermal sensations are members of the relatively slowly conducting Aδ and C classes. However, not all Aδ and C axons carry pain and temperature information; some respond to light touch in a manner similar to what was described for low-threshold mechanoreceptors.

Unlike the case for low-threshold mechanoreceptors in which morphologically distinct receptors correspond to response properties, the Aδ and C axons conveying pain and temperature information appear to originate mostly as “free nerve endings.” (This description is not entirely accurate; the endings are mostly but not entirely covered by Schwann cells.) Despite the lack of distinct morphological specialization associated with their endings, Aδ and C axons constitute a heterogeneous population that is differentially sensitive to a variety of tissue-damaging or thermal stimuli (or both). This ability to sense tissue-damaging stimuli (mechanical, thermal, or chemical) is mediated by what are called nociceptors. These receptors share some features with low-threshold mechanoreceptors but are distinct in many ways, such as the ability to become sensitized (see later). Indeed, there appear to be a significant number of C fibers that are silent or unresponsive to any stimuli until first sensitized.

The first functional distinction that may be made in the pain system is between Aδ and C axons. Aδ axons conduct signals faster than C fibers do and are thought to underlie what is called first pain, whereas C fibers are responsible for second pain. Thus after a damaging stimulus, one first feels an initial sharp, pricking, highly localized sensation (first pain) followed by a duller, more diffuse, burning sensation (second pain). Experiments in which Aδ or C fibers were selectively activated demonstrated that activity in Aδ fibers produces sensations similar to first pain and that activity in C fibers produces second pain–like sensations.

Each fiber class in turn forms a heterogeneous group with regard to sensitivity to stimuli. Thus afferents are classified according to both size and their sensitivity to mechanical, thermal, and chemical stimuli. Fibers may have a low or high threshold to mechanical stimulation or be completely insensitive to it. Thermal sensitivity has been classified as responsiveness to warmth, noxious heat, cool, and noxious cold. Note that 43°C and 15°C are the approximate limits above and below which, respectively, thermal stimuli are sensed as painful. Chemical sensitivity to a variety of irritating compounds has been tested, including capsaicin (found in chili peppers), mustard oil, and acids.

Afferent fibers may be sensitive to one or more types of stimuli and have been named accordingly. For example, C fibers sensitive only to high-intensity (damaging) mechanical stimuli are called C mechanosensitive fibers, whereas those sensitive to heat and mechanical stimuli are labeled C mechanoheat-sensitive fibers (also called polymodal fibers). Other identified fiber types include Aδ and C cold-sensitive, Aδ mechanosensitive, and mechanoheat-sensitive fibers. Thus there is quite a variety of afferent types; however, the most common afferent type is the C polymodal fiber, which accounts for nearly half of the cutaneous C fibers. Surprisingly the second most common type is the mechanoheatinsensitive afferent (i.e., an afferent that is not sensitive to noxious stimuli until sensitized—see later).

The central portion of the Aδ and C axons carrying pain and temperature information from the body terminates in the dorsal horn of the spinal cord. The Aδ fibers target lamina I (dorsalmost part of dorsal horn), V (base of dorsal horn), and X (area surrounding central canal) of the gray matter, whereas the C fibers terminate in lamina I and II. The distinct termination patterns of the Aδ and C fibers in the spinal cord suggest that the messages they are carrying to the CNS are kept separated, and this is consistent with our ability to feel two distinct types of pain.

The primary afferent termination patterns in the spinal cord are also important because they may help determine the possible interactions pain fibers can have with other afferents and with descending control systems (see later). Indeed, the gate control theory of pain refers to the phenomenon that

CHAPTER 7 The Somatosensory System innocuous stimuli, such as rubbing a hurt area, can block or reduce painful sensations. Such stimulation activates the large-diameter (Aα and Aβ) fibers, and their activity leads to release of GABA and other neurotransmitters by inter-neurons within the dorsal horn. GABA then acts by both presynaptic and postsynaptic mechanisms to shut down the activity of spinothalamic tract cells. Presynaptically, GABA activates both GABAA and GABAB receptors, which leads to partial depolarization of the presynaptic terminal and blocking of Ca++ channels, respectively. Both actions will decrease release of transmitter by the afferent terminal and thereby lessen excitation of the tract cell (see section on presynaptic inhibition).

Nociceptive and thermoreceptive information that originates from regions of the head is processed in a fashion similar to that for the trunk and limbs. The primary afferent fibers of nociceptors and thermoreceptors in the head enter the brainstem through the trigeminal nerve (some also enter through the facial, glossopharyngeal, and vagus nerves). Of note, the trigeminal distribution includes both tooth and headache pain. These fibers then descend through the brainstem as far as the upper cervical spinal cord via the descending tract of the trigeminal nerve. Some mechanoreceptive afferent fibers also join the descending tract of the trigeminal nerve. Axons in the descending tract synapse on second-order neurons in the descending nucleus of the trigeminal nerve.

Elderlypeoplearesometimessusceptibletoaconditionofchronicpainknownastrigeminal neuralgia. Peoplewiththisconditionexperiencespontaneousepisodesofsevere,oftenlancinatingpaininthedistributionofoneormorebranchesofthetrigeminalnerve.Frequentlythepainistriggeredbyweakmechanicalstimulationinthesameregion.Amajorcontributingfactortothispainfulstateappearstobemechanicaldamagetothetrigeminalganglionbyanarterythatimpingesontheganglion.Surgicaldisplacementofthearterycanoftenresolvethecondition.

The central pain pathways include the spinothalamic, spinoreticular, and spinomesencephalic tracts. The spinothalamic tract is the most important sensory pathway for somatic pain and thermal sensations from the body (see

Fig. 7.1A ). It also contributes to tactile sensation. The spinothalamic tract originates from second-order neurons located in the spinal cord (primarily laminae I and IV to VI). The axons of these cells cross to the opposite side of the cord at or near to their level of origin. They then ascend to the brain in the ventral part of the lateral funiculus and subsequently through the brainstem to the thalamus, where they terminate on third-order neurons (thalamocortical neurons), as described earlier. Spinothalamic cells conveying pain and temperature target the VPI portion of the ventroposterior complex (although some also end in the

Multireceptive spinothalamic VPL), the posterior nucleus, and the intralaminar nuclei tract cells of the thalamus. Nociceptive signals are then forwarded to 120 several cortical areas, including not only the somatosensory cortex but also cortical areas that are involved in affective responses, such as the cingulate gyrus and the insula, which have limbic system functions (see

Fig. 7.5

Most spinothalamic tract cells receive excitatory input from nociceptors in the skin, but many can also be excited by noxious stimulation of muscle, joints, or viscera. Few receive input only from viscera. Effective cutaneous stimuli include noxious mechanical, thermal (hot or cold), and chemical stimuli. Thus different spinothalamic tract cells respond in a manner appropriate for signaling noxious, thermal, or mechanical events. A

Some nociceptive spinothalamic tract cells receive convergent excitatory input from several different classes of cutaneous sensory receptors. For example, a given spino

Fig. 7.7A ). Such neurons are called wide–dynamic range cells or multirecep-120 tive cells because they are activated by stimuli with a wide range of intensities. Wide–dynamic range neurons signal mainly noxious events; weak responses to tactile stimuli appear to be ignored by the higher centers. However, in certain pathological conditions, these neurons may be suf ficiently activated by tactile stimuli to evoke a sensation of pain, possibly as a result of activity in sensitized afferents that were previously silent. This would explain some pain states in which activation of mechanoreceptors causes pain (mechanical allodynia). Other spinothalamic tract cells are activated only by noxious stimuli. Such neurons are often called high-threshold or nociceptive-specific cells (see

Fig. 7.7B

Because cells signaling visceral input also typically convey information from cutaneous receptors, the brain may misidentify the source of the pain. This phenomenon is called referred pain. A typical example is when the heart muscle becomes ischemic and pain is felt in the chest wall and left arm.

Neurotransmitters released by nociceptive afferents that activate spinothalamic tract cells include the excitatory amino acid glutamate and any of several peptides, such as SP, CGRP, and vasoactive intestinal polypeptide (VIP). Glutamate appears to act as a fast transmitter by its action on non-NMDA excitatory amino acid receptors. However, with repetitive stimulation, glutamate can also act through NMDA receptors. Peptides appear to act as neuromodulators. For example, through a combined action with an excitatory amino acid such as glutamate, SP can produce a long-lasting increase in the responses of spinothalamic tract cells; this enhanced responsiveness is called central sensitization. CGRP seems to increase release of SP and prolong the action of SP by inhibiting its enzymatic degradation.

Spinothalamic tract cells often have inhibitory receptive fields. Inhibition may result from weak mechanical stimuli, but usually the most effective inhibitory stimuli •Fig. 7.7 A,Responsesofawide–dynamicrangeormultireceptivespinothalamictractcell.B,Responsesofahigh-thresholdspinothalamictractcell.Graphsshowresponsestogradedintensitiesofmechanicalstimulation.Brushstimulusisacamel’shairbrushrepeatedlystrokedacrossthereceptivefield.Pressureisappliedbyattachmentofanarterialcliptotheskin.Thisisamarginallypainfulstimulustoahuman.Pinchisachievedbyattachmentofastiffarterialcliptotheskinandisdistinctlypainful.Squeezeisappliedbycompressingafoldofskinwithforcepsandisdamagingtotheskin.

are noxious ones. The nociceptive inhibitory receptive fields may be very large and may include most of the body and face. Such receptive fields may account for the ability of various physical manipulations, including transcutaneous electrical nerve stimulation and acupuncture, to suppress pain. Neurotransmitters that can inhibit spinothalamic tract cells include the inhibitory amino acids GABA and glycine, as well as monoamines and the endogenous opioid peptides.

Spinoreticular tract neurons frequently have large, sometimes bilateral receptive fields, and effective stimuli include noxious ones. These dorsal horn neurons target multiple regions in the medullary and pontine reticular formation. The reticular formation, which projects to the intralaminar complex of the thalamus and thereby to wide areas of the cerebral cortex, is involved in attentional mechanisms and arousal (see

Chapter 10).

The reticular formation also gives rise to descending reticulospinal projections, which contribute to the descending systems that control transmission of pain.

Many cells of the spinomesencephalic tract respond to noxious stimuli, and the receptive fields may be small or large. The terminations of this tract are in several midbrain nuclei, including the periaqueductal gray, which is an important component of the endogenous analgesia system. Motivational responses may also result from activation of the periaqueductal gray matter. For example, stimulation in the periaqueductal gray matter can cause vocalization and aversive behavior. Information from the midbrain is relayed not only to the thalamus but also to the amygdala. This provides one of several pathways by which noxious stimuli can trigger emotional responses.

Pain and temperature information originating from the face and head is conveyed along analogous ascending central pathways, as is such information from the body. Neurons in the descending trigeminal nucleus transmit pain and temperature information to specific nuclei (VPM, VPI) of the contralateral thalamus via the ventral trigeminothalamic tract, which runs in close association with the medial lemniscus. The descending nucleus also projects to the intralaminar complex and other thalamic nuclei in a fashion similar to that of the spinothalamic tract. The thalamic nuclei in turn project to the somatosensory cerebral cortex for sensory discrimination of pain and temperature and to other cortical regions responsible for motivational-affective responses.

Effects of Interruption of the Spinothalamic Tract and Lesions of the Thalamus on Somatosensory Sensation

When the spinothalamic tract and accompanying ventral spinal cord pathways are interrupted, both the sensory-discriminative and the motivational-affective components of pain are lost on the contralateral side of the body. This result motivated development of the surgical procedure known as anterolateral cordotomy, which was used to treat pain in many individuals, especially those suffering from cancer. This operation is now used infrequently because of improvements in drug therapy and because pain often returns months to years after an initially successful cordotomy. Return of pain may reflect either extension of the disease or development of a central pain state. In addition to loss of pain sensation, anterolateral cordotomy produces loss of cold and warmth sensation on the contralateral side of the body. Careful testing may reveal a minimal tactile deficit as well, but the intact sensory pathways of the dorsal part of the spinal cord provide sufficient tactile information that any loss caused by interruption of the spinothalamic tract is insignificant.

Destruction of the VPL or VPM nuclei diminishes sensation on the contralateral side of the body or face. The sensory qualities that are lost reflect those that are

CHAPTER 7 The Somatosensory System transmitted mainly by the dorsal column–medial lemniscus pathway and its trigeminal equivalent. The sensory-discriminative component of pain sensation is also lost. However, the motivational-affective component of pain is still present if the medial thalamus is intact. Presumably, pain persists because of the spinothalamic and spinoreticulothalamic projections to this part of the thalamus. In some individuals a lesion of the somatosensory thalamus results in a central pain state known as thalamic pain. Patients with thalamic pain report that even the slightest touch feels painful, although the intensity of the touch is lower than the threshold of any pain receptor. It is thought that their pain sensitivity is due to a post-lesion sprouting of low-threshold dorsal column system fibers that synapse onto surviving thalamic neurons that normally mediate only pain. Pain that is indistinguishable from thalamic pain can also be produced by lesions in the brainstem or cortex.

Pain sometimes occurs in the absence of nociceptor stimulation. This type of pain is most likely to occur after damage to peripheral nerves or to parts of the CNS that are involved in transmitting nociceptive information. Pain caused by damage to neural structures is called neuropathic pain. Neuropathic pain states include peripheral neuropathic pain, which may follow damage to a peripheral nerve, and central neuropathic pain, which sometimes occurs after damage to CNS structures.

Examples of pain secondary to damage to a peripheral nerve are causalgia and phantom limb pain. Causalgia may develop after traumatic damage to a peripheral nerve. Even though evoked pain is reduced, severe pain may develop in the area innervated by the damaged nerve. This pain may be very difficult to treat, even with strong analgesic drugs. The pain is caused in part by spontaneous activity that develops in dorsal root ganglion cells; such activity may be attributed to upregulation of Na+ channels. In some cases the pain seems to be maintained by sympathetic neural activity, because a sympathetic nerve block may alleviate the pain. Sympathetic involvement may relate to the sprouting of damaged sympathic postganglionic axons into the dorsal root ganglia, and it may be accompanied by upregulation of adrenoreceptors in primary afferent neurons. Phantom limb pain follows traumatic amputation in some individuals. Such phantom pain is clearly not caused by activation of nociceptors in the area in which pain is felt, because these receptors are no longer present.

Lesions of the thalamus or at other levels of the spinothalamocortical pathway may cause central pain, which is a severe spontaneous pain. However, interruption of the nociceptive pathway by the same lesion may simultaneously prevent or reduce the pain evoked by peripheral stimulation. The mechanism of such trauma-induced pain caused by neural damage is poorly understood. The pain appears to depend on changes in the activity and response properties of more distant neurons in the nociceptive system.

Transduction in the Somatosensory System

Unraveling the transduction processes for the somatosensory system has proven difficult because of a variety of factors. The heart of the transduction process occurs at the specialized endings of the peripheral branch of the axon of the sensory neuron (dorsal root ganglia and trigeminal ganglia cells). These endings have channels that are activated by mechanical, thermal, and/or chemical stimuli and that allow the current flow that underlies the local generator potential (see

Fig. 5.13 ). The generator potential then triggers spikes in the afferent fiber (see

Fig. 5.14 ). However, the fact is that somatosensory axons are found throughout the body at low densities, making purification of the proteins difficult (e.g., compare with the retina, in which photoreceptors are packed at high density onto a small surface).

Complicating matters, the generator potential can be modified by a host of voltage-gated channels, both excitatory (e.g., Na+ and Ca++ channels) and inhibitory (K+ channels). Moreover, as described earlier, in many cases the axon terminal is encapsulated (e.g., Pacinian corpuscles), and the properties of this accessory structure modify the transduction process. They may do so passively, as a result of the mechanical characteristics of the capsule, or actively by having the accessory cells release transmitter in response to a stimulus. An example of the latter is Merkel cells, where recent evidence suggests the Merkel cells and the axon innervating them both have mechanosensitive channels and the Merkel cells release neurotransmitter.

Despite these complications, using a number of approaches, our knowledge of somatosensory transduction processes has begun to increase rapidly.

Until recently, ASIC (acid-sensing ion channel) proteins, which belong to the DEG/ENaC family, had been thought to be the channel proteins underlying the cutaneous mechanoactivated currents, because homologs of these proteins underlie the touch sensation in invertebrates such as Caenorhabditis elegans, and because some ASIC proteins are highly expressed in dorsal root ganglion cells. However, the mechanoactivated currents in dorsal root ganglion cells are not affected by knockdown of the ASIC proteins, indicating that they are not the channel underlying cutaneous mechanotransduction. Nevertheless, touch and pain sensitivity is altered in such knockdown mutants, so they may still play a modulatory role in the transduction process.

Currently, Piezo2 is thought to be channel protein underlying the transduction for cutaneous mechanical rapidly adapting responses, because it forms a nonselective cation pore that opens in response to mechanical stimuli. Moreover, the activation and inactivation kinetics of this channel are consistent with its causing the rapidly adapting mechanoactivated current, and it is blocked by agents (gadolinium, ruthenium red) that block these rapidly adapting currents. Furthermore, transfection of ganglion cells with small interfering (si)RNA for Piezo2 blocks the rapid mechanoactivated current in these cells. Lastly, messenger (m)RNA expression of Piezo2 has been shown in dorsal root ganglion cells.

Both low-threshold (light touch) and nociceptive-type ganglion cells have been found to express Piezo2, indicating that it plays a role in both innocuous and painful touch sensation. The Piezo proteins (Piezo1 and Piezo2) are both found in a variety of organs and thus may underlie visceral sensation as well. Piezo2 has been found in the main proprioceptors (muscle spindles and Golgi tendon organs [see ]) and appears to be the main mechanotransducer protein there as well.

The receptor that binds capsaicin (the molecule in chili peppers responsible for their spiciness) has been identified, and either it or one of a family of related proteins has been found to be expressed in populations of dorsal root ganglion cells. These proteins belong to the TRP (transient receptor potential) protein family and are currently the most likely candidates for being the transducers of thermal sensations.

It is important to note that many ion channels (and other proteins, e.g., enzymes) are sensitive to temperature; however, in the case of TRP channels, temperature is acting directly as the gating mechanism. The temperatures at which specific TRP channels are active are indicated by arrows in

Fig. 7.8 (bottom), in which the direction of each •Fig. 7.8 Temperaturedependenceoffiringratesindifferentthermo-sensitiveafferents.BelowthefiringratecurvesareshowntherangesoverwhichthedifferentTRPchannelsareactivated.Thedirectionofincreasingactivationisindicatedbyanarrowineachcase.Notehowinsomecasestherangeoverwhichanafferentisactive(topgraphs)correspondswelltotheactivationrangeofasingleTRPchannel,thussuggestingthattheafferentwouldneedtoexpressonlyasingletypeofchannel.InothercasestheactiverangeoftheafferentsuggeststhatmultipleTRPchannelswouldbeneededtounderliethecompleteresponsivenessoftheafferent.

arrow indicates which temperatures cause greater activation. For comparison,

Fig. 7.8 also plots the firing rates of several thermosensitive fibers as a function of temperature. Note how the response ranges of the afferents largely match up with those of individual heat-sensitive channels. The cold fibers, however, show firing over a wider range than any one TRP channel does. One possible explanation for this discrepancy is that dorsal root ganglion cells may express multiple classes of TRP, which would enable them to respond over a wider range of physiological temperatures.

MembersoftheTRPproteinfamilywerefirstidentifiedinDrosophila andwerefoundtobepartofthephototransductionprocessinDrosophila photoreceptors.Thusthename(TRP) referstothefactthatamutationinthegeneleadstoatransientdepolarizingresponsetoalightstimulusinsteadofthenormalsustainedresponse.Onthebasisofsequencehomology,anumberofgenesencodingTRPproteinshavebeenfoundinmammals(27inhumansalone),whicharecurrentlydividedintosevensubfamilies.TRPchannelsarecationpermeableandhaveastructuresimilartovoltage-gatedK+ channels.Theyarehomotetramersorheterotetramers.Eachsubunithassixtransmembranedomains.TRPproteinsappeartohaveavarietyoffunctions(e.g.,phototransduction,chemotransduction,andmechanotransduction)andareexpressedinanumberofcelltypes.Thoselistedin

Table7.1 appeartoactastemperaturesensorswithdistinctthermalsensitivitiesthatspantherangeofphysiologicallyrelevanttemperatures.

Modulation of the Transduction Process

As with the low-threshold mechanoreceptors for innocuous touch sensations, activation of the various nociceptor

Thefourthletterinthenameidentifiesthesubfamilyandwaschosenbecauseofthefirstmemberofthesubfamilyidentified:V,vanilloid;M,melastatin;A,ankyrin-like.Eachoftheproteinslistedisexpressedinatleastsomedorsalrootganglioncells,buttheyarealsoexpressedinothercelltypes.

CHAPTER 7 The Somatosensory System transduction proteins leads to a generator potential that causes spiking of the afferent, which transmits information to the CNS. In addition, activation of nociceptors also leads to local release of various chemical compounds, including tachykinins (substance P [SP]) and calcitonin gene–related protein (CGRP). These substances and others released from the damaged cells cause neurogenic inflammation (edema and redness of the surrounding skin).

In addition to causing a local reaction, these substances may serve to activate the insensitive or silent nociceptors mentioned earlier, such that they can henceforth respond to any subsequent damaging stimuli. Sensitization of silent nociceptors has been suggested to underlie allodynia (elicitation of painful sensations by stimuli that were innocuous before an injury) and hyperalgesia (increase in the level of pain felt to already painful stimuli). Thermal hyperalgesia is due to sensitization of TRPV1 channels. The Piezo2 current is increased by substances known to cause mechanical hyperalgesia and allodynia, suggesting changes in this current underlie these phenomena.

Centrifugal Control of Somatosensation

Sensory experience is not just the passive detection of environmental events. Instead, it more often depends on exploration of the environment. Tactile cues are sought by moving the hand over a surface. Visual cues result from scanning targets with the eyes. Thus sensory information is often received as a result of activity in the motor system. Furthermore, transmission in pathways to the sensory centers of the brain is regulated by descending control systems. These systems allow the brain to control its input by filtering the incoming sensory messages. Important information can be attended to and unimportant information can be ignored.

The tactile and proprioceptive somatosensory pathways are regulated by descending pathways that originate in the S-I and motor regions of the cerebral cortex. For example, cortical projections to the dorsal column nuclei help control the sensory input that is transmitted by the dorsal column– medial lemniscus pathway.

Of particular interest is the descending control system that regulates transmission of nociceptive information. This system presumably suppresses excessive pain under certain circumstances. For example, it is well known that soldiers on the battlefield, accident victims, and athletes in competition often feel little or no pain at the time a wound occurs or a bone is broken. At a later time, pain may develop and become severe. Although the descending regulatory system that controls pain is part of a more general centrifugal control system that modulates all forms of sensation, the pain control system is so important medically that it is distinguished as a special system called the endogenous analgesia system.

Several centers in the brainstem and pathways descending from these centers contribute to the endogenous analgesia system. For example, stimulation in the midbrain •Fig. 7.9 A, Modelsofdescendingcontrolofascendingpainpathways.A,Schematicshowingtheascendingnociceptivepathwaythatconveyspaininformationtothebrain,representedbythespinothalamictract(STT),andthedescendingpathwaythatmodulates(gates)theflowofinformationintoit.Thedescendingpathwaystartsintheperiaqueductalgray(PAG),whichprojectsviatherostroventromedial(RVM)medullatothedorsalhorn.ActivationofthePAGorRVMblockstransmissionofpaininformationatthespinalcordlevel.B,Thestandardmodelofdescendingcontrolofpainperception.TonicallyactiveGABAergicinterneuronsinthePAGandRVMinhibittheprojectioncells(notethatthePAGhaslocalGABAergicinterneuronsbutalsoreceivesGABAergicterminalsfromotherbrainregions,andbothmaybeinvolvedintonicallyinhibitingthePAGprojectioncells).OpioidsappeartoinhibittheGABAergicinterneurons,thusdisinhibitingtheprojectionneurons,whoseincreasedactivityleadstoblockingtheactivationofSTTcellsbynociceptivedorsalrootganglion(DRG)cells.C,Parallelinhibition-excitationmodel.SomestudiessuggestbothexcitatoryandinhibitorycellsprojectfromthePAGandtheRVM.Inthismodel,gatingoftransmissionofpaininformationwouldbeduetoabalanceoftheexcitatoryandinhibitoryactivityinthedescendingpathways.Notethattheexcitatoryandinhibitorypathwaysareshownonoppositesidesjustforclarity.D,Possiblepresynapticandpostsynapticsitesofactionofenkephalin(Enk).ManyoftheterminalsfromthedescendingpathwaysforpainmodulationreleasebothGABAandenkephalin(EnK),whichappearstohavebothpre-andpostsynapticsitesofaction.ThepresynapticactionmightpreventreleaseofsubstanceP(SubP.)fromnociceptors.(A-C,RedrawnfromLauBK,VaughanCW.Curr Opin Neurobiol 2014;29:159.D,RedrawnfromHenryJL.In:PorterR,O’ConnorM[eds]:CibaFoundationSymposium91.London:Pitman;1982.) periaqueductal gray, locus coeruleus, or medullary raphe nuclei inhibits nociceptive neurons at the spinal cord and brainstem level, including spinothalamic and trigeminothalamic tract cells (

Fig. 7.9A-C ). Other inhibitory pathways originate in the sensorimotor cortex, hypothalamus, and reticular formation.

The endogenous analgesia system can be subdivided into two components: one component uses endogenous opioid peptides as neurotransmitters and the other does not. Endogenous opioids are neuropeptides that activate one of several types of opiate receptors. Some of the endogenous

STT relay cell afferent Circulating opioid binding sites Opiate receptor Sub P. Enk Substance P receptor opioids include enkephalin, dynorphin, and β-endorphin. Opiate analgesia can generally be prevented or reversed by the narcotic antagonist naloxone. Therefore naloxone is frequently used to determine whether analgesia is mediated by an opioid mechanism.

The opioid-mediated endogenous analgesia system can be activated by exogenous administration of morphine or other opiate drugs. Thus one of the oldest medical treatments of pain depends on the triggering of a sensory control system. Opiates typically inhibit neural activity in nociceptive pathways. Two sites of action have been proposed for opiate inhibition, presynaptic and postsynaptic (see

Fig. 7.9D ). The presynaptic action of opiates on nociceptive afferent terminals is thought to prevent release of excitatory transmitters such as SP. The postsynaptic action of opiates produces an inhibitory postsynaptic potential. How can an inhibitory neurotransmitter activate descending pathways? One hypothesis is that the descending analgesia system is under tonic inhibitory control by inhibitory interneurons in both the midbrain and medulla. The action of opiates would inhibit the inhibitory inter-neurons and thereby disinhibit the descending analgesia pathways.

Some endogenous analgesia pathways operate by neurotransmitters other than opioids and thus are unaffected by naloxone. One way of engaging a nonopioid analgesia

CHAPTER 7 The Somatosensory System pathway is through certain forms of stress. The analgesia thus produced is a form of stress-induced analgesia.

Many neurons in the raphe nuclei use serotonin as a neurotransmitter. Serotonin can inhibit nociceptive neurons and presumably plays an important role in the endogenous analgesia system. Other brainstem neurons release catecholamines, such as norepinephrine and epinephrine, in the spinal cord. These catecholamines also inhibit nociceptive neurons; therefore catecholaminergic neurons may contribute to the endogenous analgesia system. Furthermore, these monoamine neurotransmitters interact with endogenous opioids. Undoubtedly, many other substances are involved in the analgesia system. In addition, there is evidence for the existence of endogenous opiate antagonists that can prevent opiate analgesia.

1.

Sensory neurons have cell bodies in sensory nerve ganglia: (1) dorsal root ganglia for neurons innervating the body and (2) cranial nerve ganglia for neurons innervating the face, oral and nasal cavities, and dura, except for proprioceptive neurons, which are in the trigeminal mesencephalic nucleus. They connect peripherally to a sensory receptor and centrally to second-order neurons in the spinal cord or brainstem.

2.

Skin contains low-threshold mechanoreceptors, thermoreceptors, and nociceptors. Muscle, joints, and viscera have mechanoreceptors and nociceptors. Low-threshold mechanoreceptors may be rapidly or slowly adapting. Thermoreceptors include cold and warm receptors. Aδ and C nociceptors detect noxious mechanical, thermal, and chemical stimuli and may be sensitized by release of chemical substances from damaged cells. Peripheral release of substances, such as peptides, from nociceptors themselves may contribute to inflammation.

3.

Large primary afferent fibers enter the dorsal funiculus through the medial part of the dorsal root; collaterals synapse in the deep dorsal horn, intermediate zone, and ventral horn. Small primary afferent fibers enter the spinal cord through the lateral part of the dorsal root; collaterals synapse in the dorsal horn.

4.

Ascending branches of large primary afferent fibers synapse on second-order neurons in the dorsal column nuclei. These second-order neurons project via the medial lemniscus to the contralateral thalamus and synapse on third-order neurons of the VPL nucleus. The equivalent trigeminal pathway is relayed by the main sensory nucleus to the contralateral VPM nucleus.

5.

The dorsal column spinal cord pathways signal the sensations of flutter-vibration, touch-pressure, and proprioception. They also contribute to visceral sensation, including visceral pain.

6.

The spinothalamic tract includes nociceptive, thermoreceptive, and tactile neurons; its cells of origin are mostly in the dorsal horn, and the axons cross, ascend in the ventrolateral funiculus, and synapse in the VPL, VPI, and posterior and intralaminar nuclei of the thalamus. The equivalent trigeminal pathway is relayed by the descending trigeminal nucleus and projects to the contralateral VPM and intralaminar nuclei.

7.

The spinothalamic relay in the VPL, VPM, and VPI nuclei helps account for the sensory-discriminative aspects of pain. Parallel nociceptive pathways in the ventrolateral funiculus are the spinoreticular and spinomesencephalic tracts; these tracts and the spinothalamic projection to the medial thalamus contribute to the motivational-affective aspects of pain.

8.

Referred pain is explained by convergent input to spinothalamic tract cells from the body wall and from viscera.

9.

The VPL and VPM nuclei are somatotopically organized and contain inhibitory circuits. These nuclei contain multiple somatotopic maps, one for each somatosensory submodality. The somatosensory cortex includes the S-I and S-II regions; these regions are also somatotopically organized.

10.

The S-I cortex contains columns of neurons with similar receptive fields and response properties. Some S-I neurons are involved in feature extraction.

11.

Transmission in somatosensory pathways is regulated by descending control systems. The endogenous analgesia system regulates nociceptive transmission, and it uses transmitters such as endogenous opioid peptides, norepinephrine, and serotonin.

Additional Readings Lau BK, Vaughan CW. Descending modulation of pain: the GABA disinhibition hypothesis of analgesia. Curr Opin Neurobiol. Coste B, Xiao B, Santos JS, et al. Piezo proteins are pore-2014;29:159-164. forming subunits of mechanically activated channels. Nature. Squire L, Berg D, Bloom FE, et al., eds. Fundamental Neuroscience. 4th ed. Waltham, MA: Academic Press; 2013.

2012;483:176-181.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

What is the dark current, and how does the absorption of a photon change it?

2.

What are the synaptic pathways for the central and surround portions of the receptive field of an on-center bipolar cell? Of an off-center bipolar cell?

3.

What are the receptive field properties of simple and complex cells in the visual cortex?

4.

What is the frequency theory of sound encoding? Why is the place theory also required?

5.

What are the stimuli that are normally transduced by the hair cells in the semicircular canals and otolith organs?

6.

What are the functional consequences of the differing numbers of different receptor molecules between olfactory and gustatory receptor cells?

he evolution of vertebrates shows a trend called cephalization in which special sensory organs develop in the heads of animals, along with the corresponding development of the brain. These special sensory systems, which include the visual, auditory, vestibular, olfactory, and gustatory systems, detect and analyze light, sound, and chemical signals in the environment, as well as signal the position and movement of the head. The stimuli transduced by these systems are most familiar to humans when they provide conscious awareness of the environment, but they are equally important as the sensory basis for reflexive and subconscious behavior.

The Visual System

Vision is one of the most important special senses in humans and, along with audition, is the basis for most human communication. The visual system detects electromagnetic waves between 400 and 750 nm long as visible light, which enters the eye and impinges on photoreceptors in a specialized sensory epithelium, the retina.

The photoreceptors, rods and cones, can distinguish two aspects of light: its brightness (or luminance) and its wavelength (or color). Rods have high sensitivity for detecting low light intensities but do not provide well-defined visual images, nor do they contribute to color vision. Rods operate best under conditions of reduced lighting (scotopic vision). Cones, in contrast, are not as sensitive to light as rods are and thus operate best under daylight conditions (photopic vision). Cones are responsible for high visual acuity and color vision.

The retina is an outgrowth of the thalamus. Thus information processing within the retina is performed by retinal neurons, and the output signals are carried to the brain by the axons of retinal ganglion cells in the optic nerves. There is a partial crossing of these axons in the optic chiasm that causes all input from one side of the visual space to pass to the opposite side of the brain. Posterior to the optic chiasm, the axons of retinal ganglion cells form the optic tracts and synapse in nuclei of the brain. The main visual pathway in humans targets the lateral geniculate nucleus (LGN) of the thalamus and this nucleus, through the visual radiations, projects the visual information to the visual cortex. Other visual pathways project to the superior colliculus, pretectum, and hypothalamus, structures that participate in orientation of the eyes, control of pupil size, and circadian rhythms, respectively.

Structure of the Eye

The wall of the eye is composed of three concentric layers (

Fig. 8.1 ). The outer layer, or the fibrous coat, includes the transparent cornea, with its epithelium, and the opaque sclera. The middle layer, or vascular coat, includes the iris and the choroid. The iris contains both radially and circularly oriented smooth muscle fibers, which make up the pupillary dilator and constricter muscles, respectively. The choroid is rich in blood vessels that support the outer layers of the retina, and it also contains pigment. The innermost layer of the eye, the retina, is embryologically derived from the diencephalon and therefore is part of the central nervous system (CNS). The functional part of the retina covers the entire posterior aspect of the eye except for the optic nerve head, or optic disc, which is where the optic nerve axons leave the retina. Because there are no receptors at this location, it is often referred to as the anatomical “blind spot” (see

Fig. 8.1

A number of functions of the eyes are under muscular control. Externally attached extraocular muscles aim the eyes toward an appropriate visual target (see ). These muscles are innervated by the oculomotor nerve •Fig. 8.1 Illustrationofaviewofahorizontalsectionoftherighteye.(RedrawnfromWallGL.The Vertebrate Eye and Its Adaptive Radiation. BloomfieldHills,MI:CranbrookInstituteofScience;1942.)IrisPosterior chamber Anterior chamber Limbus Sclera Choroid Retina Optic disc Fovea Vitreous humor Optic nerve Temporal retina Nasal retina Ciliary body Zonule fibers Nodal point Lens Aqueous humor Cornea (cranial nerve [CN] III), the trochlear nerve (CN IV), and the abducens nerve (CN VI). Several muscles are also found within the eye (intraocular muscles). The muscles in the ciliary body control lens shape and thereby the focus of images on the retina. The pupillary dilator and sphincter muscles in the iris control the amount of light entering the eye, in a way similar to that of the diaphragm of a camera. The dilator is activated by the sympathetic nervous system, whereas the sphincter and ciliary muscles are controlled by the parasympathetic nervous system (through the oculomotor nerve; see

Light enters the eye through the cornea and passes through a series of transparent fluids and structures that are collectively called the dioptric media. These fluids and structures consist of the cornea, aqueous humor, lens, and vitreous humor (see

Fig. 8.1 ). The aqueous humor (located in the anterior and posterior chambers) and the vitreous humor (located in the space behind the lens) help maintain the shape of the eye.

Although the geometrical optic axis of the human eye passes through the nodal point of the lens and reaches the retina at a point between the fovea and the optic disc (see

Fig. 8.1 ), the eyes are oriented by the oculomotor system to a point, called the fixation point, on the visual target. Light from the fixation point passes through the nodal point of the lens and is focused on the fovea. Light from the remainder of the visual target falls on the retina surrounding the fovea.

Normally, light from a visual target is focused sharply on the retina by the cornea and lens, which bend or refract the light. The cornea is the major refractive element of the eye, with a refractive power of 43 diopters (D). However, unlike the cornea, the lens can change shape and vary its refractive power between 13 and 26 D. Thus the lens is responsible for adjusting the optical focus of the eye. Suspensory ligaments (or zonule fibers) attach to the wall of the eye at the ciliary body (see

Fig. 8.1 ) and hold the lens in place. When the muscles in the ciliary body are relaxed, the tension exerted by the suspensory ligaments flattens the lens. When the ciliary muscles contract, the tension on the suspensory ligaments is reduced; this process allows the somewhat elastic lens to assume a more spherical shape. The ciliary muscles are activated by the parasympathetic nervous system via the oculomotor nerve.

In this way, the lens allows the eye to focus on, or accommodate to, either near or distant objects. For instance, when light from a distant visual target enters a normal eye (one with a relaxed ciliary muscle), the target image is in focus on the retina. However, if the eye is directed at a nearby visual target, the light is initially focused behind the retina (i.e., the image at the retina is blurred) until accommodation aA diopter is a unit of measurement of optical power that is equal to the reciprocal of the focal length measured in meters. Thus it is a unit of reciprocal length, and a 2-D lens would bring parallel rays of light into focus at a distance of 0.5 m.

CHAPTER 8 The Special Senses occurs; that is, until the ciliary muscle contracts, causing the lens to become more spherical, the increased convexity causes the lens to refract the light waves more strongly, bringing the image into focus on the retina.

Proper imaging of light on the retina depends not only on the lens and cornea but also on the iris, which adjusts the amount of light that can enter the eye through the pupil. In this regard, the pupil is analogous to the aperture in a camera, which also controls the depth of field of the image and the amount of spherical aberration produced by the lens. When the pupil is constricted, the depth of field is increased, and the light is directed through the central part of the lens, where spherical aberration is minimal. Pupillary constriction occurs reflexively when the eye accommodates for near vision or adapts to bright light, or both. Thus when a person reads or does other fine visual work, the quality of the image is improved by adequate light.

Layers of the Retina

The 10 layers of the retina are shown in

Fig. 8.2 . The outermost portion is the pigmented epithelium (layer 1), which is just inside the choroid. The pigment cells have

Asanindividualages,theelasticityofthelensgraduallydeclines.Asaresult,accommodationofthelensfornearvisionbecomesprogressivelylesseffective,aconditioncalledpresbyopia. Ayoungpersoncanchangethepowerofthelensbyasmuchas14D.However,bythetimethatapersonreaches40yearsofage,theamountofaccommodationhalves,andafter50years,itcandecreaseto2Dorless.Presbyopiacanbecorrectedbyconvexlenses.

Defectsinfocuscanalsobecausedbyadiscrepancybetweenthesizeoftheeyeandtherefractivepowerofthedioptricmedia.Forexample,inmyopia (near-sightedness),theimagesofdistantobjectsarefocusedinfrontoftheretina.Concavelensescorrectthisproblem.Conversely,inhypermetropia (far-sightedness),theimagesofdistantobjectsarefocusedbehindtheretina;thisproblemcanbecorrectedwithconvexlenses.Inastigmatism, anasymmetryexistsintheradiiofcurvatureofdifferentmeridiansofthecorneaorlens(orsometimesoftheretina).Astigmatismcanoftenbecorrectedwithlensesthatpossesscomplementaryradiiofcurvature.

•Fig. 8.2 Layers of the Retina. Lightimpingingontheretinawouldbecomingfromthetopofthefigure,andwouldpassthroughallthesuperficiallayerstoreachthephotoreceptorrodsandcones.9 Nerve fiber layer Axons at surface of retina passing via optic nerve, chiasm and tract to lateral geniculate body Ganglion cell Bipolar cell Amacrine cell Horizontal cell Rod Cone Choroid Pigment cells Müller cell (supporting glial cell) 8 Ganglion cell layer 7 Inner plexiform layer 5 Outer plexiform layer 6 Inner nuclear layer 4 Outer nuclear layer 2 Photoreceptor layer 1 Pigment epithelium 3 Outer limiting membrane 10 Inner limiting membrane Inner segment Outer segment tentacle-like processes that extend into the photoreceptor layer (layer 2) and surround the outer segments of the rods and cones. These processes prevent transverse scatter of light between photoreceptors. In addition, they serve a mechanical function in maintaining contact between layers 1 and 2 so that the pigmented epithelium can (1) provide nutrients and remove waste from the photoreceptors; (2) phagocytose the ends of the outer segments of the rods, which are continuously shed; and (3) reconvert metabolized visual pigment into a form that can be reused after it is transported back to the photoreceptors.

Retinal glial cells, known as Müller cells, play an important role in maintaining the internal geometry of the retina. Müller cells are oriented radially, parallel to the light path through the retina. The outer ends of Müller cells form tight junctions with the inner segments of the photoreceptors, and these numerous connections have the appearance of a continuous layer, the outer limiting membrane (layer 3 of the retina).

Thejunctionbetweenlayers1and2oftheretinainadultsrepresentsthesurfaceofcontactbetweentheanteriorandposteriorwallsoftheembryonicopticcupduringdevelopmentandisstructurallyweak.Retinaldetachmentisseparationatthissurfaceandcancauselossofvisionbecauseofdisplacementoftheretinafromthefocalplaneoftheeye.Itcanalsoleadtothedeathofphotoreceptorcells,whicharemaintainedbythebloodsupplyofthechoroid(thephotoreceptorlayeritselfisavascular).Deteriorationofthepigmentedepitheliumcanalsoresultinmaculardegeneration,acriticallossofhigh-acuitycentralandcolorvisionthatdoesnotaffectperipheralvision.

Inside the external limiting membrane is the outer nuclear layer (layer 4) that contains the cell bodies and nuclei of the rods and cones. The outer plexiform layer (layer 5) contains synapses between the photoreceptors and retinal interneurons, including bipolar cells and horizontal cells, whose cell bodies are found in the inner nuclear layer (layer 6). This layer also contains the cell bodies of other retinal interneurons (the amacrine and interplexiform cells) and the Müller cells.

The inner plexiform layer (layer 7) contains synapses between the retinal neurons of the inner nuclear layer, including the bipolar and amacrine cells, and the ganglion cells, whose cell bodies lie in the ganglion cell layer (layer 8). As previously mentioned, the ganglion cells are the output cells of the retina; it is their axons that transmit visual information to the brain. These axons form the optic fiber layer (layer 9), pass along the inner surface of the retina while avoiding the fovea, and enter the optic disc, where they leave the eye as the optic nerve. The portions of the ganglion cell axons that are in the optic fiber layer remain unmyelinated, but they become myelinated after they reach the optic disc. The lack of myelin where the

Folding of membrane •Fig. 8.3 Rods and Cones. Thedrawingsatthebottom showthegeneralfeaturesofarodandacone.Theinsets showtheoutersegments.

axons cross the retina helps permit light to pass through the inner retina with minimal distortion.

The innermost layer of the retina is the inner limiting membrane (layer 10) formed by the end-feet of Müller cells.

Structure of Photoreceptors: Rods and Cones

Each rod or cone photoreceptor cell is composed of a cell body (in layer 4), an inner and an outer segment that extend into layer 2, and a set of synaptic terminals that synapse in layer 5 onto other retinal cells (Fig. 8.3 ). The outer segments of cones are not as long as those of rods, and they contain stacks of disc membranes formed by infoldings of the plasma membrane. The outer segments of rods are longer, and they contain stacks of membrane discs that float freely in the outer segment, having completely disconnected from the plasma membrane when formed at the base. Both sets of discs are rich in visual pigment molecules, but rods have a greater visual pigment density, which partly accounts for their greater sensitivity to light. A single photon can elicit a rod response, whereas several hundred photons may be required for a cone response.

The outer segments of the photoreceptors are connected by a modified cilium to the inner segments, which contain a number of organelles, including numerous mitochondria. The inner segments are the sites where the visual pigment is synthesized before it is incorporated into the membranes of the outer segment. In rods, the pigment is inserted into new membranous discs, which are then displaced distally until they are eventually shed at the apex of the outer segment, where they undergo phagocytosis by cells of the pigmented

CHAPTER 8 The Special Senses •Fig. 8.4 Graph of a Plot of the Density of Cones and Rods as a Function of Retinal Eccentricity From the Fovea. Notethatconedensitypeaksatthefovea,roddensitypeaksatabout20degreeseccentricity,andnophotoreceptorsarefoundattheopticdisc,wheretheganglioncellaxonsleavetoformtheopticnerve.(DatafromCornsweetTN.Visual Perception. NewYork:AcademicPress;1970.) epithelium. This process determines the rod-like shape of the outer segments of rods. In cones, the visual pigment is inserted randomly into the membranous folds of the outer segment, and shedding, comparable to that seen in rods, does not take place.

Regional Variations in the Retina

The macula lutea is the area of central vision and is characterized by a slight thickening and a pale color. The thickness is due to the high concentration of photoreceptors and interneurons, which are needed for high-resolution vision. It is pale because both optic nerve fibers and blood vessels are routed around it.

The fovea, which is a depression in the macula lutea, is the region of the retina with the very highest visual resolution and, as noted previously, the light from the fixation point is focused on the fovea. (A major function of eye movements is to bring objects of interest into view on the fovea.) The retinal layers in the foveal region are unusual because several of them appear to be pushed aside into the surrounding macula. Consequently, light can reach the foveal photoreceptors without having to pass through the inner layers of the retina, and both image distortion and light loss are minimized. The fovea has cones with unusually long and thin outer segments, which allows for high packing density. In fact, cone density is maximal in the fovea, providing for high visual resolution, as well as high quality of the image (

Fig. 8.4

The optic disc, where ganglion cell axons leave the retina, lacks photoreceptors and therefore lacks photosensitivity. Thus it is a so-called blind spot in the visual surface of the retina (see

Figs. 8.4 8.9 ). A person is normally unaware of the blind spot because the corresponding part of the visual field can be seen by the contralateral eye and because of the psychological process in which incomplete visual images tend to be completed perceptually.

Asmentioned,theaxonsofretinalganglioncellscrosstheretinaintheopticfiberlayer(layer9)toentertheopticnerveattheopticdisc.Theseaxonsintheopticfiberlayerpassaroundthemaculaandfovea,asdothebloodvesselsthatsupplytheinnerlayersoftheretina.Theopticdisccanbevisualizedonphysicalexaminationwithanophthalmoscope. Thenormalopticdischasaslightdepressioninitscenter.Changesintheappearanceoftheopticdiscareimportantclinically.Forexample,thedepressionmaybeexaggeratedbylossofganglioncellaxons(optic atrophy), ortheopticdiscmayprotrudeintothevitreousspacebecauseofedema(papilledema) thatresultsfromincreasedintracranialpressure.

To be detected by the retina, light energy must be absorbed. This is the job primarily of the rods and cones (a small class of ganglion cells are also photosensitive) and is accomplished by visual pigment molecules located in their outer segments. For both rods and cones, the pigment molecule consists of a chromophore, 11-cis retinal, bound to an opsin protein. The visual pigment found in the outer segments of rods is rhodopsin, or visual purple (so named because it has a purple appearance when light has been absorbed). It absorbs light best at a wavelength of 500 nm. Three variants of visual pigment, resulting from the binding of different opsins to retinal, are found in cones (in most species, each cone expresses one of the three cone pigments). The cone pigments absorb best at 419 nm (blue), at 533 nm (green), and at 564 nm (red). However, the absorption spectrum of these visual pigments is broad so that they overlap considerably (

Fig. 8.5).

•Fig. 8.5 The Spectral Sensitivity of the Three Types of Cone Pigments and of the Rod Pigment (Rhodopsin) in the Human Retina. Notethatthecurvesoverlapandthattheso-calledblueandredconesactuallyabsorbmaximallyinthevioletandyellowranges,respectively.(DatafromSquireLR,etal[eds].Fundamental Neuroscience. SanDiego,CA:AcademicPress;2002.)

Despite the differences in spectral sensitivity, the transduction process is similar in rods and cones. The absorption of a photon by a visual pigment molecule leads to the isomerization of 11-cis retinal to all-trans retinal, release of the bond with the opsin, and conversion of retinal to retinol. These changes trigger a second messenger cascade that leads to a change in the electrical activity of the rod or cone (discussed later in this section).

The separation of all-trans retinal from opsin also causes both the loss of its ability to absorb light and bleaching (i.e., the visual pigment loses its color). In both rods and cones, regeneration of the visual pigment molecule is a multistep process: the all-trans retinal is transported to the retinal pigmented cell layer, where it is reduced to retinol, isomerized, and esterified back to 11-cis retinal. It is then transported back to the photoreceptor layer, taken up by outer segments, and recombined with opsin to regenerate the visual pigment molecule, which can again absorb light. There is evidence that cones also use a second pathway to regenerate visual pigment. This pathway is much more rapid and involves transport of the retinal molecule to and from the Müller cells (see

Fig. 8.2 ) rather than the pigmented epithelial cells. The potential importance of this more rapid pathway is discussed in the section “

Ultimately, the transduction process triggered by absorption of photons causes the photoreceptor to hyperpolarize. To understand this action and its consequences fully, it is necessary to know the baseline state of the photoreceptor in the dark (i.e., before it absorbs a photon). In darkness, photoreceptors are slightly depolarized (≈−40 mV) in relation to most neurons because cyclic guanosine monophosphate (cGMP)–gated cation channels in their outer segments are open (Fig. 8.6A ). These channels allow a steady influx of Na+ and Ca2+ . The resulting current is known as the dark current, and the depolarization it causes leads to the tonic release of the neurotransmitter glutamate at the photoreceptor’s synapses.

When light is absorbed in a rod (an equivalent sequence happens in cones), photoisomerization of rhodopsin activates a G protein called transducin (see

Fig. 8.6B ). This G protein, in turn, activates cyclic guanosine monophosphate phosphodiesterase, which is associated with the rhodopsin-containing discs, hydrolyzes cGMP to 5′-GMP, and lowers the cGMP concentration in the rod cytoplasm. The reduction in cGMP leads to closing of the cGMP-gated cation channels, hyperpolarization of the rod cell membrane, and a reduction in the release of neurotransmitters. Thus cGMP acts as a “second messenger” to translate the absorption of a photon by rhodopsin into a change in membrane potential.

In sum, in all photoreceptors (cones undergo a process analogous to that described for rod transduction), capture of light energy leads to (1) hyperpolarization of the photoreceptor and (2) a reduction in the release of neurotransmitters. Because of the very short distance between the site of transduction and the synapse, the modulation of neurotransmitter release is accomplished without the generation of an action potential.

Adaptation refers to the ability of the retina to adjust its sensitivity according to ambient light. This ability allows the retina to operate efficiently over a wide range of lighting conditions, and it reflects a switching between the use of the cone and rod systems for brightand low-light conditions, respectively.

CHAPTER 8 The Special Senses •Fig. 8.6 A, Drawingofarodwiththeflowofcurrentinthedark.WiththeassistanceoftheNa+,K+pump,therodiskeptdepolarized.B, SequenceofthesecondmessengereventsthatfollowtheabsorptionoflightthroughthereductionofcGMP.BecausecGMPmaintainsopenNa+channelsinthedark,theresultsoflightabsorptionaretheclosingoftheNa+channelsandhyperpolarizationoftherod.cGMP,cyclicguanosinemonophosphate;GC,guanylatecyclase;GTP,guanosinetriphosphate;PDE,phosphodiesterase;Rh,rhodopsin;T,transducin.Passive Na+ influx Na+ current Rh T + + + – PDE GC GMP Light Disc A B cGMP GTP Hyperpolarization Membrane Membrane Na+ Na+ Ca++ K+ Active Na+ efflux

Rhodopsincontainsachromophorecalledretinal, whichisthealdehydeofretinol, orvitaminA.Retinolisderivedfromcarotenoids,suchasβ-carotene,theorangepigmentfoundincarrots.Likeothervitamins,retinolcannotbesynthesizedbyhumans;instead,itisderivedfromfoodsources.IndividualswithaseverevitaminAdeficiencysufferfrom“nightblindness,”aconditioninwhichvisionisdefectiveinlow-lightsituations.

Theextraordinarysensitivityofrods,whichcansignalthecaptureofasinglephoton,isenhancedbyanamplificationmechanisminwhichphotoactivationofonlyonerhodopsinmoleculecanactivatehundredsoftransducinmolecules.Inaddition,eachphosphodiesterasemoleculehydrolyzesthousandsofcGMPmoleculespersecond.Similareventsoccurincones,butthemembranehyperpolarizationoccursmuchmorequicklythaninrodsandrequiresthousandsofphotons.

As described previously, absorption of a photon causes 11-cis retinal to be converted to all-trans retinal, which then splits from the opsin (bleaching). The visual pigments in rods and cones are bleached at a similar rate; however, regeneration of the visual pigment occurs much more rapidly in cones than in rods. This difference is, at least in part, due to the cones’ ability to utilize a second pathway for regeneration (see previous section). This more rapid regeneration of visual pigment prevents cones from becoming unresponsive in bright light conditions. In contrast, the slowness of the regeneration of rhodopsin molecules means that at light levels not much above those found in evening hours, essentially all of the rhodopsin molecules are bleached. Thus in bright-light conditions, only the cone system is functioning, and the retina is said to be light-adapted.

When entering a darkened movie theater, a person can observe evidence of the existing light adaptation (decreased light sensitivity in association with the reduced amount of rhodopsin) in the inability to see the empty seats (or much else). The gradual return of the ability to see the seats while the person remains in the theater reflects the slow regeneration of rhodopsin and recovery of function of the rod system, a process known as dark adaptation.

This process refers to the gradual increase in light sensitivity of the retina when in low-light conditions. Rods adapt to darkness slowly as their rhodopsin levels are restored, and, indeed, it may take more than 30 minutes for the retina to become fully dark-adapted. In contrast, cones adapt rapidly to darkness, but their adapted threshold is relatively high, and so they do not function when the ambient light level is low. Thus within 10 minutes in a dark room, rod vision is more sensitive than cone vision and becomes the main system for seeing.

In sum, in the dark-adapted state, primarily rod vision is operative, and thus visual acuity is low and colors are not distinguished (this is called scotopic vision). However, when light levels are higher (e.g., when the movie is projected) and cone function resumes (this is called photopic vision), visual acuity and color vision are restored. There is an intermediate range of light levels at which rod and cones are both functional (mesopic vision).

The visual pigments in the cone outer segments contain different opsins. As a result of these differences, the three types of cones absorb light best at different wavelengths. Although the cone pigments have maximum efficiency closer to violet, green, and yellow wavelengths, they are referred to as blue, green, and red pigments, respectively (see

Fig. 8.5). The differences in the cone absorption spectra underlie humans’ ability to see colors, as opposed to only shades of gray.

According to the trichromacy theory, the differences in absorption efficiency of the cone visual pigments are presumed to account for color vision because a suitable mixture of three colors can produce any other color. However, a neural mechanism must also exist for the analysis of color brightness because the amount of light absorbed by a visual pigment, as well as the subsequent response of the cell, depends on both the wavelength and the intensity of the light (see

Fig. 8.5 ). Two or three of the cone pigments may absorb a particular wavelength of light, but the amount absorbed by each differs according to its efficiency at that wavelength. If the intensity of the light is increased (or decreased), all will absorb more (or less), but the ratio of absorption among them will remain constant. Consequently, there must be a neural mechanism to compare the absorption of light of different wavelengths by the different types of cones for the visual system to distinguish different colors. At least two different kinds of cones are required for color vision. The presence of three kinds decreases the ambiguity in distinguishing colors when all three absorb light, and it ensures that at least two types of cones will absorb most wavelengths of visible light.

The opponent process theory is based on observations that certain pairs of colors seem to activate opposing neural processes. Green and red are opposed, as are yellow and blue, as well as black and white. For example, if a gray area is surrounded by a green ring, the gray area appears to acquire a reddish color. Furthermore, a greenish red or a bluish yellow color does not exist. These observations are supported by findings that neurons activated by green wavelengths are inhibited by red wavelengths. Similarly, neurons excited by blue wavelengths may be inhibited by yellow wavelengths. Neurons with these characteristics are present both in the retina and at higher levels of the visual pathway and seem to serve to increase the ability to see the contrast between opposing colors.

A diagram of the basic circuitry of the retina is shown in

Fig. 8.7 . Several features of this circuitry are noteworthy. Input to the retina is provided by light striking the photoreceptors. The output is carried by axons of the retinal ganglion cells to the brain. Information is processed within the retina by the interneurons. The most direct pathway through the retina is from a photoreceptor to a bipolar cell and then to a ganglion cell (see

Fig. 8.7).

More indirect pathways that provide for intraretinal signal processing involve photoreceptors, bipolar cells, amacrine cells, and ganglion cells, as well as horizontal cells to provide lateral interactions between adjacent pathways.

Observationsoncolorblindnessareconsistentwiththetrichromacytheory.Incolorblindness,ageneticdefect(sex-linkedrecessive),oneormoreconemechanismsarelost.Peoplewithnormalcolorvisionaretrichromatsbecausetheyhavethreeconemechanisms.Individualswholackoneoftheconemechanismsarecalleddichromats.Whenthelong-wavelengthconemechanismisabsent,theresultingconditioniscalledprotanopia;absenceofthemedium-wavelengthsystemcausesdeuteranopia;andabsenceoftheshort-wavelengthsystemcausestritanopia.Monochromatslacktwoormoreconemechanisms.

Rod and cone pathways have several important functional differences in their phototransduction mechanisms and their retinal circuitry. As described previously, rods have more visual pigment and a better signal amplification system than cones do, and there are many more rods than cones. Thus rods function better in dim light (scotopic vision), and loss of rod function results in night blindness. In addition, all rods contain the same visual pigment, so they cannot signal color differences. Furthermore, many rods converge onto individual bipolar cells and the results are very large receptive fields and low spatial resolution. Finally, in bright light, most rhodopsin is bleached, so that rods no longer function under photopic conditions.

Cones have a higher threshold to light and thus are not activated in dim light after dark adaptation. However, they operate very well in daylight. They provide high-resolution vision because only a few cones converge onto individual bipolar cells in cone pathways. Moreover, no convergence occurs in the fovea, where the cones make one-to-one connections to bipolar cells. As a result of the reduced convergence, cone pathways have very small receptive fields and can resolve stimuli that originate from sources very close to each other. Cones also respond to sequential stimuli with good temporal resolution. Finally, cones have three

CHAPTER 8 The Special Senses •Fig. 8.7 Basic Retinal Circuitry. Thearrow attheleftindicatesthedirectionoflightthroughtheretina.Photoreceptors(R)synapseonthedendritesofbipolarcells(B)andhorizontalcells(H)intheouterplexiformlayer.Thehorizontalcellsmakereciprocalsynapticconnectionswithphotoreceptorcellsandareelectricallycoupledtootherhorizontalcells.Bipolarcellsreachsynapseonthedendritesofganglioncells(G)andontheprocessesofamacrinecells(A)intheinnerplexiformlayer.Amacrinecellsconnectwithganglioncellsandotheramacrinecells.

different visual pigments and therefore provide for color vision. Loss of cone function results in functional blindness; rod vision is not sufficient for normal visual requirements.

The receptive field of an individual photoreceptor is circular. Light in the receptive field hyperpolarizes the photoreceptor cell and cause it to release less neurotransmitter. The receptive fields of photoreceptors and retinal interneurons determine the receptive fields of the retinal ganglion cells onto which their activity converges. The characteristics of the receptive fields of retinal ganglion cells constitute an important step in visual information processing because all the information about visual events that is conveyed to the brain is contained in ganglion cell activity.

The bipolar cell, which receives input from a photoreceptor, can have either of two types of receptive fields, as shown in

Fig. 8.8.

Both are described as having a center-surround organization in which the light that strikes the central region of the receptive field either excites or inhibits the cell, whereas the light that strikes a region that surrounds the central portion has the converse effect. The receptive field with a centrally located excitatory region surrounded by an inhibitory annulus is called an on-center, off-surround receptive field (see

Fig. 8.8A ). Bipolar cells with such a receptive field are described as “on” bipolar cells. The other type of receptive field has an off-center, on-surround arrangement, which characterizes “off” bipolar cells (see

Fig. 8.8F

The center response of a bipolar cell receptive field is due to only the photoreceptors that directly synapse with the bipolar cell. Photoreceptor cells respond to light with hyperpolarization and a decrease in glutamate release and respond to the removal of light with depolarization and increased glutamate release. This implies that the difference in the center responses of “on” and “off ” bipolar cells lies in their response to glutamate. In fact, off-center bipolar 136 SECTION2Berne & Levy Physiology A On-center field 0 Sec 0.5 1.0 1.5 Central spot of light Light Peripheral spot F Off-center field B On-center cell responses C Central illumination D Annular illumination E Diffuse illumination 0 Sec 0.5 1.0 1.5 H Central illumination I Annular illumination J Diffuse illumination G Off-center cell responses

Fig. 8.8 Thereceptivefieldsofon-center(A) andoff-center(F) bipolarcellsand,belowthem,thereceptivefieldsofganglioncellsB throughE andG throughJ towhichtheyareconnected.Ganglioncellresponsestocentralspots(upper recording)andperipheralspots(lower recording)areshowninB and

G.

Alsoshownareresponsestocentral(C andH),surround(D andG),anddiffusewhole-field(E and

J)illuminationintheirreceptivefields.Theganglioncellsandtheon-centerandoff-centerbipolarcellsprovidinginputtotheseganglioncellshavesimilarreceptivefields,butwhereasganglioncellsincreaseordecreasetheirspikefrequency,bipolarcellsdepolarizeorhyperpolarize,withoutgeneratingactionpotentials.(RedrawnfromSquireLRetal[eds].Fundamental Neuroscience. SanDiego,CA:AcademicPress;2002.) cells have ionotropic glutamate receptor channels that open in response to glutamate, and thus they are excited by the removal of light stimuli from the center of their receptive field. In contrast, on-center bipolar cells have metabotropic glutamate receptors that close their channels in response to glutamate. They are depolarized by light on the center of their receptive field, because the reduced release of glutamate by the photoreceptors results in more open metabotropic channels. Thus on-center bipolar cells are excited by light stimulation of the center of their receptive fields.

The antagonistic surround response of bipolar cells is due to photoreceptors that surround those that synapse directly on them. These photoreceptors (which also connect directly with their own bipolar cells) synapse with horizontal cells that participate in complex triadic synapses with many photoreceptors and bipolar cells. The pathway through •Fig. 8.9 Relationshipsamongavisualtarget(long arrow, top),imagesontheretinasofthetwoeyes(middle), andprojectionsoftheganglioncellscarryingvisualinformationabouttheseimages(bottom). Thetargetimageissolargethatitextendsintothemonocularsegmentsoftheeyes,whereonesideofitisseenbyonlytheipsilateraleye.Notehowtheaxonsaresortedinthechiasmsothatallinformationabouttheleftvisualfieldofbotheyesisconveyedtotherightsideofthebrainandallinformationabout therightvisualfieldisconveyedtotheleftside.

continued object movement within the field will not be signaled. P cells respond differently to different wavelengths of light. Because there are blue, green, and red cones, many combinations of color properties are possible, but in fact P cells have been shown to have opposing responses only to red and green or only to blue and yellow (a combination of red and green). These mechanisms can greatly reduce the ambiguity of color detection caused by the overlap in cone color sensitivity and may provide a substrate for the opponency process observations.

M cells, on the other hand, respond with phasic bursts of action potentials to the redistribution of light, such as would be caused by the movement of an object within their large receptive fields. M cells are not sensitive to differences in wavelength but are more sensitive to luminance than P cells are.

Thus the output of the retina consists primarily of ganglion cell axons from (1) sustained, linear P cells with small receptive fields that convey information about color, form, and fine details and (2) phasic, nonlinear M cells with larger receptive fields that convey information about illumination and movement. Both exist in on-center and off-center varieties (

Fig. 8.8

The Visual Pathway

Retinal ganglion cells transmit information to the brain by way of the optic nerve, optic chiasm, and optic tract.

Fig.

8.9 shows the relationships among a visual target, the retinal images of the target in the two eyes, and the projections of retinal ganglion cells to the two hemispheres of the brain. The eyes and the optic nerves, chiasm, and tract are viewed from above.

The visual target, an arrow, is in the visual fields of both eyes (see

Fig. 8.9 ) and, in this case, is so long that it extends into the monocular segments of each retina (i.e., one end of the target can be seen by only one eye and the other end by only the other eye). The shaded circle at the center of the target represents the fixation point. The image of the target on the retinas is reversed by the lens system. The left

CHAPTER 8 The Special Senses half of the visual target is imaged on the nasal retina of the left eye and the temporal retina of the right eye. Thus the left visual field is seen by the left nasal retina and the right temporal retina. Similarly, the right half of the visual target is imaged on and seen by the left temporal retina and the right nasal retina. The lens system also causes an inversion in the vertical axis, with the upper visual field imaged on the lower retina and vice versa.

The axons of retinal ganglion cells may or may not cross in the optic chiasm, depending on the location of the ganglion cell in the retina (see

Fig. 8.9 ). Axons from the temporal portion of each retina pass through the optic nerve, the lateral side of the optic chiasm, and the ipsilateral optic tract and terminate ipsilaterally in the brain. Axons from the nasal portion of each retina pass through the optic nerve, cross to the opposite side in the optic chiasm, and then pass through the contralateral optic tract to end in the contralateral side of the brain. As a result of this arrangement, objects in the left field of vision are represented in the right side of the brain, and those in the right field of vision are represented in the left side of the brain.

Retinal ganglion cell axons can synapse in several parts of the brain, but the main target for vision is the lateral geniculate nucleus (LGN) of the thalamus. There is a point-to-point projection from the retina to the LGN. The LGN thus has a retinotopic map. Cells that represent a particular retinal location are aligned along projection lines that can be drawn across the layers of the LGN.

The projection from each eye is distributed to three of the layers of the LGN, one of the magnocellular layers (layers 1 and 2 receive M cell input) and two of the parvocellular layers (layers 3 to 6 receive P cell input). Color-coded ganglion cells project to groups of cells between the major layers, the intralaminar zones. Thus the properties of LGN neurons are very similar to those of retinal ganglion cells. For example, LGN neurons can be classified as P or M cells, and they have on-center or off-center receptive fields.

The LGN also receives input from the visual areas of the cerebral cortex, the thalamic reticular nucleus, and several nuclei of the brainstem reticular formation. The activity of LGN projection neurons is inhibited by interneurons both in the LGN and in the thalamic reticular nucleus. These cells use GABA as their inhibitory neurotransmitter. In addition, the activity of LGN neurons is influenced by corticofugal pathways and by brainstem neurons that transmit signals via monoamine neurotransmitters. These control systems filter visual information and may be important for selective attention.

The LGN projects to the primary visual cortex or striate cortex by way of the visual radiations. The visual radiation fibers carrying information derived from the lower half of the appropriate hemiretinas (and therefore the contralateral upper visual field) project to the lingual gyrus, which lies

Fig.

8.9 ).Forexample,atinylesionintheretinawouldresultinablindspot(scotoma) inthateye,whereasasimilarlesioninthestriatecortexwouldproducecorrespondingscotomasinbotheyes.Interruptionoftheopticnerveononesideproducesblindnessinthateye.Damagetotheopticnervefibersastheycrossintheopticchiasmresultsinlossofvisioninbothtemporalfieldsofvision;thisconditionisknownasbitemporal hemianopsia andoccursbecausethecrossingfibersoriginatefromganglioncellsinthenasalhalvesofeachretina.Alesionoftheentireoptictract,LGN,visualradiation,orvisualcortexononesidecauseshomonymous hemianopsia, whichislossofvisionintheentirecontralateralvisualfield.Partiallesionsresultinpartialvisualfielddefects.Forexample,alesioninthelingualgyruscausesanupperhomonymous quadrantanopsia, whichinthiscaseislossofvisioninthecontralateral,uppervisualfield.

on the medial surface of the occipital lobe, just below the calcarine sulcus. Axons in the visual radiation that represent the contralateral lower visual field project to the adjacent cuneus gyrus, which lies just above the calcarine sulcus. Together, the portions of these two gyri that line and border the calcarine sulcus constitute the primary visual cortex (or Brodmann area 17;

Fig. 8.10

Like the LGN, the striate cortex contains a retinotopic map. The representation of the macula occupies the most posterior and largest part of both gyri, and progressively more peripheral areas of the retina are projected to more anterior parts of these gyri. Overall, there is an orderly mapping of retinal loci across the surface of the striate cortex (see

Fig. 8.10

The geniculostriate pathway ends chiefly in layer 4 of the striate cortex (

Fig. 8.11 ), whereas the projection from the intralaminar LGN terminates in so-called blobs in layers 2 and 3. Similarly, axons that represent one eye or the other terminate within layer 4C in alternate adjacent patches that define ocular dominance columns. Cortical neurons in such a column respond preferentially to input from one eye. Near the border between two ocular dominance columns, neurons respond about equally to input from the two eyes.

The receptive fields of neurons in the striate cortex, aside from the monocular cells in layer 4C, are more complex than those of LGN neurons. Neurons in other layers may be binocular and respond to stimulation of both eyes, although the input from one eye often dominates (see ). In addition, cortical neurons outside layer 4C often show orientation selectivity (i.e., they respond best when the stimulus, such as a bar or an edge, is oriented and positioned in a particular way;

Fig. 8.12 ). These “simple cells” appear to be responding as though they received input from cells whose concentric center-surround receptive fields were arranged in such a way that their “on” centers were aligned in a row flanked by antagonistic regions. “Complex” cortical •Fig. 8.10 Theleftvisualfieldisrelayed(viatheLGNandvisualradiation)totheprimaryvisualcortexoftherighthemisphere,asapoint-to-pointretinotopicmap.Therepresentationofeachpartofvisualspaceisproportionaltothenumberofafferentaxonswithreceptivefieldsinthatpartofspace.Asaresult,theareaofmacularrepresentation(neartheoccipitalpole)islargerthanthatfortherestofthebinocularandmonocularfields.Notethatthelowerhalfofthefieldisrepresentedinthecuneusgyrusabovethecalcarinesulcusandtheupperhalfofthefieldinthelingualgyrusbelowthesulcus.(RedrawnfromPurvesD,etal[eds].Neuroscience. 3rded.Sunderland,MA:Sinauer;2004.) •Fig. 8.11 DiagramofvisualinformationflowintothevisualcortexfromtheLGNanditsprojectiontotheextrastriatecortex,tothesuperiorcolliculus(SC),andbacktotheLGN.M,magnocellularpath;P,parvocellularpath.(RedrawnfromSquireLR,etal[eds].Fundamental Neuroscience. SanDiego,CA:AcademicPress;2002.)LGN m cell LGN p cell LGN intralaminar p cell Cortex Cortex SC LGN 4B 2+3 5 6 4C˜4C°Blobs (2+3) neurons are similar to “simple” cells in that they are orientation specific, but instead of having flanking excitatory and inhibitory zones, they respond best to a particular stimulus orientation anywhere in their receptive field. They may also display direction selectivity; that is, they may respond when the stimulus is moved in one direction but not when it is moved in the opposite direction (see

Fig. 8.12 ). The receptive field of a “complex” cell may be thought of as a composite of adjacent “simple” cells with similar orientation selectivity. Because such neurons in a particular zone of the cortex all tend to have the same orientation selectivity, they are considered to form an orientation column (Fig. 8.13

As already discussed, color vision may depend on the presence in the retina of three different types of cones, as well as neurons in the visual pathway that show spectral opposition. Retinal ganglion cells, LGN neurons, and some P cells display spectral opponent properties. The spectral opponent neurons in the striate cortex are found in cortical blobs, and these show double-opponency, in which both the center and the surround portions respond antagonistically

CHAPTER 8 The Special Senses •Fig. 8.12 Simple and Complex Receptive Fields in the Visual Cortex Can Be Generated From Multiple Inputs With Concentric Fields. A andB representon-centerandoff-centerinput,respectively,fromtheretina.Ifthreeon-centercells(A) withadjacentreceptivefieldsconvergedontoonecorticalneuron(E), thatneuron,asimplecell,wouldrespondbesttoalongbarstimulusataspecificlocationandorientation(C). Forthreeoff-centerinputs(B), theresultingreceptivefieldisshowninD. Theconvergenceofmultiplesimplecellsontoanothercorticalneuron(F) wouldresultinacomplexcellthatrespondsbesttoabarstimuluswithaverticalorientationthatcanbeplacedanywherewithinitsreceptivefield.(RedrawnfromSquireLR,etal[eds].Fundamental Neuroscience. SanDiego,CA:AcademicPress;2002.) to two colors. Such a cell, whose center responds to red but not green (R+G−) and whose surround portion responds to green but not red (R−G+), is shown in

Fig. 8.13A . The relationships between the ocular dominance and orientation columns and the cortical color blobs are shown in

Fig. 8.13B

In animal studies, at least 25 different visual areas have been identified in the cerebral cortex, in addition to the striate cortex (Brodmann area 17, or V1). The extrastriate areas include several parallel pathways of visual processing. The P pathway originates with P cells and functions in the recognition of form and color. Structures in the P pathway include LGN layers 3 to 6, layer 4Cβ of the striate cortex, V4 (Brodmann area 19), and several areas in the inferotemporal region (

Fig. 8.14 ). Processing of form includes recognition of complex visual patterns, such as faces. Color information is processed separately from form. The M pathway originates with M cells and functions in motion detection and control of eye movement. Cortical structures in the M pathway include layers 4B and 4Cα of the striate cortex and areas MT (medial temporal) and

I Cortical blobs, concerned with color Stimulus duration Orientation columns I C C •Fig. 8.13 A, Thereceptivefieldandresponsesofadouble-opponent(R+G−/R−G+)neuroninablobofthestriatecortexasitrespondstovariouscombinationsofredandgreenbars.Thebest“on”responseistoaredbarflankedbytwogreenbars.B, Diagramofthecolumnararrangementofthevisualcortex.OculardominancecolumnsareindicatedbyI(foripsilateral)andC(forcontralateral).Orientationcolumnsareindicatedbythesmallercolumnsmarkedwithshortbarsatvaryingangles.ThecorticalblobscontainneuronslikethatinA andhavespectralopponent-receptivefields.

•Fig. 8.14 DistributionofPandMcellinfluencesondifferentareasofthevisualcortex.IT,inferotemporalarea;MST,medialsuperiortemporalarea;MT,medialtemporalarea;V1,striatecortex;V2andV4,higherordervisualareas.MT7 V4 MST Visual cortex Laminae 3-6 Laminae 1,2 Dorsal LGNM-cell pathway P-cell pathway IT V2 V1

MST (medial superior temporal) on the lateral aspect of the temporal lobe, as well as Brodmann area 7a of the parietal lobe (see

Fig. 8.14

Both P and M pathways contribute to depth perception or stereopsis, which is dependent on slight differences in the retinal images formed in the two eyes. Stereopsis is useful only for relatively nearby objects. However, in such cases, these disparities provide visual cues about depth. Interestingly, the anatomy of the visual pathways indicates that depth perception must be a cortical function because it depends on convergent input from the two eyes and the left/right eye inputs are parallel but segregated in the LGN and in Layer 4 of striate cortex.

The separation of M and P pathways from the retina through the thalamus and all the cortical regions raises the issue of how all the parts are combined to account for the clear, coherent images of events, objects, and persons that humans perceive. It seems unlikely that all the components that represent a percept, such as parts of a face and whether that face belongs to a familiar person, are somehow converged onto a single neuron that recognizes it. Rather, complex percepts probably arise from the coordinated activity of large sets of neurons across multiple regions of the CNS. The process by which a “binding” of such disparate neuronal elements into a percept is unclear, but one working hypothesis is that it may be accomplished by the temporal synchronization of many anatomically distributed neural events.

Lesionsoftheextrastriatevisualcortexcanproducevariousdeficits.Bilaterallesionsoftheinferotemporalcortexcanresultincorticalcolorblindness(achromatopsia) orinaninabilitytorecognizefaces,evenofclosemembersofthefamily(prosopagnosia). AlesioninareaMTorMSTcaninterferewithmotiondetectionandeyemovements.

CHAPTER 8 The Special Senses •Fig. 8.15 Twopuretonesareshownbythesolid anddashed lines. Frequencyisdeterminedfromthewavelengthasindicated.Amplitudeisthepeak-to-peakchangeinsoundpressure.Thetwotoneshavethesamefrequencyandamplitudebutdifferinphase.

The superior colliculus of the midbrain is a layered structure that is important for certain types of eye movements (see ). The three most superficial layers are involved exclusively in visual processing, whereas the deeper layers receive multimodal input from the somatosensory and auditory systems, as well as the visual system, particularly from cortical areas involved in eye movement.

Another retinal projection is to the pretectum, which bilaterally activates parasympathetic preganglionic neurons in the Edinger-Westphal nucleus that cause pupillary constriction in the pupillary light reflex. The pretectal areas are also interconnected through the posterior commissure, and thus the reflex causes both ipsilateral (direct) and contra-lateral (consensual) pupillary constriction when a light is shown in one eye.

The visual pathways also include connections to nuclei that serve functions other than vision. For example, a retinal projection to the suprachiasmatic nucleus of the hypothalamus controls circadian rhythmicity (see

The Auditory and Vestibular Systems

The peripheral parts of the auditory and vestibular systems share components of the bony and membranous labyrinths, use hair cells as mechanical transducers, and transmit information to the CNS through the vestibulocochlear nerve (CN VIII). However, the CNS processing and sensory functions of the auditory and vestibular systems are distinct. The function of the auditory system is to transduce sound. This allows us to recognize environmental cues and to communicate with other organisms. The most complex auditory functions are those involved in language. The function of the vestibular system is to provide the CNS with information related to the position and movements of the head in space. Control of eye movement by the vestibular system is discussed in

Sound is produced by compression and decompression waves in air or in other elastic media, such as water. Sound frequency is measured in cycles per second, or hertz (Hz). Each pure tone results from a sinusoidal wave at a particular frequency and is characterized not only by its frequency but also, instantaneously, by its amplitude and phase (

Fig. 8.15 ). Most naturally occurring sound, however, is a mixture of pure tones. Noise is unwanted sound and may have any composition of pure tones. Sound propagates at about 335 m/sec in air. The waves are associated with certain pressure changes, called sound pressure. The unit of sound pressure is Newtons per meter squared (N/m2), but sound pressure is more commonly expressed as the sound pressure level (SPL). The unit of SPL is the decibel (dB):

Equation 8.1 where P is sound pressure and PR is a reference pressure (0.0002 dyne/cm2, the absolute threshold for human hearing at 1000 Hz). A sound with intensity 10 times greater would be 20 dB; one 100 times greater would be 40 dB.

The normal young human ear is sensitive to pure tones with frequencies that range between about 20 and 20,000 Hz. The threshold for detection of a pure tone varies with its frequency (

Fig. 8.16 ). The lowest thresholds for human hearing are, for pure tones, approximately 3000 Hz. The threshold at these frequencies is approximately −3 to −5 dB, in comparison with the reference 0 dB at 1000 Hz. In reference to this scale, normal speech has an intensity of about 65 dB, and its main frequencies fall in the range of 300 to 3500 Hz. Sounds that exceed 100 dB can damage the peripheral auditory apparatus, and those higher than 120 dB can cause pain and permanent damage. As people age, their thresholds at high frequencies rise, thereby reducing their ability to hear such tones, a condition called presbycusis.

• Fig. 8.16 Sound Threshold Intensities at Different Frequencies. The bottom curve indicates the absolute intensity needed to detect a sound. The dashed curve represents the threshold for functional hearing.Thetop curveindicateslevelsatwhichsoundispainfuland damaging.

The Ear

The peripheral auditory apparatus is the ear, which can be subdivided into the external ear, the middle ear, and the inner ear (Fig. 8.17

The external ear includes the pinna and the external auditory meatus (auditory canal). The auditory canal contains glands that secrete cerumen, a waxy protective substance. The pinna helps direct sounds into the auditory canal and plays a role in sound localization. The auditory canal transmits the sound pressure waves to the tympanic membrane. In humans, the auditory canal has a resonant frequency of about 3500Hz, and this resonance contributes to the low perceptual threshold for sounds in that range.

The external ear is separated from the middle ear by the tympanic membrane (see

Fig. 8.17A ). The middle ear contains air. Three ossicles are present and serve to link the tympanic membrane to the oval window of the inner ear.

and organ of Corti Scala tympani Scala vestibuli • Fig. 8.17 Ear and Cochlear Structure. A, Location of the right human cochlea in relation to the vestibular apparatus, the middle ear, and the external ear. B,Relationshipsbetweentheouter, middle, and inner ear spaces; the cochlea is depicted unrolled for clarity.

CHAPTER 8 The Special Senses 145 • Fig. 8.17, cont’d C, Drawing of a cross-section through the cochlea. The organ of Corti (see

Fig.

8.18A and B ) is outlined.

Adjacent to the oval window is the round window, another membrane-covered opening between the middle ear and inner ear (see

Fig. 8.17A and B

The ossicles include the malleus, the incus, and the stapes. The stapes has a footplate that inserts into the oval window. Behind the oval window is a fluid-filled component of the inner ear, the vestibule. It is continuous with a tubular structure known as the scala vestibuli. Inward movement of the tympanic membrane by a sound pressure wave causes the chain of ossicles to push the footplate of the stapes into the oval window (see

Fig. 8.17B ). This movement of the stapes footplate in turn displaces the fluid within the scala vestibuli. The pressure wave that ensues within the fluid is transmitted through the basilar membrane of the cochlea to the scala tympani (described later), and it causes the round window to bulge into the middle ear.

The tympanic membrane and the chain of ossicles serve as an impedance-matching device. The ear must detect sound waves traveling in air, but the neural transduction mechanism depends on movement in the fluid-filled cochlea, where acoustic impedance is much higher than that of air. Therefore, without a special device for impedance matching, most sound reaching the ear would simply be reflected, as are voices from shore when a person is swimming under water. Impedance matching in the ear depends on (1) the ratio of the surface area of the large tympanic membrane advantage of the lever system formed by the ossicles. This impedance matching is sufficient to increase the efficiency of energy transfer by nearly 30 dB in the range of hearing from 300 to 3500 Hz.

The middle ear also serves other functions. Two muscles are found in the middle ear: the tensor tympani attached to the malleus and the stapedius attached to the stapes. When these muscles contract, they damp movements of the ossicles and decrease the sensitivity of the acoustic apparatus. This action can protect the acoustic apparatus against damaging sounds that can be anticipated. However, a sudden explosion can still damage the acoustic apparatus because reflex contraction of the middle ear muscles does not occur quickly enough. The chamber of the middle ear connects to the pharynx through the eustachian tube. Pressure differences between the external ear and middle ear can be equalized through this passage. If fluid collects in the middle ear, as during an infection, the eustachian tube may become blocked. The resulting pressure difference between the external ear and middle ear can produce painful displacement of the tympanic membrane and, in extreme cases, cause rupture of the tympanic membrane. Unequalized pressure changes as a result of flying or diving can also cause discomfort.

to that of the smaller oval window and (2) the mechanical

The inner ear includes the bony and membranous labyrinths. The bony labyrinth is a complex but continuous series of spaces in the temporal bone of the skull, whereas the membranous labyrinth consists of a series of soft tissue spaces and channels lying inside the bony labyrinth. The cochlea and the vestibular apparatus are formed from these structures. The cochlea is a spiral-shaped organ (see

Fig. 8.17A ). In humans, the spiral consists of 23 4 turns from a broad base to a narrow apex, although its internal lumen is small at the base and wide at the top. The apex of the cochlea faces laterally (see

Fig. 8.17A ). The bony labyrinth component of the cochlea is subdivided into several chambers. The vestibule is the space facing the oval window (see

Fig. 8.17A ). Continuous with the vestibule is the scala vestibuli, the spiral-shaped chamber that extends to the apex of the cochlea, where it meets and merges with the scala tympani at the helicotrema. The scala tympani is another spiral-shaped space that winds back down the cochlea and ends at the round window (see

Fig. 8.17B ). Separating the two, except at the helicotrema, is the scala media enclosed in the membranous labyrinth.

The scala media, or cochlear duct (see

Fig. 8.17B and ), is a membrane-bound spiral tube that extends along the cochlea, between the scala vestibuli and scala tympani. One wall of the scala media is formed by the basilar membrane, another by Reissner’s membrane, and the third by the stria vascularis (see

Fig. 8.17C

The spaces within the cochlea are filled with fluid. The fluid in the bony labyrinth, including the scala vestibuli and scala tympani, is perilymph, which closely resembles cerebrospinal fluid. The fluid in the membranous labyrinth, including the scala media, is endolymph, which is very different from perilymph. Endolymph, generated by the stria vascularis, contains high [K+] (about 145 mM) and low [Na+] (about 2 mm) and has a high positive potential (about +80 mV) with regard to the perilymph. As a result, a very large potential gradient (about 140 mV) exists across the membranes of the hair cell cilia that extend into the endolymph. (These hair cells, which are the sensory receptors for sound, are discussed in more detail later.)

The neural apparatus responsible for transduction of sound is the organ of Corti (see

Fig. 8.17C ), which is located within the cochlear duct. It lies on the basilar membrane and consists of several components, including three rows of outer hair cells, a single row of inner hair cells, a gelatinous tectorial membrane, and a number of types of supporting cells. The organ of Corti in humans contains 15,000 outer and 3500 inner hair cells. The rods of Corti help provide a rigid scaffold. Located on the apical surface of the hair cells are stereocilia, which can be described as nonmotile cilia that contact the tectorial membrane.

The organ of Corti is innervated by nerve fibers of the cochlear division of the vestibulocochlear nerve (CN VIII). The 32,000 auditory afferent fibers in humans originate in sensory ganglion cells in the spiral ganglion. These nerve fibers penetrate the organ of Corti and terminate at the bases of the hair cells (

Fig. 8.18

Fig. 8.17C ). Approximately 90% of the fibers end on inner hair cells, and the remainder end on outer hair cells. Thus approximately 10 afferent fibers supply each inner hair cell, whereas other afferent fibers diverge to supply about five outer hair cells each. The inner hair cells clearly provide most of the neural information about acoustic signals that the CNS processes for hearing. The sensory function of the outer hair cells is less clear.

In addition to afferent fibers, the organ of Corti is supplied by efferent fibers, most of which terminate on the outer hair cells. These cochlear efferent fibers originate in the superior olivary nucleus of the brainstem and are often called olivocochlear fibers. The length of the outer hair cells varies; this characteristic suggests that changes in outer hair cell length may affect the sensitivity, or “tuning,” of the inner hair cells. The cochlear efferent fibers may control outer hair cell length. Such a mechanism could conceivably influence the sensitivity of the cochlea and the way that the brain recognizes sound. Other efferent fibers that end on cochlear afferent fibers may be inhibitory, and they may help improve frequency discrimination.

Sound is transduced by the organ of Corti. Sound waves that reach the ear cause the tympanic membrane to oscillate, and these oscillations are transmitted to the scala vestibuli by the ossicles. This creates a pressure difference between the scala vestibuli and the scala tympani (see

Fig. 8.17B ) that serves to displace the basilar membrane and, with it, the organ of Corti (see

Fig. 8.18A and B ). Because of the shear forces set up by the relative displacement of the basilar and tectorial membranes, the stereocilia of the hair cells bend. Upward displacement bends the stereocilia toward the tallest cilium, which depolarizes the hair cells; downward deflection bends the stereocilia in the opposite direction, which hyperpolarizes the hair cells.

Acommoncauseofdeafnessisthedestructionofhaircellsbyloudsounds.Haircellscanbedestroyed,forexample,byexposuretoindustrialnoiseorbylisteningtoloudmusic.Typically,haircellsincertainpartsofthecochleaareselectivelydamagedbyexposuretohighlevelsofsoundatparticularfrequencies(aspredictedbytheplacetheory),andthushearingmaybelostoveradiscretefrequencyrange.Presbycusis,orthelossofhigh-frequencyhearingwithage,isprobablyincreasedbythelossofhaircellsasaresultoflong-termnoiseexposureinurbanenvironments.

In view of the wide range of frequencies and amplitudes of sound stimuli, it is no surprise that hair cell transduction must provide for a fast response. The fast response to deflection of the cilia is based on direct opening of ion channels by so-called tip links that connect the tip of each stereocilium with the shaft of the next taller one (see

CHAPTER 8 The Special Senses •Fig. 8.18 DetailoftheorganofCortiatrest(A) andwithupwardmovementofthebasilarmembrane(B). Theupwardmovementcausesthestereociliatobendbecauseofshearforcesproducedbyrelativedisplacementofthehaircellsandthetectorialmembrane.C, Diagramofahaircellwithtiplinkconnectionsbetweenthehaircellciliatoshowhowshearforcesopenmechanoreceptorchannelsand depolarizethehaircell.

Fig. 8.18C ). With deflection, the tip links are subjected to a lever action that transiently opens the channels, allows the entry of K+ (because of the high [K+] and high potential in endolymph), and depolarizes the hair cell. Several mechanisms have been proposed to account for the equally important rapid adaptation necessary for a high-frequency response. A “spring” response by the tip links would allow the attachment point of the tip link to be moved along the stereocilium’s shaft to reset the mechanical leverage of the tip link. In addition, it has been observed that Ca++ can enter and bind to the open channel, change it to require greater opening force, and thereby reduce the statistical probability of opening.

The potential gradient that induces movement of ions into hair cells includes both the resting potential of the hair cells and the positive potential of the endolymph. As noted previously, the total gradient across the apical membrane of hair cells is about 140 mV. Therefore, a change in K+ conductance in the apical membranes of hair cells results in a rapid current flow that produces the receptor potential in these cells. This current flow can be recorded extracellularly as a cochlear microphonic potential, an oscillatory event that has the same frequency as the acoustic stimulus. The cochlear microphonic potential represents the sum of the receptor potentials of a number of hair cells.

Hair cells, like retinal photoreceptors, release an excitatory neurotransmitter (probably glutamate) when depolarized. The neurotransmitter produces an excitatory postsynaptic potential (EPSP) in the cochlear afferent nerve fibers with which the hair cell synapses. In summary, sound is transduced when oscillatory movements of the basilar membrane cause transient changes in the transmembrane voltage of the hair cells and, finally, the generation of action potentials in cochlear afferent nerve fibers. The activity of a large number of cochlear afferent fibers in the auditory nerve can be recorded extracellularly as a compound action potential.

On the basis of differences in width and tension, investigators originally concluded that different parts of the basilar membrane have different resonant frequencies.

•Fig. 8.19 Different Frequencies of Sound Result in Different Amplitudes of Displacement at Different Sites Along the Organ of Corti. A, Travelingwaveproducedinthebasilarmembranebyasoundof200Hz.Thecurvesata,b,c,anddrepresentdisplacementofthebasilarmembraneatdifferenttimes,andthedashed line istheenvelopeformedbythepeaksofthewaveatdifferenttimes.Maximumdeflectionoccursatabout29mmfromtheovalwindow.B, Envelopesoftravelingwavesproducedbyseveralfrequenciesofsound.Notethatthemaximumdisplacementvarieswithfrequencyandisclosesttothestapeswhenthefrequencyishighest.(RedrawnfromvonBekesyG.Experiments in Hearing. NewYork:McGraw-Hill;1960.)

For example, the basilar membrane is about 100 µm wide at the base and 500 µm wide at the apex. It also has higher tension at the base. Thus, the investigators predicted that the base would vibrate at higher frequencies than would the apex, as do the shorter strings of musical instruments. Experiments have shown that the basilar membrane moves as a whole in traveling waves (

Fig. 8.19 ), but displacement of the basilar membrane is maximal nearer the base of the cochlea during high-frequency tones and maximal nearer the apex during low-frequency tones.

In effect, the basilar membrane serves as a frequency analyzer; it distributes the stimulus along the organ of Corti, and different hair cells respond differentially to particular 50 Hz frequencies of sound. This is the basis of the place theory of hearing. In addition, hair cells located at different places along the organ of Corti may be tuned to different frequencies because of variations in their stereocilia and biophysical properties. As a result of these factors, the basilar mem brane and organ of Corti have a so-called tonotopic map (

Fig. 8.20).

Neurotransmitter release by hair cells in the organ of Corti can evoke action potentials in the primary afferent fibers of the cochlear nerve. Afferent fibers in the vestibulocochlear nerve (CN VIII) are bipolar cells with a myelin sheath around the cell bodies, as well as around the axons. The cell bodies are in the spiral ganglion, their peripheral processes synapse at the base of hair cells, and their central processes 400 Hz synapse in the cochlear nuclei of the brainstem.

acteristic frequency. The characteristic frequency can be determined from a tuning curve for the fiber (

Fig. 8.21 ). A tuning curve is a plot of the threshold for activation of the nerve fiber by different sound frequencies. The major factor that influences the activity of individual afferent fibers is the location along the basilar membrane of the hair cells that they innervate. The location of those hair cells is important because for any given sound frequency, there is Distance from stapes (mm) a site of maximum displacement of the basilar membrane B as the pressure wave travels along its length (see

Fig. 8.19 ). Typically, tuning curves are sharp near the characteristic

CHAPTER 8 The Special Senses

Base20,0007,0005,0001,0004,0003,0006004002002,0001,500800Apex •Fig. 8.20 Thetonotopicmapofthecochlea.(RedrawnfromStuhlmanO.An Introduction to Biophysics. NewYork:JohnWiley&Sons;1943.) frequency, but they broaden at high sound pressure levels. Tuning curves can have excitatory and inhibitory areas (see

Fig. 8.21A ). The sharpness of the excitatory regions may reflect inhibitory processes.

The different features of an acoustic stimulus are encoded in the discharges of cochlear nerve fibers. Duration is signaled by the duration of activity; intensity is signaled both by the amount of neural activity and by the number of fibers that discharge. For low-frequency sounds, the frequency is signaled by the tendency of an afferent fiber to discharge in phase with the stimulus (phase locking; see

Fig. 8.22A ). If the tone is much more than 1 kHz, a single fiber cannot discharge with every cycle, but phase locking can also occur for sounds with periods shorter than the absolute refractory period of the afferent fiber. This allows the CNS to detect higher frequency information from the activity of a population of afferent fibers, each of which discharges in phase with the stimulus and which, as a group, signal the frequency of the stimulus (see

Fig. 8.22B ). This observation is the basis of the frequency theory of hearing.

For still higher frequencies (>5000 Hz), the place theory dominates: the CNS interprets sounds that activate afferent fibers supplying hair cells near the base of the cochlea as being of high frequency. Thus both the place and the frequency theories are necessary to explain the frequency coding of sound (duplex theory) across the entire range from 20 to 20,000 Hz.

2 A 7 10 154 –80 –60 –40 –20 0 Frequency (kHz) E I I Threshold intensity (dB) B 0.2 1 2052 100.5 Frequency (kHz) –100 –80 –60 –40 –20 0 Threshold intensity (dB) •Fig. 8.21 Tuning Curves of Neurons in the Auditory System. Tuningcurvescanbeconsideredasreceptivefieldplots.A, Tuningcurvewithcentralexcitatoryfrequencies(E)andflankinginhibitoryfrequencies(I).B, Tuningcurvesforcochlearnervefibers.(A, RedrawnfromArthurRM,etal.J Physiol [Lond] 1971;212:593.B, RedrawnfromKatsuiY.In:RosenblithWA[ed].Sensory Communication. Cambridge,MA:MITPress;1961.)

Animportant,althoughrelativelyuncommon,conditionthatcaninterruptthefunctionofcochlearnervefibersisanacoustic neuroma, atumorofSchwanncellsofthevestibulocochlearnerve(CNVIII).Asthetumorgrows,irritationofcochlearnervefibersmaycausearingingsoundintheaffectedear(tinnitus). Eventually,conductionincochlearnervefibersisblocked,andtheearbecomesdeaf.Thetumormaybeoperablewhilestillsmall;therefore,earlydiagnosisisimportant.Ifthetumorisallowedtoenlargesubstantially,itcouldinterrupttheentirevestibulocochlearnerveandcausevestibularaswellasauditorydifficulties.Itcouldalsoimpingeonordistortneighboringcranialnerves(e.g.,V,VII,IX,andX),anditcouldproducecerebellarsignsbycompressingthecerebellarpeduncles.

•Fig. 8.22 A, Atlowfrequencies,individualauditoryafferentfiberscanrespondateachcycletothesignalfrequency.B, Athigherfrequencies,eachafferentfibergeneratesanactionpotentialonlyatcertaincycles,limitedbyitsmaximumfiringfrequency.However,theoverallpopulationofafferentfiberscanstillsignalstimulusfrequencybytheiraggregatefiringfrequency.

Cochlear afferent fibers synapse on neurons of the dorsal and ventral cochlear nuclei. The neurons in these nuclei have axons that contribute to the central auditory pathways. Some of the axons from the cochlear nuclei cross to the contralateral side and ascend in the lateral lemniscus, the main ascending auditory tract. Others connect with various ipsilateral or contralateral nuclei, such as the superior olivary nuclei, which project through the ipsilateral and contralateral lateral lemnisci. Each lateral lemniscus ends in an inferior colliculus. Neurons of the inferior colliculus project to the medial geniculate nucleus of the thalamus, which gives rise to the auditory radiation. The auditory radiation ends in the primary auditory cortex (Brodmann areas 41 and 42), located on the superior surface of the temporal lobe.

The input from each ear is bilaterally represented in the ascending auditory system pathway at the level of the lateral lemniscus and above. Thus the representation of auditory space is complex, even at the brainstem level. Consequently, unilateral deafness may occur with isolated lesions of the cochlear nuclei or more peripheral structures. Central lesions do not cause unilateral deafness, although they may interfere with overall sensitivity to speech or with sound localization.

Functional Organization of the Central Auditory System

The responses of neurons in several structures that belong to the auditory system can be described by tuning curves (see

Fig. 8.21B ). By plotting the distribution of the characteristic frequencies of neurons within a nucleus or in the auditory cortex, a tonotopic map may be revealed in which neurons are ordered according to their “best” frequencies. Tonotopic maps have been found in the cochlear nuclei, superior olivary complex, inferior colliculus, medial geniculate nucleus, and auditory cortex. A given auditory structure may, in fact, contain several tonotopic maps.

Most auditory neurons at levels above the cochlear nuclei respond to stimulation of either ear (i.e., they have binaural receptive fields). Binaural receptive fields contribute to sound localization. A human can distinguish sounds originating from sources separated by as little as 1 degree. The auditory system uses several clues to judge the origin of sounds, including differences in the time (or phase) of arrival of the sound at the two ears and differences in sound intensity on the two sides of the head.

For example, neurons in the medial superior olivary nucleus have medial and lateral dendrites. The synapses on the medial dendrites are largely excitatory, and they originate from the contralateral ventral cochlear nucleus. Those on the lateral dendrites are mostly inhibitory and come from the ipsilateral ventral cochlear nucleus. Differences in the phase of the sound reaching the two ears affect the strength and timing of the excitation and inhibition reaching a particular medial superior olivary neuron. The lateral superior olivary nucleus processes differences in the sound intensity that reaches the two ears to provide information about the source of the sound. The activity of superior olivary neurons can provide information about sound localization.

Several features of the primary auditory cortex resemble those of other primary sensory areas. Not only are sensory maps—in this case, tonotopic maps—present in the auditory cortex but also this cortical region performs feature extraction. Neurons in the primary auditory cortex form isofrequency columns (in which the neurons in the column have the same characteristic frequency), and they also form alternating columns, known as summation and suppression columns. Neurons in summation columns are more responsive to binaural than to monaural input. Neurons in suppression columns are less responsive to binaural than to monaural stimulation, and, accordingly, the response to one ear is dominant. Some neurons are selective for the direction of frequency change.

Bilateral lesions of the auditory cortex have some effect on the ability to distinguish the frequency or intensity of different sounds, and they reduce the abilities to localize sound and to understand speech. Unilateral lesions, however, have little effect, especially if the nondominant (for language) hemisphere is involved (see ). Evidently, frequency discrimination depends on activity at lower levels of the auditory pathway, possibly the inferior colliculus.

Twosimpletestsareoftenusedclinicallytodistinguishthemostimportanttypesofdeafness:conduction loss andsensorineural loss. Conductionhearinglossoccursindisordersoftheexternalear(e.g.,earcanalblockedbycerumen)ormiddleear(e.g.,ruptureoftheeardrum).Sensorineuralhearinglossreflectsdisordersoftheinnerear,thecochlearnerve,orcentralconnections.

TheWeber test isusedtoevaluatethemagnitudeofconductionhearingloss.Inthistest,thebaseofavibratingtuningforkisplacedagainstthemiddleoftheperson’sforehead,andthepersonisaskedtolocalizethesound.Normally,thesoundisnotlocalizedtoaparticularear.However,ifthepersonhasconductivehearingloss(e.g.,duetoapuncturedtympanicmembrane,fluidinthemiddleear,otosclerosis,orlossofcontinuityoftheossicularchain),thesoundislocalizedtothedeafearbecauseitisconductedtothecochleathroughbone.Thesoundisalsoconductedtothecochleaoftheundamagedear,butbone-conductedsounddoesnotactivatetheorganofCortiaswellasdoessoundconductednormallythroughthetympanicmembraneandossiclechain.OnereasonwhythesoundintheWebertestisnotlocalizedtothenormalearmaybethathearinginthenormalearisinhibitedbytheambientsoundlevel(auditory masking). Conversely,inpeoplewithsensorineuralhearingloss(e.g.,duetodamagetotheorganofCorti,thecochlearnerve,orthecochlearnuclei),thesoundislocalizedtothenormalside.

IntheRinne test, avibratingtuningforkisplacedagainstthebonebehindtheperson’sear,andthepersonisaskedtoindicatewhenthesounddiesout.Thetuningforkisthenheldneartheexternalauditorymeatusofthatear.Inpeoplewithnormalhearing,thesoundisagainheardbecausethesoundismoreeffectivelytransmittedtothecochleainair(i.e.,airconduction> boneconduction).Iftheconductionmechanismisdamaged,thesoundisnotheardwhenthetuningforkisheldneartheexternalauditorymeatus.Boneconductioninthiscaseisbetterthanairconduction.Ifthehearinglossissensorineural,thesoundisheardagainwhenthetuningforkisplacedbytheexternalauditorymeatusbecausewithsensorineuralhearingloss,theinnerearandcochlearnervearelessabletotransmitimpulsesregardlessofwhetherthesoundvibrationsreachthecochleaviaairorbone.Thusbecauseairconductionismoreeffectivethanboneconduction,theboneconductionpatternseenwithsensorineuralhearinglossisthesameasinanormalear.

The Vestibular System

The vestibular system detects angular and linear accelerations of the head. Signals from the vestibular system allow the body to make adjustments in posture that maintain balance and trigger head and eye movements to stabilize the visual image on the retina. The following description of the vestibular system emphasizes the sensory aspects of vestibular function, and it introduces the central vestibular pathways. The role of the vestibular apparatus in motor control is discussed in

The Vestibular Apparatus

Structure of the Vestibular Labyrinth

The vestibular apparatus, like the cochlea, consists of a component of the membranous labyrinth located within

CHAPTER 8 The Special Senses the bony labyrinth. The vestibular apparatus on each side is composed of three semicircular canals and two otolith organs (

Fig. 8.23

Fig. 8.17A).

These structures contain endolymph and are surrounded by perilymph. The semicircular canals are named the horizontal, anterior, and posterior canals. The otolith organs are the utricle and the saccule (together indicated as “Vestibule” in

Fig. 8.17A ). Each semicircular canal has a swelling called an ampulla at the point where it joins the utricle. The saccule connects with the cochlea, through which endolymph (produced by the stria vascularis of the cochlea) can reach the vestibular apparatus.

The three semicircular canals on one side are matched with corresponding coplanar semicircular canals on the other side. The horizontal canals on each side of the head correspond, as do the anterior canal on one side and the posterior canal on the other side (see

Fig. 8.23B ). This arrangement allows the sensory epithelia, in corresponding pairs of canals on the two sides, to cooperate in sensing acceleration of the head about three nearly orthogonal axes in space. The horizontal canals are not truly horizontal; rather, they lie in the horizontal plane if the head is tilted down 30 degrees in relation to the horizon.

The ampulla of each of the semicircular canals contains a sensory epithelium called a crista ampullaris, or ampullary crest (

Fig. 8.24 ). An ampullary crest consists of a ridge, transverse to the long axis of the canal, that is covered by epithelium containing vestibular hair cells. These hair cells are innervated by primary afferent fibers of the vestibular nerve, which is a subdivision of the vestibulocochlear nerve (CN VIII).

Like cochlear hair cells, each vestibular hair cell contains a set of stereocilia on its apical surface. However, unlike cochlear hair cells, vestibular hair cells also contain a large single kinocilium. The cilia on ampullary hair cells are embedded in a gelatinous structure called the cupula. The cupula and the crista occlude the lumen of the ampulla completely. Movement of endolymph, produced by angular acceleration of the head about an axis perpendicular to the plane of the canal, deflects the cupula and consequently bends the cilia on the hair cells. The cupula has the same specific gravity as endolymph, and thus it is unaffected by linear acceleratory forces, such as gravity.

The sensory epithelia of the otolith organs are called the macula utriculi and the macula sacculi (

Fig. 8.25 ). The utricle is oriented nearly horizontally; the saccule is oriented vertically. Their hair cells are embedded in the epithelium that overlies each macula. As in the ampullary crests, the stereocilia and kinocilia of the macula project into a gelatinous mass. However, the gelatinous mass in the macula contains numerous otoliths (“ear stones”) composed of calcium carbonate crystals. Together, the gelatinous mass and its otoliths are known as an otolithic membrane. The otoliths increase the specific gravity of the otolithic membrane to about twice that of the endolymph. Hence, the otolithic membrane tends to move when subjected to acceleration, whether linear (such as that produced by gravity) or angular, particularly when the center of rotation is outside the head.

• Fig. 8.23 A, Lateral view of the right semicircular canals of a rhesus monkey that were dissected after being filled with plastic. Note the ampullae associated with each canal. Scale is in millimeters. B,Overheadviewofthebaseoftheskullshowingtheorientationofstructuresoftheinnerear.Coplanar pairsofsemicircularcanalsincludethehorizontalcanals,aswellastheanteriorandcontralateralposterior canals.(A,CourtesyofDr.JohnSimpson,New YorkUniversity SchoolofMedicine.B,Redrawnfrom Haines DE [ed]. Fundamental Neuroscience for Basic and Clinical Applications. 3rd ed. Philadelphia: ChurchillLivingstone;2006.) •Fig. 8.24 Drawing of an Ampullary Crest Inside an Ampulla.Thestereocilia andthekinocilium of eachhaircellextendintothecupula,whichextendsacrosstheentirecross-sectionoftheampulla.Head movement(acceleration)totherightwouldresultinendolymphpressuretotheleftanddeflectionofthe cupulatotheleft.

Innervation of Sensory Epithelia of the  Vestibular Apparatus

The cell bodies of the primary afferent fibers of the vestibular nerve are located in the Scarpa ganglion. The neurons are bipolar, and their cell bodies, as well as axons, are myelinated. Peripherally, the vestibular nerve gives off separate • Fig. 8.25 Structure of one of the otolith organs, the saccule. Note the orderly variation in kinocilium orientation, as well as their mirror symmetry with regard to the striola. (Redrawn from Lindeman HH. Adv Otorhinolaryngol 1973;20:405.)

CHAPTER 8 The Special Senses 153 branches to each of the vestibular epithelia; centrally, it accompanies the cochlear and facial nerves as they enter the internal auditory meatus of the skull.

Like cochlear hair cells, vestibular hair cells are functionally polarized, and the transduction mechanism is presumed to be similar. When the stereocilia are bent toward the longest cilium (in this case, the kinocilium), conductance of the apical membrane increases for cations and, because of the high K+ concentration of the endolymph, K+ enters, and the vestibular hair cell is depolarized (

Fig. 8.26 ). Conversely, when the cilia are bent away from the kinocilium, the hair cell is hyperpolarized. The hair cell releases an excitatory neurotransmitter (either glutamate or aspartate) tonically, so that the afferent fiber on which it synapses has a resting discharge. When the hair cell is depolarized, more neurotransmitter is released, and the discharge rate of the afferent fiber increases. Conversely, when the hair cell is hyperpolarized, less neurotransmitter is released, and the firing rate of the afferent fiber slows.

Angular accelerations of the head produce minute movement of the endolymph in relation to the head (

Fig. 8.27 ). This happens because the inertia of the endolymph causes it to resist the initial acceleration of the membranous labyrinth. This lag pushes on the cupula, causes the cilia to bend, and consequently changes the discharge rates of the vestibular afferent fibers. All the cilia in a given ampullary crest are oriented in the same direction. In the horizontal canal, the cilia are oriented toward the utricle, and in the other ampullae, they are oriented away from  the utricle.

• Fig. 8.26 Functional Polarization of Vestibular Hair Cells. When the stereocilia are bent toward the kinocilium, the hair cell is depolarized, and the afferent fiber is excited. When the stereocilia are bent away from the kinocilium, the hair cell is hyperpolarized, and the afferent discharge slows or stops. (Redrawn from Kandel ER, Schwartz JH. Principles of Neural Science. New York: Elsevier; 1981.)

Turning motion of head

AmpullaAxis of

Axis of hair cells •Fig. 8.27 Effect of Leftward Head Movement on the Activity of Vestibular Afferent Fibers Supplying Hair Cells in the Horizontal Semicircular Canals. The perspective in the figure is from above the head looking down. In this case, head rotation (acceleration) to the left causes endolymph pressure to the right and the result is increased output from the left canal and decreased output from the right canal. Small arrows intheampullaindicatethefunctionalpolarityofthehaircells.Thelarge curved arrow atthetopindicatesmovementofthehead;smaller curved arrows indicaterelativemovementoftheendolymph.

The way in which angular acceleration of the head affects the discharge of vestibular afferent fibers is exemplified by the activity that originates from the horizontal canals.

Fig.

8.27 shows the horizontal canals and utricle, as seen from above as the head is rotated (accelerated) to the left. As acceleration to the left begins, the inertia of the endolymph in the horizontal canals increases pressure toward the right side. This causes the cilia to bend on hair cells of the ampulla of the left horizontal canal toward the utricle and bends the cilia of the right canal away from the utricle. These actions increase the firing rate in the afferent fibers on the left and decrease the firing rate of the afferent fibers on the right. Once the head is moving at a constant velocity of rotation (i.e., no acceleration), there would be no force on either cupula, and therefore the hair cells of both canals would be firing as they do at rest. However, when the indicated rotation is stopped, the inertia of the endolymph creates a force on both cupulas, but in the direction opposite to that caused by the original acceleration. This results in an increase in the discharge rate of afferent fibers on the right side and a decrease in the discharge rate on the left. This postrotatory effect is of functional and clinical significance.

Unlike the hair cells in the ampullary crests, not all the hair cells in the otolith organs are oriented in the same •Fig. 8.28 Functional Polarization of Hair Cells in the Otolith Organs. A, Thesaccule.B, Theutricle.Thestriolaineachcaseisindicatedbythedotted line. (RedrawnfromSpoendlinHH.In:WolfsonRJ[ed].The Vestibular System and Its Diseases. Philadelphia:UniversityofPennsylvaniaPress;1966.)

Irritationofthevestibularlabyrinth,asinMeniere’s disease, canresultinrhythmicconjugatedeviationsoftheeyes,followedbyquickreturnsaccades.Thisconditionisknownasnystagmus (see

Chapter9).Theseeye movementsareaccompaniedbyasenseofvertigo andoftennausea. Thebraininterpretsadifferenceintheinputfromthetwosidesofthevestibularsystemasheadmotion.Irritation(ordestruction)ofonelabyrinthproducesanasymmetryofinputthatresultsinabnormaleyemovementandassociatedpsychologicaleffects.

direction. Instead, they are oriented in relation to a ridge, called the striola, along the otolith organ (see

Fig. 8.25 ). In the utricle, the hair cells on either side of the striola are polarized toward the striola, whereas in the saccule they are polarized away from the striola. Because the striola in each otolith organ is curved, there are hair cells with all orientations in the plane of the organ (

Fig. 8.28 ). In any particular orientation of the head, the cilia of the hair cells are bent to varying extents according to their orientation in relation to the gravitational vector. This results in a particular pattern of input from the otolith organs to the CNS. When the head is tilted to a new position, the orientation of the otolithic membranes in relation to the gravitational vector changes, and so the cilia of the hair cells are bent in a new way. This change in the bending of the cilia of the hair cells changes the pattern of input from the otolith organs to the CNS and creates the sensation of movement, as well as possibly triggering various reflexes. Similarly, a linear acceleration caused by other forces, such as might occur in a fall or the angular acceleration when a car turns around a curve (angular accelerations have linear centripetal and instantaneous tangential components), also affects output from the otolith organs.

The vestibular afferent fibers project to the brainstem through the vestibular nerve. As previously mentioned, the cell bodies of these afferent fibers are located in the Scarpa ganglion. These primary afferent fibers terminate in the vestibular nuclei (see

Fig. 4.6

D to E), which are located in the rostral medulla and caudal pons and in specific regions of the cerebellum, most prominently in the nodulus.

The vestibular nuclei give rise to various projections, including projections through the medial longitudinal fasciculus (see

Fig. 4.6C to E ) to the oculomotor nuclei. Therefore, it is not surprising that the vestibular nuclei exert powerful control over eye movements (the vestibule-ocular reflex). Other projections give rise to the lateral and medial vestibulospinal tracts, which, respectively, provide for the activation of trunk and neck muscles and thereby contribute to equilibrium and to head movements (vestibulocolic reflex). There are vestibular pathways to the cerebellum, the reticular formation, and the contra-lateral vestibular complex, as well as to the thalamus. The pathway to the thalamus, via a projection to the cerebral cortex, mediates conscious sensation of vestibular activity. Vestibular reflexes and clinical tests of vestibular function are described in

The Chemical Senses

The senses of gustation (taste) and olfaction (smell) help detect chemical stimuli that are present either in food and drink or in the air. In the evolution of humans, these chemical senses apparently did not have the survival value of some of the other senses, but they contribute considerably to quality of life and food selection, and they are important stimulants of digestion. In other animals, the chemical senses have greater survival value, and their activation evokes a number of social behaviors, including mating, territoriality, and feeding.

The stimuli that we commonly know as tastes are actually mixtures of five elementary taste qualities: salty, sweet, sour, bitter, and umami.

Taste stimuli that are particularly effective in eliciting these sensations are, respectively, sodium chloride, sucrose, hydrochloric acid, quinine, and mono-sodium glutamate. Umami has been described as having a proteinaceous, meaty character.

bThe existence of a sixth, taste, fat (free fatty acids), is currently being debated.

CHAPTER 8 The Special Senses

The sensation of taste depends on the activation of chemoreceptors located in taste buds. A taste bud consists of a group of 50 to 150 receptor cells, as well as supporting cells and basal cells (Fig. 8.29A ). The chemoreceptor cells synapse at their bases with primary afferent nerve fibers, and their apices have microvilli that extend toward a taste pore. Chemoreceptor cells live only about 10 days. They are continuously replaced by new chemoreceptor cells that differentiate from basal cells located near the base of the taste bud.

Chemoreceptor molecules, each specialized for one type of taste stimulus, sit on the microvilli of chemoreceptor cells and detect molecules that diffuse into the taste pore from the overlying mucus of the tongue, part of which originates from glands adjacent to the taste buds. Some stimuli can pass directly into the cell to depolarize it (Na+ for salty and H+ for sour) or open cation channels to generate a receptor potential (also salty and sour), whereas others (sucrose, quinine, and glutamate for sweet, bitter, and umami) activate a second messenger that can either open cation channels or directly activate intracellular Ca++ stores (see

Fig. 8.29B ). In each case, depolarization of the receptor results in the release of an excitatory neurotransmitter and, consequently, action potentials in the primary afferent nerve fiber that are transmitted to the CNS.

Coding of taste, however, is not based entirely on the selectivity of the chemoreceptors for the different primary qualities because each cell responds to a range of stimuli, although most intensely to one. Because most natural tastes have chemicals that effect responses from a number of chemoreceptors, recognition of taste quality appears to depend on the patterned input from a population of chemoreceptors, each responding differentially to the components of the stimulus. The intensity of the stimulus is reflected in the total amount of activity evoked.

Distribution and Innervation of Taste Buds

Taste buds are located on different types of taste papillae found on the tongue, palate, pharynx, and larynx. Types of taste papillae include fungiform and foliate papillae on the anterior and lateral aspects, respectively, of the tongue and circumvallate papillae on the base of the tongue (see

Fig. 8.29C).

The circumvallate papillae may contain several hundred taste buds. The tongue in humans may have several thousand taste buds. The sensitivity of different regions of the tongue for different taste qualities varies slightly because taste buds responding to each type of taste are widely distributed. The taste buds are innervated by three cranial nerves. The chorda tympani branch of the facial nerve (CN VII) innervates taste buds on the anterior two thirds of the tongue, and the glossopharyngeal nerve (CN IX) innervates taste buds on the posterior third of the tongue (see

Fig. 8.29C ). The vagus nerve (CN

X) innervates a few taste buds in the larynx and upper esophagus.

The cell bodies of taste fibers in cranial nerves VII, IX, and X are located in the geniculate, petrosal, and nodose ganglia, respectively. The central processes of the afferent fibers enter the medulla, join the solitary tract, and synapse in the nucleus of the solitary tract (see

Fig. 4.6

D to E). In some animals, including several rodent species, the second-order taste neurons of the solitary nucleus project rostrally to the ipsilateral parabrachial nucleus. The parabrachial nucleus then projects to the small-celled (parvocellular) part of the ventroposterior medial (VPMpc) nucleus of the thalamus. In monkeys, the solitary nucleus projects directly to the VPMpc nucleus. The VPMpc nucleus is connected to two different gustatory areas of the cerebral cortex: one in the face area of the S1 cortex and the other in the insula. An unusual feature of the central gustatory pathway is that it is predominantly an uncrossed pathway (unlike the central somatosensory pathways, which are predominantly crossed).

The sense of smell is much better developed in some animals (macrosmatic animals) than in humans. The ability of dogs to track other animals on the basis of odor is legendary, as is the use of pheromones by insects to attract mates. However, olfaction contributes to humans’ emotional life, and odors can effectively conjure up memories. It also helps

Release of • Fig. 8.29 A, A taste bud is shown with the taste pore at the top and its innervation below. B, basal cell; R, ciliated taste receptor cells; S, supporting cells. B, Taste receptor cell showing second messenger, ligand-gated, and direct depolarization resulting in depolarization of the cell.

• Fig. 8.29, cont’d C, Distribution of the taste buds on the tongue and their innervation. (Redrawn from Squire LR, et al [eds]. Fundamental Neuroscience. San Diego, CA: Academic Press; 2002.) people avoid consuming spoiled food and detect dangerous situations. For example, an unpleasant odorant is added to odorless, colorless natural gas so that people can easily detect a leak.

Odor has more primary qualities than taste does.

As many as 1000 different odor receptors are coded in the human genome, and although only approximately 350 types are functional, they represent the largest population of G protein–coupled receptors in the genome. The olfactory mucosa also contains somatosensory receptors of the trigeminal nerve. When performing clinical tests of olfaction, clinicians must avoid activating these somatosensory receptors with thermal or noxious stimuli, such as the ammonia used in “smelling salts.”

The olfactory chemoreceptor cells are located in the olfactory mucosa, a specialized part of the nasopharynx. Olfactory chemoreceptors are bipolar nerve cells (

Fig. 8.30 ). The cThe conscious perception of flavor, particularly of foods, is the result of both olfactory and gustatory input based on directly inhaled odor, taste from the food as it is macerated in the mouth, and retronasal odor from the volatile molecules that are released by maceration and pass up into the nasal cavity from the pharynx.

CHAPTER 8 The Special Senses 157 nonmotile cilia on the apical surface of these cells contain chemoreceptors that detect odorant chemicals dissolved in the overlying mucus layer. From its opposite side, the cell gives off an unmyelinated axon that joins other olfactory nerve filaments and penetrates the base of the skull through openings in the cribriform plate of the ethmoid bone. These olfactory nerves synapse in the olfactory bulb, a portion of the cerebral hemisphere of the brain located at the base of the cranial cavity, just below the frontal lobe (

Fig. 8.31

Humans have about 10 million olfactory chemoreceptors. Like taste cells, olfactory chemoreceptors have a short life span (about 60 days), and they are also continuously replaced. However, olfactory receptor cells are true neurons and, as such, are the only neurons that are continuously regenerated throughout life.

The olfactory mucosa is exposed to odorant molecules by ventilatory air currents or from the oral cavity during feeding. Sniffing increases the influx of odorants. The odorants are temporarily bound in mucus to an olfactory binding protein that is secreted by a gland in the nasal cavity.

Olfactory coding resembles taste coding in that most natural odors are complex and consist of many molecules that excite a wide variety of olfactory chemoreceptors. Coding for a particular perceived odor depends on the responses of many olfactory chemoreceptors, and the strength of the odorant is represented by the overall amount of afferent neural activity.

The initial synapse of the olfactory pathway is located in the olfactory bulb, which is a specialized portion of the cerebral cortex and located on the underside of the frontal lobe. It contains mitral cells, interneurons (granule cells; periglomerular cells), and distinct synaptic clusters (glomeruli; see

Fig. 8.31 ) in which theses interact with olfactory afferent fibers. As the olfactory afferent fibers reach the olfactory bulb from the olfactory mucosa, they branch as they approach an olfactory glomerulus to synapse on the dendrites of mitral cells. Each glomerulus is the target of thousands of olfactory afferent fibers, but all the afferent fibers to a single glomerulus convey input from the one type of olfactory receptor. This is all the more remarkable because olfactory receptor cells are being regenerated continuously and new axons must therefore navigate their way to a correct glomerulus.

The granule and periglomerular cells are inhibitory inter-neurons. They form dendrodendritic reciprocal synapses with the dendrites of mitral cells. Activity in a mitral cell depolarizes these inhibitory cells, and they in turn inhibit the original and adjacent glomeruli. Because each glomerulus is specialized by being the target of afferent fibers for a unique combination of odor qualities, this appears to be a way of enhancing stimulus contrast, much the way horizontal cells do in the retina. In addition, it provides a mechanism for adaptation to continuous stimulation.

• Fig. 8.30 Olfactory chemoreceptors and supporting cells. (Redrawn from de Lorenzo AJD. In: Zotterman Y [ed]. Olfaction and Taste. Elmsford, NY: Pergamon; 1963.) • Fig. 8.31 Drawing of a Sagittal Section Through an Olfactory Bulb, Showing Terminations of the Olfactory Chemoreceptor Cells in the Olfactory Glomeruli and the Intrinsic Neurons of the Olfactory Bulb. The axons of the mitral cells are shown exiting in the olfactory tract to the right. (Modified from House EL, Pansky B. A Functional Approach to Neuroanatomy. 2nd ed. New York: McGraw-Hill; 1967.)

CHAPTER 8 The Special Senses

The axons of mitral cells leave the olfactory bulb and enter the olfactory tracts. From here, the olfactory connections become highly complex. Within the olfactory tracts is a nucleus, called the anterior olfactory nucleus, that receives input from the olfactory bulb and projects to the contralateral olfactory bulb through the anterior commissure. As each olfactory tract approaches the base of the brain, it splits into the lateral and medial olfactory striae. Axons of the lateral olfactory stria synapse in the primary olfactory cortex, which includes the prepiriform cortex (and, in many animals, the piriform lobe). The medial olfactory stria includes projections to the amygdala, as well as to the basal forebrain. These structures are portions of, or directly connected to, the limbic system (see

Of note is that the olfactory pathway is the only sensory system that does not have an obligatory synaptic relay in the thalamus before signals reach the cortex. However, olfactory information does reach the mediodorsal nucleus of the thalamus, and it is then transmitted to the prefrontal and orbitofrontal cortex. The functional roles of olfaction, in addition to the conscious perception of odor, include providing much of the subtleties of taste by enhancing the 1.

Light enters the eye through the cornea and lens and is focused on the retina, which lines the back of the eye. The cornea is the most powerful refractive surface, but the lens has a variable power that allows images of near objects to be focused on the retina. The iris regulates depth of field and the amount of illumination that enters the eye.

2.

The outer segments of the photoreceptor cells transduce light. Photoreceptors synapse on retinal bipolar cells, which in turn synapse on other interneurons and on ganglion cells. The ganglion cells project to the brain through the optic nerve. The optic disc, where the optic nerve leaves the retina, contains no photoreceptors and is therefore a blind spot. The portion of the retina with the highest degree of spatial resolution is the fovea and the surrounding macula.

3.

Rod photoreceptors have high sensitivity, do not discriminate among colors, and function best under low light levels. Cone photoreceptors have lower sensitivity but higher spatial resolution. Color vision relies on the three types of cones that have different spectral sensitivities.

4.

Bipolar cells and many ganglion cells have concentric receptive fields with an on-center/off-surround or off-center/on-surround organization. Horizontal cells mediate this center-surround antagonism. Photoreceptor, bipolar, and horizontal cells respond to stimulation by modulating their membrane potential and their release of neurotransmitters,

Olfactionisnotgenerallyexaminedinaroutineneurologicalexamination.However,smellcanbetestedbyhavingthepatientinhaleandidentifyanodorant.Onenostrilshouldbeexaminedatatimewhiletheothernostrilisoccluded.Strongodorants,suchasammonia,shouldbeavoidedbecausetheyalsoactivatetrigeminalnervefibers.Smellsensationcanbelost(anosmia) afterabasalskullfractureorafterdamagetooneorbotholfactorybulbsortractsbyatumor(suchasanolfactory groove meningioma).Concussionscancauseanosmiabecausethesuddenmovementofthebraininsidetheskullcanshearthesmallunmyelinatedolfactorynervefibers.Anauraofadisagreeableodor,oftenthesmellofburningrubber,occursduringuncinate seizures, whichareepilepticseizuresthatoriginateinthemedialtemporallobe.

narrow range of gustatory receptors with the wide repertoire of olfactory receptors. In addition, via its intimate connections with limbic and, by extension, hypothalamic structures, it provides input to subconscious mechanisms related to emotions, memory, and sexual behavior.

but ganglion cells respond by generating action potentials.

5.

The axons of ganglion cells in the temporal retina project to the brain ipsilaterally; those in the nasal retina cross in the optic chiasm. Because the lens inverts the image that falls on the retina, each side of the visual field is projected to the contralateral side of the brain for both eyes. In the lateral geniculate nucleus (LGN) of the thalamus, the input from each eye terminates in separate layers, and the M ganglion cells (sensitive to movement) and P ganglion cells (sensitive to detail and color) project to separate layers as well.

6.

The LGN projects to primary visual (striate) cortex via the visual radiation and terminates largely in layer 4, where there is an orderly retinotopic map. Within the map, information from each eye maps to alternating adjacent points to create ocular dominance columns that extend vertically in the cortex. Striate cortical neurons outside of layer 4 respond best to bar or edge stimuli oriented in a particular way. Cells that “prefer” a particular stimulus orientation are grouped in orientation columns.

7.

The extrastriate visual areas have different functions. Some in the inferotemporal cortex are influenced chiefly by P cells, and they function in form detection, color vision, and face discrimination. M cells influence regions of the middle temporal and parietal cortex, which function in motion detection and the control of eye movements.

8.

A pure tone is characterized in terms of its amplitude, frequency, and phase. Natural sounds are combinations of pure tones. Sound pressure is measured in decibels (dB), in relation to a reference level.

9.

The pinna and auditory canal convey airborne sound waves to the tympanic membrane. The three small bones (ossicles) of the middle ear transmit the vibrations of the tympanic membrane to the oval window of the fluid-filled inner ear. Hearing is most sensitive at about 3000 Hz because of the dimensions of the auditory canal and the mechanics of the ossicles.

10.

The cochlea of the inner ear has three main compartments: the scala vestibuli, the scala tympani, and the intervening scala media (cochlear duct). The cochlear duct is bounded on one side by the basilar membrane, on which lies the organ of Corti, the sound transduction mechanism.

11.

When the basilar membrane oscillates in response to pressure waves introduced into the scala vestibuli at the oval window, the stereocilia of the hair cells of the organ of Corti are subjected to shear forces, which open mechanoreceptor K+ channels. This results in a membrane conductance change that modulates the release of neurotransmitters on to cochlear nerve fibers.

12.

High-frequency sounds best activate the hair cells near the base of the cochlea, and low-frequency sounds activate cells near the apex. Such a tonotopic organization is also present in central auditory structures, including the cochlear nuclei, superior olivary complex, inferior colliculus, medial geniculate nucleus, and primary auditory cortex.

13.

Auditory processing at many sites in the central auditory pathway contributes to sound localization, frequency and intensity analysis, and speech recognition.

14.

The vestibular apparatus is part of the inner ear. It includes three semicircular canals (horizontal, anterior, and posterior) and two otolith organs (utricle and saccule) on each side. These transduce, respectively, angular and linear accelerations of the head. The three semicircular canals are mutually orthogonal, so they can resolve angular acceleration of the head about any axis of rotation.

15.

In each semicircular canal, there are sensory hair cells whose cilia extend into a cupula, which blocks the cross-section of the endolymph-filled canal. Angular head acceleration displaces the endolymph and the cupula, bending the cilia. If the stereocilia bend toward the kinocilium, the hair cell is depolarized, which causes an increase in the firing rate in the afferent fiber.

16.

In the otolith organs, the cilia project into an otolithic membrane. Acceleration of the head, as with linear movement, or change in position in relation to gravity displaces the otolithic membrane (because of the mass of the otoliths) and changes the firing patterns of the hair cells, depending on their orientation.

17.

Central vestibular pathways include afferent connections to the vestibular nuclei and the cerebellum. Activation of the vestibular afferent fibers is detected by the brain as head acceleration or position change and is relayed via the vestibular nuclei to pathways that mediate compensatory eye movements, neck movements, and adjustments to posture.

18.

Taste buds contain chemoreceptor cells arranged around a taste pore. Taste buds are located on several kinds of papillae on the tongue and in the pharynx and larynx. Five types of taste-receptor cells detect the five elementary qualities of taste: salty, sweet, sour, bitter, and umami. Complex flavors are signaled by the patterned activity of multiple classes of taste receptor and by central correlation with accompanying olfactory input.

19.

Afferent taste fibers synapse in the nucleus of the solitary tract. The thalamic relay is via a part of the ventroposterior medial nucleus to the taste-receiving areas located in the S1 cortex and the insula.

20.

Odors are detected by olfactory chemoreceptor cells, which are continuously regenerated in the olfactory mucosa. These cells are true neurons that are endowed with a wide array of G protein–coupled receptors that enable the detection of hundreds of odor molecules.

21.

Individual olfactory axons project to olfactory glomeruli, specific for each stimulus type, in the olfactory bulb. They synapse on the dendrites of mitral cells, which have reciprocal synapse with inhibitory interneurons. This synaptic organization in the glomerulus underlies stimulus adaptation and contrast enhancement.

Squire L, Berg D. Fundamental Neuroscience. 4th ed. New York: Academic Press; 2012.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

What is a motor neuron, and how are α and γ motor neurons different?

2.

What is a motor unit? How does the “size principle” apply to the orderly recruitment of motor units?

3.

What is a reflex, and why are reflexes useful for clinical and scientific understanding?

4.

What information about the state of the muscle is sensed by the muscle spindles, and what afferent fibers convey this information to the central nervous system (CNS)?

5.

How do γ motor neurons modulate the responses of the muscle spindle?

6.

What are the pathways and functions of the basic spinal reflexes?

7.

What is a central pattern generator, and what types of movements can it be used for?

8.

What distinguishes the pathways of the medial and lateral descending pathways in motor control?

9.

What is decerebrate rigidity, and what are its implications for the control of muscle tone?

10.

What distinguishes the cortical motor areas from each other?

11.

What motor parameters are coded for in the activity of neurons in motor cortex?

12.

How does the organization of the mossy and olivocerebellar (climbing) fiber afferent systems to the cerebellum differ in their origins, topography, and synaptic connections.

13.

What is the geometric relationship between the major cellular elements of the cerebellar cortex?

14.

What are simple and complex spikes in Purkinje cells?

15.

What are the direct and indirect pathways in the basal ganglia, and how does their activity influence movement?

16.

How is the balance of activity between the direct and indirect pathways altered in Parkinson’s disease and Huntington’s disease?

17.

How do the vestibuloocular and optokinetic reflexes act to stabilize gaze? How do they complement each other?

18.

What are the roles of saccades and smooth pursuit movements in visual tracking?

19.

What is nystagmus, and what types of sensory stimulation can drive nystagmus in a normal individual?

20.

What is the somatotopic organization of the different CNS regions involved in motor control.

ovements are the major way in which humans interact with the world. Most activities— including running, reaching, eating, talking, writing, and reading—ultimately involve motor acts. Thus motor control is a major task of the central nervous system (CNS), and from an evolutionary perspective, it is probably the reason that nervous systems first arose. Not surprisingly, a large amount of the CNS is devoted to motor control, which can be defined as the generation of signals to coordinate contraction of the musculature of the body and head, either to maintain a posture or to make a movement (transition between two postures).

Because large amounts of the nervous system are involved in motor control, damage or diseases of the nervous system often result in motor abnormalities. Conversely, particular motor symptoms help determine the location of the damaged or malfunctioning region; thus assessment of motor function is an important clinical tool.

In this chapter, each major CNS area involved in motor control is described, starting with the spinal cord and continuing with the brainstem, cerebral cortex, cerebellum, and basal ganglia. Eye movements are discussed at the end of the chapter because of their importance and the specialized circuits involved in their generation. Each CNS area is described separately; however, CNS regions do not function in isolation, and most movements result from the coordinated action of multiple brain regions. For example, even spinal reflexes, which are mediated by local circuits in the spinal cord, can be modified by descending motor commands, and virtually all voluntary movements, which arise from cerebral activity, are ultimately generated by activation of the spinal cord circuitry (or analogous brainstem nuclei for muscles in the head and face).

Principles of Spinal Cord Organization

The spinal cord has a cylindrical shape in which the white matter is located superficially and the gray matter is found deep to the white matter shell. The gray matter forms a continuous column that runs the length of the cord. However, the nerve roots that enter and exit the spinal cord bundle into discrete nerves, which form the basis for naming the •Fig. 9.1 Musculotopic Organization of Motor Neurons in the Ventral Horn of the Spinal Cord. A, Schematicviewofthecervicothoracicspinalcordandassociatedcross-sections,showingtheloca-tionsofmotorneuronsthatinnervateaflexor(blue dots) andanextensor(red dots). B, Spinalcordcross-section,withlocationsofdifferentmusclesrepresentedbyadrawingofthearm.(RedrawnfromPurvesD,etal[eds].Neuroscience. 3rded.Sunderland,MA:Sinauer;2004.)B Distal muscles Proximal muscles specific levels (“segments”) of the spinal cord (8 cervical, 12 thoracic, 5 lumbar, 5 sacral, and 1 coccygeal). When viewed in cross-section, the gray matter column typically has an “H” or butterfly shape. The “butterfly wings” are divided into dorsal and ventral horns that are separated by an intermediate zone (

Fig. 9.1 ) (at some spinal cord levels, a small lateral horn is also present; see ). The ventral horn is where motor neurons reside, and thus it has primarily a motor function. Correspondingly, it is the main target of descending motor pathways from the brain. In contrast, the dorsal horn is the major recipient of incoming sensory information and the main source of ascending sensory pathways (e.g., the spinothalamic tract; see

Chapter 7).

The description of the motor function of the spinal cord therefore begins with the properties of motor neurons and their organization in the ventral horn. Similar organizational principles hold for cranial nerve nuclei that are involved in controlling the musculature of the head and face (e.g., the facial and motor trigeminal nuclei).

A motor neuron is a neuron that projects to muscle cells. Because motor neurons represent the only route for CNS activity to control muscle activity, motor neurons have been termed the final common pathway. There are both somatic motor neurons and autonomic motor neurons. However, in this chapter, the term motor neuron refers only to somatic motor neurons; autonomic motor neurons are discussed in . Somatic motor neurons innervate the skeletal (striated) muscles of the body. Two main classes are distinguished on the basis of their axonal diameters: α and γ motor neurons.

The α motor neurons are large, multipolar neurons that range in size up to 70 µm in diameter (see

Fig. 4.10A ). Their axons leave the spinal cord through the ventral roots and the brainstem via several cranial nerves and are distributed to the appropriate skeletal muscles via peripheral nerves. The α motor neuron axon also projects to other neurons by giving off collateral axons before leaving the CNS. The main axon terminates by synapsing onto the extrafusal muscle fibers. These synapses are called neuromuscular junctions or end plates. Extrafusal fibers are large muscle fibers that make up the bulk of a skeletal muscle and essentially generate its contractile force (a muscle also contains intrafusal fibers whose functions are detailed later in this chapter; also see

A key functional aspect of the motor neuron projection pattern out of the CNS is that each neuron’s axon innervates only one muscle, but it branches to innervate multiple fibers within that muscle. Moreover, each extrafusal muscle fiber in mammals is supplied by only one α motor neuron. Thus a motor unit can be defined as an α motor neuron and all of the skeletal muscle fibers that its axon supplies. The motor unit can be regarded as the basic unit of movement because when an α motor neuron discharges under normal circumstances, all of the muscle fibers of the motor unit contract. That is, the safety factor (see ) of the neuromuscular junction is greater than 1, and so each action potential in the motor neuron axon triggers an action potential in every muscle fiber of the motor unit.

Of importance is that the average size of the motor unit (i.e., the number of muscle fibers innervated by an axon) varies between muscles, depending on how fine a control of the muscle is required. For finely controlled muscles, such as the eye muscles, an α motor neuron may supply only a few muscle fibers. However, in a proximal limb muscle, such as the quadriceps femoris, a single α motor neuron may innervate thousands of muscle fibers.

The muscle fibers that belong to a given motor unit are called a muscle unit. All the muscle fibers in a muscle unit are of the same histochemical type (i.e., they are all either slow-twitch [type I] or fast-twitch [type IIA or IIB] fibers). For an in-depth description of muscle fiber types, see . Of importance in this chapter is that a number of physiological properties are correlated with this histochemical classification scheme. In particular, slow-twitch fibers, which contract and relax slowly as implied by their name, also generate low-force levels but essentially never fatigue. In contrast, the fast-twitch fiber contract and relax rapidly, generate higher levels of force, and fatigue at varying rates.

The first motor units to be activated in many cases, either by voluntary effort or during reflex action or just

CHAPTER 9 Organization of Motor Function to maintain posture, are those with the smallest motor axons. These motor units contain slow-twitch fibers and thus generate the smallest contractile force, allowing the initial contraction to be finely graded. These units tend to be active much of the time, if not continuously, and so their lack of fatigability makes good functional sense.

As more motor units are recruited for a motor act, motor neurons with progressively larger axons become involved, and these axons synapse onto the fast-twitch fibers. Thus they generate progressively larger amounts of tension. The most powerful such motor units are typically recruited only for tasks requiring large amounts of force (e.g., sprinting, jumping, and lifting a heavy weight), tasks that people can perform for only for short periods of time.

The orderly recruitment of motor units helps the CNS generate a large range of forces and also maintain relatively precise control at the different force levels. This recruitment pattern is called the size principle because the motor units are recruited in order of motor neuron axon size. The size principle depends on the fact that small motor neurons are activated more easily than are large motor neurons. Recall that if an excitatory synapse is active, it opens channels in the postsynaptic membrane and causes an excitatory postsynaptic current. The same size excitatory postsynaptic current generates a larger potential change at the initial segment of an axon of a small motor neuron than it does at a larger motor neuron, simply as a consequence of Ohm’s law (V = IR) and the fact that smaller motor neurons have higher membrane resistance than larger motor neurons do. Thus because excitatory postsynaptic potentials in the CNS are small and need to summate to reach threshold for triggering spikes, as the level of synaptic bombardment rises from zero, the resulting depolarization will reach spiking threshold in smaller motor neurons first. As the size principle is usually obeyed, this assumption generally appears to hold; however, there can be exceptions, and in these cases, the descending motor pathways presumably must provide differing levels of synaptic drive to the different-sized motor neurons.

The γ motor neurons are smaller than α motor neurons; they have a soma diameter of about 35 µm. The γ motor neurons that project to a particular muscle are located in the same regions of the ventral horn as the α motor neurons that supply that muscle. γ Motor neurons do not supply extrafusal muscle fibers; instead, they synapse on specialized striated muscle fibers called intrafusal muscle fibers, which traverse receptors called muscle spindles that are embedded in skeletal muscles. The function of γ motor neurons is to regulate the sensitivity of these receptors (discussed later).

Topographic Organization of Motor Neurons in the Ventral Horn

The spatial distribution of motor neurons in the spinal cord is highly organized. (This is also true in the cranial nerve nuclei.) A given skeletal muscle is supplied by a group

Aclinicallyusefulwaytomonitortheactivityofmotorunitsiselectromyography. Anelectrodeisplacedwithinaskeletalmuscletorecordthesummedactionpotentialsoftheskeletalmusclefibersofamuscleunit.Ifnospontaneousactivityisnoted,thepatientisaskedtocontractthemusclevoluntarilytoincreasetheactivityofmotorunitsinthemuscle.Astheforceofvoluntarycontractionincreases,moremotorunitsarerecruited.Inadditiontotherecruitmentofmoremotorneurons,contractilestrengthincreaseswithincreasesintherateofdischargeoftheactiveα motorneurons.Electromyographyisusedforvariouspurposes.Forexample,theconductionvelocityofmotoraxonscanbeestimatedasthedifferenceinlatencyofmotorunitpotentialswhenaperipheralnerveisstimulatedattwositesseparatedbyaknowndistance.Anotheruseistoobservefibrillationpotentialsthatoccurwhenmusclefibersaredenervated.Fibrillationpotentialsarespontaneouslyoccurringactionpotentialsinsinglemusclefibers.Thesespontaneouspotentialscontrastwithmotorunitpotentials,whicharelargerandhavealongerdurationbecausetheyrepresenttheactionpotentialsinasetofmusclefibersthatbelongtoamotorunit.

of α motor neurons, called a motor nucleus, located in the ventral horn. Each such motor nucleus takes the form of a rostrocaudally running column that can span several spinal cord levels (see

Fig. 9.1A ). Motor neurons that supply the axial musculature collectively form a column of cells that extends the length of the spinal cord. In the cervical and lumbosacral enlargements, these cells are located in the most medial part of the ventral horn; at other levels, they essentially form the entire ventral horn. The motor neurons innervating the limb muscles are in the cervical and lumbosacral enlargements, where they form columns that are lateral to those for the axial muscles. Motor neurons to muscles of the distal part of the limb are located most laterally, whereas those that innervate more proximal muscles are located more medially (

Fig. 9.1B). Also, motor neurons to flexors are dorsal to those that innervate extensors. Note that the α and γ motor neurons to a given muscle are found intermixed within the same motor neuron column.

The interneurons that connect with the motor neurons in the enlargements are also similarly topographically organized. In general, interneurons that supply the limb muscles are located mainly in the lateral parts of the deep dorsal horn and the intermediate region between the dorsal and ventral horns. Those that supply the axial muscles, however, are located in the medial part of the ventral horn. All of these interneurons receive synaptic connections from primary afferent fibers and from the axons of pathways that descend from the brain, and thus they are part of both spinal reflex arcs and descending motor control pathways.

An important aspect of interneuronal systems is that the laterally placed interneurons project ipsilaterally to motor neurons that supply the distal or the proximal limb muscles, whereas the medial interneurons project bilaterally. This arrangement of the lateral interneurons allows the limbs to be controlled independently. In contrast, the bilateral arrangement of the medial interneurons allows bilateral control of motor neurons to the axial muscles to provide postural support to the trunk and neck.

Although motor neurons are the final common pathway from the CNS to muscles and thus shape how neuronal activity is transformed into muscular contraction, each motor neuron directly acts on only a single muscle. Normal movements (or postures), however, are rarely, if ever, caused by the isolated contraction of an individual muscle. Rather, they reflect the coordinated activity of large groups of muscles. For example, elbow flexion involves an initial burst of activity in flexor muscles, such as the biceps, and in relaxation of extensor muscles, such as the triceps. This activity is then succeeded by a burst of activity of the triceps and then a second burst of activity in the biceps to stop the flexion movement at the desired position. Furthermore, other muscles are also activated during the elbow flexion to maintain overall balance and posture.

As the elbow flexion example shows, different roles are played by each muscle during a movement. The muscle that initiates, and is the prime cause of, the movement is called the agonist. Muscles that act similarly to the agonist are called synergists, whereas muscles whose activity opposes the action of the agonist are antagonists. In addition, muscles can act as fixators to immobilize a joint and in postural roles. Moreover, the relationship two muscles have to each other may depend on the specific movement being performed. For example, during elbow flexion, the triceps acts an antagonist to the biceps. In contrast, during supination of the forearm without rotation occurring about the elbow, the biceps (which also acts to supinate the forearm) is again an agonist, but the role of the triceps is that of an elbow fixator.

Thus motor control requires flexibly linking (and unlinking) the activity of groups of motor neurons that connect to different muscles. The circuits of the spinal cord are a major mechanism used by the CNS for this aspect of motor control. Indeed, descending pathways from the brain target primarily the interneurons of the spinal cord, although there are some descending axons that synapse directly onto motor neurons.

Spinal cord circuitry has several levels of organization. The most basic is the segmental level: that is, a circuit that is largely confined to a single or several neighboring segments and that is repeated again at many levels. The basic spinal reflexes covered below (i.e., the myotatic, inverse myotatic, and flexion reflexes) are mediated by such circuits. Superimposed on this segmental organization is the propriospinal system, which is a series of neurons whose axons run up and down the spinal cord to interconnect the different levels of the cord. This system allows the coordination of activity at different spinal levels, which is important for behavior involving both the forelimbs and the hind limbs, such as •Fig. 9.2 Muscle Proprioceptors. Skeletalmusclescontainsensoryreceptorsembeddedwithinthemuscle(spindles)andwithintheirtendons(Golgitendonorgans).A, Schematicviewofamuscle,showingthearrangementofaspindleinparallelwithextrafusalmusclefibersandatendonorganinserieswithmusclefibers.B, Structureandinnervation(motorandsensory)ofamusclespindle.C, Structureandinnervationofatendonorgan.A B C Ib afferent neuron Muscle fibers Tendon 250 µm Bag fibers Chain fibersGolgi tendon organ

The neural innervation of an intrafusal fiber differs significantly from that of an extrafusal fiber, which is innervated by a single motor neuron. Intrafusal fibers are multiply innervated and receive both sensory and motor innervation. The sensory innervation typically includes a single group Ia afferent fiber and a variable number of group II afferent fibers (see

Fig. 9.2B). Group Ia fibers belong to the class of sensory nerve fibers with the largest diameters and conduct at 80 to 120 m/sec; group II fibers are intermediate in size and conduct at 35 to 75 m/sec. A group Ia afferent fiber forms a spiral-shaped termination, referred to as a primary ending, on each of the intrafusal muscle fibers in the spindle. Thus primary endings are found on both types of nuclear bag fibers and on nuclear chain fibers. The group II afferent fiber forms a secondary type ending on nuclear chain and bag2 fibers, but not on bag1 fibers. The primary and secondary endings have mechanosensitive channels that are sensitive to the level of tension on the intrafusal muscle fiber.

The motor supply to a muscle spindle consists of two types of γ motor axons (see

Fig. 9.2B ). Dynamic γ motor axons end on nuclear bag1 fibers, and static γ motor axons end on nuclear chain and bag2 fibers.

Muscle spindles respond to changes in muscle length because they lie in parallel with the extrafusal fibers and therefore are also stretched or shortened along with the extrafusal fibers. Because intrafusal fibers, like all muscle fibers, display spring-like properties, a change in their length changes the tension that they are under, and this change is sensed by mechanoreceptors of the group Ia and group II spindle afferent fibers. The nonselective cation channel Piezo2 has been identified as the principal transduction channel that allows spindle sensory afferent fibers to sense changes in mechanical stress that occur when a muscle changes length.

Fig. 9.3 shows the changes in activity of the afferent fibers of a muscle spindle when the muscle is stretched. It is clear that group Ia and group II fibers respond differently to stretch. Group Ia fibers are sensitive both to the amount of muscle stretch and to its rate, whereas group II fibers respond chiefly to the amount of stretch. Thus when a muscle is stretched to a new longer length, group II firing increases in proportion to the amount of stretch (see

Fig.

9.3 , left), and when the muscle is allowed to shorten, its firing rate decreases proportionately (see

Fig. 9.3 , right). Group Ia fibers show this same static-type response, and thus under steady-state conditions (i.e., constant muscle length), their firing rate reflects the amount of muscle stretch, similar to that of group II fibers.

While muscle length is changing, however, group Ia firing also reflects the rate of stretch or shortening that the muscle is undergoing. Its activity overshoots during muscle stretch and undershoots (and possibly ceases) during muscle shortening. These are called dynamic responses. This dynamic sensitivity also means that the activity of group Ia fibers is much more sensitive to transient and oscillatory stretches, such as shown in the middle diagrams of

Fig. 9.3 . In particular, the tap profile is what occurs when a reflex hammer is used to hit the muscle tendon and thereby cause a brief stretching of the attached muscle. The change in muscle length is too brief for significant changes in group II firing to occur, but because the magnitude of the rate of change (slopes of the tap profile) is so high with this stimulus, large dynamic responses are elicited in the group Ia fibers. Thus the functionality of reflex arcs involving group Ia afferent fibers is what is being assessed when a reflex hammer is used to tap on tendons.

γ Motor Neurons Adjust the Sensitivity of the Spindle

Up to this point, we have described only how muscle spindles behave when there are no changes in γ motor neuron activity. The efferent innervation of muscle spindles is extremely important, however, because it determines the sensitivity of muscle spindles to stretch. For example, in

Fig. 9.4A, the activity of a muscle spindle afferent fiber is shown during

CHAPTER 9 Organization of Motor Function •Fig. 9.3 Responses of a Primary Ending (Group Ia) and a Secondary Ending (Group II) to Changes in Muscle Length. Notethedifferenceindynamicandstaticresponsivenessoftheseendings.Thewaveformsatthetoprepresentthechangesinmusclelength.ThemiddleandbottomrowsshowthedischargesofagroupIafiberandagroupIIfiber,respectively,duringthevariouschangesinmusclelength.

•Fig. 9.4 The Activity of γ Motor Neurons Can Counteract the Effects of Unloading on the Discharge of a Muscle Spindle Afferent Fiber. A, Theactivityofamusclespindleafferentfiberduringsteadystretch.B, Stimulationofα motorneuronat0mseccausescontractionoftheextrafusalfibers,whichleadstomuscleshorteningandincreasedmuscletensionbutunloadingofthetensionacrossthemusclespindle,whichinturninducestheafferentfibertostopfiring.Uponrelaxation,themusclereturnstoitsoriginallength,andtensionisrestoredontheintrafusalfibers,causingthereturnofactivityinthegroupIaafferentfiber.C, Coactivationofα andγ motorneuronscausesshorteningofbothextrafusalandintrafusalfibers.Thusthereisnounloadingofthespindle,andtheafferentfibermaintainsitsspontaneousactivity.(RedrawnfromKufflerSW,NichollsJG.From Neuron to Brain. Sunderland,MA:Sinauer;1976.) a steady stretch. If only the extrafusal muscle fibers were may stop discharging and become insensitive to further to contract (this can be done experimentally by selective decreases in muscle length. However, the unloading of the stimulation of α motor neurons; see

Fig. 9.4B ), the muscle spindle can be prevented if α and γ motor neurons are spindle would be unloaded by the resultant shortening of stimulated simultaneously. Such combined stimulation the muscle. If this happens, the muscle spindle afferent fiber causes the intrafusal muscle fibers of the spindle to shorten along with the extrafusal muscle fibers, maintaining the baseline tension on the equatorial portion of the intrafusal fibers (see

Fig. 9.4C •Fig. 9.5 Effects of Static and Dynamic γMotor Neurons on the Responses of a Primary Ending to Muscle Stretch. A, Thetimecourseofthestretch.B, ThedischargeofgroupIafibersintheabsenceofγmotorneuronactivity.C, Stimulationofastaticγmotoraxon.D, Stimulationofadynamicγmotoraxon.(RedrawnfromCroweA,MatthewsPBC.J Physiol. 1964;174:109.)

Note that only the two polar regions of the intrafusal muscle contract; the equatorial region, where the nuclei are located, does not contract because it has little contractile protein. Nevertheless, when the polar regions contract, the equatorial region elongates and regains its sensitivity. Conversely, when a muscle relaxes (α motor neuron activity drops) and thus elongates (if its ends are being pulled), a concurrent decrease in γ motor neuron activity allows the intrafusal fibers to relax (and thus elongate) as well and thereby prevent the tension on the central portion of the intrafusal fiber from reaching a level at which firing of the afferent fibers is saturated. Thus the γ motor neuron system allows the muscle spindle to operate over a wide range of muscle lengths while retaining high sensitivity to small changes in length.

For voluntary movements, descending motor commands from the brain in fact typically activate α and γ motor neurons simultaneously, presumably to maintain spindle sensitivity as just described. This has two important functions. By maintaining the muscle spindle’s sensitivity as the muscle changes length, the spindle remains capable of sensing, and signaling to the CNS, any disturbances to the ongoing movement that cause an unexpected stretch of the muscle, and this in turn allows the CNS to initiate both reflex (see next section) and voluntary corrections. Second, if the spindle were to become unloaded during the movement, this would oppose the intended movement by decreasing the excitatory drive, via the group Ia reflex arc (see next section), to the α motor neurons driving the agonist muscles.

As mentioned earlier, there are two types of γ motor neurons: dynamic and static (see

Fig. 9.2).

This allows the CNS to have very precise control over the sensitivity of the muscle spindle. Dynamic γ motor axons end on nuclear bag1 fibers, and static γ motor axons synapse on nuclear chain and bag2 fibers. Thus when a dynamic γ motor neuron is activated, the response of the group Ia afferent fiber is enhanced, but the activity of the group II afferent fibers is unchanged; when a static γ motor neuron discharges, the responsiveness of the group II afferent fibers and the static responsiveness of the group Ia afferent fibers are increased. The effects of stimulating the static and dynamic fibers on a group Ia afferent fiber’s response to stretch are illustrated in

Fig. 9.5 . Descending pathways can preferentially influence dynamic or static γ motor neurons and thereby alter the nature of reflex activity in the spinal cord and also, presumably, the functioning of the muscle spindle during voluntary movements.

The Phasic (or Ia) Stretch Reflex

The reflex arc responsible for the phasic stretch reflex is depicted in

Fig. 9.6 ; the rectus femoris muscle serves as an example. A rapid stretch of the rectus femoris muscle strongly activates the group Ia fibers of the muscle spindles, which then convey this signal into the spinal cord. In the spinal cord, each group Ia afferent fiber branches many times to form excitatory synapses directly (monosynaptically) on virtually all α motor neurons that supply the same (also known as the homonymous) muscle and with many α motor neurons that innervate synergists, such as the vastus intermedius muscle in this case, which also acts to extend the leg at the knee. If the excitation is powerful enough, the motor neurons discharge and cause a contraction of the muscle. Note that the group Ia fibers do not contact the γ motor neurons, possibly to avoid a positive-feedback loop situation. This selective targeting of α motor neurons is exceptional in that most other reflex and descending pathways target both α and γ motor neurons.

Other branches of group Ia fibers end on a variety of interneurons; however, one type, the reciprocal Ia inhibitory interneuron (black cell in

Fig. 9.6 ), is particularly important with regard to the stretch reflex. These interneurons are identifiable because they are the only inhibitory interneurons •Fig. 9.6 Reflex Arc of the Stretch Reflex. Thepathwaybacktotherectusfemorisinthisarccontainsasinglesynapsewithinthecentralnervoussystem;hence,itisamonosynapticreflex.Theinterneuron,showninblack, isagroupIainhibitoryinterneuron.DRG,dorsalrootganglion.

that receive input from both the group Ia afferent fibers and Renshaw cells (see

Fig. 9.12 ). They end on α motor neurons that innervate the antagonist muscles—in this case, the hamstring muscles, including the semitendinosus muscle—which act to flex the knee. Other branches of the group Ia afferent fibers synapse with yet other neurons that originate ascending pathways that provide various parts of the brain (particularly the cerebellum and cerebral cortex) with information about the state of the muscle.

The organization of the stretch reflex arc guarantees that one set of α motor neurons is activated and the opposing set is inhibited. This arrangement is known as reciprocal innervation. Although many reflexes involve such reciprocal innervation, this type of innervation is not the only possible organization of a motor control system; descending motor pathways can override such patterns.

The stretch reflex is quite powerful, in large part because of its monosynaptic nature. The power of this reflex also derives from the essentially maximal convergence and divergence that exist in this pathway, which is not apparent from the circuit diagrams, such as

Fig. 9.6 , that are typically used to illustrate reflex pathways. That is, each group Ia fiber contacts virtually all homonymous α motor neurons, and each such α motor neuron receives input from every spindle in that muscle. Although its monosynaptic nature makes the group Ia reflex rapid and powerful, it also means that there is relatively little opportunity for direct control of

CHAPTER 9 Organization of Motor Function activity flow through its reflex arc. The CNS overcomes this problem by controlling muscle spindle sensitivity via the γ motor neuron system as described previously.

The Tonic Stretch Reflex

The tonic stretch reflex can be elicited by passive bending of a joint. This reflex circuit includes both group Ia and group II afferent fibers from muscle spindles. Group II fibers make monosynaptic excitatory connections with α motor neurons, but they also excite them through disynaptic and polysynaptic pathways. Normally, there is ongoing activity in the group Ia and group II afferent fibers that helps maintain a baseline rate of firing of α motor neurons; therefore, the tonic stretch reflex contributes to muscle tone. Its activity also contributes to the ability to maintain a posture. For example, if the knee of a soldier standing at attention begins to flex because of fatigue, the quadriceps muscle is stretched, a tonic stretch reflex is elicited, and the quadriceps contracts more, thereby opposing the flexion and restoring the posture.

The foregoing discussion suggests that stretch reflexes can act like a negative-feedback system to control muscle length. By following the stretch reflex arc, it is possible to see that changes in its activity act to oppose changes in muscle length from a particular equilibrium point. For example, if the muscle’s length is increased, there will be an increase in firing by group Ia and group II fibers, which excites homonymous α motor neurons and leads to contraction of the muscle and reversal of the stretch. Similarly, passive shortening of the muscle unloads the spindles and leads to a decrease in the excitatory drive to the motor neurons and thus relaxation of the muscle. So how are humans able to rotate their joints? It is partly because the γ motor neurons are coactivated during a movement and thereby shift the equilibrium point of the spindle and partly because the gain or strength of the reflex is low enough that other input to the motor neuron can override the stretch reflex.

The inverse myotatic reflex acts to oppose changes in the level of force in the muscle. Just as the stretch reflex can be thought of as a feedback system to regulate muscle length, the inverse myotatic, or group Ib, reflex can be thought of as a feedback system to help maintain force levels in a muscle. With the upper part of the leg as an example, the group Ib reflex arc is depicted in

Fig. 9.7.

The arc starts with the Golgi tendon organ receptor, which senses the tension in the muscle. Golgi tendon organs are located at the junction of the tendon and the muscle fibers and thus lie in series with the muscle fibers, in contrast to the parallel arrangement of the muscle spindles (see

Fig. 9.2 ). Golgi tendon organs have a diameter of about 100 µm and a length of about 1 mm. A Golgi tendon organ is innervated by the terminals of group Ib afferent fibers. These terminals wrap about bundles of collagen fibers in the tendon of a muscle (or in tendinous inscriptions within the muscle).

Hyperactivestretchreflexescanleadtotremorsandclonus,whicharetypesofinvoluntaryrhythmicmovements.Althoughthenegative-feedbackactionofthestretchreflexcanhelpstabilizethelimbataparticularposition,ifanexternalperturbationtothelimboccurs,theconductiondelaybetweentheinitiatingstimulus(musclestretch)andtheresponse(musclecontraction)cancausethestretchreflexcircuittobeasourceofinstabilitythatleadstorhythmicmovements.Specifically,clonusiselicitedbyasustainedstretchofamuscleinapersonwhohasspinalcorddamage.Normally,animposedsustainedstretchonamuscleelicitsanincreaseingroupIaandgroupIIfiberactivity,whichafteradelaycausesacontractioninthemusclethatopposesthestretchbutdoesnotcompletelyreturnthemuscletoitsinitiallengthbecausethegainofthestretchreflexismuchless than1.*

Thispartialcompensation,inturn,leadstoadecreaseingroupIaandgroupIIfiberactivity,whichcausesthelimbtolengthenagain,butnotfully.ThislengtheningonceagainincreasesgroupIaandgroupIIfiberactivity,andsoon.Thedelayisessentialinsettingupthisoscillationbecauseitcausesthefeedbacksignaltocontinueevenafterthemusclehascompensatedandthusresultsinanovercompensationthatleadstothenextovercorrection.However,becausethereflexgainisnormallymuchlessthan1,thisoscillationnormallydiesoutquickly(theovercompensationsdecreaseinamplituderapidly),andthemusclecomestorestatanintermediatelength.Incontrast,whendescendingmotorpathwaysaredamaged,theresultingchangesinspinalcordconnectivityandincreasesinneuronalexcitabilityresultinahyperactivereflex(whichisequivalenttoraisingthegainofthestretchreflexcloseto1).Inthiscase,thesuccessiveovercompensationsaremuchlarger,andanovertbuttransientoscillationcanbeobserved(clonus).Ifthegainequals1,theclonusdoesnotdieoutbutratherpersistsforaslongastheinitialstretchstimulusismaintained.

*Ingeneral,gain ofasystemisdefinedasitsoutputforagiveninput.Inthiscase,theinputtothesystemistheimposedstretch,andtheoutputismovementcausedbythestretchreflex–evokedcontraction.

Because of their in-series relationship to the muscle, Golgi tendon organs can be activated either by muscle stretch or by muscle contraction. In both cases, however, the actual stimulus sensed by the Golgi tendon organ is the force that develops in the tendon to which it is linked. Thus the response to stretch is the result of the spring-like nature of the muscle (i.e., by Hooke’s law, the force on a spring is proportional to how much it is stretched).

To distinguish between the responsiveness of the muscle spindles and Golgi tendon organs, the firing patterns of group Ia and group Ib fibers can be compared when a muscle is stretched and then held at a longer length (Fig.

9.8 ). The firing rate of the group Ia fibers maintains its increase until the stretch is reversed. In contrast, the group Ib fiber shows an initial large increase in firing, reflecting the increased tension on the muscle caused by the stretch, but then shows a gradual return toward its initial firing rate as the tension on the muscle is lowered because of cross-bridge recycling and the resultant lengthening of the •Fig. 9.7 Reflex Arc of the Inverse Myotatic Reflex. Theinterneuronsincludebothexcitatory(white) andinhibitory(black) interneurons.Thisisanexampleofadisynapticreflex.

•Fig. 9.8 Changes in Group Ia and Group Ib Firing Rates When Muscle Is Stretched to a New Length. Afteratransientburst,thefiringrateofthegroupIafiberremainsconstantatanewhigherlevelthatisproportionaltotheincreaseinlength(compareblue lines in upper and lower graphs).Incontrast,thegroupIbfibershowsaninitialrapidincreaseinfiringfollowedbyaslowdecreasebacktowarditsoriginallevel(lower graph, red line) andhasafiringprofilethatmatchesthetensionlevelinthemusclecausedbythestretch(upper graph, red line).

sarcomeres. Therefore, Golgi tendon organs signal force, whereas spindles signal muscle length. Further evidence of this distinction is that group Ib firing is correlated with force level during isometric contraction even though muscle length and therefore group Ia activity are unchanged.

CHAPTER 9 Organization of Motor Function •Fig. 9.9 The Reflex Arc of the Flexion Reflex. Black interneuronsareinhibitory,andwhite onesareexcitatory.FRA,flexionreflexafferentfiber.++––FRASemitendinosusNociceptorFlexionExtensionRectusfemoris

The group Ib afferent fibers branch as they enter the spinal cord and end on interneurons. There are no mono-synaptic connections to α motor neurons. Rather, the group Ib afferent fibers synapse onto two classes of interneurons: interneurons that inhibit α motor neurons that supply the homonymous muscle (in this case the rectus femoris muscle) and excitatory interneurons that activate α motor neurons to the antagonist (the semitendinosus muscle). Because there are two synapses in series in the CNS, this is a disynaptic reflex arc. Because of these connections, group Ib fiber activity should have the opposite action of the group Ia stretch reflex during passive stretch of the muscle, which explains the group Ib reflex’s other name, the inverse myotatic reflex.

Functionally, however, the two reflex arcs can act synergistically, as the following example shows. Recall that the Golgi tendon organs monitor force levels across the tendon that they supply. If during maintained posture (such as standing at attention) knee extensors (such as the rectus femoris muscle) begin to fatigue, the force pulling on the patellar tendon declines. The decline in force reduces the activity of Golgi tendon organs in this tendon. Because the group Ib reflex normally inhibits the α motor neurons to the rectus femoris muscle, reduced activity of the Golgi tendon organs enhances the excitability of (i.e., disinhibits) the α motor neurons and thereby helps reverse the decrease in force caused by the fatigue. Simultaneously, bending of the knee stretches the knee extensors and activates the afferent fibers from the muscle spindles, which then excite the same α motor neurons. Thus coordinated action of afferent fibers from both the muscle spindle and Golgi tendon organ help oppose the decrease in contraction of the rectus femoris muscle due to fatigue and thereby work together to maintain the standing posture.

The flexion reflex starts with activation of one or more of a variety of sensory receptors, including nociceptors, whose signals can be carried to the spinal cord via a variety of afferent fibers, including group II and group III fibers, collectively called the flexion reflex afferent (FRA) fibers. In flexion reflexes, afferent volleys (1) cause excitatory inter-neurons to activate the α motor neurons that supply the flexor muscles in the ipsilateral limb and (2) cause inhibitory interneurons to inhibit the α motor neurons that supply the antagonistic extensor muscles (

Fig. 9.9).

This pattern of activity causes one or more joints in the stimulated limb to flex. In addition, commissural interneurons evoke the opposite pattern of activity in the contralateral side of the spinal cord (see

Fig. 9.9 ), which results in extension of the opposite limb, the crossed extension reflex. For lower limbs in humans (or for both forelimbs and hind limbs in quadrupeds), the crossed extension part of the reflex helps in maintaining balance by enabling the contralateral limb to be able to support the additional load that is transferred to it when the flexed limb is lifted.

Afterdamagetothedescendingmotorpathways,hyperactivestretchreflexesmayresultinspasticity,inwhichthereislargeresistancetopassiverotationofthelimbs.Inthiscondition,itmaybepossibletodemonstratewhatiscalledtheclasp-knife reflex. Whenspasticityispresent,attemptstorotatealimbaboutajointinitiallymeethighresistance.However,iftheappliedforceisincreased,therecomesapointatwhichtheresistancesuddenlydissipatesandthelimbrotateseasily.Thischangeinresistanceiscausedbyreflexinhibition.ThegroupIbreflexarcsuggeststhatrisingactivityinthispathwaycouldunderliethesuddenreleaseofresistance,andindeed,theclasp-knifereflexwasonceattributedtotheactivationofGolgitendonorganswhenthesereceptorswerethoughttohaveahighthresholdtomusclestretch.However,thetendonorganshavesincebeenshowntobeactivatedatverylowlevelsofforceandarenolongerthoughttocausetheclasp-knifereflex.Itisnowthoughtthatthisreflexiscausedbytheactivationofotherhigh-thresholdmusclereceptorsthatsupplythefasciaaroundthemuscle.Signalsfromthesereceptorscausetheactivationofinterneuronsthatleadtoinhibitionofthehomonymousmotorneurons.

Because flexion typically brings the affected limb in closer to the body and away from a painful stimulus, flexion reflexes are a type of withdrawal reflex. In

Fig. 9.9 , the neural circuit of the flexion reflex is shown for neurons that affect only the knee joint. Actually, however, considerable divergence of the primary afferent and interneuronal pathways occurs in the flexion reflex. In fact, all the major joints of a limb (e.g., hip, knee, and ankle) may be involved in a strong flexor withdrawal reflex. Details of the flexor withdrawal reflex vary, depending on the nature and location of the stimulus.

The interneurons subserving flexion reflexes also appear to be part of the central pattern generator (CPG) for generating locomotion and thus are an example of how reflex circuits are used for multiple purposes. A CPG is a set of neurons and circuits capable of generating the rhythmic activity that underlies motor acts, even in the absence of sensory input. For example, activation of the FRA inter-neurons leads to a pattern of flexor excitation and extensor inhibition on one side and the converse pattern on the opposite side, and if the FRA interneurons on each side of the spinal cord alternated in being active, a stepping pattern would emerge. That is, walking motion could result from alternately activating the FRA interneurons on each side. Note that such a rhythmic activity pattern in the FRA circuits need not be dependent on activity from the FRA fibers themselves (e.g., they could be activated by descending pathways from the brain).

To show that these circuits are actually involved in generating the locomotion rhythm, spinal cord preparations were made that showed spontaneous locomotion (i.e., if the brainstem is transected and weight is supported, the spinal cord circuits can generate activity that causes the limbs to perform a normal locomotion sequence). In one such preparation, the electromyographic signals from the flexors and extensors of a limb were recorded, and the FRA fibers then stimulated to demonstrate the effect on locomotion rhythm (

Fig. 9.10 ). Before any stimulus, a spontaneous alternating pattern of flexor and extensor electromyographic (EMG) activity exists. If the FRA fibers were not involved in the locomotion circuit, or at least were not a critical part of the circuits responsible for generating the rhythm (see

Fig.

9.10B ), the stimulus would be expected to produce only a transient response (i.e., a single burst in the flexor EMG record and brief inhibition of activity in the extensor EMG record) but to have no long-term effect on ongoing EMG pattern. Such a transient response is observed (see

Fig. 9.10A ; EMG records just after the stimulus). However, the stimulus also causes a permanent, approximately 180degree phase shift in locomotor rhythm, as can shown from a comparison of the times of contractions before and after the stimulus. The dashed vertical lines indicate the times at which a flexor EMG response would be expected if the stimulus had produced no phase shift from the EMG activity pattern. Before the stimulus, each vertical line is aligned with the onset of a flexor EMG burst, whereas after the stimulus, each vertical line occurs at the end of the flexor burst. Therefore, the stimulus affected the locomotor CPG itself and the FRA interneurons are a critical part of this CPG (see

Fig. 9.10C

A second important point illustrated by this experiment is that the locomotion CPG (and CPGs in general) can be influenced by strong afferent fiber activity. The afferent fiber’s influence ensures that the pattern generator adapts to changes in the terrain as locomotion proceeds. Such changes may occur rapidly during running, and locomotion must then be adjusted to ensure proper coordination.

Determining Spinal Cord Organization Through the Use of Reflexes

Convergence and divergence are important aspects of reflex pathways and of neuronal circuits in general. Several examples of these phenomena have been described in the previous discussion of the reflexes. Reflexes can be used to identify and characterize these phenomena in the spinal cord. For example, convergent input can be demonstrated through the phenomenon of spatial facilitation, which is illustrated in

Fig. 9.11

In this example, a monosynaptic reflex is elicited by electrical stimulation of the group Ia fibers in each of two nerves (see

Fig. 9.11A ). The reflex response is characterized by a recording of the discharges of α motor axons from the appropriate ventral root (as a compound action potential). When nerve A is stimulated, a small compound action potential is recorded as reflex A. Similarly, when nerve B is stimulated, reflex B is recorded.

Fig. 9.11B depicts the motor neurons contained within the motor nucleus.

CHAPTER 9 Organization of Motor Function •Fig. 9.10 Phase Reset of Locomotion Rhythm by Flexion Reflex Afferent (FRA) Fiber Stimulation Helps Identify Neuronal Components of the Underlying Central Pattern Generator (CPG). A, EMGrecordsfromkneeflexorandextensormuscles.Notetherhythmicalternatingpatternbeforeapplicationofthestimulus.Thesolid vertical lines beloweachtraceindicatethetimesatwhichflexorcontractionisinitiated.Thedashed vertical lines indicatethetimesatwhichflexorcontractionwouldhavebeeninitiatedifthestimuluscausednolastingeffectontherhythmicpattern.B andC, TwopossiblemodelsfortheCPGunderlyingthelocomotorrhythmdepictedinA. B, TheFRAinterneurons(INs)intheCPGarenotshown.C, TheFRAinterneuronsareshown.ThedatashowninA supportthemodelshowninC. MN,motoneuron.(DatafromHultbornH,etal.Ann N Y Acad Sci. 1998;860:70.)

The α motor neurons in the discharge zones are activated above threshold when each nerve branch is stimulated separately. Thus a distinct pair of α motor neurons spike when each nerve is stimulated alone. In addition, each of these motor neuron pairs is surrounded by a subliminal fringe of eight additional motor neurons that are excited but not sufficiently to trigger spikes. When the two nerves are stimulated at the same time, a much larger reflex discharge is recorded (compare RA and RB with RA + RB recordings at the right of

Fig. 9.11B ). As the figure demonstrates, this reflex represents the discharge of seven α motor neurons: the four that spiked after the singular stimulation of each nerve (two per nerve) and three additional α motor neurons (located in the facilitation zone) that are made to discharge only when the two nerves are stimulated simultaneously because they lie in the subliminal fringe for both nerves.

A similar effect could be elicited by repetitive stimulation of one of the nerves, provided that the stimuli occur close enough together that some of the excitatory effect of the first volley still persists after the second volley arrives. This effect is called temporal summation. Both spatial summation and temporal summation depend on the properties of the excitatory postsynaptic potentials evoked in α motor neurons by the group Ia afferent fibers (see

Fig. 6.8).

Convergence can also lead to inhibitory interactions between stimuli, a phenomenon called occlusion. If a volley in one of the two nerves in

Fig. 9.11 reaches the motor nucleus at a time when the motor neurons are highly excitable, the reflex discharge is relatively large (see

Fig. 9.11C ). A similar volley in the other nerve might also produce a large reflex response. However, when the two nerves are excited simultaneously, the reflex can be less than the sum of the two independently evoked reflexes if the cells reaching threshold to activation of either of the two nerves alone overlap significantly. In this case, each afferent nerve activates 7 α motor neurons, but the volleys in the two nerves together cause only 12 α motor neurons to discharge because two motor neurons lie in the individual discharge zones of both afferent nerves.

The phenomena of spatial and temporal summation and occlusion can also be used to demonstrate interactions between spinal cord neurons and the various reflex circuits. To start, a monosynaptic reflex discharge can be evoked by stimulation of the group Ia afferent fibers in a muscle nerve. This is a test of the reflex excitability of a population of α motor neurons. The discharges of either extensor or flexor α motor neurons can be recorded if the proper muscle nerve to be stimulated is chosen. Other kinds of afferent fibers •Fig. 9.11 Spatial Facilitation. A, Arrangementforusingelectricallyevokedafferentvolleysandrecordingsfrommotoraxonsinaventralroottostudyreflexes.B, Experimentinwhichcombinedstimulationofafferentfibersintwomusclenervesresultedinspatialsummation(RAandRB).Thedischargezones(pink areas) encloseα motorneuronsthatareactivatedabovethresholdwheneachnervebranchisstimulatedseparately.InC, thecombinedvolleyscausedocclusion.(RedrawnfromEyzaguirreC,FidoneSJ.Physiology of the Nervous System. 2nded.Chicago:Mosby–YearBook;1975.) are then stimulated along with the homonymous group Ia afferent fibers from the muscle to demonstrate whether the response to the group Ia stimulation changes. For example, stimulation of group Ia afferent fibers in the nerve to the antagonist muscles produces inhibition of the response to the homonymous group Ia stimulation (which is mediated by the reciprocal group Ia inhibitory interneuron described previously).

As another example, if the small afferent fibers of a cutaneous nerve are stimulated to evoke a flexion reflex, the responses to group Ia stimulation of the α motor neurons that innervate the extensor muscles are inhibited (and those of α motor neurons that innervate flexor muscles are potentiated).

As a final example, stimulation of a ventral root causes inhibition of group Ia responses and inhibits the reciprocal group Ia inhibition. Because the ventral root contains only motor neuron axons, this result implies the presence of axon collaterals that excite inhibitory interneurons that feed back onto the same motor neuron population (

Fig. 9.12 ). These interneurons are named Renshaw cells. Because ventral root stimulation also inhibits the group Ia inhibition of antagonist motor neurons, but no other classes of interneurons, the reciprocal group Ia interneurons are uniquely inhibited by ventral root stimulation (and activated by group Ia stimulation). Experiments like these described have been used to provide a detailed knowledge of the circuitry of the spinal cord.

•Fig. 9.12 Renshaw Cell (RC) Connections With Motor Neurons and Group Ia Inhibitory Interneurons. ThecircuitsshownmediategroupIareciprocalinhibitionofantagonistmuscles(inthiscase,anextensor)andinhibitionofthisreciprocalinhibitionbyRenshawcells.NotethatequivalentnumbersofRenshawcellsandgroupIainhibitoryinterneuronsareassociatedwithextensormotorneuronsandgroupIainputfromspindlesinextensormuscles,buttheyarenotshownforsimplicity.Orange cells areinhibitory,andblue andgreen cells areexcitatory.

Classification of Descending Motor Pathways

Descending motor pathways were traditionally subdivided into pyramidal and extrapyramidal pathways. This terminology reflects a clinical dichotomy between pyramidal tract disease and extrapyramidal disease. In pyramidal tract disease, the corticospinal (pyramidal) tract is interrupted. The signs of this disease were originally attributed to the loss of function of the pyramidal tract (so named because the corticospinal tract passes through the medullary pyramid). However, in many cases of pyramidal tract disease, the functions of other pathways are also altered, and most signs of pyramidal tract disease (see the later section “

Caused by Lesions of Descending Motor Pathways ”) are apparently not caused solely by loss of the corticospinal tract but also reflect damage to additional motor pathways. The

CHAPTER 9 Organization of Motor Function term extrapyramidal is even more problematic. Thus this classification system is not used in this book.

Another way of classifying the motor pathways is based on their sites of termination in the spinal cord and the consequent differences in their roles in the control of movement and posture. The lateral pathways terminate in the lateral portions of the spinal cord’s gray matter (

Fig. 9.13 ). The lateral pathways can excite motor neurons directly, although interneurons are their main target. They influence reflex arcs that control fine movement of the distal ends of limbs, as well as those that activate supporting musculature in the proximal ends of limbs. The medial pathways end in the medial ventral horn on the medial group of interneurons (see

Fig. 9.13 ). These interneurons connect bilaterally with motor neurons that control the axial musculature and thereby contribute to balance and posture. They also contribute to the control of proximal limb muscles. In this book, the terms lateral and medial are used to classify the descending motor pathways. However, even this terminology is not perfect, partly because although motor neuron cell bodies form localized columns, motor neuron dendritic trees are rather large and typically span most of the ventral horn. Thus any motor neuron can potentially receive input from so-called medial or lateral system pathways.

The Lateral System

The corticospinal and corticobulbar tracts originate from a wide region of the cerebral cortex. This region includes the primary motor, premotor, supplementary, and cingulate motor areas of the frontal lobe and the somatosensory cortex of the parietal lobe. The cells of origin of these tracts include both large and small pyramidal cells of layer V of the cortex, including the giant pyramidal cells of Betz. Although Betz cells are a defining feature of the primary motor cortex, they represent a small minority (<5%) of the cells that contribute to these tracts, in part because they are found only in the primary motor cortex, and even there they represent a minority of the cells contributing to the tracts. These tracts leave the cortex and enter the internal capsule, then traverse the midbrain in the cerebral peduncle, pass through the basilar pons, and emerge to form the pyramids on the ventral surface of the medulla (see

Fig. 9.13A). The corticobulbar axons leave the tract as it descends in the brainstem and terminate in the motor nuclei of the various cranial nerves. The corticospinal fibers continue caudally, and in the most caudal region of the medulla, about 90% of them cross to the opposite side. They then descend in the contralateral lateral funiculus as the lateral corticospinal tract. The lateral corticospinal axons terminate at all spinal cord levels, primarily on interneurons, but also on motor neurons. The remaining uncrossed axons continue caudally in the ventral funiculus on the same side as the ventral corticospinal tract, which belongs to the medial system. Many of these fibers ultimately decussate (cross) at the spinal cord level at which they terminate.

•Fig. 9.13 Descending Motor Pathways. Majorpathwaysconnectingthecorticalandbrainstemmotorareastothespinalcordareshown.A, Lateralsystempathways,corticospinal(red) andrubrospinal(blue) pathways.NotethattheventralcorticospinalpathwayispartofthemedialsystembutisshowninA forsimplicity.B, Medialsystempathways,medullary(blue) andpontine(green) reticulospinalandlateralvestibulospinal(red) pathways.C-B,corticobulbar;C-P,corticopontine;C-S,corticospinal;LCST,lateralcorticospinaltract;VCST,ventralcorticospinaltract.

The lateral corticospinal tract is a relatively minor tract in lower mammals but is quantitatively and functionally very important in primates, particularly in humans, in which it contains more than 1 million axons. This number still represents a relatively small proportion of the outflow from the cortex because there are approximately 20 million axons in the cerebral peduncles. Nevertheless, the corticospinal pathway is critical for the fine independent control of finger movement, inasmuch as isolated lesions of the corticospinal tract typically lead to a permanent loss of this ability, even though other movement abilities are often recovered with such lesions. Indeed, in primates, corticospinal synapses directly onto motor neurons are particularly prevalent for the motor neurons controlling finger muscles and are probably the basis of the ability to make independent, finely controlled finger movements.

The corticobulbar tract, which projects to the cranial nerve motor nuclei, has subdivisions that are comparable with the lateral and ventral corticospinal tracts. For example, part of the corticobulbar tract ends contralaterally in the portion of the facial nucleus that supplies muscles of the lower part of the face and in the hypoglossal nucleus. This component of the corticobulbar tract is organized like the lateral corticospinal tract. The remainder of the corticobulbar tract ends bilaterally.

The rubrospinal tract originates in the magnocellular portion of the red nucleus, which is located in the midbrain tegmentum. These fibers decussate in the midbrain, descend through the pons and medulla, and then take up a position just ventral to the lateral corticospinal tract in the spinal cord. They preferentially affect motor neurons controlling distal musculature, as do the corticospinal fibers. Red nucleus neurons receive input from the cerebellum and from the motor cortex; thus making this an area of integration of activity from these two motor systems.

The Medial System

The ventral corticospinal tract and much of the corticobulbar tract can be regarded as medial system pathways. These tracts end on the medial group of interneurons in the spinal cord and on equivalent neurons in the brainstem. The axial muscles are controlled by these pathways. These muscles often contract bilaterally to provide postural support or some other bilateral function, such as swallowing or wrinkling of the brow.

Other medial system pathways originate in the brainstem. These include the pontine and medullary reticulospinal tracts, the lateral and medial vestibulospinal tracts, and the tectospinal tract.

The cells that give rise to the pontine reticulospinal tract are in the medial pontine reticular formation. The tract descends in the ventral funiculus, and it ends on the

CHAPTER 9 Organization of Motor Function ipsilateral medial group of interneurons. Its function is to excite motor neurons to the proximal extensor muscles to support posture.

The medullary reticulospinal tracts arise from neurons of the medial medulla, particularly those of the gigantocellularis reticular nucleus. The tracts descend bilaterally in the ventral lateral funiculus, and they end mainly on interneurons associated with cell groups of medial motor neurons. The function of the pathway is mainly inhibitory.

The lateral vestibulospinal tract originates in the lateral vestibular nucleus, also known as Deiter’s nucleus. This tract descends ipsilaterally through the ventral funiculus of the spinal cord and ends on interneurons associated with the medial motor neuron groups. The lateral vestibulospinal tract excites motor neurons that supply extensor muscles of the proximal part of the limb that are important for postural control. In addition, this pathway inhibits flexor motor neurons because it also excites the reciprocal group Ia interneurons that receive group Ia input from extensor muscles, which in turn inhibit flexor motor neurons. The excitatory input to the lateral vestibular nucleus is from both the semicircular canals and the otolith organs, whereas the inhibitory input is from the Purkinje cells of the anterior vermis region of the cerebellar cortex. An important function of the lateral vestibulospinal tract is to assist in postural adjustments after angular and linear accelerations of the head.

The medial vestibulospinal tract originates from the medial vestibular nucleus. This tract descends in the ventral funiculus of the spinal cord to the cervical and midthoracic levels, and it ends on the medial group of interneurons. Sensory input to the medial vestibular nucleus from the labyrinth is chiefly from the semicircular canals. This pathway thus mediates adjustments in head position in response to angular acceleration of the head.

The Tectospinal Tract

The tectospinal tract originates in the deep layers of the superior colliculus. The axons cross to the contralateral side, just below the periaqueductal gray matter. They then descend in the ventral funiculus of the spinal cord to terminate on the medial group of interneurons in the upper cervical spinal cord. The tectospinal tract regulates head movement in response to visual, auditory, and somatic stimuli.

In addition to the lateral and medial systems, less specifically organized systems descend from the brainstem to the spinal cord. These include several pathways in which monoamines serve as synaptic transmitters.

The locus coeruleus and the nucleus subcoeruleus are nuclei located in the rostral pons, and they are composed of norepinephrine-containing neurons. These nuclei project widely throughout the CNS, and their projection to the spinal cord travels in the lateral funiculus. Their terminals are on interneurons and motor neurons. The dominant effect of the pathway is inhibitory.

The raphe nuclei of the medulla also project widely throughout the CNS and give rise to several raphe-spinal pathways. With regard to motor function, the ventral horn projection may enhance motor activity.

In general, the monoaminergic pathways act to alter the responsiveness of spinal cord circuits, including the reflex arcs. In this way, they induce widespread changes in excitability rather than discrete movements or specific changes in behavior.

Motor Deficits Caused by Lesions of Descending Motor Pathways

A common cause of motor impairment in humans is interruption of the cerebral cortical efferent fibers in the internal capsule; such interruptions occur in capsular strokes. The resulting disorder is often termed a pyramidal tract syndrome, or upper motor neuron disease, although these names are misnomers. Motor changes characteristic of this disorder include (1) increased phasic and tonic stretch reflexes (spasticity); (2) weakness, usually of the distal muscles, especially the finger muscles; (3) pathological reflexes, including the sign of Babinski (dorsiflexion of the big toe and fanning of the other toes when the sole of the foot is stroked); and (4) a reduction in superficial reflexes, such as the abdominal and cremasteric reflexes. Of importance is that if only the corticospinal tract is interrupted, as can occur with a lesion of the medullary pyramid, most of these signs are much reduced or absent. In this situation, the most prominent deficits are weakness of the distal muscles, especially those of the fingers, and a Babinski sign. Spasticity does not occur; instead, muscle tone may actually decrease. Evidently, the presence of spasticity requires the disordered function of other pathways, such as the reticulospinal tracts, as would occur after loss of the descending cortical influence to the brainstem nuclei of origin of these tracts.

The effects of interruption of the medial system pathways are quite different from those produced by corticospinal tract lesions. The main deficits associated with medial system interruption are an initial reduction in the tone of postural muscles and loss of righting reflexes. Long-term effects include locomotor impairment and frequent falling. However, manual manipulation of objects is perfectly normal.

The Decerebrate Preparation

The decerebrate preparation has been useful for experimentally investigating how various descending pathways interact with the spinal cord circuitry. Surgical decerebration is achieved either by transection of the midbrain, often at an intercollicular level, or by occlusion of the blood vessels feeding this area. In the latter case, a lesion also occurs in the anterior vermis of the cerebellum, an important distinction.

With the intercollicular transection, some descending pathways, such as those originating in the cerebral cortex, are interrupted, whereas others, such as those originating in the brainstem, remain intact.

However, remember that the corticospinal tract is only one component of the cortical descending fibers. Many other cortical fibers project to locations throughout the brainstem, including the nuclei of origin for the medial descending pathways. Loss of these cortical control systems results in altered activity in the intact descending pathways. As a result, affected animals show hypertonia and suppression of some spinal reflexes, such as the flexion reflex, and exaggeration of others, such as the stretch reflex; this condition is called decerebrate rigidity. Decerebrate animals maintain a posture that has been called exaggerated standing. Human patients with brainstem damage may also develop a decerebrate state that has many of the same reflex features as animal preparations. The prognosis in such patients is poor if signs of decerebration appear.

Loss of descending control on the reticular formation results in increased activity in the pontine reticulospinal pathway and decreased activity in the medullary reticulospinal pathway. Such increase and decrease in activity, respectively, produces increased excitation and decreased inhibition (disinhibition) of the motor neurons, which explains the observed rigidity. Interestingly, this hypertonia can be relieved by cutting the dorsal roots, which indicates that the reticulospinal tracts have a major effect on γ motor neurons. This is because γ motor neuron activity alters muscle stiffness by increasing muscle spindle sensitivity and thereby causes increased activity in the group Ia and group II afferent fibers, which travel through the dorsal roots into the spinal cord in order to innervate the α motor neurons.

When vessel occlusion is used to generate the decerebrate state, the lateral vestibulospinal tract becomes hyperactive because of damage to Purkinje cells in the anterior vermis of the cerebellum, which provide the major inhibitory projection to the lateral vestibular nucleus. This hypertonia is actually not lost after transection of the dorsal roots, which implies that the lateral vestibulospinal tract is acting to a significant extent directly on α motor neurons (either monosynaptically or via interneurons).

Brainstem Control of Posture and Movement

The importance of motor control pathways that originate in the brainstem is evident from observations of the extensor hypertonus and increased phasic stretch reflexes that occur in decerebrate animals. Particular brainstem systems have been identified as influencing posture and locomotion. Brainstem circuits are also critically involved in the control of eye movement; these circuits are discussed in a separate section at the end of the chapter.

Several reflex mechanisms are evoked when the head is moved or the neck is bent. There are three types of postural reflexes: vestibular reflexes, tonic neck reflexes, and righting reflexes. The sensory receptors responsible for these reflexes include the vestibular apparatus (see ), which is stimulated by head movement, and stretch receptors in the neck.

The vestibular reflexes constitute one class of postural reflex. Rotation of the head activates sensory receptors of the semicircular canals (see ). In addition to generating eye movement, the sensory input to the vestibular nuclei results in postural adjustments. Such adjustments are mediated by commands transmitted to the spinal cord through the lateral and medial vestibulospinal tracts and the reticulospinal tracts. The lateral vestibulospinal tract activates extensor muscles that support posture. For instance, if the head is rotated to the left, postural support is increased on the left side. This increased support prevents the person from falling to the left as the head rotation continues. A person who has any disease that eliminates labyrinthine function in the left ear tends to fall to the left. Conversely, a person with a disease that irritates (stimulates) the left labyrinth tends to fall to the right. The medial vestibulospinal tract causes contractions of neck muscles that oppose the induced movement (vestibulocollic reflex).

Tilting the head also changes the linear acceleration on individual hair cells of the otolith organs of the vestibular apparatus. The resulting changes in hair cell activity can produce eye movement and postural adjustment. For example, when a quadruped, such as a cat, tilts the head and body forward (without bending the neck and consequently without evoking the tonic neck reflexes), the result is extension of the forelimbs and flexion of the hind limbs. This vestibular action tends to restore the body toward its original orientation. Conversely, if the quadruped tilts the head and body backward (without bending the neck), the forelimbs flex and the hind limbs extend. Otolithic organs also contribute to the vestibular placing reaction. If an animal, such as a cat, is dropped, stimulation of the utricles leads to extension of the forelimbs in preparation for landing.

The tonic neck reflexes are another type of positional reflex. These reflexes are activated by the muscle spindles found in neck muscles. These muscles contain the largest concentration of muscle spindles of any muscle in the body. If the neck is bent (without tilting of the head), the neck muscle spindles evoke tonic neck reflexes without interference from the vestibular system. When the neck is extended, the forelimbs extend and the hind limbs flex. The opposite effects occur when the neck is flexed. Note that these effects are opposite those evoked by the vestibular system. Furthermore, if the neck is bent to the left, the extensor muscles in the limbs on the left contract more, and the flexor muscles in the limbs on the right side relax.

CHAPTER 9 Organization of Motor Function

The third class of postural reflex is the righting reflexes. These reflexes tend to restore an altered position of the head and body toward normal. The receptors responsible for righting reflexes include the vestibular apparatus, the neck stretch receptors, and mechanoreceptors of the body wall.

Brainstem Control of Locomotion

The spinal cord contains neural circuits that serve as central pattern generators for locomotion, as discussed earlier. These CPG circuits produce very regular rhythmic output that characterizes stereotyped behavior, such as walking. The irregularities of real-world environments, however, often require modification of this stereotyped output (e.g., if you are walking and see a hole in the floor where you are about to step, you can extend the forward swing of your leg past the hole onto solid ground beyond it).

Such modifications can be the result of sensory input to the spinal cord, as shown in

Fig. 9.10 , in which stimulation of FRA fibers in a peripheral nerve caused a phase shift in the locomotor pattern. They can also be the result of descending commands along the motor pathways discussed earlier. In this case, sensory data (e.g., visual) can be used by the brain to make anticipatory modifications in CPG activity so that potential obstacles can be avoided. In addition, people can voluntarily control activation, or shutdown, of the CPG (i.e., deciding consciously when to start and stop walking). Such voluntary regulation of spinal CPGs originates in the cerebral cortex; however, much of the cortical influence on locomotion appears to be mediated via projections to brainstem regions known as locomotor regions. A locomotor region can be defined as a brain area that, when stimulated, leads to sustained locomotion.

There are several such locomotor regions in the brain-stem, and they are located at different levels ranging from the subthalamus to the medulla and are connected with each other. The best known is the midbrain locomotor region, which is thought to organize commands to initiate locomotion. It is located in the midbrain at the level of the inferior colliculus. Voluntary activity that originates in the motor cortex can trigger locomotion by the action of corticobulbar fibers projecting to the midbrain locomotor region. The commands are relayed through the reticular formation and then to the spinal cord via the reticulospinal tracts.

Motor Control by the Cerebral Cortex

Thus far in this chapter, emphasis has been on reflexes and relatively automatic types of movement. We now discuss the neural basis for more complex, goal-directed voluntary movement. Such movement often varies when repeated and is frequently initiated as a result of cognitive processes rather than in direct response to an external stimulus. Thus it requires the participation of motor areas of the cerebral cortex.

•Fig. 9.14 Motor Areas of the Frontal Cortex. A andB, Lateralandmedialviewsofahemisphere,showingthemajorcorticalmotorareas.FEF,frontaleyefield.Theinset inB showsthewallsofthecingulatesulcus,whichcontainthecingulatemotorareas.NumbersinparenthesesareBrodmannareanumbersforthecingulatemotorareas(CMAs).C, Diagramshowinginterconnectionsofthemotorareas.PMA,premotorarea;SMA,supplementarymotorarea.

First, consider what is necessary to generate a voluntary movement. For example, to make a reaching movement with your arm, you must first identify the target (or goal) and locate it in external space. Next, a limb trajectory must be determined on the basis of an internal representation of your arm and, in particular, your hand in relation to the target. Finally, a set of forces necessary to generate the desired trajectory must be computed. This process is often thought of as a series of transformations between coordinate systems. For example, the location of a visually identified target is measured in a retinotopic space, but its location is perceived in an external or world space (i.e., the position of a nonmoving target is perceived as stable, even when the eye, and thus the target’s image on the retina, changes). Next, calculation of a trajectory would involve a body-or hand-centered system, and finally, forces must ultimately be computed in a muscle-based reference frame.

These steps form a linear sequence, and traditionally it was thought that a hierarchy of motor areas carried out the successive steps. For example, the target of the movement was thought to be identified by pooling of sensory information in the posterior parietal cerebral cortex (Fig.

9.14A ). This information would then be transmitted to the supplementary motor and premotor areas, where a motor plan would be developed and then forwarded to the primary motor cortex, whose activity would be related to the final execution stage (e.g., generation of appropriate force levels). The motor cortex would then transmit commands, via the descending pathways discussed earlier, to the spinal cord and brainstem motor nuclei.

Although there is significant evidence in support of this hierarchical view of the generation of voluntary movement by the cortical motor system, more recent results have suggested a different conception: namely, that the various motor areas should be thought of as forming a parallel distributed network rather than a strict hierarchy (see

Fig.

9.14C ). For example, each cortical motor area makes its own significant contribution to the descending motor pathways; the primary motor cortex contributes only approximately half the fibers in the corticospinal tract that arise from the frontal lobe. Moreover, the various motor areas are all bidirectionally connected to each other, and results of the single-unit recording studies described later suggest that each of the areas plays a role in several of the stages of planning and executing a movement. This debate forms one of the themes of the following discussion because in its various guises, the distributed network versus hierarchical organization debate has been ongoing for decades and will probably continue for some time.

The motor areas in the cerebral cortex were originally defined on the basis of experiments in which electrical stimuli applied to the cortex evoked discrete, contralateral movement. Movement, however, can also be evoked when other cortical areas are stimulated more intensely. Thus motor areas are defined as those from which movement can be evoked by the lowest stimulus intensity. On the basis of these stimulation studies, the effects produced by lesions, results of anatomical experiments, electrophysiological recordings, and modern imaging studies in humans, many “motor” areas of the cerebral cortex have been recognized (see

Fig. 9.14 ), including the primary motor cortex in the precentral gyrus, the premotor area just rostral to the primary motor cortex, the supplementary motor cortex on the medial aspect of the hemisphere, and three cingulate motor areas located on the walls of the cingulate sulcus in the frontal lobe. There are also cortical regions in other lobes whose activity is related specifically to eye movement (see the section

Somatotopic Organization of Cortical Motor Areas

The primary motor cortex (or just motor cortex) can be defined as the region of cortex from which movements are elicited with the least amount of electrical stimulation. It is essentially congruent with the Brodmann cytoarchitectonic area 4 (see

Fig. 10.3). In humans it is located on the parts of the precentral gyrus that form the rostral wall of the central sulcus and the caudal half of the apex of the gyrus. On the basis of initial mapping studies, which were done with surface stimulation, the motor cortex was described as having a topographic organization that parallels that of the somatosensory cortex. The face, body, and upper limb were represented on the lateral surface with the face located inferiorly, near the lateral fissure, the torso most superiorly, and the lower extremity mostly on the medial aspect of the hemisphere. This somatotopic organization is often represented as a figurine or in a graphic form called a motor homunculus (

Fig. 9.15B ). The distortion of the various body parts in the homunculus indicates approximately how much of the cortex is devoted to their motor control. This simple homunculus was likened to a piano keyboard and fit well with traditional conceptions of the motor cortex being the final cortical stage and acting as a relay for sending motor commands to the spinal cord.

Beginning in the 1960s and 1970s, investigators in mapping studies began using microelectrodes inserted to the deep, or output, layers of the cortex to apply stimuli. With this technique, called intracortical microstimulation, much lower stimulus intensities could be used to evoke movements and thus allowed higher resolution mapping of the motor cortex, which revealed a much more complex topography than was previously imagined (see

CHAPTER 9 Organization of Motor Function

Fig. 9.15C ). Movement about each joint was found to be evoked by many noncontiguous columns throughout wide regions of the motor cortex. Thus cell columns related to movement about a particular joint are actually interspersed among columns that control movement about many other joints. In sum, although the motor cortex may have large subdivisions corresponding to a limb or the head, within each such area there is a complex intermingling of cell columns that control the muscles within that body part.

Such mixing of cell columns makes functional sense because most movement requires the coordinated action of muscles throughout a limb and most connectivity in the cortex is localized (i.e., axon collaterals that connect different cell columns are primarily confined to a 1to 3-mm region surrounding the column from which they originate). Thus when multiple cell columns that control movement about a joint are present and intermixed with columns controlling movement about other joints, multijoint movement can be generated as a whole.

Although the topographic map of the motor cortex is in part anatomically determined by the topography of the corticospinal pathway, it is also a dynamic map. Axon collaterals link the different cell columns, so that activity in one column could potentially lead to movement about multiple joints. In fact, this can happen, but these intercolumnar connections are modulated by inhibitory interneurons that transmit or secrete gamma-aminobutyric acid (GABA). This was shown by locally blocking GABA in one region of the motor cortex and then stimulating the neighboring region. Before the block, stimuli evoked contractions of one set of muscles, but once inhibition was blocked, contractions were also evoked in muscles controlled by the region that was no longer inhibited (

Fig. 9.16 ). Functional connections between cell columns can be controlled on a millisecond time scale, and depending on their state, the motor cortex map can be radically changed. Longer term plastic changes are also known to occur; for example, the use (or disuse) of a body part can affect the size of its somatotopic representation.

The supplementary motor area (SMA) is located mainly on the medial surface of the hemisphere, just anterior to the primary motor cortex, and corresponds to the medial portion of Brodmann area 6 (see

Fig. 9.14). It is subdivided into two regions: the more posterior part is referred to as the SMA proper (or just SMA), and the anterior portion is called the pre-SMA. The SMA proper is similar to the other motor areas already listed: it contains a complete somatotopic map, it contributes to the corticospinal tract, and it is interconnected with the other motor areas. In contrast, the pre-SMA is not strongly connected with the other motor areas and spinal cord but rather is connected to the prefrontal cortex.

The results of stimulation studies show that as in the motor cortex, there is a complete somatotopic map in the SMA. Stimulation of the SMA can evoke isolated movement

Rostral wall of central sulcus •Fig. 9.15 Traditional and Modern Views of Motor Cortex Musculotopic Organization. A, Lateralviewofthecerebrum,showingaplaneofsectionthroughtheprecentralgyrus(primarymotorcortex)thatcorrespondstothesectionshowninB. B, Classicviewofmotorcortexmusculotopy(“muscularhomunculus”).C, Modernviewofmotorcortexorganizationinwhicheachbodypartisrepresentedmultipletimesacrossseveraldiscreteregions.

CHAPTER 9 Organization of Motor Function •Fig. 9.16 Dynamic Nature of a Motor Cortex Musculotopic Map. InhibitoryGABAergicinterneuronsplayanimportantroleinshapingmotorresponsestostimulationofeachregionofthemotorcortex.A, Schematicviewofexcitatoryconnectionsbetweentworegionsofprimarymotorcortexandlocalinhibitoryneuronswithinasingleregion.B, Schematicviewofaratbrain,indicatingmotorcortex(MC)regionswhereelectricalstimuliwereappliedtoevokemovements(Vibregion)andbicucullinewasappliedtoblockGABAergicsynapses(intheFLregion).FL,forelimb;Vib,vibrissa.C, ForelimbEMGrecordsshowingresponsetostimulationoftheVibregionbeforeandduringtheapplicationofbicucullineandafteritwaswashedout.NotethatVibstimulationevokedvibrissaemovementinallconditionsbutevokedforelimbmovementonlywheninhibitoryinterneuronsinFLwereblocked.(DatafromJacobsK,DonoghueJ.Science. 1991;251:944.) about single joints, similar to that after stimulation of the motor cortex, but stimulation must be of higher intensity and longer duration; moreover, the evoked movements are often more complex than those evoked by stimulation of the motor cortex. However, longer duration stimulation of the primary motor cortex can also evoke complex, apparently purposeful movement sequences, and so the distinction is not absolute. In addition, stimulation of the SMA can produce vocalization or complex postural movements, but it can also have the opposite result: namely, a temporary arrest of movement or speech. Removal of the supplementary motor cortex retards movement of the opposite extremities and may result in forced grasping movements with the contralateral hand.

This area lies rostral to the primary motor cortex and is contained in Brodmann area 6 on the lateral surface of the brain (see

Fig. 9.14 ). It can be distinguished from the primary motor cortex by the higher stimulus intensities needed to evoke movement. The premotor area has been divided into two functionally distinct subdivisions: dorsal and ventral. Like the motor cortex, both subdivisions are somatotopically organized and both contribute to the corticospinal tract. The dorsal division (PMd) contains a relatively complete map representing the leg, trunk, arm, and face. In contrast, the somatotopic map of the ventral division (PMv) is mostly limited to the arm and face, with only a small leg representation. Thus the PMv appears to be specialized for control of upper limb and head movement. A second difference between the subdivisions is that the PMd contains a large representation of the proximal muscles, whereas the PMv has a large representation of the distal muscles.

These motor areas are located within the cingulate sulcus at approximately the same anterior-posterior level as the SMA. There are three cingulate motor areas (dorsal, ventral, and rostral; see

Fig. 9.14B ). Each contains a somatotopic map and contributes to the corticospinal tract. Microstimulation in these areas evokes movement similar to that evoked by motor cortex stimulation, except that, again, higher stimulus intensities are needed. Single-cell recordings during movements have shown that the spontaneous activity of neurons in the cingulate motor areas is related to the preparation and execution of movements.

Connections of the Cortical Motor Areas

The motor areas of the cortex receive input from a number of sources, cortical and subcortical; however, the largest source of synapses in an area is the area itself: specifically, the local intrinsic connections. Moreover, all the motor areas described earlier are bidirectionally connected to each other with high topographic specificity (see

Fig. 9.14C ). For example, the arm regions of the primary motor cortex and the cingulate motor areas project to each other. Ascending pathways relay sensory information to the thalamus. This information can reach the motor cortex directly from the thalamus or indirectly by way of the somatosensory cortex. Both somatosensory information and visual information are conveyed to the motor areas from the posterior parietal cortex. The motor areas of the cortex also receive information through circuits that interconnect them with the other major brain regions involved in motor control: namely, the cerebellum and basal ganglia. These two structures project to distinct parts of the thalamus (the ventral lateral and ventral anterior nuclei), which then project to the cortical motor areas.

The output of the cortical motor areas to the spinal cord and brainstem is conducted through several descending pathways. These pathways include not only direct projections through the corticospinal and corticobulbar tracts (to the cranial nerve nuclei) but also indirect projections to the red nucleus and to various nuclei in the reticular formation. Descending projections from these brainstem sites were reviewed in the section .” Control of head and neck muscles is mediated by projections to the various cranial nerve nuclei. The motor regions also project to the cerebellum and basal ganglia, thus completing neuroanatomical loops with these structures. The major connection to the cerebellum is via the corticopontine projections to the basilar pontine nuclei, which in turn project to the cerebellum. In addition, the cortical motor areas project, mostly via disynaptic pathways that synapse in the midbrain, to the inferior olivary nucleus, another important precerebellar area. The cortical motor regions project directly to the striatum of the basal ganglia. Finally, there are major projections to the thalamus by which the cortex regulates the information that it receives.

Activity of Motor Cortex Cells

The role of individual motor cortex neurons in the control of movement has been extensively investigated in trained monkeys. In these experiments, discharges from a neuron in the primary motor cortex are recorded during the execution of a previously learned simple movement, such as wrist flexion, made immediately in response to a sensory cue (Fig.

9.17 ). Motor cortex neurons were found to change their firing rates before initiation of the movement, and the onset of this change was correlated with the reaction time (i.e., the time from the cue to onset of the movement). Moreover, in this task, the change in firing of motor cortex neurons was often correlated with the contractile force of the muscle that generates the movement and with the rate of change in force rather than with the position of the joint. These findings suggest that these cells are involved in the final stages of planning and executing movements, which is consistent with the hierarchical view of the cortical motor areas.

However, even in these early experiments, the firing rates of some motor cortex cells appeared to relate to earlier planning stages. Moreover, even when a monkey was trained to withhold the movement for a certain period after the cue, the firing rates of motor cortex neurons still changed despite the absence of any movement. Such “set-related” activity has been amply confirmed in a variety of other tasks and suggests that motor cortex activity may be involved in the earlier planning stages along with activity in other motor areas of the cortex. It also suggests the possibility that other, perhaps subcortical, systems may be needed to generate a trigger signal for the initiation of movement.

In subsequent studies, researchers have used tasks in which animals were trained to move a manipulandum (a device with a handle to hold and a small circle on the end) to capture lighted targets on a surface in front of them (Fig.

9.18A ). These experiments demonstrated that cells in the arm region of the motor cortex showed changes in their firing rates in response to movement in many different directions and thus were described as broadly tuned (see

Fig.

9.18B ). That is, a cell that showed a maximal increase for movement in one particular direction, called its preferred direction, would also show somewhat smaller increases or even decreases for movement in other directions (see

Fig.

9.18C ). Moreover, the preferred directions of the different cells were uniformly distributed across all 360 degrees of possible movement directions.

These results implied that a particular cell is probably involved in most arm movements, but they also raised the issue of how precise movements could be made with such broadly tuned cells. It was suggested that although changes in the activity of individual cells could not precisely predict or specify the direction of the upcoming movement, the net activity of the population could. To test this idea, models were made in which the activity of each cell is represented as a vector (see

Fig. 9.18D ). The direction of each cell vector is determined by the preferred direction of the cell, and the magnitude of the vector for a particular movement is proportional to the firing rate of the cell during the time preceding the movement. The individual cell vectors (see

Fig. 9.18D , black lines) from hundreds of cells can then be vectorially summed to get a resultant or population vector (see

Fig. 9.18D , red lines) that accurately predicts the upcoming movement.

One of the difficulties in assessing the relationship between firing of cortical cells and various movement parameters, such as force, velocity, displacement, and target location, is that these parameters are normally correlated with each other. Therefore, variations of the tasks described earlier have been used to decorrelate these various parameters (e.g., using weights to vary the force needed to make a movement without changing the displacement, as illustrated in

Fig. 9.17A , or rotating the starting position of the wrist so that different muscles are required to generate the same trajectory in external space). The results of these experiments showed that the activity of motor cortex cells may be related to each of the various motor planning stages. Furthermore, the activity of a single cell may be correlated with one parameter initially and then switch as the time for onset of movement approaches.

Activity in the premotor and supplementary motor areas is in many ways similar to that in the primary motor cortex. Cells in these areas show activity related to upcoming

CHAPTER 9 Organization of Motor Function flexion movement results from relaxation of antagonist •Fig. 9.17 A, Experimentalarrangementforrecordingfromacorticospinalneuronwhileamonkeyperformstrainedwristmovements.Astimulationelectrodeisusedtoelicitantidromicspikesthatareusedtoidentifythemotorcortexneuronspecificallyasapyramidaltractneuron.Stimuliarenotappliedwhilethemonkeyisperformingmovements.B, Thepyramidaltractneuron(PTN)dischargesbeforetheonsetofmovementorEMGactivitywhenflexorsneedtogenerateforce(noloadandflexorloadconditions).Moreover,thefiringrateiscorrelatedwiththelevelofflexorforcethatisneeded.Intheextensorloadcondition,flexorsdonotneedtocontracttogeneratemovement,andthusthereisnoactivityinthisPTN.Thetoptraceshowswristmovement,whichisessentiallyidenticalforallthreeexperimentalconditions.Thusthiscell’sactivityinvolvesencodingforcemagnitudeanddirection,butnotdisplacement.(Figure basedonworkofEvartsandcolleagues.) movement, and the activity is correlated with movement parameters, such as displacement, force, and target location, just as primary motor cortex activity can be, which is consistent with the distributed network view of the cortical motor areas. There do, however, appear to be some real differences between the areas as well, although these differences may be more quantitative than qualitative. For example, the percentage of cells in the premotor and supplementary motor areas that show activity related to earlier motor planning stages is higher than that of such cells in the primary motor cortex. In addition, the premotor and supplementary motor areas can be distinguished from each other by the apparently greater involvement of the premotor area in movements made to external cues (such as in the task shown in

Fig.

9.18 ) and the greater involvement of the supplementary motor area in movements made in response to internal cues (i.e., self-initiated). Research has also revealed that each of these areas is functionally heterogeneous and can therefore be further subdivided; however, such details are beyond the scope of this discussion.

Motor Control by the Cerebellum

Overview of the Role of the Cerebellum in Motor Control

In the early 1900s, scientists showed that damage to the cerebellum led to deficits in motor coordination. That is, damage or loss of the cerebellum does not lead to paralysis, loss of sensation, or an inability to understand the nature of a task; rather, it leads to an inability to perform movements well. Yet it has been hard to define the precise role or 45 135 225 315° Direction of movement • Fig. 9.18 A, Experimental setup in which a monkey holds onto the arm of the apparatus and captures light spots with the distal end of the arm. The monkey first captures the central light spot and then captures whichever of the surrounding targets that becomes illuminated. B, Raster plots showing the activity of one motor cortex cell during movement in eight different directions. T indicates the time at which the target light turns on, whereas M indicates the time at the onset of movement, which is at the center of each raster. Each mark on a raster represents a spike of a motor cortex cell, and each row of marks shows the cell’s activity during one trial. C, Cosine function was fit to the firing level as a function of the direction of movement. The horizontal bar indicates the average spontaneous firing rate in the absence of an upcoming movement. Note that for most directions, the activity in the periods just before and during movement changed significantly from baseline.

•Fig. 9.18, cont’d D, Vectormodelofpopulationactivityinthemotorcortex.Black lines representindividualcellvectors.Whenallofthemaresummedforaparticulardirectionofmovement,theresultingpopulationvector(red) pointsinessentiallythedirectionoftheupcomingmovement.(B andC, ModifiedfromGeorgopoulosAP,etal.J Neurosci. 1982;2:1527.D, ModifiedfromGeorgopoulosAP,etal.InMassionJ,etal[eds].Experimental Brain Research Series, vol.7:Neuronal Coding of Motor Performance. Berlin:Springer-Verlag;1983.) roles of the cerebellum in generating movement, although, paradoxically, scientists have more detailed knowledge of its deceptively simple anatomic and physiological organization than of any other CNS region. The cerebellum is proposed to play a critical role in the learning and execution of both voluntary and certain reflex movements. However, hypotheses about these roles face significant challenges that prevent their complete acceptance. In this section, the behavioral effects of damaging the cerebellum are discussed, followed by a description of its connectivity, both intrinsic and with the rest of the CNS, and then finally a discussion of its activity.

Behavioral Consequences of Cerebellar Damage

Damage to one side of the cerebellum impairs motor function on the ipsilateral side of the body. This reflects a double crossing of most cerebellum-related output as it travels to the motor neurons. The first crossing typically occurs in the cerebellar efferent pathway, whereas the second crossing takes place in the descending motor pathways. For example, the cerebellum projects to the contralateral motor cortex, via the thalamus, and the corticospinal pathway recrosses the midline at the lower medulla.

The specific motor deficits that result from cerebellar lesions depend on which functional component of the

CHAPTER 9 Organization of Motor Function cerebellum is most affected. If the flocculonodular lobe is damaged, the motor disorders resemble those produced by a lesion of the vestibular apparatus; such disorders include difficulty in balance and in gait and often nystagmus. If the vermis is affected, the motor disturbance affects the trunk, and if the intermediate region or hemisphere is involved, motor disorders occur in the limbs. The part of the limbs affected depends on the site of damage; hemispheric lesions affect the distal muscles more than paravermal lesions do.

Types of motor dysfunction in cerebellar disease include disorders of coordination, equilibrium, and muscle tone. Incoordination is called ataxia and is often manifested as dysmetria, a condition in which errors in the direction and force of movement prevent a limb from being moved smoothly to a desired position. Ataxia may also be manifested as dysdiadochokinesia, in which rapid alternating of supination and pronation of the arm is difficult to execute. When more complicated movement is attempted, decomposition of movement occurs, in which the movement is accomplished in a series of discrete steps rather than as a smooth sequence. An intention tremor appears when the subject is asked to touch a target; the affected hand (or foot) develops a tremor that increases in magnitude as the target is approached. When equilibrium is disturbed, impaired balance may be seen, and the individual tends to fall toward the affected side and may walk with a wide-based stance (gait ataxia). Speech may be slow and slurred; such a defect is called scanning speech. Muscle tone may be diminished (hypotonia), except for lesions of the anterior vermis (see earlier section on decerebrate rigidity); the diminished tone may be associated with a pendular knee jerk. This can be demonstrated by eliciting a phasic stretch reflex of the quadriceps muscle by striking the patellar tendon. The leg continues to swing back and forth because of the hypotonia, in contrast to the highly damped oscillation in a normal person.

These disorders reflect, in part, abnormal timing of muscle contractions. Normally, limb movements involve precisely timed EMG bursts in both agonist and antagonist muscles. There is an initial agonist burst followed by a burst in the antagonist and, finally, a second agonist burst. With cerebellar damage, the relative timing of these bursts is abnormal (

Fig. 9.19

The cerebellum (“little brain”) is located in the posterior fossa of the cranium, just below the occipital lobe, and is connected to the brainstem via three cerebellar peduncles (superior, middle, and inferior). From the outer surface, only the cortex is visible. Deep to the cortex is the white matter of the cerebellum, and buried within the white matter are the four cerebellar nuclei: proceeding medially to laterally, the fastigial, globose, emboliform, and dentate nuclei. The middle two nuclei are often grouped together and referred to as the interpositus nucleus. For the most part, cerebellar afferent fibers to the cortex and nuclei enter the cerebellum •Fig. 9.19 Disruption of Cerebellar Activity Alters the Timing of EMG Responses During Movement. Thecerebellarnucleiwerecooledtoblocktheirfunctioningtemporarilywhilemonkeysperformedmovementsabouttheirelbow.LossofcerebellaractivitydisruptstherelativetimingofagonistandantagonistEMGbursts.Thisleadstoabnormalaccelerationofthelimbandamovementtrajectorythatovershootsthetargetposition(hypermetria).(DatafromFlamentD,HoreJ.J Neurophysiol. 1986;55:1221.) via the inferior and middle peduncles, and efferent fibers from the cerebellar nuclei leave via the superior peduncle.

The cerebellar cortex is subdivided into three rostrocaudally arranged lobes: the anterior lobe, the posterior lobe, and the flocculonodular lobe (

Fig. 9.20A ). The cerebellar lobes are separated by two major fissures, the primary fissure and the posterolateral fissure, and each lobe is made up of one or more lobules. Each lobule of the cerebellar cortex is composed of a series of transverse folds called folia.

The cerebellar cortex has also been divided into longitudinal compartments (see

Fig. 9.20B and C). Initially, the cerebellar cortex was divided into three such compartments: the vermis, which lies on the midline; the paravermis, which lies adjacent to both sides of the vermis; and the lateral hemispheres. These regions have now been subdivided into many further compartments on the basis of myeloarchitectonics (patterns of axonal bundles in the white matter) and the expression patterns of specific molecules, such as aldolase C. Although the functional significance of these compartments is not fully known, the topography of cerebellar afferent fibers, particularly the olivocerebellar system, is precisely aligned with them, and the receptive field properties of cerebellar Purkinje cells also tend to follow this organizational scheme.

There are two major classes of cerebellar afferent systems: mossy fibers and olivocerebellar fibers. Mossy fibers are named for their distinctive appearance in the cerebellar cortex: as a mossy fiber courses through the granule layer, on occasion it swells and sends out a bunch of short twisted branchlets. These entities are called rosettes and are points of synaptic contact between these fibers and neurons in the granule cell layer. Mossy fibers arise from many sources, including the spinal cord (the spinocerebellar pathways), dorsal column nuclei, trigeminal nucleus, nuclei in the reticular formation, primary vestibular afferent fibers, vestibular nuclei, cerebellar nuclei, and the basilar pontine nuclei. The details of specific mossy fiber projection patterns are beyond the scope of this chapter; however, several general points are worth noting: 1.

Mossy fibers are excitatory.

2.

They convey exteroceptive and proprioceptive information from the body and head and form at least two somatotopic maps of the body across the cerebellar cortex. However, like those of the motor cortex, these maps are fractured in the sense that contiguous body regions are not necessarily represented on contiguous areas of the cerebellar cortex; rather, the maps are complicated mosaics.

3.

Mossy fibers conveying vestibular information are restricted to the flocculonodular lobe and regions of the vermis. As a result, the flocculonodular lobe and regions of the vermis are sometimes referred to as the vestibulocerebellum. However, these same regions also receive a variety of other information (e.g., visual, neck, oculomotor), and so their function is not exclusively vestibular.

4.

The largest sources of mossy fibers are the basilar pontine nuclei, which serve to relay information from areas throughout much of the cerebral cortex.

5.

Mossy fibers enter the cerebellum via all three cerebellar peduncles and provide collateral fibers to the cerebellar nuclei before heading up to the cortex. In sum, via the mossy fiber system, the cerebellum receives a wide variety of sensory information, as well as descending motor-related and cognitive-related activity. In contrast to the diverse origins of mossy fibers, olivocerebellar fibers all originate from a single nucleus: the inferior olivary nucleus, which is located in the rostral medulla, just dorsal and lateral to the pyramids. Almost all the olivary neurons are projection cells whose axons leave the nucleus without giving off collaterals and then cross the brainstem to enter the cerebellum primarily via the inferior cerebellar peduncle. Like mossy fibers, olivocerebellar axons are excitatory and send collaterals to the cerebellar nuclei as they ascend through the cerebellar white matter to the cortex. In the cerebellar cortex, olivocerebellar axons may synapse with basket, stellate, and Golgi cells, but they form a special synaptic arrangement with Purkinje cells. Each Purkinje cell receives input from only a single climbing fiber, which “climbs” up its proximal dendrites and makes hundreds of excitatory synapses. (The terminal portion of the olivocerebellar axon is referred to as a climbing fiber.) Conversely, each olivary axon branches to form about 10 to 15 climbing fibers.

CHAPTER 9 Organization of Motor Function •Fig. 9.20 Anatomic Divisions of the Cerebellum. A, Schematicmidsagittalviewofthefoldingofthecortexintolobe,lobules,andfolia.B, Schematicviewofanunfoldedferretcerebellarcortextoillustrateearliercompartmentationschemesforsubdividingthecerebellarcortexintothree(vermis,paravermis,andhemisphere)andthensevenlongitudinallyrunningzones(A,B,C1,C2,C3,D1,D2).Thelight yellow portionofeachhemisphereindicatesanareaforwhichnodatawereavailable.

C, Schematicviewofanunfoldedratcerebellum,showingitssubdivisionintomorethanI 1+2+3+3−3b−2b−*2a−5a−4−d−d−6−1−a−c−5−5−e2−e1−f−Par 4−5a−4a−4b−3−3−2−1−*3b+4+a+a+a+2+*2b+d+d+c+5+5+4+5a+6+4b+6+3+3+2+2+1+1+7+e2+*f+4+4+e1+b+2−1−b−III II IV V VII VIII IXa CP Cr IIb Cr IIa Cr Ic Cr Ib Cr Ia Sim b Sim a pf DPFL VPFL FL 5 mm 1 mm Rostral Left Right Caudal IXb IXc Xa Xb Vld Vla Vlb Vlc C •Fig. 9.20, cont’d 20compartments,accordingtostainingformolecularmarkers:inthiscase,zebrinII(aldolaseC).Letters and numbers on the right half ofthecerebellumindicatethezebrincompartmentnumber.Roman numerals down the center indicatecerebellarlobules.Names on left hemisphere indicatenamesofcerebellarlobules.CP,copulapyramis;Cr,crus;DPFL,dorsalparaflocculus;FL,flocculus;Par,paramedian;pf,primaryfissure;Sim,simplex;VPFL,ventralparaflocculus.(B, ModifiedfromVoogdJ.InLlinásRR(ed).Neurobiology of Cerebellar Evolution and Development. Chicago:AmericanMedicalAssociation;1969.C, CourtesyofDr.IzumiSugihara.)

The inferior olivary nucleus is a distinctive brain region for several reasons. As already noted, its neurons are virtually all projection cells, and so there is little local chemical synaptic interaction between the cells. Instead, olivary neurons are electrically coupled to each other by gap junctions. In fact, the inferior olivary nucleus has the highest density of neuronal gap junctions in the CNS. This allows olivary neurons to have synchronized activity that gets transmitted to the cerebellum. Afferent fibers to the inferior olivary nucleus may be divided into two main classes: those transmitting excitatory input, which arises from many regions throughout the CNS, and those transmitting inhibitory GABAergic input from the cerebellar nuclei and a few brainstem nuclei. Although these afferent fibers can modulate the firing rates of olivary neurons (as is typical in most brain regions), the membrane properties of olivary neurons limit this modulation to a range of a few hertz and endows these neurons with the potential to be intrinsic oscillators. Instead of just modulating firing rates, olivary afferent activity also acts to modify the effectiveness of the electrical coupling between olivary neurons and thus changes the patterns of synchronous activity delivered to the cerebellum. Afferent activity may also modulate expression of the oscillatory potential of olivary neurons. Thus the inferior olivary nucleus appears to be organized to generate patterns of synchronous activity across the cerebellar cortex. The functional significance of these patterns remains controversial. One hypothesis is that they provide a gating signal for synchronizing motor commands to various muscle combinations.

CHAPTER 9 Organization of Motor Function •Fig. 9.21 Three-Dimensional View of the Cerebellar Cortex, Showing Some of the Cerebellar Neurons. Thecutfaceattheleftisalongthelongaxisofthefolium;thecutfaceattherightisatrightanglestothelongaxis.BC,basketcell;BCA,basketcellaxon;CF,climbing(olivocerebellar)fiber;CN,cerebellarnuclearcell;GC,Golgicell;Glm,glomerulus;GrC,granulecell;MF,mossyfiber;PC,Purkinje cell;PF,parallelfiber;SC,stellatecell.

Cellular Elements and Efferent Fibers of the Cortex

Despite its enormous expansion throughout vertebrate evolution, the basic anatomical organization of the cerebellar cortex has remained nearly invariant. The circuitry is also among the most regular and stereotyped of any brain region. The cerebellar cortex contains eight different neuronal types: Purkinje cells, Golgi cells, granule cells, Lugaro cells, basket cells, stellate cells, unipolar brush cells, and candelabrum cells. These cells are found in all regions of the cerebellar cortex, with the exception of unipolar brush cells, which are limited mainly to cerebellar areas receiving vestibular input. These eight cell types are distributed among the three layers that make up the cerebellar cortex of higher vertebrates (

Fig. 9.21 ). The outer or superficial layer is the molecular layer; stellate and basket cells are found there. The deepest layer is the granule cell layer; this layer has the highest cellular density in the CNS and contains granule, Golgi, and unipolar brush cells. Separating the molecular and granule cell layers is the Purkinje cell layer, formed by Purkinje cell somata, which are arranged as a one-cell-thick sheet of cells. Candelabrum cells are also located in this layer. Lugaro cells are situated slightly deeper at the upper border of the granule cell layer.

The sole efferent fiber from the cortex is the Purkinje cell axon, which also has local collaterals and is GABAergic and inhibitory. Thus the remaining seven cell types are local interneurons. Of these, the stellate, basket, Golgi, Lugaro, and candelabrum cells are also inhibitory GABAergic neurons, whereas the granule and unipolar brush cells are excitatory.

Microcircuitry of the Cortex

The dendrites, axons, and patterns of synaptic connections of most neurons within the cerebellar cortex are organized with regard to the transverse (short) and longitudinal (long) axes of the folium (Fig. 9.21 ). In the vermis, where the folia run perpendicular to the sagittal plane, these axes lie in the sagittal and coronal planes, respectively. In the hemispheres, where the folia are oriented at various angles with regard to the sagittal plane, this correspondence is lost, and the local axes of the folia must then serve as the reference axes.

The Purkinje cell dendritic tree is the largest in the CNS. It extends from the Purkinje cell layer through the molecular layer to the surface of the cerebellar cortex and for several hundred microns along the transverse axis of the folium but for only 30 to 40 µm in the longitudinal direction. Thus it is like a flat pancake that lies in a plane parallel to the transverse axis of the folium. Accordingly, a set of Purkinje cell dendritic trees can be thought of as a stack of pancakes, with the stack running along the longitudinal axis of the folium.

•Fig. 9.22 Functional Connectivity of the Cerebellar Cortex. Becauseofthegeometricorganizationofthecerebellarcorticalcircuits,thefunctionalconnectivityofthecellularelementscanbedeterminedelectrophysiologically.Thefiguredepictsaclassicparadigminwhichstimulationofthecerebellarcortexactivatesabeamofparallelfibers(orange). Recordingsfromthestellateandbasketcells(green cells) andPurkinjecells(PCs;orange cells)inlinewiththisbeamshowthattheyareexcitedbytheparallelfibers.Incontrast,Purkinjecellsflankingthebeamreceiveonlyinhibition(purple areas) asaresultoftheperpendicularspatialrelationshipoftheparallelfibersandthestellateandbasketcellaxons.

The dendritic trees of the molecular layer interneurons (stellate and basket cells) are oriented in a manner similar to that of the Purkinje cell dendritic tree, although they are much less extensive. The axons of stellate and basket cells run transversely across the folium and form synapses with Purkinje cells. Stellate and basket cells synapse onto Purkinje cell dendrites. In addition, basket cells make synapses on the Purkinje cell soma and form a basket-like structure around the base of the soma, which gives the basket cell its name.

Granule cells are small neurons with four to five short unbranched dendrites, each ending in a claw-like expansion that synapses with a mossy fiber rosette and with terminals from Golgi cell axons in a complex arrangement known as a glomerulus. The axons of granule cells ascend through the Purkinje cell layer to the molecular layer, where they bifurcate and form parallel fibers. The parallel fibers run parallel to the cerebellar surface along the longitudinal axis of the folium (perpendicular to the planes of the Purkinje, stellate, and basket cell dendritic trees) and form excitatory synapses with the dendrites of the Purkinje, Golgi, stellate, and basket cells.

The orthogonal relationship between the parallel fibers and the dendritic trees of the Purkinje cells and molecular layer interneurons (basket and stellate cells) has significant functional consequences. This arrangement allows maximal convergence and divergence to occur. A single parallel fiber, which can be up to 6 mm long, passes through more than 100 Purkinje cell dendritic trees (and also interneuron dendrites); however, it has the chance to make only one or two synapses with any particular cell because it crosses through the short dimension of the dendritic tree. Conversely, a given Purkinje cell receives synapses from on the order of 100,000 parallel fibers. Thus a beam of parallel fibers can be excited experimentally, which excites a row of Purkinje cells and interneurons that are in line with this beam (

Fig. 9.22 ). In addition, because the axons of the interneurons run perpendicular to the parallel fibers, this beam of excitation is flanked by inhibition. Although this classic electrophysiological experiment clearly demonstrates the functional connectivity of the cerebellar cortex, whether such beams of excitation occur normally remains a controversial question.

The Golgi cells are inhibitory interneurons in the granule cell layer. The geometric organization of their axonal and dendritic arbors is an exception to the orthogonal and planar organization of the cortex in that their dendrites and axons carve out approximately conical territories: like two cones, tip to tip, in which the soma is at the point where the two cone tips meet. The dendritic tree forms the upper cone, which often extends into the molecular layer, and the axon forms the lower one. Golgi cells are excited by mossy and olivocerebellar fibers and by granule cell axons (parallel fibers) and inhibited by basket, stellate, and Purkinje cell axon collaterals. They in turn inhibit granule cells. Thus they participate both in feedback (when excited by parallel fibers) and in feedforward (when excited by mossy fibers) inhibitory loops that control activity in the mossy fiber–parallel fiber pathway to the Purkinje cell.

Lugaro cells have fusiform somata from which emerge two relatively unbranched dendrites, one from each side, that run along the transverse axis of the folium for several hundred microns, usually just under the Purkinje cell layer. Purkinje cell axon collaterals provide the main input to these neurons, and granule cell axons add minor input. The axon terminates mainly in the molecular layer on basket, stellate, and possibly Purkinje cells. Thus these cells appear to sample the activity of Purkinje cells and provide both positive-feedback signals (they inhibit the interneurons that inhibit Purkinje cells) and negative-feedback signals (they directly inhibit the Purkinje cell).

Unipolar brush cells have only a single dendrite that ends as a tight bunch of branchlets that resemble a brush. These cells receive excitatory input from mossy fibers and inhibitory input from Golgi cells. It is thought that they synapse with granule and Golgi cells, which would make these cells an excitatory feedforward link in the mossy fiber–parallel fiber pathway.

Candelabrum cells are GABAergic cells located in the Purkinje layer. Their dendrites and axons terminate in the molecular layer, where the axonal arborization pattern resembles a candelabrum.

The cerebellar nuclei are the main targets of the cerebellar cortex. This projection is topographically organized in such a way that each longitudinal strip of cortex targets a specific region of the cerebellar nuclei. The gross pattern is that the vermis projects to the fastigial and vestibular nuclei, the paravermal region projects to the interpositus, and the lateral hemisphere projects to the dentate nucleus.

The cerebellar nuclear neurons in turn provide the output from the cerebellum to the rest of the brain (with the primary exception of Purkinje cells that project to the

CHAPTER 9 Organization of Motor Function vestibular nuclei). In discussing the output of the cerebellar nuclei, it is useful to group the nuclear cells according to whether they are GABAergic because the GABAergic cells project back to the inferior olivary nucleus and form a negative-feedback loop to one of the cerebellum’s principal afferent sources. Of importance is that GABAergic cells project to the specific part of the inferior olivary nucleus from which they receive input and from which their overlying longitudinal strip of cortex receives climbing fibers. Thus the cerebellar cortex, cerebellar nuclei, and inferior olivary nucleus are functionally organized as a series of closed loops. The non-GABAergic, excitatory nuclear cells project to a variety of targets from the spinal cord to the thalamus. In general, each nucleus gives rise to crossed ascending and descending projections that leave the cerebellum via the superior cerebellar peduncle. The fastigial nucleus also gives rise to significant uncrossed fibers, as well as a second crossed projection called the uncinate gyrus, or hook bundle, that leaves via the inferior cerebellar peduncle.

Although there are differences in the specific targets of each nucleus, in general, the ascending cerebellar projections target midbrain structures, such as the red nucleus and superior colliculus, and the ventral lateral nucleus of the thalamus, which connects to the primary motor cortex and thereby links the cerebellum to motor areas of the cerebrum. (The cerebral motor areas are likewise linked to the cerebellum by multiple pathways, including ones that relay in the basilar pons and inferior olivary nucleus.) It should also be mentioned that ascending cerebellar projections, particularly from the dentate nucleus, also target nonmotor regions of the cerebrum, particularly in the frontal lobe. The descending fibers target mainly the basilar pontine nuclei, inferior olivary nucleus, and several reticular nuclei. Lastly, a small cerebellospinal pathway arises principally from the fastigial nucleus. In addition, the fastigial nucleus has significant projections to the vestibular nuclei.

Activity of Purkinje Cells in the Cerebellar Cortex in the Context of Motor Coordination

Mossy fiber input to the cerebellar cortex, via their excitation of granule cells, causes a Purkinje cell to discharge single action potentials, referred to as simple spikes (

Fig. 9.23 ). The spontaneous simple spike firing rate of a Purkinje cell typically is between 20 and 100 Hz but can be modulated over a much wider range (from 0 to >200 Hz), depending on the relative balance of excitation from parallel fiber input and inhibition from cerebellar cortex interneurons. Thus this activity reflects the state of the cerebellar cortex. Interestingly, evidence from studies performed in the early 2000s indicates that the spontaneous levels of simple spike activity vary systematically across the cerebellar cortex: Firing rates in zebrin-negative regions average twice those of the zebrin-positive regions. The full significance of this discovery is not known, but it suggests that despite the anatomical uniformity of the cerebellar cortical circuits, they might be functionally quite distinct.

•Fig. 9.23 Responses of a Purkinje Cell to Excitatory Input, Recorded Extracellularly. A, Granulecells,viatheirascendingaxonsandparallelfibers,excitePurkinjecellsandtriggersimplespikes.B, Climbingfiberactivityleadstohigh-frequency(≈500-Hz)burstsofspikesknownascomplex spikes inPurkinjecells.Notethatthespikesfollowingtheinitialonearesmallerandreferredtoasspikelets.

In contrast, a climbing fiber discharge causes a high-frequency burst of action potentials, called a complex spike (see

Fig. 9.23 ), in an all-or-none manner because of the massive excitation that is provided by the single climbing fiber that a Purkinje cell receives. This excitation is so powerful that there is essentially a one-to-one relationship between climbing fiber discharge and a complex spike. Thus complex spikes essentially override what is happening at the cortex level and reflect the state of the inferior olivary nucleus. The average firing rate of a spontaneous complex spike is only about 1 Hz.

Because the climbing fibers generate complex spikes at such a low frequency, they do not substantially change the average firing rates of Purkinje cells, and as a consequence, it is commonly argued that they have no direct role in shaping the output of the cerebellar cortex and are therefore not involved in ongoing motor control. Instead, it is commonly thought that their function is to alter the responsiveness of Purkinje cells to parallel fiber input. In particular, under certain circumstances, complex spike activity produces a prolonged depression in synaptic efficacy of parallel fibers, termed long-term depression (LTD). This phenomenon is the proposed mechanism by which climbing fibers act in motor-learning hypotheses. According to typical hypotheses about it, the parallel fiber system and hence simple spikes are involved in generating ongoing movement, and when there is a mismatch between the intended and actual movement, this error activates the inferior olivary nucleus and complex spikes result, which then lead to LTD of the active parallel fiber synapses. This adjustment in synaptic weight changes the motor output in the future. If this change results in a properly executed movement, activation of the inferior olivary nucleus does not occur, and the motor program is unchanged, but if there is still an error, the olivocerebellar system triggers additional complex spikes that cause further changes in synaptic efficacy, and so on. Major challenges to this view are that motor learning can occur when LTD is chemically blocked and that learned behavior can remain after removal of portions of the cerebellum in which the memory is supposedly stored.

An alternative view is that the olivocerebellar system is directly involved in motor control (note that this does not preclude a role in motor learning as well) and, in particular, helps in the timing of motor commands. This view follows from the types of motor deficits observed in cerebellar damage and accounts for the special properties of the inferior olivary nucleus mentioned earlier: namely, that it can generate rhythmic, synchronous complex spike discharges across populations of Purkinje cells. These complex spikes would then produce synchronized inhibitory postsynaptic currents (IPSPs) on cerebellar nuclear neurons as a result of the convergence present in the Purkinje cell axon to the cerebellar nuclear projection. Because of the membrane properties of cerebellar nuclear neurons, these synchronized IPSPs could have a qualitatively different effect on nuclear cell firing than would the IPSPs caused by simple spikes, which are more numerous but largely asynchronous. Specifically, they could trigger large precisely timed changes in the nuclear cell activity that would then be transmitted to other motor systems as a gating signal. In fact, voluntary movements appear to be composed of a series of periodic accelerations that reflect a central oscillatory process. However, determining whether the olivocerebellar system helps time motor commands requires further evidence.

Motor Control by the Basal Ganglia

The basal ganglia are deep nuclei in the cerebrum. Like the cerebellum, a major function of the basal ganglia is to regulate motor activity and we will focus on that role in this section. However, it is worth noting that they, like the cerebellum, also contribute to affective and cognitive functions. To understand basal ganglia function in motor control, the following discussion is organized around two major themes: (1) the connections between basal ganglia and cortex form loops through which activity flows and (2) there are two functionally distinct pathways through the basal ganglia, the direct and indirect pathways.

Organization of the Basal Ganglia and Related Nuclei

The basal ganglia include the caudate nucleus, the putamen, and the globus pallidus (

Fig. 9.24 ). The term striatum, derived from the striated appearance of these

CHAPTER 9 Organization of Motor Function •Fig. 9.24 Components of Basal Ganglia and Other Closely Associated Brain Regions. Themaincomponentsofthebasalgangliaarethecaudatenucleus,putamen,globuspallidus,andsubstantianigraparsreticulata.Majorportionsofthebasalgangliaconnectwithmotorareasinthefrontalcortex,viatheventralanteriorandventrallateralthalamicnuclei,andwiththesuperiorcolliculus.Inputfromthesubstantianigraparscompactaiscriticalfornormalbasalgangliafunction.GPe,externalsegmentofglobuspallidus;GPi,internalsegmentofglobuspallidus.

nuclei, refers only to the caudate nucleus and putamen. The striations are produced by the fiber bundles formed by the anterior limb of the internal capsule as it separates the caudate nucleus and putamen. The globus pallidus typically has two parts: an external segment and an internal segment. The combination of putamen and globus pallidus is often referred to as the lentiform nucleus.

Associated with the basal ganglia are several thalamic nuclei. These include the ventral anterior (VA) and ventral lateral (VL) nuclei and several components of the intralaminar complex. Other associated nuclei are the subthalamic nucleus of the diencephalon and the substantia nigra of the midbrain (see

Fig. 9.24 ). The substantia nigra (“black substance”) derives its name from its content of melanin pigment. Many of the neurons in the pars compacta of this nucleus contain melanin, a byproduct of dopamine synthesis. The other subdivision of the substantia nigra is the pars reticulata. This structure can be regarded as an extension of the internal segment of the globus pallidus because these nuclei have an identical origin and analogous connections.

Connections and Operation of the Basal Ganglia

With the exception of the primary visual and auditory cortices, most regions of the cerebral cortex project topographically to the striatum. The corticostriatal projection arises from neurons in layer V of the cortex. Glutamate appears to be the excitatory neurotransmitter of these neurons. The striatum then influences neurons in the nuclei of the thalamus by two pathways: direct and indirect (

Fig.

9.25A ). The thalamic neurons in turn excite neurons of the cerebral cortex thereby forming closed loops with most of the cortex. Several distinct loops have been identified on the basis of cortical regions and function; however, here we will focus on the motor-related loops as a model for basal ganglia operation (

Fig. 9.25A

The overall action of the direct pathway through the basal ganglia to motor areas of the cortex is to enhance motor activity. In the direct pathway, the striatum projects to •Fig. 9.25 Functional Connectivity of the Basal Ganglia for Motor Control. A, Connectionsbetweenvariousbasalgangliacomponentsandotherassociatedmotorareas.TheexcitatorycorticalinputtothecaudateandputameninfluencesoutputfromtheGPiandthesubstantianigraparsreticulata(SNpr)viaadirectandanindirectpathway.Thetwoinhibitorystepsintheindirectpathwaymeanthatactivitythroughthispathwayhasaneffectonbasalgangliaoutputtothethalamusandsuperiorcolliculusoppositethatofthedirectpathway.Dopamine(DA)isaneuromodulatorthatactsonD1andD2receptorsonstriatalneuronsthatparticipateinthedirectandindirectpathways,respectively.B, ChangesinactivityflowthatoccurinParkinson’sdisease,inwhichthesubstantianigraparscompacta(SNpc)degenerates.C, ChangesinactivityflowinHuntington’sdisease,inwhichinhibitorycontroloftheGPeislost.Plus symbols (+)andminus symbols (−),respectively,indicatetheexcitatoryorinhibitorynatureofasynapticconnection.glu,glutamate;GPe,externalglobuspallidus;GPi,internalglobuspallidus;VA/VL,ventralanterior/ventrallateralnucleiofthethalamus.

the internal segment of the globus pallidus (GPi). This projection is inhibitory, and the main transmitter is GABA. The GPi projects to the VA and VL nuclei of the thalamus. These connections also function with GABA and are inhibitory. The VA and VL nuclei send excitatory connections to the prefrontal, premotor, and supplementary motor cortex. This input to the cortex influences motor planning, and it also affects the discharge of corticospinal and corticobulbar neurons.

The direct pathway appears to function as follows: Neurons in the striatum have little background activity, but during movement they are activated by their input from the cortex. In contrast, neurons in the GPi have a high level of background activity. When the striatum is activated, its inhibitory projections to the globus pallidus slow the activity of pallidal neurons. However, the pallidal neurons themselves are inhibitory, and they normally provide tonic inhibition of neurons in the VA and VL nuclei of the thalamus. Therefore, activation of the striatum causes disinhibition of neurons of the VA and VL nuclei. When disinhibited, the VA/VL neurons increase their firing rates, exciting their target neurons in the motor areas of the cerebral cortex. Because the motor areas evoke movement by activating α and γ motor neurons in the spinal cord and brainstem, the basal ganglia can regulate movement by enhancing the activity of neurons in the motor cortex.

The overall effect of the indirect pathway is to reduce the activity of neurons in motor areas of the cerebral cortex. The indirect pathway involves inhibitory connections from the striatum to the external segment of the globus pallidus (GPe), which in turn sends an inhibitory projection to the subthalamic nucleus and to the GPi. The subthalamic nucleus then sends an excitatory projection back to the GPi (see

Fig. 9.25A

In this pathway, pallidal neurons in the external segment are inhibited by the GABA released from striatal terminals in the globus pallidus. The GPe normally releases GABA in the subthalamic nucleus and thereby inhibits the subthalamic neurons. Therefore, striatal inhibition of the GPe results in the disinhibition of neurons of the subthalamic nucleus. The subthalamic neurons are normally active, and they excite neurons in the GPi by releasing glutamate. When the neurons of the subthalamic nucleus become more active because of disinhibition, they release more glutamate in the GPi. This transmitter excites neurons in the GPi and consequently activates inhibitory projections that affect the VA and VL thalamic nuclei. The activity of the thalamic neurons consequently decreases, as does the activity of the cortical neurons that they influence.

The direct and indirect pathways thus have opposing actions; an increase in the activity of either one of these pathways might lead to an imbalance in motor control. Such imbalances, which are typical of basal ganglion diseases, may alter the motor output of the cortex.

CHAPTER 9 Organization of Motor Function

Actions of Neurons in the Pars Compacta of the Substantia Nigra on the Striatum

Dopamine is the neurotransmitter used by neurons of the substantia nigra pars compacta. In the nigrostriatal pathway, release of dopamine has an overall excitatory action on the direct pathway and an inhibitory action on the indirect pathway. This is, however, a modulatory type of effect; that is, dopamine is apparently causing its action not by triggering spikes directly but rather by altering the striatal cells’ response to other transmitters. The different actions on the direct and indirect pathways result from the expression of different types of dopamine receptors (D1 and D2) by the spiny projection cells of the striatum that contribute to the direct and indirect pathways. D1 receptors are found on striatal cells that form the direct pathway by projecting to the GPi, whereas D2 receptors are found on striatal cells that participate in the indirect pathway and project to the GPe. The overall consequence of dopamine release in both cases is facilitation of activity in the motor areas of the cerebral cortex.

Subdivision of the Striatum Into Striosomes and Matrix

On the basis of the associated neurotransmitters, the stria-tum has been subdivided into zones called striosomes and matrix. The cortical projections related to motor control end in the matrix area. The limbic system projects to the striosomes. Striosomes are thought to synapse in the pars compacta of the substantia nigra and to influence the dopaminergic nigrostriatal pathway.

Role of the Basal Ganglia in Motor Control

The basal ganglia influence the cortical motor areas. Therefore, the basal ganglia have an important influence on the lateral system of motor pathways. Such an influence is consistent with some of the movement disorders observed in diseases of the basal ganglia. However, the basal ganglia must additionally regulate the medial motor pathways because diseases of the basal ganglia can also affect the posture and tone of proximal muscles.

The deficits seen in the various basal ganglia diseases include abnormal movement (dyskinesia), increased muscle tone (cogwheel rigidity), and slowness in initiating movement (bradykinesia). Abnormal movement includes tremor, athetosis, chorea, ballism, and dystonia. The tremor of basal ganglion disease is a 3-Hz “pill-rolling” tremor that occurs when the limb is at rest. Athetosis consists of slow, writhing movement of the distal parts of the limbs, whereas chorea is characterized by rapid, flicking movement of the extremities and facial muscles. Ballism is associated with violent, flailing movement of the limbs (ballistic movement). Finally, dystonic movements are slow involuntary movements that may cause distorted body postures.

Parkinson’s disease is a common disorder characterized by tremor, rigidity, and bradykinesia. This disease is caused by loss of neurons in the pars compacta of the substantia nigra. Consequently, the striatum suffers a severe loss of dopamine. Neurons of the locus coeruleus and the raphe nuclei, as well as other monoaminergic nuclei, are also lost. The loss of dopamine diminishes the activity of the direct pathway and increases the activity of the indirect pathway (see

Fig. 9.25B ). The net effect is an increase in the activity of neurons in the internal segment of the globus pallidus. This results in greater inhibition of neurons in the VA and VL nuclei and less pronounced activation of the motor cortical areas. The consequence is slowed movement (bradykinesia).

Before the dopaminergic neurons are completely lost, administration of levodopa can relieve some of the motor deficits in Parkinson’s disease. Levodopa is a precursor of dopamine, and it can cross the blood-brain barrier. Currently, the possibility of transplanting dopamine-synthesizing neurons into the striatum is being explored. Future research will no doubt focus on the potential for human embryonic stem cells to play such a therapeutic role.

Another basal ganglia disturbance is Huntington’s disease, which results from a genetic defect that involves an autosomal dominant gene. This defect leads to the preferential loss of striatal GABAergic and cholinergic neurons that project to the GPe as part of the indirect pathway (and also degeneration of the cerebral cortex, with resultant dementia). Loss of inhibition of the GPe presumably leads to diminished activity of neurons in the subthalamic nucleus (see

Fig. 9.25C ). Hence, the excitation of neurons of the GPi would be reduced. This would disinhibit neurons in the VA and VL nuclei. The resulting enhancement of activity in neurons in the motor areas of the cerebral cortex may help explain the choreiform movements of Huntington’s disease. The rigidity in Parkinson’s disease may in a sense be the opposite of chorea because overtreatment of patients with Parkinson’s disease with levodopa can result in chorea.

Hemiballism is caused by a lesion of the subthalamic nucleus on one side of the brain. In this disorder, involuntary, violent flailing movements of the limbs may occur on the side of the body contralateral to the lesion. Because the subthalamic nucleus excites neurons of the GPi, a lesion of the subthalamic nucleus would reduce the activity of these pallidal neurons. Therefore, neurons in the VA and VL nuclei of the thalamus would be less inhibited, and the activity of neurons in the motor cortex would be increased.

In all these basal ganglia disorders, the motor dysfunction is contralateral to the diseased component. This is understandable because the main final output of the basal ganglia to the body is mediated by the corticospinal tract.

Eye movement has a number of features that distinguish it from other motor behavior. In comparison with the movement that limbs, with their multiple joints and muscles, can perform, eye movement is relatively simple. For example, each eye is controlled by only three agonistantagonist muscle pairs: the medial and lateral recti, the superior and inferior recti, and the superior and inferior oblique muscles. These muscles allow the eye to rotate about three axes. Assuming that the head is in an upright position, the axes are the vertical axis, a horizontal axis that runs left to right, and the torsional axis (which is directed along the axis of sight). The medial and lateral recti control movement about the vertical axis; the other four muscles generate movement about the horizontal and torsional axes. Another simplifying feature is that there are no external loads for which to be compensated. Furthermore, eye movement appears to be separable into a few distinct types, with each type being controlled by its own specialized circuitry. Thus eye movement offers a number of advantages as a model system for studying motor control. Moreover, deficits in eye movement provide important clinical clues to the diagnosis of neurological problems. We first review the different eye movement types and then discuss the neural circuitry underlying their generation.

Types of Eye Movement

Eye movement probably first evolved to hold the image of the external world still. (In contrast, limb movements evolved to generate changes in the position of the limb with regard to the external world.) The reason is that visual acuity degrades rapidly when there is eye movement in relation to the external world (i.e., the visual scene slips across the retina). A major cause of such slippage is movement of the head. The vestibuloocular reflex (VOR) is one of the main mechanisms by which head movement is compensated in order to maintain stability of the visual scene on the retina.

To maintain a stable visual scene on the retina, the VOR produces movements of the eyes that are equal and opposite the movement of the head. This reflex is initiated by stimulation of the receptors (hair cells) in the vestibular system (see ). Recall that the vestibular organs are sensitive to head acceleration, not visual cues, and thus the VOR occurs in both the light and dark. Functionally, it is what is called an open-loop system in that it generates an output (eye movement) in response to a stimulus (head acceleration), but its immediate behavior is not regulated by feedback about the success or failure of its output. It is worth noting, however, that in the light at least, any failure by the VOR to match eye and head rotation results in what is called retinal slip (i.e., slip of the visual image across the retina), and this error signal can be fed back to the VOR circuits by other neuronal pathways and over time can lead to adjustments in the strength of the VOR to eliminate the error. This adaptation of the VOR is a major model for studying plasticity in the brain.

As stated, acceleration signals initiate the VOR. The output of the VOR, however, must be a change in eye position in the orbit. Thus the problem to be solved by the nervous system is to translate the acceleration signals sensed by the vestibular organs into correct positional signals for the eyes. Mathematically, this can be thought of as a double integration. The first integration occurs in the vestibular receptor apparatus because although the hair cells respond to head acceleration, the signals in the vestibular afferent fibers are proportional to head velocity (at least for most stimuli that are encountered physiologically). The second integration, from velocity to position, occurs in the CNS in circuits described later.

The head can move in six different ways, often referred to as six degrees of freedom: three translational and three rotational. To compensate for these different types of movement, there are both translational and angular VORs, as well as separate subsystems for handling movement about different directions (e.g., rotation about a vertical or a horizontal axis).

The optokinetic reflex (OKR) is a second mechanism by which the CNS stabilizes the visual scene on the retina, and it often works in conjunction with the VOR. Whereas the VOR is activated only by head motion, the OKR is activated by movement of the visual scene, whether caused by motion of the scene itself or by head motion. Specifically, the sensory stimulus for this reflex is slip of the visual scene on the retina as detected by motion-sensitive retinal ganglion cells. An example of the former occurs when you are sitting in a train and a train on the adjacent track begins moving: Your eyes rotate to keep the image of the neighboring car stable. This often leads to a sensation that you are moving (this is not entirely surprising because OKR circuits feed into the same circuits as used by the vestibular system).

The OKR can work in conjunction with the VOR to stabilize the visual image and is particularly important for maintaining a stable image when head movements are slow because the VOR works poorly in these conditions.

In animals whose eyes have a fovea, it becomes particularly advantageous to be able to move the eye in relation to the world (i.e., the main visual scene) so that objects of importance can be focused onto the fovea and scrutinized with this high-resolution part of the retina. Two classes of eye movement underlie this ability: saccadic and smooth pursuit. Very rapid discrete movements that bring a particular region of the visual world onto the fovea are called saccades. For example, to read this sentence, you are making a series of saccades to bring successive words onto your fovea to be read. However, even in animals that lack a fovea, the eyes make saccades, and thus saccades may also be used to rapidly scan the visual environment.

Saccades are extremely rapid eye movements. In humans, eye velocity during a saccade can reach 800 degrees/second, in comparison with movement velocity of less than 10 degrees/second generated in response to typical VOR and

CHAPTER 9 Organization of Motor Function

OKR stimuli (velocities of up to ≈120 degrees/second can be produced by OKR stimuli in humans; however, they are still much slower than the maximal saccade velocities). Saccades can be made voluntarily or reflexively. Moreover, although they are usually made in response to visual targets, they can also be made toward auditory or other sensory cues, in the dark, or toward memorized targets.

Interestingly, visual processing appears to be suppressed just before and during saccades, particularly in the magnocellular visual pathway that is concerned with visual motion. This phenomenon is known as saccadic suppression and may function to prevent sensations of sudden, rapid movement of the visual world that would result during a saccade in the absence of such suppression. The mechanisms underlying saccadic suppression are not fully known, but in areas of the cortex related to visual processing, the responsiveness of the cells to visual stimuli is reduced and altered during saccades.

Once a saccade has brought a moving object of interest onto the fovea, the smooth pursuit system allows the person to keep it stable on the fovea despite its continued motion. This ability appears to be limited to primates and allows prolonged continuous observation of a moving object. Note that in some respects, smooth pursuit might seem similar to the OKR; in fact, there may not be an absolute difference because as the target size grows, the distinction between target and background is lost; however, for small moving targets, smooth pursuit requires suppression of the OKR. You can see the effect of this suppression by moving your finger back and forth in front of this text while tracking it with your eyes. Your finger will be in focus, but the words on this page will be part of the background scene and will become illegible as they slip along your retina.

When there is a prolonged OKR or VOR stimulus (e.g., if you keep turning in one direction), these reflexes will initially counterrotate the eyes in an attempt to maintain a stable image on the retina, as described earlier. However, with a prolonged stimulus, the eyes will reach their mechanical limit, no further compensation will be possible, and the image will begin to slip on the retina. To avoid this situation, a fast saccade-like movement of the eyes occurs in the opposite direction, essentially resetting the eyes to begin viewing the visual scene again. Then the slow OKR-or VOR-induced counterrotation will start anew. This alternation of slow and fast movement in opposite directions is nystagmus and can be recorded on a nystagmogram (

Fig.

9.26 ). Thus nystagmus can be defined as oscillatory or rhythmic movements of the eye in which there is a fast phase and a slow phase. The nystagmus is named according to the direction of the fast phase because the fast phase is more easily observed.

In addition to being induced physiologically by VOR or OKR stimuli, nystagmus can result from damage to the vestibular circuits, either in the periphery (e.g., cranial nerve •Fig. 9.26 Nystagmogram Showing Eye Movements That Occur During Nystagmus. Theplotshowsaleftnystagmusbecausethefastphaseisdirectedtowardtheleft(downwardonthegraph).

VIII) or centrally (e.g., vestibular nuclei), and can be an informative diagnostic symptom.

Conjugate eye movement is movement of both eyes in the same direction and in an equal amount. Such coordination allows a target to be maintained on both foveae during eye movement and is necessary to maintain binocular vision without diplopia (double vision). However, when objects are close (<30 m), maintaining a target on both foveae requires non-identical movements of the two eyes. Such disjunctive, or vergence, movements are also necessary for fixation of both eyes on objects that are approaching or receding. Stimuli that trigger vergence movements are diplopia and blurry images. It should be noted that when tracking an approaching object in addition to convergence movements, the lens accommodates for near vision, and pupillary constriction occurs.

Motor Neurons of the Extraocular Muscles

Three cranial nerve nuclei supply the extraocular muscles: oculomotor, trochlear, and abducens nuclei. These three nuclei are sometimes referred to collectively as the oculomotor nuclei; however, the context (the specific nucleus or all three) should be clear. Motor neurons for the ipsilateral medial and inferior recti, ipsilateral inferior oblique, and contralateral superior rectus muscles reside in the oculomotor nucleus; those for the contralateral superior oblique muscle reside in the trochlear nucleus; and those for the ipsilateral lateral rectus muscle are located in the abducens nucleus. These motor neurons form some of the smallest motor units (1 : 10 nerve-to-muscle ratio), which is consistent with the very fine control needed for precise eye movement.

An important point regarding motor neurons innervating the extraocular muscles is that most have spontaneous activity when the eye is in the primary position (looking straight ahead), and their firing rate is correlated with eye position and velocity. This spontaneous activity allows the antagonist muscle pairs to act in a push-pull manner, which increases the responsiveness of the system. That is, as motor neurons innervating one muscle are activated and cause increased contraction, those innervating its antagonist are inhibited, which leads to relaxation.

In addition to motor neurons, the abducens nuclei have internuclear neurons. These neurons project, via the medial longitudinal fasciculus, to medial rectus motor neurons in the contralateral oculomotor nucleus. As described later, this projection facilitates the coordinated action of the medial and lateral recti muscles that is needed for conjugate movements, such as those that occur in the VOR.

Circuits Underlying the Vestibuloocular Reflex

The VOR acts to counter head motion by causing rotation of the eyes in the opposite direction. There are separate circuits for rotational and translational movement of the head. The sensors for the former are the semicircular canals, and the sensors for the latter are the otoliths (the utricle and saccule). The circuits for the angular VOR are more straightforward (but still complex), and this section focuses on these pathways to illustrate how this reflex works; however, the basic scheme is the same: Vestibular afferent fibers go to vestibular nuclei, the vestibular nuclei in turn project to the various oculomotor nuclei, and motor neurons in the oculomotor nuclei give rise to axons that innervate the extraocular muscles. What varies are the specific vestibular and oculomotor nuclei that are involved.

With regard to the angular VOR pathways, the pathway for generating horizontal eye movement originates in the horizontal canals, and the analogous one for vertical movement originates in the anterior and posterior canals.

Fig.

9.27A shows the basic circuit for the horizontal VOR. Note that only the major central circuits originating in the left horizontal canal and vestibular nuclei are shown; however, mirror image pathways arise from the right canal and vestibular nuclei. Vestibular afferent fibers involved in the horizontal VOR pathway synapse primarily in the medial vestibular nucleus, which projects to the abducens nucleus bilaterally; inhibitory neurons project ipsilaterally, and excitatory ones project contralaterally. Control of the medial rectus muscle is achieved by abducens internuclear neurons that project from the abducens to the part of the oculomotor nucleus controlling the medial rectus muscle. Note the double decussation of this pathway results in aligning of the responses of functional synergists (e.g., the left medial rectus with the right lateral rectus).

The vertical VOR pathway involves primarily the superior vestibular nucleus, which has direct bilateral projections to the oculomotor nucleus.

Consider what happens in the horizontal canal pathway when there is head rotation to the left, as shown in

Fig.

9.27B.

Leftward head rotation would cause the visual image to slip to the right. However, compensation by the VOR would be triggered by depolarization of the hair cells of the left canal in response to the angular acceleration

CHAPTER 9 Organization of Motor Function •Fig. 9.27 Circuits Underlying the Horizontal Vestibuloocular Reflex (VOR). A, Thevestibularnucleireceiveexcitatoryinputfromtheafferentfibersofthehorizontalcanalandprojecttotheabducens(cranialnerveVI)nucleus.Thisnucleusinnervatesthelateralrectusmuscleandprojectstothecontralateraloculomotor(cranialnerveIII)nucleus,whichcontrolsthemedialrectusmuscle.Excitatoryneuronsareshowninred; inhibitoryones,inblue. Notethatonlythemajorpathwaysoriginatingintheleftvestibularnucleiareshown.Forclarity,onlythebeginningsofmirrorimagepathwaysfromtherightvestibularnucleiareshown(dotted lines). B, FlowofactivityintheVORcircuitryinducedbyleftwardheadrotation.Increasedaxonalthicknessindicatesincreasedactivity;thinneraxonsindicatedecreasedactivityincomparisonwithlevelsatrest(A). Notethatleftwardrotationcausesbothanincreaseinactivityoftheleftvestibularafferentfibersandadecreaseinactivityoftherightones.MLF,mediallongitudinalfasciculus;vestibularnuclei:I,inferior;L,lateral;M,medial;S,superior.

Fig. 8.27 ). The depolarized hair cells cause increased activity in the left vestibular afferent fibers and thereby excite neurons of the left medial vestibular nucleus. These include excitatory neurons that project to the contralateral abducens nucleus and synapse with both motor neurons and internuclear neurons. Excitation of the motor neurons leads to contraction of the right lateral rectus muscle and rotation of the right eye to the right, whereas excitation of the internuclear neurons of the right abducens nucleus leads to excitation of the medial rectus motor neurons in the left oculomotor nucleus, thus causing the left eye to rotate to the right as well.

Along the pathway starting with the inhibitory vestibular neurons that project from the left medial vestibular nucleus to the ipsilateral abducens nucleus, the activity of these cells leads to inhibition of motor neurons to the left lateral rectus muscle and motor neurons to the right medial rectus muscle (the latter via internuclear neurons to the right oculomotor nucleus). Consequently, these muscles relax, thereby facilitating rotation of the eyes to the right. Thus the eye is being pulled by the increased tension of one set of muscles and “pushed” by the release of tension in the antagonist set of muscles.

Note again that the mirror image pathways originating from the right canal have been left out of

Fig. 9.27 for clarity, but the changes in activity through them with leftward head rotation would be exactly the opposite, and thus they would function synergistically with those that are shown. As an exercise, work out the resulting changes in activity through these circuits. Remember that leftward head rotation hyper-polarizes the hair cells of the right canal, thereby leading to a decrease in right vestibular afferent activity and disfacilitation of the right vestibular nuclear neurons.

Now, consider the commissural fibers that connect the two medial vestibular nuclei are excitatory but end on local inhibitory interneurons of the contralateral vestibular nucleus and thus inhibit the projection neurons of that nucleus. This pathway reinforces the actions of the contra-lateral vestibular afferent fibers on their target vestibular nuclear neurons. In the aforementioned example, commissural cells in the left vestibular nucleus are activated and therefore cause active inhibition of the right medial vestibular nuclei projection neurons, which reinforces the disfacilitation caused by the decrease in right afferent activity. In fact, this commissural pathway is powerful enough to modulate the activity of the contralateral vestibular nuclei even after unilateral labyrinthectomy, which destroys the direct vestibular afferent input to these nuclei.

Of importance is that superimposed on the brainstem circuits is the cerebellum. Parts of the vermis and flocculonodular lobe receive primary vestibular afferent fibers or secondary vestibular afferent fibers (axons of the vestibular nuclear neurons), or both, and in turn project back to the vestibular nuclei directly and via a disynaptic pathway involving the fastigial nucleus. The exact role of these cerebellar circuits in generating the VOR is much debated, but they are critical inasmuch as damage to them leads to abnormal eye movement, such as spontaneous nystagmus, and other symptoms of vestibular dysfunction.

Whenalabyrinthisirritatedinoneear,asinMeniere’s disease, orwhenalabyrinthisrenderednonfunctional,asmayhappenasaresultofheadtraumaordiseaseofthelabyrinth,thesignalstransmittedthroughtheVORpathwaysfromthetwosidesbecomeunbalanced.Vestibularnystagmuscanthenresult.Forexample,irritationofthelabyrinthoftheleftearcanincreasethedischargeofafferentfibersthatsupplythelefthorizontalsemicircularduct.Thesignalproducedresemblesthatnormallygeneratedwhentheheadisrotatedtotheleft.Becausethestimulusisongoing,aleftnystagmusresults,withaslowphasetotheright(causedbytheVORpathway)andafastphasetotheleft.Destructionofthelabyrinthintherightearproduceseffectssimilartothoseinducedbyirritationoftheleftlabyrinth.Interestingly,thenystagmusistemporary,whichshowstheabilityofthesecircuitstoadaptovertime.

Circuits Underlying the Optokinetic Reflex

The stimulus eliciting the OKR is visual (retinal slip), and so photoreceptors are the start of the reflex arc. Key brainstem centers for this reflex lie in the tegmentum and pretectal region of the rostral midbrain. They are the nucleus of the optic tract (NOT) and a group of nuclei collectively known as the accessory optic nuclei (AON). Direction-selective, motion-sensitive retinal ganglion cells are a major afferent source carrying visual information to these nuclei. In addition, input comes from primary and higher order visual cortical areas in the occipital and temporal lobes. These latter afferent sources are particularly important in primates and humans. Cells of the NOT and AON have large receptive fields, and their responses are selective for the direction and speed of movement of the visual scene. Of interest is that the preferred directions of movement of the NOT/AON cells correspond closely to motion caused by rotation about axes perpendicular to the semicircular canals, thereby facilitating coordination of the VOR and OKR to produce stable retinal images.

The efferent connections of these nuclei are numerous and complex and not fully understood. There are polysynaptic pathways to the oculomotor and abducens nuclei and monosynaptic input to the vestibular nuclei, which allow interaction with the VOR. There are projections to various precerebellar nuclei, including the inferior olivary nucleus and basilar pontine nuclei. These pathways then loop through the flocculus and back to the vestibular nuclei. In sum, via several pathways operating in parallel, activity ultimately arrives at the various oculomotor nuclei whose motor neurons are activated, and proper counterrotation of the eyes results.

Saccades are generated in response to activity in the superior colliculus or the cerebral cortex (frontal eye fields and

ClinicaltestingoflabyrinthinefunctionisdoneeitherbyrotatingthepatientinaBáránychairtoactivatethelabyrinthsinbothearsorbyintroducingcoldorwarmwaterintotheexternalauditorycanalofoneear(caloric test). WhenapersonisrotatedinaBáránychair,nystagmusdevelopsduringtherotation.Thedirectionofthefastphaseofthenystagmusisinthesamedirectionastherotation.Whentherotationofthechairishalted,nystagmusintheoppositedirectiondevelops(postrotatorynystagmus)becausestoppingarotationhasthesameeffectasacceleratingintheoppositedirection.

Thecalorictestismoreusefulbecauseitcandistinguishbetweenmalfunctionofthelabyrinthsonthetwosides.Theneckisbentbackwardabout60degreessothatthetwohorizontalcanalsareessentiallyvertical.Ifwarmwaterisintroducedintotheleftear,theendolymphintheouterportionoftheloopoftheleftsemicircularcanaltendstoriseasthespecificgravityoftheendolymphdecreasesbecauseofheating.Thissetsupaconvectionflowofendolymph,andasaresult,thekinociliaoftheleftampullarycresthaircellsaredeflectedtowardtheutricle,asiftheheadhadrotatedtotheleft;thedischargeoftheafferentfibersthatsupplythiscanalincreases;andnystagmusoccurswiththefastphasetowardtheleft.Thenystagmusproducesasensationthattheenvironmentisspinningtotheright,andthepersontendstofalltotheright.Theoppositeeffectsareproducedifcoldwaterisplacedintheear.AmnemonicexpressionthatcanhelpinrememberingthedirectionofthenystagmusinthecalorictestisCOWS (“coldopposite,warmsame”).Inotherwords,coldwaterinoneearresultsinafastphaseofnystagmustowardtheoppositeside,andwarmwatercausesafastphasetowardthesameside.

posterior parietal areas). Activity in the superior colliculus is related to computation of the direction and amplitude of the saccade. Indeed, the deep layers of the superior colliculus contain a topographic motor map of saccade locations. From the superior colliculus, information is forwarded to distinct sites for control of horizontal and vertical saccades, referred to as the horizontal and vertical gaze centers, respectively. The horizontal gaze center consists of neurons in the paramedian pontine reticular formation, in the vicinity of the abducens nucleus (

Fig. 9.28A ). The vertical gaze center is located in the reticular formation of the midbrain: specifically, the rostral interstitial nucleus of the medial longitudinal fasciculus and the interstitial nucleus of Cajal. Because the circuitry and operation of the horizontal gaze center are better understood than those of the vertical gaze center, it is discussed here in detail. However, cells showing analogous activity patterns have been described in the vertical gaze center.

Fig. 9.28A is an overview of the neural circuitry by which saccades are generated, and

Fig. 9.28B shows the activity of certain types of neurons found in the gaze center that are responsible for horizontal saccades. Each horizontal gaze center has excitatory burst neurons that project to motor neurons in the ipsilateral abducens nucleus and to the internuclear neurons (which excites medial rectus motor

CHAPTER 9 Organization of Motor Function neurons in the contralateral oculomotor nucleus). It also has inhibitory burst neurons that inhibit the contralateral abducens. These burst neurons are capable of extremely high bursts of spikes (up to 1000 Hz). Moreover, the gaze center has neurons showing tonic activity and burst-tonic activity.

Normally, both inhibitory and excitatory burst neurons are inhibited by omnipause neurons located in the nucleus of the dorsal raphe. When a saccade is to be made, activity from the frontal eye fields or the superior colliculus, or both, leads to inhibition of the omnipause cells and excitation of the burst cells on the contralateral side. The resulting high-frequency bursts in the excitatory burst neurons provide a powerful drive to motor neurons of the ipsilateral lateral rectus and contralateral medial rectus muscles (see

Fig. 9.28A ); at the same time, inhibitory burst neurons enable relaxation of the antagonists. The initial bursts of these neurons allow strong contraction of the appropriate extraocular muscles, which overcomes the viscosity of the extraocular muscle and enables rapid movement to occur.

Smooth pursuit involves tracking a moving target with the eyes (Fig. 9.29 ). Visual information about target velocity is processed in a series of cortical areas, including the visual cortex in the occipital lobe, several temporal lobe areas, and the frontal eye fields. In the past, the frontal eye fields were thought to be related only to control of saccades, but more recent evidence has shown that there are distinct regions within the frontal eye fields dedicated to either saccade production or smooth pursuit. Indeed, there may be two distinct cortical networks, each specialized for one of these types of eye movement. Cortical activity from multiple cortical areas is fed to the cerebellum via parts of the pontine nuclei and nucleus reticularis tegmenti pontis. Specific areas in the cerebellum—namely, parts of the posterior lobe vermis, the flocculus, and the paraflocculus—receive this input, and they in turn project to the vestibular nuclei. From the vestibular nuclei, activity can then be forwarded to the oculomotor, abducens, and trochlear nuclei, as was described for the VOR earlier.

The neural circuits underlying vergence movements are not well known. There are premotor neurons (neurons that feed onto motor neurons) located in the brainstem areas surrounding the various oculomotor nuclei. In some cortical visual areas and the frontal eye fields, there are neurons whose activity is related to the disparity of the image on the two retinas or to the variation of the image during vergence movements. How vergence signals in these cortical areas feed into the brainstem premotor neurons is not clear. The cerebellum also appears to play a role in vergence movements because cerebellar lesions impair this type of eye movement. Note that lesions of the medial longitudinal fasciculus that result in a loss of the VOR do not compromise vergence.

•Fig. 9.28 Horizontal Saccade Pathways. A, Circuitdiagramofthemajorpathways.EBN,excitatoryburstneuron;FEF,frontaleyefield;IBN,inhibitoryburstneuron;LBN,longleadburstneuron;OPN,omnipauseneuron;PPRF,paramedianpontinereticularformation.B, Firingpatternsofsomeoftheneuronsinvolvedinmakingsaccades.Excitationofburstneuronsoftherighthorizontalgazecentercausesabducensmotorneuronsontherightandmedialrectusmotorneuronsonthelefttobeactivated.Theascendingpathwaytotheoculomotornucleusisthroughthemediallongitudinalfasciculus.Thelefthorizontalgazecenterissimultaneouslyinhibited.

CHAPTER 9 Organization of Motor Function •Fig. 9.29 Smooth Pursuit Pathways. Thestimulusforsmoothpursuiteyemovementisamovingvisualtarget.Thiscausesactivitytoflowthroughthecircuitrydiagramedinthefigureandleadstomaintenanceofthefoveaonthetarget.FEF,frontaleyefield;LGN,lateralgeniculatenucleus;MSTandMT,higherordervisualassociationareas;MVN,medialvestibularnucleus;NRTP,nucleusreticularistegmentipontis;SEF,supplementaryeyefield;V1,primaryvisualcortex.Retina LGN V1 MT MST Pontine nuclei FEF SEF NRTP Flocculus paraflocculus Vermis MVN y-group Fastigial nucleus Oculomotor nuclei Cortical areas involved in processing object motion Brainstem and cerebellar areas involved in transforming sensory-related signal into motor command Motor commands to eye muscles 1.

The extrafusal skeletal muscle fibers are innervated by α motor neurons. A motor unit consists of a single α motor neuron and all the muscle fibers with which it synapses. Motor unit size varies greatly among muscles; small motor units allow finer control of muscle force.

2.

The size principle refers to the orderly recruitment of α motor neurons according to their size, from smallest to largest. Because smaller motor neurons connect to weaker motor units, the relative fineness of motor control is similar for weak and strong contractions.

3.

A reflex is a simple, stereotyped motor response to a stimulus. A reflex arc includes the afferent fibers, interneurons, and motor neurons responsible for the reflex.

4.

Muscle spindles are complex sensory receptors found in skeletal muscle. They lie parallel to extrafusal muscle fibers, and they contain nuclear bag and nuclear chain intrafusal muscle fibers. By being in parallel to the main muscle, the spindle can detect changes in muscle length.

5.

Group Ia afferent fibers form primary endings on nuclear bag1, bag2, and chain fibers, and group II fibers form secondary endings on nuclear chain and bag2 fibers.

6.

Primary endings demonstrate both static and dynamic responses that signal muscle length and rate of change in muscle length. Secondary endings demonstrate only static responses and signal only muscle length.

7.

The intrafusal muscle fibers associated with muscle spindles are innervated by γ motor neurons. Contraction of intrafusal fibers does not directly cause significant changes in muscle tension or length; however, when the level of tension in these fibers is adjusted, γ motor neurons influence the sensitivity of the muscle spindle to stretch.

8.

Golgi tendon organs are located in the tendons of muscles and are thus arranged in series with the muscle. They are supplied by group Ib afferent fibers. Their in-series relationship means that tendon organs can detect the force level generated by the muscle, whether it is due to passive stretch or to active contraction of the muscle.

9.

The phasic stretch (or myotatic) reflex includes (1) a monosynaptic excitatory pathway from group Ia afferent fibers in muscle spindles to α motor neurons that supply the same and synergistic muscles and (2) a disynaptic inhibitory pathway to antagonistic motor neurons.

10.

The inverse myotatic reflex is evoked by Golgi tendon organs. Afferent volleys in group Ib fibers from a given muscle cause disynaptic inhibition of α motor neurons to the same muscle, and they excite α motor neurons to antagonist muscles.

11.

The flexion reflex is an important protective response because it acts to withdraw a limb from damaging stimuli. The reflex is evoked by volleys in afferent fibers that supply various receptors, particularly nociceptors. Via polysynaptic pathways, these volleys cause excitation of flexor motor neurons and inhibition of extensor motor neurons ipsilaterally. Concurrently, the opposite pattern of action (inhibition of flexor and excitation of extensor motor neurons) occurs contralaterally and is referred to as the crossed extension reflex.

12.

Descending pathways can be subdivided into (1) a lateral system, which ends on motor neurons to limb muscles and on the lateral group of interneurons, and (2) a medial system, which ends on the medial group of interneurons.

13.

The lateral system includes the lateral corticospinal tract and part of the corticobulbar tract. These pathways influence the contralateral motor neurons that supply the musculature of the limbs, especially of the digits, and the muscles of the lower part of the face and the tongue.

14.

The medial system includes the ventral corticospinal, lateral and medial vestibulospinal, reticulospinal, and tectospinal tracts. These pathways affect mainly posture and provide the motor background for movement of the limbs and digits.

15.

Locomotion is triggered by commands relayed through the midbrain locomotor center. However, central pattern generators formed by spinal cord circuits and influenced by afferent input provide for the detailed organization of locomotor activity.

16.

Voluntary movements depend on interactions among motor areas of the cerebral cortex, the cerebellum, and the basal ganglia.

17.

Motor areas of the cerebral cortex are arranged as a parallel distributed network, in which each contributes to the various descending motor pathways. The areas primarily involved in body and head movement include the primary motor cortex, the premotor area, the supplementary motor cortex, and the cingulate motor areas. The frontal eye fields are important for eye movement and help initiate voluntary saccades.

18.

Individual corticospinal neurons discharge before voluntary contractions of related muscles occur. The discharges are typically related to contractile force rather than to joint position. However, the activity of an individual neuron may encode different parameters of a movement at different times in relation to the execution of that movement.

19.

The population activity of motor cortex neurons can be used to predict the direction of upcoming movements.

20.

The cerebellum influences the rate, range, force, and direction of movements. It also influences muscle tone and posture, as well as eye movement and balance.

21.

The intrinsic circuitry of the cerebellum is remarkably uniform. Differences in function of different parts of the cerebellum arise largely as a result of differing afferent sources and efferent targets.

22.

Anatomical and physiological techniques have shown that the cerebellar cortex may be divided into many functionally distinct, longitudinally running compartments.

23.

Most of the input to the cerebellum is through pathways that end as mossy fibers. Mossy fibers excite granule cells, which in turn can evoke single action potentials, called simple spikes, in Purkinje cells, whose axons form the only output pathway from the cerebellar cortex.

24.

The projections of the inferior olivary nucleus to the cerebellum end as climbing fibers and are the only source of them. Each Purkinje cell receives massive input from just one climbing fiber. As a result, each climbing fiber discharge produces a high-frequency burst of several action potentials, known as a complex spike, in the Purkinje cell.

25.

Although complex spike activity is relatively rare in comparison to simple spike activity, complex spikes are precisely synchronized across populations of Purkinje cells, and because of the convergence of these cells onto cerebellar nuclear neurons, this synchronization may allow complex spike activity to significantly affect cerebellar output. Synchronization of complex spikes is the result of electrical coupling of inferior olivary neurons by gap junctions.

26.

The basal ganglia include several deep telencephalic nuclei (including the caudate nucleus, putamen, and globus pallidus). The basal ganglia interact with the cerebral cortex, subthalamic nucleus, substantia nigra, and thalamus.

27.

Activity transmitted from the cerebral cortex through the basal ganglia can either facilitate or inhibit the thalamic neurons that project to motor areas of the cortex, depending on the balance between direct and indirect basal ganglia pathways. When there is an imbalance of these two pathways, hyperkinetic or hypokinetic disorders occur.

CHAPTER 9 Organization of Motor Function 28. Some types of eye movement help stabilize the view of the visual world. This is critical because visual acuity drops dramatically when the visual world moves, or slips, across the retina. Vestibuloocular and optokinetic movements help stabilize the visual world on the retina by compensating for movement of the head or external world (or both). Smooth pursuit movements allow tracking of a visual target so that it remains centered on the foveae.

Eccles JC, Ito M, Szentagothai J. The Cerebellum as a Neuronal Machine. New York: Springer; 2013. (Original work published 1967.)

Shadmehr R, Wise SP. The Computational Neurobiology of Reaching and Pointing: A Foundation for Motor Learning. Cambridge, MA: Bradford Books; 2004.

Shepherd GM. Spinal cord. In: Synaptic Organization of the Brain. 5th ed. Oxford, UK: Oxford University Press; 2003: Chapter 3.

29.

Saccades act to move a specific part of the visual scene to the fovea, the retinal area of highest acuity, for detailed inspection.

30.

There are specialized circuits and areas in the brainstem for control of vertical and horizontal eye movements. These areas are used both by the cortex (when voluntary eye movements are made) and by the sensory input that initiates reflexive eye movement.

Shepherd GM. Cerebellum. In: Synaptic Organization of the Brain. 5th ed. Oxford, UK: Oxford University Press; 2003: Chapter 7.

Shepherd GM. Basal ganglia. In: Synaptic Organization of the Brain. 5th ed. Oxford, UK: Oxford University Press; 2003: Chapter 9.

Xiao J, Cerminara NL, Kotsurovskyy Y, et al. Systematic regional variations in Purkinje cell spiking patterns. PLoS ONE. 2014;9: e105633.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

What is the basic layering pattern of the neocortex, and how do cortical inputs and outputs align with this layering pattern? What is the functional significance of the variation in the layering pattern between cortical areas?

2.

What are the major functions of each of the lobes of the cerebrum?

3.

How does the electroencephalogram (EEG) reflect cortical activity? What are evoked potentials?

4.

How does cerebral dominance correlate with language and hand preference?

5.

What is aphasia, and what is compromised in the different types of aphasia?

6.

How do synaptic and cellular processes support learning and memory? How is memory distributed in the brain?

7.

What role does plasticity play in neural development and in response to damage of the nervous system?

n earlier chapters, the interaction of the nervous system with the body and the outside world was discussed in terms of the transduction and analysis of sensory events, the organization of motor function, and relatively simple central processes that link them, such as reflexes (e.g., the stretch reflex and the vestibuloocular reflex). The nervous system has other capabilities, so-called integrative or higher functions, that are less directly tied to specific sensory modalities or motor behavior. These functions, in particular, require interactions between different parts of the cerebral cortex, and, as is being increasingly recognized, between the cerebral cortex and other parts of the brain. The neural basis for some of these higher functions is discussed in this chapter. Because these functions (as well as sensory perception and voluntary motor function) are so highly dependent on the cerebral cortex, its basic organization is described first.

The Cerebral Cortex

The human cerebral cortex occupies a volume of about 600 cm3 and has a surface area of 2500 cm2. The surface of the cortex is highly convoluted and folded into ridges known as gyri. Gyri are separated by grooves called sulci (if shallow) or fissures (if deep; see

Fig. 4.7 ). This folding greatly increases the surface area of cortex that can be fit into the limited and fixed volume within the skull. Indeed, most of the cortex cannot be seen from the brain surface because of this folding.

The cerebral cortex can be divided into the left and right hemispheres and subdivided into a number of lobes (Fig.

10.1

Fig. 4.7 ), including the frontal, parietal, temporal, and occipital lobes. The frontal and parietal lobes are separated by the central sulcus; both are separated from the temporal lobe by the lateral fissure. The occipital and parietal lobes are separated (on the medial surface of the hemisphere) by the parieto-occipital fissure (see

Fig.

10.1 ). Buried within the lateral fissure is another lobe, the insula (see

Fig. 4.6A). A group of structures that make up the limbic lobe is on the medial aspect of the hemisphere, and its largest part, the hippocampal formation, is folded into the parahippocampal gyrus of the temporal lobe and cannot be seen from the surface of the brain.

Activity in the two hemispheres of the cerebral cortex is coordinated by interconnections through the cerebral commissures. The bulk of the cortex is connected through the massive corpus callosum (see

Figs. 4.9 10.1), and parts of the temporal lobes connect through the anterior commissure.

There are three types of cerebral cortex: neocortex, archicortex, and paleocortex. The neocortex has six cortical layers (Fig. 10.2 ). In contrast, the archicortex has only three layers, and the paleocortex has four to five layers. In humans, approximately 90% of the cerebral cortex is neocortex.

The Neocortex

Neuronal Cell Types in the Neocortex

A number of different neuronal cell types in the neocortex have been described (see

Fig. 10.2 ). Pyramidal cells are the most abundant cell type and account for approximately 75% of neocortical neurons. Stellate cells and various other types of nonpyramidal neurons make up the balance. Pyramidal cells have a large triangular cell body, a long •Fig. 10.1 Lateral(A) andmedial(B) illustrationsofthelefthemisphereofthehumancerebrumwiththemajorfeatureslabeledandthelobesindicatedbycolor.R,G,B,andSindicate,respectively,therostrum,genu,body,andspleniumofthecorpuscallosum.(FromHainesDE[ed].Fundamental Neuroscience for Basic and Clinical Applications. 3rded.Philadelphia:ChurchillLivingstone;2006.)

I. Molecular layer

II. External granular layer

III.External pyramidal layer

IV.

V.

VI.Polymorphic layer Outer band of Baillarger

Inner band of Baillarger

Fig. 10.2 An Area of Neocortex Stained by Three Different Methods. TheNisslstain(center) showsthecellbodiesofallneuronsandrevealshowdifferenttypesaredistributedamongthesixlayers.TheGolgistain(left) showsonlyasampleoftheneuronalpopulationbutrevealsdetailsoftheirdendrites.TheWeigertstainformyelin(right) demonstratesverticallyorientedbundlesofaxonsenteringandleavingthecortexandhorizontallycoursingfibersthatinterconnectneuronswithinalayer.(FromBrodmann

K.

Zellenbaues. Leipzig:JABarth;1909.) apical dendrite directed toward the cortical surface, and several basal dendrites. The cell’s axon emerges from the body opposite the apical dendrite, and those from the larger pyramidal cells project into the subcortical white matter. The axon may give off collateral branches as it descends through the cortex. The neurotransmitter of pyramidal cells is an excitatory amino acid (glutamate or aspartate). Stellate cells, often called granule cells, are interneurons with local connections. They have a small soma and numerous branched dendrites, although many have an apical dendrite and thus look like small pyramidal cells. Some are excitatory interneurons; these cells are abundant in layer IV of the cortex (described in the next section). Their axons remain in the same cortical region, and many ascend into the upper cortical layers. Some stellate cells are inhibitory inter-neurons whose neurotransmitter is gamma-aminobutyric acid (GABA).

Cytoarchitecture of Cortical Layers

Each of the six layers of the neocortex has a characteristic cellular content (see

Fig. 10.2 ). Layer I (molecular layer) has few neuronal cell bodies and contains mostly axon terminals synapsing on apical dendrites. Layer II (external granular layer) contains mostly stellate cells. Layer III (external pyramidal layer) consists mostly of small pyramidal cells. Layer IV (internal granular layer) contains mostly stellate cells and a dense matrix of axons. Layer V (internal pyramidal layer) is dominated by large pyramidal cells, the main source of cortical efferents to most subcortical regions. Layer VI (multiform layer) contains pyramidal, fusiform, and other types of cells.

Most input to the cortex from other regions of the central nervous system (CNS) is relayed by neurons in the thalamus, the paleocortex has four to five layers. The paleocortex is located at the border between the archicortex and neocortex.

In humans, the hippocampal formation is part of the archicortex. It is folded into the temporal lobe and can be viewed only when the brain is dissected. The hippocampal cortex has three layers: the molecular, pyramidal cell, and polymorphic layers. They resemble layers I, V, and VI of the neocortex. The white matter covering the hippocampus is called the alveus, which contains hippocampal afferent and efferent fibers. The efferent axons coalesce to form the fornix (Fig. 10.4

Functions of the Lobes of the Cerebral Cortex

There is no exact correspondence between the folds (lobes and gyri) of the cerebral cortex and function; nevertheless, some with the individual lobes of the cerebral hemispheres have a general association with function that helps clarify cortical organization.

One of the main functions of the frontal lobe is motor behavior. As discussed in , the motor, premotor, cingulate motor, and supplementary motor areas are located •Fig. 10.3 Brodmann’sareasinthehumancerebralcortex.(RedrawnfromCrosbyEC,etal.Correlative Anatomy of the Nervous System. NewYork:Macmillan;1962.)6312578910113825273029262834363523312433321237201919181817B •Fig. 10.4 Thehippocampusandtheamygdalaarelocatedonthemedialaspectofthetemporallobe.Thefornix,themajoroutputpathwayfromthehippocampus,projectstothemammillarybody,whichinturnconnectstotheanteriornucleusofthethalamusviathemammillothalamictract.Alsoillustratedarethecingulategyrus,thebasalforebrainarea(septalnuclei,bednucleusofthestriaterminalis,nucleusaccumbens),andtheprefrontalcortex.(FromPurvesD.Sleepandwakefulness.In:PurvesD,etal[eds].Neuroscience. 3rded.Sunderland,MA:Sinauer;2004.) in the frontal lobe, as is the frontal eye field. These areas are crucial for planning and executing motor behavior. Broca’s area, essential for the generation of speech, is located in the inferior frontal gyrus of the dominant hemisphere for human language (almost always the left hemisphere, as explained later). In addition, the more anterior prefrontal cortex in plays a major role in personality and emotional behavior.

Bilateral lesions of the prefrontal cortex may be produced either by disease or by a surgical frontal lobotomy. Such lesions produce deficits in attention, difficulty in planning and problem solving, and inappropriate social behavior. Aggressive behavior is also lessened and the motivational-affective component of pain is reduced although pain sensation remains. Frontal lobotomies are rarely performed today because modern drug therapies provide more focal and effective management for mental illness and chronic pain.

The parietal lobe contains the somatosensory cortex (see ) and the adjacent parietal association cortex. The parietal association cortex gets information from somatosensory, visual, and auditory cortices and is involved in the processing, perception, and integration of sensory information. Connections with the frontal lobe allow somatosensory information to aid in voluntary motor activity. Somatosensory, visual, and auditory information can also be transferred to language centers, such as Wernicke’s area, as described later. Lesions in the left parietal lobe can result in Gerstmann’s syndrome, which includes a person’s inability to name his or her fingers (or those of another) and a loss of the ability to perform numerical calculations. The right parietal lobe is involved in determining spatial context. Localized lesions can result in the neglect syndrome, in which the patient seems unaware of the left side of his or her body and of persons, objects, and events on his or her left. (See an “

In the Clinic ” box later in this chapter.)

The major function of the occipital lobe is visual processing and perception (see ). The primary visual cortex (Brodmann area 17) lines the calcarine sulcus and is flanked by secondary (Brodmann area 18) and tertiary (Brodmann area 19) visual cortices. Lesions of these areas in the cuneus gyrus result in blindness in the lower contralateral visual field; those in the lingual gyrus result in blindness in the upper contralateral visual field. Connections to the frontal eye fields affect direction of gaze, and projections to the midbrain assists in the control of convergent eye

Twoareasimportantforplanningandexecutingmotortasksaretheparietal cortex andthefrontal cortex, theformerbecauseitintegratessensoryinformationneededtodefinethecontextofatask(see ).Mirrorneuronshavebeenfoundinboththeinferior parietal andtheinferior frontal cortices of macaques. Thesecellsrespondduringperformanceofaspecificmotortaskandalsoduringobservationofthesametaskperformedbyanotheranimal.Becausethesemirrorcellsseemtoencodeforandrespondtoveryspecificandparticulartasks,ithasbeenspeculatedthattheymayunderliesuchfunctionsasunderstandingtheintentionsofothersandempathy,aswellastheabilitytolearntasksfromobservation.Inhumans,EEGactivityconsistentwiththebehaviorofsuchmirrorneuronshasbeenlocalizedtotheinferior frontal andsuperior parietal lobes. Autism,whichinvolvesaninabilityto“read”theintentionsandemotionsofothers,hasbeenlinkedtoalackofmirrorneuronsbyEEGevidence.

movements, pupillary constriction, and accommodation, all of which occur when the eyes adjust for near vision.

The temporal lobe has many different functions, including the processing and perception of sounds and vestibular information and higher order visual processing (see ). For example, the infratemporal cortex, on its inferior surface, is involved in the recognition of faces. In addition, Meyer’s loop, which forms part of the optic pathway, passes through the temporal lobe. As a result, temporal lobe lesions can damage vision in upper part of the visual field. Similarly, a portion of Wernicke’s area, essential for the understanding of language, lies in the posterior region of the temporal lobe.

The limbic system dominates the medial temporal lobe (see

Fig. 10.4 ), and it participates in emotional behavior and in learning and memory (see the “Learning and Memory” section). The limbic system helps control emotional behavior, in part by an influence on the hypothalamus via the Papez circuit. This circuit projects from the cingulate gyrus to the entorhinal cortex and hippocampus, and from there, via the fornix, to the mammillary bodies in the hypothalamus. The mammillothalamic tract then connects the hypothalamus with the anterior thalamic nuclei, which project back to the cingulate gyrus (see

Fig. 10.4 ). In addition, the hippocampus and amygdala are connected to the prefrontal cortex, the basal forebrain, and the anterior cingulate cortex.

Bilateral temporal lobe lesions can produce Klüver-Bucy syndrome, which is characterized by loss of the ability to recognize the meaning of objects from visual cues (visual agnosia); a tendency to examine all objects, even dangerous ones, orally; attention to irrelevant stimuli; hypersexuality; a change in dietary habits; and decreased emotionality.

Although this syndrome was originally described as following large lesions of most or all of the temporal lobe, more recent studies have highlighted the role of the amygdala. The amygdala conditions the association of fear with painful stimuli and may trigger, via connections to the medial frontal cortex and anterior cingulate gyrus, emotional or avoidance responses when these stimuli recur. In addition, the amygdala projects to the nucleus accumbens, a region of the basal ganglia which has been called a “reward center.” The nucleus accumbens signals pleasurable events in response to dopaminergic input from the ventral tegmental area of the brainstem and is also the most common site of the plaques and tangles associated with Alzheimer’s disease.

Thefunctionsofthedifferentlobesofthecerebralcortexhavebeendefinedaccordingtoboththeeffectsoflesionsproducedbydiseaseorthoseofsurgicalinterventionstotreatdiseaseinhumansandfromexperimentsonanimals.Inanotherapproach,thephysicalmanifestationsofepileptic seizures havebeencorrelatedwiththebrainlocationsthatgiverisetoseizures(epileptic seizure foci). Forexample,epilepticfociinthemotorcortexcausemovementsonthecontralateralside;theexactmovementsrelatetothesomatotopiclocationoftheseizurefocus.Seizuresthatoriginateinthesomatosensorycortexcauseanepileptic aura inwhichatouchsensationisperceived.Similarly,seizuresthatstartinthevisualcortexcauseavisualaura(scintillations,colors),thoseintheauditorycortexcauseanauditoryaura(humming,buzzing,ringing),andthoseinthevestibularcortexcauseasensationofspinning.Complexbehaviorresultsfromseizuresthatoriginateintheassociationareasofthetemporallobe;inaddition,amalodorousauramaybeperceivediftheolfactorycortexisinvolved (uncinate fit).

The Electrical Activity of the Cortex

An electroencephalogram (EEG) is a recording of the neuronal electrical activity of the cerebral cortex made by electrodes placed on the skull. EEG waves normally reflect the summed extracellular currents that result from the generation of synaptic potentials in the pyramidal cells and are thus a type of field potential. Because the currents generated by a single cell are too small to be detected as discrete events by an electrode on the skull (to record the activity of a single neuron, a microelectrode must be placed within microns of the neuron), the EEG waves reflect the combined activity of many pyramidal cells. Moreover, for the activity of a group of neurons to generate an event detectable on EEG, they must be oriented so that their individual currents summate to produce a detectable field. The arrangement of pyramidal neurons, with their apical dendrites aligned in parallel to form a dipole sheet, is particularly favorable for generating large field potentials. One pole of this sheet is oriented toward the cortical surface and the other toward the subcortical white matter. Thus the currents generated •Fig. 10.5 Electroencephalographictracingsduringdrowsiness;stages1,2,and4ofslow-wave(non–rapideyemovement[non-REM])sleep;andREMsleep.(ModifiedfromShepherdGM.Neurobiology. London:OxfordUniversityPress;1983.) by cortical pyramidal cells have a similar orientation and can therefore summate to produce a field potential that can be detected. The need for summation also explains why EEG signals reflect primarily synaptic potentials rather than action potentials: electrical events must overlap in time in order to sum, and synaptic potentials have much longer durations than do action potentials.

The sign of an EEG wave can be positive or negative, but its direction alone does not indicate whether pyramidal cells are being excited or inhibited. For instance, a negative EEG potential may be generated at the surface of the skull (or cortex) by excitation of apical dendrites or by inhibition near the somas. Conversely, a positive EEG wave can be produced by inhibition of apical dendrites or by excitation near the somas.

A normal EEG tracing consists of waves of various frequencies. The dominant frequencies depend on several factors, including the state of wakefulness, the age of the subject, the location of the recording electrodes, and the absence or presence of drugs or disease. When a normal awake adult is relaxed with the eyes closed, the dominant frequencies of the EEG recorded over the parietal and occipital lobes are about 8 to 12 Hz, the alpha rhythm. If the subject is asked to open the eyes, the wave becomes less synchronized, and the dominant frequency increases to 13 to 30 Hz, which is called the beta rhythm. The delta (0.5 to 2 Hz) and theta (3 to 7 Hz) rhythms are observed during sleep (see the following discussion;

Fig. 10.5 ). Also, brief EEG waves do exist and, because of their shape, are sometimes referred to as spikes, but this does not imply that they are associated with action potentials.

An EEG change that can be elicited by a stimulus is called a cortical evoked potential. A cortical evoked potential is best recorded from the part of the skull located over the cortical area being activated. For example, a visual stimulus results in an evoked potential that can be recorded best over the occipital bone, whereas a somatosensory evoked potential is recorded most effectively near the junction of the frontal and parietal bones. Evoked potentials reflect the activity in large numbers of cortical neurons. They may also reflect activity in subcortical structures.

Evoked potentials are small in comparison with the size of the EEG waves. However, their appearance can be enhanced by a process called signal averaging. In this process, the stimulation is repeated, and the EEGs recorded during each trial are electronically averaged. With each repetition of the stimulus, the evoked potential occurs at a fixed time after the stimulus. When the records are averaged, the components of the EEG that have a random temporal association with the stimulus cancel each other, whereas the evoked potentials sum.

Evokedpotentialsareusedclinicallytoassesstheintegrityofasensorypathway,atleasttotheleveloftheprimarysensoryreceivingarea.Thesepotentialscanberecordedincomatoseindividuals,aswellasininfantstooyoungtoundergoasensoryexamination.Theinitialpartsoftheauditoryevokedpotentialactuallyreflectactivityinthebrainstem;therefore,thisevokedpotentialcanbeusedtoassessthefunctionofbrainstemstructures.

Sleep and wakefulness are among the many functions of the body that show circadian (about 1-day) periodicity. Characteristic changes in the EEG can be correlated with changes in the behavioral state during the sleep-wake cycle. Beta wave activity dominates in an awake, aroused individual. The EEG is said to be desynchronized; it displays low-voltage, high-frequency activity. In relaxed individuals with their eyes closed, the EEG is dominated by alpha waves (see

Fig. 10.5 ). A person falling asleep passes sequentially through four stages of slow-wave sleep (called stages 1 through 4) over a period of 30 to 45 minutes (see

Fig. 10.5 ). In stage 1, alpha waves are interspersed with lower frequency waves called theta waves. In stage 2, the waves slow further, but the slow-wave activity is interrupted by sleep spindles, which are bursts of activity at 12 to 14 Hz, and by large K complexes (large, slow potentials). Stage 3 sleep is associated with delta waves and with occasional sleep spindles. Stage 4 is characterized by delta waves without spindles.

During slow-wave sleep, the muscles of the body relax, but the posture is adjusted intermittently. The heart rate and blood pressure decrease, and gastrointestinal motility increases. The ease with which individuals can be awakened decreases progressively as they pass through these sleep stages. As individuals awaken, they pass through the sleep stages in reverse order.

About every 90 minutes, slow-wave sleep changes to a different form of sleep, called rapid eye movement (REM) sleep. In REM sleep, the EEG again becomes desynchronized. The low-voltage, fast activity of REM sleep resembles that seen in the EEG from an aroused subject (see

Fig.

10.5 , bottom trace). Because of the similarity of the EEG to that of an awake individual and the difficulty awaking the person, the term paradoxical sleep characterizes this type of sleep. Muscle tone is completely lost, but phasic contractions occur in a number of muscles, most notably the eye muscles. The resulting rapid eye movements are basis of the name for this type of sleep. Many autonomic changes also take place. Temperature regulation is lost, and meiosis occurs. Penile erection may occur during this type of sleep. Heart rate, blood pressure, and respiration change intermittently. Several episodes of REM sleep occur each night. Although it is difficult to arouse a person from REM sleep, internal arousal is common. Most dreaming occurs during REM sleep.

Thesleep-wakecyclehasanendogenousperiodicityofabout25hours,butitnormallybecomesentrainedtotheday-nightcycle.Thesourceofcircadianperiodicityappearstobethesuprachiasmaticnucleusofthehypothalamus.Thisnucleusreceivesprojectionsfromtheretina,anditsneuronsseemtoformabiologicalclockthatadaptstothelight-darkcycle.However,theentrainmentcanbedisruptedwhenthesubjectisisolatedfromtheenvironmentorchangestimezones(jetlag).Destructionofthesuprachiasmaticnucleusdisruptsanumberofbiologicalrhythms,includingthesleep-wakecycle.

The proportion of slow-wave (non-REM) sleep to REM sleep varies with age. Newborns spend about half of their sleep time in REM sleep, whereas elderly people have little REM sleep. About 20% to 25% of the sleep of young adults is REM sleep.

The mechanism of sleep is incompletely understood. Stimulation in the brainstem in a large region known as the reticular activating system causes arousal and low-voltage, fast EEG activity. Sleep was once thought to be caused by a reduced level of activity in the reticular activating system. However, substantial data, including the observations that anesthesia of the lower brainstem results in arousal and that stimulation in the medulla near the nucleus of the solitary tract can induce sleep, suggest that sleep is an active process. Investigators have tried to find a relationship between sleep mechanisms and brainstem networks in which particular neurotransmitters, including serotonin, norepinephrine, and acetylcholine, are used; manipulations of the levels of these transmitters in the brain can affect the sleep-wake cycle. However, a detailed neurochemical explanation of the neural mechanisms of sleep is not yet available.

Similarly, the purpose of sleep is still unclear. However, it must have a high value because so much of life is spent in sleep and because lack of sleep can be debilitating. Medically important disorders of the sleep-wake cycle include insomnia, bed-wetting, sleepwalking, sleep apnea, and narcolepsy.

•Fig. 10.6 Electroencephalographic (EEG) Abnormalities in Several Forms of Epilepsy. A, EEGtracingsduringthetonic(left) andclonic(right) phasesofatonic-clonic(grandmal)seizure.B, Spikeandwavecomponentsofanabsence(petitmal)seizure.C, EEGtracinginapersonwithtemporallobeepilepsy.D, EEGtracingofafocalseizure.(RedrawnfromEyzaguirreC,FidoneSJ.Physiology of the Nervous System. 2nded.St.Louis:Mosby;1975.)

TheEEGbecomesabnormalinavarietyofpathologicalcircumstances.Forexample,duringcoma,theEEGisdominatedbydeltaactivity.Brain death isdefinedbyamaintainedflatEEGwave.

Epilepsy commonlycauses,andcanbediagnosedby,specificEEGabnormalities.Therearemanyformsofepilepsy,andexamplesofEEGpatternsfromsomeofthesetypesofepilepsyareshownin

Figure10.6 .Epilepticseizurescanbeeitherpartialorgeneralized.

Oneformofpartialseizuresoriginatesinthemotorcortexandresultsinlocalizedcontractionsofcontralateralmuscles.Thecontractionsmaythenspreadtoothermuscles;suchspreadfollowsthesomatotopicsequenceofthemotorcortex(see ).ThisstereotypicalprogressioniscalledaJacksonian march. Complexpartialseizures(whichmayoccurinpsychomotor epilepsy)originateinthelimbicstructuresofthetemporallobeandresultinillusionsandsemipurposefulmotoractivity.Duringandbetweenfocalseizures,scalprecordingsmayrevealEEGspikes(see

Fig.

10.6C andD

Generalizedseizuresinvolvewideareasofthebrainandlossofconsciousness.Twomajortypesarepetit mal and

Although right-handedness represents a sensorimotor dominance of the left hemisphere and left-handedness represents a sensorimotor dominance of the right hemisphere, cerebral dominance is assigned to the hemisphere in which language is to communicate; in humans, the left hemisphere is the dominant hemisphere in more than 90% of both rightand left-handed people. This dominance grand mal seizures. Inpetitmalepilepsy,consciousnessislosttransiently(typicallyforlessthan15seconds),andtheEEGdisplaysspike and wave activity (see

Fig.10.6B ).Ingrandmalseizures,consciousnessislostforalongerperiod,andtheaffectedindividualmayfallifstandingwhentheseizurestarts.Theseizurebeginswithageneralizedincreaseinmuscletone(tonic phase), followedbyaseriesofjerkymovements(clonic phase). Thebowelandbladdermaybeevacuated.TheEEGshowswidelydistributedseizureactivity(see

Fig.10.6A

EEGspikesthatoccurbetweenfull-blownseizuresarecalledinterictal spikes. Similareventscanbestudiedexperimentally.Thesespikesarisefromabrupt,long-lastingdepolarizations,calleddepolarization shifts, thattriggerrepetitiveactionpotentialsincorticalneurons.Thesedepolarizationshiftsmayreflectseveralchangesinepilepticfoci.SuchchangesincluderegenerativeCa++-mediateddendriticactionpotentialsincorticalneuronsandareductionininhibitoryinteractionsincorticalcircuits.ElectricalfieldpotentialsandthereleaseofK+ andexcitatoryaminoacidsfromhyperactiveneuronsmayalsocontributetotheincreasedcorticalexcitability.

has been demonstrated (1) by the effects of lesions of the left hemisphere that produce deficits in language function (aphasia) and (2) by the transient aphasia (inability to speak or write) that results when a short-acting anesthetic is introduced into the left carotid artery. Lesions of the nondominant hemisphere and injection of anesthetic into it do not usually affect language substantially.

Several areas in the left hemisphere are involved in language. Wernicke’s area is a large area in the posterior part of the superior temporal gyrus, extending from behind the auditory cortex into the parietal lobe. Another important language area, Broca’s area, is in the posterior part of the inferior frontal gyrus, close to the face representation of the motor cortex. Damage to Wernicke’s area results in receptive aphasia, in which the person has difficulty comprehending spoken and written language; however, speech production remains fluent, if meaningless. Conversely, a lesion in Broca’s area causes expressive aphasia, in which individuals have difficulty in generating speech and writing, although they can understand language relatively well.

The terms sensory aphasia and motor aphasia are often interchanged with receptive aphasia and expressive aphasia, respectively. The former terms, however, are misleading: A person with receptive aphasia may not have auditory or visual impairment, and one with expressive aphasia may have normal motor control of the muscles responsible for speech or writing. Aphasia does not depend on a deficit of sensation or of motor skill; rather, it is an inability to decode language-encoded sensory information into concepts or to encode concepts into language. However, lesions in the dominant hemisphere may be large enough to result in mixed forms of aphasia, as well as sensory changes or paralysis of some of the muscles used to express language. For example, the latter situation could occur with a lesion of the face representation portion of the motor cortex that results in an inability to manipulate the motor apparatus needed for speaking (vocal cords, jaws, tongue, lips) and would be manifest as unclear speech because of dysarthria, a mechanical deficit. An affected individual would, however, be able to write if the motor cortex serving the upper limb were unaffected.

Interhemispheric Communication and the Corpus Callosum

The two cerebral hemispheres can function somewhat independently, as in the control of one hand. However, information must be transferred between the hemispheres to coordinate activity on the two sides of the body. Much of that information is transmitted through the corpus callosum, although some is transmitted through other commissures (e.g., the anterior commissure or the hippocampal commissure).

The importance of the corpus callosum for interhemispheric transfer of information is illustrated in

Figure 10.7A.

An animal with an intact optic chiasm and corpus callosum and with the left eye closed learns a visual discrimination task (see

Fig. 10.7A).

The information is transmitted to both hemispheres through bilateral connections made by the optic chiasm or through the corpus callosum, or both. When the animal is tested with the left eye open and the right eye closed (see

Fig. 10.7A, center), the task can still be performed because both hemispheres have learned the task. If the optic chiasm is transected before the animal is trained, the result is the same (see

Fig. 10.7B). Information is presumably transferred between the two hemispheres through the corpus callosum. This finding can be confirmed by cutting both the optic chiasm and the corpus callosum before training (see

Fig. 10.7C ). Then the information is not transferred, and each hemisphere must learn the task independently.

A similar experiment was conducted in human patients who had undergone surgical transection of the corpus callosum to prevent the interhemispheric spread of epilepsy (

Fig. 10.8 ). The optic chiasm remained intact, but visual information was directed to one or the other hemisphere by the patient’s fixing vision on the central point of the screen. A picture or name of an object was then flashed to one side of the fixation point, so that visual information about the picture reached only the contralateral hemisphere. An opening beneath the screen allowed the patient to manipulate objects that could not be seen. The objects included those shown in the projected pictures. Normal individuals would be able to locate the correct object with either hand. However, patients with a transected corpus callosum could locate the correct object only with the hand ipsilateral to the projected image (contralateral to the hemisphere that received the visual information). For the hand to explore and recognize the correct object, the visual information must have access to the somatosensory and motor areas of the cortex. With the corpus callosum cut, the visual and motor areas are interconnected only on the same side of the brain.

Another test was to ask the patient to verbally identify what object was seen in the picture. The patient would make a correct verbal response to a picture that was projected to the right of the fixation point because the visual information reached only the left (language-dominant) hemisphere. However, the patient could not verbally identify a picture that was presented to the left hemifield because visual information reached only the right hemisphere.

Similar observations can be made in patients with a transected corpus callosum when different forms of stimuli are used. For example, when such patients are given a verbal command to raise the right arm, they do so without difficulty. The language centers in the left hemisphere send signals to the ipsilateral motor areas, and these signals produce the movement of the right arm. However, these patients cannot respond to a command to raise the left arm. The language areas on the left side cannot influence the motor areas on the right unless the corpus callosum is intact. Somatosensory stimuli applied to the right side of the body can be described by patients with a transected corpus callosum, but these patients cannot describe the same stimuli applied to the left side of the body. Information that reaches the right somatosensory areas of the cortex cannot reach the language centers if the corpus callosum has been cut.

In addition to language, other differences in the functional capabilities of the two hemispheres can be compared by exploring the performance of individuals with a transected corpus callosum. Such patients solve

CHAPTER 10 Integrative Functions of the Nervous System 219 • Fig. 10.7 Role of the Corpus Callosum in the Interhemispheric Transfer of Visual Information When Learning Involves One Eye.A, Discrimination depends on distinguishing between a cross and a circle. B, Discrimination is between triangles oriented with the apex up or down. C, Discrimination is between vertical and horizontal bars.

•Fig. 10.8 Illustration of Tests in a Patient With a Transected Corpus Callosum. A, Thepatientfixesonapointonarearprojectionscreen,andpicturesareprojectedtoeithersideofthefixationpoint.Thehandcanpalpateobjectsthatcorrespondtotheprojectedpictures,buttheseobjectscannotbeseen.B, Responsebythelefthandtoapictureofakeyintheleftfieldofview.However,theverbalresponseisthatthepatientseesapictureofaring.(RedrawnfromSperryRW.In:SchmittFO,WordenFG[eds].The Neurosciences: Third Study Program. Cambridge,MA:MITPress;1974.) three-dimensional puzzles better with the right than with the left hemisphere, which suggests that the right hemisphere has specialized functions for spatial tasks. Other functions that seem to be more associated with the right than the left hemisphere are facial expression, body language, and speech intonation (

Fig. 10.9 ). Patients with a transected corpus callosum lack normal interhemispheric coordination. When they are dressing, for example, one hand may button a shirt while the other tries to unbutton it. Observation of these patients indicates that the two hemispheres can operate quite independently when they are no longer interconnected. However, one hemisphere can express itself with language, whereas the other communicates only nonverbally.

Oneofthemorestrikingexamplesofinterhemisphericdifferencesisthephenomenonof“cortical neglect,” whichisaconsequenceofalesionintheparietalcortexofthenondominant(usuallyright)hemisphere.Insuchcases,thepatientignoresobjectsandindividualsintheleftvisualfield,drawsobjectsthatareincompleteontheleft,deniestheexistenceofhisorherleftarmandleg,andfailstodresstheleftsideofhisorherbody.Thepatientalsodenieshavinganysuchdifficulties(anosognosia). Althoughthepatientmayrespondtotouchandpinprickontheleftsideofthebody,heorshecannotidentifyobjectsplacedinthelefthand.Thelesionisadjacenttothefirstsomatosensory(SI)cortex,aswellasthevisualassociationcortex,anditsuggeststhatthisregionplaysaspecialroleintheperceptionofbodyimageandimmediateextrapersonalspace.Similarlesionsonthedominantsideresultonlyinlossofsomehigherordersomesthesias,suchasagraphesthesia (inabilitytoidentifycharactersdrawnonthepalm)andastereognosis (inabilitytoidentifyanobjectonlybytouch).

Major functions of the higher levels of the nervous system are learning and memory. Learning is a neural mechanism by which the organism’s behavior changes as a result of experience. Memory is the storage mechanism for what is learned.

The neural circuitry involved in memory and learning in mammals is complex and difficult to study. Alternative approaches are animal studies (especially in the simpler nervous systems of invertebrates), analysis of the functional consequences of lesions, and anatomical/physiological studies at the cellular and pathway level. For example, in the marine mollusk Aplysia, it has been possible to isolate a connection between a single sensory neuron and a motor neuron, which shows aspects of habituation (learning not to respond to repetitions of an insignificant stimulus), sensitization (increased responsiveness to innocuous stimuli that follow the presentation of a strong or noxious stimulus), and even associative conditioning (learning to respond to a previously insignificant event after it has been

Right-side olfaction Memory for shapes Stereognosis, left side Hearing (left-ear preference) Musical ability Recognition of forms, faces, and body image Left visual fieldRight visual field Mathematical ability Understanding language Hearing (right-ear preference) Motor speech Left-side olfaction Verbal memory Stereognosis, right side Right-hand skills (e.g., writing) L Left Right RLR 372 x 49 18228 •Fig. 10.9 Schematicillustrationofthefunctionalspecializationsoftheleftandrighthemispheres,asdeterminedinpatientsaftersectionofthecorpuscallosum.(ModifiedfromSiegelA,SapruHN.Essential Neuroscience. 5thed.Philadelphia:LippincottWilliams&Wilkins;2005.) paired with a significant one). In the case of habituation, the amount of transmitter released in successive responses gradually diminishes. The change involves an alteration in the Ca++ current that triggers release of neurotransmitter. The cause of this change is inactivation of presynaptic Ca++ channels by repeated action potentials. Long-term habituation can also be produced. In this case, the numbers of synaptic endings and active zones in the remaining terminals decreases.

Additional models of learning, provided by synaptic phenomena, are called long-term potentiation (LTP) and long-term depression (LTD). LTD has been studied most extensively in the cerebellum (see

Chapter 9), but it also occurs in the hippocampus and in other regions of the CNS.

LTP has been studied most intensively in slices of the hippocampus in vitro, but it has also been studied in the neocortex, cerebellum, and other parts of the CNS. Repetitive activation of an afferent pathway to the hippocampus or repetitive activation of one of the intrinsic connections increases the responses of pyramidal cells. The increased responses (the LTP) last for hours in vitro (and even days to weeks in vivo). The forms of LTP differ, depending on the particular synaptic system. The mechanism of the enhanced synaptic efficacy seems to involve both presynaptic and postsynaptic events. The neurotransmitters involved in LTP include excitatory amino acids that act on N-methyl-D-aspartate (NMDA) receptors, the responses of which are associated with an influx of Ca++ into the post-synaptic neuron. Second messenger pathways (including G proteins, Ca++/calmodulin-dependent kinase II, protein kinase G, and protein kinase C) are also involved, and these kinases cause protein phosphorylation and changes in the responsiveness of neurotransmitter receptors. A retrograde messenger, perhaps nitric oxide (or carbon monoxide), may be released from postsynaptic neurons to act on presynaptic endings in such a way that transmitter release is enhanced. Immediate-early genes are also activated during LTP. Hence, changes in gene expression may also be involved.

With regard to the stages of memory storage, a distinction between short-term memory and long-term memory is useful. Recent events appear to be stored in short-term memory by ongoing neural activity because short-term memory persists for only minutes. Short-term memory is used, for instance, to remember page numbers in a book after looking them up in the index. Long-term memory can be subdivided into an intermediate form, which can be disrupted, and a long-lasting form, which is difficult to disrupt. Memory loss, or amnesia, can be caused by a loss of memory information per se, or it can result from interference with the mechanism for accessing the information. Long-term memory probably involves structural changes because it can remain intact even after events that disrupt short-term memory.

The temporal lobes appear to be particularly important for memory because bilateral removal of the hippocampal formation can severely and permanently disrupt recent memory. Short-term and long-term memories are unaffected, but new long-term memories can no longer be established. Thus patients with such amnesia remember events before their surgery but fail to recall new events, even with multiple exposures, and must be reintroduced repeatedly to people they meet after the surgery. This loss of declarative memory involves the conscious recall of personal events, places, and general history. Such patients, however, can still learn some tasks because they retain procedural memory, which involves associational and motor skills. If such patients are given a complex task to perform (e.g., mirror writing), they not only improve during the first training session but also perform better on subsequent days despite their denial of having any earlier experience with that task. The cerebral structures involved in procedural memory are not yet defined.

Plasticity most commonly refers to the ability of the CNS to change its connectivity. Such changes can occur in various contexts, including learning and memory (see previous discussion), damage, and development. Damage to the CNS can induce remodeling of neural pathways and thereby alter behavior. Plasticity is greatest in the developing brain, but

Cellularstudiesofthehippocampusandtheentorhinalcortex(whichisadjacentandparalleltothehippocampus)havedemonstratedtheexistenceof“placecells”thatfirewhenthesubjectentersaspecificplaceinatestenvironment.Althoughtherearemanyplacecells,theyarenotdistributedinanorderlymannerthatwouldresembleatopographicmap.Placecellsappearinveryyounganimalsassoonastheyareabletoexplore.Morerecentstudiesrevealthepresenceof“gridcells,”whichalsorespondtospecificsitesbutaredistributedinhexagonalarraysthatresemblesanorderlymapoftheenvironmentalspaceintheposteriorentorhinalcortex.Althoughthiscognitivemapisfixedforanycontext,changestotheenvironmentorremovalofthepersontoanewtestenvironmentcausesthegenerationofanewandappropriatemapofgridcells.

Becausetheentorhinalcortexisamajorsourceofinputtothehippocampus,itisinterestingthatstudiesofLondon’staxidrivers—whomustdemonstrateanextremelydetailedknowledgeofthecity’sstreetsandmostefficientroutesbeforebeinglicensed—indicatethattheposteriorhippocampusintrainedandexperienceddriversislargerthanthatinbeginnersorthegeneralpopulation.“Gettinglost,”acommoncomplaintassociatedwithamnesia,maybeduetothelossofspatialmemoryskills.

some degree of plasticity remains in the adult brain, as evidenced by responses to certain manipulations, such as lesions of the brain, sensory deprivation, or even experience.

The capability for developmental plasticity may be maximal for some neural systems at a time referred to as the critical period. For example, it is possible to alter some connections formed in the visual pathways during their development by preventing one eye from providing input, but only during a specific critical period early in development. In such visually deprived animals, the visual connections become abnormal (

Fig. 10.10 ), and restoration of normal visual input after the critical period does not undo the abnormality, nor does it restore functional vision from the deprived eye. In contrast, similar visual deprivation later in life does not result in abnormal connections. The plastic changes seen in such experiments may reflect a competition for synaptic connections, whereby the less functional connections are pruned away. Research has shown a correlation between the form of a gene that modulates the efficacy of synaptic pruning and the probability of schizophrenia.

Plastic changes can also occur after injury to the brain in adults. Sprouting of new axons does occur in the damaged CNS; however, the sprouts do not necessarily restore normal function, and many neural pathways do not appear to produce sprouts. Additional knowledge concerning neural plasticity in the adult CNS is vital if medical therapy is to be improved for many diseases of the CNS and after neural trauma. Research is currently being conducted to explore •Fig. 10.10 Plasticity in the Visual Pathway as a Result of Sensory Deprivation During Development. Theoculardominancecolumnsaredemonstratedbyautoradiographyafterinjectionofaradioactivetracerintooneeye.Thetraceristransportedtothelateralgeniculatenucleusandthentransneurallytransportedtothestriatecortex.Thecortexislabeledinbandsthatalternatewithunlabeledbandswhoseinputisfromtheuninjectedeye.A, Normalpattern.B, Changedpatterninananimalraisedwithmonocularvisualdeprivation.Theinjectionwasmadeintothenondeprivedeye,andtheoculardominancecolumnsforthiseyewereclearlyexpanded.Otherexperimentsshowedthattheoculardominancecolumnsforthedeprivedeyecontracted.(A, FromHubelDH,WieselTN.Proc R Soc Lond B. 1977;198:1.B, FromLeVayS,etal.J Comp Neurol. 1980;191:1.) the potential of human embryonic stem cells for restoring CNS function.

Phantom limb sensation is an example of neural plasticity in adults. A patient whose limb has been amputated often perceives sensations on the missing limb when stimulated elsewhere on the body. Functional imaging studies suggest that this is a result of the spread of connections from the surrounding cortical territories into the cortical region that had served the amputated limb.

Itwastraditionalpolicytodelaycorrectivesurgeryforachildbornwithacongenitalcataractuntilthechildwasolderandmoreabletocopewiththestressofsurgery.However,ifthecorrectionisdeferreduntilafterthe“criticalperiod,”fullrecoveryoffunctionisunlikely.Similarly,childrenbornwithamblyopia, aconditioncharacterizedbystrabismus(crosseye)becauseofrelativeweaknessofoneoftheextraocularmuscles,tendtousetheunaffectedeyeinpreference.Inbothcases,earlysurgeryisnowcommonpracticesothatthecorticalcircuitrycanbecorrectlysculptedbybalancedinputfromthetwoeyes.

Such remapping can also occur after surgical amputation of the second and third digits of the hand. Before surgery, each of the digits was represented in discrete and somatotopically organized areas of the postcentral gyrus (SI cortex). After surgery, the area that represented the amputated digits is now mapped with an enlarged representation of the adjacent digits (

Fig. 10.11 ). Conversely, individuals born with syndactyly (fusion of two or more digits of the hand) have a single or mostly overlapping representation of these digits in the SI cortex. After corrective surgery, the independent digits come to have distinctive representations. Even more remarkable is that monkeys that were trained on a sensory discrimination task requiring repeated daily use of their fingertips showed cortical differences after training. Not only were the SI cortical territories of their fingertips larger than before training but also the number of cortically recorded receptive fields on the fingertips was likewise increased.

•Fig. 10.11 Representationofthedigitregionoftheleftfirstsomatosensory(SI)cortex(A) andreorganizationofthisrepresentation (B) afteramputationofthesecondandthirddigits.(FromHainesDE[ed].Fundamental Neuroscience for Basic and Clinical Applications. 3rded.Philadelphia:ChurchillLivingstone;2006.) 1. The cerebral cortex can be divided into lobes on the basis of the pattern of gyri and sulci. Each lobe has distinctive functions, as shown by the effects of lesions. The left cerebral hemisphere is dominant for language in most individuals. Wernicke’s area (in the posterior temporal lobe) is responsible for the understanding of language, and Broca’s area (in the inferior frontal lobe) is responsible for its expression.

2. The neocortex contains pyramidal cells and several kinds of interneurons. Specific thalamocortical afferent fibers terminate mainly in layer IV of the neocortex; diffuse thalamocortical afferent fibers synapse in layers I and VI. Axons from pyramidal cells in layer V are the major source of output to subcortical targets, including the spinal cord, brainstem, striatum, and thalamus.

3.

The cortical structure varies in different regions. Brodmann’s designations reflect these variations in cortical structure and correspond to functionally discrete areas.

4.

The EEG reflects electrical fields generated by the activity of pyramidal and varies with the state of the sleep-wake cycle, disease, and other factors. Cortical evoked potentials are stimulus-triggered changes in the EEG and are useful clinical data about sensory transmission.

5.

EEG patterns during sleep are divided into slow-wave and REM forms. Slow-wave sleep progresses through stages 1 through 4, each with a characteristic EEG pattern. Most dreams occur in REM sleep. Sleep is produced actively by a brainstem mechanism,

Squire L, Berg D. Fundamental Neuroscience. 4th ed. New York: Academic Press; 2012.

and its circadian rhythmicity is controlled by the suprachiasmatic nucleus.

6.

Information is transferred between the two hemispheres primarily through the corpus callosum. The right hemisphere is more capable than the left in spatial tasks, facial expression, body language, and speech intonation. The left hemisphere is specialized for the understanding and generation of language, for logic, and for mathematical computation.

7.

Learning and memory can be studied on the cellular level, in invertebrates, and in higher animals. Memory includes short-term (lasting minutes), recent, and long-term storage processes and a retrieval mechanism. The hippocampal formation is important for storing declarative and spatial memory.

8.

Lesion studies and behavioral studies indicate that plasticity occurs in the brain throughout life. However, there appears to be more plasticity early in life, and synaptic competition in “critical periods” is important for the establishment of neural circuitry.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

What are the similarities and differences in the general organizations of the parasympathetic and sympathetic systems?

2.

What are the respective actions of the parasympathetic and sympathetic innervation of the eye, and what symptoms arise when the parasympathetic or sympathetic innervation is lost?

3.

What are the changes in the balance of parasympathetic and sympathetic activity to the bladder that occur during micturation?

4.

What is meant by a “servomechanism”?

5.

What are the specific feedback loops that regulate body temperature, feeding and body weight, and water intake?

6.

What is the role of the hypothalamus in each of these feedback loops?

he main function of the autonomic nervous system is to assist the body in maintaining a constant internal environment (homeostasis). When internal stimuli signal that regulation of the body’s environment is required, the central nervous system (CNS) and its autonomic outflow issue commands that lead to compensatory actions. For example, a sudden increase in systemic blood pressure activates the baroreceptors, which in turn modify the activity of the autonomic nervous system so that the blood pressure is lowered toward its previous level (see

The autonomic nervous system has both sensory and motor divisions. The motor division is further divided into the sympathetic and parasympathetic divisions. Because much of the autonomic nervous system’s actions relate to control of the viscera, it is sometimes called the visceral nervous system.

In service of its homeostatic function, the autonomic nervous system mediates visceral reflexes (e.g., the gastrocolic reflex, where stomach distention triggers peristalsis in the intestines) and provides sensory information to the CNS for the perception of the state of our viscera, a percept known to anyone who has eaten too much at a meal. More generally, activation of autonomic receptors can evoke a variety of sensory experiences such as pain, hunger, thirst, nausea, and a sense of visceral distention; these perceptions can then lead to compensatory voluntary behaviors that assist in maintaining homeostasis.

In addition to its central role in homeostasis, the autonomic nervous system also participates in appropriate and coordinated responses to external stimuli that are required for the optimal functioning of the somatic nervous system in performing voluntary behaviors. For example, the autonomic nervous system helps regulate pupil size in response to different intensities of ambient light, thus helping the visual system to operate over a large range of light intensity.

In this chapter, the enteric nervous system is also considered part of the autonomic nervous system, although it is sometimes considered a separate entity (see also ). In addition, because the autonomic nervous system is under CNS control, the central components of the autonomic nervous system are discussed in this chapter. These central components include the hypothalamus and higher levels of the limbic system, which are associated with emotions (see ) and with many visceral types of behavior (e.g., feeding, drinking, thermoregulation, reproduction, defense, and aggression) that have survival value.

Organization of the Autonomic Nervous System

The sensory autonomic neurons are located in the dorsal root ganglia and in the cranial nerve ganglia. Like the other neurons of the dorsal root ganglia, they are pseudounipolar cells with a peripheral axonal branch extended to one of the viscera and a central branch that enters the CNS. With regard to autonomic motor output, both the sympathetic and parasympathetic nervous systems use a two-neuron motor pathway, which consists of a preganglionic neuron, whose cell body is located in the CNS, and a postganglionic neuron, whose cell body is located in one of the autonomic ganglia (

Figs. 11.1 11.2 ). The targets of this motor

CHAPTER 11 The Autonomic Nervous System and Its Central Control 227

Projections of Parasympathetic parasympathetic nervous system

Facial (VII) nerve, Glossopharyngeal (IX) nerve • Fig. 11.1 Schematic Illustration of the Sympathetic and Parasympathetic Pathways. Sympathetic pathways are shown in red and parasympathetic pathways in blue. Preganglionic neurons are shown in darker shades, and postganglionic neurons, in lighter shades.

pathway are smooth muscle, cardiac muscle, and glands. The enteric nervous system includes the neurons and nerve fibers in the myenteric and submucosal plexuses, which are located in the wall of the gastrointestinal tract.

Control of the sympathetic and parasympathetic nervous systems of many organs is often antagonistic. To highlight this contrast, the sympathetic and parasympathetic systems are sometimes referred to as the “fight or flight” and the “rest and digest” systems, respectively. Indeed, the fight-orflight response to a threat to the organism reflects an intense activation of the sympathetic nervous system, which leads to a variety of responses, including increased heart rate and blood pressure, redistribution of blood to the muscles, decreased peristalsis and gastrointestinal secretions, pupil dilation, and sweating.

However, under most conditions, the two parts of the autonomic control system work in a coordinated manner—sometimes acting reciprocally and sometimes synergistically—to regulate visceral function. Furthermore, not all visceral structures are innervated by both systems. For example, the smooth muscles and glands in the skin and most of the blood vessels in the body receive sympathetic innervation exclusively; only a small fraction of the blood vessels have parasympathetic innervation. Indeed, the parasympathetic nervous system innervates not the body wall but only structures in the head and in the thoracic, abdominal, and pelvic cavities.

The Sympathetic Nervous System

The sympathetic preganglionic neurons are located in the thoracic and upper lumbar segments of the spinal cord. For this reason, the sympathetic nervous system is sometimes referred to as the thoracolumbar division of the autonomic nervous system. Specifically, sympathetic preganglionic neurons are concentrated in the intermediolateral cell column (lateral horn) in the thoracic and upper lumbar segments of the spinal cord (see

Fig. 11.2). Some neurons may also be found in the C8 segment. In addition to the intermediolateral cell column, groups of sympathetic preganglionic neurons are found in other locations, including the lateral funiculus, the intermediate gray matter, and the gray matter dorsal to the central canal. Sympathetic post-ganglionic neurons are generally found in the paravertebral •Fig. 11.2 Details of the Sympathetic Pathway at a Thoracic Spinal Segment. Autonomicsensoryfibersarerepresentedbyblue lines; sympatheticfibers,byred lines; preganglionicaxons,bysolid lines, andpostganglionicaxons,bydashed lines. (RedrawnfromParentA,CarpenterMB.Carpenter’s Human Neuroanatomy. 9thed.Philadelphia:Williams&Wilkins;1996:295.)White ramus Gray ramus Blood vessel Paravertebral ganglion Prevertebral ganglion Visceral sensory fiber Postganglionic fiber Postganglionic fibers Hair glands Skin Arrector pili muscle Preganglionic fibers Lateral horn Spinal ganglion Peripheral nerve Visceral afferent fiber Digestive tube Sympathetic trunk or prevertebral ganglia. The paravertebral ganglia form two sets of ganglia, each lateral to one side of the vertebral column. The individual ganglia on each side are linked by longitudinally running axons that form a sympathetic trunk (see

Figs. 11.1 11.2 ). Prevertebral ganglia are located in the abdominal cavity and include the Celiac and Superior and Inferior Mesenteric Ganglia (see

Fig. 11.1 ). Thus paravertebral and prevertebral ganglia are located at some distance from their target organs.

The axons of preganglionic neurons are often small, myelinated nerve fibers known as B fibers (see

Table 5.1 ). However, some are unmyelinated C fibers. They leave the spinal cord in the ventral root and enter the paravertebral ganglion at the same segmental level through a white communicating ramus. White rami are found only from the levels of T1 to L2. The preganglionic axon may synapse on postganglionic neurons in the ganglion at its level of entry; may travel rostrally or caudally within the sympathetic trunk and give off collaterals to the ganglia that it passes; or may pass through the ganglion, exit the sympathetic trunk, and enter a splanchnic nerve to travel to a prevertebral ganglion (see

Figs. 11.1 11.2 ). Splanchnic nerves innervate the viscera; they contain both visceral afferents and autonomic motor fibers (sympathetic or parasympathetic).

Postganglionic neurons whose somata lie in paravertebral ganglia generally send their axons through a gray communicating ramus to enter a spinal nerve (see

Fig. 11.2 ). Each of the 31 pairs of spinal nerves has a gray ramus. Postganglionic axons are distributed through the peripheral nerves to effectors, such as piloerector muscles, blood vessels, and sweat glands, located in the skin, muscle, and joints. Postganglionic axons are generally unmyelinated (C fibers), although some exceptions exist. The names white and gray rami reflect the relative contents of myelinated and unmyelinated axons in these rami.

Preganglionic axons in a splanchnic nerve often travel to a prevertebral ganglion and synapse, or they may pass through the ganglion and an autonomic plexus and end in a more distant ganglion. Some preganglionic axons pass through a splanchnic nerve and end directly on cells of the adrenal medulla, which are equivalent to postganglionic cells.

The sympathetic chain extends from the cervical to the coccygeal levels of the spinal cord. This arrangement serves as a distribution system that enables preganglionic neurons, which are limited to the thoracic and upper lumbar segments, to activate postganglionic neurons that innervate all body segments. However, there are fewer paravertebral ganglia than there are spinal segments because some of the segmental ganglia fuse during development. For example, the superior cervical sympathetic ganglion represents the fused ganglia of C1 through C4; the middle cervical sympathetic ganglion is the fused ganglia of C5 and C6; and the inferior cervical sympathetic ganglion is a combination of the ganglia at C7 and C8. The term stellate ganglion refers to fusion of the inferior cervical sympathetic ganglion with the ganglion of T1. The superior cervical sympathetic ganglion provides postganglionic innervation to the head and neck, and the middle cervical and stellate ganglia innervate the heart, lungs, and bronchi.

In general, the sympathetic preganglionic neurons are distributed to ipsilateral ganglia and thus control autonomic function on the same side of the body. Important exceptions are the sympathetic innervation of the intestines and the pelvic viscera, which are both bilateral. As with motor neurons to skeletal muscle, sympathetic preganglionic neurons that control a particular organ are spread over several segments. For example, the sympathetic preganglionic neurons that control sympathetic functions in the head and neck region are distributed at levels C8 to T5, whereas those that control the adrenal gland are distributed at levels T4 to T12.

The Parasympathetic Nervous System

The parasympathetic preganglionic neurons are found in several of the cranial nerve nuclei of brainstem and in the sacral spinal cord (S3-S4) gray matter (see

Fig. 11.1 ). Hence, this part of the autonomic nervous system is sometimes called the craniosacral division. The cranial nerve nuclei that contain parasympathetic preganglionic neurons are the Edinger-Westphal nucleus (cranial nerve III), the superior (cranial nerve VII) and inferior (cranial nerve IX) salivatory nuclei, and the dorsal motor nucleus of the vagus and nucleus ambiguus (cranial nerve X). Postganglionic parasympathetic cells are located in cranial ganglia, including the ciliary ganglion (preganglionic input is from the Edinger-Westphal nucleus), the pterygopalatine and submandibular ganglia (input is from the superior salivatory nucleus), and the otic ganglion (input is from the inferior salivatory nucleus). The ciliary ganglion innervates the pupillary sphincter and ciliary muscles in the eye. The pterygopalatine ganglion supplies the lacrimal gland, as well as glands in the nasal and oral pharynx. The submandibular ganglion projects to the submandibular and sublingual salivary glands and to glands in the oral cavity. The otic ganglion innervates the parotid salivary gland and glands in the mouth.

Other parasympathetic postganglionic neurons are located near or in the walls of visceral organs in the thoracic, abdominal, and pelvic cavities. Neurons of the enteric plexus include cells that can also be considered parasympathetic postganglionic neurons. All of these cells receive input from the vagus or pelvic nerves. The vagus nerves innervate the heart, lungs, bronchi, liver, pancreas, and gastrointestinal tract from the esophagus to the splenic flexure of the colon. The remainder of the colon and rectum, as well as the urinary bladder and reproductive organs, is supplied by sacral parasympathetic preganglionic neurons that travel through the pelvic nerves to postganglionic neurons in the pelvic ganglia.

The parasympathetic preganglionic neurons that project to the viscera of the thorax and part of the abdomen are located in the dorsal motor nucleus of the vagus (see

Fig.

4.6E ) and the nucleus ambiguus. The dorsal motor nucleus is largely secretomotor (it activates glands), whereas the nucleus ambiguus is visceromotor (it modifies the activity of cardiac muscle). The dorsal motor nucleus supplies visceral organs in the neck (pharynx, larynx), thoracic cavity (trachea, bronchi, lungs, heart, and esophagus), and abdominal cavity (including much of the gastrointestinal tract, liver, and pancreas). Electrical stimulation of the dorsal motor nucleus results in gastric acid secretion, as well as secretion of insulin and glucagon by the pancreas. Although projections to the heart have been described, their function is uncertain. The nucleus ambiguus contains two groups of neurons: (1) a dorsal group (branchiomotor) that activates striated muscle in the soft palate, pharynx, larynx, and esophagus and (2) a ventrolateral group that innervates and slows the heart (see also

The visceral motor fibers in the autonomic nerves are accompanied by visceral afferent fibers. Most of these afferent fibers supply information that originates from sensory receptors in the viscera. The activity of these sensory receptors only rarely reaches the level of consciousness; however, these receptors initiate the afferent limb of reflex arcs. Both viscerovisceral and viscerosomatic reflexes are elicited by these afferent fibers. Even though these visceral reflexes generally operate at a subconscious level, they are very important for homeostatic regulation and adjustment to external stimuli.

The fast-acting neurotransmitters released by visceral afferent fibers are not well documented, although many of these neurons release an excitatory amino acid transmitter such as glutamate. However, visceral afferent fibers also contain many neuropeptides or combinations of neuropeptides, including angiotensin II, arginine vasopressin, bombesin, calcitonin gene–related peptide, cholecystokinin, galanin, substance P, enkephalin, oxytocin, somatostatin, and vasoactive intestinal polypeptide.

Visceral afferent fibers that can mediate conscious sensation include nociceptors that travel in sympathetic nerves, such as the splanchnic nerves. Visceral pain is caused by excessive distention of hollow viscera, contraction against an obstruction, or ischemia. The origin of visceral pain is often difficult to identify because of the diffuse nature of the pain and its tendency to be referred to somatic structures (see ). Visceral nociceptors in sympathetic nerves reach the spinal cord via the sympathetic chain, white rami, and dorsal roots. The terminals of nociceptive afferent fibers project to the dorsal horn and to the region surrounding the central canal. They activate not only local interneurons, which participate in reflex arcs, but also projection cells, which include spinothalamic tract cells that signal pain to the brain.

A major visceral nociceptive pathway from the pelvis involves a relay in the gray matter of the lumbosacral spinal cord. These neurons send axons into the fasciculus gracilis that terminate in the nucleus gracilis. Thus the dorsal columns not only contain primary afferents for somatic sensation (their main component) but also second-order neurons of the visceral pain pathway (recall that second-order axons for somatic pain travel in the lateral funiculus as part of the spinothalamic tract). Visceral nociceptive signals are then transmitted to the ventral posterior lateral nucleus of the thalamus and presumably from there to the cerebral cortex. Interruption of this pathway accounts for the beneficial effects of surgically induced lesions of the dorsal column at lower thoracic levels to relieve pain produced by cancer of the pelvic organs.

Other visceral afferent fibers travel in parasympathetic nerves. These fibers are generally involved in reflexes rather than sensation (except for taste afferent fibers; see ). For example, the baroreceptor afferent fibers that innervate the carotid sinus are in the glossopharyngeal nerve. They enter the brainstem, pass through the solitary tract, and terminate in the nucleus of the solitary tract (see

Fig. 4.6E ). These neurons connect with interneurons in the brainstem reticular formation. The interneurons, in turn, project to the autonomic preganglionic neurons that control heart rate and blood pressure (see

The nucleus of the solitary tract receives information from all visceral organs, except those in the pelvis. This nucleus is subdivided into several areas that receive information from specific visceral organs.

The Enteric Nervous System

The enteric nervous system, which is located in the wall of the gastrointestinal tract, contains about 100 million neurons. The enteric nervous system is subdivided into the myenteric plexus, which lies between the longitudinal and circular muscle layers of the gut, and the submucosal plexus, which lies in the submucosa of the gut. The neurons of the myenteric plexus primarily control gastrointestinal motility (see ), whereas those in the submucosal plexus primarily regulate body fluid homeostasis (see

The types of neurons found in the myenteric plexus include not only excitatory and inhibitory motor neurons (which can be considered parasympathetic postganglionic neurons) but also interneurons and primary afferent neurons. Afferent neurons supply mechanoreceptors within the wall of the gastrointestinal tract. These mechanoreceptors are the beginning of the afferent limb of reflex arcs within the enteric plexus. Local excitatory and inhibitory interneurons participate in these reflexes, and the output is sent through the motor neurons to smooth muscle cells. Excitatory motor neurons release acetylcholine and substance P; inhibitory motor neurons release dynorphin and vasoactive intestinal polypeptide. The circuitry of the enteric plexus is so extensive that it can coordinate the movements of an intestine that has been completely removed from the body. However, normal function requires innervation by the autonomic preganglionic neurons and regulation by the CNS.

Activity in the enteric nervous system is modulated by the sympathetic nervous system. Sympathetic post-ganglionic neurons that contain norepinephrine inhibit intestinal motility, those that contain norepinephrine and neuropeptide Y regulate blood flow, and those that contain norepinephrine and somatostatin control intestinal secretion. Feedback is provided by intestinofugal neurons that project back from the myenteric plexus to the sympathetic ganglia.

The submucosal plexus regulates ion and water transport across the intestinal epithelium and glandular secretion. It also communicates with the myenteric plexus to ensure coordination of the functions of the two components of the enteric nervous system. The neurons and neural circuits of the submucosal plexus are not as well understood as those of the myenteric plexus, but many of the neurons contain neuropeptides, and the neural networks are well organized.

The main type of neuron in autonomic ganglia is the post-ganglionic neuron. These cells receive synaptic connections from preganglionic neurons, and they project to autonomic effector cells. However, many autonomic ganglia also contain interneurons. These interneurons process information within the autonomic ganglia; the enteric plexus can be regarded as an elaborate example of this kind of processing. One type of interneuron found in some autonomic ganglia contains a high concentration of catecholamines; hence, these interneurons have been called small, intensely fluorescent (SIF) cells. SIF cells are believed to be inhibitory.

The classic neurotransmitter of autonomic ganglia, whether sympathetic or parasympathetic, is acetylcholine. The two classes of acetylcholine receptors in autonomic ganglia are nicotinic and muscarinic receptors, so named because of their responses to the plant alkaloids nicotine and muscarine. Nicotinic acetylcholine receptors can be blocked by such agents as curare or hexamethonium, and muscarinic receptors can be blocked by atropine. Nicotinic receptors in autonomic ganglia differ somewhat from those on skeletal muscle cells.

Nicotinic and muscarinic receptors both mediate excitatory postsynaptic potentials (EPSPs), but these potentials have different time courses. Stimulation of preganglionic neurons elicits a fast EPSP, followed by a slow EPSP. The fast EPSP results from activation of nicotinic receptors, which cause ion channels to open. The slow EPSP is mediated by muscarinic receptors (primarily the M2 receptor; see ) that inhibit the M current, a current produced by potassium conductance.

Neurons in autonomic ganglia also release neuropeptides that act as neuromodulators. Besides acetylcholine, sympathetic preganglionic neurons may release enkephalin, substance P, luteinizing hormone–releasing hormone, neurotensin, or somatostatin.

Catecholamines such as norepinephrine and dopamine serve as the neurotransmitters of SIF cells in autonomic ganglia.

Sympathetic postganglionic neurons typically release nor-epinephrine, which excites some effector cells but inhibits others. The receptors on target cells may be either α-or β-adrenergic receptors. These receptors are further subdivided into α1, α2, β1, β2, and β3 receptor types on the basis of pharmacological and genetic features. The distribution of these types of receptors and the actions that they mediate when activated by sympathetic postganglionic neurons are listed for various target organs in

Table 11.1 α1 receptors are located postsynaptically, but α2 receptors may be either presynaptic or postsynaptic. Receptors located presynaptically are generally called autoreceptors; they usually inhibit release of transmitter. The effects of agents that excite α1 or α2 receptors can be distinguished through the use of antagonists to block these receptors specifically. For example, prazosin is a selective α1-adrenergic antagonist, and yohimbine is a selective α2-adrenergic antagonist. The effects of α1 receptors are mediated by activation of the inositol triphosphate/diacylglycerol second messenger system (see

Chapter 3).

In contrast, α2 receptors decrease the rate of synthesis of cyclic adenosine monophosphate (cAMP) through action on a G protein.

β receptors were originally classified on the basis of the ability of antagonists to block them, but this has been supplemented by genetic studies. The β1 and β2 proteins have been much more extensively studied than has β3, but it is thought that the proteins that make up all three types of β receptors are similar, with seven membrane-spanning regions connected by intracellular and extracellular domains (see ). Agonist drugs that work on β receptors activate a G protein that stimulates adenylyl cyclase to increase the cAMP concentration. This action is terminated by the buildup of guanosine diphosphate.

Radialmuscle,irisα Contraction(mydriasis)++ —Sphinctermuscle,irisα —Contraction(miosis)+++ Ciliarymuscleβ Relaxationforfarvision+ Contractionfornearvision+++

Sinoatrialnodeβ1Increaseinheartrate++ Decreaseinheartrate;vagalarrest+++ Atriaβ1IncreaseincontractilityandconductionDecreaseincontractilityand(usually)velocity++ increaseinconductionvelocity++ Atrioventricular(AV)nodeβ1IncreaseinautomaticityandDecreaseinconductionvelocity;AVblockconductionvelocity++ +++ His-Purkinjesystemβ1IncreaseinautomaticityandLittleeffectconductionvelocity+++ Ventriclesβ1Increaseincontractility,conductionSlightdecreaseincontractilityvelocity,automaticity,andrateofidioventricularpacemakers+++

Coronaryα,β2Constriction+;dilation

Skeletalmuscleα,β2Constriction++;dilation c,e

Pulmonaryα,β2Constriction+;dilation

Abdominalviscera,renalα,β2Constriction+++;dilation + —Salivaryglandsα Constriction+++ Dilation++ Veins (Systemic) α,β2Constriction++;dilation++ —

Responses of Effector Organs to Autonomic Nerve Impulses—cont’d 11.1

Effector Organs Receptor Type Adrenergic Impulses,a Responsesb Cholinergic Impulses,a Responsesb

Bronchialmuscleβ2Relaxation+ Contraction++ Bronchialglands?Inhibition(?)Stimulation+++

Motilityandtoneα2,β2Decrease(usually) + Increase+++ Sphinctersα Contraction(usually)+ Relaxation(usually)+ SecretionInhibition(?)Stimulation+++

Motilityandtoneα2,β2Decrease + Increase+++ Sphinctersα Contraction(usually)+ Relaxation(usually)+ SecretionInhibition(?)Stimulation+++ Gallbladder and Ducts Relaxation+ Contraction+

Motilityandtoneα Increase(usually)Increase(?)Uterus α,β2Pregnant:contraction(α);Variable nonpregnant:relaxation(β)Sex Organs, Male α Ejaculation+++ Erection+++ + Generalizedsecretion+++ Spleen Capsule α,β2Contraction+++;relaxation+ —

Adrenal Medulla —SecretionofepinephrineandnorepinephrineLiver α,β2Glycogenolysis,gluconeogenesis β2Increasedsecretion+ —Fatcellsα,β1Lipolysis

FromGoodmanLS,GilmanA.The Pharmacological Basis of Therapeutics. 6thed.NewYork:Macmillan;1980.aAlongdash(—)signifiesnoknownfunctionalinnervation.bResponsesaredesignated+ to+++ toprovideanapproximateindicationoftheimportanceofadrenergicandcholinergicnerveactivityincontrolofthevariousorgansandfunctionslisted.cDilationpredominatesinsitubecauseofmetabolicautoregulatoryphenomena.dCholinergicvasodilationatthesesitesisofquestionablephysiologicalsignificance.eOvertheusualconcentrationrangeofphysiologicallyreleasedcirculatingepinephrine,aβ receptorresponse(vasodilation)predominatesinbloodvesselsofskeletalmuscleandtheliver,andanα receptorresponse(vasoconstriction)predominatesinbloodvesselsofotherabdominalviscera.Therenalandmesentericvesselsalsocontainspecificdopaminergicreceptors,activationofwhichcausesdilation,buttheirphysiologicalsignificancehasnotbeenestablished.fThesympatheticcholinergicsystemcausesvasodilationinskeletalmuscle,butthisisnotinvolvedinmostphysiologicalresponses.gIthasbeenproposedthatadrenergicfibersterminateatinhibitoryβ receptorsonsmoothmusclefibersandatinhibitoryα receptorsonparasympatheticcholinergic(excitatory)ganglioncellsoftheAuerbachplexus.hDependsonthestageofthemenstrualcycle,theamountofcirculatingestrogenandprogesterone,andotherfactors.iPalmsofthehandsandsomeothersites(“adrenergicsweating”).jThereissignificantvariationamongspeciesinthetypeofreceptorthatmediatescertainmetabolicresponses.

β receptor activity is controlled in a number of ways. It can be antagonized by the action of α1 receptors. The β receptors can also be desensitized by phosphorylation with prolonged exposure to agonists. Regulation of β receptor numbers represents a third control mechanism. For example, β receptor numbers can be decreased by being internalized. Alternatively, β receptor numbers can be increased (upregulated) in certain circumstances: for example, after denervation. Note that the number of α receptors is likewise regulated.

In addition to releasing norepinephrine, sympathetic postganglionic neurons release neuropeptides such as somatostatin and neuropeptide Y. For example, cells that release both norepinephrine and somatostatin supply the mucosa of the gastrointestinal tract, and cells that release both norepinephrine and neuropeptide Y innervate blood vessels in the gut and the limb. Another chemical mediator in sympathetic postganglionic neurons is adenosine triphosphate (ATP).

The endocrine cells of the adrenal medulla are similar in many ways to sympathetic postganglionic neurons (see also ). They receive input from sympathetic preganglionic neurons, are excited by acetylcholine, and release catecholamines. However, the cells of the adrenal medulla differ from sympathetic postganglionic neurons in that they release catecholamines into the circulation rather than into a synapse. Moreover, the main catecholamine released is epinephrine, not norepinephrine. In humans, 80% of the catecholamine released by the adrenal medulla is epinephrine, and 20% is norepinephrine.

Some sympathetic postganglionic neurons release acetylcholine rather than norepinephrine as their neurotransmitter. For example, sympathetic postganglionic neurons that innervate eccrine sweat glands are cholinergic. The acetylcholine receptors involved are muscarinic, and they are therefore blocked by atropine. Similarly, some blood vessels are innervated by cholinergic sympathetic postganglionic neurons. In addition to releasing acetylcholine, the postganglionic neurons that supply the sweat glands also release neuropeptides, including calcitonin gene–related peptide and vasoactive intestinal polypeptide.

The neurotransmitter released by parasympathetic postganglionic neurons is acetylcholine. The effects of these neurons on various target organs are listed in

Table 11.1 . Parasympathetic postganglionic actions are mediated by muscarinic receptors. On the basis of binding studies, the action of selective antagonists, and molecular cloning, five types of muscarinic receptors have been identified (see

Chapter 6).

Activation of M1 receptors enhances the secretion of gastric acid in the stomach. M2 receptors are the most abundant receptor type in smooth muscle, including smooth muscle in the intestines, uterus, trachea, and bladder. In addition, they are present in autonomic ganglia and in the heart, where they exert negative chronotropic and inotropic actions (see

Chapter 18). M 3 receptors are also present in the smooth muscle of a variety of organs, and although they are less abundant than M2 receptors, normal contractile patterns appear to require an interaction between the two types of receptors. M4 receptors, like M2 receptors, are present in autonomic ganglia and thus play a role in synaptic transmission at these sites. M5 receptors are present in the sphincter muscle of the pupil, in the esophagus, and in the parotid gland, as well as in cerebral blood vessels.

Muscarinic receptors, like adrenergic receptors, have diverse actions. Some of their effects are mediated by specific second messenger systems. For example, cardiac M2 muscarinic receptors may act by way of the inositol triphosphate system, and they may also inhibit adenylyl cyclase and thus cAMP synthesis. Muscarinic receptors also open or close ion channels, particularly K+ or Ca++ channels. This action on ion channels is likely to occur through activation of G proteins. A third action of muscarinic receptors is to relax vascular smooth muscle by an effect on endothelial cells, which produce endothelium-derived relaxing factor (EDRF). EDRF is actually nitric oxide, a gas released when arginine is converted to citrulline by nitric oxide synthase (see ). Nitric oxide relaxes vascular smooth muscle by stimulating guanylate cyclase and thereby increasing levels of cyclic guanosine monophosphate (cGMP), which in turn activates a cGMP-dependent protein kinase (see ). The number of muscarinic receptors is regulated, and exposure to muscarinic agonists decreases the number of receptors by internalization of the receptors.

Chagas disease istheresultofinfectionbytheparasiteTrypanosoma cruzi. About18millionpeopleareinfectedworldwide,andapproximately50,000dieeachyearasaresultofcomplicationsfromthedisease.Themostseriousformsinvolveenlargementoftheesophagus,colon,andheart.Lossofparasympatheticcontrolisasignificantcomponentoftheinitialstagesofthedisease;shortlyaftertheinitialinfection,theparasympatheticneuronsinnervatingtheheart,esophagus,andcolonaredestroyed,whichleadstoarrhythmias(andpotentiallysuddendeath)andaperistalsis.Chroniccardiomyopathy(malfunctionoftheheartmuscle)thatcanleadtodeathoccursinapproximately30%ofthoseinfected.Althoughthepathogenesisofthecardiomyopathyisnotfullyunderstood,oneleadingideainvolvesautoimmunity.Antibodiesagainsttheparasiticantigenshavebeenfoundtobindtotheβ-adrenergicandM2acetylcholinereceptorsintheheart.Theseantibodiesnotonlytriggerautoimmuneresponsesthatdestroyheartmusclebutalsoactasagonistsatthesereceptorsandcauseinappropriateresponsesofthecardiovascularsystemtochangingexternaldemands.

Central Control of Autonomic Function

The discharges of autonomic preganglionic neurons are controlled by pathways that synapse on autonomic preganglionic neurons. The pathways that influence autonomic activity include spinal cord and brainstem reflex pathways, as well as descending control systems originating at higher levels of the nervous system, such as the hypothalamus.

Examples of Autonomic Control of Particular Organs

The dilator and constrictor muscles of the iris, which are under the control of sympathetic and parasympathetic fibers, respectively, determine the size of the pupil. Activation of sympathetic innervation of the eye, via thoracic white rami and sympathetic trunk ganglia, dilates the pupil, which occurs during emotional excitement and also in response to painful stimulation. The neurotransmitter at the sympathetic postganglionic synapses is norepinephrine, and it acts at α receptors.

The parasympathetic nervous system exerts an action on pupillary size opposite that of the sympathetic nervous system. Whereas the sympathetic system elicits pupillary dilation, the parasympathetic system constricts the pupil. The preganglionic parasympathetic nerves that innervate the pupillary constrictor are in the Edinger-Westphal nucleus, which is in the midbrain, and travel in cranial nerve III, and so damage to this nerve can lead to a dilated pupil (mydriasis).

Sympatheticcontrolofthepupilissometimesaffectedbydisease.Forexample,interruptionofthesympatheticinnervationoftheheadandneckresultsinHorner’s syndrome. Thissyndromeischaracterizedbythetriadofmiosis(abnormalpupillaryconstriction),ptosis(causedbyparalysisofthesuperiortarsalmuscle),andanhydrosis(lossofsweating)ontheface.Enophthalmos (retractionoftheeyeintotheorbit)alsooccursinsomeanimals(rats,cats,anddogs,amongothers),butinhumansnotrueenophthalmosoccurs;however,thereisanapparentenophthalmos,anillusioncreatedbypartialclosureoftheeyelidfromtheptosis.Horner’ssyndromecanbeproducedbyalesionthat(1)destroysthesympatheticpreganglionicneuronsintheupperthoracicspinalcord,(2)interruptsthecervicalsympatheticchain,or(3)damagesthelowerbrainstemintheregionofthereticularformation,throughwhichpathwaysdescendtothespinalcordtoactivatesympatheticpreganglionicneurons.Inthelastcase,thereisalsoalossofsweatingonthesideofthebodyipsilateraltothelesion.

Pupil size is reduced by the pupillary light reflex and during accommodation for near vision. In the pupillary light reflex, light that strikes the retina is processed by retinal circuits that excite W-type retinal ganglion cells (see

Chapter 8). These cells respond to diffuse illumination. The axons of some of the W-type cells project through the optic nerve and tract to the pretectal area, where they synapse in the olivary pretectal nucleus. This nucleus contains neurons that also respond to diffuse illumination. Activity of neurons of the olivary pretectal nucleus causes pupillary constriction by means of bilateral connections with parasympathetic preganglionic neurons in the Edinger-Westphal nuclei. The reflex results in contraction of the pupillary sphincter muscles in both eyes, even when light is shone into only one eye.

The accommodation response, which is important for focusing on near objects, involves pupillary constriction, increasing the curvature of the lens, and convergence of the eyes. This response is triggered by information from M cells of the retina that is transmitted to the striate cortex through the geniculostriate visual pathway (see ). The specific stimuli that trigger accommodation are thought to be a blurred retinal image and disparity of the image between the two eyes. After the information is processed in the visual cortex, signals are transmitted directly or indirectly to the middle temporal cortex, where they activate neurons in a visual area known as MT. Area MT neurons transmit signals to the midbrain that activate parasympathetic preganglionic neurons in the Edinger-Westphal nuclei, which results in pupillary constriction. At the same time, signals are transmitted to the ciliary muscle that cause it to contract. The ciliary muscle contraction allows the lens to round up and increase its refractile power. (Convergence is a somatic response mediated by neurons in the oculomotor [cranial nerve III] nucleus of the midbrain.)

Thepupillarylightreflexissometimesabsentinpatientswithtertiary(advanced)syphilis,whichaffectstheCNS(i.e.,intheformoftabesdorsalis).Althoughthepupilfailstorespondtolight,ithasanormalaccommodationresponse.ThisconditionisknownastheArgyll Robertson pupil. Theexactmechanismiscontroversial.Oneexplanationrestsonthefactthatsomeoptictractfibersprojecttothepretectalareainthemidbrain.Thesefiberscanbedamagedinsyphiliticmeningitis,possiblybythepresenceofspirochetesinthesubarachnoidspace.NotethatthepretectalareaprojectstotheEdinger-Westphalnucleus,alsointhemidbrain,whosecellsoriginatetheparasympatheticinnervationoftheeye,whichcontrolsthepupillarysphinctermuscle.Althoughinputtotheolivarypretectalnucleusisinterrupted,theoptictractfibersprojectingtothelateralgeniculatenucleusarenotdestroyed,andthusvisionismaintained,asispupillaryconstrictionduringaccommodation.

The urinary bladder is controlled by reflex pathways in the spinal cord and also by a supraspinal center (

Fig. 11.3 ). The sympathetic innervation originates from preganglionic sympathetic neurons in the upper lumbar segments of the spinal cord. Postganglionic sympathetic axons act to inhibit the smooth muscle (detrusor muscle) throughout the body of the bladder, and they also act to excite the smooth muscle of the trigone region and the internal urethral sphincter.

•Fig. 11.3 Illustration of Descending and Efferent Pathways for Reflexes That Control the Urinary Bladder. Forclarity,onlysomeofthemajorinvolvedpathwaysareshown.(RedrawnfromdeGroatWC,BoothAM.InDyckPJ,etal[eds].Peripheral Neuropathy. 2nded.Philadelphia:WBSaunders;1984.)

The detrusor muscle is tonically inhibited during filling of the bladder, and such inhibition prevents urine from being voided. Inhibition of the detrusor muscle is mediated by the action of norepinephrine on β receptors, whereas excitation of the trigone and internal urethral sphincter is elicited by the action of norepinephrine on α receptors.

The external sphincter of the urethra also helps control voiding. This sphincter is a striated muscle, and it is innervated by motor axons in the pudendal nerves, which are somatic nerves. The motor neurons are located in the Onuf nucleus, in the ventral horn of the sacral spinal cord.

The parasympathetic preganglionic neurons that control the bladder are located in the sacral spinal cord (the S2 and S3 or S3 and S4 segments). These cholinergic neurons project through the pelvic nerves and are distributed to ganglia in the pelvic plexus and the bladder wall. Post-ganglionic parasympathetic neurons in the bladder wall innervate the detrusor muscle, as well as the trigone and sphincter. The parasympathetic activity contracts the detrusor muscle and relaxes the trigone and internal sphincter. These actions result in micturition, or urination. Some of the postganglionic neurons are cholinergic and others are purinergic (they release ATP).

Micturition is normally controlled by the micturition reflex (see

Fig. 11.3 ). Mechanoreceptors in the bladder wall are excited by both stretch and contraction of the muscles in the bladder wall. Thus as urine accumulates and distends the bladder, the mechanoreceptor afferents begin to discharge. The pressure in the urinary bladder is low during filling (5 to 10 cm H2O), but it increases abruptly when micturition begins. Micturition can be triggered either reflexively or voluntarily. In reflex micturition, bladder afferent fibers excite neurons that project to the brainstem and activate the micturition center in the rostral pons (Barrington’s nucleus). The descending projections also inhibit sympathetic preganglionic neurons that prevent voiding. When a sufficient level of activity occurs in this ascending pathway, micturition is triggered by the micturition center. Commands reach the sacral spinal cord through a reticulospinal pathway. Activity in the sympathetic projection to the bladder is inhibited, and the parasympathetic projections to the bladder are activated. Contraction of muscle in the wall of the bladder causes a vigorous discharge of the mechanoreceptors that supply the bladder wall and thereby further activates the supraspinal loop. The result is complete emptying of the bladder.

A spinal reflex pathway also exists for micturition. This pathway is operational in newborn infants. However, with maturation, the supraspinal control pathways take on a dominant role in triggering micturition. After spinal cord injury, human adults lose bladder control during the period of spinal shock (urinary incontinence). As the spinal cord recovers from spinal shock, some degree of bladder function is recovered because of enhancement of the spinal cord micturition reflex. However, the bladder has increased muscle tone and fails to empty completely. These circumstances frequently lead to urinary infections.

Autonomic Centers in the Brain

Influence over autonomic output is maintained by autonomic centers, which consist of local networks of neurons, in a variety of brain regions. The micturition center in the pons, which was just discussed, is one example. Many other autonomic centers with diverse functions exist. Vasomotor and vasodilator centers are in the medulla, and respiratory centers are in the medulla and pons. Perhaps the greatest concentration of autonomic centers is found in the hypothalamus.

The Hypothalamus and Preoptic Area

The hypothalamus is part of the diencephalon. Some of the nuclei of the hypothalamus are shown in

Fig. 11.4 . Located anteriorly from the hypothalamus are telencephalic structures: the preoptic region and septum, both of which help regulate autonomic function. Important fiber tracts that course through the hypothalamus are the fornix, the medial forebrain bundle, and the mammillothalamic tract. The fornix is used as a landmark to divide the hypothalamus into medial and lateral zones.

The hypothalamus has many functions; see for a discussion of hypothalamic control of endocrine function. Its control of autonomic function is emphasized here. In its control of autonomic function, the hypothalamus functions much like a control system that is termed, in •Fig. 11.4 Illustration of Main Nuclei of the Hypothalamus, Viewed From the Third Ventricle. Anterioristotheright.(RedrawnfromNautaWJH,HaymakerW.The Hypothalamus. Springfield,IL:CharlesCThomas;1969.) engineering, a servomechanism: that is, a system in which a particular physiological parameter is controlled through the use of negative feedback loops to maintain the parameter at a particular set point or value. The following examples illustrate this principle for body temperature, body weight and adiposity, and water intake.

Homeothermic animals maintain a relatively constant core body temperature in situations of fluctuating environmental temperatures and differing levels of bodily activity that cause endogenous heat production. This ability rests on information from three main groups of thermoreceptors located in the skin, CNS, and viscera.

Information about the external temperature is provided by thermoreceptors in the skin. Core body temperature is monitored by central thermoreceptive neurons in the preoptic area (and possibly the spinal cord), which monitor the temperature of local blood. Thermoreceptors in the viscera monitor the temperature in these organs. All of these receptors provide temperature information to the preoptic area (pathways described later), along with parts of the hypothalamus, in which this information is used to keep core body temperature constant. Thus the preoptic area and hypothalamus act together as a servomechanism with a set point at the normal body temperature.

Although the signals from each of these sources are integrated, their relative importance may shift, depending on the situation. Changes in environmental temperature evoke more rapid and much larger changes in the temperature of the skin than in the body core, and so cutaneous receptors are probably the initial and most often used mechanism for compensating for external changes in temperature. Central thermoreceptors are more important for situations with internal causes of temperature change, such as during exercise, or in which external changes temperature are so severe or prolonged that core body temperature starts to change despite the signals from peripheral thermoreceptors. Last, alteration of body temperature by ingestion of hot or cold food or liquids is detected by the visceral thermoreceptors.

Error signals (i.e., cooling and warming of the body), which represent a deviation from the set point of the servomechanism, evoke responses that tend to restore body temperature toward the set point. These responses are mediated by the autonomic, somatic, and endocrine systems.

Situations involving cooling, for example, trigger a variety of responses that increase heat production (thermogenesis) and minimize heat loss. Heat production is increased by mechanisms that include shivering thermogenesis (asynchronous contractions of skeletal muscle that increases heat production) and brown adipose tissue (BAT) thermogenesis (in BAT thermogenesis, oxidative phosphorylation is uncoupled from ATP synthesis, which allows the energy released by the reaction to be dissipated as heat instead), and increased thyroid hormone levels lead to increased metabolism. Heat loss is reduced by cutaneous vasoconstriction and by piloerection. Piloerection is effective in animals with fur but not in humans; in the latter, the result is only goose bumps. In addition, tachycardia occurs, which may help provide metabolites to be used in thermogenesis to the thermogenic tissues (fat and muscle) and help distribute the heat generated throughout the body. Finally, the perception of being cold influences the decision to initiate voluntary behaviors: in this case, possibly putting on a jacket.

Warming the body generally causes changes in the opposite direction. The activity of the thyroid gland diminishes, which leads to reduced metabolic activity and less heat production. Heat loss is increased by sweating, salivation (in some animals but not humans), and cutaneous vasodilation (because of decreased sympathetic activity). However, again tachycardia occurs, this time presumably to allow optimal perfusion of the cutaneous circulation for heat dissipation.

Early studies identified the preoptic region and anterior hypothalamus as a heat loss center and the posterior hypothalamus as a heat conservation center. For example, lesions in the preoptic region prevent sweating and cutaneous vasodilation, and if an individual with a lesion in this region is placed in a warm environment, hyperthermia occurs. Conversely, electrical stimulation of the heat loss center causes cutaneous vasodilation and inhibits shivering. In contrast, lesions in the area dorsolateral to the mammillary body interfere with heat production and conservation and can cause hypothermia when the person is in a cold environment. Electrical stimulation in this region of the brain evokes shivering.

Many details of the circuitry and physiologic processes underlying the temperature regulation responses are now known, and they indicate that the preoptic area and dorsal medial hypothalamic nucleus are key components in the regulation of body temperature. The preoptic area in particular appears to be the target of the various sources of sensory information. Cutaneous temperature information is conveyed by thermosensitive primary afferents that synapse in the dorsal horn of the spinal cord onto neurons that project up to and excite the parabrachial nucleus in the caudal midbrain. Information from visceral afferents is relayed by the solitary nucleus to the parabrachial nucleus as well. The parabrachial neurons in turn excite neurons within a specific part of the preoptic area, the median nucleus (of the preoptic area). Many median nucleus neurons are also sensitive to local changes in blood temperature and contain prostaglandin E2 (PGE2) receptor 3 (EP3), which mediates fever responses (see the following “In the Clinic” box).

Thus the median preoptic nucleus is a key component of the thermoregulatory control system in which information from the various types of thermoreceptors is integrated. Output from this nucleus is directed to the neighboring medial preoptic area, which projects to regions of the rostral medulla, both directly and via the dorsal medial hypothalamic nucleus. The rostral medulla has spinally projecting neurons that project to the lateral horn of the spinal cord, where the preganglionic sympathetic neurons are located, and the activity of these neurons regulates BAT thermogenesis and modulates cutaneous vasomotor tone. The rostral medulla also projects to the ventral horn of the spinal cord, which contains the somatic motor neurons that contract skeletal muscle and thus mediate the shivering response.

Fever,whichaccompaniessomeinfections,canbethoughtofasanelevationofthesetpointforbodytemperature.Thiselevationcanbecausedbythereleaseofpyrogens bymicroorganismsorbycellsmediatingtheinflammatoryresponse.Thepyrogen’seffecttoraisethesetpointismediatedprimarilybytheactionofprostaglandinPGE2’sbindingtoEP3receptorsonneuronsinthepreopticarea.PGE2isreleasedbyperipheraltissuesandbybloodvesselssupplyingthepreopticarea.ThebindingofPGE2toEP3receptorscausesareductionintheactivityofpreopticneurons.ThisreductioninneuronalactivityleadstoincreasedheatproductionthroughshiveringandBATthermogenesisandtoheatconservationbycutaneousvasoconstriction,thecombinedeffectofwhichistoraisebodytemperature.EvidenceofthismechanismoffeverproductionincludesstudiesinwhichinjectionofPGE2intothepreopticareainducedfeverandothersinwhichselectivedeletionoftheEP3receptorfrompreopticneuronsabolishedtheabilityofPGE2injectionstoinducefever.

Regulation of Feeding and Body Weight

Energy homeostasis is crucial for the survival of the animal. The challenge is that most cells need a continuous supply of nutrients to function, but most animals do not constantly eat; instead, they have periodic meals. Thus to achieve energy homeostasis, feeding behavior is controlled by many factors, which operate both on a short-term basis to control ingestion and on a long-term basis to control body weight in order to ensure sufficient energy stores. Both hedonic and homeostatic factors are involved; however, in this chapter, the focus is on the latter because of the central role that the hypothalamus plays in energy homeostasis.

In the short run, eating is controlled by a number of mechanisms. First, the stomach wall has stretch receptors that signal distention as food fills the stomach. These signals are conveyed by the afferents of the vagus nerve to the solitary nucleus in the medulla. From there the information is relayed to several brain areas, including the hypothalamus, either directly or via a relay in the parabrachial nucleus, to organize autonomic responses to the ingested material, and the thalamus and cortex, for conscious awareness of the fullness of the stomach. In the hypothalamus, the paraventricular, dorsomedial, and arcuate nuclei and the lateral hypothalamus are the major targets of these signals.

Sensory afferents also sense the concentrations of glucose and lipids in the intestines and the hepatic portal circulation and send this information to the solitary nucleus and, from there, to the hypothalamus, in a manner similar to that described for the stretch receptors. In addition, the stomach and gut release a number of hormones in response to feeding, including cholecystokinin, peptide YY, glucagonlike peptide-1 (GLP-1), and ghrelin. Hypothalamic cells have receptors for many of these hormones and can be influenced directly by them. In addition, cells in other brain areas have receptors for these hormones and thus may provide an indirect pathway to the hypothalamus. One such region is the area postrema, which is just dorsal to the solitary nucleus and projects to it. The area postrema is not protected by the blood-brain barrier (it is one of the circumventricular organs), and its neurons respond to cholecystokinin and GLP-1, which leads to decreased food intake.

Control of body weight over the long run is influenced by many factors and involves the interaction of the nervous and endocrine systems. In this section, the focus is on the role of hypothalamus and its control of the autonomic nervous system, which provides another example of how the hypothalamus is part of a servomechanism. In this case, adiposity is the controlled parameter. For further details on the endocrine system’s role, see

Early studies in which researchers used lesions and electrical stimulation provided evidence that the ventromedial and ventrolateral hypothalamus are involved in energy homeostasis. A lesion in the ventromedial region causes an increase in food intake (hyperphagia) that results in obesity, whereas electrical stimulation of the same region decreases feeding behavior. These lesions were also shown to alter autonomic activity, increasing parasympathetic and decreasing sympathetic tone, both of which lead to high blood insulin levels, which in turn promote energy conservation and storage (see ). These observations led to the idea that the ventromedial hypothalamus contains a satiety center. However, an alternative interpretation is that the primary controlled variable may not be simply eating behavior per se but rather body weight and, even more specifically, body fat levels (i.e., adiposity). Thus modulation of feeding behavior may be just one of several actions used to defend a body weight set point. Evidence for this is that although lesions cause an initial period of dynamic weight gain in which hyperphagia is present, this is followed by a static period in which the higher weight is maintained without hyperphagia. Moreover, animals with a lesion in the ventromedial hypothalamus that are fed a fixed (normal) amount of food to prevent hyperphagia nonetheless become obese, which implies changes in the regulation of other metabolic processes. Last, lesions of the ventromedial hypothalamus have been shown to alter levels of energy expenditure.

In contrast to lesions of the ventromedial hypothalamus, those of the lateral hypothalamus suppress food intake (hypophagia) and lead to a decrease in body weight; indeed, animals can starve to death after such lesions. Conversely, electrical stimulation of the medial forebrain bundle in the lateral hypothalamus evokes exploratory behavior and eating, if food is present. This stimulation also provides a dopamine-dependent reward that mediates the incentive effects of natural rewards (food, sex) as well as the rewarding effects of most drugs of abuse. These observations led to the view that the lateral hypothalamus contains a feeding center. This interpretation, however, is complicated by the fact that the dopaminergic axons of substantia nigra neurons pass just lateral to the lateral hypothalamus on their way to the striatum, and so loss or stimulation of these fibers could account for the effects produced in these experiments. However, lateral hypothalamic neurons have been found to synthesize peptides, such as orexin, that affect feeding behavior, and so the lateral hypothalamus probably does play a role in energy homeostasis.

In newer studies, investigators have identified a number of hormones and neuropeptides involved in feeding and control of body weight, and many of the interactions between the endocrine and nervous systems that underlie energy homeostasis have been clarified.

In normal individuals, blood insulin levels are correlated with adiposity (in addition to varying acutely with blood levels of glucose and other substances). Similarly, the level of the protein leptin, a hormone released by adipocytes (primarily those forming white adipose tissue), is correlated with adiposity. High levels of leptin inhibit food intake and stimulate catabolic processes, including loss of fat tissue, whereas low leptin levels trigger the reverse actions. Similarly, high insulin levels promote energy storage processes.

The ability of leptin and insulin to regulate body weight has been linked to their actions on the hypothalamus, particularly the arcuate nucleus, whose neurons express receptors for both hormones (see also ). Two major classes of arcuate nucleus neurons that respond to leptin and insulin have been identified. Neurons that express proopiomelanocortin (POMC) and cocaine-and amphetamine-related transcript (CART) are stimulated by leptin and insulin, and their activity leads to increased catabolism. In contrast, the activity of a second group of neurons, those that express neuropeptide Y (NPY) and agouti-related peptide (AgRP), triggers anabolic processes but is inhibited by leptin and insulin. Thus increased body fat levels lead to high leptin and insulin levels, which in turn both (1) increase the activity of POMCand CART-expressing neurons, leading to increased catabolism, and (2) decrease the activity of NPYand AgRP-expressing neurons, leading to decreased anabolism; both of which act to return body fat levels to their set point. Lowering body fat levels would result in a sequence of events opposite to that just described to increase body fat levels to their original level or set point.

The efferent limb that mediates the actions of these sets of arcuate neurons is not fully worked out. However, the arcuate nucleus projection to the paraventricular hypothalamic nucleus appears to be an important step in the pathway. Paraventricular neurons contain oxytocin. Many of them project to the posterior pituitary gland and are involved in lactation and uterine contractions during labor (see ). However, the paraventricular neurons involved in body weight regulation are a distinct subset of neurons that project down to the brainstem and spinal cord, where they probably synapse with autonomic and preautonomic nuclei that control parasympathetic vagal fibers to the pancreas, which act to stimulate insulin release, and sympathetic fibers, which act to inhibit its release.

Regulation of Water Intake

Water intake also depends on a servomechanism. Fluid intake is influenced by blood osmolality and volume (

Fig. 11.5

With water deprivation, the extracellular fluid becomes hyperosmotic, which in turn causes the intracellular fluid to become hyperosmotic. The brain contains neurons that serve as osmoreceptors for detection of increases in the osmotic pressure of extracellular fluid (see also ). The osmoreceptors appear to be located in the organum

Angiotensin II receptors Angiotensin II receptors Osmotic receptors Blood vessels Drinking circuit, including vasopressin-releasing neurons in the posterior pituitary B •Fig. 11.5 A, Structuresthoughttoplayaroleintheregulationofwaterintakeinrats.LHA,lateralhypothalamicarea.B, Neuralcircuitsthatsignalchangesinbloodosmolalityandvolume.(A, RedrawnfromShepherdGM.Neurobiology. NewYork:OxfordUniversityPress;1983.) vasculosum of the lamina terminalis, which is a circumventricular organ. Circumventricular organs surround the cerebral ventricles and lack a blood-brain barrier. The subfornical organ and the organum vasculosum are involved in thirst.

Water deprivation also causes a decrease in blood volume, which is sensed by receptors in the low-pressure side of the vasculature, including the right atrium of the heart (see also ). In addition, decreased blood volume triggers the release of renin by the kidneys. Renin breaks down angiotensinogen into angiotensin I, which is then hydrolyzed to angiotensin II (see ). This peptide stimulates drinking by an action on angiotensin II receptors in another of the circumventricular organs, the subfornical organ. Angiotensin II also causes vasoconstriction and release of aldosterone and antidiuretic hormone (ADH).

Insufficient water intake is usually a greater problem than excessive water intake. However, when more water is taken in than required, it is easily eliminated by inhibition of the release of ADH from neurons in the supraoptic nucleus at their terminals in the posterior pituitary gland (see ). As mentioned previously, signals that inhibit release of ADH include increased blood volume and decreased osmolality of extracellular fluid. Other areas of the hypothalamus, particularly the preoptic region and lateral hypothalamus, help regulate water intake, as do several structures outside the hypothalamus.

Several regions of the forebrain other than the hypothalamus also play a role in autonomic control. These regions include the central nucleus of the amygdala and the bed nucleus of the stria terminalis, as well as a number of areas of the cerebral cortex. Information reaches these higher autonomic centers from viscera through an ascending system that involves the nucleus of the solitary tract, the parabrachial nucleus, the periaqueductal gray matter, and the hypothalamus. Descending pathways that help control autonomic activity originate in such structures as the paraventricular nucleus of the hypothalamus, noradrenergic cell group A5, the rostral ventrolateral medulla, and the raphe nuclei and adjacent structures of the ventromedial medulla.

Neural Influences on the Immune System

Environmentalstresscancauseimmunosuppression,inwhich the number of helper T cells and the activity of natural killer cells are reduced. Immunosuppression can even be the result of classical conditioning. One mechanism for such an effect involves the release of corticotropin-releasing factor from the hypothalamus. Corticotropin-releasing factor causes the release of adrenocorticotropic hormone (ACTH) from the pituitary gland; release of ACTH stimulates the secretion of adrenal corticosteroids, which cause immunosuppression (see ). Other mechanisms include direct neural actions on lymphoid tissue. The immune system may also influence neural activity.

1.

The autonomic nervous system controls smooth muscle, cardiac muscle, and glands. It helps maintain homeostasis and coordinates responses to external stimuli. It has sensory and motor components, and the motor component consists of sympathetic and parasympathetic divisions. The enteric nervous system is often considered as part of the autonomic nervous system but is concerned specifically with control of the gastrointestinal tract.

2.

Autonomic motor pathways have preganglionic and postganglionic neurons. Preganglionic neurons reside in the CNS, whereas postganglionic neurons lie in peripheral ganglia. Sympathetic preganglionic neurons are located in the thoracolumbar region of the spinal cord, and sympathetic postganglionic neurons are located in paravertebral and prevertebral ganglia. Parasympathetic preganglionic neurons are located in cranial nerve nuclei or in the sacral portion of the spinal cord. Parasympathetic postganglionic neurons reside in ganglia located in or near the target organs.

3.

Autonomic afferent fibers innervate sensory receptors in the viscera. Most function to activate reflexes; for some, activation also leads to sensations that are experienced consciously.

4.

The enteric nervous system includes the myenteric and submucosal plexuses in the wall of the gastrointestinal tract. The myenteric plexus regulates motility, and the submucosal plexus regulates ion and water transport and secretion.

5.

Neurotransmitters at the synapses of preganglionic neurons in autonomic ganglia include acetylcholine (acting at both nicotinic and muscarinic receptors) and a number of neuropeptides. Interneurons in

Nakamura K. Central circuitries for body temperature regulation and fever. Am J Physiol Regul Integr Comp Physiol. 2011;301: R1207-R1228.

the ganglia release catecholamines. Norepinephrine (acting on adrenergic receptors) is the neurotransmitter generally released by sympathetic postganglionic neurons; neuropeptides are also released. Sympathetic postganglionic neurons that supply sweat glands release acetylcholine. Parasympathetic postganglionic neurons release acetylcholine (acting on muscarinic receptors).

6.

The pupil is controlled reciprocally by the sympathetic and parasympathetic nervous systems. Sympathetic activity causes pupillary dilation (mydriasis); parasympathetic activity causes pupillary constriction (meiosis).

7.

Emptying of the urinary bladder depends on parasympathetic outflow during the micturition reflex. Sympathetic constriction of the internal sphincter of the urethra prevents voiding. The micturition reflex is triggered by stretch receptors, and it is controlled in normal adults by a micturition center in the pons.

8.

The hypothalamus contains many nuclei that have a variety of functions related to regulation of basic bodily functions, including body temperature, body weight, and fluid intake.

9.

The goal of hypothalamic function is to maintain homeostasis of critical physiological parameters by acting as a servomechanism. The hypothalamus receives information about specific physiological parameters and uses this information to maintain each of these parameters at a specific set point. It does so via multiple mechanisms. This chapter illustrates how it maintains homeostasis via its control of the autonomic system.

Saper CB, Chou TC, Elmquist JK. The need to feed: homeostatic and hedonic control of eating. Neuron. 2002;36:199-211.

Squire L, Berg D. Fundamental Neuroscience. 4th ed. New York: Academic Press; 2012.

SECTION 3Muscle JAMES M. WATRAS

Upon completion of this chapter, the student should be able to answer the following questions: 1.

Describe the organization of skeletal muscle, including the structural features/proteins within the skeletal muscle fiber that link the contractile elements to the extracellular matrix and bone to effect movement. While describing the various linkages, identify congenital conditions that commonly affect particular structures and how they might contribute to a myopathy.

2.

Describe the molecular mechanisms by which an action potential in the α motor neuron in the ventral horn of the spinal column can lead to contraction of a skeletal muscle.

3.

Describe the mechanisms by which the force of skeletal muscle contraction increases.

4.

Compare skeletal muscle fiber types in terms of recruitment pattern, metabolic characteristics, contractile characteristics, and thus their suitability for various types of activity.

5.

Discuss the signaling pathways that contribute to the expression of the slow-twitch muscle phenotype versus the fast-twitch muscle phenotype.

6.

Describe general signaling pathways that contribute to hypertrophy or atrophy.

7.

Discuss mechanisms underlying the development of muscular fatigue.

8.

Describe mechanisms underlying the monosynaptic reflex.

9.

Discuss the length-tension curves and force-velocity curves for skeletal muscle, including the molecular bases of both curves.

Muscle cells are highly specialized for the conversion of chemical energy to mechanical energy. Specifically, muscle cells use the energy in adenosine triphosphate (ATP) to generate force or do work. Because work can take many forms (such as locomotion, pumping blood, or peristalsis), several types of muscle have evolved. The three basic types of muscle are skeletal muscle, cardiac muscle, and smooth muscle.

Skeletal muscle acts on the skeleton. In limbs, for example, skeletal muscle spans a joint, thereby allowing a lever action. Skeletal muscle is under voluntary control (i.e., controlled by the central nervous system) and plays a key role in numerous activities such as maintenance of posture, locomotion, speech, and respiration. When viewed under the microscope, skeletal muscle exhibits transverse striations (at intervals of 2 to 3 µm) that result from the highly organized arrangement of actin and myosin molecules within the skeletal muscle cells. Thus skeletal muscle is classified as a striated muscle. The heart is composed of cardiac muscle, and although it is also a striated muscle, it is an involuntary muscle (i.e., controlled by an intrinsic pacemaker and modulated by the autonomic nervous system). Smooth muscle (which lacks the striations evident in skeletal and cardiac muscle) is an involuntary muscle typically found lining hollow organs such as the intestine and blood vessels. In all three muscle types, force is generated by the interaction of actin and myosin molecules, a process that requires transient elevation of intracellular [Ca++].

In this chapter, attention is directed at the molecular mechanisms underlying contraction of skeletal muscle. Mechanisms for regulating the force of contraction are also addressed. To put this information into perspective, it is important to first examine the basic organization of skeletal muscle.

Organization of Skeletal Muscle

Fig. 12.1 illustrates skeletal muscles spanning the elbow joint. The muscles are attached to bone on either side of the joint. The point of attachment closest to the spine (proximal) is called the origin, whereas the point of attachment on the far side of the joint (distal) is called the insertion. These points of attachment occur through tendons (connective tissue) at the end of the muscle. Note that the point of insertion is close to the elbow joint, which enables a broad range of motion. Also note that the joint is spanned by a flexor muscle on one side and an extensor muscle on the opposite side of the joint. Thus contraction of the flexor muscle (see the biceps muscle in

Fig. 12.1 ) results in a decrease in the angle of the elbow joint (bringing the forearm closer to the shoulder), whereas contraction of the extensor muscle (see the triceps muscle in

Fig. 12.1 ) results in the reverse motion (extending the arm).

The basic structure of skeletal muscle is shown in

Fig.

12.2 . Each muscle is composed of numerous cells called • Fig. 12.1. Skeletal Muscle Attaches to the Skeleton by Way of Tendons and Typically Spans a Joint. The proximal and distal points of attachment of the tendon are termed origin and insertion, respectively. Note that the insertion is close to the joint, which allows a broad range of motion. Also note that skeletal muscles span both sides of the joint, which allows both flexion and extension of the forearm.

• Fig. 12.2. Skeletal muscle is composed of bundles of muscle fibers; each such bundle is called a fasciculus. A muscle fiber represents an individual muscle cell and contains bundles of myofibrils. The striations are due to the arrangement of thick and thin filaments. See text for details. (Redrawn from Bloom W, Fawcett DW. A Textbook of Histology. 10th ed. Philadelphia: Saunders; 1975.) muscle fibers. A connective tissue layer called the endomysium surrounds each of these fibers. Individual muscle fibers are then grouped together into fascicles, which are surrounded by another connective tissue layer called the perimysium. Within the perimysium are the blood vessels and nerves that supply the individual muscle fibers. The fascicles are joined together to form the muscle. The connective tissue sheath that surrounds the muscle is called the epimysium. At the ends of the muscle, the connective tissue layers come together to form a tendon, which attaches the muscle to the skeleton. The myotendinous junction is a specialized region of the tendon where the ends of the muscle fibers interdigitate with the tendon for the transmission of the force of contraction of the muscle to the tendon to effect movement of the skeleton (discussed later in this section). The tendon and the connective tissue layers are composed mainly of elastin and collagen fibers, and thus they also contribute to passive tension of muscle and prevent damage to the muscle fibers as a result of overstretching or contraction.

Individual skeletal muscle cells are narrow (≈10 to 80 µm in diameter), but they can be extremely long (up to 25 cm in length). Each skeletal muscle fiber contains bundles of filaments, called myofibrils, running along the axis of the cell. The gross striation pattern of the cell results from a repeating pattern in the myofibrils. Specifically, it is the regular arrangement of the thick and thin filaments within these myofibrils, coupled with the highly organized alignment of adjacent myofibrils, that gives rise to the striated appearance of skeletal muscle. Striations can be observed in intact muscle fibers and in the underlying  myofibrils.

Fig. 12.3 ). The sarcomere is demarcated by two dark lines called Z lines and represents a repeating contractile unit in skeletal muscle. The average length of a sarcomere is 2 µm. On either side of the Z line is a light band (I band) that contains thin filaments composed primarily of the protein actin. The area between two I bands within a sarcomere is the A band, which contains thick filaments composed primarily of the protein myosin. The thin actin filaments extend from the Z line toward the center of the sarcomere and overlap a portion of the thick filaments. The dark area at the end of the A band represents this region of overlap between thick and thin filaments. A light area in the center of the sarcomere is called the H band. This area represents the portion of the A band that contains myosin thick filaments but no thin actin filaments. Thus thin actin filaments extend from the Z line to the edge of the H band and overlap a portion of the thick filament in the A band. A dark line called the M line is evident in the center of the sarcomere and includes proteins that appear to be critical for organization and alignment of the thick filaments in the sarcomere.

Fig. 12.3, each myofibril in a muscle fiber is surrounded by sarcoplasmic reticulum (SR). The SR is an intracellular membrane network that plays a critical •Fig. 12.3. A, Myofibrilsarearrangedinparallelwithinamusclefiber.B, Eachfibrilissurroundedbysarcoplasmicreticulum(SR).TerminalcisternaeoftheSRarecloselyassociatedwithTtubulesandformatriadatthejunctionoftheIandAbands.TheZlinesdefinetheboundaryofthesarcomere.Thestriationsareformedbyoverlapofthecontractileproteins.Threebandscanbeobserved:theAband,Iband,andHband.AnMlineisvisibleinthemiddleoftheHband.C, Organizationoftheproteinswithinasinglesarcomere.Thecross-sectionalarrangementoftheproteinsisalsoillustrated.

role in the regulation of intracellular [Ca++]. Invaginations of the sarcolemma, called T tubules, pass into the muscle fiber near the ends of the A band (i.e., close to the SR). The SR and the T tubules, however, are distinct membrane systems: The SR is an intracellular network, whereas the T tubules are in contact with the extracellular space. A gap (≈15 nm in width) separates the T tubules from the SR. The portion of the SR nearest the T tubules is called the terminal cisternae, and it is the site of Ca++ release, which is critical for contraction of skeletal muscle (see the section “ ”). The longitudinal portions of the SR are continuous with the terminal cisternae and extend along the length of the sarcomere. This portion of the SR contains a high density of Ca++ pump protein (i.e., SERCA: Sarcoplasmic Endoplasmic Reticulum Ca++-ATPase), which is critical for reaccumulation of Ca++ in the SR and hence for relaxation of the muscle.

The thick and thin filaments are highly organized in the sarcomere of myofibrils (see

Fig. 12.3 ). As mentioned, thin actin filaments extend from the Z line toward the center of the sarcomere, whereas thick myosin filaments are centrally located and overlap a portion of the opposing thin actin filaments. The thick and thin filaments are oriented in such a way that in the region of overlap within the sarcomere, each thick myosin filament is surrounded by a hexagonal array of thin actin filaments. The Ca++-dependent interaction of the thick myosin filaments and the thin actin filaments generate the force of contraction after stimulation of the muscle (see the section “Actin-Myosin Interaction:

The thin filament is formed by aggregation of actin molecules (termed globular actin or G-actin) into a two-stranded helical filament called filamentous actin or F-actin. The elongated cytoskeletal protein nebulin extends along the length of the thin filament and may participate in regulation of the length of the thin filament. Dimers of the protein tropomyosin extend over the entire actin filament and cover myosin binding sites on the actin molecules. Each tropomyosin dimer extends across seven actin molecules, with sequential tropomyosin dimers arranged in a head-to-tail configuration. A troponin complex consisting of three subunits (troponin T, troponin I, and troponin

C) is present on each tropomyosin dimer and influences the position of the tropomyosin molecule on the actin filament and hence the ability of tropomyosin to inhibit binding of myosin to the actin filament at low cytosolic Ca concentrations (see the section “Actin-Myosin Interaction:

Cross-Bridge Formation”).

Binding of cytosolic Ca++ to troponin C promotes the movement of tropomyosin on the actin filament that exposes myosin binding sites on actin, thereby facilitating actin-myosin interaction, and hence contraction (see the section “Actin-Myosin Interaction: ”). Additional proteins associated with the thin filament include tropomodulin, α-actinin, and CapZ protein. Tropomodulin is located at the end of the thin filament, toward the center of the sarcomere, and may participate in setting the length of the thin filament.

• Fig. 12.4. The force of contraction of the muscle fiber is transmitted both longitudinally to the tendon (at the myotendinous junction) and laterally to adjacent extracellular connective tissue (at costameres). The force of contraction is transmitted from the end of the muscle fiber (M) to the tendon by connections with numerous collagen fibers (A, tip of arrow). Folds in the sarcolemma at the end of the muscle fiber (B) result in an interdigitation of the muscle fiber with the tendon, and represents the myotendinous junction. Costameres are located on the sides of the muscle fibers, and represent the bridges between the Z lines in the subsarcolemmal myofibrils and the extracellular connective tissue (C). Costameres facilitate the lateral transmission of force of contraction, which helps stabilize the sarcolemma. DGC, dystrophinassociated glycoprotein complex. (A and B, From Tidball JG. Myotendinous junction: morphological changes and mechanical failure associated with muscle cell atrophy. Exp Molec Pathol. 1984;40:1-12. C, From Hughes D, Wallace M, Baar K. Effects of aging, exercise, and disease on force transfer in skeletal muscle. Am J Physiol Endocrin Metab. 2015;309:E1-E10.)

CapZ protein and α-actinin serve to anchor the thin fila-of sarcomeres across muscles and the lateral transmission of ment to the Z line. force (described later in this section).

The thick myosin filaments are tethered to the Z lines by The force of contraction is transmitted both longitua cytoskeletal protein called titin. Titin is a very large, elastic dinally to the tendon (via myotendinous junctions) and protein (molecular weight, >3000 kDa) that extends from laterally to connective tissue adjacent to the muscle fibers the Z line to the center of the sarcomere and appears to be (via costameres). The myotendinous junction represents a important for organization and alignment of the thick fila-specialized region where the muscle fiber connects to the ments in the sarcomere. Some forms of muscular dystrophy tendon (

Fig. 12.4A and B).

Folding of the sarcolemma have been attributed to defects in titin (i.e., titinopathies). at the myotendinous junction results in an interdigitation

The cytoskeleton (including the intermediate filament of the tendon with the end of the muscle fiber, which protein desmin) participates in the highly organized align-increases the contact area between the muscle fiber and ment of sarcomeres. Desmin extends from the Z lines of the connective tissue and hence reduces the force per adjacent sarcomeres to the integrin protein complexes on unit area at the end of the muscle fiber. Some forms of the sarcolemma and thus participates in both the alignment muscular dystrophy are associated with decreased folding at the myotendinous junction. Proteins involved in longitudinal transmission of force at the myotendinous junction include the talin-vinculin-integrin protein complexes and the dystrophin-glycoprotein complexes. The myotendinous junction also contains Z disk proteins that have been implicated in signaling.

Lateral transmission of the force of contraction involves costameres, which link the Z lines of subsarcolemmal sarcomeres to extracellular matrix through a series of proteins (see

Fig. 12.4C ). This is particularly important for muscle fibers that do not extend the entire length of the muscle. The lateral transmission of force is also thought to stabilize the sarcolemma and to protect it from damage during contraction. The myotendinous junction and the costameres also contain signaling molecules.

Themusculardystrophiesconstituteagroupofgeneticallydetermineddegenerativedisorders.Duchenne’s muscular dystrophy (describedbyG.B.Duchennein1861)isthemostcommonofthemusculardystrophiesandaffects1per3500boys(3to5yearsofage).Severemusclewastingoccurs,andmostaffectedpatientsarewheelchairboundbytheageof12;manydieofrespiratoryfailureinadulthood(30to40yearsofage).Duchenne’smusculardystrophyisanX-linkedrecessivediseasethathasbeenlinkedtoadefectinthedystrophingenethatleadstoadeficiencyofthedystrophinproteininskeletalmuscle,brain,retina,andsmoothmuscle.Dystrophin isalarge(427-kDa)proteinthatispresentinlowabundance(0.025%)inskeletalmuscle.Itislocalizedontheintracellularsurfaceofthesarcolemmainassociationwithseveralintegralmembraneglycoproteins(formingadystrophin-glycoproteincomplex;

Fig.12.5 ).Thisdystrophin-glycoproteincomplexprovidesastructurallinkbetweenthesubsarcolemmalcytoskeletonofthemusclecellandtheextracellularmatrixandappearstostabilizethesarcolemmaandhencepreventcontraction-inducedinjury(rupture).Thedystrophin-glycoproteincomplexmayalsoserveasascaffoldforcellsignalingcascades.Theenzymenitricoxidesynthaseispresentinthedystrophin-glycoproteincomplex.

Althoughdefectsinthedystrophin-glycoproteincomplexareinvolvedinmanyformsofmusculardystrophy,someformsofmusculardystrophythatinvolveothermechanismshavebeenidentified.Specifically,adefectinsarcolemmarepair(attributedtoloss/mutationoftheproteindysferlin)appearstounderlieatleastoneformofmusculardystrophy(limb-girdle muscular dystrophy 2B, associatedwithmusclewastinginthepelvicregion).Defectsintheproteintitin(titinopathies)havebeenimplicatedinotherformsofmusculardystrophy(e.g.,limb-girdle muscular dystrophy 2J andtibial muscular dystrophy).Mutationsintheproteasecalpain 3 (resultinginlossofproteaseactivity)havealsobeenimplicatedinsometypesofmusculardystrophy(e.g.,limb-girdlemusculardystrophy2A),apparentlysecondarytoapoptosis.

Organization of the thick filament is shown in

Fig. 12.6 . Myosin is a large protein (≈480 kDa) that consists of six different polypeptides with one pair of large heavy chains (≈200 kDa) and two pairs of light chains (≈20 kDa). The heavy chains are wound together in an α-helical configuration to form a long rod-like segment, and the N-terminal portions of each heavy chain form a large globular head. The head region extends away from the thick filament toward the actin thin filament and is the portion of the molecule that can bind to actin. Myosin is also able to hydrolyze ATP, and ATPase activity is located in the globular head as well. Two pairs of light chains are associated with the globular head. One of these pairs of light chains, termed essential light chains, is crucial for the ATPase activity of myosin. The other pair of light chains, called regulatory light chains, can be phosphorylated by Ca++/calmodulin-dependent myosin light chain protein kinase, which can influence the interaction of myosin with actin (see the section “

Skeletal Muscle Types”).

Thus myosin ATPase activity occurs in the globular head of myosin and requires the presence of light chains (namely, the “essential” light chains).

Myosin filaments form by a tail-to-tail association of myosin molecules, which results in a bipolar arrangement of the thick filament. The thick filament then extends on either side of the central bare zone by a head-to-tail association of myosin molecules, thus maintaining the filament’s bipolar organization centered on the M line. Such a bipolar arrangement is critical for drawing the Z lines together (i.e., shortening the length of the sarcomere) during contraction. The mechanisms controlling this highly organized structure of the myosin thick filament are not clear, although the cytoskeletal protein titin is thought to participate in the formation of a scaffold for organization and alignment of the thick filament in the sarcomere. Additional proteins found in the thick filaments (e.g., myomesin and C protein) may also participate in the bipolar organization or packing of the thick filament (or both).

Control of Skeletal Muscle Activity

Skeletal muscle is controlled by the central nervous system. Specifically, each skeletal muscle is innervated by an α motor neuron. The cell bodies of α motor neurons are located in the ventral horn of the spinal cord (

Fig. 12.7 ). The motor axons exit via the ventral roots and reach the muscle through mixed peripheral nerves. The motor nerves branch in the muscle, and each branch innervates a single muscle fiber. The specialized cholinergic synapse that forms the neuromuscular junction and the neuromuscular transmission process that generates an action potential in the muscle fiber are described in

A motor unit consists of the motor nerve and all the muscle fibers innervated by the nerve. The motor unit is the functional contractile unit because all the muscle cells within a motor unit contract synchronously when the motor nerve fires. The size of motor units within a muscle varies, depending on the function of the muscle.

• Fig. 12.5. A, Organization of the dystrophin-glycoprotein complex in skeletal muscle. The dystrophinglycoprotein complex provides a structural link between the cytoskeleton of the muscle cell and the extracellular matrix, which appears to stabilize the sarcolemma and hence prevents contraction-induced injury (rupture). Duchenne’s muscular dystrophy is associated with loss of dystrophin. Numbers in dystrophin indicate hinge regions (e.g., H1, H2) and spectrin-like repeat domains (e.g., 4, 8, 12). ABD, actin-binding domain; C, carboxy terminus; CC, coiled-coil domain; DBD, dystroglycan-binding domain; N, amino terminus; nNOSµ, neuronal nitric oxide synthase µ; SBS, syntrophin-binding site; SSPN, sarcospan; Syn, syntrophin. Electron micrographs of a longitudinal view (B) and a cross-sectional view (C) show the distribution of dystrophin in skeletal muscle of a normal patient (CTRL). Another cross-sectional view (D) shows the loss of dystrophin from skeletal muscle in a patient with Duchenne’s muscular dystrophy (DMD). (A, From Allen D, Whitehead N, Froehner S. Absence of dystrophin disrupts skeletal muscle signaling: roles of Ca2+, reactive oxygen species, and nitric oxide in the development of muscular dystrophy. Physiol Rev. 2016;96:253-305. B, From Anastasi G, et al. Costameric proteins in human skeletal muscle during muscular inactivity. J Anat. 2008;213:284-295. C and D, From Beekman C, et al. A sensitive, reproducible and objective immunofluorescence analysis method of dystrophin in individual fibers in samples from patients with Duchenne muscular dystrophy. PLoS One. 2014;9[9]:e107494.)

Activation of motor units with a small number of fibers cardiac muscle, in contrast, is approximately 200 msec. facilitates fine motor control. Activation of varying numbers The short duration of the skeletal muscle action potential of motor units within a muscle is one way in which the allows very rapid contractions of the fiber and provides tension developed by a muscle can be controlled (see yet another mechanism by which the force of contrac“Recruitment” in the section “Modulation of the Force of tion can be increased. Increasing tension by repetitive ”). stimulation of the muscle is called tetany (see the section

The neuromuscular junction formed by the α motor “

Modulation of the Force of Contraction ”). neuron is called an end plate (see for details). Acetylcholine released from the α motor neuron at the neuromuscular junction initiates an action potential in the muscle fiber that rapidly spreads along its length. When an action potential is transmitted along the sarco-The duration of the action potential in skeletal muscle is lemma of the muscle fiber and then down the T tubules, less than 5 msec. The duration of the action potential in Ca++ is released from the terminal cisternae SR into the

Globular heads (S-1) with light chains Proteolytic cleavage • Fig. 12.6. Organization of a Thick Filament. A thick filament is formed by the polymerization of myosin molecules in a tail-to-tail configuration extending from the center of the sarcomere (A). An individual myosin molecule has a tail region and a cross-bridge region. The cross-bridge region is composed of an arm and globular heads (B). The globular heads contain light chains that are important for the function of myosin ATPase activity. LMM, light meromyosin; S-1 and S-2, myosin subfragments 1 and 2.

myoplasm. This release causes intracellular [Ca++] to rise, which in turn promotes actin-myosin interaction and contraction. The time course for the increase in intracellular [Ca++] in relation to the action potential and development of force is shown in

Fig. 12.8 . The action potential is extremely short-lived (≈5 msec). The elevation in intracellular [Ca++] begins slightly after the action potential and peaks at approximately 20 msec. This increase in intracellular [Ca++] initiates a contraction called a twitch.

The mechanism underlying the elevation in intracellular [Ca++] involves an interaction between protein in the T tubule and the adjacent terminal cisternae of the SR. As previously described (see

Fig. 12.3 ), the T tubule represents an invagination of the sarcolemma that extends into the muscle fiber and forms a close association with two terminal cisternae of the SR. The association of a T tubule with two opposing terminal cisternae is called a triad. Although there is a gap (≈15 nm in width) between the T tubule and the terminal cisternae, proteins bridge this gap. On the basis of their appearance on electron micrographs, these bridging proteins are called feet (

Fig. 12.9 ). These feet are the Ca++ release channels in the membrane of the terminal cisternae that are responsible for the elevation in intracellular [Ca++] in response to the action potential. Because this channel binds the drug ryanodine, it is commonly called the ryanodine receptor (RYR). RYR is a large protein (≈500 kDa) that exists as a homotetramer. Only a small portion of the RYR molecule is actually embedded in the SR membrane.

Most of the RYR molecule appears to be in the myoplasm and spans the gap between the terminal cisternae and the T tubule (

Fig. 12.10

At the T tubule membrane, the RYR is thought to interact with a protein called the dihydropyridine receptor (DHPR). DHPR is an L-type voltage-gated Ca++ channel with five subunits. One of these subunits binds the dihydropyridine class of channel-blocking drugs and appears to be crucial for the ability of the action potential in the T tubule to induce release of Ca++ from the SR. However, influx of Ca++ into the cell through the DHPR is not needed for the initiation of Ca++ release from the SR. In fact, skeletal muscle is able to contract in the absence of extracellular Ca++ or with a mutated DHPR that does not conduct Ca++ . Instead, release of Ca++ from the terminal cisternae of the SR is thought to result from a conformational change in the DHPR as the action potential passes down the T tubule, and this conformational change in the DHPR, by means of a protein-protein interaction, opens the RYR and releases Ca++ into the myoplasm.

Structural analysis, including the use of freeze-fracture techniques, provides evidence for a close physical association of DHPR and RYR (see

Fig. 12.9 ). DHPR in the T tubule membrane appears to reside directly opposite the four corners of the underlying homotetrameric RYR channel in the SR membrane. Studies have shown that the SH3-cysteine rich domain 3 protein Stac3 is also critical for coupling of the DHPR to the RYR during excitation-contraction coupling in skeletal muscle SR. Stac3 is not present in cardiac muscle, which relies on Ca++ influx through the sarcolemma to initiate Ca++ release from the RYR instead of the direct coupling of the DHPR and the RYR in skeletal muscle.

Other proteins that reside near the RYR include calsequestrin, triadin, and junctin (see

Fig. 12.10 ). Calsequestrin is a low-affinity Ca++-binding protein that is present in the lumen of the terminal cisternae. It allows Ca++ to be “stored” at high concentration and thereby establishes a favorable concentration gradient that facilitates the efflux of Ca++ from the SR into the myoplasm when the RYR opens. Triadin and junctin are in the terminal cisternae membrane and bind both RYR and calsequestrin; they could anchor calsequestrin near the RYR and thereby increase Ca++ buffering capacity at the site of Ca++ release. Histidine-rich calcium-binding protein is another low-affinity Ca++-binding protein in the SR lumen, although it is less abundant than calsequestrin. It appears to bind triadin in a Ca++-dependent manner, which raises the possibility that it has a role more important than serving simply as a Ca++ buffer. There is also evidence for the presence of store-operated Ca entry (SOCE) in skeletal muscle (e.g., via the Orai/Stim1 complex) during tetany. Inhibition of Ca influx did not affect excitation-contraction coupling but did reduce maximal tetanic tension at high rates of electrical stimulation, which suggests that there may be some extrusion of intracellular Ca during tetany, which is compensated by Ca influx to maintain maximal tetanic tension.

•Fig. 12.7. Skeletal Muscle Is Voluntary Muscle Controlled by the Central Nervous System, With Efferent Signals (i.e., action potentials) Passing Through an α Motor Neuron to Muscle Fibers. Eachmotorneuronmayinnervatemanymusclefiberswithinamuscle,althougheachmusclefiberisinnervatedbyonlyonemotorneuron(A). Ascanningelectronmicrograph(B) showsinnervationofseveralmusclefibersbyasinglemotorneuron.(B, FromBloomW,FawcettDW:A Textbook of Physiology. 12thed.NewYork:Chapman&Hall;1994.)

AvarietyofmutationalstudieshavebeenconductedtoascertaintheregionoftheDHPRthatiscriticalforopeningoftheRYR.Onepossiblesiteofinteraction(depictedin

Fig.12.10 )isthemyoplasmicloopbetweentransmembranedomainsIIandIIIintheα1subunitoftheDHPR.Thevoltage-sensingregionoftheDHPRinvolvedinintramembranouschargemovementisthoughttoresideintheS4transmembranesegmentsoftheα1subunit.GeneticmutationsintheRYRorDHPR,orinboth,havebeenassociatedwithpathologicaldisturbancesinmyoplasmic[Ca++].Suchdisturbancesincludemalignanthyperthermiaandcentralcoredisease,asdescribedlater.ThesemutationsaretypicallyobservedinthemyoplasmicportionoftheRYR,althoughmutationshavealsobeenobservedinamyoplasmicloopintheDHPR.ResultsofstudiesindicatethattheproteinStac3isalsoimportantforthecouplingoftheDHPRtotheRYRinskeletalmuscleandthatamutationinStac3thatispresentintherarecongenitaldisorderNativeAmericanmyopathyresultsinimpairedexcitation-contractioncouplinginskeletalmuscle.

a. Action potential potential Sarcolemma b. Myoplasmic [Ca++] c. Twitch force Calsequestrin Terminal cisternae of SRRYR Actin-myosin interaction DHPR T tubule Ca++ Ca++ SERCA ATP ADP + Pi 0 8040 120 160 200 •Fig. 12.8. A, StimulationofaskeletalmusclefiberinitiatesanactionpotentialinthemusclethattravelsdowntheTtubuleandinducesreleaseofCa++ fromtheterminalcisternaeofthesarcoplasmicreticulum(SR).Theriseinintracellular[Ca++]causesacontraction.AsCa++ ispumpedbackintotheSRbysarcoplasmicendoplasmicreticulumCa++-ATPase(SERCA),relaxationoccurs.DHPR,dihydropyridinereceptor;Pi,inorganicphosphate;RYR,ryanodinereceptor.B, Timecoursesoftheactionpotential,myoplasmicCa++ transient,andforceofthetwitchcontraction.

Terminal T tubule RYR DHPR cisternae of SR •Fig. 12.9. A, Electronmicrographofatriadillustratingthe“feet”(arrows) betweentheTtubuleandthesarcoplasmicreticulum(SR),whicharethoughttobetheryanodinereceptors(RYRs)intheSR.B, EachRYRintheSRisassociatedwithfourdihydropyridinereceptors(DHPRs)intheTtubule.(FromProtasiF,etal.RYR1andRYR3havedifferentrolesintheassemblyofcalciumreleaseunitsofskeletalmuscle.Biophys J. 2000;79:2494.)

Relaxation of skeletal muscle occurs as intracellular Ca++ is resequestered by the SR. Uptake of Ca++ into the SR is due to the action of a Ca++ pump (i.e., Ca++-ATPase). This pump is not unique to skeletal muscle; it is found in all cells in association with the endoplasmic reticulum. Accordingly, it is named SERCA, which stands for sarcoplasmic endoplasmic reticulum calcium ATPase. SERCA is the most abundant protein in the SR of skeletal muscle, and it is distributed throughout the longitudinal tubules and the terminal cisternae as well. It transports two molecules of Ca++ into its lumen for each molecule of ATP hydrolyzed.

Thus the Ca++ transient seen during a twitch contraction (see

Fig. 12.8 ) reflects release of Ca++ from the terminal cisternae via the RYR and reuptake primarily into the longitudinal portion of the SR by SERCA. The low-affinity Ca++-binding protein sarcalumenin is present throughout the longitudinal tubules of the SR and nonjunctional regions of the terminal cisternae and is thought to be involved in the transfer of Ca++ from sites of Ca++ uptake in the longitudinal tubules to sites of Ca++ release in the terminal cisternae. Results of studies suggest that sarcalumenin increases Ca++ uptake by SERCA, at least in part by buffering luminal

Ca++ near the pump.

The endogenous micropeptides phospholamban, sarcolipin, and myoregulin have been shown to regulate the activity of SERCA by decreasing the Ca sensitivity of Ca uptake (

Fig. 12.11 ). Protein kinase A–dependent phosphorylation of phospholamban in slow-twitch skeletal muscle has been reported to increase Ca transport in the SR, similar to the effect of phospholamban phosphorylation in the heart. Phospholamban and sarcolipin are present in slow-twitch muscle, whereas myoregulin is present in both fastand slow-twitch muscle.

*During the transport of Ca++ , SERCA exchanges two Ca++ ions for two H+ ions (i.e., H+ is pumped out of the SR).

•Fig. 12.10. Molecular Structure and Relationships Between the Dihydropyridine Receptor (DHPR) in the T Tubule Membrane and the Ryanodine Receptor (RYR) in the Sarcoplasmic Reticulum (SR) Membrane. TriadinisanassociatedSRproteinthatmayparticipateintheinteractionofRYRandDHPR.Calsequestrinisalow-affinityCa++-bindingproteinthathelpsaccumulateCa++ intheterminalcisternae.Seetextfordetails.COOH,carboxylicacid.(FromRossiAE,DirksenRT.Sarcoplasmicreticulum:thedynamiccalciumgovernorofmuscle.Muscle Nerve. 2006;33:715.) movement of the associated tropomyosin molecule toward

Actin-Myosin Interaction: the cleft of the actin filament. This movement of tropo myosin exposes myosin binding sites on the actin filament As noted, contraction of skeletal muscle requires an increase and allows a cross-bridge to form and thereby generate in intracellular [Ca++]. Moreover, the process of contraction tension (see section “

Cross-Bridge Cycling: Sarcomere is regulated by the thin filament. As shown in

Fig. 12.12 ”). Troponin C has four Ca++ binding sites. Two contractile force (i.e., tension) increases in a sigmoidal of these sites have high affinity for Ca++ but also bind manner as intracellular [Ca++] is elevated above 0.1 µM, Mg++ at rest. These sites seem to be involved in control-with half-maximal force occurring at less than 1 µM Ca++ . ling and enhancing the interaction between the troponin The mechanism by which Ca++ promotes this increase in I and troponin T subunits. The other two binding sites tension is as follows: Ca++ released from the SR binds to have lower affinity and bind Ca++ as its concentration rises troponin C. Once bound with Ca++ , troponin C facilitates after release from the SR. Binding of myosin to the actin • Fig. 12.11. Identification of three endogenous micropeptides that inhibit sarcoplasmic endoplasmic reticulum Ca++-ATPase (SERCA; A), and their predicted organization in the SR membrane (B). MLN, myoregulin; PLN, phospholamban; SCL, sarcolamban; SLN, sarcolipin. (Modified from Anderson DM, et al. A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell. 2015;160:595-606.) 0 0.01 0.1 1 10 100 • Fig. 12.12. The contractile force of skeletal muscle increases in a Ca++-dependent manner as a result of binding of Ca++ to troponin C and the subsequent movement of tropomyosin away from myosin binding sites on the underlying actin molecules. See text for details. (Redrawn from Hartshorne DJ. In Lapedes DN. ed. Yearbook of Science and Technology. New York: McGraw-Hill; 1976.) filaments appears to cause a further shift in tropomyosin. Although a given tropomyosin molecule extends over seven actin molecules, it is hypothesized that the strong binding of myosin to actin results in movement of an adjacent tropomyosin molecule, perhaps exposing myosin binding sites on as many as 14 actin molecules. This ability of one tropomyosin molecule to influence the movement  of another may be a consequence of the close proximity of adjacent tropomyosin molecules.

Cross-Bridge Cycling: Sarcomere Shortening

Once myosin and actin are bound, ATP-dependent conformational changes in the myosin molecule result in movement of the actin filaments toward the center of the sarcomere. Such movement shortens the length of the sarcomere and thereby contracts the muscle fiber. The mechanism by which myosin produces force and shortens the sarcomere is thought to involve four basic steps that are collectively termed the cross-bridge cycle (labeled a to d in

Fig. 12.13 ). In the resting state, myosin is thought to have partially hydrolyzed ATP (state a). When Ca++ is released from the terminal cisternae of the SR, it binds to troponin C, which in turn promotes movement of tropomyosin on the actin filament in such a way that myosin-binding sites on actin are exposed. This then allows the “energized”

GeneticdiseasesthatcausedisturbancesinCa++ homeostasisinskeletalmuscleincludemalignant hyperthermia, central core disease, andBrody’s disease. Malignanthyperthermiaisanautosomaldominanttraitthathaslife-threateningconsequencesincertainsurgicalinstances.AnestheticssuchashalothaneoretherandthemusclerelaxantsuccinylcholinecanproduceuncontrolledreleaseofCa++ fromtheSR,therebyresultinginskeletalmusclerigidity,tachycardia,hyperventilation,andhyperthermia.Thisconditionislethalifnottreatedimmediately.Therearecurrentlyaseriesoftests(involvingcontractileresponsesofmusclebiopsyspecimens)toassesswhetherapatienthasmalignanthyperthermia.Theincidenceofthisconditionisapproximately1per15,000childrenand1per50,000adultstreatedwithanesthetics.MalignanthyperthermiaistheresultofadefectintheSRCa++ releasechannel(RYR),whichbecomesactivatedinthepresenceoftheaforementionedanesthetics,causesthereleaseofCa++ intothemyoplasm,andhenceprolongsmusclecontraction(rigidity).ThedefectintheRYRisnotrestrictedtoasinglelocus.Insomecases,malignanthyperthermiahasbeenlinkedtoadefectintheDHPRoftheTtubule.

Centralcorediseaseisarareautosomaldominanttraitthatresultsinmuscleweakness,lossofmitochondriainthecoreofskeletalmusclefibers,andsomedisintegrationofcontractilefilaments.Itisoftencloselyassociatedwithmalignanthyperthermia,andsopatientswithcentralcorediseasearetreatedasthoughtheyaresusceptibletomalignanthyperthermiainsurgicalsituations.ItishypothesizedthatcentralcoresdevoidofmitochondriarepresentareasofelevatedintracellularCa++ secondarytoamutationintheRYR.ThelossofmitochondriaisthoughttooccurwhentheytakeuptheelevatedCa++ ,whichleadstomitochondrialCa++ overload.

Brody’sdiseaseischaracterizedbypainlessmusclecrampingandimpairedmusclerelaxationduringexercise.Whileanaffectedpersonrunsupstairs,forexample,musclesmaystiffenandtemporarilycannotbeused.Thisrelaxation myosin head to bind to the underlying actin (state b). Myosin next undergoes a conformational change termed “ratchet action” that pulls the actin filament toward the center of the sarcomere (state c). Myosin releases adenosine diphosphate (ADP) and inorganic phosphate during the transition to state c. Binding of ATP to myosin decreases the affinity of myosin for actin, thereby resulting in the release of myosin from the actin filament (state d). Myosin then partially hydrolyzes the ATP, and part of the energy in the ATP is used to recock the head and return to the resting state.

If intracellular [Ca++] is still elevated, myosin undergoes another cross-bridge cycle and produces further contraction of the muscle. The ratchet action of the cross-bridge is capable of moving the thin filament approximately 10 nm. The cycle continues until the SERCA pumps Ca++ back into the SR. As [Ca++] falls, Ca++ dissociates from troponin C, and the troponin-tropomyosin complex moves and blocks the myosin binding sites on the actin filament. If the supply of ATP is exhausted, as occurs with death, the cycle stops abnormalityisseeninmusclesofthelegs,arms,andeyelid,andtheresponseisworsenedincoldweather.Brody’sdiseasecanbeeitherautosomalrecessiveorautosomaldominantandmayinvolvemutationsinuptothreegenes;however,itisrare(affecting1per10,000,000births).ItappearstoresultfromdecreasedactivityoftheSERCA1Ca++ pumpfoundinfast-twitchskeletalmuscle(seethesection ”).ThedecreasedactivityofSERCA1hasbeenassociatedwithmutationsinthegenethatencodesSERCA1,althoughanotheraccessoryfactormaycontributetothedecreasedSRCa++ uptakeinthefast-twitchskeletalmuscleofindividualswithBrody’sdisease.

Myotoniacongenitaisalsoassociatedwithprolongedmusclecontractions(painlesscramping)aftervoluntarycontractions,asaresultofmutationsintheCLCN1 gene,whichencodesthechloridevoltage-gatedchannel1inskeletalmusclesarcolemmaandTtubules.Chlorideconductanceintheskeletalmuscleisimportantforrepolarizationandstabilizationofthemembranepotential,andsothereducedchlorideconductanceinskeletalmusclesofindividualswithmyotoniacongenitaresultsinhyperexcitabilityofthemusclefiber.Voluntarycontractionmaythereforebefollowedbyaseriesofactionpotentials(afterdepolarizations)inthemusclethatresultinprolongedcontractions(i.e.,cramping).Epinephrine(e.g.,duringstressfulsituations)oftenworsensthecondition,asshowninmyotonic(“fainting”)goats.Musclestiffnesscanberelievedbyrepeatedcontractions(i.e.,thewarm-upphenomenon),althoughthemechanismunderlyingthewarm-upphenomenonisnotknown.MutationsintheCLCN1 geneinmyotoniacongenitamaybetransmittedineitheranautosomalrecessivemanner(asinBecker’sdisease,onetypeofmyotoniacongenita)oranautosomaldominantmanner(asinThomsen’sdisease,theothertypeofmyotoniacongenita).Theprevalenceofmyotoniacongenitaisapproximately1per100,000worldwide;theincidenceishigher(≈1:10,000)innorthernScandinavia.

in state c with the formation of permanent actin-myosin complexes (i.e., the rigor state). In this state, the muscle is rigid, and the condition is termed rigor mortis.

As already noted, formation of the thick filaments involves the association of myosin molecules in a tail-totail configuration to produce a bipolar orientation (see

Fig. 12.6 ). Such a bipolar orientation allows myosin to pull the actin filaments toward the center of the sarcomere during the cross-bridge cycle. The myosin molecules are also oriented in a helical array in the thick filament in such a way that cross-bridges extend toward each of the six thin filaments surrounding the thick filament (see

Fig.

12.3 ). These myosin projections/cross-bridges can be seen on electron micrographs of skeletal muscle and appear to extend perpendicular from the thick filaments at rest. In the contracted state, the myosin cross-bridges slant toward the center of the sarcomere, which is consistent with the ratchet action of the myosin head.

The cross-bridge cycling mechanism just described is called the sliding filament theory because the myosin

Cycle stops here in relaxed living muscle (because of actions of regulatory mechanisms)

Tension Time0 G. 40 S. 90 msec L.R. 7.5 0 0.5 1.0 •Fig. 12.13. Cross-Bridge Cycle. Intherelaxedstate(statea),ATPispartiallyhydrolyzed(M•ADP•Pi).InthepresenceofelevatedmyoplasmicCa++ (stateb),myosin(M)bindstoactin(A).HydrolysisofATPiscompleted(statec)andcausesaconformationalchangeinthemyosinmoleculethatpullstheactinfilamenttowardthecenterofthesarcomere.AnewATPmoleculebindstomyosinandcausesreleaseofthecross-bridge(stated).PartialhydrolysisofthenewlyboundATPrecocksthemyosinhead,whichisnowreadytobindagainandagain.Ifmyoplasmic[Ca++]isstillelevated,thecyclerepeats.Ifmyoplasmic[Ca++]islow,relaxationresults.

cross-bridge is pulling the actin thin filament toward the center of the sarcomere, which results in an apparent “sliding” of the thin filament past the thick filament. There is, however, uncertainty about how many myosin molecules contribute to the generation of force and whether both myosin heads in a given myosin molecule are involved. It has been calculated that there may be 600 myosin heads per thick filament, with a stoichiometry of 1 myosin head per 1.8 actin molecules. As a result of steric considerations, it is unlikely that all myosin heads can interact with actin, and calculations suggest that even during maximal force generation, only 20% to 40% of the myosin heads bind to actin.

The conversion of chemical energy (i.e., ATP) to mechanical energy by muscle is highly efficient. In isolated muscle preparations, maximum mechanical efficiency (≈65% efficiency) is obtained at a submaximal force of 30% maximal tension. In humans performing steady-state ergometer exercise, mechanical efficiencies range from 40% to 57%.

Skeletal muscle fibers can be classified into two main groups according to the speed of contraction: fast-twitch •Fig. 12.14. A, Musclesvaryintermsofthespeedofcontraction.G,gastrocnemiusmuscleoftheleg;LR,lateralrectusmuscleoftheeye;S,soleusmuscleoftheleg.B, ThespeedofshorteningiscorrelatedwithmyosinATPaseactivity.N-SOL,normalsoleusmuscle(slowtwitch);N-EDL,normalextensordigitorumlongusmuscle(fasttwitch);S-EDL,self-innervatedextensordigitorumlongusmuscle(EDLmotornervetransectedandresutured);S-SOL,self-innervatedsoleusmuscle(soleusmotornervetransectedandresutured);X-EDL,cross-innervatedextensordigitorumlongusmuscle(EDLinnervatedbysoleusmotornerve);X-SOL,cross-innervatedSOLmuscle(soleusinnervatedbyEDLmotornerve).(A, FromMontcastleV[ed].Medical Physiology. 12thed.St.Louis:Mosby;1974.B, FromBárányM,CloseRI.J Physiol. 1971;213:455.) and slow-twitch muscle fibers. As shown in

Fig. 12.14A , the lateral rectus of the eye contracts very quickly in response to an action potential, reaching peak tension within 8 msec, and then relaxes quickly, which results in a short duration of contraction. The soleus muscle of the leg, in contrast, requires 90 msec to reach peak tension in response to an action potential, and then it relaxes slowly. The gastrocnemius muscle requires an intermediate time to reach peak tension (40 msec) because of the presence of both fast-twitch and slow-twitch muscle fibers in this muscle.

The difference in speed of contraction between fast-twitch and slow-twitch muscles is correlated with myosin ATPase activity (see

Fig. 12.14B ), which in turn reflects the type of myosin present in the muscle fiber (Fig. 12.15

Table 12.1 ). Thus fast-twitch muscle fibers contain myosin isoforms that hydrolyze ATP quickly, whereas slow-twitch muscle fibers contain myosin isoforms that hydrolyze ATP

MHC expression: Type I MHC expression: MHC expression: Function: antigravity, IIa, IIx IIb, IIx weight bearing, and Function: sustained Function: burst •Fig. 12.15. Comparisonofthreebasicmotorunitphenotypesinskeletalmuscleofextremitiesandtrunk.MHC,myosinheavychain.(RedrawnfromBaldwinK,HaddadF,PandorfC,etal.Alterationsinmusclemassandcontractilephenotypeinresponsetounloadingmodels:roleoftranscriptional/pretranslationalmechanisms.Front Physiol. 2013;4:284.) *Human“fastglycolytic”skeletalmusclefiberstypicallyexpressthetypeIIxmyosinisoenzymeinsteadofthetypeIIbmyosinisoenzyme.Incontrast,rodentsexpressonetypeofslow-twitchmusclefiber(typeI)andthreetypesoffast-twitchmusclefibers(expressingtypeIIa,typeIIx,ortypeIIbmyosinisoenzymes);thetypeIIxmusclefibersexpressmetabolicpropertiesintermediatebetweenthoseoftypeIIaandtypeIIbmusclefibers.ThetypeIIxmyosinisoenzymemaybecoexpressedwithtypeIIamyosinisoenzymein“fastoxidative”musclefibers.Intext,thesimpledesignationtype II fiber referstofastglycolyticmusclefibers.

slowly. These two types of myosin isoforms have the same basic structure described previously, with two heavy chains and two pairs of light chains, although they differ in amino acid composition.

It is very difficult to convert a slow-twitch muscle fiber into a fast-twitch fiber, although it can be accomplished by cross-innervation, which involves surgically interconnecting two motor neurons. As shown in

Fig. 12.14B , when the soleus muscle and extensor digitorum longus muscle underwent cross-innervation, so that contraction of the soleus muscle was controlled by the extensor digitorum motor neuron (and vice versa), the speed of the contraction and the myosin ATPase activity of the soleus muscle increased (labeled X-SOL in

Fig. 12.14 ), whereas the extensor digitorum longus exhibited a decrease in shortening velocity and myosin ATPase activity (labeled X-EDL). Thus the motor innervation of the muscle fiber plays an important role in determining which type of myosin isoform is expressed in the muscle fiber. Further study showed that the intracellular Ca concentration in the muscle (secondary to differences in the activity pattern of the motor neuron) was an important determinant of whether the muscle fiber expressed the slow myosin isoform or the fast myosin isoform (see the section

The myosin isoforms expressed in skeletal muscle can be distinguished on the basis of myosin heavy chain composition (see

Fig. 12.15

Table 12.1 ). Slow-twitch muscle fibers express type I myosin heavy chain, whereas fast-twitch skeletal muscle fibers could contain type IIa, type IIx, or type IIb myosin heavy chains. Some fast-twitch muscle fibers may contain a mixture of type II myosin isoforms.

Slow-twitch skeletal muscles are also characterized by a high oxidative capacity (see

Table 12.1 ), which in combination with the low myosin ATPase activity contribute to the fatigue resistance of slow-twitch muscle fibers. The oxidative capacity of the fast-twitch muscle fiber ranges from relatively high (in muscle fibers expressing type IIa myosin heavy chain) to low (in muscle fibers expressing type IIb myosin heavy chains). The low oxidative capacity of fast type IIb muscle fibers, coupled with the high myosin ATPase activity, increases the susceptibility of these muscle fibers to fatigue. Humans, however, rarely express the type IIb myosin heavy chain. Type IIb myosins are expressed in small animals such as rabbit and rats.

Motor units are generally composed of only one type of muscle fiber (

Table 12.2

Fig. 12.15), unless the muscle fibers are undergoing a transition. Conditions that may trigger a change in the type of myosin expressed in a muscle fiber within a motor unit include chronic conditions such as microgravity (in space flight), denervation, and chronic unloading, which are associated with severe atrophy and promote the gradual transition from the expression of slow muscle myosin (type I) in the fiber to the expression of fast muscle myosin (types IIa and IIx).

An important function of slow motor units is in the maintenance of posture (see

Fig. 12.15 ). The low ATPase activity of myosin in slow motor units, coupled with their high oxidative capacity, facilitates the ability of these slow motor units to maintain posture at low energy cost and thus resist fatigue. The smaller diameter of slow muscle fibers, and the higher capillary density in slow muscle, also helps slow muscle resist fatigue.

Fast muscle, in contrast, is recruited for activities that require faster movements, more force, or both (see

Fig.

12.15 ). Weightlifting, for example, can require a lot of power for short duration. In order to meet the demands for more force, additional motor units are recruited. In comparison with slow motor units, the fast motor units typically contain more muscle fibers (see

Table 12.2 ). Fast muscle fibers also have a larger diameter than do slow muscle fibers. Thus recruitment of fast motor units can help meet the increased demands of burst activities such as weightlifting. The high myosin ATPase activity in fast muscle fibers and the increase in diffusion distance (resulting from the large diameter of the fast muscle fibers), however, increase the susceptibility of fast muscle fibers to fatigue.

Additional differences between fast and slow muscles include the following: 1.

The motor neuron in slow muscle is more easily excited than that in fast muscle, and so slow muscles are typically recruited first. As stated previously, the high oxidative capacity of slow muscle, coupled with the low myosin ATPase activity, helps reduce the susceptibility to fatigue in slow muscle.

2.

The neuromuscular junction of fast muscle differs from that in slow muscle in terms of acetylcholine vesicle content, the amount of acetylcholine released, the density of nicotinic acetylcholine receptors, the acetylcholine esterase activity, and Na channel density, all of which endow the fast muscle with a higher safety factor for initiation of an action potential. During repetitive stimulation, however, the safety factor in fast muscle drops quickly (faster than that seen in slow muscle).

3.

The SR is more highly developed in fast muscle than in slow muscle, with higher levels of RYR, SERCA, lumenal Ca, and a higher DHPR/RYR ratio, all of which promote the development of a larger, faster intracellular Ca transient in fast muscle, which is important for quick, forceful contraction. In addition to the differences between fast and slow fibers just noted, other muscle proteins are also expressed in a fiber type–specific manner. Such proteins include the three troponin subunits, tropomyosin, and C protein. The differential expression of troponin and tropomyosin isoforms influences the dependency of contraction on Ca++ . Slow fibers begin to develop tension at lower [Ca++] than fast fibers do. This difference in sensitivity to Ca++ is related in part to the fact that the troponin C isoform in slow fibers has only a single low-affinity Ca++-binding site, whereas the troponin C of fast fibers has two low-affinity binding sites. Changes in the dependence of contraction on Ca++ , however, are not restricted to differences in the troponin C isoforms. Differences in troponin T and tropomyosin isoforms are also found. Thus regulation of the dependence of contraction on Ca++ is complex and involves contributions from multiple proteins on the thin filament. Phosphorylation of the regulatory light chain of myosin by Ca++/ calmodulin-dependent myosin light chain kinase, however, can increase Ca++ sensitivity of contraction, particularly in fast muscle fibers (partly because of the reported higher activity of myosin light chain kinase in fast muscle fibers).

•Fig. 12.16. Increasing the Frequency of Electrical Stimulation of Skeletal Muscle Results in an Increase in the Force of Contraction. ThisisattributabletoprolongationoftheintracellularCa++transientandistermedtetany. IncompletetetanyresultsfrominitiationofanotherintracellularCa++transientbeforethemusclehascompletelyrelaxed.Thusthereisasummationoftwitchforces.Seetextfordetails.Action potential Time 1 sec Myoplasmic Ca++ Incomplete tetanus Twitch force Tetanus force

Modulation of the Force of Contraction

A simple means of increasing the force of contraction of a muscle is to recruit more muscle fibers. Because all the muscle fibers within a motor unit are activated simultaneously, a muscle recruits more muscle fibers by recruiting more motor units. As already noted, muscle fibers can be classified as fast-twitch or slow-twitch. The type of fiber is determined by its innervation. Because all fibers in a motor unit are innervated by a single α motor neuron, all fibers within a motor unit are of the same type. Slow-twitch motor units tend to be small (100 to 500 muscle fibers) and are innervated by an α motor neuron that is easily excited (see

Table 12.2 ). Fast-twitch motor units, in contrast, tend to be large (containing 1000 to 2000 muscle fibers) and are innervated by α motor neurons that are more difficult to excite. Thus slow-twitch motor units tend to be recruited first. As more and more force is needed, fast-twitch motor units are recruited. The advantage of such a recruitment strategy is that the first muscle fibers recruited are those that have high resistance to fatigue. Moreover, the small size of slow-twitch motor units allows fine motor control at low levels of force. The process of increasing the force of contraction by recruiting additional motor units is termed spatial summation because forces from muscle fibers are being “summed” within a larger area of the muscle. This is in contrast to temporal summation, which is discussed later.

Action potentials in skeletal muscles are quite uniform and lead to the release of a reproducible pulse of Ca++ from the SR (

Fig. 12.16 ). A single action potential releases sufficient Ca++ to cause a twitch contraction. However, the duration of this contraction is very short because Ca++ is very rapidly pumped back into the SR. If the muscle is stimulated a second time before it is fully relaxed, the force of contraction increases (see

Fig. 12.16 , middle). Thus twitch forces are amplified as stimulus frequency increases. At a high level of stimulation, intracellular [Ca++] increases and is maintained throughout the period of stimulation (see

Fig. 12.16 , right), and the amount of force developed greatly exceeds that observed during a twitch. The response is termed tetany. At intermediate stimulus frequency, intracellular [Ca++] returns to baseline just before the next stimulus. However, there is a gradual rise in force (see

Fig. 12.16 , middle). This phenomenon is termed incomplete tetany. In both cases, the increased frequency of stimulation is said to produce a fusion of twitches.

The low force generation during a twitch, in comparison with that during tetany, may be due to the presence of a series elastic component in the muscle. Specifically, when the muscle is stretched a small amount shortly after initiation of the action potential, the muscle generates a twitch force that approximates the maximal tetanic force. This result, coupled with the observation that the size of the intracellular Ca++ transient during a twitch contraction is comparable with that during tetany, suggests that enough Ca++ is released into the myoplasm during a twitch to allow the actin-myosin interactions to produce maximal tension. However, the duration of the intracellular Ca++ transient during a twitch is sufficiently short that the contractile elements may not have enough time to fully stretch the series elastic components in the fiber and muscle. As a result, the measured tension is submaximal.

An increase in the duration of the intracellular Ca++ transient, as occurs with tetany, provides the muscle with sufficient time to completely stretch the series elastic component and thereby results in expression of the full contractile force of the actin-myosin interactions (i.e., maximal tension). Partial stretching of the series elastic component (as might be expected during a single twitch), followed •Fig. 12.17. Slow-Twitch Muscles Exhibit Tetany at a Lower Stimulation Frequency Than Do Fast-Twitch Muscles. A, Fast-twitchmotorunitinthegastrocnemiusmuscle.B, Slow-twitchmotorunitinthegastrocnemiusmuscle.C, Slow-twitchmuscleunitinthesoleusmuscle.Themotorunitswerestimulatedatthefrequenciesindicatedontheleft.Thecalibrationbarfortension(ingrams)generatedduringconcentrationisindicatedbythevertical brackets underthecurves.Notethelargeforcegeneratedbythefast-twitchmotorunit(A).(FromMontcastleV[ed].Medical Physiology. 12thed.St.Louis:Mosby:1974.) by restimulation of the muscle before complete relaxation, however, would be expected to yield an intermediate level of tension, similar to that seen with incomplete tetany. The location of the series elastic component in skeletal muscle is not known. One potential source is the myosin molecule itself. In addition, it is likely that there are other sources of the series elastic component, such as the connective tissue and titin.

The stimulus frequency needed to produce tetany depends on whether the motor unit consists of slow or fast fibers (

Fig. 12.17 ). Slow fibers can be tetanized at lower frequencies than can fast fibers. The ability of slow-twitch muscle to tetanize at lower stimulation frequencies reflects, at least in part, the longer duration of contraction seen in slow fibers. As also illustrated in

Fig. 12.17 , fast fibers develop a larger maximal force than slow fibers do because fast fibers are larger in diameter than slow fibers and there are more fibers in fast motor units than in slow motor units.

Modulation of Force by Reflex Arcs

Skeletal muscles contain sensory fibers (muscle spindles; also called intrafusal fibers) that run parallel to the skeletal muscle fibers. The muscle spindles assess the degree of stretch of the muscle, as well as the speed of contraction. In the stretch reflex, rapid stretching of the muscle (e.g., tapping the tendon) lengthens the spindles in the muscle and results in an increased frequency of action potentials in the afferent sensory neurons of the spindle. These afferent fibers in turn excite the α motor neurons in the spinal cord that innervate the stretched muscle. The result is that the reflex arc is a stretch-induced contraction of the muscle that does not require input from high centers in the brain. As the muscle shortens, efferent output is also sent to the spindle, which thereby takes the slack out of the spindle and ensures its ability to respond to stretch at all muscle lengths. By their action, muscle spindles provide feedback to the muscle in terms of its length and thus help maintain a joint at a given angle.

Golgi tendon organs are located in the tendons of muscles and provide feedback regarding contraction of the muscle. The main component of the tendon organ is an elongated fascicle of collagen bundles that is in series with the muscle fibers and can respond to contractions of individual muscle fibers. A given tendon organ may attach to several fast-twitch or slow-twitch muscle fibers (or both) and sends impulses through type Ib afferent nerve fibers in response to muscle contraction. The type Ib afferent impulses enter the spinal cord, which can promote inhibition of α motor neurons to the contracting (and synergistic) muscles while promoting excitation of α motor neurons to antagonistic muscles. The inhibitory actions are mediated through interneurons in the cord that release an inhibitory transmitter to the α motor neuron and create an inhibitory postsynaptic potential. The type Ib afferent impulses are also sent to higher centers of the brain (including the motor cortex and cerebellum). It is hypothesized that feedback from the tendon organs in response to muscle contraction may smooth the progression of muscle contraction by limiting the recruitment of additional motor units. Of interest is that the response of the tendon organ is not linearly related to force; rather, it drops off at higher levels of force, which may facilitate the recruitment of motor units at higher levels of effort.

The skeletal system supports the body in an erect posture with the expenditure of relatively little energy. Nonetheless, even at rest, muscles normally exhibit some level of contractile activity. Isolated (i.e., denervated) unstimulated muscles are in a relaxed state and are said to be flaccid. However, relaxed muscles in the body are comparatively firm. This firmness, or tone, is caused by low levels of contractile activity in some of the motor units and is driven by reflex arcs from the muscle spindles. Interruption of the reflex arc by sectioning of the sensory afferent fibers abolishes this resting muscle tone. The tone in skeletal muscle is distinct from the “tone” in smooth muscle (see

Muscle cells convert chemical energy to mechanical energy. ATP is the energy source used for this conversion. The ATP pool in skeletal muscle is small and capable of supporting only a few contractions if not replenished. This pool, however, is continually replenished during contraction, as described later, so that even when the muscle fatigues, ATP stores are only modestly decreased.

Muscle cells contain creatine phosphate, which is used to convert ADP to ATP and thus replenish the ATP store during muscle contraction. The creatine phosphate store represents the immediate high-energy source for replenishing the ATP supply in skeletal muscle, especially during intense exercise. The enzyme creatine phosphokinase catalyzes the reaction:

Although much of the creatine phosphokinase is present in the myoplasm, a small amount is located in the thick filament (near the M line). The creatine phosphokinase in the thick filament may participate in the rapid resynthesis of ATP near the myosin heads during muscle contraction. The phosphate store created, however, is only about five times the size of the ATP store and thus cannot support prolonged periods of contraction (less than a minute of maximal muscle activity). Skeletal muscle fatigue during intense exercise is associated with depletion of the creatine phosphate store, although as described subsequently, this does not necessarily imply that the fatigue is caused by depletion of the creatine phosphate store. Because the creatine phosphokinase–catalyzed reaction is reversible, the muscle cell replenishes the creatine phosphate pool during recovery from fatigue by using ATP synthesized through oxidative phosphorylation.

Muscle cells contain glycogen, which can be metabolized during muscle contraction to provide glucose for oxidative phosphorylation and glycolysis, both of which generate ATP to replenish the ATP store. Muscle cells can also take up glucose from blood, a process that is stimulated by insulin (see

Chapter 39). The cytosolic enzyme phosphory lase releases glucose 1-phosphate residues from glycogen, which are then metabolized by a combination of glycolysis (in the cytosol) and oxidative phosphorylation (in the mitochondria) to yield the equivalent of 37 mol of ATP per mole of glucose 1-phosphate. Blood glucose yields 36 mol of ATP per mole of glucose because 1 ATP is used to phosphorylate glucose at the start of glycolysis. These ATP yields, however, are dependent on an adequate oxygen supply. Under anaerobic conditions, in contrast, metabolism of glycogen and glucose yields only 3 and 2 mol of ATP per mole of glucose 1-phosphate and glucose, respectively (along with 2 mol of lactate). As discussed later, muscle fatigue during prolonged exercise is associated with depletion of glycogen stores in the muscle.

Fatty acids represent an important source of energy for muscle cells during prolonged exercise. Muscle cells contain fatty acids but can also take up fatty acids from blood. In addition, muscle cells can store triglycerides, which can be hydrolyzed when needed to produce fatty acids. The fatty acids are subjected to β oxidation within the mitochondria. For fatty acids to enter the mitochondria, however, they are converted to acylcarnitine in the cytosol and then transported into the mitochondria, where they are converted to acyl coenzyme A (CoA). Within the mitochondria, the acyl CoA is subjected to β oxidation and yields acetyl CoA, which then enters the citric acid cycle and ultimately produces ATP.

If the energy demands of exercise cannot be met by oxidative phosphorylation, an oxygen debt is incurred. After completion of exercise, respiration remains above the resting level in order to “repay” this oxygen debt. The extra oxygen consumption during this recovery phase is used to restore metabolite levels (such as creatine phosphate and ATP) and to metabolize the lactate generated by glycolysis. The increased cardiac and respiratory work during recovery also contributes to the increased oxygen consumption seen at this time and explains why more oxygen has to be “repaid” than was “borrowed.” Some oxygen debt occurs even with low levels of exercise because slow oxidative motor units consume considerable ATP, derived from creatine phosphate or glycolysis, before oxidative metabolism can increase ATP production to meet steady-state requirements. The oxygen debt is much greater with strenuous exercise, when fast glycolytic motor units are used (Fig. 12.18 ). The oxygen debt is approximately equal to the energy consumed during exercise minus that supplied by oxidative metabolism (i.e., the darkand light-colored areas in

Fig. 12.18 are approximately equal). As indicated earlier, the additional oxygen used during recovery from exercise represents the energy requirements for restoring normal cellular metabolite levels.

The ability of muscle to meet energy needs is a major determinant of the duration of the exercise. However, fatigue is not the result of depletion of energy stores. Instead, metabolic byproducts seem to be important factors in the onset of fatigue. Fatigue may potentially occur at any of the points involved in muscle contraction, from the brain to the muscle cells, as well as in the cardiovascular and respiratory systems that maintain energy supplies (i.e., fatty acids and glucose) and oxygen delivery to the exercising muscle.

Several factors have been implicated in muscle fatigue. During brief periods of tetany, the oxygen supply to the muscle is adequate as long as the circulation is intact. However, the force/stress generated during these brief

Rate of energy expenditure •Fig. 12.18. An Oxygen Debt Is Incurred by the Exercising of Muscle When the Rate of Energy Expenditure Exceeds the Rate of Energy Production by Oxidative Metabolism. Upper panel, Energyexpenditureduringstrenuousexercise.Lower panel, Energyexpenditureduringenduranceexercise.Seetextfordetails.

tetanic periods decays rapidly to a level that can be maintained for long periods (

Fig. 12.19 ). This decay represents the rapid and almost total failure of the fast motor units. The decline in force/stress is paralleled by depletion of glycogen and creatine phosphate stores and the accumulation of lactic acid. Of importance is that the decline in force/ stress occurs when the ATP pool is not greatly reduced, so that the muscle fibers do not go into rigor. In contrast, the slow motor units are able to meet the energy demands of fibers under this condition, and they do not exhibit significant fatigue, even after many hours. Evidently, some factor associated with energy metabolism can inhibit contraction (e.g., in the fast fibers), but this factor has not been clearly identified.

During intense exercise, accumulation of inorganic phosphate (Pi) and lactic acid in the myoplasm accounts for muscle fatigue. The accumulation of lactic acid, to levels as high as 15 to 26 mmol/L, decreases myoplasmic pH (from ≈7 to ≈6.2) and inhibits actin-myosin interactions. This decrease in pH reduces the sensitivity of the actin-myosin interaction to Ca++ by altering Ca++ binding to troponin C and by decreasing the maximum number of actin-myosin interactions. Pi has also been implicated as an important factor in the development of fatigue during intense exercise, inasmuch as phosphate concentrations can increase from approximately 2 mmol/L at rest to nearly 40 mmol/L in working muscle. Such an elevation in [Pi] can reduce tension by at least the following three different mechanisms: •Fig. 12.19. Aseriesofbrieftetanicstimulationsofskeletalmuscleresultinarapiddecreaseinforce(tetanicstress,exemplifiedbythe“Wholemuscle”lineinplot)thatisattributabletofatigueoffast-twitch(typeII)motorunitsinthemuscle.Undertheseconditions,however,slow-twitch(typeI)motorunitsareresistanttofatigue.3 2 1 0 10 2 3 4 5 10 20 30 Minutes Tetanus stress (kg/cm2 ) Type 1 motor units Whole muscle Type II motor units (1) inhibition of Ca++ release from the SR, (2) decrease in the sensitivity of contraction to Ca++ , and (3) alteration in actin-myosin binding. A number of other factors, including glycogen depletion from a specialized compartment, a localized increase in [ADP], extracellular elevation of [K+], and generation of oxygen free radicals, have also been implicated in various forms of exercise-induced muscle fatigue. Finally, the central nervous system contributes to fatigue, especially in how fatigue is perceived by the individual.

Regardless of whether the muscle is fatigued as a consequence of high-intensity exercise or prolonged exercise, the myoplasmic ATP level does not decrease substantially. In view of the reliance of all cells on the availability of ATP to maintain viability, fatigue has been described as a protective mechanism to minimize the risk of muscle cell injury or death. Consequently, it is likely that skeletal muscle cells have developed redundant systems to ensure that ATP levels do not drop to dangerously low levels and hence risk the viability of the cell.

Most persons tire and cease exercise long before the motor unit fatigues. General physical fatigue may be defined as a homeostatic disturbance produced by work. The basis for the perceived discomfort (or even pain) probably involves many factors. These factors may include a decrease in plasma glucose levels and accumulation of metabolites. Motor system function in the central nervous system is not impaired. Highly motivated and trained athletes can withstand the discomfort of fatigue and may exercise to the point at which some motor unit fatigue occurs. Part of the enhanced performance observed after training involves motivational factors.

Skeletal muscle fibers differentiate before they are innervated, and some neuromuscular junctions are formed well after birth. Before innervation, the muscle fibers physiologically resemble slow (type I) cells. Acetylcholine receptors are distributed throughout the sarcolemma of these uninnervated cells and are supersensitive to that neurotransmitter. An end plate is formed when the first growing nerve terminal establishes contact with a muscle cell. The cell forms no further association with nerves, and receptors to acetylcholine become concentrated in the end plate membranes. Cells innervated by a small motor neuron form slow (type I) oxidative motor units. Fibers innervated by large motor nerves develop all the characteristics of fast (type II) motor units. Innervation produces major cellular changes, including synthesis of the fast and slow myosin isoforms, which replace embryonic or neonatal variants. Thus muscle fiber type is determined by the nerves that innervate the fiber.

An increase in muscle strength and size occurs during maturation. As the skeleton grows, the muscle cells lengthen. Lengthening is accomplished by the formation of additional sarcomeres at the ends of the muscle cells (

Fig. 12.20 ), a process that is reversible. For example, the length of a cell decreases when terminal sarcomeres are eliminated, which can occur when a limb is immobilized with the muscle in a shortened position or when improper setting of a fracture causes shortening of the limb segment. Changes in muscle length affect the velocity and extent of shortening but do not influence the amount of force that can be generated by the muscle. The gradual increase in strength and diameter of a muscle during growth is achieved mainly by hypertrophy. Doubling the myofibrillar diameter by adding more sarcomeres in parallel (hypertrophy, for example) may double the amount of force generated but has no effect on the maximal velocity of shortening. Resistance exercise can promote hypertrophy by activation of the Akt-mTOR signaling pathway and simultaneous inhibition of the forkhead box O protein (FoxO)–atrogene pathway, which results in an increase in net protein synthesis (

Fig. 12.21A ). The increase in intracellular Ca and decrease in ATP concentration can also stimulate mitochondrial biogenesis and expression of a slow muscle phenotype through activation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) signaling (see

Fig. 12.21B

Skeletal muscles have a limited ability to form new fibers (hyperplasia). These new fibers result from differentiation of satellite cells that are present in the tissues. However, major cellular destruction leads to replacement by scar tissue.

Muscles not only must be used to maintain normal growth and development but must also experience loading.

•Fig. 12.20. Effects of Growth on the Mechanical Output of a Muscle Cell. Typically,skeletalmusclecellgrowthinvolveseitherlengthening(addingmoresarcomerestotheendsofthemusclefibers)orincreasingmusclefiberdiameter(hypertrophyasaresultoftheadditionofmoremyofilaments/myofibrilsinparallelwithinthemusclefiber).Theformationofnewmusclefibersiscalledmuscle hyperplasia, anditisinfrequentinskeletalmuscle.

Muscles immobilized in a cast lose mass. In addition, space flight exposes astronauts to a microgravity environment that mechanically unloads their muscles. Such unloading leads to rapid loss of muscle mass (i.e., atrophy) and weakness. Disuse atrophy appears to involve both inhibition of protein synthesis and stimulation of protein degradation (with net activation of the FoxO-atrogene pathway). Other categories of skeletal muscle atrophy include sarcopenia (which is atrophy associated with the aging process) and cachexia (which is atrophy associated with an illness).

Muscles that frequently contract to support the body typically have a high number of slow (type I) oxidative motor units. These slow motor units atrophy more rapidly than the fast (type II) motor units during prolonged periods of unloading. This atrophy of slow motor units is associated with a decrease in maximal tetanic force but also an increase in maximal shortening velocity. The increase in velocity is correlated with expression of the fast myosin isoform in these fibers. An important aspect of space medicine is the design of exercise programs that minimize such phenotypic changes during prolonged space flight.

• Fig. 12.21. Basic molecular signaling pathways involved in contributing to net protein synthesis (A) and mitochondrial biogenesis (B) in skeletal muscle in response to exercise. AKT, a serine/threonine-specific protein kinase (protein kinase B); AMPK, 5′ adenosine monophosphate–activated protein kinase; ATP, adenosine triphosphate; CaM kinase, Ca++/calmodulin-dependent protein kinase; CREB, cAMP response element binding protein; ERRα, estrogen-related receptor-α; FoxO, forkhead box O class of transcription factors; FoxO-P, phosphorylated FoxO; Glut-4, glucose transporter type 4; HDAC5, histone deacetylase 5; IGF-1, insulin-like growth factor 1; MEF2, myocyte enhancer factor 2; mTOR, mammalian target of rapamycin; Nrf1, Nrf2, and Nrf2α, nuclear respiratory factors 1, 2, and 2α; PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1α; PI3K, phosphatidylinositol-3-kinase; Tn I, troponin I. (A, From Sandri M, et al. PGC-1alpha protects skeletal muscle from atrophy by suppressing FoxO3 action and atrophy-specific gene transcription. PNAS. 2006;103:16260-16265. B, From Lin J, Handschin C, Spiegelman BM. Metabolic control through the PGC-1 family of transcription coactivators. Cell Metab. 2005;1:361-370.)

Testosterone is a major factor responsible for the greater Denervation, Reinnervation,  muscle mass in men because it has both myotrophic action ). A variety of synthetic molecules, called anabolic steroids, As already noted, innervation is crucial for the skeletal have been designed to enhance muscle growth while mini-muscle phenotype. If the motor nerve is cut, muscle fasmizing their androgenic action. These drugs are widely used ciculation occurs. Fasciculation is characterized by small, by bodybuilders and athletes in sports in which strength is irregular contractions caused by release of acetylcholine from important. The doses are typically 10-to 50-fold greater the terminals of the degenerating distal portion of the axon. than might be prescribed therapeutically for individuals Several days after denervation, muscle fibrillation begins. with impaired hormone production. Unfortunately, none Fibrillation is characterized by spontaneous, repetitive of these compounds lack androgenic effects. Hence, at the contractions. At this time, the cholinergic receptors have doses used, they induce serious hormone disturbances, spread out over the entire cell membrane, in effect reverting including depression of testosterone production. A major to their preinnervation embryonic arrangement. The muscle issue is whether these drugs do in fact increase muscle and fibrillations reflect supersensitivity to acetylcholine. Affected athletic performance in individuals with normal circulating muscles also atrophy, with a decrease in the size of the levels of testosterone. Although they have been used since muscle and its cells. Atrophy is progressive in humans, with the 1950s, the scientific facts remain uncertain, and most degeneration of some cells 3 or 4 months after denervation. experimental studies in animals have not demonstrated Most of the muscle fibers are replaced by fat and connective any significant effects on muscle development. Reports of tissue after 1 to 2 years. These changes can be reversed if studies in humans remain controversial. Proponents claim reinnervation occurs within a few months. Reinnervation increases in strength that provide advantages in world-class is normally achieved by growth of the peripheral stump of performance. Critics argue that these increases are largely motor nerve axons along the old nerve sheath. placebo effects associated with expectations and motivational Reinnervation of formerly fast (type II) fibers by a small factors. The public debate on abuse of anabolic steroids has motor axon causes that cell to redifferentiate into a slow led to their designation as controlled substances, along with (type I) fiber, and vice versa. This suggests that large and opiates, amphetamines, and barbiturates. small motor nerves differ qualitatively and that the nerves have a specific “trophic” effect on the muscle fibers. This “trophic” effect reflects the rate of fiber stimulation. For example, stimulation via electrodes implanted in the muscle can lessen denervation atrophy. More strikingly, chronic low-frequency stimulation of fast motor units causes these units to be converted to slow units. Some conversion toward a typical fast-fiber phenotype can occur when the frequency of contraction in slow units is greatly decreased by reducing the excitatory input. Excitatory input can be reduced by sectioning the appropriate spinal or dorsal root or by severing the tendon, which functionally inactivates peripheral mechanoreceptors.

The frequency of contraction determines fiber development and phenotype through changes in gene expression and protein synthesis. Fibers that undergo frequent contractile activity form many mitochondria and synthesize the slow isoform of myosin. Fibers innervated by large, less excitable axons contract infrequently. Such relatively inactive fibers typically form few mitochondria, have large concentrations of glycolytic enzymes and synthesize the fast isoform of myosin.

Fig.12.22A ).Specifically,itappearsthatstimulationofadultfast-twitchmusclecellsatafrequencyconsistentwithslow-twitchmusclecellscanactivatetheCa++-dependentphosphatasecalcineurin,whichinturncandephosphorylateNFATandresultintranslocationofNFATfromthemyoplasmtothenucleus,followedbythetranscriptionofslow-twitchmusclegenes(andinhibitionoffast-twitchmusclegenes).Inaccordancewiththismechanism,expressionofconstitutivelyactiveNFATinfast-twitchmusclepromotestheexpressionofslow-twitchmyosinwhileinhibitingtheexpressionoffast-twitchmyosin.Thetranscriptionfactormyocyte enhancing factor 2 (MEF2) hasalsobeenimplicatedinthistransitionfromfast-twitchtoslow-twitchmuscle(see

Fig.12.22B ).ActivationofMEF2isthoughttoresultfromCa++/calmodulin-dependentphosphorylationofaninhibitorofMEF2:namely,histonedeacetylase(HDAC).

Intracellular [Ca++] appears to play an important role in expression of the slow myosin isoform. Slow-twitch muscle fibers have a higher resting level of intracellular Ca++ than do fast-twitch muscle fibers. In addition, chronic electrical stimulation of fast-twitch muscle is accompanied by a 2.5fold increase in resting myoplasmic [Ca++] that precedes the increased expression of slow-twitch myosin and decreased expression of fast-twitch myosin. Similarly, chronic elevation of intracellular Ca++ (approximately fivefold) in muscle cells expressing fast-twitch myosin induces a change in gene expression from the fast muscle myosin isoform to the slow myosin isoform within 8 days. An increase in citrate synthetase activity (an indicator of oxidative capacity) and a decrease in lactate dehydrogenase activity (an indicator of glycolytic capacity) accompany this Ca++-dependent

Muscle: action potential Surface membrane •Fig. 12.22. Molecular Signaling Pathways Contributing to the Transition From Fast-Twitch Muscle to Slow-Twitch Muscle. Chronicelectricalstimulationofafast-twitchmuscleinapatternconsistentwithaslow-twitchmuscleresultsindevelopmentoftheslow-twitchmusclephenotypebecauseofdephosphorylationofthetranscriptionfactornuclearfactorfromactivatedTcells(NFAT)bytheCa++/calmodulin-dependentproteinphosphatasecalcineurin(CaN);thisinturnresultsinnucleartranslocationofNFATandexpressionofslow-twitchmusclefibergenes(A). Activationofthetranscriptionfactormyocyteenhancerfactor2(MEF2)alsoappearstocontributetothisfibertypetransition(B), inwhichactivationofMEF2involvesCa++/calmodulin-dependentphosphorylationofaninhibitor,histonedeacetylase(HDAC).CaMK,Ca++/calmodulin-dependentproteinkinase;ECC,excitation-contractioncoupling;P,phosphorylationofHDAC.(FromLiuY,etal:.Signalingpathwaysinactivity-dependentfibertypeplasticityinadultskeletalmuscle.J Muscle Res Cell Motil. 2005;26:13-21.) transition from fast-twitch to slow-twitch myosin. These Ca++-dependent changes are reversible by a reduction of intracellular [Ca++].

Response to Exercise

Exercise physiologists identify three categories of training regimens and responses: learning, endurance, and strength training (

Table 12.3).

Typically, most athletic endeavors involve elements of all three. The learning aspect of training involves motivational factors, as well as neuromuscular coordination. This aspect of training does not involve adaptive changes in the muscle fibers per se. However, motor skills can persist for years without regular training, unlike the responses of muscle cells to exercise.

All healthy persons can maintain some level of continuous muscular activity that is supported by oxidative metabolism. This level can be greatly increased by a regular exercise regimen that is sufficient to induce adaptive responses. The adaptive response of skeletal muscle fibers to endurance exercise is mainly the result of an increase in the oxidative metabolic capacity of the motor units involved. This demand places an increased load on the cardiovascular and respiratory systems and increases the capacity of the heart and respiratory muscles. The latter effects are responsible for the principal health benefits associated with endurance exercise.

Muscle strength can be increased by regular massive efforts that involve most motor units. Such efforts recruit fast glycolytic motor units, as well as slow oxidative motor units. During these efforts, blood supply to the working muscles may be interrupted as tissue pressures rise above intravascular pressure. The reduced blood flow limits the duration of the contraction. Regular maximal-strength exercise, such as weightlifting, induces the synthesis of more myofibrils and hence hypertrophy of the active muscle cells. The increased stress also induces the growth of tendons and bones.

Endurance exercise does not cause fast motor units to become slow, nor does maximal muscular effort produce a shift from slow to fast motor units. Thus any practical exercise regimen, when superimposed on normal daily activities, probably does not alter muscle fiber phenotype.

Activities such as hiking or, in particular, downhill running, in which contracting muscles are stretched and lengthened too vigorously, are followed by more pain and stiffness than after comparable exercise that does not involve vigorous muscle stretching and lengthening (e.g., cycling). The resultant dull, aching pain develops slowly and reaches its peak within 24 to 48 hours. The pain is associated with reduced range of motion, stiffness, and weakness of the affected muscles. The prime factors that cause the pain are swelling and inflammation from injury to muscle cells, most commonly near the myotendinous junction. Type II motor units are affected more than type I motor units because the maximal force is highest in large cells, in which the loads imposed are approximately 60% greater than the maximal force that the cells can develop. Recovery is slow and depends on regeneration of the injured sarcomeres.

Biophysical Properties of Skeletal Muscle

The molecular mechanisms of muscle contraction described earlier underlie and are responsible for the biophysical properties of muscle. Historically, these biophysical properties were well described before elucidation of the molecular mechanisms of contraction. They remain important ways of describing muscle function.

When muscles contract, they generate force (often measured as tension or stress) and decrease in length. In examination of the biophysical properties of muscle, one of these parameters is usually held constant, and the other is measured after an experimental maneuver. Accordingly, an isometric contraction is one in which muscle length is held constant, and the force generated during the contraction is then measured. An isotonic contraction is one in which the force (or tone) is held constant, and the change in length of the muscle is then measured.

When a muscle at rest is stretched, it resists stretch by a force that increases slowly at first and then more rapidly as the extent of stretch increases (

Fig. 12.23 ). This purely passive property is due to the elasticity of the muscle tissue. If the muscle is stimulated to contract at these various lengths, a different relationship is obtained. Specifically, contractile force increases as muscle length is increased up to a point (designated LO to indicate optimal length). As the muscle is stretched beyond LO, contractile force decreases. This length-tension curve is consistent with the sliding filament theory, described previously. At a very 0 1 201 Length (fraction of optimum) Sarcomere length (µm) •Fig. 12.23. Length-Tension Relationship in Skeletal Muscle. A, Experimentalsetupinwhichmaximalisometrictetanictensionismeasuredatvariousmusclelengths.B, Howactivetensionwascalculatedatvariousmusclelengths(i.e.,bysubtractingpassivetensionfromtotaltensionateachmusclelength).C, Plotofactivetensionasafunctionofmusclelength,withthepredictedoverlapofthickandthinfilamentsatselectedpoints.

•Fig. 12.24. Force-Velocity Relationship of Skeletal Muscle. Theexperimentalsetupisshownontheright.Theinitialmusclelengthwaskeptconstant,buttheamountofweightthatthemusclehadtoliftduringtetanicstimulationvaried.Whilethesevariousamountsofweightwerelifted,muscle-shorteningvelocitywasmeasured.Seetextfordetails.F,force;V,velocity.

long sarcomere length (3.7 µm), actin filaments no longer overlap with myosin filaments, and so there is no contraction. As muscle length is decreased toward LO, the amount of overlap increases, and contractile force progressively increases. As sarcomere length decreases below 2 µm, the thin filaments collide in the middle of the sarcomere, the actin-myosin interaction is disturbed, and hence contractile force decreases. For construction of the length-tension curves, muscles were maintained at a given length, and then contractile force was measured (i.e., isometric contraction).

Thus the length-tension relationship supports the sliding filament theory of muscle contraction.

The velocity at which a muscle shortens is strongly dependent on the amount of force that the muscle must develop (

Fig. 12.24 ). In the absence of any load, the shortening velocity of the muscle is maximal (denoted as V0). V0 corresponds to the maximal cycling rate of the cross-bridges (i.e., it is proportional to the maximal rate of energy turnover [ATPase activity] by myosin). Thus V0 for fast-twitch muscle is higher than that for slow-twitch muscle. Increasing the load decreases the velocity of muscle shortening until, at maximal load, the muscle cannot lift the load and hence cannot shorten (zero velocity). Further increases in load result in stretching the muscle (negative velocity). The maximal isometric tension (i.e., force at which shortening velocity is zero) is proportional to the number of active cross-bridges between actin and myosin, and it is usually greater for fast-twitch motor units (because of the larger diameter of fast-twitch muscle fibers and greater number of muscle fibers in a typical fast-twitch motor unit). In

Fig. 12.24 , the power-stress curve reflects the rate of work done at each load and shows that the maximal rate of work was done at a submaximal load (namely, when the force of contraction was approximately 30% of the maximal tetanic tension). To calculate the latter curve, the x-and y-coordinates were simply multiplied, and then the product was plotted as a function of the x-coordinate.

1.

Skeletal muscle is composed of numerous muscle cells (muscle fibers) that are typically 10 to 80 µm in diameter and up to 25 cm in length. The appearance of striations in skeletal muscle is due to the highly organized arrangement of thick and thin filaments in the myofibrils of skeletal muscle fibers. The sarcomere is a contractile unit in skeletal muscle. Each sarcomere is approximately 2 µm in length at rest and is bounded by two Z lines. Sarcomeres are arranged in series along the length of the myofibril. Thin filaments, containing actin, extend from the Z line toward the center of the sarcomere. Thick filaments, containing myosin, are positioned in the center of the sarcomere and overlap the actin thin filaments. Muscle contraction results from the Ca++-dependent interaction of myosin and actin, in which myosin pulls the thin filaments toward the center of the sarcomere.

2.

Contraction of skeletal muscle is under control of the central nervous system (i.e., voluntary). Motor centers in the brain control the activity of α motor neurons in the ventral horns of the spinal cord. These α motor neurons, in turn, synapse on skeletal muscle fibers. Whereas each skeletal muscle fiber is innervated by only one motor neuron, a motor neuron innervates several muscle fibers within the muscle. A motor unit refers to all the muscle fibers innervated by a single motor neuron.

3.

The motor neuron initiates contraction of skeletal muscle by producing an action potential in the muscle fiber. As the action potential passes down the T tubules of the muscle fiber, dihydropyridine receptors (DHPRs) in the T tubules undergo conformational changes that result in the opening of neighboring SR Ca++ channels called ryanodine receptors (RYRs), which then release Ca++ to the myoplasm from the SR. The increase in myoplasmic Ca++ promotes muscle contraction by exposing myosin-binding sites on the actin thin filaments (a process that involves binding of Ca++ to troponin C, followed by movement of tropomyosin toward the groove in the thin filament). Myosin cross-bridges then appear to undergo a ratchet action, with the thin filaments pulled toward the center of the sarcomere and contracting the skeletal muscle fiber. Relaxation of the muscle follows as myoplasmic Ca++ is resequestered by Ca++-ATPase (SERCA) in the SR.

4.

The force of contraction can be increased by the activation of more motor neurons (i.e., recruiting of more muscle fibers) or by an increase in the frequency of action potentials in the muscle fiber, which produces tetany. The increase in force during tetanic contractions is due to prolonged elevation of intracellular [Ca++].

5.

The two basic types of skeletal muscle fibers are distinguished on the basis of their speed of contraction (i.e., fast-twitch versus slow-twitch). The difference in speed of contraction is attributed to the expression of different myosin isoforms that differ in myosin ATPase activity. In addition to the difference in myosin ATPase activity, fast-and slow-twitch muscles also differ in metabolic activity, fiber diameter, motor unit size, sensitivity to tetany, and recruitment pattern.

6.

Typically, slow-twitch muscles are recruited before fast-twitch muscle fibers because of the greater excitability of motor neurons innervating slow-twitch muscles. The high oxidative capacity of slow-twitch muscle fibers supports sustained contractile activity. Fast-twitch muscle fibers, in contrast, tend to be large and typically have low oxidative capacity and high glycolytic capacity. The fast-twitch motor units are thus best suited for short periods of activity when high levels of force are required.

7.

Fast-twitch muscle fibers can be converted to slow-twitch muscle fibers (and vice versa), depending on the stimulation pattern. Chronic electrical stimulation of a fast-twitch muscle results in the expression of slow-twitch myosin and decreased expression of fast-twitch myosin, along with an increase in oxidative capacity. The mechanism or mechanisms underlying this change in gene expression are unknown, but the change appears to be secondary to an elevation in resting intracellular [Ca++]. The Ca++-dependent phosphatase calcineurin and the transcription factor NFAT have been implicated in this transition from the fast-twitch to the slow-twitch phenotype. Ca++/calmodulin-dependent kinase and the transcription factor MEF2 may also participate in the phenotype transition.

8.

Skeletal muscle fibers atrophy after denervation. Muscle fibers depend on the activity of their motor nerves for maintenance of the differentiated phenotype. Reinnervation by axon growth along the original nerve sheath can reverse these changes. Skeletal muscle has a limited capacity to replace cells lost as a result of trauma or disease. Inhibition of the PI3K/Akt signaling pathways and activation of the FoxO pathway appears to contribute to the decreased rate of protein synthesis and the increased rate of protein degradation (respectively) observed during disuse atrophy. The increased protein degradation during atrophy is attributed to increases in both protease activity (e.g., activation of caspase 3) and ubiquitination (through elevated levels of ubiquitin ligases).

9.

Skeletal muscle exhibits considerable phenotypic plasticity. Normal growth is associated with cellular hypertrophy, caused by the addition of more myofibrils and more sarcomeres at the ends of the cell to match skeletal growth. Strength training induces cellular hypertrophy, whereas endurance training increases the

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Baldwin KM, Haddad F, Pandorf CE, Roy RR, Edgerton VR. Alterations in muscle mass and contractile phenotype in response to unloading models: role of transcriptional/pretranslational mechanisms. Front Physiol. 2013;4:284.

Burr AR, Molkentin JD. Genetic evidence in the mouse solidifies the calcium hypothesis of myofiber death in muscular dystrophy. Cell Death Differ. 2015;22:1402-1412.

oxidative capacity of all involved motor units. Training regimens cannot alter fiber type or the expression of myosin isoforms.

10.

Muscle fatigue during exercise is not due to depletion of ATP. The mechanism or mechanisms underlying exercise-induced fatigue are not known, although the accumulation of various metabolic products (lactate, Pi, ADP) has been implicated. In view of the importance of preventing depletion of myoplasmic ATP, which would affect the viability of the cell, it is likely that multiple mechanisms may have been developed to induce fatigue and hence lower the rate of ATP hydrolysis before the individual risks injury or death of the skeletal muscle cell.

11.

When the energy demands of an exercising muscle cannot be met by oxidative metabolism, an oxygen debt is incurred. Increased breathing during the recovery period after exercise reflects this oxygen debt. The greater the reliance on anaerobic metabolism to meet the energy requirements of muscle contraction, the greater the oxygen debt.

Gordon AM, Homsher E, Regnier M. Regulation of contraction in striated muscle. Physiol Rev. 2000;80(2):853-924.

Imbrici P, Altamura C, Pessia M, Mantegazza R, Desaphy JF, Camerino DC. ClC-1 chloride channels: state-of-the-art research and future challenges. Front Cell Neurosci. 2015;9:156.

Joyner MJ, Casey DP. Regulation of increased blood flow (hyperemia) to muscles during exercise: a hierarchy of competing physiological needs. Physiol Rev. 2015;95(2):549-601.

Nelson CR, Fitts RH. Effects of low cell pH and elevated inorganic phosphate on the pCa-force relationship in single muscle fibers at near-physiological temperatures. Am J Physiol Cell Physiol. 2014;306(7):C670-C678.

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Schiaffino S, Reggiani C. Fiber types in mammalian skeletal muscles. Physiol Rev. 2011;91(4):1447-1531.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

Describe the organization of cardiac muscle and how it meets the demands of the organ.

2.

Describe the molecular mechanisms involved in excitation-contraction coupling in cardiac muscle and its suitability for this organ.

3.

Describe the molecular mechanisms that lead to an increase in the force of contraction of the heart.

4.

Discuss the length-tension relationship and the force-velocity curve for cardiac muscle, including the molecular basis for both curves.

If the student has already completed on skeletal muscle, the student will be able to compare cardiac and skeletal muscle for each of the learning objectives just listed.

he function of the heart is to pump blood through the circulatory system, and this is accomplished by the highly organized contraction of cardiac muscle cells. Specifically, the cardiac muscle cells are connected together to form an electrical syncytium, with tight electrical and mechanical connections between adjacent cardiac muscle cells. An action potential initiated in a specialized region of the heart (e.g., the sinoatrial node) is therefore able to pass quickly throughout the heart to facilitate synchronized contraction of the cardiac muscle cells, which is important for the pumping action of the heart. Likewise, refilling of the heart requires synchronized relaxation of the heart; abnormal relaxation often results in pathological conditions.

This chapter begins with a description of the organization of cardiac muscle cells within the heart, including discussion of the tight electrical and mechanical connections. The mechanisms that underlie contraction, relaxation, and regulation of the force of contraction of cardiac muscle cells are also addressed. Although cardiac muscle and skeletal muscle are both striated muscles, they are significantly different in terms of organization, electrical and mechanical coupling, excitation-contraction coupling, and mechanisms to regulate the force of contraction. These differences are also highlighted.

Basic Organization of Cardiac Muscle Cells

Cardiac muscle cells are much smaller than skeletal muscle cells. Typically, cardiac muscle cells measure 10 µm in diameter and approximately 100 µm in length. As shown in

Fig. 13.1A , cardiac cells are connected to each other through intercalated disks, which include a combination of mechanical junctions and electrical connections. The mechanical connections, which keep the cells from pulling apart when contracting, include the fascia adherens and desmosomes. Gap junctions between cardiac muscle cells, on the other hand, provide electrical connections between cells to allow propagation of the action potential throughout the heart. Thus the arrangement of cardiac muscle cells within the heart is said to form an electrical and mechanical syncytium that allows a single action potential (generated within the sinoatrial node) to pass throughout the heart so that the heart can contract in a synchronous, wave-like manner. Blood vessels course through the myocardium.

The basic organization of thick and thin filaments in cardiac muscle cells is comparable with that in skeletal muscle (see ). Electron microscopy reveals repeating light and dark bands that represent I bands and A bands, respectively (see

Fig. 13.1B

Fig.

12.3 ). Thus cardiac muscle is classified as a striated muscle. The Z line transects the I band and represents the point of attachment of the thin filaments. The region between two adjacent Z lines represents the sarcomere, which is the contractile unit of the muscle cell. The thin filaments are composed of actin, tropomyosin, and troponin and extend into the A band. The A band is composed of thick filaments, along with some overlap of thin filaments. The thick filaments are composed of myosin and extend from the center of the sarcomere toward the Z lines.

Myosin filaments are formed by a tail-to-tail association of myosin molecules in the center of the sarcomere, followed by a head-to-tail association as the thick filament extends toward the Z lines. Thus the myosin filament is polarized and poised for pulling the actin filaments toward the center of the sarcomere. A cross-section view of the sarcomere near the end of the A band shows that each thick filament is surrounded by six thin filaments, and each thin filament receives cross-bridge attachments from three thick filaments. This complex array of thick and thin filaments is characteristic of both cardiac and skeletal muscle and helps • Fig. 13.1 A, Photomicrograph of cardiac muscle cells (magnification, ×210). Intercalated disks at either end of a muscle cell are identified in the lower left portion of the micrograph. The intercalated disk physically connects adjacent myocytes and, because of the presence of gap junctions, electrically couples the cells as well so that the muscle functions as an electrical and mechanical syncytium. B, Schematic representation of the organization of a sarcomere within a cardiac muscle cell. (A, From Telser A. Elsevier’s Integrated Histology. St. Louis: Mosby; 2007. B, Redrawn from Fawcett D, McNutt NS. The ultrastructure of the cat myocardium. I. Ventricular papillary muscle. J Cell Biol. 1969;42:1-45.) stabilize the filaments during muscle contraction (see

Fig.

12.3B , for the hexagonal array of thick and thin filaments in the sarcomere of striated muscle).

Several proteins may contribute to the organization of the thick and thin filaments, including meromyosin and C protein (in the center of the sarcomere), which appear to serve as a scaffold for organization of the thick filaments. Similarly, nebulin extends along the length of the actin filament and may serve as a scaffold for the thin filament. The actin filament is anchored to the Z line by α-actinin, whereas the protein tropomodulin resides at the end of the actin filament and regulates the length of the thin filament. These proteins are present in both cardiac and skeletal muscle cells.

The thick filaments are tethered to the Z lines by a large elastic protein called titin. Although titin was postulated to tether myosin to the Z lines and thus prevent overstretching of the sarcomere, there is evidence indicating that titin may participate in cell signaling (perhaps by acting as a stretch sensor and thus modulating protein synthesis in response to stress). Such signaling by titin has been observed in both cardiac and skeletal muscle cells. Moreover, genetic defects in titin result in atrophy of both cardiac and skeletal muscle cells and may contribute to both cardiac dysfunction and skeletal muscle dystrophies (termed titinopathies). Titin is also thought to contribute to the ability of cardiac muscle to increase force upon stretch (discussed in the later section “

Although both cardiac muscle and skeletal muscle contain an abundance of connective tissue, there is more connective tissue in the heart. The abundance of connective tissue in the heart helps prevent muscle rupture (as in skeletal muscle), but it also prevents overstretching of the heart. Length-tension analysis of cardiac muscle, for example, shows a dramatic increase in passive tension as cardiac muscle is stretched beyond its resting length. Skeletal muscle, in contrast, tolerates a much greater degree of stretch before passive tension increases to a comparable level. The reason for this difference between cardiac and skeletal muscle is not known, although one possibility is that stretch of skeletal muscle is typically limited by the range of motion of the joint, which in turn is limited by the ligaments/connective tissue surrounding the joint.

The heart, on the other hand, appears to rely on the abundance of connective tissue around cardiac muscle cells to prevent overstretching during periods of increased venous return. During intense exercise, for example, venous return may increase fivefold. However, the heart is capable of pumping this extra volume of blood into the arterial system with only minor changes in the ventricular volume of the heart (i.e., end-diastolic volume increases less than 20%). Although the abundance of connective tissue in the heart limits stretch of the heart during these periods of increased venous return, additional regulatory mechanisms help the heart pump the extra blood that it receives (as discussed in the section “ ”). Conversely, if the heart were to be overstretched, the contractile ability of cardiac muscle cells would be expected to decrease (because of decreased overlap of the thick and thin filaments), which would result in insufficient pumping, increased venous pressure, and perhaps pulmonary edema.

Within cardiac muscle cells, myofibrils are surrounded by the sarcoplasmic reticulum (SR), an internal network of membranes (see

Fig. 13.1B ). This is similar to the SR in skeletal muscle except that the SR in the heart is less dense and not as well developed. Terminal regions of the SR abut the T tubule or lie just below the sarcolemma (or both) and play a key role in the elevation of intracellular [Ca++] during an action potential. The mechanism by which an action potential initiates release of Ca++ in the heart, however, differs significantly from that in skeletal muscle (as discussed in the section “ ”). The heart contains an abundance of mitochondria; up to 30% of the volume of the heart is occupied by these organelles. The high density of mitochondria provides the heart with great oxidative capacity, more so than is typical in skeletal muscle.

The sarcolemma of cardiac muscle also contains invaginations (T tubules) comparable to those seen in skeletal muscle. In cardiac muscle, however, T tubules are positioned at the Z lines, whereas in mammalian skeletal muscle, T tubules are positioned at the ends of the I bands. In cardiac muscle, the connections between the T tubules and the SR are fewer than, and not as well developed as, those in skeletal muscle.

Familial cardiomyopathic hypertrophy (FCH) occursinapproximately0.2%ofthegeneralpopulationbutisaleadingcauseofsuddendeathinotherwisehealthyadults.Ithasbeenlinkedtogeneticdefectsinavarietyofproteinsincardiacsarcomeres,includingmyosin,troponin,tropomyosin,andmyosin-bindingproteinC,astructuralproteinlocatedinthemiddleoftheAbandofthesarcomere.FCHisanautosomaldominantdisease,andtransgenicstudiesindicatethatexpressionofonlyasmallamountofthemutatedproteincanresultindevelopmentofthecardiomyopathicphenotype.Moreover,mutationofasingleaminoacidinthemyosinmoleculeissufficienttoproducecardiomyopathichypertrophy.ThepathogenesisofFCH,however,isvariable,evenwithinafamilywithasinglegenedefect,intermsofbothonsetandseverity;thisvariabilitysuggeststhepresenceofmodifyingloci.

Control of Cardiac Muscle Activity

Cardiac muscle is an involuntary muscle with an intrinsic pacemaker. The pacemaker represents a specialized cell (located in the sinoatrial node of the right atrium) that is able to undergo spontaneous depolarization and generate action potentials. Of importance is that although several cells in the heart are able to depolarize spontaneously, the fastest spontaneous depolarizations occur in cells in the sinoatrial node. Moreover, once a given cell spontaneously depolarizes and fires an action potential, this action potential is then propagated throughout the heart (by specialized conduction pathways and cell-to-cell contact). Thus depolarization from only one cell is needed to initiate a wave of contraction in the heart (i.e., a heartbeat). The mechanisms underlying this spontaneous depolarization are discussed in depth in

Fig. 16.17 , once an action potential is initiated in the sinoatrial node, it is propagated between atrial cells via gap junctions, as well as through specialized conduction fibers in the atria. The action potential can pass throughout the atria within approximately 70 msec. For the action potential to reach the ventricles, it must pass through the atrioventricular node, after which the action potential passes throughout the ventricle via specialized conduction pathways (the bundle of His and the Purkinje system) and gap junctions in the intercalated disks of adjacent cardiac myocytes. The action potential can pass through the entire heart within 220 msec after initiation in the sinoatrial node. Because contraction of a cardiac muscle cell typically lasts 300 msec, this rapid conduction promotes nearly synchronous contraction of heart muscle cells. This is a very different scenario from that of skeletal muscle, in which cells are grouped into motor units that are recruited independently as the force of contraction is increased.

Blood and extracellular fluids typically contain 1 to 2 mmol/L of free Ca++ , and it has been known since the days of the physiologist Sidney Ringer (ca. 1882) that the heart requires extracellular Ca++ to contract. Thus an isolated heart typically continues to beat when perfused with a warm (37°C), oxygenated, physiological salt solution that contains approximately 2 mmol/L Ca++ (e.g., Tyrode’s solution), but it stops beating in the absence of extracellular . This cessation of contractions in Ca++-deficient media is also observed in hearts that are electrically stimulated, which further demonstrates the importance of extracellular Ca++ for contraction of cardiac muscle. This situation is quite different from that of skeletal muscle, which can contract in the total absence of extracellular Ca++ .

Action potentials in cardiac muscle are prolonged, lasting 150 to 300 msec (

Fig. 13.2 inset), which is substantially longer than the action potentials in skeletal muscle (≈5 msec). The long duration of the action potential in cardiac muscle is due to a slow inward Ca++ current through a voltage-gated L-type calcium channel in the sarcolemma. The amount of Ca++ coming into the cardiac muscle cell is relatively small and serves as a trigger for release of Ca++ from the SR. In the absence of extracellular Ca++ , an action potential can still be initiated in cardiac muscle, although it • Fig. 13.2 Excitation-contraction coupling in the heart requires Ca++ influx through L-type calcium channels in the sarcolemma and T tubules. See text for details. Inset shows time course of action potential (AP), intracellular Ca transient (Ca), and contraction. ATP, adenosine triphosphate; NCX, sarcolemmal 3Na+-Ca++ antiporter; PLN, phospholamban; RYR, ryanodine receptor. (Modified from Bers DM. Cardiac excitation-contraction coupling. Nature. 2002;415:198-205. Inset modified from Mountcastle VB: Medical Physiology, 13 ed. St Louis, Mosby, 1974; Brooks CM, Hoffman BF, Suckling EE, Orias O: Excitability of the Heart. New York, Grune & Stratton, 1955.) is considerably shorter in duration and unable to initiate a contraction. Thus influx of Ca++ during the action potential is crucial for triggering release of Ca++ from the SR and thus initiating contraction.

The L-type calcium channel is composed of five subunits (α1, α2, β, γ, and δ). The α1 subunit is also called the dihydropyridine receptor (DHPR) because it binds the dihydropyridine class of calcium channel–blocking drugs (e.g., nitrendipine and nimodipine). Although this channel complex is present in both skeletal and cardiac muscle, it serves very different functions in the two muscle types (discussed in the next paragraph).

In each cardiac muscle sarcomere, terminal regions of the SR abut T tubules and the sarcolemma (see

Figs. 13.1B 13.2 ). These junctional regions of the SR are enriched in ryanodine receptors (RYRs; a Ca++ release channel in the SR). The RYR is a Ca++-gated calcium channel, and so influx of Ca++ during an action potential is able to initiate release of Ca++ from the SR in cardiac muscle. The amount of Ca++ released into the cytosol from the SR is much greater than that entering the cytosol from the sarcolemma, although release of Ca++ from the SR does not occur without this entry of “trigger” Ca++ . In contrast, in skeletal muscle, release of Ca++ from the SR does not involve entry of Ca++ across the sarcolemma but instead results from a voltage-induced conformational change in the DHPR. Thus excitation-contraction coupling in cardiac muscle is termed electrochemical coupling (involving Ca++-induced release of Ca++), whereas excitation-contraction coupling in skeletal muscle is termed electromechanical coupling (involving direct interactions between the DHPR in the T tubule and the RYR in the SR). The basis for this difference in Ca++ release mechanisms appears to depend on the DHPR isoform because expression of cardiac DHPR in skeletal muscle cells results in a requirement for extracellular Ca++ for contraction of these modified skeletal muscle cells.

As in skeletal muscle, contraction of cardiac muscle is regulated by thin filaments, and an elevation in intracellular [Ca++] is necessary to promote actin-myosin interaction. At low (<50 nmol/L) intracellular [Ca++], binding of myosin to actin is blocked by tropomyosin. As cytosolic [Ca++] increases during an action potential, however, binding of Ca++ to troponin C results in a conformational change in the troponin/tropomyosin complex in which tropomyosin slips into the groove of the actin filament and exposes myosin binding sites on the actin filament. As long as cytosolic [Ca++] remains elevated, and hence myosin binding sites are exposed, myosin will bind to actin, undergo a ratchet action, and contract the cardiac muscle cell. Note that because myosin binding sites on actin are blocked at low [Ca++] and exposed during a rise in intracellular [Ca++], contraction of cardiac muscle is termed thin filament regulated. This is identical to the situation in skeletal muscle; in smooth muscle, in contrast, contraction is thick filament regulated (see

During a rise in intracellular [Ca++] and exposure of myosin-binding sites on actin, the myosin cross-bridges undergo a series of steps that result in contraction of the cardiac muscle cell. At rest, the myosin molecules are energized in that they have partially hydrolyzed adenosine triphosphate (ATP) to “cock the head” and are thus ready to interact with actin. An elevation in intracellular [Ca++] then exposes myosin-binding sites on actin and thus allows myosin to bind actin (step 1). The bound myosin subsequently undergoes a powerstroke in which the actin filament is pulled toward the center of the sarcomere (step 2). Adenosine diphosphate (ADP) and inorganic phosphate (Pi) are released from the myosin head during this step as the energy from ATP is used to contract the muscle. The myosin head moves approximately 70 nm during each ratchet action (cross-bridge cycle). Binding of ATP to myosin decreases the affinity of myosin for actin and thus allows myosin to release from actin (step 3). Myosin then partially hydrolyzes the bound ATP to reenergize (“cock”) the head (step 4) and ready the cross-bridge for another cycle. This four-step cycle is identical to that described for skeletal muscle (see

Fig. 12.13).

Cardiac muscle and skeletal muscle differ, however, in the level of intracellular [Ca++] attained after an action potential and hence in the number of actin-myosin interactions. In skeletal muscle, intracellular [Ca++] rises and the number of actin-myosin interactions is high after an action potential. In cardiac muscle, the rise in intracellular [Ca++] can be regulated, which affords the heart an important means of modulating the force of contraction without recruiting more muscle cells or undergoing tetany. Recall that in the heart, all the muscle cells are activated during a contraction, and so recruiting more muscle cells is not an option. Moreover, tetany of cardiac muscle cells would prevent any pumping action and thus be fatal. Consequently, the heart relies on different means of increasing the force of contraction, including varying the amplitude of the intracellular Ca++ transient.

Relaxation of Cardiac Muscle

Relaxation of skeletal muscle simply requires reaccumulation of Ca++ by the SR through the action of the sarcoplasmic endoplasmic reticulum calcium-ATPase (SERCA), also known as the SR Ca++ pump. Although SERCA plays a key role in the decrease in cytosolic [Ca++] in cardiac muscle, the process is more complex than that in skeletal muscle because some trigger Ca++ enters the cardiac muscle cell through the sarcolemmal calcium channels during each action potential. A mechanism must therefore exist to extrude this trigger Ca++; otherwise, the amount of Ca++ in the SR would continuously increase, and Ca++ overload would result. In particular, some Ca++ is extruded from the cardiac muscle cell though the sarcolemmal 3Na+-Ca++ antiporter and a sarcolemmal Ca++ pump (Fig. 13.2 ). The extracellular [Ca++] is in the millimolar range, whereas the amount of intracellular [Ca++] is submicromolar, and so extrusion of Ca++ is accomplished against a large chemical gradient. Similarly, [Na+] is considerably higher in the extracellular media than within the cell. The antiporter uses the Na+ gradient across the cell to power the uphill movement of Ca++ out of the cell. Because three Na+ ions enter the cell in exchange for one Ca++ ion, the 3Na+-Ca++ antiporter is electrogenic and creates a depolarizing current. The sarcolemmal Ca++ pump, on the other hand, uses the energy in ATP to extrude Ca++ from the cell. Both extrusion mechanisms and SERCA thus contribute to the relaxation of cardiac muscle by decreasing cytosolic [Ca++].

Although the interaction of actin and myosin requires a relatively small increase in free intracellular [Ca++], the abundance of Ca++-binding proteins in the myoplasm necessitates a much larger increase in total intracellular [Ca++]. The resting intracellular [Ca++] is approximately 50 to 100 nmol/L; half-maximal force of contraction requires approximately 600 nmol/L of free Ca++ . However, because of Ca++-binding proteins such as parvalbumin and troponin C, the total myoplasmic concentration must increase by 70 µmol/L. As already noted, much of this increase in total myoplasmic [Ca++] occurs through release of Ca++ from the SR. In a number of species, including rabbits, dogs, cats, guinea pigs, and humans, uptake and release of Ca++ by the SR account for approximately 70% of the intracellular Ca++ transient. Thus up to 30% of the rise in intracellular [Ca++] may be attributable to influx of Ca++ through voltage-gated calcium channels in the sarcolemma, and the 3Na+-Ca++ antiporter contributes significantly to Ca++ extrusion during relaxation.

The sarcolemmal Ca++ pump is in lower abundance than the 3Na+-Ca++ antiporter but has a higher affinity for Ca++ and thus may contribute more to the regulation of resting intracellular [Ca++] (see

Fig. 13.2 ). The relative contribution of the Ca++ extrusion mechanisms, however, varies between species. For example, rat and mouse myocytes rely primarily on Ca++ reuptake by the SR (i.e., the SR accounts for 92% of Ca++ transport).

Regulation of the Force of Contraction

Because the heart represents an electrical syncytium, in which all the cardiac muscle cells contract during a single beat, it is not possible to increase the force of contraction by recruiting more muscle cells. Moreover, tetany of the heart would be lethal because it would defeat the critical pumping action of the heart. The heart has therefore developed alternative strategies to increase the force of contraction. The long duration of the action potential found in cardiac muscle, which is due to activation of the voltage-gated L-type calcium channel, results in a long refractory period, which in turn prevents tetany. Modulation of Ca++ influx through L-type calcium channels during an action potential, however, provides the heart with a mechanism to alter cytosolic [Ca++] and hence the force of contraction.

A simple means of modulating the force of contraction of cardiac muscle cells in vitro is to vary extracellular [Ca++]. As noted previously, contraction of the heart requires extracellular Ca++ . Decreasing extracellular [Ca++] from a normal range of 1 to 2 mmol/L to 0.5 mmol/L, for example, reduces the force of the contraction. This reduction in force of contraction is not associated with a change in the duration of the contraction because the kinetic characteristics of Ca++ sequestration by the SR and Ca++ extrusion have not been modified. Although this approach of varying extracellular [Ca++] to alter the force of contraction is demonstrable in vitro, it is not a common means of modulating the force of cardiac contraction in vivo.

In vivo, an increase in the size of the intracellular Ca++ transient and hence the force of contraction occurs in response to sympathetic stimulation (see the section

Chapter 18). Sympathetic stimulation often occurs during periods of excitement or fright and involves activation of β-adrenergic receptors on the heart by norepinephrine (released from nerve terminals in the heart) or epinephrine (released from the adrenal medulla into the bloodstream). As shown in

Fig. 13.3 , the β-adrenergic agonist isoproterenol results in a dramatic increase in the size of the intracellular Ca++ transient and, consequently, a more forceful contraction. An increase in the force of contraction is termed positive inotropy. Typically, the rate of relaxation accompanying this β-adrenergic stimulation also increases, which results in a shorter contraction. The increase in the rate of muscle relaxation is termed positive lusitropy. The frequency of contractions of the heart also increases with β-adrenergic stimulation and is termed positive chronotropy. Thus β-adrenergic stimulation of the heart produces stronger, briefer, and more frequent contractions.

The sympathetic nervous system is stimulated when a human or an animal becomes excited, and it is said to prepare the individual for “fight or flight.” In the case of the heart, increased levels of the adrenal medullary hormone epinephrine or the sympathetic neurotransmitter norepinephrine activate β-adrenergic receptors on the cardiac muscle cells, which in turn activates adenylate cyclase, increases cyclic adenosine monophosphate (cAMP), and thus promotes cAMP-dependent phosphorylation of numerous proteins in cardiac muscle cells (

Fig. 13.4

Both voltage-gated L-type calcium channels (responsible for the trigger Ca++) and a protein associated with SERCA, called phospholamban, are phosphorylated by cAMPdependent protein kinase. The combined action of these phosphorylations increases the amount of Ca++ in the SR. Specifically, phosphorylation of the sarcolemmal calcium channel causes more trigger Ca++ to enter the cell, and phosphorylation of phospholamban increases the activity of SERCA, thereby allowing the SR to accumulate more Ca++ before it is extruded by the 3Na+-Ca++ antiporter and the sarcolemmal Ca++ pump. The net result is that the SR releases more Ca++ into the cytosol during the next action potential, which promotes more actin-myosin interactions and hence greater force of contraction (see

Fig. 13.3 ). The increased activity of SERCA after sympathetic stimulation also results in a shortened contraction because of the rapid reaccumulation of Ca++ by the SR. This in turn allows the heart to increase its rate of relaxation. An additional consequence of sympathetic stimulation is an increase in heart rate through a direct effect on the pacemaker cells (see •Fig. 13.3 Stimulationofβ-adrenergicreceptorsintheheartincreasestheforceofcontraction.Electricalstimulationofmyocardiumresultsinatransientriseinintracellular[Ca++]andproductionofforce(A). Isoproterenol(aβ-adrenergicreceptoragonist)increasestheamplitudeoftheintracellularCa++transientandhencetheamountofforcegenerated(B). A Control B +Isoproterenol Ca Ca Force Force 0.5 sec

Additional proteins and some micropeptides also appear to be associated with SERCA and influence SR calcium transport. This includes the 34–amino acid peptide dwarf open reading frame (DWORF), which increases SERCA calcium affinity (

Fig. 13.5 ), apparently by displacing phospholamban. DWORF was identified in putative non-coding RNA.

Themechanismsunderlyingtheresponseofthehearttoβ-adrenergicstimulationarecomplexandinvolvecAMPdependentphosphorylationofseveralproteins.AnA kinase anchoring protein (AKAP) hasbeenshowntobecloselyassociatedwiththeL-typecalciumchannelintheheart,therebypositioningcAMP-dependent protein kinase closetothechannelandfacilitatingcAMP-dependentphosphorylationofthischannelduringsympatheticstimulation.HowthesecAMP-dependentphosphorylationsincreasetheamplitudeoftheintracellularCa++ transientand,insodoing,resultinamoreforceful,briefercardiaccontractionisdiscussedingeneraltermslater(seealso

Mutationsinthecardiacryanodinereceptor(RYR2)havebeenassociatedwithcardiacarrhythmias.Specifically,catecholaminergicpolymorphicventriculartachycardia(CPVT)isaninheritedautosomaldominantdiseasethatistypicallymanifestedduringchildhoodasanexercise-inducedtachycardiathatcanprogresstoarrhythmiasduringexercise(orstress)andresultinsuddendeath.Approximately40%ofpatientswithCPVTexhibitadefectinRYR2thathasbeenassociatedwithincreasedreleaseofCa++ fromtheSR.ThemutationinRYR2mayinvolvesubstitutionofahighlyconservedaminoacid,whichdiffersfrommalignanthyperthermia,inwhichsplicingerrorsordeletionswithinRYRhavebeenreported.Itishypothesizedthatduringperiodsofexerciseorstress,increasedlevelsofintracellularCa++ (becauseofthecombinedeffectsofβ-adrenergicstimulationandincreasedactivityofthemutatedRYR2)promotethedevelopmentofdelayedafterdepolarizationsandhencearrhythmias.Elevationofintracellular[Ca++]duringdiastoleisthoughttopromotethedevelopmentofdelayedafterdepolarizationsthroughactivationofthe3Na+-Ca++ antiporter,whereinCa++ extrusionduringdiastoleresultsinanetinwardcurrentsufficienttodepolarizethecelltothethresholdforanactionpotential.TreatmentofCPVTinvolvesantiadrenergictherapy(withβ-adrenergicantagonists)or(forunresponsivepatients)animplanteddefibrillator.

Stretching of the heart increases the force of contraction both in vivo and in vitro and is an intrinsic mechanism for regulating contractile force. In contrast, skeletal muscle typically exhibits maximal tension at resting length. Stretching of the heart in vivo occurs during times of increased venous return of blood to the heart (e.g., during exercise or when the heart rate is slowed, or both). The Frank-Starling law of the heart refers to this ability of the heart to increase its force of contraction when stretched, which occurs at •Fig. 13.4 Sympatheticstimulationoftheheartresultsinanincreaseincytosoliccyclicadenosinemonophosphate(cAMP)andhencephosphorylationofseveralproteinsbyproteinkinaseA(PKA).AnAkinaseanchorprotein(AKAP)adjacenttotheL-typecalciumchannelfacilitatesphosphorylationofthischannelandpossiblynearbysarcoplasmicreticulumcalciumchannels.OtherproteinsphosphorylatedbyPKAincludephospholamban(PLN)andtroponinI.Muscarinicagonists(e.g.,acetylcholine[ACh]),ontheotherhand,inhibitthissympatheticcascadebyinhibitingtheproductionofcAMPbyadenylatecyclase(AC).β-AR,β-adrenergicreceptor;ATP,adenosinetriphosphate;Gi,inhibitoryGprotein;M2Rec,muscarinicacetylcholineM2receptor;Reg,regeneratinggenereceptor.(RedrawnfromBersDM.Cardiacexcitation-contractioncoupling.Nature 2002;415:198.) times of increased venous return (Fig. 13.6A

The importance of this mechanism is that it helps the heart pump whatever volume of blood it receives. Thus when the heart receives a lot of blood, the ventricles are stretched, and the force of contraction is increased, which ensures ejection of this extra volume of blood. Stretching of cardiac muscle also increases passive tension, which helps prevent overstretching of the heart. This passive resistance in the heart is greater than that in skeletal muscle and is attributed to both extracellular matrix (connective tissue) and intracellular elastic proteins (titin).

This stretch-induced increase in force of contraction of cardiac muscle occurs over a narrow range of sarcomere lengths (ca. 1.6-2.3 um), resulting in a steep length-dependent activation of contraction. This ascending limb of the length-tension relationship in cardiac muscle is much steeper than that seen in skeletal muscle. It is important to note that this stretch-induced increase in force can occur within a singe heart beat.

The mechanism(s) underlying this stretch-induced increase in force of contraction of cardiac muscle is controversial, but appears to involve changes in the overlap of the thick and thin filaments as well as stretch-induced increase in the Ca++ sensitivity of contraction (

Fig. 13.6B of the stretch-induced increase in force of contraction has been attributed to an increase in Ca++ sensitivity, whereas the remaining 40% of the stretch-induced increase in force of contraction has been attributed to changes in the overlap of the thick and thin filaments. The changes in myofilament overlap, however, are less likely to contribute to the continued increase in force of cardiac contraction as sarcomere length increases from 2.0um to 2.3 um, as this region is thought to represent a region of optimal overlap of myofilaments (and represents a plateau in the length tension relationship in skeletal muscle).

The mechanism(s) contributing to the stretch-induced increase in Ca++ sensitivity of cardiac contraction are not clear, but appears to involve the intracellular elastic protein titin, as well as regulatory proteins (e.g., troponinC).

As in skeletal muscle, myosin uses the energy in ATP to generate force, so the ATP pool, which is small, must be continually replenished. Typically, this replenishment of ATP pools is accomplished by aerobic metabolism, including the oxidation of fats and carbohydrates. During times of ischemia, the creatine phosphate pool, which converts ADP to ATP, may decrease. As in skeletal muscle, the

In rat ventricular trabecular muscle, approximately 60% creatine phosphate pool is small.

276 SECTION3Berne & Levy Physiology ContractionSarcomereCa2˜Ca2˜Ca2˜Sarcomere RelaxationSarcoplasm MyocyteSarcoplasmic reticulum RyRSERCASERCAPLNDWORF A B C 125 100 75 50 25 0 250 200 150 100 50 0 WT Tg KO % Vmax 8.0 7.5 7.0 6.5 6.0 5.5 pCa * * WT Tg KO KCa[Ca2˜]free (nM) •Fig. 13.5 Asmall(34–aminoacid)peptideencodedbyalongnoncodingRNAappearstoimprovesarcoplasmicreticulumfunctionandmyocyteperformancebycounteringtheinhibitoryeffectofphospholamban(PLN)(asdepictedintheworkingmodelshowninpanelA).TheabilityofDWORFtoincreasecardiacSRCauptakeisshowninpanelsBandC,whereoverexpressionofDWORFincardiomyocytes(labeledTg)increasedtheCasensitivityofSRCatransport,whereasknockoutofDWORF(labeledKO)hadthereverseeffect.DWORF,dwarfopenreadingframe;KO,knockout;SERCA,sarcoplasmicendoplasmicreticulumcalcium-ATPase;Tg,transgenic;WT,wild-type.(FromNelsonBR,MakarewichCA,AndersonDM,etal.ApeptideencodedbyatranscriptannotatedaslongnoncodingRNAenhancesSERCAactivityinmuscle.Science. 2016;351:271-275.)

When cardiac muscle is completely deprived of O2 because of occlusion of a coronary vessel (i.e., stopped-flow ischemia), contractions quickly cease (within 30 seconds). This is not due to depletion of either ATP or creatine phosphate because these levels decline more slowly. Even after 10 minutes of stopped-flow ischemia, when creatine phosphate levels are near zero and only 20% of the ATP remains, reperfusion can restore these energy stores, as well as contractile ability. However, prolonging the stopped-flow ischemia for 20 minutes results in further drops in ATP, so that reperfusion has considerably less effect, with only limited restoration of ATP and creatine phosphate levels or contractile activity.

Exercise such as endurance running can increase the size of the heart as a result of hypertrophy of individual cardiac muscle cells. Concomitant with this enlarged so-called athlete’s heart is improved cardiac performance, as assessed by an increase in stroke volume, increased oxygen consumption, and preserved relaxation. Thus the athlete’s heart represents an example of “physiological hypertrophy,” with beneficial contractile effects.

In contrast, if exposed to chronic pressure overload, the heart may undergo either concentric left ventricular hypertrophy or dilated left ventricular hypertrophy, which causes impairment of function. Details regarding the morphological, functional, and mechanistic differences between these various types of hypertrophy can be found elsewhere in this textbook (see

Chapter 18).

Concentric hypertrophy is characterized by thickening of the left ventricular wall and represents a compensatory hypertrophy to the increased load. Dilated hypertrophy is characterized by increased ventricular volume (end-diastolic volume). Both concentric/compensatory left ventricular hypertrophy and dilated left ventricular hypertrophy have been shown to exhibit decreased contractile response to β-adrenergic stimulation, which limits the contractile reserve. In dilated left ventricular hypertrophy, normal •Fig. 13.6 Stretchingoftheheartincreasestheforceofcontraction(A). ThisisattributabletobothanincreaseinthemaximalforceofcontractionandanincreaseinthesensitivityofcontractiontoCa++ (B). ItreflectsanintrinsicregulatoryprocessreferredtoastheFrank-Starling law of the heart. (B,RedrawnfromDobeshD,KonhilasJ,deTombeP.Cooperativeactivationincardiacmuscle:impactofsarcomerelength.Am J Physiol Heart Circ Physiol. 2002;282:H1055-H1062.) contractile function, along with the Frank-Starling response, may also be impaired.

The cellular and molecular mechanisms underlying the development of cardiac hypertrophy are not clear, although an elevation in intracellular [Ca++] has been implicated.

The link or links between cardiac hypertrophy, decreased cardiac performance, and impaired β-adrenergic response during chronic pressure overload are unclear. Decreased cardiac performance has been attributed to dysregulation of intracellular [Ca++]. Alterations in the level, activity, and phosphorylation status of a variety of proteins, including L-type calcium channels, phospholamban, SERCA, and RYR, have all been implicated in the Ca++ dysregulation associated with a failing heart (pathological hypertrophy).

A microRNA (miR-222) has been shown to be important for cardiac growth in response to exercise. It also appeared to inhibit maladaptive remodeling of the heart after ischemia/ reperfusion injury.

Amodestelevationinintracellular[Ca++](asaresultofincreasedcontractileactivity,forexample),hasbeenproposedtoactivateaCa++/calmodulin-dependentproteinphosphatase(calcineurin) thatcandephosphorylatethetranscriptionfactornuclear factor of activated T cells (NFAT), therebyfacilitatingtranslocationofNFATtothenucleusandultimatelypromotingproteinsynthesisandthushypertrophy(

Fig.13.7 ).ActivationofCa++/calmodulin-dependentproteinkinasehasalsobeenimplicatedinactivationofthetranscriptionfactormyocyte enhancer factor 2 (MEF2) bypromotingthedissociation(nuclearexport)ofaninhibitorofMEF2(namely,histone deacetylase [HDAC]).

Theimpairedβ-adrenergicresponseofcardiacmuscleafterchronicpressureoverloadinvolves,atleastinpart,adecreaseinβ-adrenergicreceptorsbecauseofinternalization.Bothphosphatidylinositol-3-kinase (PI3K) andβ-adrenergic receptor kinase 1 havebeenimplicatedintheinternalizationofβ-adrenergicreceptors.

Highbloodpressure,defectsinheartvalves,andventricularwallsweakenedasaresultofmyocardialinfarctioncanallleadtoheartfailure,aleadingcauseofdeath.Heartfailuremaybeseenwiththickeningofthewallsoftheventricleorwithdilation(i.e.,increasedvolume)oftheventricles.

Resultsofstudiessuggestthatdilatedcardiomyopathycanbepreventedinananimalmodelbydownregulatingphospholamban.ThemechanismunderlyingthispreventiveeffectofphospholambandownregulationisthoughttoinvolveanincreaseinCa++ uptakeactivityintheSRbecausephospholambantypicallyinhibitsSERCA.IncreasedactivityofSERCAwouldfacilitaterelaxationoftheheartasaresultofrapidCa++ uptakebytheSR.Inaddition,theforceofcontractionisincreasedbecausemoreCa++ isavailableforrelease.IncreasedCa++ uptakebytheSRmayalsodecreaseactivationoftheCa++-dependentphosphatasesthathavebeenimplicatedinthedevelopmentofcardiachypertrophy.

There is evidence that cardiac hypertrophy may not be associated with some functional impairments. Intermittent aortic constrictions, for example, result in decreased β-adrenergic signaling, decreased capillary density, and decreased SERCA2 levels, without evidence of hypertrophy. Activation of PI3K appears to be involved in this response.

278 SECTION3Berne & Levy Physiology Activity Ca++ Other transducers? Other effectors? Reprogramming Gene Expression Myocyte Hypertrophy/Tissue Remodeling CalcineurinCaMK Other targets? NFAT NFAT GSK3 HDAC CsA Cabin MCIP Agonists MEF2 GATA P •Fig. 13.7 Calcium-dependentactivationofcalcineurinandcalmodulin-dependentproteinkinasehavebeenimplicatedinthedevelopmentofcardiachypertrophyandinvolveactivationofthefollowingtranscriptionfactors:nuclearfactorofactivatedTcells(NFAT),transcriptionfactorbindingtoDNAsequenceGATA(GATA),andmyocyteenhancerfactor2(MEF2).Cabin,calcineurin-bindingprotein/inhibitor;CaMK,Ca++/calmodulin-dependentproteinkinase;CsA,cyclosporine;GSK3,glycogensynthasekinase3;HDAC,histonedeacetylase;MCIP,modulatorycalcineurin-interactingprotein.(RedrawnfromOlsonEN,WilliamsRS.Calcineurinsignalingandmuscleremodeling.Cell. 2000;101:689-692.) 1.

Cardiac muscle is an involuntary, striated muscle. Cardiac muscle cells are relatively small (10 µm × 100 µm) and form an electrical syncytium with tight electrical and mechanical connections between adjacent cardiac muscle cells. Action potentials are initiated in the sinoatrial node and spread quickly throughout the heart to allow synchronous contraction, a feature important for the pumping action of the heart.

2.

Contraction of cardiac muscle involves the Ca++dependent interaction of actin and myosin filaments, as in skeletal muscle. However, unlike skeletal muscle, cardiac muscle requires an influx of extracellular Ca++ . Specifically, the influx of Ca++ during an action potential triggers release of Ca++ from the SR, which then promotes actin-myosin interaction and contraction.

3.

Relaxation of cardiac muscle involves reaccumulation of Ca++ by the SR and extrusion of Ca++ from the cell via the 3Na+-Ca++ antiporter and the sarcolemmal Ca++ pump. The Ca++ pump of the SR is associated with numerous proteins (forming a regulosome), including some endogenous micropeptide inhibitors and activators.

4.

The force of contraction of cardiac muscle is increased by stretch (Frank-Starling Law of the Heart) and by sympathetic stimulation. Skeletal muscle, in contrast, increases force by recruiting more muscle fibers or by tetany.

5.

Hypertrophy of the heart can occur in response to exercise, chronic pressure overload, or genetic mutations. The cardiac hypertrophy resulting from exercise is typically beneficial, with improved cardiac performance, increased oxygen consumption, and normal relaxation. Chronic pressure overload, on the other hand, can result in cardiac hypertrophy that is initially associated with a decreased β-adrenergic response but may progress to dilated cardiac hypertrophy, characterized by decreased contractile ability. Genetic mutations resulting in cardiac hypertrophy include familial hypertrophic

Anderson DM, Anderson KM, Chang CL, et al. A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell. 2015;160:595-606.

Endoh M. Cardiac Ca2+ signaling and Ca2+ sensitizers. Circ J. 2008;72:1915-1925.

Haghighi K, Bidwell P, Kranias EG. Phospholamban interactome in cardiac contractility and survival: a new vision of an old friend. J Mol Cell Cardiol. 2014;77:160-167.

Hidalgo C, Granzier H. Tuning the molecular giant titin through phosphorylation: role in health and disease. Trends Cardiovasc Med. 2013;23:165-171.

cardiomyopathy, in which a mutation in a single intracellular protein may alter contractile function and promote a hypertrophic response. Researchers have identified a microRNA that appears to contribute to the exercise-induced hypertrophy of the heart and to inhibit maladaptide remodeling after ischemia/reperfusion injury.

Kobirumaki-Shimozawa F, Inoue T, Shintani SA, et al. Cardiac thin filament regulation and the Frank–Starling mechanism. J Physiol Sci. 2014;64:221-232.

Marks AR. Calcium cycling proteins and heart failure: mechanisms and therapeutics. J Clin Invest. 2013;123(1):46-52.

Tao L, Bei Y, Zhang H, Xiao J, Li X. Exercise for the heart: signaling pathways. Oncotarget. 2015;6:20773-20784.

Williams GS, Boyman L, Lederer WJ. Mitochondrial calcium and the regulation of metabolism in the heart. J Mol Cell Cardiol. 2015;78:35-45.

Upon completion of this chapter the student should be able to answer the following questions: 1.

Describe the organization of smooth muscle in various tissues, and how it meets the demands of each tissue/ organ.

2.

Discuss the mechanisms that promote contraction and relaxation of smooth muscle in the vasculature and various organs.

3.

Describe the autoregulatory mechanism by which an artery can maintain relatively constant blood flow to a tissue over a broad range of perfusion pressures.

4.

Describe the basis and utility of a transition from phasic contraction to tonic contraction.

5.

Discuss the length-tension curves and force-velocity curves for smooth muscle, and the molecular basis for each of these curves.

If the student has already completed on skeletal muscle and cardiac muscle, comparison of all three tissues for each of the learning objectives listed should be possible.

onstriated, or smooth, muscle cells are a major component of hollow organs such as the alimentary canal, airways, vasculature, and urogenital tract. Contraction of smooth muscle serves to alter the dimensions of the organ, which may result in either propelling the contents of the organ (as in peristalsis of the intestine) or increasing the resistance to flow (as in vasoconstriction). The basic mechanism underlying contraction of smooth muscle involves an interaction of myosin with actin (as in striated muscle), although there are some important differences. Specifically, contraction of smooth muscle is thick-filament regulated and requires an alteration in myosin before it can interact with actin, whereas contraction of striated muscle is thin-filament regulated and requires movement of the troponin-tropomyosin complex on the actin filament before myosin can bind to actin. Smooth muscle can contract in response to either electrical or hormonal signals and exhibits the ability to remain contracted for extended periods at low levels of energy consumption, which is important for functions such as maintaining vascular tone and hence blood pressure. Thus regulation of contraction of smooth muscle is complex, sometimes involving multiple intracellular signaling cascades. In the present chapter, effort is made to identify mechanisms underlying this diverse regulation of smooth muscle contraction and, when appropriate, compare these regulatory mechanisms with those observed in striated muscle. Alterations in smooth muscle function/ regulation that have been implicated in various pathological conditions are also discussed.

Overview of Smooth Muscle

Types of Smooth Muscle

Smooth muscle has been subdivided into two groups: single unit and multiunit. In single-unit smooth muscle the smooth muscle cells are electrically coupled such that electrical stimulation of one cell is followed by stimulation of adjacent smooth muscle cells. This results in a wave of contraction, as in peristalsis. Moreover, this wave of electrical activity, and hence contraction, in single-unit smooth muscle may be initiated by a pacemaker cell (i.e., a smooth muscle cell that exhibits spontaneous depolarization). In contrast, multiunit smooth muscle cells are not electrically coupled, so stimulation of one cell does not necessarily result in activation of adjacent smooth muscle cells. Examples of multiunit smooth muscle include the vas deferens of the male genital tract and the iris of the eye. Smooth muscle, however, is even more diverse, with the single-unit and multiunit classifications representing ends of a spectrum. The terms single-unit and multiunit represent an oversimplification, however; many smooth muscles are modulated by a combination of neural elements with at least some degree of cell-to-cell coupling and locally produced activators or inhibitors that also promote a somewhat coordinated response of smooth muscles.

A second consideration when discussing types of smooth muscle is the activity pattern (

Fig. 14.1 ). In some organs the smooth muscle cells contract rhythmically or intermittently, whereas in other organs the smooth muscle cells are continuously active and maintain a level of “tone.” Smooth muscle exhibiting rhythmic or intermittent activity is termed phasic smooth muscle and includes smooth muscles in the walls of the gastrointestinal (GI) and urogenital tracts. Such phasic smooth muscle corresponds to the single-unit category described earlier because the smooth muscle cells contract in response to action potentials that propagate from cell to cell. Smooth muscle that is continuously active, on the other hand, is termed tonic smooth muscle.

Vascular smooth muscle, respiratory smooth muscle, and some sphincters are continuously active. The continuous partial activation of tonic smooth muscle is not associated with action potentials, although it is proportional to membrane potential. Tonic smooth muscle would thus correspond to the multiunit smooth muscle described earlier. Phasic and tonic contractions of smooth muscle result from interactions of actin and myosin filaments, although as discussed later in this chapter, there is a change in cross-bridge cycling kinetics during tonic contraction, such that the smooth muscle can maintain force at low energy cost.

•Fig. 14.1 Somecontractileactivitypatternsexhibitedbysmoothmuscles.Tonicsmoothmusclesarenormallycontractedandgenerateavariablesteady-stateforce.Examplesaresphincters,bloodvessels,andairways.Phasicsmoothmusclescommonlyexhibitrhythmiccontractions(e.g.,peristalsisintheGItract)butmaycontractintermittentlyduringphysiologicalactivitiesundervoluntarycontrol(e.g.,voidingofurinefromurinarybladder,swallowing).

Structure of Smooth Muscle Cells

Fig. 14.2 ). Blood vessels and airways exhibit a simple tubular structure in which the smooth muscle cells are arranged circumferentially, so contraction reduces the diameter of the tube. This contraction increases resistance to the flow of blood or air but has little effect on the length of the organ. Smooth muscle cell organization is more complex in the GI tract. Layers of smooth muscle in both circumferential and longitudinal orientations provide the mechanical action for mixing food and also propelling the luminal contents from the mouth to the anus. Coordination between these layers depends on a complex system of autonomic nerves linked by plexuses. These plexuses cells in the walls of these organs contributes to their ability to reduce internal volume to almost zero during urination are located between the two muscle layers. The smooth muscle in the walls of saccular structures such as the urinary bladder or rectum allows the organ to increase in size with accumulation of urine or feces. The varied arrangement of

Normally partially contracted (tone) Blood vessels, airways Stomach, intestines Esophagus, urinary bladder Phasically active or defecation. Smooth muscle cells in hollow organs occur in a spectrum of forms, depending on their function and mechanical loads.

In all hollow organs the smooth muscle is separated from the contents of the organ by other cellular elements, which may be as simple as vascular endothelium or as complex as the mucosa of the digestive tract. The walls of hollow organs also contain large amounts of connective tissue that bear an increasing share of the wall stress as organ volume increases.

The following sections describe the structural components that enable smooth muscle to set or alter hollow organ volume. These components include contractile and regulatory proteins, force-transmitting systems such as the •Fig. 14.2 Scanningelectronmicrographsofsmoothmuscle.A,Musculararteriolewithfusiformsmoothmusclecellsinacircularorientation(bar, 20µm).B,Superimposedimagesofcircular(below) andlongitudinal(above) layersofintestinalsmoothmusclesandwichingneuralcomponentsofthemyentericplexus(asterisk) (bar, 50mm).C,Rectangularsmoothmusclecellswiththinprojectionstoadjacentcellsinasmalltesticularduct(bar, 5µm).(FromMottaPM[ed].Ultrastructure of Smooth Muscle. Norwell,MA:KluwerAcademic;1990.) cytoskeleton, linkages between cells and the extracellular matrix, and membrane systems that transduce extracellular signals into changes in myoplasmic [Ca++].

A variety of specialized contacts exists between smooth muscle cells. Such contacts allow mechanical linkage and communication between cells. In contrast to skeletal muscle cells, which are normally attached at either end to a tendon, smooth (and cardiac) muscle cells are connected to each other. Because smooth muscle cells are anatomically arranged in series, they not only must be mechanically linked but must also be activated simultaneously and to the same degree. This mechanical and functional linkage is crucial to smooth muscle function. If such linkage did

Dense bodies c Caveolae Thick filaments Mitochondria Sarcoplasmic reticulum Thick Thin Filaments 0.1 µm0.1 µm C 0.5 mµ not exist, contraction in one region would simply stretch another region without a substantial decrease in radius or increase in pressure. The mechanical connections are provided by attachments to sheaths of connective tissue and by specific junctions between muscle cells.

Several types of junctions are found in smooth muscle. Functional linkage of the cells is provided by gap junctions (

Fig. 14.3A ). Gap junctions form low-resistance pathways between cells (see ). They also allow chemical communication by diffusion of low-molecular-weight compounds. In certain tissues, such as the outer longitudinal layer of smooth muscle in the intestine, large numbers of such junctions exist. Action potentials are readily propagated from cell to cell through such tissues.

B Surface couplings Sarcoplasmic reticulum 0.5 mµ0.2 mµ 0.5 mµ •Fig. 14.3 Junctions,membranes,andmyofilaments,insmoothmuscle.A,TransmissionelectronmicrographofGapjunctionbetweenintestinalsmoothmusclecells.B,Longitudinalviewofapulmonaryarterysmoothmusclecell.Thesarcoplasmicreticulumisstainedwithosmiumferricyanideandappearstoformacontinuousnetworkthroughoutthecellconsistingoftubules,fenestratedsheets(long arrows), andsurfacecouplingsatthecellmembrane(short arrows). C,Transversesectionofabundleofvenoussmoothmusclecellsillustratingtheregularspacingofthickfilaments(long line) andtherelativelylargenumberofsurroundingthin(actin)filaments(inset). Densebodies(arrowheads) aresitesofattachmentforthethinactinfilamentsandequivalenttotheZlinesofstriatedmuscles.Elementsofsarcoplasmicreticulum(short line) occurattheperipheryofthesecells.D,Scanningelectronmicrographoftheinnersurfaceofthesarcolemmaofanintestinalsmoothmusclecell.Longitudinalrowsofcaveolaeprojectintothemyoplasm(small, light-colored spheres), surroundedbydarkerelementsofthetubularsarcoplasmicreticulum.Theattachmentsofthinfilamentstothesarcolemmabetweentherowsofmembraneelementswereremovedduringpreparationofthespecimen.(AandD,FromMottaPM[ed].Ultrastructure of Smooth Muscle. Norwell,MA:KluwerAcademic;1990.BandC,FromSomlyoAP,SomlyoAV.Smoothmusclestructureandfunction.In:FozzardHAetal.[eds].The Heart and Cardiovascular System.2nded.NewYork:RavenPress;1992.) muscle cells. The adherens junction appears as thickened regions of opposing cell membranes that are separated by a small gap (≈60 nm) containing dense granular material. Thin filaments extend into the adherens junction to allow the contractile force generated in one smooth muscle cell to be transmitted to adjacent smooth muscle cells.

Embryonic smooth muscle cells do not fuse, and each differentiated cell has a single centrally located nucleus. Though dwarfed by skeletal muscle cells, smooth muscle cells are nevertheless quite large (typically 40–600 µm long). These cells are 2 to 10 µm in diameter in the region of the nucleus, and most taper toward their ends. Contracting cells become quite distorted as a result of the force exerted on the cell by attachments to other cells or to the extracellular matrix, and cross sections of these cells are often very irregular.

Smooth muscle cells lack T tubules, the invaginations of the skeletal muscle sarcolemma that provide electrical links to the sarcoplasmic reticulum (SR). However, the sarcolemma of smooth muscle has longitudinal rows of tiny sac-like inpocketings called caveolae (see

Fig. 14.3B–D ). Caveolae increase the surface-to-volume ratio of the cells and are often closely apposed to the underlying SR. A gap of approximately 15 nm has been observed between the caveolae and the underlying SR, comparable to the gap between the T tubules and terminal SR in skeletal muscle. Moreover, “Ca++ sparks” and a variety of Ca++-handling proteins have been observed in the vicinity of caveolae, thus raising the possibility that the caveolae and the underlying SR may contribute to regulation of intracellular [Ca++] in smooth muscle. The voltage-gated L-type Ca++ channel and the 3Na+-1Ca++ antiporter, for example, are associated with caveolae. The proteins caveolin and cholesterol are both critical for the formation of caveolae, and it is hypothesized that the caveolae reflect a specialized region of the sarcolemma that may also contain various signaling molecules in addition to the Ca++ signaling mentioned earlier.

Smooth muscle also has an intracellular membrane network of SR that serves as an intracellular reservoir for Ca++ (see

Fig. 14.3B–D ). Calcium can be released from the SR into the myoplasm when stimulatory neurotransmitters, hormones, or drugs bind to receptors on the sarcolemma. Importantly, intracellular Ca++ channels in the SR of smooth muscle include the ryanodine receptor (RYR), which is similar to that found in skeletal muscle SR, and the inositol 1,4,5-trisphosphate (InsP3)-gated Ca++ channel. The RYR is typically activated by a rise in intracellular [Ca++] (i.e., Ca++-induced release of Ca++ in response to an influx of Ca++ through the sarcolemma). The InsP3-gated Ca++ channel is activated by InsP3, which is produced when a hormone or hormones bind to various Ca++-mobilizing receptors on the sarcolemma. Intracellular [Ca++] is lowered through the action of an SR Ca++-ATPase (SERCA) and extrusion of Ca++ from the cell via a 3Na+-1Ca++ antiporter and a sarcolemmal Ca++-ATPase. The amount of SR in smooth muscle cells varies from 2% to 6% of cell volume and approximates that of skeletal muscle. Chemical signals such as InsP3 or a localized increase in intracellular [Ca++] (e.g., within the gap between the caveolae and SR) functionally link the sarcolemma and the SR.

Smooth muscle cells contain a prominent rough endoplasmic reticulum and Golgi apparatus, which are located centrally at each end of the nucleus. These structures reflect significant protein synthetic and secretory functions. The scattered mitochondria are sufficient for oxidative phosphorylation to generate the increased adenosine triphosphate (ATP) consumed during contraction.

The thick and thin filaments of smooth muscle cells are about 10,000 times longer than their diameter and are tightly packed. Therefore the probability of observing an intact filament by electron microscopy is extremely low. In contrast to skeletal muscle, which contains a transverse alignment of thick and thin filaments that results in striations, the contractile filaments in smooth muscle are not in uniform transverse alignment, and thus smooth muscle has no striations. The lack of striations in smooth muscle does not imply a lack of order. The thick and thin filaments are organized in contractile units that are analogous to sarcomeres.

The thin filaments of smooth muscle have an actin and tropomyosin composition and structure similar to that in skeletal muscle. However, the cellular content of actin and tropomyosin in smooth muscle is about twice that of striated muscle. Smooth muscle lacks troponin and nebulin but contains two proteins not found in striated muscle: caldesmon and calponin. The precise roles of these proteins are unknown, but they do not appear to be fundamental to cross-bridge cycling. It has been suggested that both calponin and caldesmon may regulate the contractility of smooth muscle. (in part by inhibiting actomyosin ATPase activity). Most of the myoplasm is filled with thin filaments that are roughly aligned along the long axis of the cell. The myosin content of smooth muscle is only a fourth that of striated muscle. Small groups of three to five thick filaments are aligned and surrounded by many thin filaments. These groups of thick filaments with interdigitating thin filaments are connected to dense bodies or areas (

Fig. 14.4

Fig. 14.3A–B ) and represent the equivalent of the sarcomere. The contractile apparatus of adjacent cells is mechanically coupled by the links between membrane-dense areas.

The cytoskeleton in smooth muscle cells serves as an attachment point for the thin filaments and permits transmission of force to the ends of the cell. In contrast to skeletal muscle, the contractile apparatus in smooth muscle is not organized into myofibrils, and Z lines are lacking. The functional equivalents of the Z lines in smooth muscle cells •Fig. 14.4 Apparentorganizationofcell-to-cellcontacts,cytoskeleton,andmyofilamentsinsmoothmusclecells.Smallcontractileelementsfunctionallyequivalenttoasarcomereunderliethesimilaritiesinmechanicsbetweensmoothandskeletalmuscle.Linkagesconsistingofspecializedjunctionsorinterstitialfibrillarmaterialfunctionallycouplethecontractileapparatusofadjacentcells.Densebodies,thefunctionalequivalentofZlinesinstriatedmuscle,areinterconnectedbyintermediatefilaments.Themyofilamentsareorientedlargelyinparallelwiththelongitudinalaxisofthecell,thoughanobliqueorientationhasbeenobservedinsomearteries.

are ellipsoidal dense bodies in the myoplasm and dense areas that form bands along the sarcolemma (see

Figs. 14.3A–B 14.4 ). These structures serve as attachment points for the thin filaments and contain α-actinin, a protein also found in the Z lines of striated muscle. Intermediate filaments with diameters between those of thin filaments (7 nm) and thick filaments (15 nm) are prominent in smooth muscle. These filaments link the dense bodies and areas into a cytoskeletal network. The intermediate filaments consist of protein polymers of desmin or vimentin.

Control of Smooth Muscle Activity

The contractile activity of smooth muscle can be controlled by numerous factors, including hormones, autonomic nerves, pacemaker activity, and a variety of drugs. Like skeletal or cardiac muscle, contraction of smooth muscle is dependent on Ca++ , and the agents just listed induce smooth muscle contraction by increasing intracellular [Ca++]. However, in contrast to skeletal or cardiac muscle, action potentials in smooth muscle are highly variable and not always needed to initiate contraction. Moreover, several agents can increase intracellular [Ca++] and hence contract smooth muscle without changing the membrane potential.

Fig. 14.5 shows various types of action potentials in smooth muscle and the corresponding changes in force. An action potential in smooth muscle can be associated with a slow twitch-like response, and the twitch forces can summate during periods of repetitive action potentials (i.e., similar to tetany in skeletal muscle). Such a pattern of activity is characteristic of single-unit smooth muscle in many viscera.

Periodic oscillations in membrane potential can occur as a result of changes in the activity of Na+,K+-ATPase in the sarcolemma. These oscillations in membrane potential can trigger multiple action potentials in the cell. Alternatively the contractile activity of smooth muscle may not be associated with generation of action potentials or even a change in membrane potential. In many smooth muscles the resting membrane potential is sufficiently depolarized (−60 to −40 mV) that a small decrease in membrane potential can significantly inhibit influx of Ca++ through voltage-gated Ca++ channels in the sarcolemma. By decreasing Ca++ influx, the force developed by smooth muscle decreases. Such a graded response to slight changes in the resting membrane potential is common in multiunit smooth muscles that maintain constant tension (e.g., vascular smooth muscle).

Contraction of smooth muscle in response to an agent that does not produce a change in membrane potential is termed pharmacomechanical coupling and typically reflects the ability of the agent to increase the level of the intracellular second messenger InsP3. Other agents result in a decrease in tension, also without a change in membrane potential. These agents typically increase levels of the intracellular second messengers cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP). The molecular mechanisms by which InsP3, cGMP, cAMP, and Ca++ alter the contractile force of smooth muscle are presented later.

Phosphorylation of a myosin light chain is required for the interaction of myosin with actin, and although Ca++dependent phosphorylation plays a key role in this process, the level of myosin phosphorylation (and hence the degree of contraction) is dependent on the relative activities of both myosin light-chain kinase (MLCK, which promotes phosphorylation) and myosin phosphatase (MP, which promotes dephosphorylation). Several agonists/hormones increase the level of myosin light-chain phosphorylation by simultaneously activating MLCK through an increase in intracellular [Ca++] and inhibiting MP through a signaling cascade involving the monomeric G protein RhoA and its effector Rho kinase (ROK). Moreover, hyperactivity of this RhoA/ROK signaling cascade has been implicated in various pathological conditions such as hypertension and vasospasm (discussed later).

Innervation of Smooth Muscle

Neural regulation of smooth muscle contraction depends on the type of innervation and neurotransmitters released, the proximity of the nerves to the muscle cells, and the •Fig. 14.5 Relationshipsbetweenmembranepotential(Em)andgenerationofforce(F)indifferenttypesofsmoothmuscle.A,Actionpotentialsmaybegeneratedandleadtoatwitchorlargersummedmechanicalresponses.Actionpotentialsarecharacteristicofsingle-unitsmoothmuscles(manyviscera).Gapjunctionspermitthespreadofactionpotentialsthroughoutthetissue.B,Rhythmicactivityproducedbyslowwavesthattriggeractionpotentials.Thecontractionsaregenerallyassociatedwithaburstofactionpotentials.Slowoscillationsinmembranepotentialusuallyreflecttheactivityofelectrogenicpumpsinthecellmembrane.C,Toniccontractileactivitymayberelatedtothevalueofthemembranepotentialintheabsenceofactionpotentials.GradedchangesinEmarecommoninmultiunitsmoothmuscles(e.g.,vascular),whereactionpotentialsarenotgeneratedandpropagatedfromcelltocell.D,Pharmacomechanicalcoupling;changesinforceproducedbytheadditionorremoval(arrows) ofdrugsorhormonesthathavenosignificanteffectonmembranepotential.

type and distribution of the neurotransmitter receptors on the muscle cell membranes (

Fig. 14.6 ). In general, smooth muscle is innervated by the autonomic nervous system. The smooth muscle in arteries is innervated primarily by sympathetic fibers, whereas the smooth muscle in other tissues can have both sympathetic and parasympathetic innervation. In the GI tract, smooth muscle is innervated by nerve plexuses that make up the enteric nervous system. The smooth muscle cells of some tissues (e.g., uterus) have no innervation.

The neuromuscular junctions and neuromuscular transmission in smooth muscle are functionally comparable to that of skeletal muscle but structurally less complex. The autonomic nerves that supply smooth muscle have a series of swollen areas, or varicosities, that are spaced at intervals along the axon. These varicosities contain vesicles for the neurotransmitter (see

Fig. 14.6).

The postsynaptic membrane of smooth muscle exhibits little specialization when compared with that of skeletal muscle (see ). The synaptic cleft is typically about 80 to 120 nm wide but can be as narrow as 6 to 20 nm or even greater than 120 nm. In synapses in which a wide synaptic cleft is found, release of neurotransmitter can affect multiple smooth muscle cells. There are a large number of neurotransmitters that affect smooth muscle activity. A partial listing is provided in

Table 14.1

Regulation of Contraction

Contraction of smooth muscle requires phosphorylation of a myosin light chain. Typically this phosphorylation occurs in response to a rise in intracellular [Ca++], either after an action potential or in the presence of a hormone/agonist. As depicted in

Fig. 14.7 , a rise in intracellular [Ca++] in smooth muscle results in the binding of 4 Ca++ ions to the protein calmodulin, and then the Ca++-calmodulin complex activates MLCK, which phosphorylates the regulatory light chain of myosin. This phosphorylation step is critical for the interaction of smooth muscle myosin with actin. In addition to this phosphorylation step in smooth muscle,

Varicosity with neurotransmitter vesicles •Fig. 14.6 Controlsystemsofsmoothmuscle.Inbothmultiunitsmoothmuscle(A)andsingleunitsmoothmuscle(B),neurallyreleasedtransmittersorcirculatingorlocallygeneratedhormonesorsignalingmoleculescaninducecontractionorrelaxationofthesmoothmuscle.Thecombinationofaneurotransmitter,hormone,ordrugwithspecificreceptorsactivatescontractionbyincreasingcellCa++ .Theresponseofthecellsdependsontheconcentrationofthetransmittersorhormonesatthecellmembraneandthenatureofthereceptorspresent.Hormoneconcentrationsdependondiffusiondistance,release,reuptake,andcatabolism.Consequently,cellslackingcloseneuromuscularcontactswillhavealimitedresponsetoneuralactivityunlesstheyareelectricallycoupledsothatdepolarizationistransmittedfromcelltocell.A,Multiunitsmoothmusclesresemblestriatedmusclesinthatthereisnoelectricalcouplingandneuralregulationisimportant.B,Single-unitsmoothmusclesarelikecardiacmuscle,andelectricalactivityispropagatedthroughoutthetissue.Mostsmoothmusclesprobablyliebetweenthetwoendsofthesingleunit–multiunitspectrum.C,Scanningmicrographofvaricosenervelyingoverratsmallintestinescalebar= 3µm.D,Fluorescentimageofcatecholaminesinsingleadrenergicaxoninguineapigmesentery.Scalebar= 10µm.E,Depictionofdistributionofsympatheticnerves(in black) invasculature.Sympatheticnervesareassociatedwithsmallarteries(SA),largearterioles(la),smallveins(SV),largevenules(lv),smallarterioles(sa),andterminalarterioles(TA).Capillaries(c),post-capillaryvenules(pcv),andsmallvenules(svl)appeartolacksympatheticinnervation.(C,FromBurnstockG:Autonomicneuralcontrolmechanisms.Withspecialreferencetotheairways.InKalinerMA,BarnesPJ(Eds.),TheAirways.NeuralControlinHealthandDisease,MarcelDekker,NewYork(1988),pp.1–22.D,FromChamleyJH,MarkGE,CampbellGR,BurnstockG.(1972)Sympatheticgangliainculture.I.Neurons.Z.Zellforsch.Mikrosk.Anat.135,287-314.) aThepredominanteffectofsympatheticstimulationissmoothmusclecontractioncausedbytheabundanceofα1-ARrelativetoβ2-ARinsmoothmuscle.bActivationofβ2-ARonsmoothmusclemodulatesthedegreeofsmoothmusclecontractionduringsympatheticstimulation.Therapeuticβ2-ARagonistsareimportantfortherelaxationofbronchialsmoothmuscleduringasthmaticattacks.cVascularsmoothmusclesarepoorlyinnervatedbytheparasympatheticsystem.Duringvagalstimulation,however,acetylcholine(ACh)canbecomeelevatedinthecoronarycirculationandresultincoronaryrelaxation(mediatedbybindingofAChtoendothelialcells).NotethatthiseffectofAChisindirectbecausebindingofAChtoendothelialcellsresultsinreleaseofthesmoothmusclerelaxantnitricoxidefromtheendothelialcells.Inregionsofthecoronarycirculationwithdamagedendothelium,bindingofAChtocoronarysmoothmusclecouldpromotecontraction(vasospasm;directeffect).dAvarietyofhormonescanelevateInsP3insmoothmuscleandtherebyresultinsmoothmusclecontraction.SuchhormonesincludeangiotensinII,vasopressin,andendothelin,alongwiththeneurotransmittersnorepinephrineandacetylcholine.Asnotedabove,however,eachhormone/transmitterbindstoaspecificreceptortype.eDuringperiodsofintensemuscularactivity,adenosinecanbereleasedfromtheworkingmuscle,diffusetotheneighboringvasculature,andpromotevasodilation.Thus,adenosineisactingasalocalfactortoincreasebloodflowtoaspecificregion(i.e.,workingmuscle).AR,adrenergicreceptor;EC,endothelialcell;InsP3,inositol1,4,5-trisphosphate;SMC,smoothmusclecell.

an ATP molecule is also needed to energize the myosin cross-bridge for the development of force.

TheentericnervoussystemcontrolsmanyaspectsofGIfunction,includingmotility.Somechildrenarebornwithoutentericnervesinthedistalportionofthecolon.Theabsenceofnervesiscausedbymutantgenesthatdisruptthesignalsnecessaryfortheembryonicnervestomigratetothecolon.Inthesechildren,normalmotilityofthecolondoesnotoccurandsevereconstipationresults.ThisconditioniscalledHirschsprung’s disease. Itcanbecorrectedbysurgicallyremovingtheportionofthecolonthatdoesnotcontainentericnerves.

Contraction of smooth muscle is thus said to be thick-filament regulated, which contrasts with the thin-filament regulation of contraction of striated muscle, where binding of Ca++ to troponin exposes myosin binding sites on the actin thin filament. The thick-filament regulation is attributable to expression of a distinct myosin isoform in smooth muscle.

The myosin cross-bridge cycle in smooth muscle is similar to that in striated muscle in that after attachment to the actin filament, the cross-bridge undergoes a ratchet action in which the thin filament is pulled toward the center of the thick filament and force is generated. ADP and Pi are released from the myosin head at this time, thereby allowing ATP to bind. ATP decreases the affinity of myosin for actin, which allows release of myosin from actin. Energy from the newly bound ATP is then used to produce a conformational change in the myosin head (i.e., recocking the head) so that the cross-bridge is ready for another contraction cycle. The cross-bridge cycle continues as long as the myosin cross-bridge remains phosphorylated. Note that although the four basic steps of the cross-bridge cycle appear to be the same for striated and smooth muscle, the kinetics of cross-bridge cycling is much slower for smooth muscle.

Cross-bridge cycling continues with the hydrolysis of 1 ATP molecule per cycle until myoplasmic [Ca++] falls. With the decrease in [Ca++], MLCK becomes inactive, and the cross-bridges are dephosphorylated by MP (see

Fig. 14.7

Fig. 14.4 , the thin filaments in smooth muscle are attached to dense bodies, and the myosin thick filaments appear to reside between two dense bodies and overlap a portion of the thin filaments, much like the overlap of thick and thin filaments in the sarcomere of striated muscle. A bipolar arrangement of myosin molecules within the thick filament is thought to allow the myosin cross-bridges to pull the actin filaments toward the center of the thick filament, thus contracting the smooth muscle and hence developing force.

From a structural standpoint, smooth muscle myosin is similar to striated muscle myosin in that they both contain a pair of heavy chains and two pairs of light chains. Despite this similarity, they represent different gene products and thus have different amino acid sequences. As noted, smooth muscle myosin, unlike skeletal muscle myosin, is unable to interact with the actin thin filament unless the regulatory light chain of myosin is phosphorylated. Moreover, the thin filament in smooth muscle lacks troponin, which plays a •Fig. 14.7 RegulationofsmoothmusclemyosininteractionswithactinbyCa++-stimulatedphosphorylation.Intherelaxedstate,cross-bridgesarepresentasahigh-energymyosin-ADP-PicomplexinthepresenceofATP.Attachmenttoactindependsonphosphorylationofthecross-bridgebyaCa++-calmodulin–dependentmyosinlight-chainkinase(MLCK).Phosphorylatedcross-bridgescycleuntiltheyaredephosphorylatedbymyosinphosphatase.Notethatcross-bridgephosphorylationataspecificsiteonamyosinregulatorylightchainrequiresATPinadditiontothatusedineachcyclicinteractionwithactin.

critical role in the thin-filament regulation of contraction in striated muscle (see

Although intracellular Ca++ is required for smooth muscle contraction, the sensitivity of contraction to Ca++ is variable. Several hormones/agonists, for example, increase the force of contraction at a given submaximal intracellular [Ca++], thereby resulting in Ca++ sensitization (

Fig.

14.8 ). Ca++ sensitization is depicted as a leftward shift in the Ca++ dependence of smooth muscle contraction (see

Fig. 14.8B ) and can occur in response to a decrease in the activity of MP activity at a given intracellular [Ca++]. Likewise an increase in MP activity at a given intracellular [Ca++] promotes a rightward shift in the Ca++ dependence of smooth muscle contraction, resulting in Ca++ desensitization (see

Fig. 14.8B ). Reciprocal changes in the activities of MP and MLCK can occur, as shown in

Fig. 14.8A , where stimulation of the ROK signaling cascade by an agonist can simultaneously inhibit MP and stimulate MLCK at a given intracellular [Ca++], thereby increasing the Ca++ sensitivity of contraction (i.e., Ca++ sensitization).

Fig.

14.8A ).RhoAactivatesRhokinase(ROK),whichinturninhibitsMPbybothdirectandindirectmechanisms.DirectinhibitionofMPbyactivatedROKinvolvesROKphosphorylationofthemyosin-bindingsubunit(MBS)ofMP.IndirectinhibitionofMPbyactivatedROKinvolvesphosphorylationofCPI-17, anendogenous17-kDaprotein,whichtheninhibitsMP.Hormones/agonistssuchascatecholamines(actingonα1-adrenergicreceptors),vasopressin,endothelin,angiotensin,andmuscarinicagonistsincreasethesensitivityofsmoothmusclecontractiontoCa++ throughactivationofRhoA/ROKsignaling.ROKcanalsobeactivatedbyarachidonicacidandinhibitedbyY-27632,ahighlyspecificinhibitor(see

Fig.14.8A ).Thoughnotshownin

Fig.14.8 ,inactiveRhoAistypicallylocatedinthecytosol,boundtoGDPandaninhibitoryprotein(Rho-GDP dissociation inhibitor [GDI]). BindingofagonisttovariousG-coupledreceptorscanactivateRhoAbystimulatingguanine nucleotide exchange factor (GEF) toyieldRhoA-GTP,whichlocalizestothesarcolemmaandactivatesROK.Conversely,stimulationofMPactivityreducestheCa++ sensitivityofcontractionofsmoothmuscle,therebypromotingrelaxation(andhencevasodilation).

HyperactivityoftheRhoA/ROKsignalingcascadehasbeenimplicatedinvariouspathologicalconditionssuchashypertensionandvasospasm.HyperactivityofRhoA/ROKinthevascularsmoothmuscleofhypertensiveanimals,forexample,wasmanifestedbyincreasedlevelsofactivatedRhoA,upregulationofROK,enhancementofagonist-inducedCa++ sensitizationofcontraction,andagreaterreductioninbloodpressurebyROKinhibitorsascomparedwithnormotensivecontrols.Asimilartrendwasobservedinhumans,inthatROKinhibitorsdecreasedforearmvascularresistanceinhypertensivepatientstoagreaterextentthaninnormotensivecontrols.ROKinhibitorshavealsobeenshowntoreverseorpreventexperimentallyinducedcerebralvasospasmandcoronaryvasospasm,aswellastheassociatedupregulationofRhoA/ROKandincreasedmyosinlight-chainphosphorylation.HyperactivityofRhoA/ROKhasadditionallybeenimplicatedinbronchialasthma,erectiledysfunction,andpretermlabor,asevidencedbytheeffectsofROKinhibitors.Inaddition,ROKinhibitorshavedecreasedvascularsmoothmuscleproliferationandreducedrestenosisafterballoonangioplastyinratcarotidartery.

During a phasic contraction, myoplasmic [Ca++], cross-bridge phosphorylation, and force reach a peak and then return to baseline (

Fig. 14.9 ). In contrast, during a tonic contraction, myoplasmic [Ca++] and cross-bridge phosphorylation decline after an initial spike but do not return to baseline levels. During this later phase, force slowly increases and is sustained at a high level (see

Fig. 14.9).

This sustained force is maintained with only 20% to 30% of the cross-bridges phosphorylated, and thus ATP utilization is reduced. The term latch state refers to this condition of tonic contraction during which force is maintained at low energy expenditure.

P P Ins(1,4,5)P3 Ca++ SR Ca++ CaM MLCK MLC MLC P GEFs Rho Rho-kinase CPI-17 CPI-17 MBS M20 cat MBS Inactive Active Contraction M20 cat MP Y27632 GTP ? AA PLC GqG12/13 •Fig. 14.8 Rho-kinase(ROK)signalinginsmoothmuscle.A,AvarietyofagonistsofG-coupledreceptorssimultaneouslystimulateInsP3productionandactivateRhoA-ROKsignaling.InsP3(Ins[1,4,5]P3)isproducedbyphospholipaseC(PLC)-mediatedhydrolysisofPIP2.InsP3increasesintracellular[Ca++]byopeningInsP3-gatedCa++ channelsinthesarcoplasmicreticulum(SR),therebyresultinginCa++-calmodulin–dependentactivationofmyosinlight-chainkinase(MLCK)andsubsequentphosphorylationofthemyosinregulatorylightchain(MLC)andpromotionofactin-myosininteraction(contraction).ActivatedRhoA(depictedasRho-GTP)stimulatesROK,whichinhibitsmyosinphosphatase(MP)byphosphorylatingthemyosin-bindingsubunit(MBS)ofMP.ROKalsoinhibitsMPindirectlybyphosphorylating/activatingCPI-17,a17-kDainhibitorofMP.TheneteffectofROKphosphorylationsisadecreaseinMPactivity,whichresultsinanincreasedlevelofmyosinlight-chainphosphorylation(MLCP)andhencegreaterforceofcontractionatagivenintracellular[Ca++](i.e.,increasedsensitivityofcontractiontoCa++).B,Ca++ sensitization referstoanincreaseintheforceofcontractionatagivenintracellular[Ca++]andisdepictedasaleftwardshiftintheCa++ dependenceofcontractionofsmoothmuscle.Ca++ sensitizationcanresultfromadecreaseinMPactivityand/oranincreaseinMLCKactivityatagivenintracellular[Ca++](asshownintheROKsignalingcascadeinpanelA).Conversely,Ca++ desensitization referstoadecreaseintheforceofcontractionatagivenintracellular[Ca++]andisdepictedasarightwardshiftintheCa++ dependenceofcontraction.AA,arachidonicacid;CaM,Ca++-calmodulincomplex;cat,catalyticsubunitofmyosinphosphatase;G12/13andGq,heterotrimericG-proteins;GEF,guanine-nucleotideexchangefactor;M20,subunitofmyosinphosphatase;MP,myosinphosphatasecomplex;Y27632,commercialinhibitorofROK.(FromFukataYetal.Trends Pharmacol Sci 2001;22:32-39.) •Fig. 14.9 Timecourseofeventsincross-bridgeactivationandcontractioninsmoothmuscle.A,AbriefperiodofstimulationisassociatedwithCa++ mobilization,followedbycross-bridgephosphorylationandcyclingtoproduceabriefphasic,twitch-likecontraction.B,Inasustainedtoniccontractionproducedbyprolongedstimulation,theCa++ andphosphorylationlevelstypicallyfallfromaninitialpeak.Forceismaintainedduringtoniccontractionsatareduced[Ca++](andhencealowlevelofmyosinlight-chainphosphorylation),withlowercross-bridgecyclingratesmanifestedbylowershorteningvelocitiesandATPconsumption.

The latch state is thought to reflect a slowing of the cross-bridge cycle, so that the myosin heads remain in contact with the actin filament for a longer time, thereby maintaining tension at low energy cost. Note that the intracellular [Ca++] falls to a low level during the tonic phase of contraction, though it is still above the resting/ basal [Ca++]. The mechanism contributing to the ability of smooth muscle to maintain force at a low intracellular [Ca++] during tonic contraction is thought to involve dephosphorylation of the myosin regulatory light chain while the myosin cross-bridge is attached to the actin filament, resulting in slowing of the rate of dissociation of the myosin from the actin, allowing the myosin to spend more time in an attached, force-generating conformation. Relaxation of smooth muscle following tonic contraction occurs when intracellular [Ca++] decreases to a level that prevents a net phosphorylation of the regulatory light chain by MLCK. It has also been proposed that caldesmon may participate in the transition to the latch state.

As already noted, ATP consumption is reduced during the latch state. Under this condition, smooth muscle uses 300fold less ATP than would be required by skeletal muscle to generate the same force. Smooth muscle, like skeletal muscle, requires ATP for ion transport to maintain the resting membrane potential, sequester Ca++ in the SR, and extrude Ca++ from the cell. All these metabolic needs are readily met by oxidative phosphorylation. Fatigue of smooth muscle does not occur unless the cell is deprived of oxygen. However, aerobic glycolysis with lactic acid production normally supports membrane ion pumps even when oxygen is plentiful.

Regulation of Myoplasmic Calcium Concentration

The mechanisms that couple activation to contraction in smooth muscle involve two Ca++ sources: one involving the sarcolemma and the other involving the SR. The sarcolemma regulates Ca++ influx and efflux from the extracellular Ca++ pool. The SR membranes determine Ca++ movement between the myoplasm and the SR pool. Skeletal muscle contraction does not require extracellular Ca++ (see ). In contrast, extracellular Ca++ is important for smooth muscle contraction. Thus regulation of myoplasmic [Ca++] involves not only the SR but also the sarcolemma (

Fig. 14.10). A number of factors can alter the myoplasmic

Na+-K+ pump 2K+ 3 Na+ Ca++ MLCK off CaCM CM ATP Receptor-activated CaCM*MLCK ATP G G Sarcoplasmic reticulum Time IP3 PIP2 3Na Cell Ca++ Neurotransmitter or hormone Neurotransmitter or hormone Ca++ channel Refilling ATP ?

•Fig. 14.10 Principalmechanismsdeterminingmyoplasmic[Ca++]insmoothmuscle.Releaseofcalciumfromthesarcoplasmicreticulum(SR)isarapidinitialeventinactivation,whereasboththeSRandthesarcolemmaparticipateinthesubsequentstimulus-dependentregulationofmyoplasmic[Ca++].Thesarcolemmaintegratesmanysimultaneousexcitatoryandinhibitoryinputstogovernthecellularresponse.Higher-orderregulatorymechanismscanaltertheactivityofvariouspumps,exchangers,orenzymes(theasterisk designateswell-establishedinstances).ThelabelATP indicatesthattheprocessrequiresATPhydrolysis;thequestion mark onthesarcolemmareferstotheCa++ pathwayimportantforrefillingtheSR(i.e.,store-operatedCa++ entry[SOCE]),whichappearstoinvolvetheinteractionofaCa++-sensitivestromalinteractionmolecule(STIM)ontheSRandthesarcolemmalCa++ channelOrai.CaCM,Ca++-calmodulincomplex;G,guaninenucleotide–bindingproteins;IP3,inositol1,4,5-trisphosphate;MLCK,myosinlight-chainkinase;PIP2,phosphatidylinositolbisphosphate;PLC,phospholipaseC.

Inappropriatecontractionofsmoothmuscleisassociatedwithmanypathologicalsituations.Oneexampleissustainedvasospasmofacerebralarterythatdevelopsseveralhoursafterasubarachnoidhemorrhage.Itisthoughtthatfreeradicalsgeneratedasaresultofthehemorrhageraisemyoplasmic[Ca++]insurroundingarterialsmoothmusclecells.Theriseinmyoplasmic[Ca++]activatesMLCK,whichleadstocross-bridgephosphorylationandcontraction.Thevasoconstrictiondeprivesotherareasofthebrainofoxygenandmayleadtopermanentinjuryordeathofsurroundingneurons.Forafewdaysthecerebralarteryremainssensitivetovasoactiveagents,andthereforetreatmentwithvasodilatorsmayrestoreflow.AnincreaseinROKactivityandMPphosphorylationhasbeenobservedduringcerebralvasospasm.AdministrationofROKinhibitorspromotesrelaxationofthevasospasmanddecreasesthelevelofmyosinlight-chainphosphorylation.Thesmoothmusclecellsceasetorespondtothevasodilatorsafterseveraldays,andtheylosecontractileproteinsandsecreteextracellularcollagen.Thelumenofthearteryremainsconstrictedasaresultofstructuralandmechanicalchangesthatdonotinvolveactivecontraction.

[Ca++] of smooth muscle. This differs from skeletal muscle, in which action potential–induced release of Ca++ from the SR fully activates the contractile apparatus.

The role of smooth muscle SR in regulating myoplasmic [Ca++] is comparable to that of skeletal muscle. Stimulation of the cell opens SR Ca++ channels, and myoplasmic [Ca++] increases rapidly. This release is not linked to voltage sensors, as is the case in skeletal muscle, but to binding of the second messenger InsP3 to receptors in the SR. InsP3 is generated by a stimulus that acts on sarcolemmal receptors that are coupled via a guanine nucleotide–binding protein (G protein) to activate phospholipase C (PLC) (see ). PLC hydrolyzes the membrane phospholipid phosphatidylinositol bisphosphate (PIP2) into InsP3 and diacylglycerol. InsP3 then diffuses to the SR and opens the InsP3-gated Ca++ channel, thereby resulting in release of Ca++ from the SR into the myoplasm. This complex process may permit graded release of Ca++ from the SR and also enable many different neurotransmitters and hormones to effect smooth muscle contraction. Calcium is reaccumulated by the SR through the activity of the SERCA, although as indicated later, extrusion of Ca++ from the smooth muscle cell also contributes to the reduction in myoplasmic [Ca++]. Refilling of the SR with Ca++ not only involves reaccumulation of cytosolic Ca++ but also depends on the extracellular [Ca++]. The dependence on extracellular [Ca++] is thought to reflect the operation of a “store-operated” Ca++ channel present in the sarcolemma at points near underlying SR called junctional SR.

A variety of hormones and neurotransmitters elevate myoplasmic [Ca++] by stimulating InsP3 production. Vascular smooth muscle, for example, is innervated by sympathetic fibers of the autonomic nervous system. These fibers use norepinephrine as a neurotransmitter, which when released binds to α1-adrenergic receptors on vascular smooth muscle cells and results in G protein–dependent activation of PLC. Activation of PLC results in production of InsP3, which activates the InsP3-gated Ca++ channel in the SR, thereby elevating myoplasmic [Ca++] and causing vasoconstriction. Other agents that promote vasoconstriction by activating the InsP3 cascade include angiotensin II and vasopressin. Development of drugs that block production of angiotensin II (e.g., angiotensin-converting enzyme [ACE] inhibitors) provides a means of promoting vasodilation that is important for individuals with hypertension or congestive heart failure. As mentioned previously, a variety of agents can produce contraction of smooth muscle without altering membrane potential (i.e., pharmacomechanical coupling). Agonist-induced activation of the InsP3 cascade represents an example of pharmacomechanical coupling. Many of the hormones/agonists that activate PLC through G protein– coupled receptors also promote sarcolemmal Ca++ influx and activation of RhoA/ROK. The net effect is a rise in intracellular [Ca++], which activates MLCK, concomitant with a rise in ROK activity, which inhibits MP, both of which act complementarily to increase the level myosin light-chain phosphorylation.

Fig.14.11 ).Specifically,EDHFappearstobeanarachidonicacidmetabolite(e.g.,epoxyeicosatrienoic acid [EET])thatisproducedbyendothelialcellsinresponsetovariousstimuliandthenreleasedtotheunderlyingvascularsmoothmuscle.EEThasbeenshowntoactivateatransient receptor channel (e.g.,TRPV4)inthesarcolemmaofsmoothmusclethatleadstotheinfluxofCa++ ,whichthenopensRYRchannelsintheSRandresultsinCa++ sparks.TheCa++ sparksinturnactivatealarge-conductanceK+ channelinthesarcolemma(BKCa),andthesmoothmusclecellbecomeshyperpolarized.HyperpolarizationinturndecreasesbasalCa++ influxthroughvoltage-gatedCa++ channelsinthesmoothmuscle,therebydecreasingintracellular[Ca++]andhencerelaxingthesmoothmuscle,asdescribedearlier.

In addition to the InsP3 receptor, the SR also contains the Ca++-gated Ca++ channel, also called the RYR, which may be activated during periods of Ca++ influx through the sarcolemma. Short-lived spontaneous opening of the RYR 11,12 EET COOH •Fig. 14.11 Anarachidonicacidmetabolite(11,12-epoxyeicosatrienoicacid[11,12EET])releasedfromendothelialcellscanopenthetransientreceptorchannelTRPV4intheunderlyingsmoothmuscletopermittheinfluxofCa++ ,whichinturninitiatesbriefopeningsoftheSRryanodinereceptor(Ca++ sparks)localizednearthesarcolemma.OpeningofCa++-activatedK+ channelsinthesarcolemmabycalciumsparksresultsinhyperpolarizationofthesmoothmuscleandhencevasodilation.BKCa,largeconductanceCa++-activatedpotassiumchannel.(FromEarleySetal.Circ Res 2005;97:1270-1279.) resulting in localized elevations in myoplasmic [Ca++] occurs in many cells, including smooth muscle. When observed with Ca++-sensitive fluorescent dyes, these spontaneous localized elevations in myoplasmic [Ca++] produce brief light flashes and as a result are named Ca++ sparks. In smooth muscle an increase in cAMP has been associated with an increase in the frequency of Ca++ sparks, particularly in situations in which the SR is in close proximity to the sarcolemma (i.e., junctional SR, perhaps near caveolae). An increase in the frequency of these sparks hyperpolarizes vascular smooth muscle by activation of a large-conductance Ca++-gated K+ channel in the sarcolemma. This hyperpolarization then decreases overall myoplasmic [Ca++], and relaxation occurs.

Calcium is extruded from the smooth muscle cell by the activity of sarcolemmal Ca++-ATPase and by a 3Na+-1Ca++ antiporter (i.e., 3 Na+ ions enter the cell for each Ca++ ion extruded). Extrusion of Ca++ from the cell competes with sequestration of Ca++ in the SR by SERCA and thus reduces the accumulation of Ca++ in the SR. When the [Ca++] in the SR decreases, the SR is thought to initiate Ca++ influx into the cell through a process called store-operated Ca++ entry (SOCE) to facilitate refilling of the SR. Specifically, stromal interaction molecule 1 (STIM1) in the SR is hypothesized to monitor SR [Ca++] and then initiate Ca++ influx through the sarcolemmal channel protein Orai through a protein-protein interaction. Thus the influx of Ca++ during SOCE is thought to occur in the confined space between the caveolae and peripheral SR of smooth muscle. Recent studies raise the possibility that STIM1-Orai–mediated SOCE may also contribute to the rise in intracellular Ca++ transient in smooth muscle following α1-adrenergic receptor stimulation. The Stim1-Orai complex has also been implicated in remodeling of smooth muscle in pathological conditions such as restenosis following balloon angioplasty.

In addition to the stimulatory effects of various agents on sarcolemma Ca++ channels and InsP3 cascades, there are several inhibitory factors that lower myoplasmic [Ca++] and thereby relax smooth muscle. For example, the dihydropyridine class of Ca++ channel blocking drugs decreases the influx of Ca++ through sarcolemmal L-type voltage-gated Ca++ channels and reduces vasomotor tone. Similarly, drugs that open K+ channels in the sarcolemma (e.g., hydralazine) promote relaxation (e.g., vasodilation) by hyperpolarizing the membrane potential, which reduces the influx of Ca++ through voltage-gated Ca++ channels. Conversely, agents that decrease K+ permeability of the sarcolemma may promote vasoconstriction by inducing membrane depolarization, which then increases influx of Ca++ through these same voltage-gated Ca++ channels. Smooth muscle also contains receptor-activated Ca++ channels. Conductance of these receptor-activated Ca++ channels is linked to receptor occupancy.

A variety of drugs and hormones relax smooth muscle by increasing the cellular concentrations of cAMP or cGMP.

Nitric oxide (NO) is produced by nerves and vascular endothelial cells and relaxes smooth muscle by increasing cGMP (

Fig. 14.12 ). Acetylcholine released from parasympathetic fibers causes vasodilation in some vascular beds as a result of stimulating the production of NO by vascular endothelial cells. Shear stress and adenosine (e.g., released from exercising muscle) may also promote NO release from vascular endothelial cells. The molecular mechanisms underlying the cGMP-dependent relaxation of vascular smooth muscle are complex and have been reported to involve (1) inhibition of InsP3 production, (2) inhibition of the InsP3 receptor, (3) activation of myosin light-chain phosphatase, and (4) activation of the Ca++-activated K+ channel (BKCa), which promotes hyperpolarization of the cells and thus inhibits Ca++ influx through voltage-gated Ca++ channels.

Similarly, elevation of cAMP in vascular smooth muscle by activation of β-adrenergic receptors or adenosine receptors promotes vasodilation through multiple mechanisms, including (1) decreased affinity of MLCK for Ca++/ calmodulin, (2) decreased cytosolic [Ca++] concentration, and/or (3) increased MP activity. The decreased affinity of MLCK for Ca++/calmodulin following elevation of cAMP involves protein kinase A (PKA)-dependent phosphorylation of MLCK. The ability of PKA to reduce cytosolic [Ca++] is complex and can involve activation of potassium channels (e.g., ATP-dependent potassium channels), resulting in hyperpolarization of the smooth muscle and hence decreased Ca++ influx through voltage-gated Ca++ channels. cAMP has also been shown to increase the frequency of Ca++ sparks in vascular smooth muscle, which as described earlier, hyperpolarizes the membrane potential by activation of Ca++-gated K+ channels, thereby reducing the influx of Ca++ through voltage-gated Ca++ channels. cAMP can also promote relaxation of smooth muscle by increasing the activity of MP (through either PKA-dependent phosphorylation of a subunit of MP or through inhibition of ROK). Inhibition of ROK can occur through PKA-dependent phosphorylation or through a signaling pathway involving the cAMP-modulated guanine nucleotide exchange factor Epac (exchange protein directly activated by cAMP)

Relaxation of smooth muscle by elevation of cAMP has afforded asthmatics a means of reversing bronchiolar constriction with use of β2-adrenergic agonists. The local vasodilatory effect of adenosine produced in working muscle during periods of intense exercise has also been attributed at least in part to elevated cAMP levels in vascular smooth muscle secondary to adenosine-induced stimulation of purinergic receptors on the sarcolemma of vascular smooth muscle. Adenosine may also activate a sarcolemmal K+ channel to induce membrane hyperpolarization, which as already noted will decrease the influx of Ca++ through voltage-gated Ca++ channels and cause vasodilation. Thus regulation of smooth muscle tone may be under the influence of not only the autonomic nervous system and circulating hormones but also neighboring endothelial cells and skeletal muscle cells via diffusible substances such as NO and adenosine.

Cat.Cat.Ca2˜Ca2˜IP3RICa2˜Ca2˜K˜CaV1.2BKCaCaMMLCK PDE5 RGS2 MYPT1 MLCP GMP pGC cGMP Hyper-polarization NucleuscGMP-A/B cGMP-A/B LZ LZ ˜˜˜°PKG1 NO sGC GTPGPCR IRAG ER Actin/myosin °˛G˜q IP3 PLC°12354 •Fig. 14.12 Signalingmechanismsbywhichnitricoxide(NO)andnitroprusside(NP)promoterelaxationofvascularsmoothmuscle.Nitricoxidestimulatessolubleguanylcyclase(sGC)inthevascularsmoothmusclecell,resultinginproductionofcGMP,activationofproteinkinaseG(PKG1),whichinturnphosphorylatesvarioussubstratesthat(1)inhibitInP3production,(2)inhibitInsP3-inducedCa++ release, (3)activatemyosinlight-chainphosphatase,(4)activatethelargeconductanceCa++-activatedK+ channel(BKCa),whichhyperpolarizesthecellandthusinhibitsthevoltage-gatedCa++ channel(Cav1.2)(5).BKCa,calcium-activatedmaxi-Kchannel;CaM,calmodulin;Cat,catalyticdomain;Cav1.2,L-typecalciumchannel;ER,endoplasmicreticulum;GPCR,Gprotein–coupledreceptor;IP3RI,inositol1,4,5-trisphosphate(IP3)receptorI;IRAG,inositoltrisphosphatereceptor-associatedcGMP-kinasesubstrate;LZ,leucine/isoleucinezipper;MLCK,myosinlight-chainkinase;MLCP,myosinlight-chainphosphatase;MYPT,regulatorymyosinphosphatasetargetingsubunit1;PDE5,phosphodiesterase5;pGC,particulateguanylylcyclase;PLCβ,phospholipaseCbeta;RGS2,regulatorofG-proteinsignaling2;sGC,solubleguanylylcyclase.(ModifiedfromSchlossmannJ,DeschM.Am J Physiol Heart Circ Physiol 2011;301:H672–H682.)

Blood flow to tissues such as the brain is maintained at a relatively constant flow over a wide range of blood pressures through a process called autoregulation. The mechanism underlying autoregulation of blood flow involves the myogenic response, wherein an increase in distending pressure in an artery results in vasoconstriction, whereas a decrease in transmural pressure results in a vasodilation (over a given range of pressures). As shown in

Fig. 14.13 , distention of the resistance artery resulted in an immediate elevation of intracellular [Ca++], followed by vasoconstriction in an effort to maintain relatively constant flow. The mechanism underlying this elevation of intracellular [Ca++] and subsequent vasoconstriction is complex. The rise in intracellular [Ca++] in response to stretch been reported to involve stretch-activated channels, InsP3-signaling, and voltage-gated Ca++ channels.

During development and growth, the number of smooth muscle cells increases (

Fig. 14.14 ). Smooth muscle tissue mass also increases if an organ is subjected to a sustained increase in mechanical work. This increase in mass is called compensatory hypertrophy. A striking example occurs with arterial smooth muscle cells (i.e., in the tunica media of the artery) in hypertensive patients. The increased mechanical load on the muscle cells appears to be the common factor that induces this hypertrophy. Chromosomal replication can result in significant numbers of polyploid muscle cells. The polyploid cells contain multiple sets of the normal number of chromosomes. They synthesize more contractile proteins and thus increase the size of the cell (see

Fig. 14.14

The myometrium, which is the smooth muscle component of the uterus, undergoes hypertrophy as parturition (birth) approaches. Hormones play an important role in this response. The smooth muscle is quiescent during pregnancy when the hormone progesterone predominates, and few gap junctions that electrically couple the smooth muscle cells are present. At term, under the dominant influence of estrogen, the myometrium undergoes marked hypertrophy. Large numbers of gap junctions form just before birth and convert the myometrium to a single-unit tissue to coordinate contraction during parturition.

The growth and development of tissues that contain smooth muscle are associated with increases in the connective tissue 1.4 1.2 1.0 0.8 0.6 0.0 •Fig. 14.14 Smoothmusclecellscarryoutmanyactivities.A,Theyretainthecapacitytodivideduringnormalgrowthorincertainpatho logicalresponsessuchasformationofatheroscleroticplaque.B,Cellsmayalsohypertrophyinresponsetoincreasedloads.Chromosomalreplicationnotfollowedbycelldivisionyieldscellswithagreatercontentofcontractileproteins.C,Smoothmusclecellsalsosynthesizeandsecretetheconstituentsoftheextracellularmatrix.

Althoughsmoothmuscleisinvolvedinphysiologicaladjustmentstoexercise,sustainedchangesinthe mechanicalloadingthatinducecellularadaptationsareusuallytheresultofapathologicalcondition(e.g.,hypertension).Afairlycommonexampleinmenisurinary bladder hypertrophy causedbybenignorcancerous enlargementoftheprostategland,whichobstructsthebladderoutlet.Theclinicalresultisdifficultyurinating, •Fig. 14.13 Myogenicresponseinvascularsmoothmuscleisanautoregulatoryresponsetomaintainconstantflowtoatissue.Anincreaseintransmuralpressure(TMP; top panel) elevatesintracellular[Ca++](middle panel), resultinginavasoconstriction(bottom panel). Notethattheincreaseintransmuralpressureinitiallystretchestheskeletalmuscleresistanceartery(bottom panel), butthisisquicklyfollowedbyvasoconstriction.(FromSchubertRetal.Cardiovasc Res 2008;77:8-18.) matrix. Smooth muscle cells can synthesize and secrete the materials that make up this matrix, including collagen, elastin, and proteoglycans (see

Fig. 14.14 ). The synthetic and secretory capacities are evident when smooth muscle cells are isolated and placed in tissue culture. The cells rapidly distentionofthebladder,andimpairedemptying.Inthissituationtheabilityofthebladdersmoothmuscletocontractanddevelopstressisdiminished.Thereasonsforthisremainunexplained,butphenotypicmodulationofthesmoothmusclecellswithalteredcontractileproteinisoformexpressionandgrossanatomicdistortionofthebladderwalloccurs.NeuromuscularchangesalsoaffectmyoplasmicCa++ mobilizationandcross-bridgephosphorylation.Fortunately,normalstructureandfunctionareusuallyrestoredaftertheobstructionisalleviated.

lose thick myosin filaments and much of the thin-filament lattice, and there is expansion of the rough endoplasmic reticulum and Golgi apparatus. The phenotypically altered cells multiply and lay down connective tissue. This process is reversible, and some degree of redifferentiation with the formation of thick filaments occurs after cell replication ceases. Determinants of the smooth muscle cell phenotype are largely unknown, but hormones and growth factors in blood, as well as mechanical loads on cells, have been implicated in the control of phenotypic modulation.

Atherosclerosis isadiseasecharacterizedbylesionslocatedinthewallofbloodvessels.Thelesionsareinducedbydisordersthatinjuretheendothelium,suchashypertension,diabetes,andsmoking.Threeformedelements(monocytes,Tlymphocytes,andplatelets)thatcirculateinthebloodstreamactonthedamagedvascularendothelium.There,theygeneratechemotacticfactorsandmitogensthatmodifythestructureofthesurroundingsmoothmusclecells.ThelatterlosemostoftheirthickandthinfilamentsanddevelopanextensiveroughendoplasmicreticulumandGolgicomplex.Thesecellsmigratetothesubendothelialspace(i.e.,thetunicamediaoftheartery),proliferate,andparticipateinformationofthefattylesionsorthefibrousplaquesthatcharacterizeatherosclerosis.InhibitionordownregulationofRhokinase(ROK)hasbeenshowntopromoteregressionofatherosclerotic-likelesionsinananimalmodel.ThemechanismormechanismsunderlyingthisbeneficialeffectofROKinhibitionareunclearbutmayberelatedtoregulationofbothendothelialpermeabilityandmonocytemigrationbyROK.Thatis,hyperactivityofROKhasbeenimplicatedinvariouspathologicalconditions,includingincreasedtransendothelialpermeability(perhapssecondarytoincreasedactomyosinactivity),whereasinhibitionofROKhasbeenshowntodecreasetransendothelialmigrationofmonocytesandneutrophils.

Biophysical Properties of Smooth Muscle

Smooth muscle contains large amounts of connective tissue composed of extensible elastin fibrils and inextensible collagen fibrils. Because this extracellular matrix can withstand high distending forces or loads, it is responsible for the passive length-tension curve measured in relaxed tissues. This ability of the matrix also limits organ volume.

When lengths are normalized to the optimal length for development of force (i.e., L0), the length-tension curves for smooth and skeletal muscle are very similar (

Fig. 14.15 ). However, the length-tension curves of striated and smooth muscle differ quantitatively. For example, smooth muscle cells shorten more than skeletal muscle cells do. In addition, smooth muscle is characteristically only partially activated, and the peak isometric force attained varies with the stimulus. In skeletal muscle the stimulus (i.e., action potential) always produces a full twitch contraction. Smooth muscle can generate active force comparable to that of skeletal muscle, even though smooth muscle contains only about a fourth as much myosin. This does not imply that the cross-bridges in smooth muscle have greater force-generating capacity. Instead, active cross-bridges in smooth muscle are much more likely to be in 2.4 2.0 1.6 1.2 0.8 0.4 0.6 0.8 1.0 1.2 Po = 2.22 ± 0.17 × 106 dynes/cm2 N = 11 •Fig. 14.15 Lengthdependenceofcontractionofsmoothmuscleshowsabell-shapedresponsesimilartothatseeninskeletalmuscle.Thesmoothmusclestripswereobtainedfromhogcarotidartery.Contractionwasinducedbypotassiumdepolarization.Optimalmusclelength(Lo)wasgraphicallydeterminedasthelengthatwhichmaximaltension(Po)wasdeveloped.Redcircles,passivetension;bluecircles,activetension,triangles,totaltension;(FromHerlihyJT,MurphyRA.Circ. Res. 1973;33:275-283.) the attached force-generating configuration because of their slow cycling kinetics.

Smooth and striated muscles both exhibit a hyperbolic dependence of shortening velocity on load. However, contraction velocities are far slower in smooth muscle than in striated muscle. One factor that underlies these slow velocities is that the myosin isoform in smooth muscle cells has low ATPase activity.

Skeletal muscle cells have a force-velocity curve in which shortening velocities are determined only by load and the myosin isoform (see ). In contrast, both force and shortening velocity, which reflect the number of cycling cross-bridges and their cycling rates, vary in smooth muscle. When activation of smooth muscle is altered, for example, by different frequencies of nerve stimulation or changing hormone concentrations, a “family” of velocity-stress curves can be derived (

Fig. 14.16 ). This implies that both cross-bridge cycling rates and the number of active cross-bridges in smooth muscle are regulated in some way,

Vo(% of max)

Velocity (% of max)

Stress (% of max) •Fig. 14.16 A,Force-velocitycurvesforfastandslowhumanskeletalmusclecellsandsmoothmuscle.B,Smoothmuscleshavevariableforce-velocityrelationshipsthataredeterminedbythelevelofCa++-stimulatedcross-bridgephosphorylation.C,Maximalshorteningvelocitieswithnoload(Vo,whichrepresentstheinterceptsontheordinateinB)aredirectlydependentoncross-bridgephosphorylationbyMLCK.D,Activeforce/stress(abscissainterceptsinB)risesrapidlywithphosphorylation;nearmaximalstress,itmaybegeneratedwithonly20%to30%ofthecross-bridgesinthephosphorylatedstate.

which is in marked contrast to striated muscle. This difference is conferred by a regulatory system that depends on phosphorylation of cross-bridges, which in turn depends on myoplasmic [Ca++]. Because myosin light-chain phosphorylation is required for actin-myosin interaction in smooth muscle, a dependence of maximal force on the degree of myosin phosphorylation is expected (i.e., phosphorylation of more myosin molecules results in more actin-myosin interactions and hence more force generated). The variation in maximal shortening velocity as a function of the degree of myosin phosphorylation may reflect dephosphorylation of the myosin light chain while the myosin is still attached to the actin, thus slowing the rate of detachment (i.e., latch state) at low levels of phosphorylation. At higher levels of phosphorylation, the likelihood of latch states would be reduced and the myosin cross-bridges would be released more quickly from actin, thereby yielding a higher shortening velocity at all loads (see

Fig. 14.16B 1.

Smooth muscle cells are linked by a variety of junctions that serve both mechanical and communication roles. These linkages are essential in cells that must contract uniformly.

2.

The sarcolemma plays an important role in Ca++ exchange between extracellular fluid and myoplasm. The sarcolemma of smooth muscle contains numerous caveolae that contribute to regulation of intracellular [Ca++] and also appear to serve as a scaffold for signaling molecules. The sarcoplasmic reticulum (SR) contains an intracellular Ca++ pool that can be mobilized to transiently increase myoplasmic [Ca++]. Myoplasmic [Ca++] is dependent on extracellular Ca++ . Transporters in the sarcolemma that regulate myoplasmic [Ca++] include receptor-mediated Ca++ channels, voltage-gated Ca++ channels, Ca++-ATPase, and the 3Na+1Ca++ antiporter. The SR also regulates myoplasmic [Ca++]. The Ca++ channels in the SR open in response to a chemical. Neurotransmitters or hormones that act via receptors in the sarcolemma can activate phospholipase C (PLC), followed by generation of the second messenger inositol 1,4-triphosphate (InsP3). InsP3 then activates InsP3-gated Ca++ channels on the SR. Many agonists that activate PLC through G protein–coupled receptors also activate the RhoA/Rho kinase (ROK) signaling cascade, thereby increasing the sensitivity of smooth muscle contraction to Ca++ . Smooth muscle SR also contains Ca++-gated Ca++ channels (ryanodine receptor [RYR]). Ca++ reaccumulates in the SR via SERCA (sarcoplasmic endoplasmic reticulum Ca++ ATPase).

3.

Smooth muscles contain contractile units that consist of small groups of thick myosin filaments that interdigitate with large numbers of thin filaments attached to Z-line equivalents termed dense bodies or membrane-dense areas. No striations are evident. Contraction is caused by a sliding filament–crossbridge mechanism.

4.

Contraction of smooth muscle is dependent on both release of Ca++ from the SR and entry of Ca++ across the sarcolemma. Smooth muscle lacks troponin. Phosphorylation of cross-bridges by a Ca++-dependent myosin light chain kinase (MLCK) is necessary for attachment to the thin filament. Dephosphorylation of an attached cross-bridge by myosin phosphatase (MP) slows its cycling rates. Higher myoplasmic [Ca++] increases the ratio of MLCK to MP activity, with the result that more of the cross-bridges remain phosphorylated throughout a cycle. This increases shortening velocities.

5.

Smooth muscle activity is controlled by nerves (principally autonomic), circulating hormones, locally generated signaling substances, junctions with other smooth muscle cells, and even junctions with other non–smooth muscle cells. A variety of hormones/ agonists increase the sensitivity of smooth muscle contraction to Ca++ by reducing the activity of MP and reciprocally increasing the activity of MLCK at a given intracellular [Ca++]. Activation of the RhoA/ROK signaling cascade contributes to this inhibition of MP and stimulation of MLCK, and hence to the increase in sensitivity of smooth muscle contraction to Ca++ . Smooth muscle also has an intrinsic ability to respond to stretch, which is important for autoregulation of blood flow to various tissues.

6.

The response to sustained or tonic stimulation is a rapid contraction followed by sustained maintenance of force with reduced cross-bridge cycling rates and ATP consumption. This behavior, called the latch state, is advantageous for muscles that may need to withstand continuous external force, such as blood vessels, which must be able to withstand blood pressure. During the latch state, ATP is consumed at less than 1/300 the rate needed to maintain the same force in skeletal muscle.

7.

The length-tension relationships, hyperbolic velocity-load relationships, power output curves, and ability to resist imposed loads are comparable to those of skeletal muscle. Shortening velocities and ATP consumption rates are very low in smooth muscle, in keeping with expression of a myosin isoform with low activity. Smooth muscles also have the unusual ability to alter velocity-stress relationships, which reflects regulation of both the number of active cross-bridges (determining force) and their average cycling rates for a given load (determining velocity).

Burnstock G, Ralevic V. Purinergic signaling and blood vessels in health and disease. Pharmacol Rev. 2014;66:102-192.

Joyner MJ, Casey DP. Regulation of increased blood flow (hyperemia) to muscles during exercise: a hierarchy of competing physiological needs. Physiol Rev. 2015;95:549-601.

Longden TA, et al. Ion channel networks in the control of cerebral blood flow. J Cereb Blood Flow Metab. 2016;36:492-512.

Momotani K, Somlyo AV. p63RhoGEF: a new switch for G q-mediated activation of smooth muscle. Trends Cardiovasc Med. 2012;22:122-127.

Roberts OL, Dart C. cAMP signalling in the vasculature: the role of Epac (exchange protein directly activated by cAMP). Biochem Soc Trans. 2014;42:89-97.

8. Smooth muscle is also a synthetic and secretory cell with a major role in formation of the extensive extracellular matrix that surrounds and links the cells. Cellular hypertrophy occurs in response to physiological needs, and smooth muscle cells retain the potential to divide.

Schlossmann J, Desch M. IRAG and novel PKG targeting in the cardiovascular system. Am J Physiol Heart Circ Physiol. 2011;301: H672-H682.

Shimokawa H, et al. RhoA/Rho-Kinase in the Cardiovascular System. Circ Res. 2016;118:352-366.

Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev. 2003;83:1325-1358.

Spinelli AM, Trebak M. ORAI channel-mediated Ca2+ signals in vascular and airway smooth muscle. Am J Physiol Cell Physiol. 2016;310:C402-C413.

Tinker A, et al. The role of ATP-sensitive potassium channels in cellular function and protection in the cardiovascular system. Br J Pharmacol. 2014;171:12-23.

SECTION 4The Cardiovascular System ACHILLES J. PAPPANO AND WITHROW GIL WIER

Overview of Circulation Regulation of the Heart and Vasculature

Chapter 16 Elements of Cardiac Function Chapter 19 Integrated Control of the Chapter 17 Cardiovascular System Properties of the Vasculature

Upon completion of this chapter, the student should be able to answer the following questions: 1.

How does the arrangement of heart and vessels enable unidirectional flow of well-oxygenated blood to the body?

2.

What is the advantage of the reciprocal relation between blood flow velocity and vascular cross-sectional area?

3.

How do the differing compositions (smooth muscle, fibrous and elastic tissue) of blood vessels contribute to their respective functions?

he circulatory system transports and distributes essential substances to tissues and removes metabolic byproducts. This system also participates in homeostatic mechanisms such as regulation of body temperature, maintenance of fluid balance, and adjustment of O2 and nutrient supply in various physiological states. The cardiovascular system, which accomplishes these tasks, is composed of a pump (the heart), a series of distributing and collecting tubes (blood vessels), and an extensive system of thin vessels (capillaries) that enable rapid exchange between the tissues and vascular channels. Blood vessels throughout the body are filled with a heterogeneous fluid (blood) that is essential for the transport processes performed by the heart and blood vessels. This chapter is a general, functional overview of the heart and blood vessels, whose functions are analyzed in much greater detail in subsequent chapters.

The Heart

The heart consists of two pumps in series: one pump propels blood through the lungs for exchange of O2 and CO2 (the pulmonary circulation) and the other pump propels blood to all other tissues of the body (the systemic circulation). Flow of blood through the heart is in one direction (unidirectional). Unidirectional flow through the heart is achieved by the appropriate arrangement of flap valves. Although cardiac output is intermittent, continuous flow to body tissues (periphery) occurs by distention of the aorta and its branches during ventricular contraction (systole) and by elastic recoil of the walls of the large arteries with forward propulsion of the blood during ventricular relaxation (diastole).

The Cardiovascular Circuit

In the normal intact circulation, the total volume of blood is constant, and an increase in the volume of blood in one area must be accompanied by a decrease in another. However, the distribution of blood circulating to the different regions of the body is determined by the output of the left ventricle and by the contractile state of the resistance vessels (arterioles) of these regions. The circulatory system is composed of conduits arranged in series and in parallel (

Fig.

15.1 ). This arrangement, which is discussed in subsequent chapters, has important implications in terms of resistance, flow, and pressure in blood vessels.

Blood entering the right ventricle via the right atrium is pumped through the pulmonary arterial system at a mean pressure about one seventh that in the systemic arteries. The blood then passes through the lung capillaries, in which CO2 in the blood is released and O2 is taken up. The O2-rich blood returns via the pulmonary veins to the left atrium, where it is pumped from the ventricle to the periphery, which thus completes the cycle.

Blood moves rapidly through the aorta and its arterial branches. As these branches approach the periphery, the branches narrow, and their walls become thinner. They also change histologically. The aorta is a predominantly elastic structure, but the peripheral arteries become more muscular until, at the arterioles, the muscular layer predominates (

Fig. 15.2

In the large arteries, frictional resistance is relatively small, and pressures are only slightly less than those in the aorta. The small arteries, on the other hand, offer moderate resistance to blood flow. This resistance reaches a maximal level in the arterioles, which are sometimes referred to as the “stopcocks” of the vascular system. Hence, the pressure drop is greatest across the terminal segment of the small arteries and the arterioles (

Fig. 15.3 ). Adjustment in the degree of contraction of the circular muscle of these small vessels allows regulation of tissue blood flow and aids in the control of arterial blood pressure.

In addition to the reduction in pressure along the arterioles, there is a change from pulsatile to steady blood flow (see

Fig. 15.3 ). Pulsatile arterial blood flow, caused by the intermittent ejection of blood from the heart, is damped • Fig. 15.3 Phasic Pressure, Velocity of Flow, and Cross-Sectional • Fig. 15.1 Schematic Diagram of the Parallel and Series Arrange-Area of the Systemic Circulation. The important features are the major ment of the Vessels That Constitute the Circulatory System. The pressure drop across the small arteries and arterioles, the inverse capillary beds are represented by thin lines connecting the arteries relationship between blood flow velocity and cross-sectional area, and (on the right) with the veins (on the left). The crescent-shaped thicken-the maximal cross-sectional area and minimal flow rate in the capilings proximal to the capillary beds represent the arterioles (resistance laries. (From Levick JR. An Introduction to Cardiovascular Physiology. vessels). (Redrawn from Green HD. In: Glasser O [ed]. Medical Physics. 5th ed. London: Hodder Arnold; 2010.)Vol 1. Chicago: Year Book; 1944.) • Fig. 15.2 Internal Diameter, Wall Thickness, and Relative Amounts of the Principal Components of the Vessel Walls of the Various Blood Vessels That Constitute the Circulatory System. Cross-sections of the vessels are not drawn to scale because of the huge range in size from aorta and venae cavae to capillary. (Redrawn from Burton AC. Relation of structure to function of the tissues of the wall of blood vessels. Physiol Rev. 1954;34:619.) at the capillary level by a combination of two factors: distensibility of the large arteries and frictional resistance in the small arteries and arterioles.

Many capillaries arise from each arteriole. The total cross-sectional area of the capillary bed is very large despite the fact that the cross-sectional area of each capillary is less than that of each arteriole. As a result, blood flow velocity becomes quite slow in the capillaries (see

Fig. 15.3 ), which is analogous to the decrease in velocity of flow in the wide regions of a river. Conditions in the capillaries are ideal for the exchange of diffusible substances between blood and tissue because capillaries consist of short tubes with walls that are only one cell thick and in which flow velocity is low.

On its return to the heart from the capillaries, blood passes through venules and then through veins of increasing size. Pressure within these vessels progressively decreases until the blood reaches the right atrium (see

Fig. 15.3 ). Near the heart, the number of veins decreases, the thickness and composition of the vein walls change (see

Fig. 15.2), the total cross-sectional area of the venous channels diminishes, and the velocity of blood flow increases (see

Fig. 15.3 ). The velocity of blood flow and the cross-sectional area at each level of the vasculature are essentially mirror images (see

Fig. 15.3

Table 15.1 ) indicate that between the aorta and the capillaries, the total cross-sectional area increases about 500-fold. The volume of blood in the systemic vascular system is greatest in the veins and venules (64%). Only 6% of total blood volume exists in the capillaries, and 14% of total blood volume is found in the aorta, arteries, and arterioles. In contrast, blood volume in the pulmonary vascular bed is about equally divided among the arterial, capillary, and venous vessels. The cross-sectional area of the venae cavae is larger than that of the aorta. Therefore, the velocity of flow is slower in the venae cavae than in the aorta (see

Fig. 15.3

Aortaandlargearteries3006.0Smallarteries4008.0Capillaries3006.0Smallveins230046.0Largeveins90018.0Total420084.0

Arteries1302.6Capillaries1102.2Veins2004.0Total4408.8Heart (End-Diastole) 3607.2

DatafromBoronWF,BoulpaepEL.Medical Physiology. 2nded.Philadelphia:ElsevierSaunders;2009.*Valuesrefertoa70-kgwoman.

Inapatientwithhyperthyroidism(Graves’ disease), basalmetabolismiselevatedandoftenassociatedwitharteriolarvasodilation.Thisreductioninarteriolarresistancediminishesthedampingeffectonpulsatilearterialpressureandismanifestedaspulsatileflowinthecapillaries,asobservedinthefingernailbedsofpatientswiththisailment.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

How does the action potential contribute to excitability and contraction in heart muscle?

2.

What is automaticity, and how does it differ from excitability? How do derangements of these properties contribute to arrhythmias?

3.

What is the structural basis of the electrocardiogram?

4.

How are the concepts of preload and afterload, developed for skeletal muscle, applied to the contraction of the heart?

5.

What is the role of Ca++ in excitation-contraction coupling?

6.

What changes occur in atrial and ventricular pressures and volumes during a cardiac cycle, and what is their temporal relation to the electrocardiogram?

7.

How does the relation between end-diastolic volume and left ventricular developed pressure define the Frank-Starling law of the heart and regulate the force of cardiac contraction?

8.

What is the pressure-volume loop of the left ventricle, and how does it define changes in left ventricular function?

9.

How is cardiac metabolism linked to O2 consumption, and how are these processes affected by changes in cardiac work?

Electrical Properties of the Heart

The cells of the heart, like neurons, are excitable and generate action potentials. These action potentials initiate contraction and thus determine the heart rate. Disorders in electrical activity can induce serious and sometimes lethal disturbances in cardiac rhythm.

In this section, the electrical properties of cardiac cells are described. In addition, how these electrical properties account for the electrocardiogram (ECG) is considered. The initiation of contraction as a result of the electrical properties of cardiac cells is considered in a later section.

The Cardiac Action Potential

Fig. 16.1 illustrates action potentials found in different cardiac cells. Two main types of action potentials occur in the heart and are depicted. One type, the fast response, occurs in normal atrial and ventricular myocytes and in the specialized conducting fibers (Purkinje fibers of the heart) and is divided into five phases. The rapid upstroke of the action potential is designated phase 0. The upstroke is followed by a brief period of partial, early repolarization (phase 1) and then by a plateau (phase 2) that persists for approximately 0.1 to 0.2 second. The membrane then repolarizes (phase 3) until the resting state of polarization (phase 4) is again attained. Final repolarization (phase 3) develops more slowly than depolarization (phase 0). The other type of action potential, the slow response, occurs in the sinoatrial (SA) node, which is the natural pacemaker region of the heart, and in the atrioventricular (AV) node, which is the specialized tissue that conducts the cardiac impulse from the atria to the ventricles. The slow-response cells lack the early repolarization phase (phase 1).

Other differences between the electrical properties of the fast-response and slow-response cells include the following: The resting membrane potential (phase 4) of the fast-response cells is considerably more negative than that of the slow-response cells. Moreover, the slope of the upstroke (phase 0), the amplitude of the action potential, and the overshoot (membrane voltage positive to 0 mV) are greater in the fast-response cells than in the slow-response cells. The action potential amplitude and the steepness of the upstroke are important determinants of propagation velocity along the myocardial fibers. In slow-response cardiac tissue, the action potential is propagated more slowly and conduction is more likely to be blocked than in fast-response cardiac tissue. Slow conduction and a tendency toward conduction block increase the likelihood of some rhythm disturbances (see the section “

The action potential initiates contraction of the myocyte. The relationship between the action potential and contraction of a cardiac myocyte is shown in

Fig. 16.2 . Rapid depolarization (phase 0) precedes cell shortening, and completion of repolarization occurs just before peak shortening. Relaxation of the muscle takes place mainly during phase 4 of the action potential. The duration of contraction usually parallels the duration of the action potential.

The various phases of the cardiac action potential are associated with changes in cell membrane permeability, mainly by Na+, K+, and Ca++ ions. Changes in cell membrane permeability alter the rate of movement of these ions across the membrane and thereby change the membrane voltage (Vm). These changes in permeability are accomplished by the opening and closing of ion channels that are specific for individual ions (see

As with all other cells in the body, the concentration of K+ inside a cardiac muscle cell (intracellular [K+]) exceeds the concentration outside the cell (extracellular [K+]). The concentration gradient is reversed for Na+ and Ca++ . Estimates of the extracellular and intracellular concentrations of Na+, K+, and Ca++ and the Nernst equilibrium potentials (see

Eq. 1.5b ) for these ions are compiled in

Table 16.1

The resting cell membrane has a relatively high permeability for K+; permeability for Na+ and Ca++ is much less. Because of the existing chemical gradient for K+ and Vm, K+ tends to diffuse from the inside to the outside of the cell.

3 Any diffusion of K+ that occurs at the resting membrane potential (i.e., during phase 4) takes place mainly through 4 specific potassium channels. Several types of potassium channels exist in cardiac cell membranes. Opening and –120

ERP RRP closing of some of these channels are regulated by Vm, whereas others are controlled by a chemical signal (e.g., the extracellular acetylcholine concentration). The specific 4 is a voltage-regulated channel that conducts the inward rectifying K+ current (iK1), which is discussed in more detail later. For now, it is necessary only to know how this current is established.

The dependence of Vm on conductance and the intracellular and extracellular concentrations of K+, Na+, and other ions are described by the chord conductance equation •Fig. 16.2 Temporalrelationshipbetweenthechangesintrans-membranepotential(top trace) andcellshortening(bottom trace) inasingleventricularmyocyte.Thevalueof0mVmembranepotentialisindicatedby“0.”(FromPappanoA.unpublishedrecord,1995.)0 50 mV 400 msec 7˜m •Fig. 16.1 Action Potentials of Cardiac Fibers. A, Fast-responsecardiacfibers.B, Slow-responsecardiacfibers.Thephasesoftheactionpotentialsarelabeled(seetextfordetails),asaretheeffectiverefractoryperiod(ERP)andtherelativerefractoryperiod(RRP).Notethatincomparisonwithfast-responsefibers,therestingpotential ofslowfibersislessnegative,theupstroke(phase0)oftheactionpotentialislesssteep,theamplitudeoftheactionpotentialissmaller,phase1isabsent,andtheRRPextendswellintophase4afterthefibershavefullyrepolarized.

Eq. 2.7 ). In a resting cardiac cell, K+ conductance (gK) is approximately 100 times greater than Na+ conductance (gNa). Therefore, Vm is similar to the Nernst equilibrium potential for K+ . As a result, alterations in extracellular [K+] can significantly change Vm: Hypokalemia causes hyperpolarization, and hyperkalemia causes depolarization. In contrast, because gNa is so small in the resting cell, changes in extracellular [Na+] do not significantly affect Vm.

Fastresponsesofcardiacmusclemaychangetoslowresponsesincertainpathologicalconditions.Forexample,incoronaryarterydisease,aregionofcardiacmusclemaybedeprivedofitsnormalbloodsupply.Asaresult,[K+]intheinterstitialfluidthatsurroundstheaffectedmusclecellsrisesbecauseK+ islostfromtheinadequatelyperfused(orischemic)cells.Theactionpotentialsinsomeofthesecellsmaythenbeconvertedfromfasttoslowresponses.Conversionfromafasttoaslowresponseasaresultofincreasinginterstitial[K+]isillustratedlaterin

Fig.16.13

Genesis of the Upstroke (Phase 0)

Any stimulus that abruptly depolarizes Vm to a critical value (the threshold) elicits an action potential. The characteristics of fast-response action potentials are shown in

Fig. 16.1A.

The rapid depolarization (phase 0) is related almost exclusively to the influx of Na+ into the myocyte as a result of a sudden increase in gNa. The action potential amplitude (the membrane potential change during phase 0) is dependent on extracellular [Na+]. When extracellular [Na+] is decreased, the amplitude of the action potential decreases, and when it is reduced from its normal value of approximately 140 mEq/L to approximately 20 mEq/L, the cell is no longer excitable.

When the resting membrane potential, Vm, is suddenly depolarized from −90 mV to the threshold level of approximately −65 mV, the cell membrane properties change dramatically. Na+ enters the myocyte through specific fast voltage-activated sodium channels that exist in the membrane (see

Fig. 5.7). These channels can be blocked by the puffer fish toxin tetrodotoxin. In addition, many drugs used to treat certain cardiac rhythm disturbances (cardiac arrhythmias) act by blocking these fast sodium channels.

The sodium channels open very rapidly, or activate (in ≈0.1 msec), thereby resulting in an abrupt increase in gNa. However, once open, the sodium channels inactivate (time course ≅ 1 to 2 msec), and gNa rapidly decreases (Fig. 16.3 ). The sodium channels remain in the inactivated state until the membrane begins to repolarize. With repolarization, the channel transitions to the closed state, from which it can then be reopened by another depolarization of Vm to the threshold. These properties of the sodium channel are the basis of the refractory period of the action potential. When the sodium channels are in the inactivated state, they cannot be reopened, and another action potential cannot be generated. During this period, the cell is said to be in the effective refractory period. This prevents a sustained, tetanic contraction of cardiac muscle, which would retard ventricular relaxation and therefore interfere with the normal intermittent pumping action of the heart. As the cell repolarizes (phase 3), the inactivated channels begin to transition to the closed state. During this period, called the relative refractory period, another action potential can be generated, but it requires a larger-than-normal depolarization of Vm. Only when Vm has returned to the resting level (phase 4) are all the sodium channels closed and thus able to be reactivated by the normal depolarization of Vm.

Anioniccurrentthroughonemembranechannelcanbemeasuredwiththepatchclamptechnique.Theindividualchannelsopenandcloserepeatedlyinarandommanner.Thisprocessisillustratedin

Fig.16.4 ,whichshowsthecurrentflowthroughsodiumchannelsinamyocardialcell.Beforethetimeindicatedbythearrow,themembranepotentialwasclampedat−85mV.Atthetimeindicatedbythearrow,thepotentialwassuddenlychangedto−45mV,atwhichvalueitwasheldfortheremainderoftherecording.

Fig.16.4 indicatesthatimmediatelyafterthemembranepotentialwasmadelessnegative,onesodiumchannelopened(1.5pAinamplitude),andthenasecondsodiumchannelopened(3pAtotalcurrentfrombothchannels),whereuponbothchannelsclosed(i.e.,thecurrentreturnedto0).Bothchannelsclosedforapproximately4or5msec,andthenbothchannelsopenedatthesametime.Thereafter,onechannelclosedandthenthesecondrapidlyclosed.Finally,afterseveralseconds,onechannelopenedandthenclosed.Thereafter,bothchannelsremainedclosedfortherestoftherecording,eventhoughthemembranevoltagewasheldconstantat−45mV.

Genesis of Early Repolarization (Phase 1)

In many cardiac cells that have a prominent plateau, phase 1 is an early, brief period of limited repolarization. This brief repolarization results in the notch between the end of the upstroke and the beginning of the plateau (see

Figs. 16.1 16.3).

Repolarization is brief because of activation of a transient outward current (ito) carried mainly by K+ . Activation of potassium channels during phase 1 causes a brief efflux of K+ from the cell because the cell interior is positively charged and intracellular [K+] greatly exceeds extracellular [K+] (see

Fig. 16.3 ). The cell is briefly and partially repolarized as a result of this transient efflux of K+ .

The size of the phase 1 notch varies among cardiac cells. It is prominent in myocytes in the epicardial and midmyocardial regions of the left ventricular wall (

Fig. 16.5 ) and in ventricular Purkinje fibers. However, the notch is negligible in myocytes from the endocardial region of the left ventricle (see

Fig. 16.5) because the density of i to channels is less in these cells. The notch is also less prominent in the presence 4 Chemical Electrostatic 3 2 1 0 Fast Na+ Channel Na+ Na+ – – – – – – – – – – – – – – – – – – – – K+ Channel (ito) K+ K+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Ca++ Ca++ Ca++ K+ K+ K+ Channels (iK, iK1) K+ K+ – – – – – – – – – – – – – – – – – – – – K+ Channels (iK, iK1) K+ K+ – – – – – – – – – – – – – – – – – – – – 0 1 3 4 2

K+ Channels (iK, iK1, ito) •Fig. 16.3 Principal Ionic Currents and Channels That Generate the Various Phases of the Action Potential in a Cardiac Cell. Phase 0, ThechemicalandelectrostaticforcesbothfavortheentryofNa+ intothecellthroughfastsodiumchannelstogeneratetheupstroke.Phase 1, BoththechemicalandelectrostaticforcesfavortheeffluxofK+ throughtransientoutwardcurrent(ito)channelstogenerateearly,partialrepolarization.Phase 2, Duringtheplateau,thenetinfluxofCa++ throughcalciumchannelsisbalancedbytheeffluxofK+ throughoutwardrectifyingcurrent(iK),inwardrectifyingcurrent(iK1),anditochannels.Phase 3, ThechemicalforcesthatfavortheeffluxofK+ throughiKandiK1channelspredominateovertheelectrostaticforcesthatfavortheinfluxofK+ throughthesesamechannels.Phase 4, ThechemicalforcesthatfavortheeffluxofK+ throughiKandiK1channelsveryslightlyexceedtheelectrostaticforcesthatfavortheinfluxofK+ throughthesesamechannels.

0 1.5 3 4.5 10 msec pA Channel #1 current Channel #2 current •Fig. 16.4 Current (in Picoamperes [pA]) Through Two Individual Sodium Channels in a Cultured Heart Cell, Recorded With the Patch Clamp Technique. Membranevoltagewasheldat−85mV,thenabruptlychangedto−45mVatthetimeindicatedbythearrow, andheldatthispotentialfortheremainderoftherecord.(RedrawnfromCachelinAB,etal.J Physiol. 1983;340:389.) of 4-aminopyridine, which blocks the potassium channels that carry ito.

Genesis of the Plateau (Phase 2)

During the action potential plateau, Ca++ enters myocardial cells through calcium channels (described in next paragraph) that activate and inactivate much more slowly than the fast sodium channels do. During the flat portion of phase 2 (see

Figs. 16.1 16.3), this influx of Ca ++ is counterbalanced by the efflux of K+ . K+ exits through channels that conduct mainly the ito, iK, and iK1 currents. The ito current is responsible for phase 1, as described previously, but it is not completely inactivated until after phase 2 has expired. The iK and iK1 are described later in this section of the chapter.

Ca++ enters the cell via voltage-regulated calcium channels, which are activated as Vm becomes progressively less negative during the action potential upstroke. Two types of calcium channels (L-type and T-type) have been identified in cardiac tissue. Some of their important characteristics are illustrated in

Fig. 16.6 . L-type channels are so designated because once open, they inactivate slowly (see

Fig. 16.6, lower panel) and enable a long-lasting Ca++ current. They are the predominant type of calcium channel in the heart, and they are activated during the action potential upstroke when Vm reaches approximately −20 mV. L-type channels are blocked by calcium channel antagonists such as verapamil, amlodipine, and diltiazem (

Fig. 16.7

T-type (or “transient”) calcium channels are much less abundant in the heart. They are activated at membrane potentials that are more negative (approximately −70 mV) than the potential that activates L-type channels (−20 mV). They also inactivate more quickly than do L-type channels (see

Fig. 16.6 ; compare the upper and lower panels).

Opening of calcium channels results in an increase in Ca++ conductance (gCa) and calcium current (iCa) soon after •Fig. 16.5 Actionpotentialsrecordedfromtheepicardial(Epi)(A), midmyocardial(Mid)(B), andendocardial(Endo)(C) regionsofthefreewalloftheleftventricle.Thepreparationsweredrivenatabasiccyclelength(BCL):thatis,atinterstimulusintervalsof300and8000msec.Thevalueof0mVmembranepotentialisindicatedby“0.”(FromLiuD-W,etal.Circ Res. 1993;72:671.) the action potential upstroke (see

Fig. 16.3 ). Because the intracellular concentration of Ca++ is much lower than the extracellular concentration of Ca++ (see

Table 16.1 ), the increase in gCa promotes the influx of Ca++ into the cell throughout the plateau. This Ca++ influx during the plateau is involved in excitation-contraction coupling, as described in the section “

Various neurotransmitters and drugs may substantially influence gCa. The adrenergic neurotransmitter norepinephrine, the β-adrenergic receptor agonist isoproterenol, and various other catecholamines enhance gCa, whereas the parasympathetic neurotransmitter acetylcholine decreases gCa. Enhancement of gCa by catecholamines is the principal mechanism by which they enhance cardiac muscle contractility.

During the plateau (phase 2) of the action potential, the concentration gradient for K+ across the cell membrane is virtually the same as it is during phase 4; at that time, however, Vm is positive. Therefore, there is a large gradient that favors efflux of K+ from the cell (see

Fig. 16.3). If g

K were the same during the plateau as it is during phase 4, efflux of K+ during phase 2 would greatly exceed the influx of Ca++ , and a sustained plateau could not be achieved.

•Fig. 16.6 Effects of Isoproterenol on the Ca++ Currents. ThecurrentswereconductedbyT-type(upper panel) andL-type(lower panel) calciumchannelsinatrialmyocytes.Upper panel, Themembranepotentialwaschangedfrom−80to−20mV.Lower panel, Themembranepotentialwaschangedfrom−30to+30mV.(RedrawnfromBeanBP.J Gen Physiol. 1985;86:1.) •Fig. 16.7 Effectsofdiltiazem,acalciumchannelantagonist,ontheactionpotentials(inmillivolts[mV])andisometriccontractileforces(inmillinewtons[mN])recordedfromapapillarymuscleinvitro.Thetrac-ingswererecordedundercontrolconditions(C)andinthepresenceofdiltiazeminconcentrationsof3,10,and30µmol/L.(RedrawnfromHirthC,etal.J Mol Cell Cardiol. 1983;15:799.)20 50 msec Action potential Force 0 mV 10 30 C and 3 C 3 10 30 mN 0.5 50 msec

However, as Vm approaches and then attains positive values near the peak of the action potential upstroke, gK suddenly decreases (

Fig. 16.8 ). The diminished K+ current associated with the reduction in gK prevents excessive loss of K+ from the cell during the plateau.

ToenhancegCa,catecholaminesfirstbindtoβ-adrenergicreceptorsinthecardiaccellmembrane.Thisinteractionstimulatesthemembrane-boundenzymeadenylatecyclase,whichraisestheintracellularconcentrationofcyclicadenosinemonophosphate(cAMP;seealso ).TheriseincAMPactivatescAMP-dependentproteinkinase,whichinturnpromotesphosphorylationofL-typecalciumchannelsinthecellmembraneandthusaugmentstheinfluxofCa++ intothecells(see

Fig.16.6 ).Conversely,acetylcholineinteractswithmuscarinicreceptorsinthecellmembranetoinhibitadenylatecyclase.Inthisway,acetylcholineantagonizestheactivationofcalciumchannelsandtherebydiminishesgCa.

Calciumchannelantagonistsaresubstancesthatblockcalciumchannels.Examplesincludethedrugsverapamil,amlodipine,anddiltiazem.ThesedrugsdecreasegCaandtherebyimpedetheinfluxofCa++ intomyocardialcells.Calciumchannelantagonistsdecreasethedurationoftheactionpotentialplateauanddiminishthestrengthofthecardiaccontraction(see

Fig.16.7 ).Calciumchannelantagonistsalsodepressthecontractionofvascularsmoothmuscleandtherebyinducegeneralizedvasodilation.Thisdiminishedvascularresistancereducesthecounterforce(afterload)thatopposesthepropulsionofbloodfromtheventriclesintothearterialsystem,asexplainedin .Hence,vasodilatordrugssuchasthecalciumchannelantagonistsareoftenreferredtoasafterload-reducing drugs.

The reduction in gK at both positive and low negative values of Vm is called inward rectification. Inward rectification is a characteristic of several K+ currents, including iK1 (

Fig. 16.9 ). For these channels, large K+ currents flow at negative values of Vm (i.e., gK is large). However, when Vm is near 0 mV or positive, as occurs during the plateau (phase 2), little or no K+ current flows (i.e., gK is small). Thus the substantial gK that prevails during phase 4 of the cardiac action potential (see

Fig. 16.8 ) is large because of the iK1 channels, but current through these channels is greatly diminished during the plateau (see

Fig. 16.9

Other potassium channels play a role in phase 2 of the action potential. These potassium channels are characterized as delayed iK channels. They are closed during phase 4 and are activated very slowly by the potentials that prevail toward the end of phase 0. Hence, activation of these channels tends to increase gK very gradually during phase 2. These channels play only a minor role during phase 2, but they contribute to the process of final repolarization (phase 3), as described in the section “

Genesis of Repolarization (Phase 3) .” There exist two types of iK channels, classified according to their rates of activation: The more slowly activating channel is designated the iKs channel, and the more rapidly activating channel is designated the iKr channel (see

Fig. 16.8). The

Nav1.5 SCN5A

Cav1.2 CACNA1C

ICa,L

IK1 Ito,1 Ito,2 IKr IKs

Kir2.1/2.2 Kv4.2/4.3 Kv1.4/1.7 •Fig. 16.8 Changes in Ion Currents During the Various Phases of the Action Potential in a Fast-Response Cardiac Ventricular Cell. Upper panels, Depolarizingioncurrents.Lower panels, Repolarizingioncurrents.TheinwardcurrentsincludethefastNa+ (iNa)andL-typeCa++ (iCa,L)currents.OutwardcurrentsareiK1,ito,andtherapid(iKr)andslow(iKs)delayedrectifierK+ currents.Theclonesandgenesfortheprincipalioniccurrentsarealsoincluded.(RedrawnfromTomaselliG,MarbánE.Cardiovasc Res. 1999;42:270.) duration of the action potential in myocytes in various regions of the ventricular myocardium is determined in part by the relative distributions of these iKr and iKs channels.

The action potential plateau persists as long as the efflux of charge carried mainly by K+ is balanced by the influx of charge carried mainly by Ca++ . The effects of altering this balance are exemplified by the action of the calcium channel antagonist diltiazem in a papillary muscle preparation (see

Fig. 16.7 ). With increasing concentrations of diltiazem, the plateau voltage becomes progressively less positive and the plateau duration diminishes. Conversely, administration of certain potassium channel antagonists prolongs the plateau substantially.

Genesis of Final Repolarization (Phase 3)

The process of final repolarization (phase 3) starts at the end •Fig. 16.9 Inward Rectified K+ of phase 2, when efflux of K+ from the cardiac cell begins to tricular Myocyte When the Potential Was Changed From a Holding exceed influx of Ca++ . As noted, at least three outward K+

Potential of −80 mV to Various Test Potentials. Positivevaluesalong currents (ito, iK, and iK1) contribute to the final repolariza tion (phase 3) of the cardiac cell (see

Figs. 16.3 16.8 inwardcurrents.Thepointatwhichthecurrenttraceintersectsthe x-axisisthereversalpotential(open circle); itdenotestheNernstThe ito, iKr, and iKs currents help initiate repolarization. equilibriumpotential(EK),atwhichpointthechemicalandelectrostaticThese currents are therefore important determinants of forcesareequal.Vm,membranepotential.(RedrawnfromGilesWR, the duration of the plateau. For example, the duration of

ImaizumiY.J Physiol [Lond]. 1988;405:123.) the plateau is substantially less in atrial myocites than in ventricular myocytes (

Fig. 16.10 ) because the magnitude •Fig. 16.10 Typical Action Potentials (in Millivolts, y-Axis). Thesepotentialswererecordedfromcellsintheventricle(A), sinoatrial(SA)node(B), andatrium(C). NotethatthetimecalibrationinB differsfromthatinA andC. Numbersonthecurvesdenotethephasesoftheactionpotential.(FromHoffmanBF,CranefieldPF.Electrophysiology of the Heart. NewYork:McGraw-Hill;1960.) of ito during the plateau is greater in atrial myocites than in ventricular myocytes. As already noted, the duration of the action potential in ventricular myocytes varies considerably with the location of these myocytes in the ventricular walls (see

Fig. 16.5). The i to and iK currents mainly account for these differences. In endocardial myocytes, in which the duration of the action potential is least, the magnitude of iK is greatest. The converse applies to the midmyocardial myocytes. The magnitude of iK and the duration of the action potential are intermediate for epicardial myocytes.

The iK1 current does not participate in the initiation of repolarization because the conductance of these channels is very small over the range of Vm values that prevail during the plateau. However, the iK1 channels contribute substantially to the rate of repolarization once phase 3 has been initiated. As Vm becomes increasingly negative during phase 3, the conductance of the channels that carry the iK1 current progressively increases and thereby accelerates repolarization (see

Fig. 16.3

Restoration of Ionic Concentrations (Phase 4)

The steady inward leak of Na+ that enters the cell rapidly during phase 0 and more slowly throughout the cardiac cycle would gradually depolarize the resting membrane voltage were it not for Na+,K+-ATPase, which is located in the cell membrane (see ). Similarly, most of the excess Ca++ ions that had entered the cell mainly during phase 2 are eliminated principally by a 3Na+-Ca++ antiporter, which exchanges three Na+ ions for one Ca++ ion. However, some of the Ca++ ions are eliminated by an ATP-driven Ca++ pump.

Cardiacionchannelsareconnectedtocellularproteinstoformmacromolecularcomplexes.Thesecomplexesareinvolvedinmodulatingthetransport,membranelocalization,operation,posttranslationalmodificationandturnoverofparticularionchannels.ThecarboxyterminalsofionchannelslinkthemwithseveralintracellularproteinssuchasPDZ(postsynapticdensity,disclarge,andzonulaoccludens-1)domainproteinswhosebindingsitesinteractwithsynapse-associatedprotein(SAP97),syntrophin,andA-kinaseanchoringprotein(AKAP5)amongothers.Differentmacromolecularcomplexesarefoundindistinctcellularlocations,andthisisthoughttounderlieionchanneldistribution.Thevoltage-sensitivesodiumchannel(Nav1.5)islinkedwithsyntrophin/dystrophinatlateralcellmembranesandwithankyrinB,plakophilin-2,andcalmodulin-dependentproteinkinaseIIattheintercalateddisk.Also,differentionchannelscanfoundinthesamecomplex.ThusNav1.5channelsandinward-rectifyingK(Kir2.1)channelscanbeconnectedinacomplex,orchannelosome, withSAP97.ThislinknotonlyaffectsthelocalizationbutalsoallowschangesintheabundanceofKir2.1channelstoproducereciprocalchangesinNav1.5abundance;theconverseisalsoobserved.ThusthecomplexcontainsKir2.1,whichsetstherestingmembranepotential,andNav1.5,whichaccountsforrapidexcitation.Colocalizationofthesetwochannelsthereforeexertsapowerfuleffectonexcitabilityanditsregulationundernormalandpathologicalconditions(arrhythmias).

Fig. 16.1A ) consist of four principal components: an upstroke (phase 0), an early partial repolarization (phase 1), a plateau (phase 2), and a final repolarization (phase 3). However, in the slow-response action potential (see

Fig. 16.1B ), the upstroke is much less steep, early repolarization (phase 1) is absent, the plateau is less prolonged and not as flat, and the transition from the plateau to the final repolarization is less distinct.

Blocking fast sodium channels with tetrodotoxin in a fast-response fiber can generate slow responses under appropriate conditions. The Purkinje fiber action potentials shown in

Fig. 16.11 clearly exhibit the two response types. In the control tracing (A), the typical fast-response action potential displays a prominent notch, as a result of ito, that separates the upstroke from the plateau. Progressively higher concentrations of tetrodotoxin produce a graded blockade of the fast sodium channels, as demonstrated in tracings B to E. The upstroke and notch are progressively less prominent in tracings B to D. In tracing E, the notch has disappeared, and the upstroke is very gradual; this action potential resembles a typical slow response.

•Fig. 16.11 Effect of Tetrodotoxin, Which Blocks Fast Sodium Channels, on the Action Potentials (Tracings A to E) Recorded in a Purkinje Fiber. Theconcentrationsoftetrodotoxinwere0mol/LintracingA,3×10−8mol/LintracingB,3×10−7mol/LintracingC,and3×10−6mol/LintracingsDandE;EwasrecordedlaterthanD.(RedrawnfromCarmelietE,VereeckeJ.Pflügers Arch. 1969;313:300.)100 mV 1 sec A B C D E

Certain cells in the heart, notably those in the SA and AV nodes, exhibit slow-response action potentials. In these cells, depolarization is achieved mainly by influx of Ca++ through L-type calcium channels instead of influx of Na+ through fast sodium channels. Repolarization is accomplished in these fibers by inactivation of the calcium channels and by the increased K+ conductance through the iK1 and iK channels (see

Fig. 16.3).

An action potential traveling along a cardiac muscle fiber is propagated by local circuit currents, much as it is in nerve and skeletal muscle fibers (see ). When the wave of depolarization reaches the end of the cell, the impulse is conducted to adjacent cells through gap junctions (see

Chapter 2).

Impulses pass more readily along the length of the cell (isotropic) than laterally from cell to cell (anisotropic) because gap junctions are preferentially located at the ends of the cell. Gap junctions are rather nonselective in their permeability by ions and have a low electrical resistance that allows ionic current to pass from one cell to another. The electrical resistance of gap junctions is similar to that of cytoplasm. The flow of charge from cell to cell follows the principles of local circuit currents and therefore allows intercellular propagation of the impulse.

Conduction of the Fast Response

In fast-and slow-response fibers, the characteristics of conduction differ. In fast-response fibers, fast sodium channels are activated when the transmembrane potential •Fig. 16.12 Theroleoflocalcurrentsinthepropagationofawaveofexcitationdownacardiacfiber.

of one region of the fiber suddenly changes from a resting value of approximately −90 mV to the threshold value of approximately −65 mV. The inward Na+ current then rapidly depolarizes the cell at that site. This portion of the fiber subsequently becomes part of the depolarized zone, and the border is displaced accordingly. The same process then begins at the new border. This process is repeated again and again, and the border moves continuously down the fiber as a wave of depolarization (

Fig. 16.12

The conduction velocity along the fiber varies directly with the action potential amplitude and the rate of change of the potential (dVm/dt) during phase 0. The action potential amplitude is the potential difference between the fully depolarized and the fully polarized regions of the cell interior. The magnitude of the local current is proportional to this potential difference (see ). Because these local currents shift the potential of the resting zone toward the threshold value, they are local stimuli that depolarize the adjacent resting portion of the fiber to its threshold potential. The greater the potential difference between the depolarized and polarized regions (i.e., the greater the action potential amplitude), the more effectively local stimuli can depolarize adjacent parts of the membrane, and the more rapidly the wave of depolarization is propagated down the fiber.

The dVm/dt during phase 0 is also an important determinant of conduction velocity. If the active portion of the fiber depolarizes gradually, the local currents between the resting region and the neighboring depolarizing region are small. The resting region adjacent to the active zone is depolarized gradually, and more time is therefore required for each new section of the fiber to reach threshold. This allows some sodium channels to inactivate.

The resting membrane potential is another important determinant of conduction velocity. Changes in the resting membrane potential influence both the amplitude of the action potential and dVm/dt, which in turn alter the conduction velocity (Fig. 16.13 ). Depolarization of Vm inactivates the fast sodium channels, which in turn decreases the amplitude of the action potential and the dVm/dt, and as a consequence conduction velocity is slowed. In addition to changes in extracellular [K+], premature excitation of a cell that has not completely repolarized also results in a •Fig. 16.13 Effect of Changes in Extracellular Potassium Concentration (Extracellular [K+]) on the Action Potentials Recorded From a Purkinje Fiber. Thestimulusartifact(StintracingD)appearsasabiphasicspiketotheleftoftheupstrokeoftheactionpotential.Thehorizontal dashed lines nearthepeaksoftheactionpotentialsdenote0mV.Whenextracellular[K+]is3mmol/L(“mM”;tracingsAandF),therestingmembranevoltage(Vm)is−82mV,andtheslopeofphase0issteep.Attheendofphase0,theovershootattainsavalueof+30mV.Hence,theactionpotentialamplitudeis112mV.Thedistancefromthestimulusartifacttothebeginningofphase0isinverselyproportionaltotheconductionvelocity.Whenextracellular[K+]isincreasedgraduallyto16mmol(tracingsBtoE),therestingVmbecomesprogressivelylessnegative.Atthesametime,theamplitudesanddurationsoftheactionpotentialsandthesteepnessoftheupstrokesalldiminish.Asaconsequence,conductionvelocitydecreasesprogressively.Atextracellular[K+]levelsof14and16mmol(tracingsDandE),therestingVmattainslevelssufficienttoinactivateallthefastsodiumchannelsandleadtothecharacteristicslow-responseactionpotentials.(FromMyerburgRJ,LazzaraR.In:FischE[ed].Complex Electrocardiography. Philadelphia:FADavis;1973.) decrease in conduction velocity. This too reflects the fact that when Vm is depolarized, more fast sodium channels are inactivated, and thus only a fraction of the sodium channels are available to conduct the inward Na+ current during phase 0.

Conduction of the Slow Response

Fig. 16.12 ) also propagate the slow response, whose conduction characteristics differ quantitatively from those of the fast response. The threshold potential is approximately −40 mV for the slow response, and conduction is much slower than for the fast response. The conduction velocities of the slow response in the SA and AV nodes are approximately 0.02 to 0.10 m/sec. The fast-response conduction velocities are approximately 0.30 to 1.00 m/sec for myocardial cells and 1.00 to 4.00 m/sec for the specialized conducting (Purkinje) fibers in the ventricles. Slow responses are more readily blocked than are fast responses; that is, conduction ceases before the impulse reaches the end of the myocardial fiber. Also, fast-response fibers can respond at repetition rates that are much faster than those of slow-response fibers.

Fig.16.13 alsotakeplaceinthecardiactissueofpatientswithcoronaryarterydisease.Whenregionalmyocardialbloodflowisdiminished,thesupplyofO2andmetabolicsubstratesdeliveredtotheischemictissuesisinsufficient.TheNa+,K+-ATPaseinthemembraneofcardiacmyocytesrequiresconsiderablemetabolicenergytomaintainthenormaltransmembranegradientsofNa+ andK+ .Whenbloodflowisinadequate,theactivityofNa+,K+-ATPaseisimpaired,andtheischemicmyocytesgainexcessNa+ andloseK+ tothesurroundinginterstitialspace.Asaconsequence,[K+]intheextracellularfluidsurroundingtheischemicmyocytesiselevated.Suchchangesinextracellular[K+]may,ifsufficientlylarge,disturbcardiacrhythmandconductioncritically(see

Fig.16.13

Because of the rapid development of artificial pacemakers and other electrical devices for correcting cardiac rhythm disturbances, detailed knowledge of cardiac excitability is essential. The excitability characteristics of various types of cardiac cells differ considerably, depending on whether the action potentials are fast or slow responses.

Once the fast response has been initiated, the depolarized cell is no longer excitable until it has partially repolarized (see

Fig. 16.1A ). In the fast response, the interval from the beginning of the action potential until the fiber is able to conduct another action potential is called the effective refractory period which extends from the beginning of phase 0 to a point in phase 3 at which repolarization has reached approximately −50 mV. At approximately this value of Vm, many of the fast sodium channels have transitioned from the inactivated state to the closed state. However, the cardiac fiber is not fully excitable until it has been completely repolarized. Before complete repolarization (i.e., during the relative refractory period), an action potential may be evoked only when the stimulus is stronger than a stimulus that could elicit a response during phase 4.

When a fast response is evoked during the relative refractory period of a previous excitation, its characteristics vary with the membrane potential that exists at the time of stimulation (

Fig. 16.14).

The later in the relative refractory period that the fiber is stimulated, the greater are the increases in the amplitude of the response and the slope of the upstroke because the number of fast sodium channels that have recovered from inactivation increases as repolarization proceeds. As a consequence, propagation velocity also increases the later in the relative refractory period that the fiber is stimulated. Once the fiber is fully repolarized, the response is constant no matter what time in phase 4 the stimulus is applied.

•Fig. 16.15 Effects of Excitation at Various Times After the Initiation of an Action Potential in a Slow-Response Fiber. Inthis fiber,excitationverylateinphase3(orearlyinphase4)inducesa small,nonpropagated(local)response(wavea).Laterinphase4,a •Fig. 16.14 Changesinactionpotentialamplitudeandupstrokeslopeasactionpotentialsareinitiatedatdifferentstagesoftherelativerefractoryperiodoftheprecedingexcitation.(RedrawnfromRosenMR,etal.Am Heart J. 1974;88:380.)

In slow-response fibers, the relative refractory period frequently extends well beyond phase 3 (see

Fig. 16.1B ). Even after the cell has completely repolarized, it may be difficult to evoke a propagated response for some time. This characteristic of slow-response fibers is called postrepolarization refractoriness.

propagatedresponse(waveb)canbeelicited,butitsamplitudeissmallandtheupstrokeisnotverysteep;thisresponseisconductedveryslowly.Stilllaterinphase4,fullexcitabilityisregained,andtheresponse(wavec)displaysnormalcharacteristics.(ModifiedfromSingerDH,etal.Prog Cardiovasc Dis. 1981;24:97.)

Fig.16.32 ),thetimingoftheseearlybeatsmaydeterminetheirclinicalconsequence.Iftheyoccurlateintherelativerefractoryperiodoftheprecedingdepolarizationorafterfullrepolarization,theprematuredepolarizationisinconsequential.However,iftheprematuredepolarizationsoriginateearlyintherelativerefractoryperiodoftheventricles,conductionoftheprematureimpulsefromthesiteoforiginisslow,andhenceacardiacimpulseismorelikelytoreexcitesomemyocardialregionthroughwhichithadpassedpreviously(aphenomenonknownasreentry).Ifthatreentryisirregular(i.e.,ifventricularfibrillationensues),theheartcannotpumpeffectively,anddeathmayresult.

Action potentials evoked early in the relative refractory period are small, and the upstrokes are not very steep (Fig.

16.15 ). The amplitudes and upstroke slopes progressively improve as action potentials are elicited later in the relative refractory period. Recovery of full excitability is much slower than recovery of the fast response. Impulses that arrive early in the relative refractory period are conducted much more slowly than those that arrive late in that period. The long refractory periods also lead to conduction blocks. Even when slow responses recur at low frequency, the fiber may be able to conduct only a fraction of these impulses; for example, in certain conditions, only alternate impulses may be propagated.

•Fig. 16.16 Effectofchangesincyclelength(CL)ontheactionpotentialduration(APD)ofPurkinjefibers.(ModifiedfromSingerD,TenEickRE.Am J Cardiol. 1971;28:381.)

Effects of Cycle Length

Cycle length is the time between successive action potentials. Changes in cycle length alter the duration of the action potential in cardiac cells (

Fig. 16.16

Fig. 16.5 ) and thus change their refractory periods. As a consequence, changes in cycle length are often important factors in the initiation or termination of certain arrhythmias (irregular heart rhythms).

The changes in action potential duration produced by stepwise reductions in cycle length from 2000 to 200 msec in a Purkinje fiber are shown in

Fig. 16.16 . Note that as cycle length diminishes, the duration of the action potential decreases. This direct correlation between action potential duration and cycle length is mediated by changes in gK that involve at least two types of potassium channels: namely, those that conduct the delayed rectifier K+ currents (iKr and iKs) and those that conduct the transient outward K+ current (ito).

The iK current is activated at values of Vm near zero, but the current activates slowly, remains activated for hundreds of milliseconds, and also inactivates very slowly. As a consequence, as the basic cycle length diminishes, each action potential tends to occur earlier in the inactivation period of the iK current initiated by the preceding action potential. Therefore, the shorter the basic cycle length, the greater the outward K+ current during phase 2 and hence the shorter the action potential duration.

The ito current also influences the relationship between cycle length and action potential duration. The ito current is also activated at near zero potential, and its magnitude varies inversely with cardiac frequency. Therefore, as cycle length decreases, the consequent increase in the outward K+ current shortens the plateau.

Natural Excitation of the Heart and the Electrocardiogram

Excitation of the heart normally occurs in an ordered manner, which allows effective pumping of blood. This ordered excitation occurs via the heart’s conduction system (

Fig. 16.17 ). The SA node is the pacemaker of the heart and initiates the spread of action potentials throughout the atria. This spread of excitation reaches the AV node, where conduction is slowed so that atrial contraction can occur and the ventricles can be adequately filled. Excitation then spreads rapidly throughout the ventricles via the Purkinje fibers so that the ventricular myocytes contract in a coordinated manner. The properties of each component of the heart’s conduction system are described in the next sections.

•Fig. 16.17 Thecardiacconductionsystem.

CHAPTER 16 Elements of Cardiac Function

The autonomic nervous system controls various aspects of cardiac function, such as the heart rate and contraction strength. However, cardiac function does not require intact innervation. Indeed, a patient with a cardiac transplant, whose heart is completely denervated, may still adapt well to stressful situations. The ability of a denervated, transplanted heart to adapt to changing conditions lies in certain intrinsic properties of cardiac tissue, especially its automaticity.

The properties of automaticity (the ability to initiate its own beat) and rhythmicity (the regularity of pacemaking activity) allow a perfused heart to beat even when it is completely removed from the body. The vertebrate heartbeat is myogenic in origin. If the coronary vasculature of an excised heart is artificially perfused with blood or an oxygenated electrolyte solution, rhythmic cardiac contractions may persist for many hours. Some cells in the atria and ventricles can initiate beats; such cells reside mainly in nodal tissues or specialized conducting fibers of the heart.

As noted, the region of the mammalian heart that ordinarily generates impulses at the greatest frequency is the SA node; it is the main cardiac pacemaker. Detailed mapping of the electrical potentials on the surface of the right atrium has revealed that two or three sites of automaticity, located 1 or 2 cm from the SA node itself, serve along with the SA node as an atrial pacemaker complex. At times, all these loci initiate impulses simultaneously. At other times, the site of earliest excitation shifts from locus to locus, depending on certain conditions, such as the level of autonomic neural activity.

In humans, the SA node is approximately 8 mm long and 2 mm thick, and it lies posteriorly in the groove at the junction between the superior vena cava and the right atrium. The sinus node artery runs lengthwise through the center of the node. The SA node contains two principal cell types: (1) small, round cells that have few organelles and myofibrils and (2) slender, elongated cells that are intermediate in appearance between the round and “ordinary” atrial myocardial cells. The round cells are probably the pacemaker cells; the slender, elongated cells probably conduct the impulses within the node and to the nodal margins.

A typical transmembrane action potential recorded from an SA node cell is depicted in

Fig. 16.10B.

In comparison with the transmembrane potential recorded from a ventricular myocardial cell (see

Fig. 16.10A ), the resting potential of the SA node cell is usually less negative, the upstroke of the action potential (phase 0) is less steep, the plateau is not sustained, and repolarization (phase 3) is more gradual. These are characteristic attributes of the slow response. Tetrodotoxin (which blocks the fast Na+ current) has no influence on the SA nodal action potential because the action potential upstroke is not produced by an inward Na+ current through fast channels. Thus tetrodotoxin has no effect on cells that exhibit the slow response.

Ordinarily,thefrequencyofpacemakerfiringiscontrolled system.Increasedsympatheticnervousactivity,throughthe releaseofnorepinephrine,raisestheheartrateprincipallyby increasingtheslopeoftheslowdiastolicdepolarization.Thismechanismofincreasingheartratefunctionsduringphysical exertion,anxiety,orcertainillnessessuchasfebrileinfectiousA diseases.Increasedvagalactivity,throughthereleaseof acetylcholine,diminishestheheartratebyhyperpolarizingthepacemakercellmembraneandreducingtheslope oftheslowdiastolicdepolarization.Thesemechanisms predominantoversympatheticactivity.Anextremeexample isvasovagalsyncope,abriefperiodoflightheadednessorlossofconsciousnesscausedbyanintenseburstofvagal activity.Thistypeofsyncopeisareflexresponsetopainor tocertainpsychologicalstimuli.

•Fig. 16.18 Mechanisms Involved in the Changes in Frequency of Pacemaker Firing. A, Areductionintheslope(fromwaveatowave b)ofslowdiastolicdepolarizationdiminishesthefiringfrequency.TP,thresholdpotential.B, Anincreaseinthethresholdpotential(fromTP-1toTP-2)oranincreaseinthemagnitudeofthemaximumdiastolicpotential(fromwavesegmentsatod)alsodiminishesthefiringfrequency.(FromHoffmanBF,CranefieldPF.Electrophysiology of the Heart. NewYork:McGraw-Hill;1960.)

The transmembrane potential during phase 4 is much less negative in SA (and AV) nodal automatic cells than in atrial or ventricular myocytes because nodal cells lack the iK1 (inward rectifying) type of potassium channel. Thus the ratio of gK to gNa during phase 4 is much less in nodal cells than in myocytes. Hence, during phase 4, Vm deviates much more from the K+ Nernst equilibrium potential (EK) in nodal cells than it does in myocytes.

The principal feature of a pacemaker cell that distinguishes it from the other cells manifests in phase 4. In nonautomatic cells, the potential remains constant during this phase, whereas a pacemaker fiber is characterized by slow diastolic depolarization throughout phase 4. Depolarization proceeds at a steady rate until a threshold is attained, and an action potential is then triggered.

Pacemaker cell frequency may be varied by a change in (1) the rate of depolarization during phase 4, (2) the maximal negativity during phase 4, or (3) the threshold potential (

Fig. 16.18). When the rate of slow diastolic depolarization is increased, the threshold potential is attained earlier, and the heart rate increases. A rise in the threshold potential delays the onset of phase 0, and the heart rate is reduced. Similarly, when the maximal negative potential is increased, more time is required to reach the threshold potential, when the slope of phase 4 remains unchanged, and the heart rate therefore diminishes.

Ionic Basis of Automaticity

Several ionic currents contribute to the slow diastolic depolarization that characteristically occurs in the automatic

ChangesinautonomicneuralactivitydonotusuallychangetheheartratebyalteringthethresholdlevelofVminthenodalpacemakercells.However,certainantiarrhythmicdrugs,suchasquinidineandprocainamide,doshiftthethresholdpotentialoftheautomaticcellstolessnegativevalues.

cells in the heart. In the pacemaker cells of the SA node, at least three ionic currents mediate the slow diastolic depolarization: (1) an outward K+ current, iK; (2) a hyperpolarization-induced inward current, if; and (3) an inward Ca++ current, ICa (

Fig. 16.19

The repetitive firing of the pacemaker cell begins with the iK current. Efflux of K+ tends to repolarize the cell after the upstroke of the action potential. K+ continues to move out well beyond the time of maximal repolarization, but its efflux diminishes throughout phase 4 (see

Fig. 16.19 ). As the current diminishes, its opposition to the depolarizing effects of the two inward currents (if and iCa) also gradually decreases. The progressive diastolic depolarization is mediated by the if and iCa currents, which oppose the repolarizing effect of the iK current.

The inward current if is activated near the end of repolarization and is carried mainly by Na+ through specific channels that differ from the fast sodium channels. The current was dubbed “funny” because its discoverers had not expected to detect an inward Na+ current in pacemaker cells at the end of repolarization. This current is activated as the membrane potential becomes hyperpolarized beyond −50 mV. The more negative the membrane potential at this time, the greater the activation of if.

The second current responsible for diastolic depolarization is the inward rectifying Ca++ current, iCa. This current is activated toward the end of phase 4 as the membrane potential reaches a value of approximately −35 mV (see

Fig. 16.19 ). Once the calcium channels are activated, influx of Ca++ into the cell increases. This influx accelerates the rate of diastolic depolarization, which then leads to the action potential upstroke. A decrease in extracellular •Fig. 16.19 Themembranepotentialchanges(blue trace, top half) thatoccurinsinoatrialnodecellsareproducedbythreeprincipalcurrents(green trace, bottom half): theinwardCa++ current(iCa);ahyperpolarization-inducedinwardcurrent(if),andanoutwardK+ current(iK).Thethinnoisygreen trace showsnetmembranecurrentandtheapproximatetimecourseoftherepolarizingoutwardK+ current(iK),thehyperpolarization-inducedinwardcurrent(if),andtheL-typeCa++ current(iCa).Thethick bold red line inthecurrenttraceindicatesthemagnitudeanddirectionofestimatedIf.(RedrawnfromvanGinnekenACG,GilesW.J Physiol. 1991;434:57.)

Theso-calledfunnycurrent(if)incardiacSAnodecellsisactivatedbyhyperpolarizationandgatedbycyclicnucleotides,anditschannelisdesignatedHCN.TherearefourmembersoftheHCN genefamily,andsuchchannelsarefoundincentralnervoussystemneuronsthatgenerateactionpotentialsrepetitively.Transmembranesegment4(S4)ofHCNhasmanypositivelychargedaminoacidsthatactasvoltagesensors,whicharealsopresentinvoltage-gatedsodium,potassium,andcalciumchannels.ThedominantchannelexpressedintheheartisderivedfromtheHCN4 gene.MutationsinaminoacidsinS4andintheS4-to-S5linkercausemarkedchangesinthevoltagedependenceofactivation,insuchawaythatgreaterhyperpolarizationisneededtoopenthechannel.Thiseffectislikethatofacetylcholine,andithasbeenpredictedthattheoccurrenceofsuchmutationsinthehumanheartcouldunderliesinusbradycardiaandsicksinussyndrome.

•Fig. 16.20 Membrane Action Potentials Recorded From an Sino-atrial Node Pacemaker Cell. TheconcentrationofCa++inthebathwasreducedfrom2to0.2mmol/L(“mM”).(ModifiedfromKohlhardtM,etal.Basic Res Cardiol. 1976;71:17.)–60 0 Ca++ 2 mM 500 msec Ca++ 0.2 mM mV

Fig. 16.20 ) or the addition of calcium channel antagonists diminishes the amplitude of the action potential and the slope of the slow diastolic depolarization in SA node cells. Research evidence indicates that additional ion currents—including a sustained (background) inward Na+ current (iNa), the T-type Ca++ current, and the Na+/Ca++ exchange current triggered by spontaneous release of Ca++ from the sarcoplasmic reticulum—may also be involved in pacemaking. These observations illustrate the many ways to sustain this vital function.

RegionsoftheheartotherthantheSAnodemayinitiatebeatsinspecialcircumstances.Suchsitesarecalledectopic foci orectopic pacemakers. Ectopicfocimaybecomepacemakerswhen(1)theirownrhythmicitybecomesenhanced,(2)therhythmicityofthehigherorderpacemakersbecomesdepressed,or(3)allconductionpathwaysbetweentheectopicfocusandregionswithgreaterrhythmicitybecomeblocked.Ectopicpacemakersmayactasasafetymechanismwhennormalpacemakingcentersfail.However,ifanectopiccenterfireswhilethenormalpacemakingcenterstillfunctions,theectopicactivitymayinduceeithersporadicrhythmdisturbances,suchasprematuredepolarizations,orcontinuousrhythmdisturbances,suchasparoxysmaltachycardias(seethesection“

The autonomic neurotransmitters affect automaticity by altering membrane ionic currents. The adrenergic transmitters increase all three currents involved in SA nodal automaticity. To increase the slope of diastolic depolarization, the augmentation of if and iCa by adrenergic transmitters must exceed the enhancement of iK by these same transmitters.

The hyperpolarization induced by acetylcholine released from vagus nerve endings in the heart is achieved by the activation of specific potassium channels: the aThe ionic basis for automaticity in AV node pacemaker cells resembles that in SA node cells. Similar mechanisms also account for automaticity in ventricular Purkinje fibers, except that the fast Na+ current rather than iCa is involved. Also, a voltageand time-dependent K+ current rather than the hyperpolarization-induced inward current if has been suggested to mediate the slow diastolic depolarization; however, this remains to be clarified.

acetylcholine-regulated potassium channels (KACh). Acetylcholine also depresses the if and iCa currents. The autonomic neural effects on cardiac cells are described in greater detail in

When the SA node or other components of the atrial pacemaker complex are excised or destroyed, pacemaker cells in the AV junction generally take over the pacemaker function for the entire heart. After some time, which may vary from minutes to days, automatic cells in the atria usually become dominant again and resume their pacemaker function. Purkinje fibers in the specialized conduction system of the ventricles also display automaticity. These fibers characteristically fire at a very slow rate. When the AV junction cannot conduct cardiac impulses from the atria to the ventricles, these idioventricular pacemakers in the Purkinje fiber network initiate the ventricular contractions, but at a frequency of only 30 to 40 beats per minute.

Ifanectopicfocusinoneoftheatriasuddenlybegantofireatahighrate(e.g.,150impulsesperminute)inanindividualwithanormalheartrateof70beatsperminute,theectopicsitewouldbecomethepacemakerfortheentireheart.Ifthatrapidectopicfocussuddenlystoppedfiring,theSAnodewouldremainbrieflyquiescentbecauseofoverdrivesuppression.TheintervalfromtheendoftheperiodofoverdriveuntiltheSAnoderesumesfiringiscalledthesinus node recovery time. Inpatientswithsicksinussyndrome,thesinusnoderecoverytimeisprolonged.Theconsequentperiodofasystole(absenceofaheartbeat)maycauselossofconsciousness.

The automaticity of pacemaker cells diminishes after these cells have been excited at a high frequency. This phenomenon is known as overdrive suppression. Because the intrinsic rhythmicity of the SA node is greater than that of the other latent pacemaking sites in the heart, firing of the SA node tends to suppress the automaticity in other loci.

Overdrive suppression results from the activity of membrane Na+,K+-ATPase. A certain amount of Na+ enters the cardiac cell during each depolarization. The more frequently the cell is depolarized, the more Na+ enters the cell per minute. At high excitation frequencies, the activity of Na+,K+-ATPase increases to extrude this larger amount of Na+ from the cell. The activity of Na+,K+-ATPase hyperpolarizes the cell because three Na+ ions are extruded by the pump in exchange for two K+ ions that enter the cell (see ). Therefore, slow diastolic depolarization requires more time to reach the firing threshold. In addition, when the overdrive suddenly ceases, the activity of Na+,K+-ATPase does not slow instantaneously but temporarily remains overactive. This continued extrusion of Na+ opposes the gradual depolarization of the pacemaker cell during phase 4, and it temporarily suppresses the cell’s intrinsic automaticity.

From the SA node, the cardiac impulse spreads radially throughout the right atrium (see

Fig. 16.17 ) along ordinary atrial myocardial fibers at a conduction velocity of approximately 1 m/second. A special pathway, the anterior interatrial myocardial band (or Bachmann’s bundle), conducts the SA node impulse directly to the left atrium. The wave of excitation proceeds inferiorly through the right atrium and ultimately reaches the AV node (see

Fig. 16.17 ), which is normally the sole entry route of the cardiac impulse to the ventricles.

SomepeoplehaveaccessoryAVpathways.Becausethesepathwaysoftenserveasapartofareentryloop(seethesection“ ”),theycanbeassociatedwithseriouscardiacrhythmdisturbances.Wolff-Parkinson-Whitesyndrome,acongenitaldisturbance,isthemostcommonclinicaldisorderinwhichabypasstractofmyocardialfibersbecomesanaccessorypathwaybetweentheatriaandventricles.Ordinarily,thesyndromecausesnofunctionalabnormality.ThedisturbanceiseasilydetectedonanECGbecauseaportionoftheventricleisexcitedviathebypasstractbeforetheremainderoftheventricleisexcitedviatheAVnodeandtheHis-Purkinjesystem.Thispreexcitationappearsasabizarreconfigurationintheventricular(QRS)complexoftheECG.Onoccasion,however,areentryloopdevelopsinwhichtheatrialimpulsetravelstotheventriclesviaoneofthetwoAVpathways(AVnodeorbypasstract)andthenbacktotheatriathroughtheotherofthesetwopathways.Continuouscirclingaroundtheloopleadstoaveryrapidrhythm(supraventriculartachycardia).Thisrapidrhythmmaybeincapacitatingbecauseitmightnotallowsufficienttimeforventricularfilling.TransientblockoftheAVnodebyaninjectionofadenosineintravenouslyorbyareflexiveincreaseinvagalactivity(bypressingontheneckoverthecarotidsinusregion)usuallyabolishesthetachycardiaandrestoresanormalsinusrhythm.

The atrial plateau (phase 2) is briefer and less developed, and repolarization (phase 3) is slower (see

Fig. 16.10 ) than in a typical ventricular fiber. The action potential duration in atrial myocytes is briefer than that in ventricular myocytes because efflux of K+ is greater during the plateau in atrial myocytes than in ventricular myocytes. The presence of an ultrarapid K+ current (IKur) in atrial myocytes, but not in ventricular myocytes, contributes to greater K+ efflux and shorter action potentials in atrial myocytes.

The atrial excitation wave reaches the ventricles via the AV node. In adult humans, this node is approximately 15 mm long, 10 mm wide, and 3 mm thick. The node is situated posteriorly on the right side of the interatrial septum near the ostium of the coronary sinus. The AV node contains the same two cell types as the SA node, but the round cells •Fig. 16.21 Membranepotentialsrecordedfromanatrioventricularnodecellundercontrolconditions(C)andinthepresenceofthecalciumchannelantagonistdiltiazematconcentrationsof0.1,0.3,and1µmol/L.(RedrawnfromHirthC,etal.J Mol Cell Cardiol. 1983;15:799.)–25 0 25 msec C 0.1 0.3 1 mV in the AV node are less abundant and the elongated cells predominate. The AV node is made up of three functional regions: the atrionodal (AN) region, or the transitional zone between the atrium and the remainder of the node; (2) the nodal (N) region, or the midportion of the AV node; and the nodal-His (NH) region, or the zone in which nodal fibers gradually merge with the bundle of His, which is the upper portion of the specialized conducting system for the ventricles (see

Fig. 16.17). Normally, the AV node and the bundle of His are the only pathways along which the cardiac impulse travels from atria to ventricles.

Several features of AV conduction are of physiological and clinical significance. The principal delay in conduction of impulses from the atria to the ventricles occurs in the AN and N regions of the AV node. Conduction velocity is actually less in the N region than in the AN region. However, the path length is substantially greater in the AN region than in the N region. Conduction times through the AN and N regions account for the delay between the start of the P wave (the electrical manifestation of atrial excitation) and the QRS complex (the electrical manifestation of ventricular excitation) on an ECG (see the section “

Electrocardiography”).

In terms of function, the delay between atrial and ventricular excitation enables optimal ventricular filling during atrial contraction.

In the N region, slow-response action potentials prevail. The resting potential is approximately −60 mV, the upstroke velocity is low (≈5 V/sec), and the conduction velocity is approximately 0.05m/sec.

Tetrodotoxin, which blocks the fast sodium channels, has virtually no effect on action potentials in this region (or on any other slow-response fibers). Conversely, calcium channel antagonists decrease the amplitude and duration of the action potentials (

Fig.

16.21 ) and depress AV conduction.

bThe shapes of the action potentials in the AN region are intermediate between those in the N region and those in the atria. Similarly, action potentials in the NH region are transitional between those in the N region and those in the bundle of His.

CHAPTER 16 Elements of Cardiac Function

Like other slow-response action potentials, the relative refractory period of cells in the N region extends well beyond the period of complete repolarization; that is, these cells display postrepolarization refractoriness (see

Fig. 16.15 ). As the heart rate increases, the time between successive atrial depolarizations is decreased, and conduction through the AV junction slows. Abnormal prolongation of the AV conduction time is called a first-degree AV block (see the section ”). Most of the prolongation of AV conduction induced by an increase of atrial frequency takes place in the N region of the AV node.

Impulses tend to be blocked in the AV node at stimulation frequencies that are easily conducted in other regions of the heart. If the atria are depolarized at a high repetition rate, only a fraction (e.g., half) of the atrial impulses might be conducted through the AV junction to the ventricles. The conduction pattern in which only a fraction of the atrial impulses are conducted to the ventricles is called a second-degree AV block (see the section ”). This type of block may protect the ventricles from excessive contraction frequencies, wherein the filling time between contractions might be inadequate.

Retrograde conduction can occur through the AV node. However, the conduction time is significantly longer, and the impulse is blocked at lower repetition rates when the impulse is conducted in the retrograde instead of the ante-grade direction. In addition, the AV node is a common site for reentry (see the section “

As in the SA node, the autonomic nervous system regulates AV conduction. Weak vagal activity may simply prolong the AV conduction time. Thus for any given atrial cycle length, the atrium-to-His or atrium-to-ventricle conduction time is prolonged by vagal stimulation. Stronger vagal activity may cause some or all of the impulses arriving from the atria to be blocked in the node. The conduction pattern in which none of the atrial impulses reaches the ventricles is called a third degree, or complete, AV block (see the section ”). The vagally induced delay or absence of conduction through the AV node occurs mainly in the N region. This effect of vagal stimulation reflects the action of acetylcholine to hyperpolarize the membrane of the conducting fibers in the N region (Fig. 16.22 ). The greater the hyperpolarization at the time of arrival of the atrial impulse, the more impaired the AV conduction.

Cardiac sympathetic nerves, in contrast, facilitate AV conduction. They decrease the AV conduction time and enhance the rhythmicity of latent pacemakers in the AV junction. The norepinephrine released at the postganglionic sympathetic nerve terminals increases the amplitude and slope of the upstroke of the AV nodal action potentials, principally in the AN and N regions of the node.

The bundle of His passes subendocardially down the right side of the interventricular septum for approximately 1 cm •Fig. 16.22 Effects of a Brief Vagal Stimulus (St) on the Transmembrane Potential. Thisresponsewasrecordedfromanatrioventricular(AV)nodalfiber(red trace). Compoundactionpotentialswererecordedfromtheatrium(A1,A2,A3;upper blue trace)andfromtheHis-Purkinjesystem(H;lower blue trace).Notethatshortlyaftervagalstimulation,themembraneofthefiberwashyperpolarized.Theatrialexcitation(A2)thatarrivedattheAVnodewhenthecellwashyperpolarizedfailedtobeconducted,asdenotedbytheabsenceofadepolarizationintheHiselectrogram(H).Theatrialexcitationsthatpreceded(A1)andfollowed(A3)excitationA2wereconductedtotheHisbundleregion.(RedrawnfromMazgalevT.etal.Am J Physiol. 1986;251:H631.) and then divides into the right and left bundle branches (see

Fig. 16.17 ). The right bundle branch, a direct continuation of the bundle of His, proceeds down the right side of the interventricular septum. The left bundle branch, which is considerably thicker than the right, arises almost perpendicular from the bundle of His and perforates the interventricular septum. On the subendocardial surface of the left side of the interventricular septum, the left bundle branch splits into a thin anterior division and a thick posterior division.

Conductionofimpulsesintherightorleftbundlebranchorineitherdivisionoftheleftbundlebranchmaybeimpaired.Conductionblocksmaydevelopinoneormoreoftheseconductionpathwaysasaconsequenceofcoronaryarterydiseaseordegenerativeprocessesassociatedwithaging,andtheygiverisetocharacteristicECGpatterns.Blockofeitherofthemainbundlebranchesisknownasright orleft bundle branch block. Blockofeitherdivisionoftheleftbundlebranchiscalledleft anterior orleft posterior hemiblock.

The right bundle branch and the two divisions of the left bundle branch ultimately subdivide into a complex network of conducting fibers, called Purkinje fibers, that spread out over the subendocardial surfaces of both ventricles. Purkinje fibers have abundant, linearly arranged sarcomeres, as do myocytes. However, the transverse (T) tubular system, which is well developed in myocytes, is absent in the Purkinje fibers of many species. Purkinje fibers are the broadest cells in the heart: 70 to 80 µm in diameter, in comparison with diameters of 10 to 15 µm for ventricular myocytes. Partly because of the large diameter of the Purkinje fibers, conduction velocity (1 to 4 m/second) in these fibers exceeds that in any other fiber type within the heart. The increased conduction velocity enables rapid activation of the entire endocardial surface of the ventricles.

The action potentials recorded from Purkinje fibers resemble those of ordinary ventricular myocardial fibers. However, because of the long refractory period of Purkinje fiber action potentials, many premature excitations of the atria are conducted through the AV junction but are then blocked by the Purkinje fibers. Blockade of these atrial excitations prevents premature contraction of the ventricles. This function of protecting the ventricles against the effects of premature atrial depolarization is especially pronounced at slow heart rates because the action potential duration, and hence the effective refractory period, of the Purkinje fibers vary inversely with the heart rate (see

Fig. 16.16 ). At slow heart rates, the effective refractory period of the Purkinje fibers is especially prolonged.

In contrast to Purkinje fibers, the effective refractory period of AV node cells does not change appreciably over the normal range of heart rates and actually increases at very rapid heart rates. Therefore, when the atrium is excited at high repetition rates, it is the AV node that normally protects the ventricles from these excessively high frequencies.

The first portions of the ventricles to be excited by impulses arriving from the AV node are the interventricular septum (except the basal portion) and the papillary muscles. The activation wave spreads into the substance of the septum from both its left and right endocardial surfaces. Early contraction of the septum makes it more rigid and allows it to serve as an anchor point for contraction of the remaining ventricular myocardium. Furthermore, early contraction of the papillary muscles prevents eversion of the AV valves into the atria during ventricular systole.

The endocardial surfaces of both ventricles are activated rapidly, but the wave of excitation spreads from endocardium to epicardium at a slower velocity (≈0.3 to 0.4 m/sec). The epicardial surface of the right ventricle is activated earlier than that of the left ventricle because the right ventricular wall is appreciably thinner than the left. In addition, the apical and central epicardial regions of both ventricles are activated somewhat earlier than their respective basal regions. The last portions of the ventricles to be excited are the posterior basal epicardial regions and a small zone in the basal portion of the interventricular septum.

The conditions necessary for reentry (defined in the following box) are illustrated in

Fig. 16.23 . In each of the four panels, a single bundle of cardiac fibers splits into a left branch and a right branch. A connecting bundle runs cSimilar directional changes in the refractory period also occur in ventricular myocytes in response to changes in heart rate.

•Fig. 16.23 The Role of Unidirectional Block in Reentry. A, Anexcitationwavetravelingdownasinglebundle(S)offiberscontinuesdowntheleft(L)andright(R)branches.Thedepolarizationwaveenterstheconnectingbranch(C)frombothendsandisextinguishedatthezoneofcollision.B, ThewaveisblockedintheLandRbranches.C, AbidirectionalblockexistsintheRbranch.D, AunidirectionalblockexistsintheRbranch.Theantegradeimpulseisblocked,buttheretrogradeimpulseisconductedthroughandreenterstheSbundle.

between the two branches. Normally, the impulse moving down the single bundle is conducted along the left and right branches (see

Fig. 16.23A ). As the impulse reaches the link to the connecting bundle (

Fig. 16.23C), it enters from both sides and becomes extinguished at the point of collision. The impulse from the left side cannot proceed because the tissue beyond is absolutely refractory; it has just been depolarized from the other direction. The impulse also cannot pass through the connecting bundle from the right for the same reason.

Fig. 16.23B shows that the impulse cannot complete the circuit if an antegrade block exists in the left and right branches of the fiber bundle. Also, if a bidirectional block exists at any point in the loop (e.g., branch R in

Fig.

16.23C ), the impulse also cannot reenter.

Incertainconditions,acardiacimpulsemayreexcitesomemyocardialregionthroughwhichithadpassedpreviously.Thisphenomenon,knownasreentry, isresponsibleformanyclinicalarrhythmias(disturbancesincardiacrhythm).Thereentrymaybeorderedorrandom.Inorderedreentry,theimpulsetraversesafixedanatomicalpath,whereasinrandomreentry,thepathcontinuestochange.

A necessary condition for reentry is that at some point in the loop the impulse can pass in one direction but not in the other. This phenomenon is called unidirectional block. As shown in

Fig. 16.23D , the impulse may travel down

CHAPTER 16 Elements of Cardiac Function the left branch normally but is blocked in the antegrade direction in the right branch because of some pathological change in the myocardial cells in that branch. The impulse that was conducted down the left branch and through the connecting branch of the connecting bundle may then be able to penetrate the depressed region in the right branch from the retrograde direction, even though the antegrade impulse had been blocked previously at this same site. Why is the antegrade impulse blocked but not the retrograde impulse? The reason is that the antegrade impulse arrives at the depressed region in the right branch earlier than the retrograde impulse because the path length of the ante-grade impulse is very short, whereas the retrograde impulse traverses a much longer path. Therefore, the antegrade impulse may be blocked simply because it arrives at the depressed region during its effective refractory period. If the retrograde impulse is delayed sufficiently, the refractory period may have ended in the affected region, and the impulse can then be conducted back through this region and return to the single bundle.

Although unidirectional block is a necessary condition for reentry, it alone cannot cause reentry. For reentry to occur, the effective refractory period of the reentered region must be shorter than the conduction time around the loop. In

Fig. 16.23D , if the tissue just beyond the depressed zone in the right branch is still refractory from the ante-grade depolarization, the retrograde impulse will not be conducted into the single bundle branch. Therefore, the conditions that promote reentry are those that prolong the conduction time or shorten the effective refractory period.

The functional characteristics of the various components of the reentry loops responsible for specific cardiac arrhythmias are diverse. Some loops are large and involve entire specialized conduction bundles, whereas others are microscopic. The loop may include myocardial fibers, specialized conducting fibers, nodal cells, and junctional tissues in almost any conceivable arrangement. In addition, the various cardiac cells in the loop may be normal or abnormal.

The propagation velocity along a multicellular cardiac conduction fiber is normally facilitated by gap junctions that lie between consecutive conducting fibers. Variations in the protein structure of the connexins in the gap junctions can affect the propagation velocity along these fibers. The chemical structure of the specific connexins can vary locally in cardiac tissues and, as a result, can establish local variations in propagation velocity. Such topical variations in velocity might include regions of unidirectional block that induce reentrant rhythm disturbances.

Triggered activity is so named because it is always coupled to a preceding action potential. Because reentrant activity is also coupled to a preceding action potential, the arrhythmias induced by triggered activity are usually difficult to distinguish from those induced by reentry. Triggered activity is caused by afterdepolarizations, of which two types •Fig. 16.24 Effect of Pacing at Different Cycle Lengths (CL) on Cesium-Induced Early Afterdepolarizations (EADs) in a Purkinje Fiber. A, EADsarenotevident.B, EADsfirstappear(arrows). ThethirdEADreachesthresholdandtriggersanactionpotential(third arrow). C, EADsthatappearaftereachdrivendepolarizationtriggeranactionpotential.D, Triggeredactionpotentialsoccurinsalvos.(ModifiedfromDamianoBP,RosenM.Circulation. 1984;69:1013.) are recognized: early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs). EADs may appear either at the end of the action potential plateau (phase 2) or approximately midway through repolarization (phase 3), whereas DADs occur near the very end of repolarization or just after full repolarization (phase 4).

EADs are more likely to occur when the prevailing heart rate is slow; a rapid heart rate suppresses EADs (

Fig. 16.24 ). EADs are also more likely to occur in cardiac cells with prolonged action potentials than in cells with shorter action potentials. For example, EADs can be induced more readily in myocytes from the midmyocardial region of the ventricular walls than in myocytes from the endocardial or epicardial regions because of the longer action potential of midmyocardial myocytes (see

Fig. 16.5 ). Certain antiarrhythmic drugs, such as quinidine, prolong the action potential. As a consequence, such drugs increase the likelihood that EADs may occur. Hence, some antiarrhythmic drugs are also proarrhythmic.

Insomecases,mutationsintheconnexin40gene(GJA5) underliethedevelopmentofatrialfibrillation.Thismutationimpairstheassemblyofgapjunctionsinmyocytesandthereforereduceselectricalcouplingofthecells,asdescribedbyGollobMHandcolleagues(2006).

The direct correlation between a cell’s action potential duration and its susceptibility to EADs is related to the time required for calcium channels in the cell membranes to recover from inactivation. When action potentials are sufficiently prolonged, the calcium channels that were activated at the beginning of the plateau have sufficient time to recover from inactivation and thus may be reactivated before the cell fully repolarizes. This secondary activation could then trigger an EAD.

ThepathologicalsignificanceofEADswasrecognizedinconnectionwiththecongenitalanddrug-inducedlongQTsyndrome.Astheactionpotentiallengthens,EADsoccurandcausetriggeredautomaticity.Intheelectrocardiogram,thisproblemismanifestedaspolymorphicventriculartachycardia,alsocalledtorsades de pointes. Episodesoftorsadesdepointescanbeself-limitedbutmayprogresstoventricularfibrillationandsuddendeath.Hypokalemiaandbradycardia(see

Fig.16.24) areclinicalhallmarksofthiscondition.RestoringextracellularK+ tonormallevelsandincreasingheartratearetwoapproachesusedtoovercomethepropensityfordevelopmentoftorsadesdepointes.

In contrast to EADs, DADs are more likely to occur when the heart rate is high (Fig. 16.25 ). DADs are associated with elevated intracellular [Ca++]. The amplitudes of DADs are increased by interventions that raise intracellular [Ca++], such as increasing extracellular [Ca++] and administering toxic amounts of digitalis glycosides. The elevated levels of intracellular Ca++ provoke the oscillatory release of Ca++ from the sarcoplasmic reticulum. Hence, in myocardial cells, DADs are accompanied by small rhythmic changes in the force developed. The high intracellular [Ca++] also activates certain membrane channels that allow the passage of Na+ and K+ . The net flux of these cations constitutes a transient inward current (iti), that contributes to the appearance of DADs. The elevated intracellular [Ca++] may also activate the 3Na+-Ca++ antiporter. This electrogenic antiporter, which moves three Na+ ions into the cell for each Ca++ ion that it releases, also creates a net inward cation current that contributes to the appearance of DADs.

The ECG enables physicians to infer the course of cardiac electrical impulses by recording the variations in electrical potential at various loci on the surface of the body. By analyzing the details of these fluctuations in electrical potential, the physician gains valuable insight into (1) the anatomical orientation of the heart; (2) the relative sizes of its chambers; (3) various disturbances in rhythm and conduction; (4) the extent, location, and progress of ischemic damage to the myocardium; (5) the effects of •Fig. 16.25 Transmembrane Action Potentials Recorded From Purkinje Fibers. Acetylstrophanthidin,adigitalisglycoside,wasaddedtothebath,andsequencesofsixdrivenbeats(denotedbythedots)wereeachproducedatabasiccyclelength(BCL)of800(A), 700(B), 600(C), and500(D) msec.NotethatdelayedafterpotentialsoccurredafterthedrivenbeatsandthattheseafterpotentialsreachedthresholdafterthelastdrivenbeatinB toD. (FromFerrierGR,etal.Circ Res. 1973;32:600.) altered electrolyte concentrations; and (6) the influence of certain drugs (notably digitalis, antiarrhythmic agents, and calcium channel antagonists). Because electrocardiography is an extensive and complex discipline, only the elementary principles are considered in this section.

P-R QRS Q P R S T QT ST 0.1 sec •Fig. 16.26 Importantdeflectionsandintervalsofatypicalscalarelectrocardiogram.

In electrocardiography, a lead is the electrical connection from the patient’s skin to a recording device (electrocardiograph) that measures the electrical activity of the heart. The system of leads used to record routine ECGs is oriented in certain planes of the body. The diverse electrical events that exist in the heart at any moment can be represented by a three-dimensional vector (a quantity with magnitude and direction). A system of recording leads oriented in a given plane detects only the projection of the three-dimensional vector on that plane. The potential difference between two recording electrodes represents the projection of the vector on the line between the two leads. Components of vectors projected on such lines are not vectors but scalar quantities (having magnitude but not direction). Hence, a recording of changes in the difference in potential between two points on the skin surface over time is called a scalar ECG.

A scalar ECG detects temporal changes in the electrical potential between some point on the surface of the skin and an indifferent electrode or between pairs of points on the skin surface. The cardiac impulse progresses through the heart in a complex three-dimensional pattern. Hence, the precise configuration of the ECG varies from individual to individual, and in any given individual, the pattern varies with the anatomical location of the leads. The graphic display of the electrical impulse recorded in an ECG is called a tracing.

In general, a tracing consists of P, QRS, and T waves (

Fig. 16.26). The P wave reflects the spread of depolarization through the atria, the QRS wave (or complex) reflects depolarization of the ventricles, and the T wave represents repolarization of the ventricles (repolarization of the atria occurs, and is therefore masked, during ventricular depolarization). The PR interval (or more precisely, the PQ interval) is a measure of the time from the onset of atrial activation to the onset of ventricular activation; it normally ranges from 0.12 to 0.20 second. A large fraction of this time involves passage of the impulse through the AV conduction system. Pathological prolongations of the PR interval are associated with disturbances in AV conduction. Such disturbances may be produced by inflammatory, circulatory, pharmacological, or neuronal mechanisms.

The configuration and amplitude of the QRS complex vary considerably among individuals. The duration is usually between 0.06 and 0.10 second. An abnormally prolonged QRS complex may indicate a block in the normal conduction pathways through the ventricles (such as a block of the left or right bundle branch). During the ST interval, the entire ventricular myocardium is depolarized. Therefore, the ST segment normally lies on the isoelectric line. Any appreciable deviation of the ST segment from the isoelectric line may indicate ischemic damage to the myocardium. The QT interval, sometimes referred to as the period of “electrical systole” of the ventricles, is closely correlated with the mean action potential duration of the ventricular myocytes. The duration of the QT interval is approximately 0.4 second, but it varies inversely with the heart rate, mainly because the duration of the myocardial cell’s action potential varies inversely with the heart rate (see

Fig. 16.16

In most leads, the T wave is deflected in the same direction from the isoelectric line as the major component of the QRS complex, although biphasic (i.e., oppositely directed) T waves are perfectly normal in certain leads. Deviation of the T wave and QRS complex in the same direction from the isoelectric line indicates that the repolarization process is proceeding in a direction counter to that of the depolarization process. T waves that are abnormal either in direction or in amplitude may indicate myocardial damage, electrolyte disturbances, or cardiac hypertrophy.

The original ECG lead system was devised by Willem Einthoven at the beginning of the 20th century. In this system, the vector sum of all cardiac electrical activity at any moment is called the resultant cardiac vector. This directional electrical force is considered to lie in the center of an equilateral triangle whose apices are located in the left and right shoulders and the pubic region (

Fig. 16.27 ). This triangle, called Einthoven’s triangle, is oriented in the frontal plane of the body. Hence, only the projection of the resultant cardiac vector on the frontal plane is detected by this system of leads. For convenience, the electrodes are connected to the right and left forearms rather than to the corresponding shoulders because the arms represent simple electrical extensions of leads from the shoulders. Similarly, the leg represents an extension of the lead system from the pubis, and thus the third electrode is generally connected to an ankle (usually the left one).

Certain conventions dictate the manner in which these standard limb leads are connected to the electrocardiograph. Lead I records the potential difference between the left arm and the right arm. The connections are such that when the •Fig. 16.27 Einthoven’striangle,illustratingtheelectrocardiographicconnectionsforstandardlimbleadsI,II,andIII.

potential at the left arm (VLA) exceeds the potential at the right arm (VRA), the tracing is deflected upward from the isoelectric line. In

Fig. 16.27

Fig. 16.28 , this arrangement of connections for lead I is designated by a plus sign at the left arm and by a minus sign at the right arm. Lead II records the potential difference between the right arm and the left leg, and the tracing is deflected upward when the potential at the left leg (VLL) exceeds VRA. Finally, lead III registers the potential difference between the left arm and the left leg, and the tracing is deflected upward when VLL exceeds VLA. These connections were chosen arbitrarily so that the QRS complexes are upright in all three standard limb leads in most normal individuals.

If the frontal projection of a resultant cardiac vector at some moment is represented by an arrow (tail negative, head positive), as in

Fig. 16.27 , the potential difference, VLA − VRA, recorded in lead I is represented by the component of the vector projected along the horizontal line between the left arm and the right arm, also shown in

Fig. 16.27 . If the vector makes an angle (θ) of 60 degrees with the horizontal line (as in

Fig. 16.28A), the deflection recorded in lead I is upward because the positive arrowhead lies closer to the left arm than to the right arm. The deflection in lead II is also upright because the arrowhead lies closer to the left leg than •Fig. 16.28 MagnitudeanddirectionoftheQRScomplexesinlimbleadsI,II,andIIIwhenthemeanelectricalaxis(θ)is60degrees(A), 120degrees(B), and0degrees(C).

to the right arm. The magnitude of the lead II deflection is greater than that in lead I because in this example, the direction of the vector parallels that of lead II; therefore, the magnitude of the projection on lead II exceeds that on lead I. Similarly, in lead III, the deflection is upright and its magnitude equals that in lead I.

If the vector in

Fig. 16.27A is the result of electrical events that occur during the peak of the QRS complex, the orientation of this vector is said to represent the mean electrical axis of the heart in the frontal plane. The positive rotatory direction of this axis is assumed to be in the clockwise direction from the horizontal plane (contrary to the usual mathematical convention). In normal individuals, the average mean electrical axis is approximately +60 degrees (as in

Fig. 16.28A ). Therefore, QRS complexes are usually upright in all three leads and largest in lead II.

If the mean electrical axis shifts substantially to the right (as in

Fig. 16.28B , in which θ= 120 degrees), projections of the QRS complexes on the standard leads change considerably. In this case, the largest upright deflection is in lead III, and the deflection in lead I is inverted because the arrowhead is closer to the right arm than to the left arm. Such a shift is termed right axis deviation and occurs with hypertrophy (i.e., increased thickness) of the right ventricle. When the axis shifts to the left, as occurs with hypertrophy of the left ventricle (see

Fig. 16.28C , where θ= 0 degrees), the largest upright deflection is in lead I, and the QRS complex in lead III is inverted.

In addition to limb leads I, II, and III, other limb leads that are also oriented in the frontal plane are routinely recorded in patients. These leads are (1) aVR, for which the right arm is defined as the positive lead and the middle of the heart is defined as the negative lead (i.e., the left arm and ankle leads are connected together); (2) aVL, for which the left arm is the positive lead and the middle of the heart is defined as the negative lead (i.e., the right arm and ankle leads are connected together); and (3) aVF, for which the ankle (foot) lead is defined as positive and the middle of the heart is defined as the negative lead (i.e., the two arm leads are connected together). The axes of these leads form angles of +90 degrees for aVF, −30 degrees for aVL, and −150 degrees for aVR (all with respect to the horizontal axis).

Leads can also be applied to the surface of the chest, so-called precordial leads, to determine the projections of the cardiac vector on the sagittal and transverse planes of the body. These precordial leads are recorded from six selected points on the anterior and lateral surfaces of the chest in the vicinity of the heart. The leads extend from the right border of the sternum in the fourth intercostal space (lead V1) to under the left arm (midaxillary line) in the fifth intercostal space (lead V6). Each precordial lead (V1 to V6) is defined as a positive lead, whereas the middle of the heart is defined as the negative lead. Detailed analysis of the ECG, as detected by the various lead systems just described, is beyond the scope of this book. Interested students are referred to textbooks on electrocardiography for more information.

Changesinthemeanelectricalaxismayoccuriftheanatomicalpositionoftheheartisalteredoriftherelativemassoftherightandleftventriclesisabnormal,asitisincertaincardiovasculardisturbances.Forexample,theaxistendstoshifttowardtheleft(morehorizontal)inshort,stockyindividualsandtowardtheright(morevertical)intall,thinpersons.Inaddition,inleftorrightventricularhypertrophy(increasedmyocardialmassofeitherventricle),theaxisshiftstowardthehypertrophiedside.

Cardiac arrhythmias are disturbances in either impulse initiation or impulse propagation. Disturbances in impulse initiation include those that arise from the SA node and •Fig. 16.29 Electrocardiographic Tracings of Heart Rhythms. A, Normalsinusrhythm.B, Sinus tachycardia.C, Sinusbradycardia.

those that originate from various ectopic foci. The principal disturbances in impulse propagation are reentrant rhythms and conduction blocks.

Mechanisms that vary the firing frequency of cardiac pacemaker cells were described previously. Changes in the firing rate of the SA node are usually produced by cardiac autonomic nerves. When the firing rate of the SA node is decreased, the heart rate also decreases (bradycardia). Conversely, increased SA node firing results in elevation of the heart rate (tachycardia). Examples of ECGs of sinus tachycardia and sinus bradycardia are shown in

Fig. 16.29 . The P, QRS, and T deflections are all normal, but duration of the cardiac cycle (the PP interval) is altered. The changes in cardiac frequency are characteristically gradual. A rhythmic variation of the PP interval at the respiratory frequency (i.e., a respiratory sinus arrhythmia) is a normal, common occurrence (see

Fig. 18.7

Various physiological, pharmacological, and pathological processes can impede transmission of an impulse through the AV node. The site of block can be localized more precisely by an electrocardiogram of the His bundle (Fig.

16.30 ). To obtain such tracings, an electrode catheter is inserted into a peripheral vein and advanced centrally into the right side of the heart until the electrode lies in the AV junctional region. When the electrode is properly positioned, a distinct deflection (H in

Fig. 16.30 ) is registered as the cardiac impulse passes through the bundle of His. The time intervals required for propagation from the atrium to the bundle of His and from the bundle of His to the ventricles (His-to-ventricle interval) may be measured accurately. Abnormal prolongation of the atrium-to-His or His-to-ventricle interval indicates block above or below the bundle of His, respectively.

Premature depolarizations occur occasionally in most normal individuals, but they arise more commonly in certain abnormal conditions. Such depolarizations may originate in the atria, AV junction, or ventricles. One type of premature depolarization follows a normally conducted depolarization at a constant time interval (the coupling interval). If the normal depolarization is suppressed in some way (e.g., by vagal stimulation), the premature depolarization is also abolished. Such premature depolarizations are called coupled extrasystoles, or simply extrasystoles, and they

P A H V T QRS 0.2 sec •Fig. 16.30 ElectrocardiogramoftheHisbundle(lower tracing, retouched)andleadIIrecordingofthescalarelectrocardiogram(upper tracing). ThedeflectionH,whichrepresentsconductionoftheimpulseoverthebundleofHis,isclearlyvisiblebetweentheatrial andtheventricular(V)deflections.TheconductiontimefromtheatriatothebundleofHisisdenotedbytheA-Hinterval;thatfromthebundleofHistotheventricles,bytheH-Vinterval.(CourtesyofDr.

J.

Edelstein.)

ThreedegreesofAVblockcanbedistinguished,asshownin

Fig.16.31 .First-degreeAVblockischaracterizedbyaprolongedPRinterval.Inmostcasesoffirst-degreeblock,theatrium-to-HisintervalisprolongedandtheHis-toventricleintervalisnormal.Hence,thedelayinafirst-degreeAVblockislocatedabovetheHisbundle(i.e.,intheAVnode).Insecond-degreeAVblock,allQRScomplexesareprecededbyPwaves,butnotallPwavesarefollowedbyQRScomplexes.TheratioofPwavestoQRScomplexesisusuallytheratiooftwosmallintegers(suchas2:1,3:1,or 3:2).ThesiteofblockmaybelocatedaboveorbelowtheHisbundle.Ablockbelowthebundleisusuallymoreseriousthanoneabovethebundlebecausetheformerismorelikelytoevolveintoathird-degreeblock.Anartificialpacemakerisfrequentlyimplantedwhentheblockisbelowthebundle.Third-degreeAVblockisoftenreferredtoascomplete heart block becausetheimpulseiscompletelyunabletotraversetheAVconductionpathwayfromatriatoventricles.ThemostcommonsitesofcompleteblockaredistaltothebundleofHis.Incompleteheartblock,theatrialandventricularrhythmsareentirelyindependent.Becauseoftheslowventricularrhythmthatresults,thevolumeofbloodpumpedbytheheartisofteninadequate,especiallyduringmuscularexercise.Third-degreeblockisfrequentlyassociatedwithsyncope(pronouncedlightheadedness),whichiscausedprincipallybyinsufficientcerebralbloodflow.Third-degreeblockisoneofthemostcommonconditionsthatnecessitatetheimplantationofartificialpacemakers.

•Fig. 16.31 Atrioventricular (AV) Blocks. A, First-degreeblock;thePRintervalis0.28second(normal, <0.20sec).B, Second-degreeblock(ratioofPwavestoQRScomplexes,2:1).C, Third-degreeblock;notethedissociationbetweenthePwavesandtheQRScomplexes.

•Fig. 16.32 Premature Atrial Depolarization and Premature Ventricular Depolarization. Theprematureatrialdepolarization(A; secondbeat)ischaracterizedbyaninvertedPwave(justbelowthesecond“P”)andnormalQRScomplexesandTwaves.Theintervalaftertheprematureatrialdepolarizationisnotmuchlongerthantheusualintervalbetweenbeats.Thebriefrectangulardeflectionjustbeforethelastatrialdepolarizationisastandardizationsignal.Theprematureventriculardepolarization(B) ischaracterizedbybizarre,invertedQRScomplexesandelevatedTwavesandisfollowedbyacompensatorypause.

generally reflect a reentry phenomenon. A second type of premature depolarization occurs as the result of enhanced automaticity in some ectopic focus. This ectopic center may fire regularly, and a zone of tissue that conducts unidirectionally may protect this center from being depolarized by the normal cardiac impulse. If this premature depolarization occurs at a regular interval or at an integral multiple of that interval, the disturbance is called parasystole.

A tracing of a premature atrial depolarization is shown in

Fig. 16.32A . With a premature atrial depolarization, the normal interval between beats is shortened. In addition, the configuration of the premature P wave differs from that of the other normal P waves because the course of atrial excitation, which originates at some ectopic focus in the atrium, differs from the normal spread of excitation, which originates at the SA node. The QRS complex of the premature depolarization is generally normal because the ventricular excitation spreads over the usual pathways.

A tracing of a premature ventricular depolarization is shown in

Fig. 16.32B . Propagation of the impulse is abnormal, and the configuration of the QRS complex and T wave is entirely different from the normal ventricular deflections because the premature excitation originates at some ectopic focus in the ventricles. The time interval between the premature QRS complex and the preceding normal QRS complex is shortened, whereas the interval after the premature QRS complex and the next normal QRS complex is prolonged by a compensatory pause. The interval from the QRS complex just before the premature excitation to the QRS complex just after it is virtually equal to the duration of two normal cardiac cycles.

•Fig. 16.33 Paroxysmaltachycardias.A, supraventriculartachycardia;PwavesprecedeeachQRScomplex.B, ventriculartachycardia;Pwavesnotreadilyobserved.

As noted, a compensatory pause usually follows a premature ventricular depolarization. This pause occurs because the ectopic ventricular impulse does not disturb the natural rhythm of the SA node. There are two possible reasons for this: The ectopic ventricular impulse is not conducted in a retrograde direction through the AV conduction system, or the SA node had already fired at its natural interval before the ectopic impulse could have reached and depolarized it prematurely. Likewise, the SA nodal impulse generated just before or after the ventricular extrasystole generally does not affect the ventricle because the AV junction and perhaps also the ventricles are still refractory from the premature ventricular excitation.

Fig.16.33A )areusuallyindistinguishable,andthereforethetermparoxysmal supraventricular tachycardia referstobothtypes.Inthistachycardia,theimpulseoftencirclesareentryloopthatincludesatrialandAVjunctionaltissue.TheQRScomplexesarefrequentlynormalbecauseventricularactivationproceedsovertheusualpathways.Asitsnameimplies,paroxysmalventriculartachycardiaoriginatesfromanectopicfocusintheventricles.TheECGischaracterizedbyrepeatedbizarreQRScomplexesthatreflecttheabnormalintraventricularimpulseconduction(see

Fig.16.33B ).Paroxysmalventriculartachycardiaismuchmoreominousthansupraventriculartachycardiabecausetheformerisfrequentlyaprecursorofventricularfibrillation,alethalarrhythmiadescribedinthenextsection.

In contrast to the gradual rate changes that characterize sinus tachycardia, tachycardias that originate from an ectopic focus typically begin and end abruptly. Such ectopic tachycardias are generally called paroxysmal tachycardias. Episodes of paroxysmal tachycardia may persist for only a few beats or for many hours or days, and episodes often recur. Paroxysmal tachycardias may result from (1) rapid firing of an ectopic pacemaker, (2) triggered activity secondary to afterpotentials that reach threshold, or (3) an impulse that circles a reentry loop repetitively.

Under certain conditions, cardiac muscle undergoes an irregular type of contraction that is ineffectual in propelling blood. Such an arrhythmia is termed fibrillation, and the disturbance may involve either the atria or the ventricles. Fibrillation probably represents a reentry phenomenon in which the reentry loop fragments into multiple, irregular circuits.

The electrocardiographic changes in atrial fibrillation are shown in

Fig. 16.34A . This arrhythmia occurs in various types of chronic heart disease. The atria do not contract and relax sequentially during each cardiac cycle, and thus they do not contribute to ventricular filling. Instead, the atria undergo a continuous, uncoordinated rippling motion. P waves do not appear on the ECG; instead, the tracing shows continuous irregular fluctuations in potential called f waves. The AV node is activated at intervals that may vary considerably from cycle to cycle. Hence, no constant interval occurs between successive QRS complexes or between successive ventricular contractions. Because the strength of ventricular contraction depends on the interval between beats (see ), the volume and rhythm of the pulse are irregular. In many affected patients, the atrial reentry loop and the pattern of AV conduction are more regular than they are in atrial fibrillation. The rhythm is then referred to as atrial flutter.

•Fig. 16.34 Atrial(A)andventricular(B)fibrillation.

Atrialfibrillationandflutterarenotusuallylife-threatening;somepeoplewiththesedisturbancescanfunctionnormally.However,becausetheatriadonotcontractandrelaxrhythmically,bloodclotstendtoformintheatria.Suchclots,ifdislodged,maythentraveltothepulmonaryorsystemicvascularbeds.Patientswithatrialfibrillationorflutteraregenerallytreatedwithanticoagulantdrugssuchasdicumaroltopreventtheformationofsuchclots.Ventricularfibrillation,incontrast,leadstolossofconsciousnesswithinafewseconds.Theirregular,continuous,uncoordinatedtwitchingoftheventricularmusclefiberspumpsnoblood.Deathensuesunlessimmediateresuscitationisachievedortherhythmspontaneouslyrevertstonormal,whichrarelyoccurs.Ventricularfibrillationmaysupervenewhentheentireventricle,orsomeportionofit,isdeprivedofitsnormalbloodsupply.Itmayalsooccurasaresultofelectrocutionorinresponsetocertaindrugsandanesthetics.OntheECG(see

Fig.16.34B ),thefluctuationsinpotentialarehighlyirregular.

Ventricular fibrillation is often initiated when a premature impulse arrives during the vulnerable period of the cardiac cycle. This period coincides with the downslope of the T wave on the ECG. During this period, the excitability of cardiac cells varies spatially. Some fibers are still in their effective refractory periods, others have almost fully recovered their excitability, and still others are able to conduct impulses, but only at very slow conduction velocities. As a consequence, the action potentials are propagated over the chambers in many irregular wavelets that travel along circuitous paths and at various conduction velocities. As a region of cardiac cells becomes excitable again, it is ultimately activated by one of the wave fronts traveling around the chamber. Hence, the process is self-sustaining.

•Fig. 16.35 Electrocardiogramsrecordedfromanormalsubject(A) andfromapatientwithlongQTsyndrome(B). Q SQ S P PT T R R 1 sec. Normal Q SQ S P P T R R Long QT A B

Insomeindividuals,theintervalbetweentheQRScomplexandtheTwaveisabnormallyprolonged,aconditiontermedlong QT syndrome (Fig.16.35 ).SeveralcongenitalformsoflongQTsyndromehavebeenidentifiedinhumansubjects.TwoofthemanygenesthathavebeenidentifiedasabasisforthissyndromearetheHERG gene(aK+ channelgene),locatedonchromosome7,andtheSCN5A gene(aNa+ gene),locatedonchromosome3.PatientswithcongenitalformsoflongQTsyndromemayhaveperiodicepisodesofsyncope(fainting),andapproximately10%ofpediatricsubjectswiththisdisordermaydiesuddenly,withoutanyprecedingsymptoms.Insomeindividualswithsilentionchannelmutations,longQTsyndromemaynotbeevidentuntiladrugistakenthataffectstheionchannelinvolved.Manydrugs,includingseveralantiarrhythmicagents,havebeenidentifiedascausingacquiredlongQTsyndrome.

Atrial fibrillation may be changed to a normal sinus rhythm by drugs that prolong the refractory period. As the cardiac impulse completes the reentry loop, it may then encounter refractory myocardial fibers. When atrial fibrillation does not respond adequately to drugs, electrical defibrillation may be used to correct this condition.

Dramatic therapy is required for ventricular fibrillation. Conversion to a normal sinus rhythm is accomplished by means of a strong electrical current that places the entire myocardium briefly in a refractory state. Techniques have been developed to administer the current safely through the intact chest wall. In successful cases, the SA node again takes over the normal pacemaker function for the entire heart.

The Cardiac Pump

The great amount of work performed by the heart over an individual’s lifetime is impressive. A useful way to

Implantablecardioverter-defibrillator(ICD)deviceshavebeendevelopedtopreventdeathinpatientsinwhomeitherventricularfibrillationorparoxysmalventriculartachycardiahassuddenlydeveloped.Theformerislethalunlessitistreatedimmediately,andthelatteroftenleadstoventricularfibrillationandsuddendeath.TheICDdeviceisimplantedsubcutaneouslyintheleftsubclavicularregionofthechestwall.Atrialandventricularleadsenablerecordingoftherightatrialandrightventricularelectrocardiogramsandprovidetheabilityforrightatrialorrightventricularpacing,orboth.Thedefibrillationcoilintherightventricleenablestheapplicationofastrongelectricalcurrenttotheventricleandtherebyusuallyterminatesthelethalarrhythmia.

understand how the heart accomplishes its important task is to consider the relationships between the structure and function of its components.

Relationship of Heart Structure to Function

The Myocardial Cell ). Of importance is that both are striated as a result of regular arrangement of the contractile proteins actin and myosin, and generation of force and contraction of muscle fiber occur as a result of their interactions (i.e., sliding filament mechanism).

Skeletal muscle and cardiac muscle show similar length-force relationships. As shown in , this relationship for the heart may be expressed graphically, as in

Fig.

16.36, by substituting ventricular systolic pressure for force and end-diastolic ventricular volume for resting myocardial fiber (and hence sarcomere) length. The lower curve in

Fig.

16.36 represents the increment in pressure produced by each increment in volume when the heart is in diastole. The upper curve represents the peak force or pressure developed by the ventricle during systole as a function of initial fiber length (or diastolic filling pressure). This curve illustrates the Frank-Starling law of the heart.

The pressure-volume curve during diastole is initially quite flat (compliant), which indicates that large increases in volume can be accommodated with only small increases in pressure. In contrast, the development of systolic pressure is considerable at the lower filling pressures. However, the ventricle becomes much less distensible with greater filling, as evidenced by the sharp rise in the diastolic pressure curve at large intraventricular volumes.

In a normal intact heart, peak force may be attained at a filling pressure of approximately 12 mm Hg. At this intraventricular diastolic pressure, which is near the upper limit observed in a normal heart, sarcomere length is near its resting length of 2.2 µm. However, the force causes filling pressures to peak as high as 30 mm Hg. At even higher diastolic pressures (>50 mm Hg), sarcomere length is no greater than 2.6 µm. This ability of the myocardium to resist stretch at high filling pressures probably resides in the noncontractile constituents of the heart tissue (connective tissue), and it may serve as a safety factor against overloading of the heart in diastole. Usually, ventricular diastolic pressure is approximately 0 to 7 mm Hg, and the average diastolic sarcomere length is approximately 2.2 µm. Thus a normal heart operates on the ascending portion of the Frank-Starling curve, as depicted in

Fig. 16.36 •Fig. 16.36 Relationshipofmyocardialrestingfiberlength(sarco-merelength)orend-diastolicvolumetotheforcedevelopedorpeaksystolicventricularpressureduringventricularcontractioninanintactheart.(RedrawnfromPattersonSW,etal.J Physiol. 1914;48:465.)

Cardiac muscle functions as a syncytium; that is, a stimulus applied to any part of the cardiac muscle results in contraction of the entire muscle. Gap junctions with high conductance are present in the intercalated disks between adjacent cells and facilitate conduction of the cardiac impulse from one cell to the next.

Cardiac muscle must contract repetitively for a lifetime, and hence it requires a continuous supply of O2. Cardiac muscle is therefore very rich in mitochondria. The large number of mitochondria (see

Fig. 13.1

B), which have the enzymes necessary for oxidative phosphorylation, allows rapid oxidation of substrates and synthesis of ATP and thus sustains the myocardial energy requirements.

CHAPTER 16 Elements of Cardiac Function

To provide adequate O2 and substrate for its metabolic machinery, the myocardium is also endowed with a rich capillary supply, approximately one capillary per fiber. Thus diffusion distances are short, and O2, CO2, substrates, and waste material can move rapidly between the myocardial cell and capillary. The transverse (T) tubular system within myocardial cells participates in this exchange of substances between capillary blood and myocardial cells (as described later, the T tubule system also plays a key role in excitation-contraction coupling). The T tubular system is absent or poorly developed in the atrial cells of many mammals.

Results of the earliest studies on isolated hearts indicated that optimal concentrations of Na+ , K+ , and Ca++ in extracellular fluid are necessary for contraction of cardiac muscle. Without Na+ , the heart is not excitable and does not beat. As already described, the resting membrane potential is independent of the extracellular [Na+] gradient across the membrane but very much dependent on extracellular [K+]. Decreases or increases in extracellular [K+], especially if they are large or occur quickly, can lead to arrhythmias, loss of excitability of the myocardial cells, and even cardiac arrest. Ca++ is also essential for cardiac contraction. Removal of Ca++ from the extracellular fluid results in decreased contractile force and eventual arrest in diastole. Conversely, an increase in extracellular [Ca++] enhances contractile force, and very high extracellular [Ca++] induces cardiac arrest in systole (rigor). The free intracellular [Ca++] is the factor principally responsible for the contractile state of the myocardium.

The process by which the action potential of the cardiac myocyte leads to contraction is termed excitation-contraction coupling (see also

Chapter 13). Cardiac muscle is excited when a wave of excitation spreads rapidly along the myocardial sarcolemma from cell to cell via gap junctions. Excitation also spreads into the interior of the cells via the T tubules, which invaginate the cardiac fibers at the Z lines. Electrical stimulation at the Z line or the application of Ca++ to the Z lines in a skinned cardiac fiber (whose sarcolemma is removed) elicits localized contraction of the adjacent myofibrils. During the plateau (phase 2) of the action potential, permeability of the sarcolemma by Ca++ increases. Ca++ flows down its electrochemical gradient and enters the cell through calcium channels in the sarcolemma and in the T tubules.

During the action potential, Ca++ enters the cell via calcium channels (L-type). However, the amount of Ca++ that enters the cell interior from the extracellular/ interstitial fluid is not sufficient to induce contraction of the myofibrils. Instead, it acts as a trigger (trigger Ca++) to release Ca++ from the sarcoplasmic reticulum, where the intracellular Ca++ is stored (see

Fig. 13.2 ). Ca++ leaves the sarcoplasmic reticulum through Ca++ release channels, which are called ryanodine receptors because the channel protein binds ryanodine avidly. Cytoplasmic [Ca++] increases from a resting level of approximately 10−7 mol to levels of approximately 10−5 mol during excitation. This Ca++ then binds to the protein troponin C. The Ca++–troponin C complex interacts with tropomyosin to unblock active sites between the actin and myosin filaments. This unblocking initiates cross-bridge cycling and hence contraction of the myofibrils.

Mechanisms that raise cytosolic [Ca++] increase the force developed, and those that lower cytosolic [Ca++] decrease the force developed. For example, catecholamines increase the movement of Ca++ into the cell by phosphorylation of the sarcolemmal calcium channels via a cAMP-dependent protein kinase (see

Fig. 13.4). This in turn causes the release of more Ca++ from the sarcoplasmic reticulum, and as a result, contractile force increases. Increasing extracellular [Ca++] increases the amount of Ca++ that enters the cell via the calcium channels and thereby increases contractile force as just described. Reducing the Na+ gradient across the sarcolemma also increases contractile force, an effect mediated by the 3Na+-Ca++ antiporter that normally extrudes Ca++ from the cell (see

Fig. 13.2 ). For example, reducing extracellular [Na+] causes less Na+ to enter the cell in exchange for Ca++ , which results in an increase in intracellular [Ca++] and thus contractile force. Raising intracellular [Na+] has a similar effect. In fact, this is the mechanism by which cardiac glycosides increase contractile force. Cardiac glycosides inhibit Na+,K+-ATPase and thereby raise intracellular [Na+] in the cells. The elevated cytosolic [Na+] reverses the direction of the 3Na+-Ca++ antiporter, and therefore less Ca++ is removed from the cell. The increase in intracellular [Ca++] results in an increase in contractile force. Finally, contractile force is diminished when intracellular [Ca++] is decreased by a reduction in extracellular [Ca++], by an increase in the Na+ gradient across the sarcolemma, or by the administration of a calcium channel antagonist that prevents Ca++ from entering the myocardial cell.

At the end of systole, the influx of Ca++ stops, and the sarcoplasmic reticulum is no longer stimulated to release Ca++ . In fact, the sarcoplasmic reticulum avidly takes up Ca++ by means of Ca++-ATPase. The Ca++-ATPase in the sarcoplasmic reticulum is similar to but distinct from the Ca++-ATPase found in the sarcolemma. Cytosolic [Ca++] is also reduced during diastole through the action of the 3Na+-Ca++ antiporter in the sarcolemma, as well as by a sarcolemmal Ca++-ATPase (see

Fig. 13.2

Both cardiac contraction and relaxation are accelerated by catecholamines (see

Fig. 13.3 ). When catecholamines bind to their receptor (β1-adrenoceptor), adenylate cyclase is activated, thereby increasing intracellular cAMP levels, which then leads to activation of cAMP-dependent protein kinase A. Protein kinase A has multiple effects in the cell. As already described, it phosphorylates the calcium channel in the sarcolemma and causes increased entry of Ca++ into the cell, thus increasing the force of contraction. In addition, protein kinase A phosphorylates other proteins that facilitate relaxation. One such protein is phospholamban. Phospholamban normally inhibits the Ca++-ATPase of the sarcoplasmic reticulum. However, when phospholamban is phosphorylated, its inhibitory action is reduced, and uptake of Ca++ into the sarcoplasmic reticulum is enhanced. The increased activity of the Ca++-ATPase in the sarcoplasmic reticulum decreases intracellular [Ca++], thereby causing relaxation. Protein kinase A also phosphorylates troponin I, which in turn inhibits binding of Ca++ by troponin C. As a result, tropomyosin returns to its position of blocking the myosin-binding sites on the actin filaments, and relaxation results.

Contraction of cardiac muscle is influenced by both preload and afterload (

Fig. 16.37 ). Preload is the force that stretches the relaxed muscle fibers, thereby increasing their resting length (see

Fig. 16.37B ). In the left ventricle, for example, the blood filling and thus the stretching of the wall during diastole represents the preload. Afterload is the force added to the muscle (see

Fig. 16.37C ) against which the contracting muscle must act (see

Fig. 16.37D ). Again from the perspective of the left ventricle, afterload is the pressure in the aorta that must be overcome by the contracting left ventricular muscle to open the aortic valve and eject the blood.

Preload can be increased by greater filling of the left ventricle during diastole (i.e., increasing end-diastolic volume). From lower initial end-diastolic volumes, increments in filling pressure during diastole increase resting fiber length and elicit a greater systolic pressure during the subsequent contraction (see

Fig. 16.36 ). Systolic pressure increases until a maximal systolic pressure is reached at the optimal preload (see

Fig. 16.36 ). If diastolic filling continues beyond this point, the pressure developed will no longer increase. At very high filling pressures, peak pressure development in systole is actually reduced.

At a constant preload, higher systolic pressure can be reached during ventricular contractions by an increase in the afterload (e.g., increasing aortic pressure by restricting the runoff of arterial blood to the periphery). Incremental increases in afterload produce progressively higher peak systolic pressures. However, if the afterload continues to increase, it becomes so great that the ventricle can no longer generate enough force to open the aortic valve. At this point, ventricular systole is totally isometric (i.e., there is no ejection of blood), and thus no change occurs in ventricular volume during systole. The maximal pressure developed by the left ventricle under these conditions is the maximal isometric force that the ventricle is capable of generating at a given preload. At preloads below the optimal filling volume, an increase in preload can yield greater maximal isometric force (see

Fig. 16.36

Preload and afterload depend on certain characteristics of the vascular system and the behavior of the heart. With regard to the vasculature, the degree of venomotor tone and peripheral resistance influences preload and afterload. With regard to the heart, a change in rate or stroke volume can also alter preload and afterload. Hence, cardiac and •Fig. 16.37 Preload and Afterload in a Papillary Muscle. A, Restingstage(noload)intheintactheartjustbeforeopeningoftheAVvalves.B, Preload(PL)intheintactheartattheendofventricularfilling.C, Supportedpreloadplusafterload(AL)intheintactheartjustbeforeopeningoftheaorticvalve.D, Liftingpreloadplusafterloadintheintactheartduringventricularejection,withadecreaseinventricularvolume.PL+ AL,totalload.

vascular factors interact with each other to affect preload and afterload (see

Contractility defines cardiac performance at a given preload and afterload. Contractility determines the change in peak isometric force (isovolumic pressure) at a given initial fiber length (end-diastolic volume). Contractility can be augmented by drugs, such as norepinephrine or digitalis, or by an increase in contraction frequency (tachycardia). The increase in contractility (positive inotropic effect) produced by these interventions is reflected by incremental increases in the force developed and in the velocity of contraction.

Indices of Contractility

A reasonable index of myocardial contractility can be derived from the contour of ventricular pressure curves (

Fig. 16.38 ). A hypodynamic heart is characterized by elevated end-diastolic pressure, slowly rising ventricular pressure, and a somewhat reduced ejection phase (curve C in

Fig. 16.38 ). A hyperdynamic heart (curve B in

Fig.

16.38 ) is characterized by reduced end-diastolic pressure, fast-rising ventricular pressure, and a brief ejection phase. The slope of the ascending limb of the ventricular pressure curve indicates the maximal rate of force development by the ventricle. The maximal rate of change in pressure with time—that is, the maximum dP/dt—is illustrated by the tangents to the steepest portion of the ascending limbs of the ventricular pressure curves in

Fig. 16.38 . The slope of the ascending limb is maximal during the isovolumic phase of systole (see

Fig. 16.38 ). At any given degree of ventricular filling, the slope provides an index of the initial contraction velocity and hence an index of contractility.

Similarly, the contractile state of the myocardium can be calculated from the velocity of blood flow that occurs initially in the ascending aorta during the cardiac cycle (

Fig.

16.39 ). In addition, the ejection fraction, which is the ratio of the volume of blood ejected from the left ventricle per beat (stroke volume) to the volume of blood in the left ventricle at the end of diastole (end-diastolic volume), is widely used clinically as an index of contractility.

•Fig. 16.38 Leftventricularpressurecurveswithtangentsdrawntothesteepestportionsoftheascendinglimbstoindicatemaximalratesofchangeinpressurewithtime(dP/dtvalues).A(blue curve), control;B(dashed red curve), hyperdynamicheart,aswithadministrationofnorepinephrine;C(green dashed curve), hypodynamicheart,asincardiacfailure.160 120 80 40 0 0 0.2 0.4 0.6 Time (sec) Left ventricular pressure (mm Hg) Max dP/dt B A C

The atria are thin-walled, low-pressure chambers that function more as large-reservoir conduits of blood for their respective ventricles than as important pumps for the forward propulsion of blood. The ventricles comprise a continuum of muscle fibers originating from the fibrous skeleton at the base of the heart (chiefly around the aortic orifice). These fibers sweep toward the cardiac apex at the epicardial surface. They pass toward the endocardium and gradually undergo a 180-degree change in direction to lie parallel to the epicardial fibers and to form the endocardium and papillary muscles.

At the apex of the heart, the fibers twist and turn inward to form papillary muscles. At the base of the heart and around the valve orifices, these myocardial fibers form a thick, powerful muscle mass that not only decreases the ventricular circumference to implement the ejection of blood but also narrows the AV valve orifices, which aids in closure of the valve. Ventricular ejection is also accomplished by a decrease in the longitudinal axis as the heart begins to narrow toward the base. The early contraction of the apical part of the ventricles, coupled with the approximation of the ventricular walls, propels the blood toward the ventricular outflow tracts. The right ventricle, which develops a mean pressure that is approximately one seventh that developed by the left ventricle, is considerably thinner than the left ventricle.

The cardiac valve leaflets consist of thin flaps of flexible, tough, endothelium-covered fibrous tissue that are firmly attached at the base to the fibrous valve rings. Movement of the valve leaflets is essentially passive, and the orientation of the cardiac valves is responsible for the unidirectional flow of blood through the heart. There are two types of valves in the heart: atrioventricular and semilunar (

Figs. 16.40 16.41

The tricuspid valve, located between the right atrium and the right ventricle, is made up of three cusps, whereas the mitral valve, which lies between the left atrium and the left ventricle, has two cusps. The total area of the cusps of each AV valve is approximately twice that of the respective AV orifice, and so considerable overlap of the leaflets occurs when the valves are in the closed position. Attached to the free edges of these valves are fine, strong ligaments (chordae tendineae cordis) that arise from the powerful papillary muscles of the respective ventricles. These ligaments prevent the valves from becoming everted during ventricular

Atrial systoleIsovol. contractionRapid ejectionReduced ejectionIsovol. relaxationRapid ventricular filling 0 0.1 0.2 0.3 0.4 0.60.5 0.7 0.8 Time (sec) Ventricular systole S P P R c a 4 1 2 3 v T Q Left atrial pressure Mitral valve opens Mitral valve closes Left ventricular pressure Aortic valve opens Aortic valve closes Aortic pressure Heartsounds VenouspulseElectrocardiogram •Fig. 16.39 Leftatrial,aortic,andleftventricularpressurepulsescorrelatedintimewithaorticflow,ventricularvolume,heartsounds,venouspulse,andtheelectrocardiogramforacompletecardiaccycleinahumansubject.Isovol,isovolumic.

systole.

In a normal heart, the valve leaflets remain relatively close together during ventricular filling. The partial approximation of the valve surfaces during diastole is caused by eddy currents that prevail behind the leaflets and by tension that is exerted by the chordae tendineae cordis and papillary muscles.

CHAPTER 16 Elements of Cardiac Function •Fig. 16.40 Illustrationofaheartsplitperpendiculartotheinterventricularseptumtodepicttheanatomicalrelationshipsoftheleafletsoftheatrioventricularandaorticvalves.Pulmonary artery Left atrium Left auricular appendage Mitral valve—anterior cusp Pulmonary veins Superior vena cava Aorta Aorta Mitral valve— posterior cusp Right ventricle Left ventricle Left ventricle Papillary muscle Papillary muscles appendage Orifices of coronary arteries Posterior cusp Anterior cusp •Fig. 16.41 Illustrationoffourcardiacvalvesasviewedfromthebaseoftheheart.Notehowtheleafletsoverlapintheclosedvalves.Anterior cusp Right cusp Left cusp Posterior cusp Anulus fibrosus Medial cusp valve

The pulmonic and aortic valves are located between the right ventricle and the pulmonary artery and between the left ventricle and the aorta, respectively. These valves consist of three cup-like cusps that are attached to the valve rings (see

Figs. 16.40 16.41 ). At the end of the reduced ejection phase of ventricular systole, blood flow briefly reverses toward the ventricles. This reversal of blood flow snaps the cusps together and prevents regurgitation of blood into the ventricles. During ventricular systole, the cusps do not lie back against the walls of the pulmonary artery and aorta; instead, they float in the bloodstream at a point approximately midway between the vessel walls and their closed position. Behind the semilunar valves are small outpocketings (sinuses of Valsalva) of the pulmonary artery and aorta. In these sinuses, eddy currents develop, which tend to keep the valve cusps away from the vessel walls. Furthermore, the orifices of the right and left coronary arteries are behind the right and the left cusps, respectively, of the aortic valve. Were it not for the presence of the sinuses of Valsalva and the eddy currents developed therein, the coronary ostia could be blocked by the valve cusps, and coronary blood flow would cease.

The Pericardium

The pericardium invests the entire heart and the cardiac portion of the great vessels, and it is reflected onto the cardiac surface as the epicardium. The sac normally contains a small amount of fluid, which provides lubrication for the continuous movement of the enclosed heart. The pericardium is not very distensible; it strongly resists a large, rapid increase in cardiac size and hence prevents sudden overdistention of the chambers of the heart. However, in congenital absence of the pericardium or after its surgical removal, cardiac function is not seriously affected. Nevertheless, with the pericardium intact, an increase in diastolic pressure in one ventricle increases the pressure and decreases the compliance of the other ventricle.

Four sounds are usually generated by the heart, but only two are ordinarily audible through a stethoscope. With electronic amplification, the less intense sounds can be detected and recorded graphically as a phonocardiogram. This means of registering faint heart sounds helps delineate the precise timing of the heart sounds in relation to other events in the cardiac cycle.

The first heart sound is initiated at the onset of ventricular systole (

Fig. 16.42 ) and reflects closure of the AV valves. It is the loudest and longest of the heart sounds, has a crescendo-decrescendo quality, and is heard best over the apical region of the heart. The tricuspid valve sounds are heard best in the fifth intercostal space just to the left of the sternum; the mitral sounds are heard best in the fifth intercostal space at the cardiac apex.

•Fig. 16.42 Thefirstandsecondheartsoundsofthephonocardio-gram(bottom tracing) areshowninregardtotheirrelationshiptotheP,R,andTwavesoftheelectrocardiogram(top tracing). 0 0.2 1.2 1.40.8 1.00.4 0.6 1.6 Time (sec) Phonocardiogram ElectrocardiogramP R T 2 1

The second heart sound, which occurs with abrupt closure of the semilunar valves (see

Fig. 16.42 ), is composed of higher frequency vibrations (higher pitch) and is of shorter duration and lower intensity than is the first heart sound. The portion of the second sound caused by closure of the pulmonic valve is heard best in the second thoracic interspace just to the left of the sternum, whereas that caused by closure of the aortic valve is heard best in the same intercostal space but to the right of the sternum. The aortic valve sound is generally louder than the pulmonic valve, but in cases of pulmonary hypertension, the reverse is true. The nature of the second heart sound changes with respiration. During expiration, a single heart sound is heard that reflects simultaneous closing of the pulmonic and aortic valves. However, during inspiration, closure of the pulmonic valve is delayed, mainly as a result of increased blood flow from an inspiration-induced increase in venous return.

With this delayed closure of the pulmonic valve, the second heart sound can be heard as two components; this is termed physiological splitting of the second heart sound.

A third heart sound is sometimes heard in children with thin chest walls or in patients with left ventricular failure. It consists of a few low-intensity, low-frequency vibrations heard best in the region of the cardiac apex. The vibrations occur in early diastole and are caused by the abrupt cessation of ventricular distention and by the deceleration of blood entering the ventricles. A fourth, or atrial, sound consists of a few low-frequency oscillations. This sound is occasionally heard in individuals with normal hearts. It is caused by the oscillation of blood and cardiac chambers as a result of atrial contraction.

The Cardiac Cycle

The phase between the start of ventricular systole and opening of the semilunar valves (when ventricular pressure rises abruptly) is called the isovolumic (literally, “same volume”) contraction period. This term is appropriate because ventricular volume remains constant during this brief period (see

Fig. 16.39 ). The onset of isovolumic contraction also coincides with the peak of the R wave on an ECG, initiation of the first heart sound, and the earliest rise in ventricular pressure on the ventricular pressure curve after atrial contraction.

Opening of the semilunar valves marks the onset of the ventricular ejection phase, which may be subdivided dWith inspiration, intrathoracic pressure is reduced (see

Chapter 21), which then increases venous blood flow to the right atrium.

Inoverloadedhearts,asincongestiveheartfailure,whenventricularvolumeisverylargeandtheventricularwallsarestretchedmaximally,athirdheartsoundisoftenheard.Athirdheartsoundinpatientswithheartdiseaseisusuallyagravesign.Whenthethirdandfourth(atrial)soundsareaccentuated,asoccursincertainabnormalconditions,tripletsofsoundsresemblingthesoundofagallopinghorse(calledgallop rhythms)mayoccur.Mitralinsufficiencyandmitralstenosisproduce,respectively,systolicanddiastolicmurmursthatareheardbestatthecardiacapex.Aorticinsufficiencyandaorticstenosis,incontrast,produce,respectively,diastolicandsystolicmurmursthatareheardbestinthesecondintercostalspacejusttotherightofthesternum.Thecharacteristicsofthemurmursserveasanimportantguideinthediagnosisofvalvulardisease.

into an earlier, shorter phase (rapid ejection) and a later, longer phase (reduced ejection). The rapid ejection phase is distinguished from the reduced ejection phase by three characteristics: (1) a sharp rise in ventricular and aortic pressure that terminates at peak ventricular and aortic pressure, (2) an abrupt decrease in ventricular volume, and (3) a pronounced increase in aortic blood flow (see

Fig. 16.39 ). The sharp decrease in left atrial pressure at the onset of ventricular ejection results from descent of the base of the heart and consequent stretching of the atria. During the reduced ejection period, runoff of blood from the aorta to the peripheral blood vessels exceeds the rate of ventricular output, and aortic pressure therefore declines. Throughout ventricular systole, the blood returning from the peripheral veins to the atria produces a progressive increase in atrial pressure.

During the rapid ejection period, left ventricular pressure slightly exceeds aortic pressure and aortic blood flow accelerates (continues to increase), whereas during the reduced ventricular ejection phase, aortic pressure is higher and aortic blood flow decelerates. This reversal of the ventricular-aortic pressure gradient in the presence of continuous flow of blood from the left ventricle to the aorta is the result of storage of potential energy in the stretched arterial walls. This stored potential energy causes blood flow from the left ventricle into the aorta to decelerate. The peak of the flow curve coincides with the point at which the left ventricular pressure curve intersects the aortic pressure curve during ejection. Thereafter, flow decelerates (continues to decrease) because the pressure gradient has been reversed.

Fig. 16.39 shows a tracing of a venous pulse curve recorded from a jugular vein. Three waves are apparent. The a wave occurs with the rise in pressure caused by atrial contraction. The c wave is caused by impact of the common carotid artery with the adjacent jugular vein and, to some extent, by the abrupt closure of the tricuspid valve in early ventricular systole. The v wave reflects the rise in pressure associated with atrial filling. Except for the c wave, the

CHAPTER 16 Elements of Cardiac Function venous pulse curve closely resembles the left atrial pressure curve.

At the end of ventricular ejection, a volume of blood approximately equal to that ejected during systole remains in the ventricular cavities. This residual volume is fairly constant in normal hearts. However, residual volume decreases somewhat when the heart rate increases or when peripheral vascular resistance has diminished.

Closure of the aortic valve produces the characteristic incisura (notch) on the descending limb of the aortic pressure curve, and it also produces the second heart sound (with some vibrations evident on the atrial pressure curve). The incisura marks the end of ventricular systole. The period between closure of the semilunar valves and opening of the AV valves is termed isovolumic relaxation. It is characterized by a precipitous fall in ventricular pressure without a change in ventricular volume.

The major portion of ventricular filling occurs immediately after the AV valves open. At this point, the blood that returned to the atria during the previous ventricular systole is abruptly released into the relaxing ventricles. This period of ventricular filling is called the rapid filling phase. In

Fig.

16.39 , the onset of the rapid filling phase is indicated by the decrease in left ventricular pressure below left atrial pressure. This pressure reversal opens the mitral valve. The rapid flow of blood from atria to relaxing ventricles produces transient decreases in atrial and ventricular pressures and a sharp increase in ventricular volume.

The rapid ventricular filling phase is followed by a phase of slow ventricular filling called diastasis. During diastasis, blood returning from the peripheral veins flows into the right ventricle and blood from the lungs flows into the left ventricle. This small, slow addition to ventricular filling is indicated by gradual rises in atrial, ventricular, and venous pressures and in ventricular volume (see

Fig. 16.39

Anincreaseinmyocardialcontractility,asproducedbycatecholaminesorbydigitalisinapatientwithafailingheart,maydecreaseresidualventricularvolumeandincreasethestrokevolumeandejectionfraction.Inseverelyhypodynamicanddilatedhearts,residualvolumecanbecomemuchgreaterthanstrokevolume.

The onset of atrial systole occurs soon after the beginning of the P wave (atrial depolarization) of the ECG. The transfer of blood from atrium to ventricle achieved by atrial contraction completes the period of ventricular filling.

Atrial systole is responsible for the small increases in atrial, ventricular, and venous pressure, as well as in ventricular volume (see

Fig. 16.39 ). Throughout ventricular diastole, atrial pressure barely exceeds ventricular pressure. This small pressure difference indicates that the pathway through the open AV valves during ventricular filling has low resistance.

Because there are no valves at the junction of the venae cavae and right atrium or at the junction of the pulmonary veins and left atrium, atrial contraction may force blood in both directions. However, little blood is actually pumped back into the venous tributaries during the brief atrial contraction, mainly because of the inertia of the inflowing blood.

The contribution of atrial contraction to ventricular filling is governed to a great extent by the heart rate and the position of the AV valves. At slow heart rates, filling practically ceases toward the end of diastasis, and atrial contraction contributes little additional filling. During tachycardia, however, diastasis is abbreviated and the atrial contribution can become substantial. Should tachycardia become so severe that the rapid filling phase is attenuated, atrial contraction assumes great importance in rapidly propelling blood into the ventricle during this brief period of the cardiac cycle. If the period of ventricular relaxation is so brief that filling is seriously impaired, even atrial contraction cannot provide adequate ventricular filling. The consequent reduction in cardiac output may result in syncope (fainting).

Atrialcontractionisnotessentialforventricularfilling,ascanbeobservedinpatientswithatrialfibrillationorcompleteheartblock.Inatrialfibrillation,theatrialmyofiberscontractinacontinuous,uncoordinatedmannerandthereforecannotpumpbloodintotheventricles.Incompleteheartblock,theatriaandventriclesbeatindependentlyofeachother.However,ventricularfillingmaybenormalinpatientswiththesetwoarrhythmias.Incertaindiseasestates,theAVvalvesmaybemarkedlynarrowed(stenotic).Insuchconditions,atrialcontractionplaysamuchmoreimportantroleinventricularfillingthanitdoesinanormalheart.

The changes in left ventricular pressure and volume throughout the cardiac cycle are summarized in

Fig. 16.43 . Diastolic filling starts when the mitral valve opens (point A in

Fig. 16.43 ), and it terminates when the mitral valve closes (point C). The initial decrease in left ventricular pressure (from points A to B), despite the rapid inflow of blood from the left atrium, is attributed to progressive ventricular relaxation and distensibility. During the remainder of diastole (from points B to C), the increase in ventricular pressure reflects ventricular filling and changes in the passive elastic characteristics of the ventricle. Note that only a small increase in pressure accompanies the substantial increase in ventricular volume during diastole (from points B to C).

•Fig. 16.43 Schematizedpressure-volumeloopoftheleftventricleforasinglecardiaccycle.

The small pressure increase reflects the compliance of the left ventricle during diastole. The small increase in pressure just before the mitral valve closes (to the left of point C) is caused by the contribution of atrial contraction to ventricular filling. With isovolumic contraction (from points C to D), pressure rises steeply, but ventricular volume does not change because the mitral and aortic valves are both closed. When the aortic valve opens (point D), and during the first (rapid) phase of ejection (from points D to E), the large reduction in volume is associated with a steady increase in ventricular pressure. This reduction in volume is followed by reduced ejection (from points E to F) and a small decrease in ventricular pressure. Closure of the aortic valve (point F) is followed by isovolumic relaxation (from points F to A), which is characterized by a sharp drop in pressure. Ventricular volume does not change during the interval between closing of the aortic valve and opening of the mitral valve (from points F to A) because both the mitral and aortic valves are closed. The mitral valve opens (point A) to complete one cardiac cycle.

Several key cardiovascular system parameters are evident on a left ventricular pressure-volume loop (P-V loop), or may be calculated from it. End-diastolic volume is obtained at mitral valve closure (point C in

Fig. 16.43 ) and the end-systolic volume at mitral valve opening (point A). The stroke volume is then apparent as the “width” of the P-V loop and is calculated as follows:

The preload of the left ventricle, considered here as left ventricular end-diastolic pressure, is the pressure coordinate when the mitral valve closes (point C in

Fig. 16.43 ). The •Fig. 16.44 A, Leftventricularpressure-volumeloopsrecordedinahuman.Theleftventriclewassubjectedtodifferentpreloadsbytransientocclusionofbloodflowintheinferiorvenacava.Aspreload(leftventricularend-diastolicpressure[LVEDP])wasdecreased,bothend-diastolicvolumeandend-systolicvolumedecreased,butend-diastolicvolumedecreasedmore,whichresultedindecreasedstrokevolume.B, CardiacfunctioncurveswhenstrokevolumeisplottedasafunctionofLVEDPforanormalheartinthebasalstate(blue line), aheartwithincreasedcontractility(red line), andaheartwithreducedcontractility(green line). Atanygivenpreload,strokevolumeishigherinheartswithincreasedcontractilityandlowerinheartswithdecreasedcontractilitythanstrokevolumeinthenormalbasalstate.(A, FromSenzakiH,etal.Single-beatestimationofend-systolicpressure-volumerelationinhumans.Anewmethodwiththepotentialfornoninvasiveapplication.Circulation. 1996;94[10]:2497-2506.) approximate “diastolic” arterial blood pressure may be read when the aortic valve opens (point D), and the approximate “systolic” arterial blood pressure is read during systole (point E). Left ventricular P-V loops recorded from a human (

Fig.

16.44A ) are similar to the schematized version (see

Fig.

16.43 ). The slope of the end-systolic P-V relation (line extending from the dot in

Fig. 16.44A ) defines contractility; a steeper slope indicates increased contractility. In this subject, partial occlusion of the inferior vena cava reduced the preload of the left ventricle on successive beats (as the inflow of blood to the left ventricle was reduced, as a consequence of the reduced return of blood to the right ventricle) and the effect was successively smaller stroke volume of the left ventricle.

This illustrates, in humans, the operation of the Frank-Starling law of the heart (

Chapter 13), whereby changes in preload (left ventricular volume) change myocardial fiber length, and thus the strength of the subsequent contraction, as well as stroke volume produced. This important phenomenon is characterized by the cardiac function curve (see

Fig. 16.44B).

As preload increases, stroke volume increases (solid blue line). If contractility of the heart is increased, as in the action of norepinephrine, the slope of the end-systolic P-V relation becomes steeper, and the entire cardiac function curve shifts upward (solid red line), which reflects the fact that the ventricle is now able to produce a larger stroke volume at a given preload. The increased stroke volume at any given preload is largely produced by a decreased end-systolic volume: Hearts with increased contractility are able to “squeeze down” to a greater extent. Conversely, if the ventricle is damaged, as after cardiac ischemia, or if its contractility is otherwise reduced from normal (as after calcium channel blockade), the cardiac function curve is shifted downward (green line), which reflects a reduced stroke volume at any given preload. The cardiac function curve is also known as ventricular function curves or Starling curves. To assess the integrated functioning of the cardiovascular system ( ), cardiac output, rather than stroke volume, is usually measured; preload of the heart is considered to be the filling pressure of the right ventricle, typically measured as mean right atrial pressure (Pra), or central venous pressure.

Measurement of Cardiac Output

The Fick Principle

In 1870 the German physiologist Adolph Fick contrived the first method for measuring cardiac output in intact animals and people. The basis for this method, called the Fick principle, is simply an application of the law of conservation of mass. The principle is derived from the fact that the quantity of O2 delivered to the pulmonary capillaries via

Terminal bronchiole To pulmonary veins From pulmonary artery Pulmonary artery q1 q2 O2 consumption 250 mL O2 /min [O2 ] pv 0.20 mL O2 /mL blood [O2 ] pa 0.15 mL O2 /mL blood q1 + q2 = q3 Alveolus q3 Pulmonary vein Alveoli •Fig. 16.45 Schema Illustrating the Fick Principle for Measuring Cardiac Output. Thechangeincolorfrompulmonaryarterytopulmonaryveinrepresentsthechangeincolorofthebloodasvenousbloodbecomesfullyoxygenated.

the pulmonary artery, plus the quantity of O2 that enters the pulmonary capillaries from the alveoli, must equal the quantity of O2 that is carried away by the pulmonary veins. The Fick principle is depicted schematically in

Fig.

16.45.

The rate of O2 delivery to the lungs (q1) equals the O2 concentration in pulmonary arterial blood ([O2]pa), multiplied by pulmonary arterial blood flow (Q), which equals cardiac output; that is,

Equation 16.1

If q2 is the net rate of O2 uptake by the pulmonary capillaries from the alveoli, then at steady state, q2 equals the O2 consumption of the body. The rate at which O2 is carried away by the pulmonary veins (q3) equals the O2 concentration in pulmonary venous blood ([O2]pv), multiplied by total pulmonary venous flow, which is virtually equal to pulmonary arterial blood flow (Q); that is,

Equation 16.2

From the law of conservation of mass,

Equation 16.3

Therefore,

Equation 16.4

When this is solved for cardiac output,

Equation 16.5

Eq. 16.5 is the statement of the Fick principle.

To determine cardiac output by this method, three values must be known: (1) O2 consumption of the body, (2) the O2 concentration in pulmonary venous blood ([O2]pv), and (3) the O2 concentration in pulmonary arterial blood ([O2]pa). O2 consumption is computed from measurements of the volume and O2 content of expired air over a given interval. Because the O2 concentration of peripheral arterial blood is essentially identical to that in the pulmonary veins, [O2]pv is determined with a sample of peripheral arterial blood withdrawn by needle puncture. The compositions of pulmonary arterial blood and mixed systemic venous blood are virtually identical to one another. Samples for O2 analysis are obtained from the pulmonary artery or right ventricle through a catheter. A very flexible catheter with a small balloon near the tip can be inserted into a peripheral vein. As the flexible tube is advanced, the flowing blood carries it toward the heart. By monitoring the pressure changes, the physician can advance the catheter tip into the pulmonary artery.

With the values depicted in

Fig. 16.45 , cardiac output can be calculated as follows: If the O2 consumption is 250 mL/minute, the arterial (pulmonary venous) O2 content is 0.20 mL of O2 per milliliter of blood, and the mixed venous (pulmonary arterial) O2 content is 0.15 mL of O2 per milliliter of blood, cardiac output equals 250/ (0.20 − 0.15) = 5000 mL/minute.

The Fick principle is also used to estimate the O2 consumption of organs when blood flow and the O2 content of arterial and venous blood can be determined. Algebraic rearrangement reveals that O2 consumption equals blood flow multiplied by the difference in the arteriovenous O2 concentration. For example, if blood flow through one kidney is 700 mL/minute, the arterial O2 content is 0.20 mL of O2 per milliliter of blood, and the renal venous O2 content is 0.18 mL of O2 per milliliter of blood, the rate of O2 consumption by that kidney must be 700 (0.20 − 0.18) = 14 mL of O2 per minute.

In the clinic, cardiac output is most commonly measured noninvasively with Doppler echocardiography. By this method, the velocity of blood in the ascending aorta is measured. Obtaining the cross-sectional area of the aorta (also measured by echocardiography) allows the volume of blood ejected in a single beat (i.e., stroke volume) to be determined (see

Eq. 17.1 ). Multiplying stroke volume by the heart rate then yields a value for cardiac output in liters per minute.

Consumption of O2 by the heart depends on the amount and type of activity that the heart performs. Under basal conditions, myocardial O2 consumption (V˜O2) is approximately 8 to 10 mL/minute/100 g of heart. It can increase severalfold during exercise and decrease moderately in such conditions as hypotension and hypothermia. The O2 content of cardiac venous blood is normally low (≈5 mL/ dL), and the myocardium can receive little additional O2 by further extraction of O2 from coronary blood. Therefore, increased O2 demands of the heart must be met mainly by an increase in coronary blood flow (see ). In experiments in which the heartbeat is arrested but coronary perfusion is maintained, O2 consumption falls to 2 mL/ minute/100 g or less, which is still six to seven times greater than the O2 consumption of resting skeletal muscle.

Cardiac work has external and internal components. Left ventricular work per beat (stroke work) is approximately equal to the product of stroke volume and the mean aortic pressure against which the blood is ejected by the left ventricle. External cardiac work (We) may be defined as follows:

Equation 16.6

That is, each small increment in volume that is pumped, (dV) is multiplied by the associated pressure (P), and the products (PdV) are integrated over the time interval of interest (t2 − t1) to calculate total work. Added to this is kinetic work due to the velocity (v) of blood flow and the density (ρ) of blood. The mean pressure during expulsion is used to simplify this to

Equation 16.7

At resting levels of cardiac output, the kinetic energy component is negligible. However, with high cardiac output, as in strenuous exercise, the kinetic energy component can account for up to 50% of total cardiac work.

The internal work (Wi) of the heart can be written as

Equation 16.8 where α is a proportionality constant that converts T dt into units of work, T is wall tension, and dt is time. Clinically, V˜O2 and left ventricular power are difficult to measure; however, both are closely related to the systolic pressure–time index, the integral of left ventricular pressure, and time during systole. Such measurements are important because internal work is a large determinant of myocardial O2 need. An alternative approach to evaluate cardiac work and its relation to O2 consumption has been developed in which P-V loops (see

Fig. 16.44 ) are examined in conditions with varied preload and afterload; contractility is maintained constant.

Simultaneously halving aortic pressure and doubling cardiac output, or vice versa, result in the same value for cardiac work. However, the O2 requirements are greater for any given amount of cardiac work when a major proportion of the work is pressure work, as opposed to volume work. An increase in cardiac output at a constant aortic pressure (volume work) is accomplished with only a small increase in left ventricular O2 consumption, whereas increased

CHAPTER 16 Elements of Cardiac Function arterial pressure at constant cardiac output (pressure work) is accompanied by a large increase in myocardial O2 consumption. Thus myocardial O2 consumption may not be well correlated with overall cardiac work. The magnitude and duration of left ventricular pressure are correlated with left ventricular O2 consumption. The work of the right ventricle is one-seventh that of the left ventricle because pulmonary vascular resistance is much less than systemic vascular resistance.

The efficiency of the heart may be calculated as the ratio of the work accomplished to the total energy used. If the average O2 consumption is assumed to be 9 mL/minute/100 g for the two ventricles, a 300-g heart will consume 27 mL of O2 per minute. This value is equivalent to 130 small calories when the respiratory quotient is 0.82. Together, the two ventricles do approximately 8 kg-m of work per minute, which is equivalent to 18.7 small calories. Therefore, the gross efficiency of the heart is approximately 14%:

Equation 16.9

Thegreaterenergydemandofpressureworkthanofvolumeworkisclinicallyimportant,especiallyinaorticstenosis.Inthiscondition,leftventricularO2consumptionisincreased,mainlybecauseofthehighintraventricularpressuredevelopedduringsystole.However,coronaryperfusionpressure(andhenceO2supply)iseithernormalorreducedbecauseofthepressuredropacrossthenarroworificeofthediseasedaorticvalve.

The actual gross mechanical efficiency of the heart is slightly higher (18%) than the value calculated and is determined through subtracting the O2 consumption of the nonbeating (asystolic) heart (≈2 mL/minute/100 g) from the total cardiac O2 consumption in the calculation of efficiency. The efficiency of the heart as a pump is relatively low. During physical exercise, efficiency improves because mean blood pressure does not change appreciably, whereas cardiac output and work increase considerably, without a proportional increase in myocardial O2 consumption. Of interest is that the chemical efficiency of the heart is rather high, as indicated by the estimate of 60% for the efficiency of generating ATP from oxidative phosphorylation. The energy expended in cardiac metabolism that does not contribute to the propulsion of blood through the body appears in the form of heat. The energy of flowing blood is also dissipated as heat.

Myocardial Adenosine Triphosphate and Its Relation to Mechanical Function

The chemical energy that fuels cardiac contractile work and relaxation is derived from ATP hydrolysis (

Fig. 16.46 •Fig. 16.46 Overall Scheme for Production and Utilization of Adenosine Triphosphate (ATP) Within a Cardiac Myocyte. Pathwaysforutilizationofglucose,fattyacids(FA),andlactateareindicated,asaretherequirementsforO2andH+bytheelectrontransportchaininmitochondria.ADP,adenosinediphosphate;CoA,coenzymeA;CPT-I,carnitinepalmitoyltransferase;G-6-P,glucose-6-phosphate;GLUT,glucosetransporter;NADH,nicotinamideadenosinedehydrogenase;PDH,pyruvatedehydrogenase;Pi,inorganicphosphate;SR,sarcoplasmicreticulum;TG,triglyceride.ATPase FACPT-I Cardiomyocyte Citric Acid CycleCO2 Acetyl-CoA Acetyl-CoA NADH NADH Lactate GLUT Glycogen Cytosol Contractile work, SR Ca++ uptake, lon homeostasis G-6-P PDH Pyruvate Electron Transport Chain TGATP ATP ATP ADP + Pi ADP + Pi ADP + Pi H+ O2 Mitochondrion — GLYCOLYSIS Fatty Acid ˛-Oxidation

The healthy heart has a relatively constant level of ATP (≈5µmol/g wet weight) despite an extremely high rate of ATP hydrolysis (≈0.3µmol/g−1/second−1). The tissue content of ATP is low in relation to the rate of breakdown and production; complete turnover of the myocardial ATP content occurs approximately every 12 seconds in the heart at rest. ATP hydrolysis (see

Fig. 16.46) provides energy for contractile work (actin-myosin interaction and cell shortening), pumping Ca++ back into the sarcoplasmic reticulum at the end of systole and maintaining normal ion gradients (low Na+ and high K+ in the cell). Approximately two thirds of the ATP hydrolyzed by the heart is used to fuel contractile work, and the remaining third is used for ion pumps and “housekeeping” functions such as synthesis of proteins and nucleic acids. The Ca++-ATPase of the sarcoplasmic reticulum is the primary ion pump consuming ATP. This process occurs during the end of systole, when cytosolic Ca++ is rapidly sequestered into the sarcoplasmic reticulum to initiate diastolic relaxation. In the healthy heart, the rate of ATP hydrolysis is exquisitely matched to the rate of ATP resynthesis. There is no significant change in the concentration of ATP or ADP even with the onset of heavy exercise.

ATP resynthesis occurs primarily through oxidative phosphorylation in mitochondria (>98%) and, to a small degree, through glycolysis (<2%). Oxidative phosphorylation requires O2 and H2. The O2 is delivered to the myocardium and consumed in the mitochondria to make H2O, and the H2 is from the metabolism of carbon fuels (mainly fatty acids, glucose, and lactate) and the generation of the reduced form of nicotinamide adenine dinucleotide (NADH). Pyruvate dehydrogenase (PDH) regulates the oxidation of glucose and lactate. The activity of PDH is inhibited by product inhibition from acetyl–coenzyme A and NADH. Also, PDH activity is inhibited when phosphorylated by PDH kinase and activated when dephosphorylated by PDH phosphatase. ATP is formed from ADP and inorganic phosphate with the use of H+ . Of importance is that the rates of ATP formation and breakdown depend on an adequate delivery of O2 to the myocardium, which is a function of myocardial blood flow and oxygenation of arterial blood. An increase in ATP breakdown in the myocardium, such as occurs when heart rate, systolic blood pressure, and contractility are increased (as during exercise), necessitates an increase in O2 delivery to the myocardium so that the mitochondria can generate sufficient ATP by oxidative phosphorylation to meet the demand for ATP. Thus the rate of myocardial O2 consumption is tightly linked to the work rate (or power) of the myocardium.

The heart is versatile in its use of substrates, and within certain limits, uptake of a particular substrate is directly proportional to its arterial concentration. The use of one substrate by the heart is also influenced by the presence or absence of other substrates. For example, the addition of lactate to the blood that perfuses a heart metabolizing glucose leads to a reduction in glucose uptake and vice versa. At normal blood concentrations, glucose and lactate are consumed at approximately equal rates.

In contrast, uptake of pyruvate is very low, as is its arterial concentration. For glucose, the threshold concentration is approximately 4 mmol/L. Below this blood level, no glucose is taken up by the myocardium. Insulin reduces the glucose threshold and increases the rate of glucose uptake by the heart. Cardiac utilization of lactate occurs in most circumstances; insulin does not affect its uptake by the myocardium. Under hypoxic conditions, glucose utilization is facilitated by an increase in the rate of transport across the myocardial cell wall. However, lactate cannot be metabolized by the hypoxic heart and is produced by the heart under anaerobic conditions. Associated with lactate production by the hypoxic heart is the breakdown of cardiac glycogen.

Of the total cardiac O2 consumption, only 35% to 40% can be accounted for by the oxidation of carbohydrate. Thus the heart derives the major part of its energy from the oxidation of noncarbohydrate sources: namely, esterified and nonesterified fatty acids, which account for approximately 60% of the myocardial O2 consumption in people in the postabsorptive state. Various fatty acids have different thresholds for myocardial uptake, but these

CHAPTER 16 Elements of Cardiac Function acids are generally used in direct proportion to their arterial concentration. Ketone bodies, especially acetoacetate, are readily oxidized by the heart, and they are a major source of energy in diabetic acidosis. As is true of carbohydrate substrates, use of a specific noncarbohydrate is influenced by the presence of other substrates, whether noncarbohydrate or carbohydrate. Therefore, within certain limits, the heart preferentially uses the substrate that is available in the largest concentration. The oxidation of amino acids makes a small contribution to myocardial energy expenditure.

Normally, the heart derives its energy by oxidative phosphorylation, in which each mole of glucose yields 36 mol of ATP. However, during hypoxia, glycolysis takes over, and 2 mol of ATP is provided by each mole of glucose; β-oxidation of fatty acids is also curtailed. If hypoxia is prolonged, cellular creatine phosphate and eventually ATP are depleted.

In ischemia, lactic acid accumulates and decreases intracellular pH. This condition inhibits glycolysis, fatty acid use, and protein synthesis, and therefore it results in cellular damage and eventually necrosis of myocardial cells.

1. The transmembrane action potentials recorded from cardiac myocytes may contain the following five phases: Phase 0: The action potential upstroke is initiated when a suprathreshold stimulus rapidly depolarizes the membrane by activating the fast sodium channels. Phase 1: The notch is an early partial repolarization that is achieved by the efflux of K+ through channels that conduct the transient outward current (ito). Phase 2: The plateau represents a balance between the influx of Ca++ through calcium channels and the efflux of K+ through several types of potassium channels. Phase 3: Final repolarization is initiated when the efflux of K+ exceeds the influx of Ca++ . The resultant partial repolarization rapidly increases K+ conductance and restores full repolarization.

Phase 4: The resting potential of the fully repolarized cell is determined by conductance of the cell membrane to K+ , mainly through iK1 channels.

2. Fast-response action potentials are recorded from atrial and ventricular myocardial fibers and from ventricular specialized conducting (Purkinje) fibers. Such an action potential is characterized by a large amplitude, a steep upstroke, and a relatively long plateau. The effective refractory period of fast-response fibers begins at the upstroke of the action potential and persists until midway through phase 3. The fiber is relatively refractory during the remainder of phase 3, and it regains full excitability soon after it is fully repolarized (phase 4).

3.

Slow-response action potentials are recorded from normal SA and AV nodal cells and from abnormal myocardial cells that have been partially depolarized. The action potential is characterized by a less negative resting potential, a smaller amplitude, a less steep upstroke, and a shorter plateau than is typical of the fast-response action potential. The upstroke in slow-response fibers is produced by the activation of calcium channels. Slow-response fibers become absolutely refractory at the beginning of the upstroke, and partial excitability may not be regained until very late in phase 3 or until after the fiber is fully repolarized.

4.

Normally, the SA node serves as the cardiac pacemaker to initiate the cardiac impulse. This impulse is propagated from the SA node to the atria and ultimately reaches the AV node. After a delay in the AV node, the cardiac impulse is propagated throughout the ventricles. Ectopic foci in the atrium, the AV node, or the His-Purkinje system may initiate propagated cardiac impulses if the normal pacemaker cells in the SA node are suppressed or if the rhythmicity of the ectopic automatic cells is abnormally enhanced.

5.

Under certain abnormal conditions, afterdepolarizations may be triggered by an otherwise normal action potential. EADs arise early in phase 3 of a normal action potential. They are more likely to occur when the basic cycle length of the initiating beats is very long and when the cardiac action potentials are abnormally prolonged. DADs appear late in phase 3 or in phase 4. They are more likely to occur when the basic cycle length of the initiating beats is short and when the cardiac cells are overloaded with Ca++ .

6.

Reentrant arrhythmias occur when a cardiac impulse traverses a loop of cardiac fibers and reenters previously excited tissue, when the impulse is conducted slowly around the loop, and when the impulse is blocked unidirectionally in some section of the loop.

7.

The ECG, which is recorded from the surface of the body, traces the conduction of the cardiac impulse throughout the heart. The ECG may be used to detect and analyze certain cardiac arrhythmias, such as altered SA rhythms, AV conduction blocks, premature depolarizations, ectopic tachycardias, and atrial and ventricular fibrillation.

8.

On excitation, voltage-gated calcium channels open to admit extracellular Ca++ into the cardiac myocytes. The influx of Ca++ triggers the release of Ca++ from the sarcoplasmic reticulum. The elevated intracellular [Ca++] elicits contraction of the myofilaments. Relaxation is accomplished through the restoration of resting cytosolic [Ca++] by the pumping of Ca++ back into the sarcoplasmic reticulum and the exchange of Ca++ for extracellular Na+ across the sarcolemma. Velocity and force of contraction are functions of intracellular [Ca++]. Force and velocity are inversely related, and so with no load, velocity is maximal. In

Abriel H, Rougier J-S, Jalife J. Ion channel macromolecular complexes in cardiomyocites: Roles in sudden cardiac death. Circ Res. 2015;116:1971.

Bers DM, Guo T. Calcium signaling in cardiac ventricular myocytes. Ann N Y Acad Sci. 2005;1047:86. Cannell MB, Kong CH. Local control in cardiac E-C coupling. J Mol Cell Cardiol. 2012;52:298.

an isovolumic contraction, no external shortening occurs.

9.

In ventricular contraction, preload is stretch of the fibers by blood during ventricular filling. Afterload is the arterial pressure against which the ventricle ejects the blood. An increase in myocardial fiber length, as occurs with augmented ventricular filling (preload) during diastole, produces a more forceful ventricular contraction. This relationship between fiber length and strength of contraction is known as the Frank-Starling law of the heart.

10.

Contractility is an expression of cardiac performance at a given preload and afterload. Contractility can be modulated by the autonomic nervous system.

11.

To determine cardiac output, according to the Fick principle, the O2 consumption of the body (q2) and the oxygen content of arterial blood ([O2]a) and mixed venous blood ([O2]v) are measured. Cardiac output = q2/([O2]a − [O2]v). It can also be measured noninvasively by Doppler echocardiography.

12.

In the normal heart, the rate of ATP hydrolysis is matched by the rate of ATP synthesis. ATP is generated by oxidative phosphorylation of fatty acids and glucose, a process that requires O2. When the rate of ATP hydrolysis increases, as during exercise, the consumption of O2 for oxidative phosphorylation also increases. The rate of O2 utilization is thereby coupled to the rate of cardiac work. The myocardium functions only aerobically, and in general it uses substrates in proportion to their arterial concentration.

Gima K, Rudy Y. Ionic current basis of electrocardiographic waveforms. Circ Res. 2003;90:889.

Priori SG. The fifteen years of discoveries that shaped molecular electrophysiology: time for appraisal. Circ Res. 2010;107: 451.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

What physical properties of blood vessels and blood determine hemodynamics, and how are they defined by Poiseuille’s law?

2.

How is arterial compliance related to stroke volume and pulse pressure? How does arterial compliance affect the arterial pulse wave and cardiac work?

3.

What are mean, systolic, diastolic, and pulse pressures, and how are they measured?

4.

What vessels constitute the microcirculation? How is pulsatile blood flow in large arteries converted into steady flow in the microcirculation?

5.

What are the hydrostatic and osmotic factors that underlie Starling’s hypothesis for capillary function?

6.

How do intrinsic and extrinsic factors modulate peripheral circulation, and how do these factors affect blood flow in particular organs?

7.

How does the myogenic hypothesis account for autoregulation of blood flow? What is the effect of tissue metabolism on autoregulation?

he vasculature consists of a closed system of tubes or vessels that distributes blood from the heart to the tissues and returns blood from the tissues to the heart. It can be divided into three components: the arterial system, which takes blood from the heart and distributes it to the tissues; the venous system, which returns blood from the tissues to the heart; and the microcirculation, which separates the arterial and venous systems and is the site where nutrients and cellular waste products are exchanged between blood and tissues. These components of the vasculature are described in this chapter. In addition, the properties of blood flow to specific vascular beds and tissues are considered. As an introduction to this material, the physics of blood/fluid flow through the vasculature (i.e., hemodynamics) is reviewed.

The physics of fluid flow through rigid tubes provides a basis for understanding the flow of blood through blood vessels, even though the blood vessels are not rigid tubules (i.e., they are distensible) and blood is not a simple homogeneous fluid. Knowledge of these physical principles underlies understanding of the interrelationships among velocity of blood flow, blood pressure, and the dimensions of the various components of the systemic circulation.

Velocity of the Bloodstream

Velocity, as relates to fluid movement, is the distance that a particle of fluid travels with regard to time, and it is expressed in units of distance per unit time (e.g., centimeters per second). Flow, in contrast, is the rate of displacement of a volume of fluid, and it is expressed in units of volume per unit time (e.g., cubic centimeters per second). In a rigid tube, velocity (v) and flow (Q) are related to one another by the cross-sectional area (A) of the tube:

Equation 17.1

The interrelationships among velocity, flow, and area are shown in

Fig. 17.1 . Because conservation of mass requires that the fluid flowing through a rigid tube be constant, the velocity of the fluid varies inversely with the cross-sectional area. Thus fluid flow velocity is greatest in the section of the tube with the smallest cross-sectional area and slowest in the section of the tube with the greatest cross-sectional area.

Fig. 15.3 , velocity decreases progressively as blood traverses the arterial system. In the capillaries, velocity decreases to a minimal value. As the blood then passes centrally through the venous system toward the heart, velocity progressively increases again. The relative velocities in the various components of the circulatory system are related only to the respective cross-sectional areas.

The total energy in a hydraulic system consists of three components: pressure, gravity, and velocity. The velocity of blood flow can have an important effect on the pressure within the tube. Consider the effect of velocity on pressure in a tube with different cross-sectional areas (

Fig. 17.2 ). In this system, the total energy remains constant. The total pressure within the tube equals the lateral (static) pressure plus the dynamic pressure. The gravitational component can be neglected because the tube is horizontal. The total pressures in segments A, B, and C are equal, provided that •Fig. 17.1 Asfluidflowsthroughatubeofvariablecross-sectionalarea(A),thelinearvelocity(v)variesinverselywiththecross-sectionalarea.Q,flow.

˜v2/2 = 3.8 mm Hg 15 mm Hg 3.8 mm Hg •Fig. 17.2 Inanarrowsection(B)ofatube,thelinearvelocity(v)andhencethedynamiccomponentofpressure(ρv2/2)aregreaterthaninthewidesections(AandC),ofthesametube.Ifthetotalenergyisvirtuallyconstantthroughoutthetube(i.e.,iftheenergylossbecauseofviscosityisnegligible),thelateralpressureinthenarrowsectionislowerthanthelateralpressureinthewidesectionsofthetube(seetheheightsofthecolumnoffluidabovecompartmentsA,B,andCeach,whichreflectpressure).

the energy loss from viscosity is negligible (i.e., this fluid is an “ideal fluid”). The effect of velocity on the dynamic component (Pdyn) can be estimated as follows:

Equation 17.2 where ρ is the density of the fluid (grams per cubic centimeters) and v is velocity (centimeters per second). Assume that the fluid has a density of 1 g/cm3. In section A in

Fig. 17.2 , the lateral pressure is 100 mm Hg; note that 1 mm Hg equals 1330 dynes/cm2. According to

Eq. 17.2, P dyn = 5000 dynes/cm2, or 3.8 mm Hg. In the narrow section B of the tube, where the velocity is twice as high, Pdyn = 20,000 dynes/ cm2, or 15 mm Hg. Thus the lateral pressure in section B is 15 mm Hg lower than the total pressure, whereas the lateral pressures in sections A and C are only 3.8 mm Hg lower. In most arterial locations, the dynamic component is a negligible fraction of the total pressure. However, at sites of an arterial constriction or obstruction, the high flow velocity is associated with large kinetic energy, and the dynamic pressure component may therefore increase significantly. Hence, the pressure would be reduced, and perfusion of distal segments would be correspondingly decreased. This example helps explain how pressure changes in a vessel that is narrowed by atherosclerosis or spasm of the blood vessel •Fig. 17.3 Laminar and Turbulent Flow. A, Whenflowislaminar,allelementsofthefluidmoveinstreamlinesthatareparalleltotheaxisofthetube;thefluiddoesnotmoveinaradialorcircumferentialdirection.Thelayeroffluidincontactwiththewallismotionless;thefluidthatmovesalongthecentralaxisofthetubehasthemaximalvelocity.B, Inturbulentflow,theelementsofthefluidmoveirregularlyinaxial,radial,andcircumferentialdirections.Vorticesfrequentlydevelop.

wall: that is, in narrowed sections of a tube, the dynamic component increases significantly because the flow velocity is associated with large kinetic energy.

The most fundamental law that governs the flow of fluids through cylindrical tubes was derived empirically by the French physiologist Jean Léonard Marie Poiseuille in the 1840s. He was interested primarily in the physical determinants of blood flow, but he replaced blood with simpler liquids in his measurements of flow through glass capillary tubes. His work was so precise and important that his observations have been designated Poiseuille’s law.

Poiseuille’s law applies to the steady (i.e., nonpulsatile) laminar flow of newtonian fluids through rigid cylindrical tubes. A newtonian fluid is one whose viscosity remains constant, and laminar flow is the type of motion in which the fluid moves as a series of individual layers, with each layer moving at a velocity different from that of its neighboring layers (

Fig. 17.3A ). In the case of laminar flow through a tube, the fluid consists of a series of infinitesimally thin concentric tubes sliding past one another, of which the central tube has the highest velocity. The velocities of the concentric laminae decrease parabolically towards the vessel wall. Despite the differences between the vascular system (i.e., flow is pulsatile, the vessels are not rigid cylinders, and blood is not a newtonian fluid), Poiseuille’s law does provide valuable insight into the determinants of blood flow through the vascular system. In certain unusual situations, however, flow can become turbulent (see

Fig. 17.3B ), rather than laminar. Under these conditions, vortices (swirls) are present, and the distribution of flow velocities is chaotic. This condition is described in more detail later in this chapter.

Poiseuille’s law describes the laminar flow of fluids through cylindrical tubes in terms of pressure, the dimensions of the tube, and the viscosity of liquid:

Equation 17.3

Pi − Po = pressure gradient from the inlet (i) of the tube to the outlet (o) r = radius of the tube η= viscosity of the fluid l = length of the tube

As is clear from the equation, flow through the tube increases as the pressure gradient is increased, and it decreases as either the viscosity of the fluid or the length of the tube increases. The radius of the tube is a critical factor in determining flow because it is raised to the fourth power.

Resistance to Flow

In electrical theory, Ohm’s law is that the resistance (R) equals the ratio of voltage drop (E) to current flow (I).

Equation 17.4

Similarly, in fluid mechanics, hydraulic resistance (R) may be defined as the ratio of the pressure drop (Pi − Po,) to flow (Q):

Equation 17.5

For the steady, laminar flow of a newtonian fluid through a cylindrical tube, the physical components of hydraulic resistance may be appreciated by the rearranging of Poiseuille’s law to yield the hydraulic resistance equation:

Equation 17.6

Thus when Poiseuille’s law applies, the resistance to flow depends on only the dimensions of the tube and the characteristics of the fluid.

The principal determinant of resistance to blood flow through any vessel is the caliber of the vessel because resistance varies inversely as the fourth power of the radius of the tube. In

Fig. 17.4 , the resistance to flow through small blood vessels is measured, and the resistance per unit length of vessel (R/l) is plotted against the vessel diameter. As shown, resistance is highest in the capillaries (diameter of 7 µm), and it diminishes as the vessels increase in diameter 1000 100 10 1 0.1 0.01 58 5850 5042 4234 3426 2618 187 Vessel diameter (µm) R/l(mm Hg/[mm3/sec])/µm Capillaries Arterial side Venous side •Fig. 17.4 Resistance Per Unit Length (R/l) of Individual Small Blood Vessels. Thecapillaries,withadiameterof7µm,aredenotedbythevertical dashed line. Resistancesofarteriolesareplottedtotheleftoftheverticaldashedline,andresistancesofvenules,totherightoftheverticaldashedline.Forbothtypesofvessels,theresistanceperunitlengthisinverselyproportionaltothefourthpowerofthevesseldiameter.(RedrawnfromLipowskyHH,etal.Circ Res. 1978;43:738.) on the arterial and venous sides of the capillaries. Values of R/l are virtually inversely proportional to the fourth power of the diameter (or radius) of the larger vessels on both sides of the capillaries.

Changes in vascular resistance occur when the caliber of vessels changes. The most important factor that leads to a change in vessel caliber is contraction of the circular smooth muscle cells in the vessel wall. Changes in internal pressure also alter the caliber of blood vessels and therefore alter the resistance to blood flow through these vessels. Blood vessels are elastic tubes. Hence, the greater the transmural pressure (i.e., the difference between internal and external pressure) across the wall of a vessel, the greater the caliber of the vessel and the less its hydraulic resistance.

It is apparent from

Fig. 15.3 that the greatest drop in pressure occurs in the very small arteries and arterioles. However, capillaries, which have a mean diameter of approximately 7 µm, have the greatest resistance to blood flow. Nevertheless, of all the different varieties of blood vessels that lie in series with one another (as in

Fig. 15.3 ), the arterioles, not the capillaries, have the greatest resistance. This seeming paradox is related to the relative numbers of parallel capillaries and parallel arterioles: There are far more capillaries than arterioles in the systemic circulation, and total resistance across the many capillaries arranged in parallel is much less than total resistance across the fewer arterioles arranged in parallel. In addition, arterioles have a thick coat of circularly arranged smooth muscle fibers that can vary the lumen radius. Even small changes in radius alter resistance greatly, as can be seen from the hydraulic •Fig. 17.5 Forresistances(R1,R2,andR3)arrangedinseries,totalresistance(Rt)equalsthesumoftheindividualresistances.P,pressure;Q,flow.

Eq. 17.6 ), wherein R varies inversely with r4.

In the cardiovascular system, the various types of vessels listed along the horizontal axis in

Fig. 15.3 lie in series with one another. The individual members of each category of vessels are ordinarily arranged in parallel with one another (see

Fig. 15.1 ). Thus capillaries are in most instances parallel elements throughout the body, except in the renal vasculature (in which the peritubular capillaries are in series with the glomerular capillaries) and the splanchnic vasculature (in which the intestinal and hepatic capillaries are aligned in series with each other). The total hydraulic resistance of components arranged in series or in parallel can be derived in the same manner as those for analogous combinations of electrical resistance.

Resistance of Vessels in Series

In the system depicted in

Fig. 17.5 , three hydraulic resistances, R1, R2, and R3, are arranged in series. The pressure drop across the entire system (i.e., the difference between inflow pressure [Pi] and outflow pressure [Po]) consists of the sum of the pressure drops across each of the individual resistances (equation a in

Fig. 17.5 ). In the steady state, the flow (Q) through any given cross-section must equal the flow through any other cross-section. When each component in equation (a) is divided by Q (equation [b] in

Fig. 17.5 ), it is evident from the definition of resistance (

Eq. 17.5 ) that for resistances in series, the total resistance (Rt) of the entire system equals the sum of the individual resistances; that is,

Equation 17.7

Resistance of Vessels in Parallel

For resistances in parallel, as illustrated in

Fig. 17.6 , inflow and outflow pressure is the same for all tubes. In steady state, the total flow (Qt) through the system equals the sum of the flows through the individual parallel elements (equation [a] in

Fig. 17.5).

Because the pressure gradient (Pi − Po) is identical for all parallel elements, each term •Fig. 17.6 Forresistances(R1,R2,andR3)arrangedinparallel,thereciprocalofthetotalresistance(Rt)equalsthesumofthereciprocalsoftheindividualresistances.P,pressure;Q,flow.

in equation (a) may be divided by that pressure gradient to yield equation (b). From the definition of resistance, equation (c) in

Fig. 17.5 may be derived. According to this equation, for resistances in parallel, the reciprocal of the total resistance (Rt) equals the sum of the reciprocals of the individual resistances; that is,

Equation 17.8

In a few simple illustrations, some of the fundamental properties of parallel hydraulic systems become apparent. For example, if the resistances of the three parallel elements in

Fig. 17.6 were all equal, then

Equation 17.9

Therefore, from

Eq. 17.8,

Equation 17.10

When the reciprocals of these terms, are equated,

Equation 17.11

Thus the total resistance is less than the individual resistances. For any parallel arrangement, the total resistance must be less than that of any individual component. For example, consider a system in which a tube with very high resistance is added in parallel to a low-resistance tube. The total resistance of the system must be less than that of the low-resistance component by itself because the high-resistance component affords an additional pathway, or conductance, for flow of fluid.

Consider the physiological relationship between the total peripheral resistance (TPR) of the entire systemic vascular bed and the resistance of one of its components, such as the renal vasculature. TPR is the ratio of the arteriovenous (AV) pressure difference (arterial pressure [Pa] − venous pressure [Pv]) to the flow through the entire systemic vascular bed (i.e., the cardiac output [Qt]). For example, the renal vascular resistance (Rr) would be the ratio of the same AV pressure difference (Pa − Pv) to renal blood flow (Qr).

In an individual with an Pa of 100 mm Hg, a peripheral Pv of 0 mm Hg, and a cardiac output of 5000 mL/min, TPR is 0.02 mm Hg/mL/minute, or 0.02 peripheral resistance units (PRUs). Normally, the rate of blood flow through one kidney would be approximately 600 mL/ minute. Renal resistance would therefore be 100 mm Hg ÷ 600 mL/minute, or 0.17 PRUs, which is 8.5 times greater than the TPR. In an organ such as the kidney, which weighs only approximately 1% as much as the whole body, the vascular resistance is much greater than that of the entire systemic circulation. Hence, it is not surprising that the resistance to flow would be greater for a component organ, such as the kidney, than for the entire systemic circulation because the systemic circulation has not only one kidney but also many more alternative pathways for blood to flow.

Fig. 17.3A ), a thin layer of fluid in contact with the tube wall adheres to the wall and hence is motionless. The layer of fluid just central to the external lamina must shear against this motionless layer, and therefore that layer moves slowly but with a finite velocity. Similarly, the next more central layer moves still more rapidly; the longitudinal velocity profile is that of a paraboloid (see

Fig.

17.3A ). The fluid elements in any given lamina remain in that lamina as the fluid moves longitudinally along the tube. The velocity at the center of the stream is maximal and equal to twice the mean velocity of flow across the entire cross-section of the tube.

Irregular motions of the fluid elements may develop in the flow of fluid through a tube; such flow is called turbulent. In this condition, fluid elements do not remain confined to a specific laminae; instead, rapid, radial mixing occurs (see

Fig. 17.3B ). Greater pressure is necessary to force a given flow of fluid through the same tube when the flow is turbulent than when it is laminar. In turbulent flow, the pressure drop is approximately proportional to the square of the flow rate, whereas in laminar flow, the pressure drop is proportional to the first power of the flow rate. Hence, to produce a given flow, a pump such as the heart must do considerably more work if turbulent flow develops.

Whether turbulent or laminar flow exists in a tube under given conditions may be predicted on the basis of a dimensionless number called Reynold’s number (NR). This number represents the ratio of inertial to viscous forces. For a fluid flowing through a cylindrical tube,

Equation 17.12 where ρ= fluid density, D = tube diameter, v = mean velocity, and η= viscosity. When NR is 2000 or less, the flow is usually laminar; when NR is 3000 or greater, the flow is turbulent; and when NR is between 2000 and 3000, the flow is transitional between laminar and turbulent.

Eq. 17.12 indicates that high fluid densities, small tube diameters, high flow velocities, and low fluid viscosities predispose to turbulence. In addition to these factors, abrupt variations in tube dimensions or irregularities in the tube walls may produce turbulence.

Shear Stress on the Vessel Wall

As blood flows through a vessel, it exerts a force on the vessel wall parallel to the wall. This force is called a shear stress (τ). Shear stress is directly proportional to the flow rate and viscosity of the fluid:

Equation 17.13

Turbulenceisusuallyaccompaniedbyaudiblevibrations.Turbulentflowwithinthecardiovascularsystemmaybedetectedthroughastethoscopeduringphysicalexamination.Whentheturbulenceoccursintheheart,theresultantsoundistermedamurmur; whenitoccursinavessel,thesoundistermedabruit. Insevereanemia,functionalcardiacmurmurs(murmursnotcausedbystructuralabnormalities)arefrequentlydetectable.Thephysicalbasesforsuchmurmursresidesare(1)thereducedviscosityofbloodinanemiaand (2)thehighflowvelocitiesassociatedwiththehighcardiacoutputthatusuallyprevailsinanemicpatients.Bloodclots,orthrombi,aremorelikelytodevelopinturbulentflowthaninlaminarflow.Aproblemwiththeuseofartificialvalvesinthesurgicaltreatmentofvalvularheartdiseaseisthatthrombimayoccurinassociationwiththeprostheticvalve.Thethrombimaybedislodgedandoccludeacrucialbloodvessel.Itisimportanttodesignsuchvalvestoavertturbulenceandtoincludeanticoagulantsasapartoftherapy.

Incertaintypesofarterialdisease,particularlyhypertension,thesubendotheliallayersofvesselstendtodegeneratelocally,andsmallregionsoftheendotheliummaylosetheirnormalsupport.Theviscousdragonthearterialwallmaycauseatearbetweenanormallysupportedregionandanunsupportedregionoftheendotheliallining.Bloodmaythenflowfromthevessellumenthroughtheriftintheliningandbecomedissectedbetweenthevariouslayersoftheartery.Suchalesioniscalledadissecting aneurysm. Itoccursmostoftenintheproximalportionsoftheaortaandisextremelyserious.Onereasonforitspredilectionforthissiteisthehighvelocityofbloodflow,withassociatedlargeshearratevaluesattheendothelialwall.Shearstressatthevesselwallalsoinfluencesmanyothervascularfunctions,suchasthepermeabilityofthevesselwallsbylargemolecules,thebiochemicalactivityofendothelialcells,theintegrityoftheformedelementsinblood,andbloodcoagulation.Anincreaseinshearstressontheendothelialwallisalsoaneffectivestimulusforthereleaseofnitricoxide(NO)fromvascularendothelialcells;NOisapotentvasodilator(seethesection“ •Fig. 17.7 Therelativeviscosityofwholebloodincreasesataprogressivelygreaterrateasthehematocritratioincreases.Foranygivenhematocritratio,theapparentviscosityofbloodislowerwhenmeasuredinabiologicalviscometer(suchasahindlegbloodvessel)thaninaconventionalcapillarytubeviscometer.(RedrawnfromLevyMN,ShareL.Circ Res. 1953;1:247.)9010 1 2 3 4 5 6 7 8 7030 50 Hematocrit ratio Relative viscosity Hind leg Capillary tube viscometer

Rheologic Properties of Blood

The viscosity of a given newtonian fluid at a specified temperature stays constant over a wide range of tube dimensions and flows. However, for a nonnewtonian fluid such as blood, viscosity may vary considerably as a function of tube dimensions and flows. Therefore, the term viscosity does not have a unique meaning for blood. The term apparent viscosity is frequently used for the derived value of blood viscosity obtained under the particular conditions of measurement.

Rheologically, blood is a suspension of formed elements, principally erythrocytes, in a relatively homogeneous liquid, the blood plasma. Because blood is a suspension, the apparent viscosity of blood varies as a function of the hematocrit (ratio of the volume of red blood cells to the volume of whole blood). The viscosity of plasma is 1.2 to 1.3 times that of water. The upper curve in

Fig. 17.7 shows that the apparent viscosity of blood with a normal hematocrit ratio of 45% is 2.4 times that of plasma.

In severe anemia, blood viscosity is low. As the hematocrit increases, the slope of the curve increases progressively; it is especially steep at the upper range of erythrocyte concentrations (see

Fig. 17.7

For any given hematocrit, the apparent viscosity of blood depends on the dimensions of the tube used in estimating the viscosity.

Fig. 17.8 demonstrates that the apparent viscosity of blood diminishes progressively as tube diameter decreases to less than approximately 0.3 mm. The diameters of the blood vessels with the highest resistance, the arterioles, are considerably less than this critical value. This phenomenon

Fig. 17.7 also illustrates that the apparent viscosity of blood, when measured in living tissues, is considerably less than the apparent viscosity of the same blood measured in a conventional capillary tube viscometer.

0.60 6 5 4 3 2 1 0.50.20.1 0.40.3 Tube diameter (mm) Relative viscosity •Fig. 17.8 Therelativeviscosityofbloodrelativetothatofwaterincreasesasafunctionoftubediameteruptoadiameterofapproximately0.3mm.(RedrawnfromFåhraeusR,LindqvistT.Am J Physiol. 1931;96:562.) therefore reduces the resistance to flow in blood vessels that possess the greatest resistance. The influence of tube diameter on apparent viscosity is explained in part by the actual change in blood composition as it flows through small tubes. The composition of blood changes because the red blood cells tend to accumulate in the faster axial stream, whereas plasma tends to flow in the slower marginal layers. Because the axial portions of the bloodstream contain a greater proportion of red cells and this axial portion moves at greater velocity, the red blood cells tend to traverse the tube in less time than plasma does. Furthermore, the hematocrit of the blood contained in small blood vessels is lower than that in blood in large arteries or veins.

The physical forces responsible for the drift of erythrocytes toward the axial stream and away from the vessel walls when blood is flowing at normal rates are not fully understood. One factor is the great flexibility of red blood cells. At low flow rates, like those in the microcirculation, rigid particles do not migrate toward the central axis of a tube, whereas flexible particles do. The concentration of flexible particles near the tube’s central axis is enhanced by an increase in the shear rate.

The apparent viscosity of blood diminishes as the shear rate is increased (

Fig. 17.9 ), a phenomenon called shear thinning. The greater the amount of flow, the greater the rate that one lamina of fluid shears against an adjacent lamina. The greater tendency for erythrocytes to accumulate in the axial laminae at higher flow rates is partly responsible for this nonnewtonian behavior. However, a more important factor is that at very slow flow rates, the suspended cells tend to form aggregates; such aggregation increases blood viscosity. As flow is increased, this aggregation decreases, and so does the apparent viscosity of blood (see

Fig. 17.9

The tendency for erythrocytes to aggregate at low flow rates depends on the concentration of the larger protein molecules in plasma, especially fibrinogen. For this reason, changes in blood viscosity with flow rate are much more pronounced when the concentration of fibrinogen is high. In addition, at low flow rates, leukocytes tend to adhere to the endothelial cells of the microvessels and thereby increase the apparent viscosity of the blood.

•Fig. 17.9 Decreaseintheviscosityofblood(cp,centipoise)atincreasingratesofshear(sec−1).Theshearrateisthevelocityofonelayeroffluidinrelationtothatoftheadjacentlayersandisdirectionallyrelatedtotherateofflow.(RedrawnfromAminTM,SirsJA.Q J Exp Physiol. 1985;70:37.)2400 7 6 5 4 3 18060 120 Shear rate/sec–1 Viscosity (cp) •Fig. 17.10 Effectoftheplasmafibrinogenconcentrationontheflexibilityofhumanerythrocytes.(RedrawnfromAminTM,SirsJA.Q J Exp Physiol. 1985;70:37.)40 8 6 4 2 0 31 2 Fibrinogen concentration (mg/mL) Erythrocyte flexibility (% min–1)

The deformability of erythrocytes is also a factor in shear thinning, especially when the hematocrit is high. The mean diameter of human red blood cells is approximately 7 µm, but they are able to pass through openings with a diameter of only 3 µm. As blood with densely packed erythrocytes flows at progressively greater rates, the erythrocytes become more and more deformed. Such deformation diminishes the apparent viscosity of blood. The flexibility of human erythrocytes is enhanced as the concentration of fibrinogen in plasma increases (

Fig. 17.10 ). If the red blood cells become hardened, as they are in certain spherocytic anemias, shear thinning may diminish.

The Arterial System

The systemic and pulmonary arterial systems distribute blood to the capillary beds throughout the body. The arterioles are high-resistance vessels of this system that regulate the distribution of flow to the various capillary beds. The aorta, the pulmonary artery, and their major branches have a large amount of elastin in their walls, which makes these vessels highly distensible (i.e., compliant). This distensibility serves to dampen the pulsatile nature of blood flow that results as the heart pumps blood intermittently. When blood is ejected from the ventricles during systole, these vessels distend, and during diastole, they recoil and propel the blood forward (Fig. 17.11 ). Thus the intermittent output of the heart is converted to a steady flow through the capillaries.

The elastic nature of the large arteries also reduces the work of the heart. If these arteries were rigid rather than compliant, the pressure would rise dramatically during systole. This increased pressure would require the ventricles to pump against a large load (i.e., afterload) and thus increase the work of the heart. Instead, as blood is ejected into these vessels, they distend, and the resultant increase in systolic pressure, and thus the work of the heart, are reduced.

Aspeopleage,theelastincontentofthelargearteriesisreducedandreplacedbycollagen.Thisreducesarterialcompliance(

Fig.17.12 ).Thuswithage,systolicpressureincreases,asdoesthedifferencebetweensystolicanddiastolicbloodpressure,calledthepulse pressure (describedinthenextsection).

Determinants of Arterial Blood Pressure

Arterial blood pressure is routinely measured in patients, and it provides a useful estimate of their cardiovascular status. Arterial pressure can be defined as mean arterial pressure (Pa), which is the pressure averaged over time, and as systolic (maximal) and diastolic (minimal) arterial pressure within the cardiac cycle (

Fig. 17.13 ). The difference between systolic and diastolic pressure is termed pulse pressure.

The determinants of arterial blood pressure are arbitrarily divided into “physical” and “physiological” factors. The two

Systole Arterial blood flows through the

Diastole Arterial blood continues to flow through capillaries throughout systole.

the capillaries throughout diastole.

A When the arteries are normally compliant, a B During ventricular diastole the previously substantial fraction of the stroke volume is stretched arteries recoil. The volume of blood stored in the arteries during ventricular systole. that is displaced by the recoil furnishes The arterial walls are stretched. continuous capillary flow throughout diastole.

Left atrium Left atrium Left ventricle Left ventricle Aorta Aorta Capillaries Capillaries Systole A volume of blood equal to the entire stroke volume must flow through the capillaries during systole. Diastole Flow through the capillaries ceases during diastole.

C When the arteries are rigid, virtually none D Rigid arteries cannot recoil appreciably during of the stroke volume can be stored in the diastole. arteries.

•Fig. 17.11 Whenarteriesarenormallycompliant(A andB),bloodflowsthroughthecapillariesthroughoutthecardiaccycle.Whenthearteriesarerigid,bloodflowsthroughthecapillariesduringsystole (C),butflowceasesduringdiastole(D).

physical factors, or fluid mechanical characteristics, are fluid volume (i.e., blood volume) within the arterial system and the static elastic characteristics (compliance) of the system. The physiological factors are cardiac output (which equals heart rate × stroke volume) and peripheral resistance.

To estimate Pa from an arterial blood pressure tracing, the area under the pressure curve is divided by the time interval involved (see

Fig. 17.13 ). Alternatively, Pa can be approximated from the measured values of systolic pressure (Ps) and diastolic pressure (Pd) by means of the following formula:

Equation 17.14

Consider that Pa depends on only two physical factors: mean blood volume in the arterial system and arterial compliance (

Fig. 17.14 ). Arterial volume (Va), in turn, depends on the rate of inflow, (Qh) into the arteries from the heart (cardiac output) and on the rate of outflow (Qr) from the arteries through the resistance vessels (peripheral runoff). These relationships are expressed mathematically as

Equation 17.15 where dVa/dt is the change in arterial blood volume per unit of time. If Qh exceeds Qr, arterial volume increases, the arterial walls are stretched further, and pressure rises. The converse happens when Qr exceeds Qh. When Qh equals Qr, Pa remains constant. Thus increases in cardiac output raise Pa, as do increases in peripheral resistance. Conversely, decreases in cardiac output or peripheral resistance decrease Pa.

Arterial pulse pressure is systolic pressure minus diastolic pressure. It is principally a function of just one physiological factor, stroke volume, which determines the change in arterial blood volume (a physical factor) during ventricular systole. This physical factor, in addition to a second physical factor (arterial compliance), determines the arterial pulse pressure (see

Fig. 17.14

As described previously, Pa depends on cardiac output and peripheral resistance. During the rapid ejection phase of systole, the volume of blood introduced into the arterial system exceeds the volume that exits the system through the arterioles. Arterial pressure and volume therefore peak; the peak arterial pressure is systolic pressure. During the remainder of the cardiac cycle (i.e., ventricular diastole), exceeds cardiac ejection. The resultant decrement in arterial blood volume thus causes pressure to fall to a minimum, which is diastolic pressure.

Fig. 17.15 illustrates the effect cardiac ejection is zero, and peripheral runoff now greatly of stroke volume on pulse pressure when arterial compliance is constant.

•Fig. 17.12 Pressure-volumerelationshipsofaortasobtainedat autopsyfromhumansindifferentagegroups(denotedbythenumbers•Fig. 17.13 Arterial Systolic, Diastolic, Pulse, and Mean Pres-attherightendofeachofthecurves).Notethatcompliance(ΔV/ΔP)sure. Meanarterialpressure(P)representstheareaunderthearterialdecreaseswithage.(RedrawnfromHallockP,BensonIC.J Clin Invest. pressurecurve(dark red) dividedbythedurationofthecardiaccycle1937;16:595.)(t2− t1).

•Fig. 17.14 Thetwophysicaldeterminantsofpulsepressurearearterialcompliance(Ca)andthechangeinarterialvolume.Thetwophysiologicaldeterminantsofmeanarterialpressure(P)arecardiacoutputandtotalperipheralresistance.

Arterial compliance (Ca), the ratio of blood volume to mean blood pressure (see

Eq. 19.1 ), also affects pulse pressure. This relationship is illustrated in

Fig. 17.16 . When cardiac output and TPR are constant, a decrease in arterial compliance results in an increase in pulse pressure. Diminished arterial compliance also imposes a greater workload on the left ventricle (i.e., increased afterload), even if stroke volume, TPR, and Pa are equal in the two individuals.

As previously discussed, if the heart rate and stroke volume remain constant, an increase in TPR causes Pa to increase.

•Fig. 17.15 Effect of a Change in Stroke Volume on Pulse Pressure in a System in Which Arterial Compliance Remains Constant Over the Prevailing Range of Pressures and Volumes. Alargerincrementinbloodvolume,whereby(V4− V3)> (V2− V1),resultsingreatermeanbloodpressure(PB > PA)andagreaterpulsepressure,sothat(P4− P3)> (P2− P1).

When arterial compliance is constant, an increase in TPR leads to proportional increases in systolic and diastolic pressure so that the pulse pressure is unchanged (Fig. 17.17A ). However, arterial compliance is not linear. As Pa increases and the artery is stressed, compliance decreases (see

Fig.

17.17B ). Because of the decrease in arterial compliance with increased Pa, pulse pressure increases when Pa is elevated.

Effect of Arterial Compliance on Myocardial Energy Consumption

The increased cardiac energy requirement imposed by a rigid arterial system is illustrated in

Fig. 17.18 . In the data depicted in

Fig. 17.18 , the cardiac output from the left ventricle either was allowed to flow through the natural route (the aorta) or was directed through a stiff plastic tube to the peripheral arteries. In this experiment, the TPR values were virtually identical, regardless of which pathway •Fig. 17.16 Foragivenvolumeincrement(V2− V1),reducedarterialcompliance(complianceB[LowCa]< complianceA[HighCa])resultsinincreasedpulsepressure,whereby(P4− P1)> (P3− P2).Pa,meanarterialpressure.

•Fig. 17.17 Comparisonoftheeffectsofagivenchangeinperipheralresistanceonpulsepressure(P)whenthepressure-volumecurveforthearterialsystemiseitherrectilinear(A) orcurvilinear(B). Theincrementinarterialvolumeisthesameforbothconditions;thatis,(V4−V3)=(V2−V1).PressureABVolumeP1V1V2V3V4P3P6P4P2–P5–PressureP1V1V2V3V4P3P6P4P2–P5– •Fig. 17.18 Therelationshipbetweenmyocardialoxygencon-sumption(1mL/100g/beat)andstrokevolume(inmilliliters)inananesthetizeddogwhosecardiacoutputcouldbepumpedbytheleftventricleeitherthroughtheaortaorthroughastiffplastictubetotheperipheralarteries.(ModifiedfromKellyRP,TuninR,KassDA.Circ Res. 1992;71:490.)Plastic tubing Native aorta Stroke volume (mL) 5 0 0.05 O 2 consumption (mL) O2/100 g/beat)0.1 10 15 was selected. The results showed that for any given stroke volume, myocardial oxygen consumption was substantially greater when the blood was diverted through the plastic tubing than when it flowed through the aorta. The increased oxygen consumption indicates that the left ventricle has to expend significantly more energy to pump blood through a less compliant conduit than through a more compliant conduit.

Arterialpulsepressureprovidesvaluableinformationaboutaperson’sstrokevolume,providedthatarterialcomplianceisessentiallynormal.Patientswhohaveseverecongestiveheartfailureorwhohavesufferedaseverehemorrhagearelikelytohaveaverylowarterialpulsepressurebecausetheirstrokevolumesareabnormallysmall.Conversely,individualswithlargestrokevolumes,asinaorticvalveregurgitation,arelikelytohaveanincreasedarterialpulsepressure.Similarly,well-trainedathletesatresttendtohavelargestrokevolumesbecausetheirheartratesareusuallylow.Theprolongedventricularfillingtimesintheseindividualsinducetheventriclestopumpalargestrokevolume,andhencetheirpulsepressureislarge.

The radial stretch of the ascending aorta brought about by left ventricular ejection initiates a pressure wave that is propagated down the aorta and its branches. The pressure wave travels much faster (≈4 to 12 m/second) than the blood itself does. This pressure wave is the “pulse” that can be detected through palpation of a peripheral artery.

Inchronichypertension,aconditioncharacterizedbyapersistentelevationinTPR,thearterialpressure-volumecurveresemblesthatshownin

Fig.17.17B . BecausearteriesbecomesubstantiallylesscompliantwhenParises,anincreaseinTPRcausessystolicpressuretobemoreelevatedthandiastolicpressure.Diastolicpressureiselevatedinsuchindividualsbutordinarilynotmorethan10to40mmHgabovetheaveragenormallevelof80mmHg.Notuncommonly,however,systolicpressureiselevatedby50to100mmHgabovetheaveragenormallevelof120mmHg.

Thevelocityofthepressurewavevariesinverselywitharterialcompliance.Ingeneral,transmissionvelocityincreaseswithage,whichconfirmstheobservationthatthearteriesbecomelesscompliantwithadvancingage.Velocityalsoincreasesprogressivelyasthepulsewavetravelsfromtheascendingaortatowardtheperiphery.Thisincreaseinvelocityreflectsthedecreaseinvascularcomplianceinthemoredistalportionsthaninthemoreproximalportionsofthearterialsystem.

ThePacontourbecomesdistortedasthewaveistransmitteddownthearterialsystem.Thisdistortioninthepressurewavecontourofthehumanarterialtreeisdemonstratedasafunctionofageandofrecordingsitein

Fig.17.19 .Dampingofthehigh-frequencycomponentsofthearterialpulseiscausedlargelybytheviscoelasticpropertiesofthearterialwalls.Thepulsepressurewavetravelsmorerapidlyinolderpeoplethanintheyoungerpeople,asaconsequenceofreducedcompliance.Severalfactors—includingwavereflectionandresonance,vasculartapering,andpressure-inducedchangesintransmissionvelocity—contributetopeakingofthePawave.

Most commonly, blood pressure is estimated indirectly by means of a sphygmomanometer. In hospital intensive care units, needles or catheters may be introduced into the peripheral arteries of patients to measure arterial blood pressure directly by means of strain gauges. When blood pressure readings are taken from the arm, systolic pressure may be estimated by palpation of the radial artery at the wrist (palpatory method). While pressure in the cuff exceeds the systolic level, no pulse is perceived. As pressure falls just below the systolic level (

Fig. 17.20A ), a spurt of blood passes through the brachial artery under the cuff during the peak of systole, and a slight pulse is felt at the wrist.

The auscultatory method is a more sensitive and therefore more precise technique for measuring systolic pressure, and it also enables diastolic pressure to be estimated. The practitioner listens with a stethoscope applied to the skin of the antecubital space over the brachial artery. While the pressure in the cuff exceeds systolic pressure, the brachial artery is occluded, and no sounds are heard (see

Fig.

17.20B ). When the inflation pressure falls just below the systolic level (120 mm Hg in

Fig. 17.20A ), a small spurt of blood escapes the occluding pressure of the cuff, and slight tapping sounds (called Korotkoff sounds) are heard with each heartbeat. The pressure at which the first sound is detected •Fig. 17.19 Pulse Pressure Curves Recorded From Various Sites in the Arterial Trees of Humans at Different Ages. Inthe24-yearold,thearterialpulsedisplaysstrikingchangesinthepulsepressureamplitudeandcontourasitpassesdownthearterialtree.Thepulsepressurewaveinthe68-year-oldshowslittleamplificationandisrelativelyunchangedasthepulsetravelsbecausethereislesswavereflection.(ReproducedbypermissionofHodderEducationfromNicholsWW,O’RourkeM,eds.McDonald’s Blood Flow in Arteries: Theoretical, Experimental and Clinical Principles. 5thed.London:Arnold;2005.) represents systolic pressure. It usually corresponds closely to the directly measured systolic pressure. As the inflation pressure of the cuff continues to fall, more blood escapes under the cuff per beat and the sounds become louder. When the inflation pressure approaches the diastolic level, the Korotkoff sounds become muffled. When the inflation pressure falls just below the diastolic level (80 mm Hg in

Fig. 17.20A ), the sounds disappear; the pressure reading at this point indicates diastolic pressure. The origin of the Korotkoff sounds is related to the discontinuous spurts of blood that pass under the cuff and meet a static column of blood beyond the cuff; the impact and turbulence generate audible vibrations. Once the inflation pressure is less than diastolic pressure, flow is continuous in the brachial artery, and sounds are no longer heard (see

Fig. 17.20C

The Venous System

Veins are elements of the circulatory system that return blood to the heart from tissues. Moreover, veins constitute a very large reservoir that contains up to 70% of the blood in the circulation. The reservoir function of veins makes them able to adjust the volume of blood returning to the heart,

Theankle-brachialindex(ABI)istheratioofsystolicbloodpressuresattheankle(dorsalispedisartery)tothatinthebrachialartery.TheABI,whichisobtainedbysimplemeasurements,isanindicatorofpossibleperipheralarterydisease.TheABIhasalsobeenproposedasapredictorofriskforcardiovascularandcerebrovasculardisease.PeoplewithanormalABIratioof1.1to1.4havealowerincidenceofeithercoronaryorcerebrovasculareventsthandothosewitharatioof0.9orlower.Inaddition,astherateofABIincreaseswithtime,theincidencesofcardiovascularmorbidityandmortalityalsoincrease.

or preload, so that the needs of the body can be matched when cardiac output is altered (see

Chapter 19). This high capacitance is an important property of veins.

The hydrostatic pressure in postcapillary venules is approximately 20 mm Hg, and it decreases to approximately 0 mm Hg in the thoracic venae cavae and right atrium. Hydrostatic pressure in the thoracic venae cavae and right atrium is also termed central venous pressure. Veins are very distensible and have very low resistance to blood flow. Such low resistance allows movement of blood from

Consider that the arterial blood pressure is being measured in a patient whose blood pressure is 120/80 mm Hg. The pressure (represented by the oblique line) in a cuff around the patient’s arm is allowed to fall from greater than 120 mm Hg (point B) to below 80 mm Hg (point C) in about 6 seconds.

When the cuff pressure exceeds the systolic arterial pressure (120 mm Hg), no blood progresses through the arterial segment under the cuff, and no sounds can be detected by a stethoscope bell placed on the arm distal to the cuff.

When the cuff pressure falls below the diastolic arterial pressure, arterial flow past the region of the cuff is continuous, and no sounds are audible. When the cuff pressure is between 120 and 80 mm Hg, spurts of blood traverse the artery segment under the cuff with each heartbeat, and the Korotkoff sounds are heard through the stethoscope.

•Fig. 17.20 A toC, Measurementofarterialbloodpressurewithasphygmomanometer.

peripheral veins to the heart with only small reductions in central venous pressure. Moreover, veins control filtration and absorption by adjusting postcapillary resistance (see the section “ ”) and assist in the cardiovascular adjustments that accompany changes in body position.

The ability of veins to participate in these various functions depends on their distensibility, or compliance. Venous compliance varies with the position in the body in such a way that veins in the lower limb are less compliant than those at or above the level of the heart. Veins in the lower limbs are also thicker than those in the brain or upper limbs. The compliance of veins, like that of arteries, decreases with age, and the vascular thickening that occurs is accompanied by a reduction in elastin and an increase in collagen content.

Variations in venous return are achieved by adjustments in venomotor tone, respiratory activity (see

Chapter 19), and orthostatic stress or gravity.

Gravitational forces influence the amount of blood in the venous system and therefore may profoundly affect cardiac output. For example, soldiers standing at attention for a long time may faint because gravity causes blood to pool in the dependent blood vessels, which reduces cardiac output. Warm ambient temperatures interfere with the compensatory vasomotor reactions, and the absence of muscular activity exaggerates these effects. Gravitational effects are amplified in airplane pilots during pullout from dives. The centrifugal force in the footward direction may be several times greater than the force of gravity. Pilots characteristically black out momentarily during the pullout maneuver as blood is drained from the cephalic regions and pooled in the lower parts of the body.

Some explanations have been advanced to explain the gravitationally induced reduction in cardiac output, but they are inaccurate. For example, it has been argued that when an individual is standing, the force of gravity impedes venous return to the heart from the dependent regions of the body. This explanation is incomplete because it does not account for the gravitational counterforce on the arterial side of the same vascular circuit, and this counterforce facilitates venous return. Moreover, it does not account for the effect of gravity in causing venous pooling. When a person is standing upright, gravity causes blood to accumulate in the lower extremities and distend both the arteries and veins. Because venous compliance is so much greater than arterial compliance, this distention occurs more on the venous side than on the arterial side of the circuit.

The hemodynamic effects of such venous distention (venous pooling) resemble those caused by the hemorrhage of an equivalent volume of blood from the body. When an adult shifts from a supine position to a relaxed standing position, 300 to 800 mL of blood pools in the legs. This pooling may reduce cardiac output by approximately 2 L/min. The compensatory adjustments made to assume a standing position are similar to the adjustments to blood loss (see also ): There are reflex increases in heart rate and cardiac contractility. In addition, both arterioles and veins constrict; the arterioles are affected to a greater extent than are the veins.

When a recumbent person stands but remains at rest, the pressure in the veins rises in the dependent regions of the body (

Fig. 17.21 ). The Pv in the legs increases gradually and does not reach an equilibrium value until almost 1 minute after the person begins standing. The slowness of this rise in Pv is attributable to the venous valves, which allow flow only toward the heart. When a person stands, the valves prevent blood in the veins from falling toward the feet. Hence, the column of venous blood is supported at numerous levels by these valves. Because of these valves, the venous column can be thought of as consisting of many discontinuous segments. However, blood continues to enter the column from many venules and small tributary veins, and the pressure continues to rise. As soon as the pressure in one segment exceeds that in the segment just above it, the intervening valve is forced open. Ultimately, all the valves are open, and the column is continuous.

•Fig. 17.21 Meanpressures(±95%confidenceintervals)inthefootveinsofhumansubjectsduringquietstanding,duringwalking,andduringrunning.(FromStickC,etal.J Appl Physiol. 1992;72:2063.)100 80 60 40 20 0 Venous pressure (mm Hg) Walking (3 km/hr) Walking (6 km/hr) Running (10 km/hr) Standing

Someofthedrugsusedtotreatchronichypertensioninterferewiththereflexadaptationtostanding.Similarly,astronautsexposedtoweightlessnesslosetheiradaptationstogravityafterafewdaysinspace,andtheyexperiencepronounceddifficultieswhentheyfirstreturntoearth.Whensuchastronautsandotherindividualswithimpairedreflexadaptationsstand,theirbloodpressuremaydropsubstantially.Thisresponseiscalledorthostatic hypotension, whichmaycauselightheadednessorfainting.

Thesuperficialveinsintheneckordinarilyarepartiallycollapsedwhenanormalindividualissittingorstanding.Venousreturnfromtheheadisconductedlargelythroughthedeepercervicalveins,whichareprotectedfromcollapsebecausetheyaretetheredtosurroundingstructures.Whencentralvenouspressureisabnormallyelevated,thesuperficialneckveinsaredistended,andtheydonotcollapseevenwhenthepersonsitsorstands.Suchcervicalvenousdistentionisanimportantclinicalsignofcongestiveheartfailure.

Theauxiliarypumpingmechanismgeneratedbyskeletalmusclecontractionsismuchlesseffectiveinpeoplewithvaricoseveinsintheirlegs.Thevalvesinthesedefectiveveinsdonotfunctionproperly,andthereforewhenthelegmusclescontract,thebloodinthelegveinsisforcedinboththeretrogradeandantegradedirections.Thuswhenanindividualwithvaricoseveinsstandsorwalks,Pvintheanklesandfeetisexcessivelyhigh.Theconsequenthighcapillarypressureleadstotheaccumulationofedematousfluidintheanklesandfeet.

Precise measurement reveals that the final level of Pv in the feet during quiet standing is only slightly greater than that in a static column of blood extending from the right atrium to the feet. This finding indicates that the pressure drop caused by blood flow from the foot veins to the right atrium is very small. Because of this very low resistance, all the veins can be viewed as having a common venous compliance in the model of the circulatory system illustrated in . When an individual who has been standing quietly begins to walk, Pv in the legs decreases appreciably (see

Fig. 17.21 ). Because of the intermittent venous compression exerted by the contracting leg muscles, and because of the operation of the venous valves, blood is forced from the veins toward the heart. Hence, muscular contraction lowers the mean Pv in the legs and serves as an auxiliary pump. Furthermore, muscular contraction prevents venous pooling and lowers capillary hydrostatic pressure. In this way, muscular contraction reduces the tendency for edematous fluid to collect in the feet during standing.

The circulatory system supplies the tissues with blood in amounts that meet the body’s requirements for O2 and nutrients. The capillaries, whose walls consist of a single layer of endothelial cells, allow rapid exchange of gases, water, and solutes with interstitial fluid. The muscular arterioles, which are the major resistance vessels, regulate regional blood flow to the capillary beds. Venules and veins serve primarily as collecting channels and storage vessels. The lymphatic system is composed of lymphatic vessels, nodes, and lymphoid tissue. This system collects the fluid and proteins that have escaped from blood and transports them back into the veins for recirculation in blood. In this section, the network of the smallest blood vessels of the body, as well as the lymphatic vessels, are examined in detail.

The microcirculation is defined as the circulation of blood through the smallest vessels of the body: arterioles, capillaries, and venules. Arterioles (5 to 100 µm in diameter) have a thick smooth muscle layer, a thin adventitial layer, and an endothelial lining (see

Fig. 15.2 ). Arterioles give rise directly to capillaries (5 to 10 µm in diameter) or, in some tissues, to metarterioles (10 to 20 µm in diameter), which then give rise to capillaries (

Fig. 17.22 ). Metarterioles can bypass the capillary bed and connect to venules, or they can connect directly to the capillary bed. Arterioles that give rise directly to capillaries regulate flow through these capillaries by constriction or dilation. The capillaries form an interconnecting network of tubes with an average length of 0.5 to 1 mm.

Functional Properties of Capillaries

In metabolically active organs, such as the heart, skeletal muscle, and glands, capillary density is high. In less active tissues, such as subcutaneous tissue or cartilage, capillary density is low. Capillary diameter also varies. Some capillaries have diameters smaller than those of erythrocytes. Passage through these tiny vessels requires the erythrocytes to become temporarily deformed. Fortunately, normal erythrocytes are quite flexible.

Blood flow in capillaries depends chiefly on the contractile state of arterioles. The average velocity of blood flow •Fig. 17.22 Composite Schematic Illustration of the Microcirculation. Thecircular structures onthearterioleandvenulerepresentsmoothmusclefibers,andthebranching solid lines representsympatheticnervefibers.Thearrows indicatethedirectionofbloodflow.AV,arteriovenous.

in capillaries is approximately 1 mm/second; however, it can vary from zero to several millimeters per second in the same vessel within a brief period. These changes in capillary blood flow may be random or rhythmic. The rhythmic oscillatory behavior of capillaries is caused by contraction and relaxation (vasomotion) of the precapillary vessels (i.e., the arterioles and small arteries).

Vasomotion is an intrinsic contractile behavior of vascular smooth muscle and is independent of external input. Changes in transmural pressure (intravascular pressure minus extravascular pressure) also influence the contractile state of precapillary vessels. An increase in transmural pressure, caused either by an increase in Pv or by dilation of arterioles, results in contraction of the terminal arterioles. A decrease in transmural pressure causes precapillary vessel relaxation. Humoral and possibly neural factors also affect vasomotion. For example, when increased transmural pressure causes the precapillary vessels to contract, the contractile response can be overridden and vasomotion abolished. This effect is accomplished by metabolic (humoral) factors when the O2 supply becomes too low for the requirements of parenchymal tissue, as occurs in skeletal muscle during exercise.

Although a reduction in transmural pressure relaxes the terminal arterioles, blood flow through the capillaries cannot increase if the reduction in intravascular pressure is caused by severe constriction of the upstream microvessels. Large arterioles and metarterioles also exhibit vasomotion. However, their contraction usually does not completely occlude the lumen of the vessel and arrest blood flow, whereas contraction of the terminal arterioles may arrest blood flow. Thus the flow rate in capillaries may be altered by contraction and relaxation of small arteries, arterioles, and metarterioles.

Blood flow through the capillaries has been called nutritional flow because it provides for exchange of gases and solutes between blood and tissue. Conversely, blood flow that bypasses the capillaries as it passes from the arterial to the venous side of the circulation via metarterioles has been termed nonnutritional, or shunt, flow (see

Fig. 17.22 ). In some areas of the body (e.g., fingertips, ears), true AV shunts exist (see

Fig. 17.37 ). However, in many tissues, such as muscle, anatomical shunts are lacking. Even in the absence of these shunts, nonnutritional flow can occur. In tissues with metarterioles, nonnutritional flow may be continuous from arteriole to venule during low metabolic activity, when many precapillary vessels are closed. When metabolic activity increases in these tissues, more precapillary vessels open to allow capillary perfusion.

True capillaries lack smooth muscle and are therefore incapable of active constriction. Nevertheless, the endothelial cells that form the capillary wall contain actin and myosin, and they can alter their shape in response to certain chemical stimuli.

Because of its narrow lumen (i.e., small radius), a thin-walled capillary can withstand high internal pressures without bursting. This property can be explained in terms of the law of Pierre-Simon Laplace:

Equation 17.16

T = tension in the vessel wall r = radius of the vessel

Laplace’s equation applies to very thin-walled vessels, such as capillaries. Wall tension opposes the distending force (ΔPr) that tends to pull apart a theoretical longitudinal slit in the vessel (

Fig. 17.23 ). Transmural pressure in a blood vessel in vivo is essentially equal to intraluminal pressure because extravascular pressure is generally negligible. To calculate wall tension, pressure in mm Hg is converted to dynes per square centimeter according to the equation P = hρg, where h is the height of an Hg column in centimeters, ρ is the density of Hg in g/cm3, and g is gravitational acceleration in cm/s2. For a capillary with a pressure of 25mmHg and a radius of 5 × 10−4 cm, the pressure (2.5 cm Hg × 13.6 g/cm3 × 980 cm/sec2) is 3.33 × 104 •Fig. 17.23 Diagram of a Small Blood Vessel to Illustrate the Law of Laplace. T=Pr,whereP=intraluminalpressure,r=radiusofthevessel,andT=walltensionastheforceperunitlengthtangentialtothevesselwall.Walltensionpreventsrupturealongatheoreticallongitudinalslitinthevessel.

dyne/cm2. Wall tension is then 16.7 dyne/cm. For an aorta with a pressure of 100 mm Hg and a radius of 1.5 cm, wall tension is 2 × 105 dyne/cm. Thus at the pressures normally found in the aorta and capillaries, the wall tension of the aorta is approximately 12,000 times greater than that of the capillaries. In a person standing quietly, capillary pressure in the feet may reach 100 mm Hg. Even under such conditions, capillary wall tension increases to a value that is still only one three-thousandth of the wall tension in the aorta at the same internal pressure.

The diameter of the resistance vessels (arterioles) is determined from the balance between the contractile force of the vascular smooth muscle and the distending force produced by intraluminal pressure. The greater the contractile activity of the vascular smooth muscle of an arteriole, the smaller its diameter. In small arterioles, contraction can continue to the point at which the vessel is completely occluded. Occlusion is caused by infolding of the endothelium and by trapping of blood cells in the vessel.

With a progressive reduction in intravascular pressure, vessel diameter decreases (as does vessel wall tension, according to the law of Laplace) and blood flow eventually ceases, although pressure within the arteriole is still greater than tissue pressure. The pressure that causes flow to cease has been called the critical closing pressure, and its mechanism is still unclear. The critical closing pressure is low when vasomotor activity is reduced by inhibition of sympathetic nerve activity in the vessel and is increased when vasomotor tone is enhanced by activation of the vascular sympathetic nerve fibers.

Iftheheartbecomesgreatlydistendedwithbloodduringdiastole,asmayoccurwithcardiacfailure,itfunctionslessefficiently.Toejectagivenvolumeofbloodperbeat,moreenergyisrequired(walltensionmustbegreater)forthedistendedheartthanforanormalundilatedheart.ThelessefficientpumpingofadistendedheartisanexampleofLaplace’slaw,accordingtowhichthetensioninthewallofavesselorchamber(inthiscase,theventricles)equalstransmuralpressure(pressureacrossthewall,ordistendingpressure)multipliedbytheradiusofthevesselorchamber.Laplace’srelationshipordinarilyappliestoinfinitelythin-walledvessels,butitcanbeappliedtothespherical,dilatedheartifcorrectionismadeforwallthickness.Undertheseconditions,theequationisσ=ΔPr/2w,whereσ= wallstress,ΔP= transmuralpressuredifference,r= radius,andw= wallthickness.

Vasoactive Role of the Capillary Endothelium

The endothelium is an important source of substances that cause contraction or relaxation of vascular smooth muscle. One of these substances is prostacyclin, or prostaglandin I2 (PGI2). PGI2 can relax vascular smooth muscle via an increase in cyclic adenosine monophosphate (cAMP;

Fig. 17.24 ). PGI2 is formed in the endothelium from muscle; its action is not endothelium mediated. Vasodilator agents such as adenosine, H+, CO2, and K+ may be released from parenchymal tissue and act locally on resistance vessels (see

Fig. 17.24

Lumen Endothelium Interstitial space Relaxation Basement membrane ATP ADP AMP Adenosine Parenchymal tissue cAMP RelaxationcGMP NO NP ACh GCyc L-arg. Vascular smooth muscle Cyc OxPGI2Syn PGI2 AA H+, CO2, K+

Injurytotheendotheliumofbloodvesselsprecedesatherosclerosis.Theprotective(antiatherogenic)effectoftheendotheliumresidesinseveralproperties.Forexample,theendotheliumregulatesadhesionofleukocytestothevesselwall,suppressestheproliferationofvascularsmoothmusclecells,maintainsavesselliningthatresiststheformationofthrombi,andregulatesvascularsmoothmuscletone.AllthesefunctionsinvolvetheactionofNO.Asindicatedpreviously,productionofNOisregulatedbymanysubstancesandbyshearstressactingonthevesselwall.

•Fig. 17.24 Endothelium-Mediated and Non–Endothelium-Mediated Vasodilation. Prostacyclin(PGI2)isformedfromarachidonicacid(AA)bytheactionofcyclooxygenase(CycOx)andprostacyclinsynthase(PGI2Syn)intheendotheliumandelicitsrelaxationoftheadjacentvascularsmoothmuscleviaincreasesincAMP.Stimulationoftheendothelialcellswithacetylcholine(ACh)orotheragents(seetext)resultsintheformationandreleaseofanendothelium-derivedrelaxingfactoridentifiedasnitricoxide(NO).NOstimulatesguanylylcyclase(GCyc)toincreasecGMPinthevascularsmoothmuscletoproducerelaxation.Thevasodilatornitroprusside(NP)actsdirectlyonvascularsmoothmuscle.Substancessuchasadenosine,H+ ,CO2,andK+ canariseintheparenchymaltissueandelicitvasodilationbydirectactiononvascularsmoothmuscle.ADP,adenosinediphosphate;AMP,adenosinemonophosphate;ATP,adenosinetriphosphate;cAMP,cyclicadenosinemonophosphate;cGMP,cyclicguanosinemonophosphate;L-arg,L-arginine.

arachidonic acid, and the process is catalyzed by PGI2 synthase. The mechanism that triggers synthesis of PGI2 is not known. However, PGI2 may be released by an increase in shear stress caused by accelerated blood flow. The primary function of PGI2 is to inhibit platelet adherence to the endothelium and platelet aggregation and thus prevent intravascular clot formation. PGI2 also causes relaxation of vascular smooth muscle.

Of far greater importance in endothelium-mediated vascular dilation is the formation and release of nitric oxide (NO), a component of endothelium-derived relaxing factor (see

Fig. 17.24 ). When endothelial cells are stimulated by acetylcholine or other vasodilator agents (e.g., adenosine triphosphate [ATP], bradykinin, serotonin, substance P, histamine), NO is released. These agents do not cause vasodilation in blood vessels lacking the endothelium. NO (synthesized from L-arginine) activates guanylyl cyclase in vascular smooth muscle to increase the concentration of cyclic guanosine monophosphate (cGMP), which produces relaxation by decreasing myofilament sensitivity to [Ca++]. Release of NO can be stimulated by the shear stress of blood flow on the endothelium. The drug nitroprusside also increases cGMP by acting directly on vascular smooth

Acetylcholine also stimulates the release of an endothelium-dependent hyperpolarizing factor that underlies the relaxation of adjacent smooth muscle. Although arachidonic acid metabolites have been suggested, the factor remains unknown. Moreover, how the factor reaches vascular smooth muscle (diffusion through the extracellular space or passage via myoepithelial junctions) is unclear. Nevertheless, there are diverse ways by which endothelial cells communicate with vascular smooth muscle.

The endothelium can also synthesize endothelin, a potent vasoconstrictor peptide. Endothelin affects vascular tone and blood pressure and may be involved in pathological states, including atherosclerosis, pulmonary hypertension, congestive heart failure, and renal failure.

Passive Role of the Capillary Endothelium

Solvent and solute move across the capillary endothelial wall by three processes: diffusion, filtration, and pinocytosis. Diffusion is the most important process for transcapillary exchange, and pinocytosis is the least important.

Diffusion. Under normal conditions, only approximately 0.06 mL of water per minute moves across the capillary wall per 100 g of tissue as a result of filtration. In contrast, 300 mL of water per minute per 100 g of tissue moves across the capillary wall by diffusion. Thus diffusion is the key factor in providing exchange of gases, substrates, and waste products between capillaries and tissue cells.

The process of diffusion is described by Fick’s law (see also

Equation 17.17

J = quantity of a substance moved per unit time

A = cross-sectional area of the diffusion pathway

ΔC = concentration gradient of the solute •Fig. 17.25 Flowand Diffusion-Limited Transport From Capillaries (Cap) to Tissue. A, Flow-limitedtransport.Thesmallestwater-solubleinerttracerparticles(blue dots) reachnegligibleconcentrationsafterpassingonlyashortdistancedownthecapillary.Largerparticles(brown dots) withsimilarpropertiestravelfartheralongthecapillarybeforereachinganinsignificantintracapillaryconcentration.Bothsubstancescrosstheinterstitialfluid(ISF)andreachtheparenchymaltissue(Cell).Becauseoftheirsize,moreofthesmallerparticlesaretakenupbythetissuecells.Thelargestparticles(red circles) cannotpenetratethecapillaryporesandhencedonotescapefromthecapillarylumenexceptbypinocytoticvesicletransport.Anincreaseinthevolumeofbloodfloworanincreaseincapillarydensityincreasestissuesupplyofthediffusiblesolutes.Notethatcapillarypermeabilityisgreateratthevenousendofthecapillary(alsointhevenule,notshown)becauseofthelargernumberofporesinthisregion.B, Diffusion-limitedtransport.Whenthedistancebetweencapillariesandparenchymaltissueislargeasaresultofedemaorlowcapillarydensity,diffusionbecomesalimitingfactorinsolutetransportfromcapillarytotissue,evenathighratesofcapillaryflow.

For diffusion across a capillary wall, Fick’s law can also be expressed as

Equation 17.18

P = capillary permeability by the substance

Co = concentration of the substance outside the

Ci = concentration of the substance inside the capillary

The PS product provides a convenient expression of available capillary surface area because the intrinsic permeability of the capillary is rarely altered much under physiological conditions. However, in pathological conditions, as with a bee sting, capillary permeability may be altered.

In capillaries, diffusion of lipid-insoluble molecules is restricted to water-filled channels or pores. Movement of solute across the capillary endothelium is complex and involves corrections for attractions between solute and solvent molecules, interactions between solute molecules, pore configuration, and charge on the molecules in relation to charge on the endothelial cells. Such solute motion is not simply a matter of random thermal movement of molecules that seemingly run down a concentration gradient. For small molecules, such as water, NaCl, urea, and glucose, the capillary pores offer little restriction to diffusion (i.e., they have a low reflection coefficient; see the section “ ”). Diffusion of these substances is so rapid that the mean concentration gradient across the capillary endothelium is extremely small. The larger the lipid-insoluble molecules are, the more restricted is their diffusion through capillaries. Diffusion eventually becomes minimal when the molecular weight of the molecules exceeds approximately 60,000. With small molecules, the only limitation to net movement across the capillary wall is the rate at which blood flow transports the molecules to the capillary. Transport of these molecules is said to be flow limited.

With flow-limited small molecules, the concentration of the molecule in blood reaches equilibrium with its concentration in interstitial fluid at a location near the origin of the capillary from its parent arteriole. Its concentration falls to negligible levels near the arterial end of the capillary (

Fig.

17.25A ). If the flow is large, the small molecule can still be present at a distant locus downstream in the capillary. A somewhat larger molecule moves farther along the capillary before it reaches an insignificant concentration in blood. Furthermore, the number of still larger molecules that enter the arterial end of the capillary but cannot pass through the capillary pores equals the number that leaves the venous end of the capillary (see

Fig. 17.25A

With large molecules, diffusion across the capillaries becomes the limiting factor (diffusion limited); that is, the permeability of a capillary to a large solute molecule limits its transport across the capillary wall. Diffusion of small lipid-insoluble molecules is so rapid that diffusion limits blood-tissue exchange only when distances between capillaries and parenchymal cells are great (e.g., as in tissue edema or very low capillary density; see

Fig. 17.25B

Movement of lipid-soluble molecules across the capillary wall is not limited to capillary pores (only ≈0.02% of the capillary surface); it also occurs directly through the lipid membranes of the entire capillary endothelium. Consequently, lipid-soluble molecules move rapidly between blood and tissue. The degree of lipid solubility (oil-to-water partition coefficient) provides a good index of the ease of transfer of lipid molecules through the capillary endothelium.

Both O2 and CO2 are lipid soluble, and they readily pass through endothelial cells. Calculations based on (1) the diffusion coefficient for O2, (2) capillary density and diffusion distances, (3) blood flow, and (4) tissue O2 consumption indicate that the O2 supply of normal tissue at rest and during activity is not limited by diffusion or by the number of open capillaries.

Measurements of the partial pressure of O2 (PO2) and O2 saturation of blood in microvessels indicate that in many tissues, O2 saturation at the entrance of capillaries has decreased to approximately 80% as a result of diffusion of O2 from arterioles and small arteries. Moreover, CO2 loading and the resulting intravascular shifts in the oxyhemoglobin dissociation curve occur in the precapillary vessels. Hence, in addition to gas exchange at the capillaries, O2 and CO2 pass directly between adjacent arterioles and venules and possibly between arteries and veins (countercurrent exchange). The countercurrent exchange represents a diffusional shunting of gas away from the capillaries; this shunting may limit the supply of O2 to the tissue at low blood flow rates.

Capillary Filtration. The permeability of the capillary endothelial membrane is not uniform. For example, liver capillaries are quite permeable, and albumin escapes from them at a rate several times greater than that from the less permeable muscle capillaries. Furthermore, permeability is not uniform along the length of the capillary. The venous ends are more permeable than the arterial ends, and permeability is greatest in the venules, a property attributed to the greater number of pores in these regions.

Where does filtration occur? Some water passes through the capillary endothelial cell membranes, but most flows through apertures (pores) in the endothelial walls of the capillaries (

Figs. 17.26 17.27 ). The pores in skeletal and cardiac muscle capillaries have diameters of approximately 4 nm. There are clefts between adjacent endothelial cells in cardiac muscle, and the gap at the narrowest point is approximately 4 nm. The clefts (pores) are sparse and represent only approximately 0.02% of the capillary surface area. Pores are absent in cerebral capillaries, where the blood-brain barrier blocks the entry of many small molecules.

In addition to clefts, some of the more porous capillaries (e.g., those in the kidneys and intestines) contain fenestrations 20 to 100 nm wide, whereas other capillaries (e.g., those in the liver) have a discontinuous endothelium (see

Fig. 17.27 ). Fenestrations and discontinuous endothelia allow the passage of molecules that are too large to pass through the intercellular clefts of the endothelium.

The direction and magnitude of water movement across the capillary wall can be estimated as the algebraic sum of the hydrostatic and osmotic pressure that exists across the wall. An increase in intracapillary hydrostatic pressure favors movement of fluid from the vessel interior to the interstitial space, whereas an increase in the concentration

CHAPTER 17 Properties of the Vasculature of osmotically active particles within vessels favors movement of fluid into the vessels from the interstitial space (

Fig. 17.28

Hydrostatic Forces. Hydrostatic pressure (blood pressure) within capillaries is not constant. Instead, it depends on arterial and venous pressure and on precapillary resistance (in the arterioles) and postcapillary resistance (in the venules and small veins). An increase in arterial or venous pressure elevates capillary hydrostatic pressure, whereas a reduction in arterial or venous pressure has the opposite effect. An increase in arteriolar resistance or closure of arteries reduces capillary pressure, whereas a greater resistance to flow in venules and veins increases capillary pressure.

Hydrostatic pressure is the principal force in capillary filtration. A given change in Pv produces a greater effect on capillary hydrostatic pressure than does the same change in Pa. Approximately 80% of an increase in Pv is transmitted back to the capillaries.

Capillary hydrostatic pressure (Pc) varies from tissue to tissue. Average values, obtained from direct measurements in human skin, are approximately 32 mm Hg at the arterial end of capillaries and approximately 15 mm Hg at the venous end of capillaries at the level of the heart (see

Fig. 17.28 ). As discussed previously, when a person stands, hydrostatic pressure increases in the legs and decreases in the head.

Tissue pressure, or, more specifically, interstitial fluid pressure (Pi) outside the capillaries, opposes capillary filtration. The difference between Pc and Pi constitutes the driving force for filtration. Normally, Pi is close to zero, and so Pc essentially represents the hydrostatic driving force.

Osmotic Forces. The key factor that restrains fluid loss from capillaries is the osmotic pressure of plasma proteins (such as albumin). This osmotic pressure is called colloid osmotic pressure or oncotic pressure (πp). The total osmotic pressure of plasma is approximately 6000 mm Hg (reflecting the presence of electrolytes and other small molecules, as well as plasma proteins), whereas oncotic pressure is only approximately 25 mm Hg. This low level of oncotic pressure is an important factor in fluid exchange across the capillary because plasma proteins are essentially confined to the intravascular space, whereas electrolytes are virtually equal in concentration on both sides of the capillary endothelium. The relative permeability of solute by water influences the actual magnitude of osmotic pressure. The reflection coefficient (σ) is the relative impediment to the passage of a substance through the capillary membrane. The reflection coefficient of water is 0, and that of albumin (to which the endothelium is essentially impermeable) is 1. Filterable solutes have reflection coefficients between 0 and 1. In addition, different tissues have different reflection coefficients for the same molecule. Hence, movement of a given solute across the endothelial wall varies with the tissue. The actual oncotic pressure of the plasma (πp) is defined by the following equation (see also

Equation 17.19 1 µm 0.5 µm 0.1 µm0.1 µm GJTJ CT Nu PC V BM * V A B C D •Fig. 17.26 A, Electronmicrographofacross-sectionofacapillaryinamouseventricle.Theluminaldiameterisapproximately4µm.Inthissection,thecapillarywallisformedbyasingleendothelialcell(Nu,endothelialnucleus).Thethinpericapillaryspaceisoccupiedbyapericyte(PC)andaconnectivetissue(CT)cell(“fibroblast”),whichformsafunctionalcomplex(arrow)withitself.V,plasmalemmalvesicles.B, DetailoftheendothelialcellinA showingplasmalemmalvesicles(V)attachedtotheendothelialcellsurface.Thesevesiclesareespeciallyprominentinvascularendotheliumandareinvolvedintransportofsubstancesacrossthebloodvesselwall.Notethecomplexalveolarvesicle(asterisk). BM,basementmembrane.C, Junctionalcomplexinacapillaryofamouseheart.“Tight”junctions(TJ)typicallyforminthesesmallbloodvesselsandappeartoconsistoffusionsbetweenapposedendothelialcellsurfacemembranes.D, Interendothelialjunctioninamusculararteryofapapillarymuscle.Althoughtightjunctionssimilartothoseofcapillariesarefoundintheselargebloodvessels,extensivejunctionsthatresemblegapjunctionsintheintercalateddisksbetweenmyocardialcellsoftenappearinarterialendothelium(exampleshownatGJ).

CHAPTER 17  Properties of the Vasculature 365

Junction of two Discontinuousendothelial cells endothelium • Fig. 17.27 Illustration of an electron micrograph of a capillary in cross-section.

With prolonged standing, particularly when associated with elevation of Pv in the legs (such as that caused by pregnancy and congestive heart failure), filtration across capillaries is greatly enhanced, exceeding the capacity of the lymphatic system to remove the filtrate from the interstitial space and thus leading to edema.

The concentration of plasma proteins may change in several pathological states and thus alter the osmotic force and movement of fluid across the capillary membrane. The plasma protein concentration is increased in conditions of dehydration (e.g., water deprivation, prolonged sweating, severe vomiting, diarrhea). In this condition, less water moves by osmotic force from the tissues to the vascular compartment, thereby decreasing the volume of the interstitial fluid. In contrast, the plasma protein concentration is reduced in some renal diseases because of its loss in urine, and edema may occur.

When capillary injury is extensive, as in severe burns, intravascular fluid and plasma protein leak into the interstitial space in the damaged tissues. The protein that escapes from the vessel lumen increases the oncotic pressure of the interstitial fluid. This greater osmotic force outside the capillaries leads to additional fluid loss and possibly to severe dehydration.

Balance of Hydrostatic and Osmotic Forces. The pressure sure and the role of these forces in regulating fluid passage across the capillary endothelium were expounded by Frank

Starling in 1896. This relationship constitutes Starling’s • Fig. 17.28 Schematic representation of the factors responsible for filtration and absorption across the capillary wall and the formation of lymph.

Albumin is the most important plasma protein that determines oncotic pressure. Its molecular weight is 69,000 D. Albumin exerts an osmotic force greater than can be accounted for solely on the basis of its concentration in plasma. Therefore, it cannot be replaced on a mole-bymole basis by inert substances of appropriate molecular size, such as dextran. This additional osmotic force becomes disproportionately great at high concentrations of albumin (as in plasma), and this force is weak to absent in dilute solutions of albumin (as in interstitial fluid). The reason for this activity of albumin is its negative charge at normal blood pH and the attraction and retention of cations (principally Na+) in the vascular compartment (Gibbs-Donnan effect).

hypothesis. It can be expressed as follows:

Equation 17.20 k = filtration constant for the capillary membrane πi= interstitial fluid oncotic pressure Filtration occurs when the algebraic sum is positive; absorption occurs when it is negative.

Traditionally, filtration was thought to occur at the arterial end of the capillary, and absorption was thought to occur at its venous end because of the gradient of hydrostatic pressure along the capillary. This scheme is true for an idealized capillary (see

Fig. 17.28 ). However, in well-perfused capillaries, arteriolar vasoconstriction can reduce Pc in such a way that absorption at the arteriolar end can occur transiently. With continued vasoconstriction, absorption diminishes with time because Pi increases. In some vascular beds (e.g., the renal glomerulus), hydrostatic pressure in the capillary is high enough to cause filtration along the entire length of the capillary. In other vascular beds (e.g., the intestinal mucosa), the hydrostatic and oncotic forces are such that absorption occurs along the whole capillary.

In the steady-state Pa, Pv, postcapillary resistance, hydrostatic and oncotic pressure of interstitial fluid, and oncotic pressure of plasma are relatively constant. Hence, in the normal state, filtration and absorption across the capillary wall are well balanced. However, a change in precapillary resistance influences fluid movement across the capillary wall. Vasoconstriction reduces net filtration, and vasodilation increases filtration.

Inthelungs,meancapillaryhydrostaticpressureisonlyapproximately8mmHg(see ).Becauseplasmaoncoticpressureis25mmHgandpressureoftheinterstitialfluidinthelungsisapproximately15mmHg,thenetforceslightlyfavorsnetabsorption(i.e.,fluidleavestheinterstitialspace).Despitenetabsorption,pulmonarylymphisformed.Thislymphconsistsoffluidthatisosmoticallywithdrawnfromthecapillariesbythesmallamountofplasmaproteinthatescapesthroughthecapillaryendothelium.Inpathologicalconditions,suchasleftventricularfailureormitralvalvestenosis,pulmonarycapillaryhydrostaticpressuremayexceedplasmaoncoticpressure.Whenthisoccurs,itmaycausepulmonaryedema,aconditioninwhichexcessivefluidaccumulatesinthepulmonaryinterstitium.Thisfluidaccumulationseriouslyinterfereswithgasexchangeinthelungs.

Capillary Filtration Coefficient. The rate of fluid movement (Qf) across the capillary membrane depends not only on the algebraic sum of the hydrostatic and osmotic forces across the endothelium (ΔP) but also on the area (Am) of the capillary wall available for filtration, the distance (Δx) across the capillary wall, the viscosity (η) of the filtrate, and the filtration constant (k) of the membrane. These factors may be expressed as follows:

Equation 17.21

This expression, which describes the flow of fluid through the membrane pores, is essentially Poiseuille’s law for flow through tubes.

Because the thickness of the capillary wall and the viscosity of the filtrate are relatively constant, they can be included in the filtration constant k. If the area of the capillary membrane is not known, the rate of filtration can be expressed per unit weight of tissue. Hence, the equation can be simplified as

Equation 17.22 where kt is the capillary filtration coefficient for a given tissue and the units for Qf are milliliters per minute per 100 g of tissue.

In any given tissue, the filtration coefficient per unit area of capillary surface, and hence capillary permeability, is not changed by various physiological conditions, such as arteriolar dilation and capillary distention, or by such adverse conditions as hypoxia, hypercapnia, or reduced pH. When capillaries are injured (as by toxins or severe burns), significant amounts of fluid and protein leak out of the capillaries into the interstitial space. This increase in capillary permeability is reflected by an increase in the filtration coefficient.

Because capillary permeability is constant under normal conditions, the filtration coefficient can be used to determine the relative number of open capillaries (i.e., the capillary surface area available for filtration in tissue). For example, the increased metabolic activity of contracting skeletal muscle relaxes the precapillary resistance vessels and hence opens more capillaries. This process, called capillary recruitment, increases the filtering surface area.

Disturbances in Hydrostatic-Osmotic Balance. Relatively small changes in Pa may have little effect on filtration. The change in pressure may be countered by adjustments in precapillary resistance vessels (autoregulation; see ) so that hydrostatic pressure remains constant in the open capillaries. However, a severe reduction in Pa usually evokes arteriolar constriction mediated by the sympathetic nervous system. This response may occur in hemorrhage, and it is often accompanied by a fall in Pv. These changes reduce capillary hydrostatic pressure. However, the lowering of blood pressure in hemorrhage causes a decrease in blood flow (and hence in O2 supply) to the tissue, with the result that vasodilator metabolites accumulate and relax the arterioles. Precapillary vessel relaxation also occurs because of the reduced transmural pressure (autoregulation; see ). Consequently, absorption predominates over filtration, and fluid moves from the interstitium into the capillary. These responses to hemorrhage constitute one of the compensatory mechanisms used by the body to restore blood volume (see

An increase in Pv alone, as occurs in the feet when a person stands up, would elevate capillary pressure and enhance filtration. However, the increase in transmural pressure closes precapillary vessels (myogenic mechanism; see ), and hence the capillary filtration coefficient actually decreases. This reduction in capillary surface available for filtration prevents large amounts of fluid from leaving the capillaries and entering the interstitial space.

In a healthy individual, the filtration coefficient (kt) for the whole body is approximately 0.006 mL/minute/100 g of tissue/mm Hg. For a 70-kg man, an elevation in Pv of 10 mm Hg for 10 minutes would increase filtration from capillaries by 420 mL. Edema does not usually occur because the fluid is returned to the vascular compartment by the lymphatic vessels. When edema develops, it usually appears in the dependent parts of the body, where the hydrostatic pressure is greatest, but its location and magnitude are also determined by the type of tissue. Loose tissues, such as the subcutaneous tissue around the eyes or in the scrotum, are more prone than firm tissues, as in a muscle, or encapsulated structures, as in a kidney, to collect larger quantities of interstitial fluid.

Pinocytosis. Some transfer of substances across the capillary wall can occur in tiny pinocytotic vesicles. These vesicles (see

Figs. 17.26 17.27 ), formed by the pinching off of the endothelial cell membrane, can take up substances on one side of the capillary wall, move them across the cell by kinetic energy, and deposit their contents on the other side. This process is termed transcytosis. The amount of material transported in this way is very small in relation to that moved by diffusion. However, pinocytosis may be responsible for the movement of large (30-nm) lipid-insoluble molecules between blood and interstitial fluid. The number of pinocytotic vesicles in endothelium varies among tissues (amount in muscle > amount in lung > amount in brain), and the number increases from the arterial end to the venous end of the capillary.

The terminal vessels of the lymphatic system consist of a widely distributed, closed-end network of highly permeable lymphatic capillaries. These lymphatic capillaries resemble blood capillaries, with two important differences: tight junctions are not present between endothelial cells, and fine filaments anchor lymphatic vessels to the surrounding connective tissue. With muscular contraction, these fine strands pull on the lymphatic vessels to open spaces between the endothelial cells and enable the entrance of protein and large particles into the lymphatic vessels. The lymphatic capillaries drain into larger vessels that finally enter the right and left subclavian veins, where they connect with the respective internal jugular veins.

Only cartilage, bone, epithelia, and tissues of the central nervous system lack lymphatic vessels. These vessels return the plasma capillary filtrate to the circulation. This task is accomplished by means of tissue pressure, and it is facilitated by intermittent skeletal muscle activity, lymphatic vessel contractions, and an extensive system of one-way valves. In this regard, lymphatic vessels resemble veins, although the larger lymphatic vessels do have thinner walls than do the corresponding veins, and they contain only a small amount of elastic tissue and smooth muscle.

The volume of fluid transported through the lymphatic vessels in 24 hours is approximately equal to the body’s total plasma volume. The lymphatic vessels return all of the proteins filtered back to the blood; these proteins account for approximately one fourth to half of the circulating plasma proteins in the blood. The lymphatic vessels are the only means by which the protein that leaves the vascular compartment can be returned to blood. Net backward diffusion of protein into the capillaries cannot occur against the large protein concentration gradient. If the protein were not removed by the lymph vessels, it would accumulate in interstitial fluid and act as an oncotic force that draws fluid from the blood capillaries and produces edema.

In addition to returning fluid and protein to the vascular bed, the lymphatic system filters the lymph at the lymph nodes and removes foreign particles such as bacteria. The

CHAPTER 17 Properties of the Vasculature largest lymphatic vessel, the thoracic duct, not only drains the lower extremities but also returns the protein lost through the permeable liver capillaries. Moreover, the thoracic duct carries substances absorbed from the gastrointestinal tract. The principal substance is fat, in the form of chylomicrons.

Lymph flow varies considerably. The flow from resting skeletal muscle is almost nil, and it increases during exercise in proportion to the degree of muscular activity. It is increased by any mechanism that enhances the rate of blood capillary filtration; such mechanisms include increased capillary pressure or permeability and decreased plasma oncotic pressure. When the volume of interstitial fluid exceeds the drainage capacity of the lymphatic vessels, or when the lymphatic vessels become blocked, interstitial fluid accumulates and gives rise to clinical edema.

Functional Anatomy of Coronary Vessels

The right and left coronary arteries arise at the root of the aorta behind the right and left cusps of the aortic valve, respectively. These arteries provide the entire blood supply to the myocardium. The right coronary artery supplies mainly the right ventricle and atrium. The left coronary artery, which divides near its origin into the anterior descending and the circumflex branches, supplies mainly the left ventricle and atrium. There is some overlap between the regions supplied by the left and right arteries. In humans, the right coronary artery is dominant (supplying most of the myocardium) in approximately 50% of individuals. The left coronary artery is dominant in another 20%, and the flow delivered by each main artery is approximately equal in the remaining 30%. The epicardial distribution of the coronary arteries and veins is illustrated in

Fig. 17.29

Coronary arterial blood passes through the capillary beds; most of it returns to the right atrium through the coronary sinus. Of the coronary arteries, epicardial arteries are largest (2 to 5 mm in diameter), large arterioles are medium in size (1.0 to 0.5 mm in diameter), and small arterioles are smallest (<0.1 mm in diameter). Some of the coronary venous blood reaches the right atrium via the anterior coronary veins. In addition, vascular communications directly link the myocardial vessels with the cardiac chambers; these communications are the arteriosinusoidal, arterioluminal, and thebesian vessels. The arteriosinusoidal channels consist of small arteries or arterioles that lose their arterial structure as they penetrate the chamber walls, where they divide into irregular, endothelium-lined sinuses. These sinuses anastomose with other sinuses and with capillaries, and they communicate with the cardiac chambers. The arterioluminal vessels are small arteries or arterioles that open directly into the atria and ventricles. The thebesian vessels are small veins that connect capillary beds directly with the cardiac chambers and also communicate with the cardiac veins. All the minute vessels of the myocardium communicate in the form of an extensive

Posterior descending branch of right coronary artery • Fig. 17.29 Illustrations of the anterior and posterior surfaces of the heart, depicting the location and distribution of the principal coronary vessels.

plexus of subendocardial vessels. However, the myocardium does not receive significant nutritional blood flow directly from the cardiac chambers.

The primary factor responsible for perfusion of the myocardium is aortic pressure. Changes in aortic pressure generally evoke parallel directional changes in coronary blood flow. This is caused in part by changes in coronary perfusion pressure. However, the major factor in the regulation of coronary blood flow is a change in arteriolar resistance engendered by changes in the metabolic activity of the heart. When the metabolic activity of the heart increases, coronary resistance decreases; when cardiac metabolism decreases, coronary resistance increases (see

Blood flow in the heart is autoregulated. If a cannulated coronary artery is perfused by blood from a pressure-controlled reservoir, perfusion pressure can be altered without a change in aortic pressure and cardiac work. The relationship between initial and steady-state blood flow is shown in the experiment depicted in

Fig. 17.30 . This is an example of autoregulation of blood flow, which is mediated by a myogenic mechanism in large and small arterioles ( ). The metabolic activity of cardiac muscle in small arterioles and the endothelium modulate autoregulation. The coronary circulation adjusts serial resistances within the microvasculature thereby adapting blood flow to O2 requirements. Blood pressure is kept within narrow 0.2 0.4 0.6 0.8 1 Time (sec) •Fig. 17.30 Pressure-Flow Relationships in the Coronary Vascular

Bed. Asaorticpressurewasheldconstant,thecardiacoutput,heart rate,andcoronaryarteryperfusionpressurewereabruptlyincreased ordecreasedfromthecontrollevel,whichisindicatedbythepointatwhichthetwolinescross.Thesolid circles representtheflowsthat resultedimmediatelyafterthechangeinperfusionpressure;theopen circles representthesteady-stateflowsatthenewpressures.Thereisatendencyforflowtoreturntowardthecontrollevel(autoregulationofbloodflow),andthisismostprominentovertheintermediatepressurerange(≈60to180mmHg).(FromBerneRM,RubioR.Coronarycirculation.InPageE,ed.Handbook of Physiology: Section 2: The Cardiovascular System: The Heart. Vol1.Bethesda,MD:AmericanPhysiologicalSociety;1979.) limits by baroreceptor reflex mechanisms. Hence, changes in coronary blood flow are caused mainly by changes in the diameter of coronary resistance vessels in response to the metabolic demands of the heart.

•Fig. 17.31 Comparison of Phasic Coronary Blood Flow in the Left and Right Coronary Arteries. Extravascularcompressionissogreatduringearlyventricularsystolethatthedirectionofbloodflowinthelargecoronaryarteriessupplyingtheleftventricleisbrieflyreversed.Maximalinflowintheleftcoronaryarteryoccursinearlydiastole,whentheventricleshaverelaxedandextravascularcompressionofthecoronaryvesselsisvirtuallyabsent.Afteraninitialreversalinearlysystole,bloodflowintheleftcoronaryarteryfollowstheaorticpressureuntilearlydiastole,whenitrisesabruptlyandthendeclinesslowlyasaorticpressurefallsduringtheremainderofdiastole.

In addition to providing the pressure to move blood through the coronary vessels, the heart also affects its blood supply by the squeezing effect (extravascular compression) of the contracting myocardium on its own blood vessels. The patterns of flow in the left and right coronary arteries are shown in

Fig. 17.31 . In the left ventricle, coronary perfusion pressure is the difference between aortic diastolic pressure and left ventricular end-diastolic pressure.

Left ventricular myocardial pressure (pressure within the wall of the left ventricle) is highest near the endocardium and lowest near the epicardium. This pressure gradient does not normally impair endocardial blood flow because the greater blood flow to the endocardium during diastole compensates for the greater blood flow to the epicardium during systole. Measurements of coronary blood flow indicate that the epicardial and endocardial halves of the left ventricle receive approximately equal blood flow under normal conditions. Because extravascular compression is greatest at the endocardial surface of the ventricle, the equality of epicardial and endocardial blood flow indicates that the tone of the endocardial resistance vessels is less than that of the epicardial vessels.

Theminimalextravascularresistanceandabsenceofleftventricularworkduringdiastolecanbeusedtoimprovemyocardialperfusioninpatientswithadamagedmyocardiumandlowbloodpressure.Inamethodcalledcounterpulsation, aninflatableballoonisinsertedintothethoracicaortathroughafemoralartery.Theballoonisinflatedduringeachventriculardiastoleanddeflatedduringeachsystole.Thisprocedureenhancescoronarybloodflowduringdiastolebyraisingdiastolicpressureatatimewhencoronaryextravascularresistanceislowest.Furthermore,itreducescardiacenergyrequirementsbyloweringaorticpressure(afterload)duringventricularejection.

The flow pattern in the right coronary artery is similar to that in the left coronary artery (see

Fig. 17.31 ). In contrast to the left ventricle, reversal of blood flow does not occur in the right ventricle in early systole because pressure in the thin right ventricle is lower during systole. Hence, systolic blood flow constitutes a much greater proportion of total coronary inflow than it does in the left coronary artery.

•Fig. 17.32 A, Unmaskingoftherestrictingeffectofventricularsystoleonmeancoronarybloodflowbyinductionofventricularfibrillationduringperfusionoftheleftcoronaryarteryatconstantpressure.Withtheonsetofventricularfibrillation,coronarybloodflowincreasesabruptlybecauseextravascularcompressionisremoved.Flowthengraduallyreturnstowardandoftenfallsbelowtheprefibrillationlevel.Thisincreaseincoronaryresistancethatoccursdespitetheremovalofextravascularcompressiondemonstratestheheart’sabilitytoadjustitsbloodflowtomeetitsenergyrequirements.B, EffectofcardiacsympatheticnervestimulationoncoronarybloodflowandonbloodO2tension(PO2)inthecoronarysinusinafibrillatingheartduringperfusionoftheleftcoronaryarteryatconstantpressure.(BerneRM.Unpublishedobservations.)

The extent to which extravascular compression restricts coronary inflow can be readily observed when the heart is suddenly arrested in diastole or with the induction of ventricular fibrillation.

Fig. 17.32A depicts mean left coronary flow when the vessel was perfused with blood at a constant pressure from a reservoir. When ventricular fibrillation was electrically induced, blood flow increased immediately and substantially. A subsequent increase in coronary resistance over a period of many minutes reduced myocardial blood flow to below the level that existed before induction of ventricular fibrillation (see

Fig. 17.32B , just before stellate ganglion stimulation).

When diastolic pressure in the coronary arteries is abnormally low (as in severe hypotension, partial coronary artery occlusion, or severe aortic stenosis), the ratio of endocardial to epicardial blood flow falls below a value of 1. This ratio indicates that blood flow to the endocardial regions is more severely impaired than that to the epicardial regions of the ventricle. There is also an increase in the gradient of myocardial lactic acid and myocardial adenosine concentrations from epicardium to endocardium. For this reason, the myocardial damage observed in atherosclerotic heart disease (e.g., after coronary occlusion) is greatest in the inner wall of the left ventricle.

Tachycardia and bradycardia have dual effects on coronary blood flow. A change in heart rate mainly alters diastole. In tachycardia, the proportion of time spent in systole, and consequently the period of restricted inflow, increases. However, this mechanical effect is overridden by the dilation of coronary resistance vessels associated with the increased metabolic activity of the more rapidly beating heart. With bradycardia, the opposite occurs: Coronary inflow is less restricted (more time spent in diastole), but so are the metabolic (O2) requirements of the myocardium.

Stimulation of cardiac sympathetic nerves markedly increases coronary blood flow. However, the increase in flow is associated with an increased heart rate and more forceful systole. The stronger contraction and the tachycardia tend to restrict coronary flow. The increase in myocardial metabolic activity, however, tends to dilate coronary resistance vessels. The increase in coronary blood flow evoked by cardiac sympathetic nerve stimulation reflects the sum of these factors. In perfused hearts in which the mechanical effect of extravascular compression is eliminated by cardiac arrest or by ventricular fibrillation, an initial coronary vasoconstriction of the coronary vessels is often observed. After this initial vasoconstriction, the metabolic effect evokes vasodilation (see

Fig. 17.32B

Furthermore, when β-adrenergic receptor blockade eliminates the positive chronotropic and inotropic effects, activation of the cardiac sympathetic nerves increases coronary resistance. These observations indicate that the direct action of the sympathetic nerve fibers on the coronary resistance vessels is vasoconstriction.

Both α-adrenergic receptors (constrictors) and β2adrenergic receptors (dilators) are present on the coronary vessels. Coronary resistance vessels also participate in the baroreceptor and chemoreceptor reflexes, and the sympathetic constrictor tone of the coronary arterioles can be modulated by such reflexes. Nevertheless, coronary resistance is predominantly under local nonneural control.

Vagus nerve stimulation causes slight dilation of the coronary resistance vessels, and activation of the carotid and aortic chemoreceptors can cause a slight decrease in coronary resistance via the vagus nerves to the heart. Failure of strong vagal stimulation to increase coronary blood flow is not due to lack of muscarinic receptors on the coronary resistance vessels because intracoronary administration of acetylcholine elicits marked vasodilation. In the human heart, acetylcholine caused vasodilation when administered directly into the left anterior descending coronary artery of subjects with no evidence of coronary artery disease. However, acetylcholine caused vasoconstriction in the coronary artery of subjects whose endothelium had been damaged and rendered dysfunctional by atherosclerosis.

A striking characteristic of the coronary circulation is the close relationship between the level of myocardial metabolic activity and the magnitude of coronary blood flow (Fig.

17.33 ). This relationship is also found in a denervated heart and in a completely isolated heart, either in the beating state or in the fibrillating state. Ventricles can fibrillate for many hours when the coronary arteries are perfused with arterial blood from some external source. As already noted, a fibrillating heart uses less O2 than a pumping heart does, and blood flow to the myocardium is reduced accordingly.

The mechanisms that link the cardiac metabolic rate and coronary blood flow remain unsettled. However, it appears that a decrease in the ratio of O2 supply to O2 demand releases vasodilator substances from the myocardial cells into the interstitial fluid, where they relax the coronary resistance vessels. Decreases in arterial blood O2 content or in coronary blood flow and increases in metabolic rate all decrease the O2 supply/demand ratio (

Fig. 17.34 ). As a consequence, substances are released that dilate the arterioles and thereby adjust the O2 supply to the O2 demand. A decrease in O2 demand diminishes the release of vasodilators and enables greater expression of basal tone.

Numerous metabolites participate in the vasodilation that accompanies increased cardiac work. Accumulation of vasoactive metabolites can also account for the increase in blood flow that results from a brief period of ischemia (i.e., reactive hyperemia; see ). The duration of the enhanced coronary flow after release of the briefly occluded vessel is, within certain limits, proportional to the duration of the period of occlusion. Among the factors implicated in reactive hyperemia are ATP-sensitive potassium (KATP) channels, NO, CO2, H+, K+, hypoxia, H2O2, and adenosine.

Of these agents, the key factors appear to be adenosine, NO, opening of the KATP channels, and H2O2. The contributions of each of these agents and their interaction under basal conditions and during increased myocardial •Fig. 17.33 RelationshipbetweenmyocardialO2consumptionandcoronarybloodflowduringavarietyofinterventionsthatincreaseordecreasethemyocardialmetabolicrate.(FromBerneRM,RubioR:Coronarycirculation.InPageE,ed.Handbook of Physiology: Section 2: The Cardiovascular System: The Heart. Vol1.Bethesda,MD:AmericanPhysiologicalSociety;1979.)18 16 14 12 10 8 6 4 2 20 30 40 50 60 70 80 90 100 110 120 Coronary blood flow (mL/min/100 g) Myocardial oxygen consumption(mL/min/100 g) •Fig. 17.34 ImbalanceintheO2supply–O2demandratioalterscoronarybloodflowbytherateofreleaseofavasodilatormetabolitefromcardiomyocytes.Adecreaseintheratioelicitsanincreaseinvasodilatorrelease,whereasanincreaseintheratiohastheoppositeeffect.

372 SECTION4Berne & Levy Physiology •Fig. 17.35 Schematic Representation of Factors That Increase (+) or Decrease (−) Coronary Vascular Resistance. Intravascularpressure(arterialbloodpressure)stretchesthevesselwall.KATPchannels,adenosinetriphosphate–sensitivepotassiumchannels;NO,nitricoxide;PCO2,partialpressureofcarbondioxide;PO2,partialpressureofoxygen.˜ Myocardial contractile activity Autonomic control Sympathetic nerves: °-Receptors ˛-Receptors Vagus nerves Oxygen supply Myogenic mechanism ˜ Metabolism ˝ Myocardial PO2 KATP channels H2O2 NO Adenosine – + +± + – – – Other metabolic factors PO2, PCO2, H+, K+ Systolic compression activity are complex. A reduction in oxidative metabolism in vascular smooth muscle reduces ATP synthesis, which in turn opens KATP channels and causes hyperpolarization. This change in potential reduces entry of Ca++ and relaxes coronary vascular smooth muscle to increase flow. A reduction in ATP also opens KATP channels in cardiac muscle and generates an outward current that reduces action potential duration and limits Ca++ entry during phase 2 of the action potential. This action may be protective during periods of imbalance between O2 supply and demand. In addition, as cardiac work increases, H2O2 production rises, which activates Kv1.5 channels and thereby causes hyperpolarization of muscle membrane and relaxation of vascular smooth muscle. Moreover, the release of NO and adenosine dilates the arterioles and thereby adjusts the O2 supply to the O2 demand. At low concentrations, adenosine appears to activate endothelial KATP channels and to enhance release of NO. Conversely, at higher concentrations, adenosine acts directly on vascular smooth muscle by activating KATP channels. Decreased O2 demand would sustain the ATP level, as well as reduce the amount of vasodilator substances released, and allows greater expression of basal tone. If production of all these agents is inhibited, coronary blood flow is reduced, both at rest and during exercise. Furthermore, contractile dysfunction and signs of myocardial ischemia become evident.

According to the adenosine hypothesis, a reduction in myocardial O2 tension produced by inadequate coronary blood flow, hypoxemia, or increased metabolic activity of the heart leads to release of adenosine from the myocardium. Adenosine enters the interstitial fluid space to reach the coronary resistance vessels and induces vasodilation by activating adenosine receptors. However, it cannot be responsible for the increased coronary flow observed during prolonged enhancement of cardiac metabolic activity because release of adenosine from cardiac muscle is transitory. Factors that alter coronary vascular resistance are illustrated in

Fig. 17.35

Effects of Diminished Coronary Blood Flow

Most of the O2 in coronary arterial blood is extracted during one passage through the myocardial capillaries. Thus the supply of O2 to myocardial cells is flow limited; any substantial reduction in coronary blood flow curtails O2 delivery to the myocardium because O2 extraction is nearly maximal even when blood flow is normal.

A reduction in coronary flow that is neither too prolonged nor too severe to induce myocardial necrosis can nonetheless cause substantial (but temporary) dysfunction of the heart. A relatively brief period of severe ischemia followed by reperfusion can result in pronounced mechanical dysfunction (myocardial stunning). However, the heart eventually recovers fully from the dysfunction. The pathophysiological basis for myocardial stunning appears to be intracellular Ca++ overload, initiated during the period of ischemia, combined with the generation of OH− and superoxide free radicals early in the period of reperfusion. These changes impair the responsiveness of myofilaments to Ca++ .

In the normal human heart, there are virtually no functional intercoronary channels. Abrupt occlusion of a coronary artery or one of its branches leads to ischemic necrosis and eventual fibrosis of the areas of myocardium supplied by the occluded vessel. However, if a coronary artery narrows slowly and progressively over a period of days or weeks, collateral vessels develop and may furnish sufficient blood to the ischemic myocardium to prevent or reduce the extent of necrosis. Collateral vessels may develop between branches of occluded and nonoccluded arteries. They originate from preexisting small vessels that undergo proliferative changes of the endothelium and smooth muscle. These changes may occur in response to wall stress and to chemical agents, including vascular endothelial growth factors (VEGFs) released by the ischemic tissue. The VEGFs, of which there are at least five in mammals, are glycoproteins. The VEGFs induce angiogenesis, elicit vasodilation and increase endothelial permeability. By causing vasodilation, VEGFs enable perfusion of more capillaries and increase capillary permeability by opening tight junctions between endothelial cells and by adding fenestrations.

Myocardial stunning, prolongedventriculardysfunctionwithoutmyocardialnecrosis,maybeevidentinpatientswhohavesufferedanacutecoronaryarteryocclusion.Ifthepatientistreatedsufficientlyearlybycoronarybypasssurgeryorballoonangioplasty,andifadequatebloodflowisrestoredtotheischemicregion,themyocardialcellsinthisregionmayrecoverfully.However,formanydaysorevenweeks,thecontractilityofthemyocardiumintheaffectedregionmaybegrosslysubnormal.

Prolongedreductionsincoronarybloodflow(myocardialischemia)maycriticallyandpermanentlyimpairthemechanicalandelectricalbehavioroftheheart.Diminishedcoronarybloodflowasaconsequenceofcoronaryarterydisease(usuallycoronaryatherosclerosis)isoneofthemostcommoncausesofseriouscardiacdisease.Theischemiamaybeglobal(affectsanentireventricle)orregional(affectssomefractionoftheventricle).TheimpairmentinmechanicalcontractionoftheaffectedmyocardiumisproducednotonlybythediminisheddeliveryofO2andmetabolicsubstratesbutalsobytheaccumulationofpotentiallyharmfulsubstances(e.g.,K+ ,lacticacid,H+)inthecardiactissues.Ifthereductionincoronaryflowtoanyregionoftheheartissufficientlysevereandprolonged,necrosisoftheaffectedcardiaccellsresults.

Myocardial hibernation describesthephenomenoninwhichcellularmetabolismisdownregulatedincellswhosefunctionisimpairedbyinadequatedeliveryofO2andnutrients.Myocardialhibernationoccursmainlyinpatientswithcoronaryarterydisease.Thecoronarybloodflowinsuchpatientsisdiminishedpersistentlyandsignificantly,andthemechanicalfunctionoftheheartisimpaired.Ifcoronarybloodflowisrestoredtonormalbybypasssurgeryorangioplasty,mechanicalfunctionreturnstonormal.

The O2 and nutrient requirements of the skin are relatively small. Unlike other body tissues, the supply of O2 and nutrients is not the chief factor in the regulation of cutaneous blood flow. The primary function of the cutaneous circulation is to maintain a constant body temperature. Thus the skin undergoes wide fluctuations in blood flow, depending on whether the body needs to lose or conserve heat. Changes in ambient and internal body temperature activate mechanisms responsible for alterations in skin blood flow.

Numeroussurgicalattemptshavebeenmadetoenhancethedevelopmentofcoronarycollateralvessels.However,thetechniquesuseddonotincreasethecollateralcirculationoverandabovethatproducedbycoronaryarterynarrowingalone.Whendiscreteocclusionsorseverenarrowingoccursincoronaryarteries,asincoronaryatherosclerosis,thelesionscanbebypassedwithanarteryoraveingraft.Frequently,thenarrowsegmentcanbedilatedbyinsertionofaballoon-tippedcatheterintothediseasedvesselviaaperipheralarteryandtheninflationoftheballoon.Distentionofthevesselbyballooninflation(angioplasty)canproducelastingdilationofanarrowed coronaryartery(Fig.17.36 ),particularlywhenadrug-elutingstent(thedrugshelppreventrestenosis)isinsertedduringangioplasty.

Manydrugsareavailableforuseinpatientswithcoronaryarterydiseasetorelieveanginapectoris,thechestpainassociatedwithmyocardialischemia.Thesecompoundsincludeorganicnitrates/nitrites,calciumchannelantagonists,andβ-adrenoceptorantagonists.Organicnitrates/nitritesaremetabolizedtoNO.NOdilatesthegreatveinstoreducevenousreturn(preload),therebyreducingcardiacwork(see )andmyocardialO2requirements.Inaddition,NOdilatesthecoronaryarteriestoincreasecollateralflow.Ofimportanceisthatorganicnitrates/nitritesdonotinterferewithcoronaryautoregulation.Calciumchannelantagonistsalsocausevasodilation;noneselectivelydilatesthecoronaryvessels.Theβ-adrenoceptorantagonistsreducetheheartratetoindirectlyincreasecoronaryflowandopposethereflextachycardiathathasbeenobservedwithorganicnitrates/nitrites.

Inpatientswithmarkednarrowingofacoronaryartery,administrationofdipyridamole,avasodilator,canfullydilatenormalvesselbranchesthatareparalleltothenarrowedsegmentandtherebyreducethepressureonthepartiallyoccludedvessel.Thereducedpressureonthenarrowedvesselfurthercompromisesbloodflowtotheischemicmyocardium.Thisphenomenon,knownascoronary steal, occursbecausedipyridamoleactsbyblockingthecellularuptakeandmetabolismofendogenousadenosine.Ofnoteisthatdipyridamoleinterfereswithcoronaryautoregulation.

Regulation of Skin Blood Flow

The skin contains essentially two types of resistance vessels: arterioles and arteriovenous anastomoses. AV anastomoses shunt blood from the arterioles to the venules and venous plexuses; hence, they bypass the capillary bed. Such anastomoses are found in the fingertips, palms of the hands, toes, soles of the feet, ears, nose, and lips. AV anastomoses differ morphologically from arterioles; the anastomoses are either short and straight or long coiled vessels, approximately 20 to 40 µm in luminal diameter, and they have thick muscular walls richly supplied with nerve fibers (

Fig. 17.37 ). These vessels are almost exclusively under sympathetic neural control, and they dilate maximally when their nerve

Fig. 17.36 A, Angiogram(withintracoronaryradiopaquedye)ofmarkednarrowingoftheleftanteriordescendingbranchoftheleftcoronaryartery(white arrow). B, Thesamesegmentofthecoronaryartery(black arrow) afterangioplastyandinsertionofadrug-elutingstent.(CourtesyofDr.MichaelAzrin.)

Fig. 17.37 Arteriovenous (AV) Anastomosis in the Ear Injected With Berlin Blue Dye. A,artery;V,vein;arrowpointstoanAVanasto-mosis.ThewallsoftheAVanastomosisinthefingertipsarethickerandmorecellular.(FromPritchardMML,DanielPM.J Anat. 1956;90:309.) supply is interrupted. Conversely, reflex stimulation of the sympathetic fibers to these vessels may constrict them and obliterate the vascular lumen. Although AV anastomoses do not exhibit basal tone, they are highly sensitive to vasoconstrictor agents such as epinephrine and norepinephrine. Furthermore, AV anastomoses are not under metabolic control, and they do not show reactive hyperemia or auto-regulation of blood flow. Thus regulation of blood flow through these anastomotic channels is governed principally by the nervous system in response to reflex activation by temperature receptors or from higher centers of the central nervous system.

Most of the resistance vessels in the skin exhibit some basal tone and are under dual control of the sympathetic nervous system and local regulatory factors. However, neural control predominates. Stimulation of sympathetic nerve fibers

Thefingersandtoesofsomeindividualsareverysensitivetocold.Onexposuretocold,thearteriolestothefingersandtoesconstrict.Theconsequentischemiaresultsinlocalizedblanchingoftheskinassociatedwithtingling,numbness,andpain.Theblanchingisfollowedbycyanosis(adarkbluecoloroftheskin)andlaterbyrednessasthearterialspasmsubsides.Thecauseofthiscondition,calledRaynaud’sdisease,isunknown.

induces vasoconstriction, and cutting of the sympathetic nerves induces vasodilation. After chronic denervation of the cutaneous blood vessels, the degree of tone that existed before denervation is gradually regained over a period of several weeks. This restoration of tone is accomplished by an enhancement of basal tone. Denervation of the skin vessels results in enhanced sensitivity to catecholamines in circulation (denervation hypersensitivity).

Parasympathetic vasodilator nerve fibers do not innervate cutaneous blood vessels. However, stimulation of the sweat glands, which are innervated by sympathetic cholinergic fibers, dilates the skin resistance vessels. Sweat contains an enzyme that lyses a protein (kallidin) in the tissue fluid to produce bradykinin, a polypeptide with potent vasodilator properties. Bradykinin, formed locally, dilates the arterioles and increases blood flow to the skin.

Certain skin vessels, particularly those in the head, neck, shoulders, and upper part of the chest, are regulated by higher centers in the brain. Blushing, in response to embarrassment or anger, and blanching, in response to fear or anxiety, are examples of cerebral inhibition and stimulation, respectively, of the sympathetic nerve fibers to the affected cutaneous regions.

In contrast to AV anastomoses in the skin, the resistance vessels display autoregulation of blood flow and reactive hyperemia. If the arterial inflow to a limb is stopped briefly by inflation of a blood pressure cuff, the skin becomes bright red below the point of vascular occlusion when the cuff is subsequently deflated. The increased cutaneous blood flow (reactive hyperemia) is also manifested by distention of the superficial veins in the affected extremity.

The Role of Temperature in the Regulation of Skin Blood Flow

The primary function of the skin is to maintain a constant internal environment and protect the body from adverse changes. Ambient temperature is one of the most important external variables with which the body must contend. Exposure to cold elicits a generalized cutaneous vasoconstriction that is especially pronounced in the hands and feet. This response is chiefly mediated by the nervous system. Arrest of the circulation to a hand by a pressure cuff plus immersion of that hand in cold water induces vasoconstriction in the skin of the other extremities that are exposed to room temperature. When the circulation to the chilled hand is not occluded, the reflex-generalized vasoconstriction is caused in part by the cooled blood that returns to the general circulation. This returned blood then stimulates the temperature-regulating center in the anterior hypothalamus, which then activates heat preservation centers in the posterior hypothalamus to evoke cutaneous vasoconstriction.

The skin vessels of the cooled hand also respond directly to cold. Moderate cooling or a brief exposure to severe cold (0°C to 15°C) constricts the resistance and capacitance vessels, including the AV anastomoses. Prolonged exposure to severe cold evokes a secondary vasodilator response. Prompt vasoconstriction and severe pain are elicited by immersion of the hand in ice water. However, this response is soon followed by dilation of the skin vessels, with reddening of the immersed part and alleviation of the pain. With continued immersion of the hand, alternating periods of constriction and dilation occur, but the skin temperature rarely drops as much as it did in response to the initial vasoconstriction. Prolonged severe cold, of course, damages tissue. The rosy faces of people exposed to a cold environment are examples of cold-induced vasodilation. However, blood flow through the skin of the face may be greatly reduced despite the flushed appearance. The red color of the slowly flowing blood is mainly caused by reduced O2 uptake by the cold skin and the cold-induced shift of the oxyhemoglobin dissociation curve to the left (see

Direct application of heat to the skin not only dilates the local resistance and capacitance vessels and the AV anastomoses but also reflexively dilates blood vessels in other parts of the body. The local effect is independent of the vascular nerve supply, whereas the reflex vasodilation is a combined response to stimulation of the anterior hypothalamus by the returning warmed blood and stimulation of cutaneous heat receptors in the heated regions of the skin.

The close proximity of the major arteries and veins allows countercurrent heat exchange between them. Cold blood that flows in veins from a cooled hand toward the heart takes up heat from adjacent arteries; this warms the venous blood and cools the arterial blood. Heat exchange takes place in the opposite direction when the extremity is exposed to heat. Thus heat conservation is enhanced during exposure of extremities to cold environments, and heat conservation is minimized during exposure of the extremities to warm environments.

Skin Color: Relationship to Skin Blood Volume, Oxyhemoglobin, and Blood Flow

Skin color is determined mainly by the pigment content. However, the degree of pallor or ruddiness is mainly a function of the amount of blood in the skin, except when the skin is very dark. With little blood in the venous plexus, the skin appears pale, whereas with moderate to large quantities of blood in the venous plexus, the skin displays a color. This color may be red, blue, or some shade between, depending

CHAPTER 17 Properties of the Vasculature on the degree of oxygenation of the blood. A combination of vasoconstriction and reduced hemoglobin can impart an ashen gray color to the skin. A combination of venous engorgement and reduced hemoglobin content can impart a dark purple hue.

Skin color provides little information about the rate of cutaneous blood flow. Rapid blood flow may be accompanied by skin pallor when the AV anastomoses are open, and slow blood flow may be associated with skin ruddiness when the skin is exposed to cold.

The rate of blood flow in skeletal muscle varies directly with the contractile activity of the tissue and the type of muscle. Blood flow and capillary density are greater in red muscle (slow-twitch muscle with high oxidative capacity) than in white muscle (fast-twitch muscle with low oxidative capacity). In resting muscle, the precapillary arterioles contract and relax intermittently. Thus at any given moment, most of the capillary bed is not perfused, and total blood flow through quiescent skeletal muscle is low (1.4 to 4.5 mL/ minute/100 g). During exercise, the resistance vessels relax, and muscle blood flow may increase to 15 to 20 times the resting level, depending on the intensity of the exercise.

Regulation of Skeletal Muscle Blood Flow

Neural and local factors regulate muscle circulation. Physical factors such as Pa, tissue pressure, and blood viscosity influence muscle blood flow. However, another physical factor, the squeezing effect of the active skeletal muscle, affects blood flow in the vessels. With intermittent contractions, inflow is restricted, and as previously described, venous outflow is enhanced. The venous valves prevent backward flow of blood between contractions and thereby aid in the forward propulsion of blood. With strong sustained contractions, as occur during exercise, the vascular bed can be compressed to the point at which blood flow actually ceases temporarily.

The resistance vessels of muscle possess a high degree of basal tone; they also display tone in response to continuous low-frequency activity in the sympathetic vasoconstrictor nerve fibers. The basal firing frequency of sympathetic vasoconstrictor fibers is only approximately 1 to 2 per second, and maximal vasoconstriction occurs at frequencies of approximately 10 per second.

Vasoconstriction evoked by sympathetic nerve activity is caused by the local release of norepinephrine. Intra-arterially injected norepinephrine elicits only vasoconstriction (α1adrenergic receptor). In contrast, low doses of epinephrine produce vasodilation (β2-adrenergic receptor), whereas large doses cause vasoconstriction.

Baroreceptor reflexes greatly influence the tonic activity of the sympathetic nerves. An increase in carotid sinus pressure causes the muscle vascular bed to dilate, whereas a decrease in carotid sinus pressure elicits vasoconstriction (

Fig. 17.38 ). When sympathetic constrictor tone is high, the decrease in blood flow evoked by common carotid artery occlusion is small, but the increase in flow after the release of occlusion is large. The vasodilation produced by baroreceptor stimulation is caused by inhibition of sympathetic vasoconstrictor activity.

The resistance vessels in skeletal muscle contribute significantly to maintenance of arterial blood pressure because skeletal muscle constitutes a large fraction of the body’s mass, and the muscle vasculature thus constitutes the largest vascular bed. Participation of the skeletal muscle vessels in vascular reflexes is important in maintaining normal arterial blood pressure.

A comparison of the sympathetic neural effects on the blood vessels of muscle and skin is summarized in

Fig.

17.39.

Note that the lower the basal tone of the skin vessels, the greater their constrictor response; also note the absence of active cutaneous vasodilation.

In active skeletal muscle, blood flow is regulated by meta- bolic factors. In resting muscle, neural factors predominate,

Fig. 17.39 ). Cutting of the sympathetic nerves to muscle abolishes the neural component of vascular tone, and it unmasks the intrinsic basal tone of the blood vessels. The neural and local mechanisms that regulate blood flow oppose each other, and during muscle contraction, the local vasodilator mechanism supervenes. However, during exercise, strong sympathetic nerve stimulation slightly attenuates the vasodilation induced by locally released metabolites.

•Fig. 17.38 Evidence of Participation of the Muscle Vascular Bed in Vasoconstriction and Vasodilation Mediated by the Carotid Sinus Baroreceptors After Occlusion and Release of the Common Carotid Artery. Inthispreparation,thesciaticandfemoralnervesconstitutedtheonlydirectinnervationofthehindlegmusclemass.Themusclewasperfusedwithbloodataconstantpressure.(RedrawnfromJonesRD,BerneRM.Am J Physiol. 1963;204:461.)

Blood reaches the brain through the internal carotid and vertebral arteries. The vertebral arteries join to form the basilar artery, which, in conjunction with branches of the •Fig. 17.39 Basaltoneandtherangeofresponseofresistancevesselsinmuscle(dashed lines) andskin(shaded areas) tostimulationandsectionofsympatheticnerves.Peripheralresistanceisplottedonalogarithmicscale.(RedrawnfromCelanderO,FolkowB.Acta Physiol Scand. 1953;29:241.) internal carotid arteries, forms the circle of Willis. Arteries on the brain surface differ from those that penetrate the brain parenchyma. Pial arteries and arterioles have an extrinsic innervation (e.g., via superior cervical ganglion, sphenopalatine nerves, trigeminal nerve); parenchymal arterioles have an intrinsic innervation (via cerebral neurons). Pial arteries have more smooth muscle cells than do parenchymal arterioles. Also, pial arteries and arterioles have collateral branches, whereas parenchymal arterioles do not. Therefore, parenchymal arterioles regulate blood flow to discrete cortical regions, and their occlusion can reduce blood flow significantly.

The cerebral circulation is unique because it lies within a rigid structure, the cranium. Any increase in arterial inflow must be associated with a comparable increase in venous outflow because the intracranial contents cannot be compressed. The volume of blood and extravascular fluid can vary considerably in most body tissues. In the brain, however, the volume of blood and extravascular fluid is relatively constant; a change in one of these fluid volumes must be accompanied by a reciprocal change in the other. The rate of cerebral blood flow is maintained within a narrow range; in humans, it averages 55 mL/minute/100 g of brain tissue.

Regulation of Cerebral Blood Flow

At rest, the brain consumes 20% of total body oxygen and 25% of total body glucose. Of all body tissues, the brain is the least tolerant of ischemia. Interruption of cerebral blood flow for as little as 5 seconds results in loss of consciousness. Ischemia that lasts just a few minutes may cause irreversible tissue damage. Fortunately, regulation of the cerebral circulation is primarily under the direction of the brain itself. Local regulatory mechanisms and reflexes that originate in the brain tend to maintain a relatively constant cerebral circulation in the presence of such adverse effects as sympathetic vasomotor nerve activity, circulating humoral vasoactive agents, and changes in arterial blood pressure. Under certain conditions, the brain also regulates its blood flow by initiating changes in systemic blood pressure.

Changes in cerebral blood flow are associated with “functional recruitment” of capillaries. Thus the rate of flow through each capillary is adjusted to meet the needs of the organ. In “capillary recruitment,” in contrast, more capillaries are open to accommodate greater blood flow.

The brain has several protective mechanisms that regulate blood flow. These mechanisms include the blood-brain barrier, extrinsic regulation of central cardiovascular centers, intrinsic control (autoregulation) of circulation, and functional hyperemia, in which blood flow increases to a brain region that is active.

The blood-brain barrier regulates ion and nutrient transport between the blood and the brain and also limits the entry of harmful substances from the blood into the brain. The •Fig. 17.40 Diagram of a Neurovascular Unit With an Astrocyte Linking a Neuron to an Arteriole of the Brain Microcirculation.

Arteriolartoneismodulatedbyvascularsmoothmuscleandbytheactionofpericytes.Theendothelialcellrestrictsdiffusionofsubstancesbyvirtueoftightjunctions.Theneurovascularunitisacomponentoftheblood-brainbarrierandalsoservesasaregulatorofbloodflowduringneuronalactivity.

blood-brain barrier includes tight junction proteins (junctional adhesion molecule-1, occludins, claudins), which are connected to the endothelial cell cytoskeleton to form a barrier that opposes paracellular movement of substances from blood to brain. In addition, the blood-brain barrier includes the neurovascular unit (microcirculation, pericytes, the extracellular matrix, astrocytes, and neurons;

Fig.

17.40 ). Pericytes regulate blood flow by adjusting vascular diameter, and they secrete angiopoetin, a growth factor that stimulates the expression of occludins in endothelial cells. Occludins are prominently expressed in brain endothelial cells, in contrast to their sparse distribution in nonneural endothelium. The neurovascular unit regulates blood flow and capillary permeability. Thus the neurovascular unit is involved in pathological states, including hypoxia, neurodegenerative diseases, and inflammation, that are characterized by dysfunction of the blood-brain barrier.

The extrinsic innervation of cerebral (pial) vessels consists of components of the autonomic nervous system. Cervical sympathetic nerve fibers that accompany the internal carotid and vertebral arteries into the cranial cavity innervate the cerebral vessels. In comparison to other vascular beds, sympathetic control of the cerebral vessels is weak, and the contractile state of the cerebrovascular smooth muscle depends primarily on local metabolic factors. The density of α1-adrenergic receptors is less than in other vascular beds. Cerebral vessels receive parasympathetic fibers from the facial nerve that produce a slight vasodilation

Controls: rest Sens 1 Sens 2 •Fig. 17.41 Effects of Different Stimuli on Regional Blood Flow in the Contralateral Human Cerebral Cortex. Sens1,low-intensityelectricalstimulationofthehand;Sens2,high-intensity(painful)electricalstimulationofthehand.Otherstimuliareasnoted.(RedrawnfromIngvarDH.Brain Res. 1976;107:181.) on stimulation. The sympathetic nervous system exerts the most prominent effect on cerebral blood flow during pathophysiological conditions.

In general, total cerebral blood flow is relatively constant and is autoregulated. Autoregulation of cerebral blood flow involves interplay among myogenic, metabolic, and neural mechanisms much as described for peripheral vessels (see ). However, regional blood flow in the brain is associated with regional neural activity. For example, movement of one hand results in increased blood flow only in the hand area of the contralateral sensorimotor and premotor cortex. Talking, reading, and other stimuli to the cerebral cortex are also associated with increased blood flow in the appropriate regions of the contralateral cortex (

Fig.

17.41 ). Glucose uptake also corresponds to regional cortical neuronal activity. Thus when the retina is stimulated by light, uptake of glucose is enhanced in the visual cortex.

The neurovascular unit plays an integral role in the discrete regulation of blood flow. Production of vasoactive compounds couples increased neuronal activity to greater uptake of oxygen and glucose. Within the neurovascular unit, astrocytes link neurons with the microcirculation (see

Fig. 17.40 ). At one pole, astrocytes surround presynaptic and postsynaptic neurons at synapses. At the other pole, astrocytes converge on vascular smooth muscle and endothelial cells of cerebral vessels. When activated by the neurotransmitter glutamate or acetylcholine, astrocytes produce inositol trisphosphate (IP3), which causes the release of Ca++, which, in turn, activates large conductance potassium (BKCa) channels. The released K+ raises extracellular [K+] to 8 to 15 mEq/L in the space between the astrocyte and arteriolar smooth muscle. The elevated extracellular [K+] causes hyperpolarization of smooth muscle by activating Na+/K+-ATPase and also by increasing the conductance of inward-rectifying K+ channels. The hyperpolarization reduces Ca++ entry into vascular smooth muscle because the membrane potential is shifted away from the threshold. Hence, the parenchymal arteriole dilates, and blood flow increases.

With regard to K+ , stimuli such as hypoxia, electrical stimulation of the brain, and seizures elicit rapid increases in cerebral blood flow, and they are associated with increases in perivascular K+ . The increments in K+ are similar to those that produce pial arteriolar dilation when K+ is applied topically to these vessels. When extracellular K+ exceeds 15 mEq/L, smooth muscle cells depolarize, and Ca++ entry is increased to cause contraction and vasoconstriction. Thus the extracellular [K+] has a dual effect on smooth muscle function that is derived from its actions on Na+/K+-ATPase, K+ conductance, and the K+ concentration gradient.

The cerebral vessels are also regulated by CO2. Increases in arterial blood CO2 tension (PaCO2) elicit marked cerebral vasodilation; for example, inhalation of 7% CO2 increases cerebral blood flow twofold. Conversely, decreases in PaCO2, caused by hyperventilation, diminish cerebral blood flow. CO2 evokes changes in arteriolar resistance by altering perivascular pH. When PaCO2 and the HCO3 − concentration are independently changed, pial vessel diameter and blood flow are inversely related to pH, regardless of the level of PaCO2 or [HCO3 −]. Acidosis initiates a marked vasodilation of brain arterioles. The vasodilation is mediated by a very localized release of Ca++ from the endoplasmic reticulum (Ca++ “sparks”). This local Ca++ signal activates large conductance BKCa channels; the ensuing hyperpolarization stabilizes the vascular smooth muscle cell and opposes vasoconstriction.

Elevationinintracranialpressure,causedbyabraintumor,resultsinanincreaseinsystemicbloodpressure.Thisresponse,calledCushing’s phenomenon, isevokedbyischemicstimulationofvasomotorregionsinthemedulla.Cushing’sphenomenonhelpsmaintaincerebralbloodflowinconditionssuchasexpandingintracranialtumors.

Carbon dioxide diffuses into vascular smooth muscle from brain tissue or from the lumen of blood vessels, whereas H+ in blood is prevented from reaching arteriolar smooth muscle by the blood-brain barrier. Hence, cerebral vessels dilate when the [H+] of cerebrospinal fluid is increased, but these vessels dilate only minimally in response to an increase in the [H+] of arterial blood. Chemical regulation of cerebral blood flow by PaCO2 is impaired in humans with endothelial dysfunction (e.g., diabetes, hypertension); the relative roles of H+ and NO in response to changes of PaCO2 are not clear.

Potassium concentration also affects cerebral blood flow. Hypoxia, electrical stimulation of the brain, and seizures elicit rapid increases in cerebral blood flow and in perivascular [K+]. The increases in [K+] are similar in magnitude to those that produce pial arteriolar dilation when K+ is applied topically to these vessels. However, the increase in [K+] is not sustained throughout the period of cerebral stimulation. Thus only the initial increase in cerebral blood flow can be attributed to the release of K+ .

Adenosine also has a major effect on cerebral blood flow. Adenosine levels in the brain increase in response to ischemia, hypoxemia, hypotension, hypocapnia, electrical stimulation of the brain, and induced seizures. Topically applied adenosine is a potent dilator of the pial arterioles. Any intervention that either reduces the O2 supply to the brain or increases the O2 requirements of the brain results in the rapid (within 5 seconds) formation of adenosine in cerebral tissue. Unlike the changes in pH or [K+], the adenosine concentration in the brain increases with initiation of the change in O2 supply, and it remains elevated throughout the period of O2 imbalance. The adenosine that is released into cerebrospinal fluid during cerebral ischemia becomes incorporated into adenine nucleotides in cerebral tissue. These local factors, including pH, K+, and adenosine, act in concert to adjust cerebral blood flow to the metabolic activity of the brain. The cerebral circulation displays reactive hyperemia

CHAPTER 17 Properties of the Vasculature and excellent autoregulation when arterial blood pressure is between 60 and 160 mm Hg. Mean arterial pressures below 60 mm Hg result in reduced cerebral blood flow and then syncope, whereas mean pressures above 160 mm Hg may lead to increased permeability of the blood-brain barrier and consequently to cerebral edema. Hypercapnia or any other potent vasodilator abolishes autoregulation of cerebral blood flow. Autoregulation of cerebral blood flow is probably mediated by a myogenic mechanism that is modulated by a metabolic component.

The gastrointestinal tract is supplied by the celiac, superior mesenteric, and inferior mesenteric arteries. The superior mesenteric artery carries more than 10% of the cardiac output. Small mesenteric arteries form an extensive vascular network in the submucosa of the gastrointestinal tract. The arterial branches penetrate the longitudinal and circular muscle layers of the tract, and they give rise to thirdand fourth-order arterioles. Some third-order arterioles in the submucosa supply the tips of the villi (

Fig. 17.42

The direction of blood flow in the capillaries and venules in a villus is opposite that in the main arteriole (see

Fig.

17.42 ). This arrangement is a countercurrent exchange system. Effective countercurrent exchange enables diffusion of O2 from arterioles to venules. At low blood flow rates, a substantial portion of the O2 may be shunted from arterioles to venules near the base of the villus. This reduces the O2 supply to the mucosal cells at the tip of the villus. When intestinal blood flow is very low, shunting of O2 is so great that extensive necrosis of the intestinal villi takes place.

Neural control of the mesenteric circulation is almost exclusively sympathetic. Increased sympathetic activity, through α1-adrenergic receptors, constricts the mesenteric arterioles and capacitance vessels. These receptors are preeminent in the mesenteric circulation. However, β2-adrenergic receptors are also present, and so the agonist isoproterenol causes vasodilation.

In response to aggressive behavior or to artificial stimulation of the hypothalamic “defense” area, pronounced vasoconstriction occurs in the mesenteric vascular bed. This vasoconstriction shifts blood flow from the less important intestinal circulation to the more crucial skeletal muscles, heart, and brain.

Autoregulation of blood flow is not as well developed in the intestinal circulation as in other vascular beds. The principal mechanism responsible for autoregulation is metabolic, although a myogenic mechanism probably also

Simple columnar epithelium with goblet cells •Fig. 17.42 Microcirculation Pattern of the Small Intestine. A, Capillaryplexusesarisefromarteriolesinthevillusandalsointheintestinalcrypt.Bloodleavestheintestinalcryptviavenulesthatentertheportalcirculation.B, Lymphaticvessels(lacteals)originatewithinthevillusandeventuallyformaplexusatthebaseofthevillus.(RedrawnfromKierszenbaumA.Histology and Cell Biology: An Introduction to Pathology. Philadelphia:Mosby;2002.) ). The adenosine concentration in mesenteric venous blood rises fourfold after brief arterial occlusion. It also rises during enhanced metabolic activity of the intestinal mucosa, such as during absorption of food. Adenosine, a potent vasodilator in the mesenteric vascular bed, may be the principal metabolic mediator of autoregulation. However, [K+] and altered plasma osmolality may also contribute to autoregulation.

Oxygen consumption by the small intestine is more rigorously controlled than is blood flow. Experiments have shown that O2 uptake of the small intestine remains constant when arterial perfusion pressure is varied between 30 and 125 mm Hg.

Food ingestion increases intestinal blood flow. Secretion of certain gastrointestinal hormones contributes to this hyperemia. Gastrin and cholecystokinin augment intestinal blood flow, and they are secreted when food is ingested. Absorption of food also affects intestinal blood flow. Undigested food has no vasoactive influence, whereas several products of digestion are potent vasodilators. Among the various constituents of chyme, the principal mediators of mesenteric hyperemia are glucose and fatty acids.

Normally, blood flow to the liver is approximately 25% of cardiac output. Hepatic blood flow is supplied by two sources: the portal vein (≈75%) and the hepatic artery. Because portal venous blood has already passed through the gastrointestinal capillary bed, much of the O2 of the portal vein blood flow has already been extracted. The hepatic artery delivers the remaining 25% of the blood, which is fully saturated with O2. Hence, approximately three fourths of the O2 used by the liver is derived from hepatic arterial blood.

The small branches of the portal vein and hepatic artery give rise to terminal portal venules and hepatic arterioles (

Fig. 17.43 ). These terminal vessels enter the hepatic acinus (the functional unit of the liver) at its center. Blood flows from these terminal vessels into the sinusoid capillaries, which constitute the capillary network of the liver. The sinusoid capillaries radiate toward the periphery of the acinus, where they connect with the terminal hepatic venules. Blood from these terminal venules drains into progressively larger branches of the hepatic veins, which are tributaries of the inferior vena cava.

•Fig. 17.43 Microcirculation of the Hepatic Acinus. Arrows indicatethedirectionofbloodflowfromtheterminalportionsofthehepaticarteryandportalveintothesinusoidcapillaries.Themixtureofarterialandvenousbloodflowsintothecentralveinandthenpassesintothesublobularvein.(RedrawnfromRossMH,PawlingW.Histology:A Text and Atlas: With Correlated Cell and Molecular Biology. Philadelphia:LippincottWilliams&Wilkins;2006.) Periportal space (space of Mall) Capillary Terminal branch of hepatic artery Terminal branch of portal vein Portal canal Arteriosinusoidal branch Lymphatic vessel Bile ductule Inlet venule Hepatic sinusoid Terminal hepatic venule (central vein) Sublobular vein

Mean blood pressure in the portal vein is approximately 10 mm Hg, and mean blood pressure in the hepatic artery is approximately 90 mm Hg. The resistance of the vessels upstream to the hepatic sinusoid capillaries is considerably greater than that of the downstream vessels. Consequently, the pressure in the sinusoid capillaries is only 2 or 3 mm Hg greater than that in the hepatic veins and inferior vena cava. The ratio of presinusoidal to postsinusoidal resistance is much greater in the liver than in almost any other vascular bed. Hence, drugs and other interventions that alter presinusoidal resistance usually affect pressure in the sinusoid capillaries and fluid exchange across the sinusoidal wall only slightly. However, changes in hepatic and central venous pressure are transmitted almost quantitatively to the hepatic sinusoid capillaries, and they profoundly affect the transsinusoidal exchange of fluids.

Regulation of Flow

Blood flow in the portal venous and hepatic arterial systems varies reciprocally. When blood flow is curtailed in one system, flow increases in the other but does not fully compensate for the decreased flow in the first system.

The portal venous system is not autoregulated. As portal Pv and flow are raised, resistance either remains constant or decreases. The hepatic arterial system is autoregulated, however, and adenosine may be involved in this adjustment of blood flow.

The liver tends to maintain constant O2 consumption because O2 extraction from hepatic blood is very efficient. As the rate of O2 delivery to the liver varies, the liver compensates by an appropriate change in the fraction of O2 extracted from blood. Such extraction is facilitated by the distance between the presinusoidal vessels at the acinar center and the postsinusoidal vessels at the periphery of the acinus (see

Fig. 17.43 ). The substantial distance between these types of vessels prevents countercurrent exchange of O2, in contrast to the countercurrent exchange that occurs in an intestinal villus.

The sympathetic nerves constrict the presinusoidal resistance vessels in the portal venous and hepatic arterial systems. Neural effects on the capacitance vessels are more important, however. The liver contains approximately 15% of the total blood volume of the body. In appropriate conditions, as in response to hemorrhage, approximately half of the hepatic blood volume can be rapidly expelled by constriction of the capacitance vessels (see also ). Hence, the liver is an important blood reservoir in humans.

Whencentralvenouspressureiselevated,asincongestiveheartfailure,largevolumesofplasmawaterdiffusefromtheliverintotheperitonealcavity;thisaccumulationoffluidintheabdomenisknownasascites. Extensivefibrosisoftheliver,asinhepaticcirrhosis,markedlyincreaseshepaticvascularresistanceandtherebyraisespressuresubstantiallyintheportalvenoussystem.Theconsequentincreaseincapillaryhydrostaticpressurethroughthesplanchniccirculationalsoleadstoextensivefluidtransudationintotheabdominalcavity.Thepressuremaylikewiserisesubstantiallyinotherveinsthatanastomosewiththeportalvein.Forexample,theesophagealveinsmayenlargeconsiderablytoformesophagealvarices.Thesevaricesmayruptureandleadtosevere,frequentlyfatalinternalbleeding.TopreventthesegraveproblemsassociatedwithelevatedportalPvincirrhosisoftheliver,ananastomosis(portacavalshunt)isofteninsertedsurgicallybetweentheportalveinandtheinferiorvenacavatolowerportalPv.

Fetal circulation differs from the circulation in postnatal infants. Of most importance is that the fetal lungs are functionally inactive, and the fetus depends completely on the placenta for O2 and nutrients. Oxygenated fetal blood from the placenta passes through the umbilical vein to the fetal liver. Approximately half the flow from the placenta passes through the liver, and the remainder bypasses the fetal liver and reaches the inferior vena cava through the ductus venosus (Fig. 17.44 ). Blood from the ductus venosus joins the blood returning from the lower part of the fetal trunk and the extremities in the inferior vena cava. This blood merges with blood from the fetal liver through the hepatic veins.

The streams of blood tend to maintain their characteristics in the inferior vena cava and are divided into two streams of unequal size by the edge of the interatrial septum (crista dividens). The larger stream, which contains mainly blood from the umbilical vein, is shunted from the inferior vena cava to the left atrium through the foramen ovale (see

Fig. 17.44 ). The other stream passes into the right atrium, where it merges with blood returning from the upper parts of the fetal body through the superior vena cava and with blood from the myocardium.

Unlike the ventricles in adults, those in a fetus operate essentially in parallel. Only a tenth of right ventricular output passes through the lungs because the pulmonary vascular resistance in the fetus is high. The remainder passes from the fetal pulmonary artery through the ductus arteriosus to the aorta at a point distal to the origins of the arteries to the fetal head and upper extremities. Blood flows from the pulmonary artery to the aorta because pulmonary vascular resistance is high, and the diameter of the ductus arteriosus is as large as that of the descending aorta.

The large volume of blood that passes through the foramen ovale into the fetal left atrium is joined by blood returning from the lungs, and it is pumped out by the left ventricle into the aorta. Most of the blood in the ascending aorta goes to the fetal head, upper thorax, and arms; the remainder joins blood from the ductus arteriosus and supplies the rest of the body. The amount of blood pumped by the left ventricle is approximately half that pumped by the right ventricle. The major fraction of the blood that passes down the descending aorta comes from the ductus arteriosus and right ventricle and flows by way of the two umbilical arteries to the placenta.

Oxygen saturation of fetal blood occurs at various loci (see

Fig. 17.44 ). Thus the fetal tissues that receive the most highly saturated blood are the liver, heart, and upper parts of the body, including the head.

At the placenta, the chorionic villi dip into the maternal sinuses, and O2, CO2, nutrients, and metabolic waste products are exchanged across the membranes. The barrier to exchange prevents equilibration of O2 between the two circulations at normal rates of blood flow. Therefore, the PO2 of the fetal blood that leaves the placenta is very low. Were it not for the fact that fetal hemoglobin has a greater affinity for O2 than adult hemoglobin does, the fetus would not receive an adequate O2 supply. The fetal oxyhemoglobin dissociation curve is shifted to the left. Therefore, at equal pressures of O2, fetal blood carries significantly more O2 than maternal blood does.

In early gestation, the high glycogen levels that prevail in cardiac myocytes may protect the heart from acute periods of hypoxia. Glycogen levels decrease in late gestation, and they reach adult levels by term.

The umbilical vessels have thick muscular walls that react to trauma, tension, sympathomimetic amines, bradykinin, angiotensin, and changes in PO2. In animals in which the umbilical cord is not tied, hemorrhage of the newborn is minimized by constriction of these large umbilical vessels in response to stretching of the umbilical arteries and by an associated increase in PO2 in systemic arteries.

Closure of the umbilical vessels increases total peripheral resistance and the arterial blood pressure of the infant. When blood flow through the umbilical vein ceases, the ductus venosus, a thick-walled vessel with a muscular sphincter, closes. The factor that initiates closure of the ductus venosus is unknown.

Ifapregnantwomanissubjectedtohypoxia,thereducedbloodPO2inthefetusevokestachycardiaandanincreaseinbloodflowthroughtheumbilicalvessels.Ifthehypoxiapersistsorifflowthroughtheumbilicalvesselsisimpaired,fetaldistressoccursandismanifestedinitiallyasbradycardia.

•Fig. 17.44 Schematic Diagram of the Fetal Circulation. Thenumbers representthepercentageofO2saturationofthebloodflowingintheindicatedbloodvessel.Fetalbloodthatleavestheplacentais80%saturated,butthesaturationofthebloodthatpassesthroughtheforamenovaleisreducedto67%.ThisreductioninO2saturationiscausedbythemixingofsaturatedbloodwithdesaturatedbloodreturningfromthelowerpartofthefetalbodyandtheliver.AdditionofthedesaturatedbloodfromthefetallungsreducestheO2saturationofleftventricularbloodto62%,whichisthelevelofsaturationofthebloodreachingthefetalheadandupperextremities.Thebloodintherightventricle—whichisamixtureofdesaturatedsuperiorvenacavalblood,coronaryvenousblood,andinferiorvenacavalblood—isonly52%saturatedwithO2.Whenthemajorportionofthisbloodtraversestheductusarteriosusandjoinsthatpumpedbytheleftventricle,theresultingO2saturationofthebloodtravelingtothelowerpartofthefetalbodyandbacktotheplacentais58%.Theinset atupperleftillustratesthedirectionofflowofamajorportionoftheinferiorvenacavalbloodthroughtheforamenovaletotheleftatrium.Arrows indicatethedirectionsofflow.(DatafromDawesGS,etal.J Physiol. 1954;126:563.)

Immediately after birth, the asphyxia caused by constriction or clamping of the umbilical vessels, together with cooling of the body, activates the respiratory center of the newborn infant. As the lungs fill with air, pulmonary vascular resistance decreases to approximately 10% of the value that existed before lung expansion. This change in vascular resistance is not caused by the presence of O2 in the lungs because the change is just as great if the lungs are filled with N2. However, filling the lungs with liquid does not reduce pulmonary vascular resistance.

After birth, left atrial pressure is raised above that in the inferior vena cava and right atrium by (1) the decrease in pulmonary resistance, with the consequent large flow of blood through the lungs to the left atrium; (2) the reduction of flow to the right atrium caused by occlusion of the umbilical vein; and (3) the increased resistance to left ventricular output produced by occlusion of the umbilical arteries. Reversal of the pressure gradient across the atria abruptly closes the valve over the foramen ovale, and the septal leaflets fuse over a period of several days.

The decrease in pulmonary vascular resistance causes the pressure in the pulmonary artery to fall to approximately half its previous level (to ≈35 mm Hg). This change in pressure, coupled with a slight increase in aortic pressure, reverses the flow of blood through the ductus arteriosus. However, within several minutes, the large ductus arteriosus begins to constrict. This constriction produces turbulent flow, which is manifested as a murmur in newborn infants. Constriction of the ductus arteriosus is progressive and usually complete within 1 to 2 days after birth. Closure of the ductus arteriosus appears to be initiated by the high PO2 of the arterial blood passing through it; pulmonary ventilation with O2 closes the ductus, whereas ventilation with air low in O2 opens this shunt vessel. Whether O2 acts directly on the ductus or through the release of a vasoconstrictor substance is not known.

At birth, the walls of the two ventricles have approximately equal thickness. In addition, the muscle layer of the pulmonary arterioles is thick; this thickness is partly responsible for the high pulmonary vascular resistance of the fetus. After birth, the thickness of the walls of the right ventricle diminishes, as does the muscle layer of the pulmonary arterioles. In contrast, the left ventricular walls become thicker. These changes progress over a period of weeks after birth and reflect the effects of different hemodynamic forces (e.g., vascular resistance) on the two ventricles. Cardiac hypertrophy underlies the increase of heart weight during the normal growth period after birth. The physical demands imposed by the developing cardiovascular system, together with increased levels of soluble factors (e.g., growth hormone, insulin-like growth factor-1), account for physiological hypertrophy by which left ventricular mass more than doubles during the period from birth to early adulthood.

Theductusarteriosusoccasionallyfailstocloseafterbirth.Inthenewborn,thiscongenitalcardiovascularabnormality,calledpatent ductus arteriosus, cansometimesbecorrectedbytheadministrationofnonsteroidalanti-inflammatoryagentssuchasibuprofen.Ifthisdoesnotresultinclosureoftheductusorifthechildisolder,closuremustbeachievedsurgically.

1.

The vascular system is composed of two major subdivisions: the systemic circulation and the pulmonary circulation. These subdivisions are in series with each other and are composed of a number of vessel types (e.g., arteries, arterioles, capillaries) that are aligned in series with one another. In general, the vessels of a given type are arranged in parallel with each other.

2.

The mean velocity (v) of blood flow in a given type of vessel is directly proportional to the total blood flow being pumped by the heart, and it is inversely proportional to the cross-sectional area of all the parallel vessels of that type.

3.

Poiseuille’s law characterizes blood flow that is steady and laminar in vessels larger than arterioles. However, blood flow is nonnewtonian in very small blood vessels (i.e., Poiseuille’s law is not applicable).

4.

Flow tends to become turbulent when (1) flow velocity is high, (2) fluid viscosity is low, (3) fluid density is great, (4) vessel diameter is large, or (5) the wall of the vessel is irregular.

5.

Arteries not only conduct blood from the heart to the capillaries but also store some of the ejected blood during each cardiac systole. Hence, blood flow continues through the capillaries during cardiac diastole. Veins return blood to the heart from the capillaries and have a relatively low resistance and high capacitance that enables them to serve as reservoirs for blood.

6.

The aging process diminishes compliance of the arteries, as well as of the veins. The less compliant the arteries are, the more work the heart must do to achieve a given cardiac output. The less compliant the veins are, the poorer is their ability to store blood.

7.

Mean arterial pressure varies directly with cardiac output and total peripheral resistance. Arterial pulse pressure varies directly with stroke volume but inversely with arterial compliance.

8.

Blood flow through capillaries is regulated chiefly by contraction of arterioles (resistance vessels). The capillary endothelium is the source of NO and PGI2, which relax vascular smooth muscles.

9.

Water and small solutes move between the vascular and interstitial fluid compartments through capillary pores mainly by diffusion but also by filtration and absorption. Molecules larger than approximately 60 kD are essentially confined to the vascular compartment. Lipid-soluble substances, such as CO2 and O2, pass directly through the lipid membranes of the capillary; the rate of transfer is directly proportional to their lipid solubility. Large molecules can move across the capillary wall in vesicles by pinocytosis. The vesicles are formed from the lipid membrane of the capillaries.

10.

Capillary filtration and absorption are described by Starling’s equation:

Filtration occurs when the algebraic sum of these terms is positive; absorption occurs when it is negative.

11. Fluid and protein that have escaped from blood capillaries enter lymphatic capillaries and are transported via the lymphatic system back to the blood vascular compartment.

12.

Physical factors that influence coronary arterial blood flow are the viscosity of the blood, frictional resistance of the vessel walls, aortic pressure, and extravascular compression of the vessels within the walls of the left ventricle. Left coronary arterial blood flow is restricted during ventricular systole by extravascular compression, and the flow is greatest during diastole, when the intramyocardial vessels are not compressed. Neural regulation of coronary arterial blood flow is much less important than metabolic regulation. Activation of the cardiac sympathetic nerves constricts the coronary resistance vessels. However, the enhanced myocardial metabolism caused by the associated increase in heart rate and contractile force produces vasodilation, which overrides the direct constrictor effect of sympathetic nerve stimulation. Stimulation of the cardiac branches of the vagus nerves causes slight dilation of the coronary arterioles. A striking parallelism exists between metabolic activity of the heart and coronary arterial blood flow. A decrease in O2 supply or an increase in O2 demand apparently releases vasodilators that decrease coronary arterial resistance. Of the known factors (CO2, O2, H+ , K+ , H2O2, adenosine) that can mediate this response, KATP channels, NO, H2O2 and adenosine are the most likely candidates, although CO2, O2, and H+ cannot be ruled out.

13.

Most of the resistance vessels in the skin are under dual control of the sympathetic nervous system and local vasodilator metabolites. The AV anastomoses found in the hands, feet, and face, however, are solely under neural control. The main function of skin blood vessels is to aid in the regulation of body temperature by constricting to conserve heat and by dilating to lose heat. Skin blood vessels dilate directly and reflexively in response to heat, and they constrict directly and reflexively in response to cold.

14.

Blood flow in skeletal muscle is regulated centrally by sympathetic nerves and locally by the release of vasodilator metabolites. In persons at rest, neural

Camici PG, d’Amati G, Rimoldi O. Coronary microvascular dysfunction: mechanisms and functional assessment. Nat Rev Cardiol. 2015;12:48.

Chiu J-J, Chien S. Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives. Physiol Rev. 2011;91:327.

Edwards G, Félétou M, Weston AH. Endothelium-derived hyper-polarization factors and associated pathways: a synopsis. Pflügers Arch. 2010;459:863.

Kara T, Narkiewicz K, Somers VK. Chemoreflexes—physiology and clinical implications. Acta Physiol Scand. 2003;177:377.

regulation of blood flow is paramount, but it yields to metabolic regulation during muscle contractions (as during exercise).

15.

Cerebral blood flow is regulated predominantly by metabolic factors, especially CO2, K+ , and adenosine. The increased regional cerebral activity produced by stimuli such as touch, pain, hand motion, talking, reading, reasoning, and problem solving are associated with enhanced blood flow in the activated area of the contralateral cerebral cortex. The neurovascular unit (microcirculation, pericytes, the extracellular matrix, astrocytes and neurons), a component of the blood-brain barrier, is thought to link brain activity with increased blood flow and oxygenation.

16.

The microcirculation in intestinal villi constitutes a countercurrent exchange system for O2. Because of the presence of this countercurrent exchange system, the villi are in jeopardy in states of low blood flow. The splanchnic resistance and capacitance vessels are very responsive to changes in sympathetic neural activity.

17.

The liver receives approximately 25% of cardiac output; approximately three fourths of this output is from the portal vein and approximately a fourth from the hepatic artery. When flow is diminished in either the portal or hepatic system, flow in the other system usually increases, but not proportionately. The liver tends to maintain constant O2 consumption, in part because its mechanism for extracting O2 from blood is so efficient. The liver normally contains approximately 15% of the total blood volume. It serves as an important blood reservoir for the body.

18.

In the fetus, a large percentage of right atrial blood passes through the foramen ovale to the left atrium, and a large percentage of pulmonary arterial blood passes through the ductus arteriosus to the aorta. At birth, the umbilical vessels, ductus venosus, and ductus arteriosus close by contraction of their muscle layers. The reduction in pulmonary vascular resistance caused by lung inflation is the main factor that reverses the pressure gradient between the atria and thereby causes the foramen ovale to close.

Ludmer PL, Selwyn AP, Shook TL, et al. Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries. N Engl J Med. 1986;315:1046.

Ohanyan V, Yin L, Bardakjian R, et al. Requisite role of Kv1.5 channels in coronary metabolic dilation. Circ Res. 2015;117:612.

Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of angiogenesis. Cell. 2011;146:873.

Resnick N, Yahav H, Shay-Salit A, et al. Fluid shear stress and the vascular endothelium: for better and for worse. Prog Biophys Mol Biol. 2003;81:177.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

How do the parasympathetic and sympathetic nervous systems regulate the functions of heart and vasculature?

2.

What factors affect the differential sympathetic regulation of resistance and capacitance vessels?

3.

How does the baroreceptor-mediated reflex mimic the operation of skeletal muscle proprioceptor reflex?

4.

What are the two major mechanisms, intrinsic to heart muscle, that regulate myocardial performance?

5.

What are the major hormones that regulate myocardial performance?

6.

How is myocardial performance affected by changes in the arterial blood concentrations of O2, CO2, and H+?

7.

What is the myogenic mechanism of vascular smooth muscle, and how does it participate in regulation of tissue blood flow?

8.

What are the humoral factors that participate in regulation of blood flow, and what are their actions?

Regulation of Heart Rate and Myocardial Performance

Cardiac output is defined as the quantity of blood pumped by the heart each minute. Cardiac output may be varied by a change in the heart rate or the volume of blood ejected from either ventricle with each heartbeat; this volume is called the stroke volume. Mathematically, cardiac output (CO) can be expressed as the product of heart rate (HR) and stroke volume (SV):

Equation 18.1

Thus to understand how cardiac activity is controlled, consider how the heart rate and stroke volume are regulated. Heart rate is regulated by the activity of the autonomic nervous system to modulate the intrinsic cardiac pacemaker. Stroke volume is determined by myocardial performance (which is determined by cardiac cell contractility) and by the hemodynamic loads on the heart. All of these determinants are interdependent, inasmuch as a change in one determinant of cardiac output almost invariably alters another.

Nervous Control of the Heart Rate

Although certain local factors, such as temperature changes and stretching of tissue, can affect the heart rate, the autonomic nervous system is the principal means by which the heart rate is controlled.

The average resting heart rate is approximately 70 beats per minute in normal adults, and it is significantly faster in children. During sleep, the heart rate decreases by 10 to 20 beats per minute. It may increase during emotional excitement, and during muscular exercise, it may increase to rates well above 150 beats per minute. In well-trained athletes, the usual resting rate is only approximately 50 beats per minute.

Both divisions of the autonomic nervous system tonically influence the cardiac pacemaker, which is normally the sinoatrial (SA) node. The sympathetic nervous system enhances automaticity, whereas the parasympathetic nervous system inhibits it. Changes in heart rate usually involve a reciprocal action of these two divisions of the autonomic nervous system. Thus the heart rate ordinarily increases with a combined decrease in parasympathetic activity and increase in sympathetic activity; the heart rate decreases with the opposite changes in autonomic neural activity.

Parasympathetic tone usually predominates in healthy, resting individuals. When a resting individual is given atropine, a muscarinic receptor antagonist that blocks parasympathetic effects, the heart rate generally increases substantially. If a resting individual is given propranolol, a β-adrenergic receptor antagonist that blocks sympathetic effects, the heart rate usually decreases only slightly (

Fig.

18.1 ). When both divisions of the autonomic nervous system are blocked, the heart rate of young adults averages approximately 100 beats per minute. The rate that prevails after complete autonomic blockade is called the intrinsic heart rate.

•Fig. 18.1 Effectsoffourequaldosesofatropine(muscarinicrecep (β-adrenergicreceptorantagonistthatblockssympatheticeffects)ontheheartratesof10healthyyoungmen.Inhalfthetrials,atropine wasgivenfirst(top curve); intheotherhalf,propranololwasgivenfirst(bottom curve). (RedrawnfromKatonaPG,etal.J Appl Physiol. •Fig. 18.2 Changesinheartrateevokedbystimulation(horizontal 1982;52:1652.) bars) ofthevagus(A) andsympatheticnerves(B). (ModifiedfromWarnerHR,CoxA.J Appl Physiol. 1962;17:349.)

The cardiac parasympathetic fibers originate in the medulla oblongata, in cells that lie in the dorsal motor nucleus of the vagus nerve or in the nucleus ambiguus (see ). In humans, centrifugal vagal fibers pass inferiorly through the neck near the common carotid arteries and then through the mediastinum to synapse with postganglionic vagal cells. These cells are located either on the epicardial surface or within the walls of the heart. Most of the vagal ganglion cells are located in epicardial fat pads near the SA and atrioventricular (AV) nodes.

The right and left vagus nerves are distributed to different cardiac structures. The right vagus nerve affects the SA node predominantly; stimulation of this nerve slows SA nodal firing and can even stop the firing for several seconds. The left vagus nerve mainly inhibits AV conduction tissue to produce various degrees of AV block (see ). However, the distribution of the efferent vagal fibers is overlapping in such a way that left vagal stimulation also •Fig. 18.3 Changes in Heart Rate When the Vagus and Cardiac

Sympathetic Nerves Are Stimulated Simultaneously. Thesympa depresses the SA node and right vagal stimulation impedes AV conduction.

The SA and AV nodes are rich in acetylcholinesterase, an enzyme that rapidly hydrolyzes the neurotransmitter acetylcholine (ACh). The effects of a given vagal stimulus decay very quickly (

Fig. 18.2A ) when vagal stimulation is discontinued because ACh is rapidly destroyed. In addition, vagal effects on SA and AV nodal function have a very short latency (≈50 to 100 msec) because the ACh released quickly activates special ACh-regulated potassium (KACh) channels in the cardiac cells. These channels open quickly because the muscarinic receptor is coupled directly to the KACh channel by a guanine nucleotide–binding protein. These two features of the vagus nerves—brief latency and rapid theticnerveswerestimulatedat0,2,and4Hzinthepresenceofvagalnervestimulation(Vag)at0,4,and8Hz.(ModifiedfromLevyMN,ZieskeH.J Appl Physiol. 1969;27:465.) decay of the response—enable them to exert beat-by-beat control of SA and AV nodal function.

Parasympathetic influences usually predominate over sympathetic effects at the SA node, as shown in

Fig. 18.3 . When the frequency of sympathetic stimulation increases from 0 to 4 Hz, the heart rate increases by approximately 80 beats per minute in the absence of vagal nerve stimulation (0 Hz). However, when the vagus nerves are stimulated at 8 Hz, increasing the sympathetic stimulation frequency from 0 to 4 Hz has only a negligible influence on heart rate.

The cardiac sympathetic fibers originate in the intermediolateral columns of the upper five or six thoracic segments and the lower one or two cervical segments of the spinal cord (see ). These fibers emerge from the spinal column through the white communicating branches and enter the paravertebral chains of ganglia. The preganglionic and postganglionic neurons synapse mainly in the stellate or middle cervical ganglia, depending on the species. In the mediastinum, postganglionic sympathetic and preganglionic parasympathetic fibers join to form a complicated plexus of mixed efferent nerves to the heart.

The postganglionic cardiac sympathetic fibers in this plexus approach the base of the heart along the adventitial surface of the great vessels. From the base of the heart, these fibers are distributed to the various chambers as an extensive epicardial plexus. They then penetrate the myocardium, usually accompanying the coronary vessels.

In contrast to abrupt termination of the response after vagal activity, the effects of sympathetic stimulation decay gradually after stimulation is stopped (see

Fig. 18.2B ). Nerve terminals take up to 70% of the norepinephrine released during sympathetic stimulation; much of the remainder is carried away by the bloodstream. These processes are slow. Furthermore, the facilitatory effects of sympathetic stimulation on the heart attain steady-state values much more slowly than do the inhibitory effects of vagal stimulation. The onset of the cardiac response to sympathetic stimulation begins slowly for two main reasons. First, norepinephrine appears to be released slowly from the sympathetic nerve terminals. Second, the cardiac effects of the neurally released norepinephrine are mediated mainly by a relatively slow second messenger system involving cyclic adenosine monophosphate (cAMP; see ). Hence, sympathetic activity alters the heart rate and AV conduction much more slowly than vagal activity does. Whereas vagal activity can exert beat-by-beat control of cardiac function, sympathetic activity cannot.

Stimulation of various brain regions can have significant effects on cardiac rate, rhythm, and contractility (see ). In the cerebral cortex, centers that regulate cardiac function are located in the anterior half of the brain, principally in the frontal lobe, the orbital cortex, the motor and premotor cortex, the anterior portion of the temporal lobe, the insula, and the cingulate gyrus. Stimulation of the midline, ventral, and medial nuclei of the thalamus elicits tachycardia. Stimulation of the posterior and posterolateral regions of the hypothalamus can also change the heart rate. Stimuli applied to the H2 field of Forel in the posterior hypothalamus evoke various cardiovascular responses, including tachycardia and associated limb movements; these changes resemble those observed during muscular exercise. Undoubtedly, the cortical and hypothalamic centers initiate the cardiac reactions that occur during excitement, anxiety, and other emotional states. The hypothalamic centers also initiate the cardiac response to alterations in environmental temperature. Experimentally induced temperature changes in the preoptic anterior hypothalamus alter the heart rate and peripheral resistance.

Stimulation of the parahypoglossal area of the medulla reciprocally activates cardiac sympathetic pathways and inhibits cardiac parasympathetic pathways. In certain dorsal regions of the medulla, distinct cardiac accelerator sites (increase the heart rate) and augmentor sites (increase cardiac contractility) have been detected in animals with transected vagus nerves. The accelerator regions are more abundant on the right side, whereas the augmentor sites are more prevalent on the left. A similar distribution also exists in the hypothalamus. Therefore, the sympathetic fibers mainly descend ipsilaterally through the brainstem.

Corticalcentershaveimportanteffectsonautonomicfunction.Theinsulaexertsdistinctregulationofthebalancebetweensympatheticandparasympatheticactionsonthecardiovascularsystem.Inpatientssubjectedtoelectricalstimulation,stimuliappliedtotheleftinsularcortexelicitpredominantlyparasympatheticresponses(bradycardiaandvasodepression),whereasstimuliappliedtotherightinsularcortexevokesympatheticactions(tachycardiaandvasopression).Aspredicted,patientswithacute,stroke-induceddamageoftheleftinsularcortexdisplayincreasedsympathetictoneandanincreasedriskofarrhythmiasandcardiovascularmortality.Whentherightinsularcortexisacutelyinvolvedinthestroke,theincidenceofcardiovascularmortality/morbidityisunchanged.

Fig. 18.4 ). Baroreceptors located in the aortic arch and carotid sinuses are responsible for this reflex (see the section ”). The inverse relationship between heart rate and arterial blood pressure is generally most pronounced over an intermediate range of arterial blood pressures. Below this intermediate range, the heart rate maintains a constant, high value; above this pressure range, the heart rate maintains a constant, low value.

The effects of changes in carotid sinus pressure on the activity in cardiac autonomic nerves are described in

Fig.

18.5 , which shows that over an intermediate range of carotid sinus pressures (100 to 180 mm Hg), reciprocal changes are evoked in efferent vagal and sympathetic neural activity. Below this range of carotid sinus pressure, sympathetic activity is intense, and vagal activity is virtually absent. Conversely, above the intermediate range of carotid sinus pressure, vagal activity is intense and sympathetic activity is minimal.

Bainbridge Reflex, Atrial Receptors, and Atrial Natriuretic Peptide

In 1915, Francis A. Bainbridge reported that infusing blood or saline into dogs accelerated their heart rate. This increase did not seem to be tied to arterial blood pressure because the heart rate rose regardless of whether arterial blood pressure did or did not change. However, Bainbridge also noted that the heart rate increased whenever central venous pressure rose sufficiently to distend the right side of the heart. This response is termed the Bainbridge reflex. Bilateral transection of the vagus nerves abolished this response.

Many investigators have confirmed Bainbridge’s observations and have noted that the magnitude and direction of the response depend on the prevailing heart rate. When the heart rate is slow, intravenous infusions of blood or electrolyte solutions usually accelerate the heart. At more rapid heart rates, however, such infusions ordinarily slow the heart. What accounts for these different responses? Increases in blood volume not only evoke Bainbridge reflex but also activate other reflexes (of note, the baroreceptor reflex). These other reflexes tend to elicit opposite changes in heart rate. Therefore, changes in heart rate evoked by an alteration in blood volume are the result of these antagonistic reflex effects (

Fig. 18.6 ). Evidently, the Bainbridge reflex predominates over the baroreceptor reflex when blood volume rises, but the baroreceptor reflex prevails over the Bainbridge reflex when blood volume diminishes.

•Fig. 18.4 Experimentaldatashowingthatheartratedecreasesasarterialpressureisincreased.(AdaptedfromCornishKG,etal.Am J Physiol. 1989;257:r595.)250 200 150 100 50 50 75 100 125 150 Arterial pressure (mm Hg) Heart rate (beats/min)

Both atria have receptors that are affected by changes in blood volume and that influence the heart rate. These receptors are located principally in the venoatrial junctions: in the right atrium at its junctions with the venae cavae and in the left atrium at its junctions with the pulmonary veins. Distention of these atrial receptors sends afferent impulses to the brainstem in the vagus nerves. The efferent impulses are carried from the brainstem to the SA node by fibers from both autonomic divisions.

The cardiac response to these changes in autonomic neural activity is highly selective. Even when the reflex increase in heart rate is large, changes in ventricular contractility are generally negligible. Furthermore, the neurally induced increase in heart rate is not usually accompanied by an increase in sympathetic activity in the peripheral arterioles.

•Fig. 18.5 Experimentaldatashowingthatincreasesincarotidsinuspressureresultindecreasedsympatheticefferentnerve(Symp)activityandincreasedcardiacvagalnerveactivity.(AdaptedfromKollaiM,KoizumiK.Pflügers Arch. 1989;413:365.)100 50 0 60 100 140 180 220 Sinus pressure (mm Hg) Vagal activity (% of max) 100 50 0 Symp. activity (% of max) •Fig. 18.6 Intravenous Infusions of Blood or Electrolyte Solutions Tend to Increase the Heart Rate Through the Bainbridge Reflex and to Decrease the Heart Rate Through the Baroreceptor Reflex. Theactualchangeinheartrateinducedbysuchinfusionsistheresultofthesetwoopposingeffects.Intravenous infusion Increases right atrial pressure Atrial receptors stimulated Bainbridge reflex – + Increases cardiac output Increases arterial pressure Baroreceptor reflex Heart rate

Stimulation of the atrial receptors increases not only the heart rate but also urine volume. Reduced activity in the renal sympathetic nerve fibers may partially account for this diuresis. However, the principal mechanism appears to be a neurally mediated reduction in vasopressin (antidiuretic hormone) secretion by the posterior pituitary gland (see ). Stretch of the atrial walls also releases atrial natriuretic peptide (ANP) from the atria.

ANP, a 28–amino acid peptide, exerts potent diuretic and natriuretic effects on the kidneys (see also ) and vasodilator effects on the resistance and capacitance vessels. Thus ANP is an important regulator of blood volume and blood pressure.

Incongestiveheartfailure,NaClandwaterareretained,mainlybecausestimulationbytherenin-angiotensinsystemincreasesthereleaseofaldosteronefromtheadrenalcortex.TheplasmalevelofANPisalsoincreasedincongestiveheartfailure.ByenhancingtherenalexcretionofNaClandwater,ANPgraduallyreducesfluidretentionandtheconsequentelevationsincentralvenouspressureandcardiacpreload.

Rhythmic variations in heart rate, occurring at the frequency of respiration, are detectable in most individuals and tend to be more pronounced in children. The heart rate typically accelerates during inspiration and decelerates during expiration (Fig. 18.7 *The myocytes of the ventricles secrete a related peptide in response to stretch. This peptide, termed brain natriuretic peptide (BNP) because of its initial discovery in the central nervous system, has actions similar to those of ANP (see •Fig. 18.7 Respiratory Sinus Arrhythmia. Notethattheheartrateincreasesduringinspirationanddecreasesduringexpiration.(ModifiedfromWarnerMR,etal.Am J Physiol. 1986;251:H1134.)50 80 110 140 Expiration Time (sec) Inspiration 3 6 9 12 150 Heart rate(beats/min)

Recordings from cardiac autonomic nerves reveal that neural activity increases in the sympathetic fibers during inspiration and increases in the vagal fibers during expiration. The heart rate response to cessation of vagal stimulation is very quick because, as already noted, ACh released from the vagus nerves is rapidly hydrolyzed by acetylcholinesterase. This short latency enables the heart rate to vary rhythmically at the respiratory frequency. Conversely, the norepinephrine released periodically at the sympathetic endings is removed very slowly. Therefore, the rhythmic variations in sympathetic activity that accompany inspiration do not induce any appreciable oscillatory changes in heart rate. Thus respiratory sinus arrhythmia is brought about almost entirely by changes in vagal activity. In fact, respiratory sinus arrhythmia is exaggerated when vagal tone is enhanced.

Fig. 18.8 ). Stretch receptors in the lungs are stimulated during inspiration, and this action leads to a reflex increase in heart rate. The afferent and efferent limbs of this reflex are located in the vagus nerves. Intrathoracic pressure also decreases during inspiration and thereby increases venous return to the right side of the heart (see ). The consequent stretch of the right atrium elicits the Bainbridge reflex. After the time delay required for the increased venous return to reach the left side of the heart, left ventricular output increases and raises arterial blood pressure. This rise in blood pressure in turn reduces the heart rate through the baroreceptor reflex.

Central factors are also responsible for respiratory cardiac arrhythmia. The respiratory center in the medulla directly influences the cardiac autonomic centers (see

Fig. 18.8). In heart-lung bypass studies, the chest is opened, the lungs are collapsed, venous return is diverted to a pump-oxygenator, and arterial blood pressure is maintained at a constant level. In such studies, rhythmic movement of the rib cage attests to the activity of the medullary respiratory centers, and is often accompanied by rhythmic changes in heart rate at the respiratory frequency. This respiratory cardiac arrhythmia is almost certainly induced by a direct interaction between the respiratory and cardiac centers in the medulla.

The cardiac response to peripheral chemoreceptor stimulation illustrates the complex interactions that may ensue when one stimulus excites two organ systems simultaneously. Stimulation of carotid chemoreceptors consistently increases ventilatory rate and depth (see ), but ordinarily it changes the heart rate only slightly. The magnitude of the ventilatory response determines whether the heart rate increases or decreases as a result of carotid chemoreceptor stimulation. Mild chemoreceptor-induced stimulation of respiration decreases the heart rate moderately; more pronounced stimulation increases the heart rate only slightly. If the pulmonary response to chemoreceptor stimulation is blocked, the heart rate response may be greatly exaggerated, as described later.

•Fig. 18.9 The Primary Effect of Stimulation of Peripheral Chemoreceptors on the Heart Rate Is to Excite the Cardiac Vagal Center in the Medulla and Thus to Decrease the Heart Rate. Peripheralchemoreceptorstimulationalsoexcitestherespiratorycenterinthemedulla.Thiseffectproduceshypocapniaandincreaseslunginflation,bothofwhichsecondarilyinhibitthemedullaryvagalcenter.Thusthesesecondaryinfluencesattenuatetheprimaryreflexeffectofperipheralchemoreceptorstimulationonheartrate.Medullary vagal center Peripheral chemoreceptors Respiratory activity Primary effect (+) Secondary effects (–) (+) (–) (–) Hypocapnia Increased lung stretch Heart rate •Fig. 18.8 Respiratorysinusarrhythmiaisgeneratedbyadirectinteractionbetweentherespiratoryandcardiaccentersinthemedulla,aswellasbyreflexesthatoriginatefromstretchreceptorsinthelungs,fromstretchreceptorsintherightatrium(theBainbridgereflex),andfrombaroreceptorsinthecarotidsinusesandaorticarch.Cardiac vagal center (medulla) Respiratory center (medulla) Change in intrathoracic pressure Change in venous return Change in arterial pressure Baroreceptor reflex Heart rate Change in lung volume (stretch receptors) Bainbridge reflex

The cardiac response to peripheral chemoreceptor stimulation is the result of primary and secondary reflex mechanisms (

Fig. 18.9). The principal effect of the primary reflex stimulation is to excite the medullary vagal center and thereby decrease the heart rate. The respiratory system mediates secondary reflex effects. The respiratory stimulation by arterial chemoreceptors tends to inhibit the medullary vagal center. This inhibition varies with the level of concomitant stimulation of respiration; small increases in respiration inhibit the vagal center slightly, whereas large increases in ventilation inhibit the vagal center more profoundly.

An example of the primary inhibitory influence is shown in

Fig. 18.10 . In this example, the lungs are completely collapsed, and blood oxygenation is accomplished with an artificial oxygenator. When the carotid chemoreceptors are stimulated, intense bradycardia and some degree of AV block ensue. Such effects are mediated primarily by efferent vagal fibers.

The pulmonary hyperventilation that is ordinarily evoked by carotid chemoreceptor stimulation influences the heart rate secondarily, both by initiating more pronounced pulmonary inflation reflexes and by producing hypocapnia (see

Fig. 18.9). Both influences tend to depress the primary cardiac response to chemoreceptor stimulation and thereby accelerate the heart rate. Hence, when pulmonary hyper- ventilation is not prevented, the primary and secondary effects neutralize each other, and carotid chemoreceptor stimulation affects the heart rate only moderately.

Sensory receptors located near the endocardial surfaces of the ventricles initiate reflex effects similar to those elicited •Fig. 18.10 Changes in Heart Rate With Carotid Chemoreceptor Stimulation During Total Heart Bypass. Thelungsremaindeflated,andrespiratorygasexchangeisaccomplishedbyanartificialoxygenator.Thelowertracingrepresentstheoxygensaturationofthebloodperfusingthecarotidchemoreceptors.Thebloodperfusingtheremainderofthebody,includingthemyocardium,isfullysaturatedwithoxygen.(ModifiedfromLevyMN,etal.Circ Res. 1966;18:67.)

Fig.18.11 wasrecordedfromaquadriplegicpatientwhocouldnotbreathespontaneouslyandrequiredtrachealintubationandartificialrespiration.Whenthetrachealcatheterwasbrieflydisconnected(nearthebeginningofthetopstripinthefigure,indicatedbythearrow)toallownursingcare,profoundbradycardiadevelopedafter9heartbeats.Thepatient’sheartratewas65beatsperminutejustbeforethetrachealcatheterwasdisconnected.Inlessthan10secondsaftercessationofartificialrespiration,hisheartratedroppedtoapproximately20beatsperminute.Thisbradycardiacouldbepreventedbyblockingtheeffectsofefferentvagalactivitywithatropine,anditsonsetcouldbedelayedconsiderablybyhyperventilationofthepatientbeforethetrachealcatheterisdisconnected.

by the arterial baroreceptors. Excitation of these endocardial receptors causes the heart rate and peripheral resistance to diminish. Other sensory receptors have been identified in the epicardial regions of the ventricles. Although all these ventricular receptors are excited by various mechanical and chemical stimuli, their exact physiological functions remain unclear.

Regulation of Myocardial Performance

Intrinsic Regulation of Myocardial Performance

As noted previously, the heart can initiate its own beat in the absence of any nervous or hormonal control. The myocardium can also adapt to changing hemodynamic conditions by means of mechanisms that are intrinsic to cardiac muscle itself. For example, racing greyhounds with •Fig. 18.11 Electrocardiogram of a 30-Year-Old Quadriplegic Man Who Could Not Breathe Spontaneously and Required Tracheal Intubation and Artificial Respiration. Thetwostripsarecontinuous.(ModifiedfromBerkJL,LevyMN.Eur Surg Res. 1977;9:75.) denervated hearts perform almost as well as those with intact innervation. Their maximal running speed decreases by only 5% after complete cardiac denervation. In these dogs, the threefold to fourfold increase in cardiac output during a race is achieved principally by an increase in stroke volume. Normally, the increase in cardiac output with exercise is accompanied by a proportionate increase in heart rate; stroke volume does not change much (see ). This adaptation in the denervated heart is not achieved entirely by intrinsic mechanisms; circulating catecholamines undoubtedly contribute. For example, if β-adrenergic receptor antagonists are given to greyhounds with denervated hearts, their racing performance is severely impaired.

Ventricularreceptorshavebeenimplicatedintheinitiationofvasovagal syncope, afeelingoflightheadednessorbrieflossofconsciousnessthatmaybetriggeredbypsychologicalororthostaticstress.Theventricularreceptorsarebelievedtobestimulatedbyreducedventricularfillingvolumeincombinationwithvigorousventricularcontraction.Inapersonstandingquietly,ventricularfillingisdiminishedbecausebloodtendstopoolintheveinsintheabdomenandlegs,asexplainedin

Chapter17.

Consequently,thereductionincardiacoutputandarterialbloodpressureleadstoageneralizedincreaseinsympatheticneuralactivitythroughthebaroreceptorreflex(see

Fig.18.5 ).Theenhancedsympatheticactivitytotheheartevokesavigorousventricularcontractionthatstimulatestheventricularreceptors.Excitationoftheventricularreceptorsinitiatestheautonomicneuralchangesthatevokevasovagalsyncope:namely,acombinationofprofound,vagallymediatedbradycardiaandgeneralizedarteriolarvasodilationmediatedbyareductioninsympatheticneuralactivity.

Theheartispartiallyorcompletelydenervatedinvariousclinicalsituations:(1)asurgicallytransplantedheartistotallydenervated,althoughtheintrinsic,postganglionicparasympatheticfiberspersist;(2)atropineblocksvagaleffectsontheheart,andpropranololblockssympatheticβ-adrenergicinfluences;(3)certaindrugs,suchasreserpine,depletecardiacnorepinephrinestoresandtherebyrestrictorabolishsympatheticcontrol;and(4)inchroniccongestiveheartfailure,cardiacnorepinephrinestoresareoftenseverelydiminished,andanysympatheticinfluencesareattenuated.

Two principal intrinsic mechanisms, the Frank-Starling mechanism and rate-induced regulation, enable the myocardium to adapt to changes in hemodynamic conditions. The Frank-Starling mechanism (Frank-Starling law of the heart) is invoked in response to changes in the resting length of myocardial fibers. Rate-induced regulation is evoked by changes in the frequency of the heartbeat.

CHAPTER 18 Regulation of the Heart and Vasculature

In the 1910s, the German physiologist Otto Frank and the English physiologist Ernest Starling independently studied the response of isolated hearts to changes in preload and afterload (see

Chapter 16).

When ventricular filling pressure (preload) is increased, ventricular volume increases progressively, and after several beats, becomes constant and larger. At equilibrium, the volume of blood ejected by the ventricles (stroke volume) with each heartbeat increases to equal the greater quantity of venous return to the right atrium.

The increased ventricular volume facilitates ventricular contraction and enables the ventricles to pump a greater stroke volume. This increase in ventricular volume is associated with an increase in length of the individual ventricular cardiac fibers. The increase in fiber length alters cardiac performance mainly by altering the number of myofilament cross-bridges that interact (see

Chapter 16).

More recent evidence indicates that the principal mechanism involves a stretch-induced change in the sensitivity of cardiac myofilaments to Ca++ (see ). There exists an optimal fiber length, however. Excessively high filling pressures that overstretch the myocardial fibers may depress rather than enhance the pumping capacity of the ventricles (see

Fig. 16.36

Starling also showed that isolated heart preparations could adapt to changes in the counterforce to the ventricular ejection of blood during systole (i.e., afterload). As the left ventricle contracts, it does not eject blood into the aorta until the ventricle has developed a pressure that just exceeds the prevailing aortic pressure (see

Fig. 16.39 ). The aortic pressure during ventricular ejection essentially constitutes the left ventricular afterload. In Starling’s experiments, arterial pressure was controlled by a hydraulic device in the tubing that led from the ascending aorta to the right atrial blood reservoir. To hold venous return to the right atrium constant, the hydrostatic level of the blood reservoir was maintained. As Starling raised arterial pressure to a new constant level, the left ventricle responded at first to the increased afterload by pumping a diminished stroke volume. Because venous return was held constant, the diminution in stroke volume was accompanied by a rise in ventricular diastolic volume, as well as by an increase in the length of the myocardial fibers. This change in end-diastolic fiber length finally enabled the ventricle to pump a normal stroke volume against the greater peripheral resistance. As mentioned, a change in the number of cross-bridges between the thick and thin filaments probably contributes to this adaptation, but the major factor appears to be a stretch-induced change in the sensitivity of the contractile proteins to Ca++ .

Cardiac adaptation to alterations in heart rate also involves changes in ventricular volume. During bradycardia, for example, the increased duration of diastole allows greater ventricular filling. The consequent increase in myocardial fiber length increases stroke volume. Therefore, the reduction in heart rate may be fully compensated by the increase in stroke volume, and cardiac output may therefore remain constant.

When cardiac compensation involves ventricular dilation, the effect of the increased size of the ventricle on the generation of intraventricular pressure must be considered. According to Laplace’s relationship (see

Chapter 17), if the ventricle enlarges, the force required by each myocardial fiber to generate a given intraventricular systolic pressure must be appreciably greater than that developed by the fibers in a ventricle of normal size. Thus more energy is required for a dilated heart to perform a given amount of external work than for a normal-sized heart to do so. Hence, computation of afterload on contracting myocardial fibers in the walls of the ventricles must account for ventricular dimensions along with intraventricular (and aortic) pressure.

The relatively rigid pericardium that encloses the heart determines the pressure-volume relationship at high levels of pressure and volume. The pericardium limits heart volume even under normal conditions, when an individual is at rest and the heart rate is slow. In patients with chronic congestive heart failure, the sustained cardiac dilation and hypertrophy may stretch the pericardium considerably. In such patients, the pericardial limitation of cardiac filling is exerted at pressures and volumes entirely different from those in normal individuals.

To assess changes in ventricular performance, the Frank-Starling mechanism is often represented by a family of ventricular function curves. To construct a control ventricular function curve, for example, blood volume is altered over a range of values, and stroke work (i.e., stroke volume × mean arterial pressure) and end-diastolic ventricular pressure are measured at each step. Similar observations are then made during the desired experimental intervention. For example, the ventricular function curve obtained during infusion of norepinephrine lies above and to the left of the control ventricular function curve (

Fig. 18.12). Clearly, for a given level of left ventricular end-diastolic pressure (an index of preload), the left ventricle performs more work during the norepinephrine infusion than during control conditions. Hence, the upward and leftward shift of the ventricular function curve signifies improved ventricular contractility. Conversely, a shift downward and to the right indicates impaired contractility and a tendency toward cardiac failure.

The Frank-Starling mechanism is well suited to match cardiac output to venous return. Any sudden, excessive output by one ventricle soon causes an increase in venous return to the second ventricle. The consequent increase in diastolic fiber length in the second ventricle augments the output of that ventricle to correspond to the output of its mate. In this way, the Frank-Starling mechanism maintains a precise balance between the output of the right and left ventricles. If the two ventricles were not arranged in series in a closed circuit, any small but maintained imbalance in output of the two ventricles would be catastrophic.

•Fig. 18.12 Aconstantinfusionofnorepinephrineshiftstheventricu-larfunctioncurveupandtotheleft.Thisshiftsignifiesanenhancementinventricularcontractility.(RedrawnfromSarnoffSJ,etal.Circ Res. 1960;8:1108.)0 5 10 15 20 25 30 35 40 Norepinephrine Control 0 10 20 30 40 50 60 Left ventricular end-diastolic pressure (cm H2O) Left ventricular stroke work (g•m)

The curves that relate cardiac output to mean atrial pressure for the two ventricles do not coincide; the curve for the left ventricle usually lies below that for the right ventricle (

Fig. 18.13). At equal right and left atrial pressures

Fig. 18.13 ), right ventricular output exceeds left ventricular output. Hence, venous return to the left ventricle (a function of right ventricular output) exceeds left ventricular output, and left ventricular diastolic volume and pressure rise. According to the Frank-Starling mechanism, left ventricular output therefore increases (from point B toward point C in

Fig. 18.13 ). Only when the output of both ventricles is identical (points A and C) is equilibrium reached. Under such conditions, however, left atrial pressure (point C) exceeds right atrial pressure (point A). This is precisely the relationship that ordinarily prevails.

Thefactthatleftatrialpressureexceedsrightatrialpressureaccountsfortheobservationthatinindividualswithcongenitalatrialseptaldefectsinwhichthetwoatriacommunicatewitheachotherviaapatentforamenovale,thedirectionofshuntflowisusuallyfromlefttotherightsideoftheheart.

•Fig. 18.13 Relationships Between the Output of the Right and Left Ventricles and Mean Pressure in the Right and Left Atria, Respectively. Atanygivenlevelofcardiacoutput,meanleftatrialpressure(e.g.,pointC)exceedsmeanrightatrialpressure(pointA).Atrial pressure Right Left A C B Cardiac output

Force0.632020 •Fig. 18.14 Changesindevelopmentofforce(y-axis)inanisolatedpapillarymuscleastheintervalbetweencontractionsisvariedfrom20secondsto0.63secondandthenbackto20seconds.(RedrawnfromKoch-WeserJ,BlinksJR.Pharmacol Rev. 1963;15:601.)

Myocardial performance is also regulated by changes in the frequency at which the myocardial fibers contract. The effects of changes in contraction frequency on the force developed in an isometrically contracting papillary muscle are shown in

Fig. 18.14 . Initially, the cardiac muscle is stimulated to contract once every 20 seconds. When the muscle is suddenly made to contract once every 0.63 seconds, the force developed increases progressively over the next several beats. At the new steady state, the force developed is more than five times greater than the force at the larger contraction interval. A return to the larger interval (20 seconds) has the opposite influence on the development of force.

The rise in the force developed when the contraction interval is decreased is caused by a gradual increase in intracellular [Ca++]. Two mechanisms contribute to the rise in intracellular [Ca++]: an increase in the number of depolarizations per minute and an increase in the inward Ca++ current per depolarization.

In the first mechanism, Ca++ enters the myocardial cell during each action potential plateau (see ). As the interval between beats is diminished, the number of plateaus per minute increases. Although the duration of •Fig. 18.15 Calcium Currents Induced in a Myocyte During the First (Labeled 1) and Seventh (Labeled 7) Depolarizations in a Consecutive Sequence of Depolarizations. Thearrows indicatethehalf-timesofinactivationofthecalciumcurrent,asobtainedfromkineticanalysis.Bytheseventhdepolarization,themaximalinwardrectifyingcalciumcurrenthadincreasedbymorethan50%,andthehalf-timeofinactivationhadincreasedby20msec.(ModifiedfromLeeKS.Proc Natl Acad Sci U S A. 1987;84:3941.) each action potential (and of each plateau) decreases as the interval between beats is reduced, the overriding effect of the increased number of plateaus per minute on the influx of Ca++ prevails, and intracellular [Ca++] increases.

In the second mechanism, as the interval between beats is suddenly diminished, the inward rectifying calcium current (iCa) progressively increases with each successive beat until a new steady state is attained at the new basic cycle length. In an isolated ventricular myocyte, influx of Ca++ into the myocyte increases on successive depolarizations (

Fig. 18.15 ). Both the increased magnitude and the slowed inactivation of iCa result in greater Ca++ influx into the myocyte during the later depolarizations than during the first depolarization. This greater Ca++ influx strengthens contraction.

Transient changes in the intervals between beats also profoundly affect the strength of contraction. When the left ventricle contracts prematurely (

Fig. 18.16 , beat A), the premature contraction (extrasystole) itself is weak, whereas contraction B (postextrasystolic contraction) after the compensatory pause is very strong. In the intact circulatory system, this response depends partly on the Frank-Starling mechanism. Inadequate time for ventricular filling just before the premature beat results in the weak premature contraction. Subsequently, the exaggerated degree of filling associated with the long compensatory pause (see

Fig.

18.16 , beat B) contributes to the vigorous postextrasystolic contraction.

The weakness of the premature beat is directly related to its degree of prematurity: The earlier the premature beat, the weaker its force of contraction. The curve that represents strength of contraction of a premature beat in relation to the coupling interval is called a mechanical restitution curve.

Fig. 18.17 shows the restitution curve obtained when the coupling intervals of test beats were isolated in an isolated ventricular muscle preparation.

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 Time (sec) •Fig. 18.16 Inanisovolumicleftventriclepreparation,aprematureventricularsystoliccontraction(beat

A)istypicallyweak,whereasthepostextrasystoliccontraction(beatB)ischaracteristicallystrong,andtheenhancedcontractilitymaydiminishoverafewcontractions(e.g.,beatC).(FromLevyMN.Unpublishedtracing.) 100 80 60 40 20 0 0 0.2 0.4 0.6 0.8 1.0 Test beat interval (sec) Test beat force (% of max) relatively large amount of Ca++ taken up by it during the time that had elapsed from the end of the last regular beat until the beginning of the postextrasystolic beat.

Extrinsic Regulation of Myocardial Performance

Although a completely isolated heart can adapt well to changes in preload and afterload, various extrinsic factors also influence the heart in an individual. Often, these extrinsic regulatory mechanisms may overwhelm the intrinsic mechanisms. The extrinsic regulatory factors may be subdivided into nervous and chemical components.

•Fig. 18.17 Force Generated During Premature Contractions in an Isolated Ventricular Muscle Preparation. Themusclewasstimulatedtocontractoncepersecond.Periodically,themusclewasstimulatedprematurely.Thescalealongthex-axisdenotesthetimebetweenthedrivenandtheprematurebeat.Thescalealongthey-axisdenotestheratioofthecontractileforceoftheprematurebeattothatofthedrivenbeat.(ModifiedfromSeedWA,WalkerJM.Cardiovasc Res. 1988;22:303.)

Restitution of the force of contraction depends on the time course of the intracellular circulation of Ca++ in cardiac myocytes during contraction and relaxation. During relaxation, the Ca++ that dissociates from the contractile proteins is taken up by the sarcoplasmic reticulum for subsequent release. However, there is a lag of approximately 500 to 800 msec before this Ca++ is available for release from the sarcoplasmic reticulum in response to the next depolarization. Thus the strength of the premature beat is reduced because the time during the preceding relaxation is insufficient to allow much of the Ca++ taken up by the sarcoplasmic reticulum to become available for release during the premature beat. Conversely, the postextrasystolic beat is considerably stronger than normal because more Ca++ is released from the sarcoplasmic reticulum as a result of the

Sympathetic nervous activity enhances atrial and ventricular contractility. The alterations in ventricular contraction evoked by electrical stimulation of the left stellate ganglion in an isovolumic left ventricle preparation are shown in

Fig.

18.18 . Note that the duration of systole is reduced and the rate of ventricular relaxation is increased during the early phases of diastole; both these effects assist ventricular filling. For any given cardiac cycle length, the abbreviated systole allows more time for diastole and hence for ventricular filling.

). Neurally released norepinephrine or circulating catecholamines interact with β-adrenergic receptors on the cardiac cell membranes (

Fig. 18.19 ). This interaction activates adenylate cyclase, which raises intracellular levels of cAMP (see ). Consequently, protein kinases that promote the phosphorylation of various proteins are activated within the myocardial cells. Phosphorylation of phospholamban facilitates reuptake of Ca++ by the sarcoplasmic reticulum, and phosphorylation of troponin I reduces the sensitivity of contractile proteins to Ca++ . These effects facilitate relaxation and reduce end-diastolic pressure (see ). Phosphorylation of specific sarcolemmal proteins also activates calcium channels in the membranes of myocardial cells.

Activation of calcium channels increases the influx of Ca++ during the action potential plateau, and more Ca++ is released from the sarcoplasmic reticulum in response to each cardiac excitation. The contractile strength of the heart is thereby increased.

Fig. 18.20 shows the correlation between the contractile force in a thin strip of ventricular muscle and the free [Ca++] (indicated by the aequorin light signal) in the myoplasm as the concentration of isoproterenol (a β-adrenergic agonist) is increased (see also

Fig. 13.3

The overall effect of increased cardiac sympathetic activity in intact animals can best be appreciated in terms of 0.1 0.2 0.3 0.4 0.5 0.6 0 0.1 0.2 0.3 0.4 0.5 0.6 Time (sec) •Fig. 18.18 Inanisovolumicleftventriclepreparation,stimulationofcardiacsympatheticnervesevokesasubstantialriseinpeakleftventricularpressureandinthemaximalratesofriseandfallinintraventricularpressure(dP/dt).(FromLevyMN.Unpublishedtracing.) families of ventricular function curves. When the frequency of electrical stimulation applied to the left stellate ganglion increases, the ventricular function curves shift progressively to the left. The changes parallel those produced by infusions of norepinephrine (see

Fig. 18.12 ). Hence, for any given left •Fig. 18.19 Interneuronal and Intracellular Mechanisms Responsible for Interactions Between the Sympathetic and Parasympathetic Systems in the Neural Control of Cardiac Function. AC,adenylatecyclase;ACh,acetycholine;ATP,adenosinetriphosphate;β,β-adrenergicreceptor;βγ,beta/gamma;cAMP,cyclicadenosinemonophosphate;GsandGi,stimulatoryandinhibitoryGproteins;M,muscarinicreceptor;NE,norepinephrine;NPY,neuropeptideY.(ModifiedfromLevyMN.In:KulbertusHE,FranckG,eds.Neurocardiology. Mt.Kisco,NY:Futura;1988.) •Fig. 18.20 Effects of Various Concentrations of Isoproterenol (Iso) on the Aequorin Light Signal (in Nanoamperes) and Contractile Force (in Millinewtons Per Square Millimeter) in a Rat Ventricular Muscle Injected With Aequorin. Theaequorinlightsignalreflectstheinstantaneouschangesinintracel-lular[Ca++].µM,micromolar.(ModifiedfromKuriharaS,KonishiM.Pflügers Arch. 1987;409:427.)0.1 sec Force Aequorin light signal 20 nA 0.2 µM Iso.0.1 µM Iso.0.05 µM Iso.Control 5 mN mm2 A B C D ventricular end-diastolic pressure, the ventricle can perform more work as sympathetic nervous activity is increased.

The vagus nerves inhibit the cardiac pacemaker, atrial myocardium, and AV conduction tissue. The vagus nerves also depress the ventricular myocardium, but the effects are less pronounced than in the atria. In pumping heart preparations, the ventricular function curve shifts to the right during vagal stimulation, an indication of reduced contractility. Vagus nerve stimulation suppresses the contractile force in the left ventricle. This is shown in pressure-volume curves obtained at constant ventricular rate (

Fig. 18.21 ). The negative inotropic effect of vagus nerve stimulation, depicted as the reduced slope of the end-systolic pressure-volume relation, is opposed by a muscarinic receptor antagonist and diminished by a β-adrenoceptor antagonist. The results indicate that vagus nerve stimulation reduces contractility in the heart and does so by at least two pathways.

Mechanisms for the vagal effects on the ventricular myocardium are shown in

Fig. 18.19 . ACh released by the vagal nerves interact with muscarinic receptors on the cardiac ventricular cell membrane to inhibit adenylate cyclase and the cAMP/protein kinase A cascade. This direct inhibition diminishes the Ca++ conductance of the cell membrane, reduces phosphorylation of the calcium channel, and hence decreases myocardial contractility. The ACh released from vagal nerves can also inhibit norepinephrine release by activating muscarinic receptors on neighboring sympathetic nerves, a mechanism of indirect inhibition. Thus vagal activity can decrease ventricular contractility partly by antagonizing any stimulatory effects that concomitant sympathetic activity may be exerting on ventricular contractility. Similarly, sympathetic nerves release norepinephrine and certain neuropeptides, including neuropeptide Y, which inhibits the release of ACh from neighboring vagal fibers (see

Fig. 18.19

The adrenal medulla is essentially a component of the auto ). The principal hormone secreted by the adrenal medulla is epinephrine; some norepinephrine is also released. The rate of secretion of these catecholamines by the adrenal medulla is regulated by mechanisms that control the activity of the sympathetic nervous system. Concentrations of catecholamines in blood thus rise under the same conditions that activate the sympathetic nervous system. However, the cardiovascular effects of circulating catecholamines are probably minimal under normal conditions. Moreover, the pronounced changes in myocardial contractility with exercise, for example, are mediated mainly by the norepinephrine released from cardiac sympathetic nerve fibers rather than by the catecholamines released from the adrenal medulla.

How adrenocortical steroids influence myocardial contractility is controversial. Cardiac muscle taken from adrenalectomized animals and placed in a tissue bath is more likely to fatigue in response to stimulation than is cardiac muscle obtained from normal animals. In some species, however, adrenocortical hormones enhance contractility. In addition, the glucocorticoid hydrocortisone potentiates the cardiotonic effects of catecholamines. This potentiation is mediated in part by the ability of adrenocortical steroids to inhibit the extraneuronal catecholamine uptake mechanisms.

•Fig. 18.21 Vagus Nerve Stimulation Reduces Ventricular Con tractility. Pressure-volumecurvesobtainedatconstantventricular ratebeforeopenheartsurgeryinhuman.A, Controlpressure-volumecurveswerecalculatedduringocclusionoftheinferiorvenacava.Theend-systolicpressure-volumerelation,definedbytheslopeofthestraight line, measuredapproximately4mmHg/mL.B, Duringstimulationoftheleftvagusnerve,theslopeoftheend-systolicpressure-volumerelationdecreasedtoapproximately3mmHg/mL,anindicationthatcontractilityhaddecreased.LV,leftventricular.(RedrawnfromLewisME,Al-KhalidiAH,BonserRS,etal.Vagusnervestimulationdecreasesleftventricularcontractilityinvivointhehumanandpigheart.J Physiol. 2001;534:547.)

Thyroid hormones enhance myocardial contractility. Rates of ATP hydrolysis and Ca++ uptake by the sarcoplasmic reticulum are increased in hyperthyroidism; the opposite effects occur in hypothyroidism. Thyroid hormones increase cardiac protein synthesis, and this response leads to cardiac hypertrophy. These hormones also affect the composition of myosin isoenzymes in cardiac muscle. By increasing isoenzymes with the greatest ATPase activity, thyroid hormones enhance myocardial contractility.

Cardiovascularproblemsarecommoninpatientswithadrenocorticalinsufficiency(Addison’sdisease).Bloodvolume

The cardiovascular changes in thyroid dysfunction also depend on indirect mechanisms. Thyroid hyperactivity tendstofall,whichmayleadtoseverehypotensionandcardiovascularcollapse,theso-calledaddisoniancrisis(see

Thyroidhormoneexertsitscardiacactionsbytwopaths:genomicandnongenomic.Thegenomicrouteinvolvesinteractionofthyroxine(T3)withnuclearreceptorsthatregulatethetranscriptionofT3-responsivegenes.Inhyperthyroidism,messengermRNAforcardiacmyocyteproteinsinvolvedinregulatingintracellular[Ca++](e.g.,sarcoplasmicendoplasmicreticulumcalcium-ATPase[SERCA],ryanodinechannel)isincreased,asareamountsofcontractileproteins(e.g.,myosinheavychain,actin,troponinI).Consequently,theratesofcontractionandrelaxationincreaseasATPhydrolysisandO2consumptionincrease.Inthehyperthyroidstate,theuseofATPislessefficient,andthefractionallossofheatisgreater.Ifuntreated,severehyperthyroidismcanresultinheartfailure.

Cardiacactivityisdepressedinpatientswithinadequatethyroidfunction(hypothyroidism).Theconverseistrueinpatientswithoveractivethyroidglands(hyperthyroidism).Characteristically,patientswithhyperthyroidismexhibittachycardia,highcardiacoutput,andarrhythmiassuchasatrialfibrillation.Insuchpatients,sympatheticneuralactivitymaybeincreased,orthesensitivityofthehearttosuchactivitymaybeenhanced.Studieshaveshownthatthyroidhormoneincreasesthedensityofβ-adrenergicreceptorsincardiactissue(seealso ).Inexperimentalanimals,thecardiovascularmanifestationsofhyperthyroidismmaybesimulatedbytheadministrationofexcessthyroxine.

increases the body’s metabolic rate, which in turn results in arteriolar vasodilation. The consequent reduction in total peripheral resistance increases cardiac output, as explained in

Insulin has a positive inotropic effect on the heart. The effect of insulin is evident even when hypoglycemia is prevented by glucose infusions and when β-adrenergic receptors are

CHAPTER 18 Regulation of the Heart and Vasculature blocked. Indeed, the positive inotropic effect of insulin is potentiated by β-adrenergic receptor antagonists. The enhanced contractility cannot be explained satisfactorily by the concomitant augmentation of glucose transport into myocardial cells.

Glucagon has potent positive inotropic and chronotropic effects on the heart. This endogenous hormone is probably not important in normal regulation of the cardiovascular system, but it has been used clinically to enhance cardiac performance. The effects of glucagon on the heart and certain metabolic effects are similar to those of catecholamines. Both glucagon and catecholamines activate adenylate cyclase to increase myocardial levels of cAMP. The catecholamines activate adenylate cyclase by interacting with β-adrenergic receptors, but glucagon activates this enzyme by a different mechanism. Nevertheless, the rise in cAMP increases influx of Ca++ through calcium channels in the sarcolemma and facilitates release and reuptake of Ca++ by the sarcoplasmic reticulum, just as catecholamines do.

The cardiovascular derangements in hypopituitarism are related principally to the associated deficiencies in adrenocortical and thyroid function. Growth hormone affects the myocardium, at least in combination with thyroxine. In hypophysectomized animals, growth hormone alone has little effect on the depressed heart, whereas thyroxine by itself restores adequate cardiac performance under basal conditions. However, when blood volume or peripheral resistance is increased, thyroxine alone does not restore adequate cardiac function, but the combination of growth hormone and thyroxine reestablishes normal cardiac performance. In certain animal models of heart failure, administration of growth hormone alone increases cardiac output and myocardial contractility.

Changes in cardiac performance as a result of stimulation of central and peripheral chemoreceptors have been described previously in this chapter. These effects usually predominate. However, O2 and CO2 do have direct effects on the myocardium.

Hypoxia has a biphasic effect on myocardial performance. Mild hypoxia stimulates performance, but more severe hypoxia depresses performance because oxidative metabolism is limited.

An increase in partial pressure of carbon dioxide (PCO2)— which results in a decrease in pH—has a direct depressant effect on the heart. This effect is mediated by changes in intracellular pH. A reduction in intracellular pH, induced by an increase in PCO2, diminishes the amount of Ca++ released from the sarcoplasmic reticulum in response to excitation. The diminished pH also decreases the sensitivity of the myofilaments to Ca++ . Increases in intracellular pH have the opposite effect; that is, they enhance sensitivity to Ca++ .

Regulation of the Peripheral Circulation

The peripheral circulation is essentially under dual control: centrally through the nervous system and locally by conditions in tissues surrounding the blood vessels. Nervous and humoral regulation of vascular smooth muscle is described in

Fig. 14.7

Table 14.1 ), in which transmitters, hormones and their receptors are discussed. Aspects of local control are discussed in , in which the relative importance of these two control mechanisms is shown to vary in different tissues.

The arterioles are involved in regulating the rate of blood flow throughout the body. These vessels offer the greatest resistance to the flow of blood pumped to the tissues by the heart, and thus these vessels are important in the maintenance of arterial blood pressure. The arteriole walls are composed in large part of smooth muscle fibers that allow the diameter of the vessel lumen to vary (see

Fig.

15.2 ). When this smooth muscle contracts strongly, the endothelial lining folds inward and completely obliterates the vessel lumen. When the smooth muscle is completely relaxed, the vessel lumen is maximally dilated. Some resistance vessels are closed at any given time. In addition, the smooth muscle in these vessels is partially contracted (which accounts for the tone of these vessels). If all the resistance vessels in the body dilated simultaneously, arterial blood pressure would fall precipitously.

Vascular smooth muscle controls total peripheral resistance, arterial and venous tone, and the distribution of blood flow throughout the body. The properties of vascular smooth muscle are discussed in . In the following sections, intrinsic and extrinsic control of vascular smooth muscle tone, and thus perfusion of peripheral tissues, is reviewed.

Intrinsic or Local Control of Peripheral Blood Flow

In certain tissues, blood flow is adjusted to the existing metabolic activity of the tissue. Furthermore, when tissue metabolism is steady, changes in perfusion pressure (arterial blood pressure) evoke changes in vascular resistance that tend to maintain a constant blood flow. This myogenic mechanism, which is illustrated graphically in

Fig. 18.22 , is commonly referred to as autoregulation of blood flow. When pressure is abruptly increased or decreased from a control pressure of 100 mm Hg, flow increases or decreases, respectively. However, even with pressure maintained at its new level, blood flow returns toward the control level within 30 to 60 seconds.

•Fig. 18.22 Pressure-flowrelationshipinthevascularbedoftheskeletalmuscle.Filled (red) circles representtheflowratesobtainedimmediatelyafterabruptchangesinperfusionpressurefromthecontrollevel(thepointwherelinescross).Open (blue) circles representthesteady-stateflowratesobtainedatthenewperfusionpressure.(RedrawnfromJonesRD,BerneRM.Circ Res. 1964;14:126.)0 20 40 60 10080 120 140 160 180 0 2 4 6 8 10 12 Perfusion pressure (mm Hg) Muscle blood flow (mL/min/100 g)

Over the pressure range of 20 to 120 mm Hg, the steady-state flow is relatively constant. Calculation of hydraulic resistance (pressure/flow) across the vascular bed during steady-state conditions shows that the resistance vessels constrict with an elevation in perfusion pressure but dilate with a reduction in perfusion pressure. This response to perfusion pressure is independent of the endothelium because it is identical in intact vessels and in vessels that have been stripped of their endothelium. According to the myogenic mechanism, vascular smooth muscle contracts in response to an increase in the pressure difference across the wall of a blood vessel (transmural pressure), and it relaxes in response to a decrease in transmural pressure. The signaling mechanisms that allow distention of a vessel to elicit contraction are unknown. However, because stretch of vascular smooth muscle has been shown to raise intracellular [Ca++], an increase in transmural pressure is believed to activate membrane calcium channels.

Transientreceptorpotential(TRP)channelshavebeenimplicatedinthemyogenicmechanism.ThesechannelsaremammalianhomologuesofaDrosophila melanogaster genethat,whenmutated,allowsonlyatransientresponsetoasustainedlightstimulus.Thepressure-inducedvasoconstrictiveresponseofanartery(myogenicresponse)appearstohavethefollowingsignalpath:pressure → increasedphospholipaseCactivity→ synthesisofdiacylglycerol→ activationofTRPchannel→ smoothmuscledepolarizationandopeningofL-typecalciumchannelsthatincreaseintracellular[Ca++]andmuscletone.Thisisameansofregulatingvascularresistance.OtherTRPchanneltypeshavebeenproposedtoparticipateinchronichypoxicpulmonaryhypertensionandinthevasoconstrictioncausedbytheα-adrenergicagonistnorepinephrine.

In normal subjects, blood pressure is maintained at a fairly constant level via the baroreceptor reflex. Hence, the myogenic mechanism may play little role in regulating blood flow to tissues under normal conditions. However, when a person changes from a lying to a standing position, transmural pressure rises in the lower extremities, and the precapillary vessels constrict in response to this imposed stretch.

, the endothelium lining the vasculature produces a number of substances that can relax (e.g., nitric oxide) or contract (e.g., angiotensin II and endothelin) vascular smooth muscle. Thus the endothelium plays an important role in regulating blood flow to specific vascular beds.

The metabolic activity of a tissue governs blood flow in that tissue. Any intervention that results in inadequate O2 supply prompts the formation of vasodilator metabolites that are released from the tissue and act locally to dilate the resistance vessels. When the metabolic rate of the tissue increases, or when O2 delivery to the tissue decreases, more vasodilator substances are released (see

Chapter 17).

Potassium, inorganic phosphate ions, and interstitial fluid osmolarity induce vasodilation. During skeletal muscle contraction, both (1) K+ and phosphate are released and (2) osmolarity is increased. Therefore, these factors may contribute to active hyperemia (increase in blood flow caused by enhanced tissue activity). However, significant increases in the phosphate concentration and in osmolarity are not always observed during muscle contraction, and they may increase blood flow only transiently. Therefore, they probably do not mediate the vasodilation observed during muscular activity.

Potassium is released at the onset of skeletal muscle contraction or with an increase in cardiac muscle activity. Hence, release of K+ could underlie the initial decrease in vascular resistance observed in response to physical exercise or to increased cardiac work. However, release of K+ is not sustained, but arteriolar dilation persists throughout the period of enhanced muscle activity. Furthermore, reoxygenated venous blood obtained from active cardiac and skeletal muscles does not elicit vasodilation when the blood is infused into a test vascular bed. It is unlikely that oxygenation of venous blood alters its K+ or phosphate content or its osmolarity and thereby neutralizes its vasodilator effect. Therefore, some agent other than K+ must mediate the vasodilation associated with metabolic activity of the tissue.

Adenosine, which contributes to the regulation of coronary blood flow, may also participate in control of the resistance vessels in skeletal muscle. In addition, some prostaglandins may be important vasodilator mediators in certain vascular beds. Many prostaglandins have thus been

CHAPTER 18 Regulation of the Heart and Vasculature proposed as mediators of metabolic vasodilation, and the relative contribution of each remains to be determined.

Metabolic control of vascular resistance by the release of a vasodilator substance requires the existence of a basal vessel tone. Tonic activity in vascular smooth muscle is readily demonstrable, but in contrast to tone in skeletal muscle, the tone in vascular smooth muscle is independent of the nervous system. Thus some metabolic factor must be responsible for maintaining this tone. The following factors may be involved: (1) the myogenic response to the stretch imposed by blood pressure, (2) the high partial pressure of oxygen in arterial blood (PaO2), or (3) the presence of Ca++ .

If arterial inflow to a vascular bed is stopped temporarily, blood flow on release of the occlusion immediately exceeds the flow that prevailed before occlusion, and the flow gradually returns to the control level. This increase in blood flow is called reactive hyperemia. This type of event provides evidence for the existence of a local metabolic factor that regulates tissue blood flow.

In the experiment shown in

Fig. 18.23 , blood flow to the leg was stopped by clamping of the femoral artery for 15, 30, and 60 seconds. Release of the 60-second occlusion resulted in a peak blood flow that was 70% greater than the control flow, and the flow returned to the control level within 110 seconds.

Within limits, peak flow and particularly the duration of reactive hyperemia are proportional to the duration of the occlusion (see

Fig. 18.23 ). If the extremity is exercised during the occlusion period, reactive hyperemia is increased. These observations and the close relationship between metabolic activity and blood flow in an unoccluded limb are consistent with the notion of a metabolic mechanism in the local regulation of tissue blood flow.

•Fig. 18.23 Graphsofreactivehyperemiainthehindlimbofthelegafter15-,30-,and60-secondocclusionofthefemoralartery.(FromBerneRM.Unpublishedobservations.)0 2 4 6 8 10 0 20 40 60 80 100 Time (min) Femoral blood flow(mL/min) 0 20 40 60 80 100 120 Femoral arterial pressure(mm Hg)

Coordination of Arterial and Arteriolar Dilation

When the vascular smooth muscle of arterioles relaxes in response to vasodilator metabolites whose release is caused by a decrease in the ratio of O2 supply to O2 demand of the tissue, resistance may diminish concomitantly in the small upstream arteries that feed these arterioles. The result is blood flow greater than that produced by arteriolar dilation alone. There are two possible mechanisms for this coordination of arterial and arteriolar dilation. First, the vasodilation in the microvessels may be propagated, and when dilation is initiated in the arterioles, it can propagate along the vessels from the arterioles back to the small arteries. Second, the metabolite-mediated dilation of the arterioles accelerates blood flow in the feeder arteries. This greater blood flow velocity increases the shear stress on the arterial endothelium, which in turn can induce flow-mediated vasodilation by release of one or more vasodilators (e.g., nitric oxide, prostacyclin, H2O2, epoxyeicosatrienoic acid).

Inthelegs,diseaseofthearterialwallscanleadtoobstructionofthearteriesandsymptoms,aconditioncalledintermittent claudication. Thesymptomsconsistoflegpainwhenthepersonwalksorclimbsstairs,andthepainisrelievedbyrest.Thediseaseiscalledthromboangiitis obliterans, anditappearsmostfrequentlyinmenwhoaresmokers.Withminimalwalking,theresistancevesselsbecomemaximallydilatedbylocalreleaseofmetabolites;whentheO2demandofthemusclesincreaseswithmorerapidwalking,bloodflowcannotincreasesufficientlytomeetthemuscleneedsforO2,andpaincausedbymuscleischemiaresults.

Extrinsic Control of Peripheral Blood Flow

Several regions in the cerebral medulla influence cardiovascular activity. Stimulation of the dorsal lateral medulla (pressor region) evokes vasoconstriction, cardiac acceleration, and enhanced myocardial contractility. Stimulation of cerebral centers caudal and ventromedial to the pressor region decreases arterial blood pressure. This depressor area exerts its effect by direct inhibition of spinal regions and by inhibition of the medullary pressor region. These areas are not true anatomical centers in which a discrete group of cells is discernible, but they constitute “physiological” centers.

The cerebrospinal vasoconstrictor regions are tonically active. Reflexes or humoral stimuli that enhance this activity increase the frequency of impulses that reach the terminal neural branches to the vessels. A constrictor neurohumor (norepinephrine) is released at the terminals to elicit a constrictive α-adrenergic effect on the resistance vessels. Inhibition of the vasoconstrictor areas diminishes the impulse frequency in the efferent nerve fibers, and vasodilation results. Thus neural regulation of the peripheral circulation is achieved mainly by alteration in the impulse frequency in the sympathetic nerves to the blood vessels. Surgical section of the sympathetic nerves to an extremity abolishes sympathetic vascular tone and thereby increases blood flow to that limb. With time, vascular tone is regained by an increase in basal (intrinsic) tone.

Both the pressor and depressor regions may undergo rhythmic changes in tonic activity that are manifested as oscillations in arterial pressure. Some rhythmic changes (Traube-Hering waves) occur at the frequency of respiration and are caused by a cyclic fluctuation in sympathetic impulses to the resistance vessels. Other fluctuations in sympathetic activity (Mayer waves) occur at a frequency lower than that of respiration.

Vasoconstrictor fibers of the sympathetic nervous system supply the arteries, arterioles, and veins; the neural influence is much less on larger vessels than on arterioles and small arteries. Capacitance vessels (veins) respond more to sympathetic nerve stimulation than do resistance vessels; the capacitance vessels are maximally constricted at a lower stimulation frequency than the are resistance vessels. However, capacitance vessels lack β-adrenergic receptors, and they respond less to vasodilator metabolites. Norepinephrine is the neurotransmitter released at the sympathetic nerve terminals in blood vessel. Factors such as circulating hormones and particularly locally released substances mediate the release of norepinephrine from the nerve terminals.

The response of the resistance and capacitance vessels to stimulation of sympathetic fibers is illustrated in

Fig.

18.24 . When arterial pressure is held constant, stimulation of sympathetic fibers reduces blood flow (constriction of resistance vessels) and decreases the blood volume of the tissue (constriction of capacitance vessels). Constriction of the resistance vessels establishes a new equilibrium of the forces responsible for filtration and absorption across the capillary wall (see

Eq. 17.20

In addition to active changes (contraction and relaxation of vascular smooth muscle) in vessel caliber, passive changes are also caused by alterations in intraluminal pressure. An increase in intraluminal pressure distends the vessels, and a decrease reduces the caliber of the vessels as a consequence of elastic recoil of the vessel walls.

At basal vascular tone, approximately a third of the blood volume of a tissue can be mobilized when the sympathetic nerves are stimulated at physiological frequencies. Basal tone is very low in capacitance vessels; if these vessels are denervated experimentally, the increases in volume evoked

Change of tissue Arterial pressureBlood flow volume (mL) (mm Hg) •Fig. 18.24 Effect of Sympathetic Nerve Stimulation (2 Hz) on Blood Flow and Tissue Volume of the Lower Limb. Theupward arrow denotesthechangeinslopeofthetissuevolumecurveatthepointatwhichthedecreaseinvolumecausedbyemptyingofcapacitancevesselsceasesandlossofextravascularfluidbecomesevident.Theabruptdecreaseintissuevolumeiscausedbymovementofbloodoutofthecapacitancevesselsandoutofthelowerlimb.Thelate,slow,progressivedeclineinvolume(totherightofthearrow)iscausedbythemovementofextravascularfluidintothecapillariesandhenceawayfromthetissue.Thelossoftissuefluidresultsfromtheloweringofthecapillaryhydrostaticpressuresecondarytoconstrictionoftheresistancevessels.(FromMellanderS.Acta Physiol Scand Suppl. 1960;50[176]:1.) by maximal doses of ACh are small. Therefore, at basal vascular tone, blood volume is close to the maximal blood volume of the tissue. More blood can be mobilized from the capacitance vessels in the skin than from those in the muscle. This disparity depends in part on the greater sensitivity of the skin vessels to sympathetic stimulation, but it also occurs because basal tone is lower in skin vessels than in muscle vessels. Therefore, in the absence of a neural influence, skin capacitance vessels contain more blood than do muscle capacitance vessels.

Physiological stimuli mobilize blood from capacitance vessels. For example, during physical exercise, activation of sympathetic nerve fibers constricts the peripheral veins and hence augments cardiac filling pressure. In arterial hypo-tension (as occurs in hemorrhage), the capacitance vessels constrict and thereby correct the decreased central venous pressure associated with blood loss.

The efferent fibers of the cranial division of the parasympathetic nervous system innervate the blood vessels of the head and some of the viscera, whereas fibers of the sacral division innervate blood vessels of the genitalia, bladder,

CHAPTER 18 Regulation of the Heart and Vasculature and large bowel. Skeletal muscle and skin do not receive parasympathetic innervation. The effect of cholinergic fibers on total vascular resistance is small because only a small proportion of the resistance vessels of the body receive parasympathetic fibers.

Inhemorrhagicshock,theresistancevesselsconstrictandtherebyassistinthemaintenanceofnormalarterialbloodpressure.Witharterialhypotension,theenhancedarteriolarconstrictionalsoleadstoasmallmobilizationofbloodfromthetissuebyvirtueofrecoilofthepostarteriolarvesselswhenintraluminalpressureisreduced.Furthermore,extravascularfluidismobilizedbecauseofgreaterfluidabsorptionintothecapillariesinresponsetotheloweredcapillaryhydrostaticpressure.

Stimulation of the parasympathetic fibers to the salivary glands induces marked vasodilation. A vasodilator polypeptide, bradykinin, formed locally from the action of an enzyme on a plasma protein substrate in the glandular lymphatic vessels, mediates this vasodilation. Bradykinin is formed in other exocrine glands, such as the lacrimal and sweat glands. Its presence in sweat may be partly responsible for the dilation of cutaneous blood vessels.

Epinephrine and norepinephrine exert a powerful effect on peripheral blood vessels. In skeletal muscle, low concentrations of epinephrine dilate resistance vessels (β2-adrenergic effect), but high concentrations produce constriction (α1adrenergic effect), as noted in

Table 14.1.

In all vascular beds, the primary effect of norepinephrine is vasoconstriction. When stimulated, the adrenal gland can release epinephrine and norepinephrine into the systemic circulation. However, under physiological conditions, the effect of catecholamine release from the adrenal medulla is less important than norepinephrine release from sympathetic nerve endings.

Table 14.1 ). Some are released from the endothelium (e.g., nitric oxide, endothelin, thromboxane A2), whereas others are derived from perivascular tissues (e.g., histamine, adenosine, angiotensin II).

Areas of the cerebral medulla that mediate sympathetic and vagal effects are under the influence of neural impulses that originate in the baroreceptors, chemoreceptors, hypothalamus, cerebral cortex, and skin. These areas of the medulla are also affected by changes in the blood concentrations of CO2 and O2.

The baroreceptors (or pressoreceptors) are stretch receptors located in the carotid sinuses and in the aortic arch (

Figs. 18.25 18.26 ). The carotid sinuses are the slightly

Fig. 18.25 Diagrammaticrepresentationofthecarotidsinusandcarotidbodyandtheirinnervation.(RedrawnfromAdamsWE.The Comparative Morphology of the Carotid Body and Carotid Sinus.

Fig. 18.26 Anteriorviewoftheaorticarchshowingtheinnervationoftheaorticbodiesandbaroreceptors.(ModifiedfromNonidezJF.Anat Rec. 1937;69:299.)

Springfield,IL:CharlesCThomas;1958.)Left vagus nerveRight vagus nerve Right inferior cervical ganglion Right stellate ganglion Recurrent branch of right vagus Vagosympathetic fibers Brachiocephalic artery Baroreceptor fibers Cardiac nerves Aorta Right coronary artery Left inferior cervical ganglion Left subclavian ansa Left subclavian artery Recurrent branch of left vagus Aortic bodies Ductus arteriosus Pulmonary artery Left stellate ganglion Vagosympathetic fibers Vagosympathetic fibers widened areas at the origins of the internal carotid arteries. Impulses that arise in the carotid sinus travel up the carotid sinus nerve (nerve of Hering) to the glossopharyngeal nerve (cranial nerve IX) and, via the latter, to the nucleus of the tractus solitarius (NTS) in the medulla. The NTS is the site of the central projections of the chemoreceptors and baroreceptors. Stimulation of the NTS inhibits sympathetic nerve outflow to the peripheral blood vessels (depressor effect), whereas lesions of the NTS produce vasoconstriction (pressor effect). Impulses that arise in the aortic arch baroreceptors reach the NTS via afferent fibers in the vagus nerves.

Baroreceptor nerve terminals in the walls of the carotid sinus and aortic arch respond to the vascular stretch and deformation induced by changes in arterial blood pressure. The frequency of firing of these nerves is enhanced by an increase in arterial blood pressure and diminished by a reduction in arterial blood pressure. An increase in impulse frequency, as occurs with a rise in arterial pressure, inhibits the cerebral vasoconstrictor regions and results in peripheral vasodilation and lowering of arterial blood pressure. Bradycardia brought about by activation of the cardiac branches of the vagus nerves contributes to this lowering of blood pressure.

The carotid sinus baroreceptors are more sensitive than those in the aortic arch. Changes in carotid sinus pressure evoke greater changes in systemic arterial pressure and peripheral resistance than do equivalent changes in aortic arch pressure.

The receptors in the carotid sinus walls respond more to pulsatile pressure than to constant pressure. This is illustrated in

Fig. 18.27 , which shows that at normal levels 0 0.5 1.0 1.5 2.0 •Fig. 18.27 Relationshipofphasicaorticbloodpressureinthefiring

CHAPTER 18 Regulation of the Heart and Vasculature of mean arterial blood pressure (≈100 mm Hg), a barrage of impulses from a single fiber of the sinus nerve is initiated in early systole by the pressure rise; only a few spikes occur during late systole and early diastole. At lower arterial pressure, these phasic changes are even more evident, but the overall discharge frequency is reduced. The blood pressure threshold for evoking sinus nerve impulses is approxi mately 50 mm Hg; maximal sustained firing is reached at approximately 200 mm Hg. Because the baroreceptors adapt, their response at any mean arterial pressure level is greater to a high pulse pressure than to a low pulse pressure.

The increases in resistance that occur in response to reduced pressure in the carotid sinus vary from one peripheral vascular bed to another. These variations allow blood flow to be redistributed. The resistance changes elicited by altering carotid sinus pressure are greatest in the femoral vessels, less in the renal vessels, and least in the mesenteric and celiac vessels.

In addition, the sensitivity of the carotid sinus reflex can be altered. Local application of norepinephrine or stimulation of sympathetic nerve fibers to the carotid sinuses enhances the sensitivity of its receptors in such a way that a given increase in intrasinus pressure produces a greater depressor response. Baroreceptor sensitivity decreases in hypertension because the carotid sinuses become stiffer as a result of the high intra-arterial pressure. Consequently, a given increase in carotid sinus pressure elicits a smaller decrease in systemic arterial pressure than it does at a normal level of blood pressure. Thus the set point of the baroreceptors is raised in hypertension in such a way that the threshold is increased and the pressure receptors are less sensitive to changes in transmural pressure. As would be expected, denervation of the carotid sinus can produce temporary and, in some instances, prolonged hypertension.

The arterial baroreceptors play a key role in short-term adjustments in blood pressure in response to relatively abrupt changes in blood volume, cardiac output, or peripheral resistance (as in exercise). However, long-term control of blood pressure—over a period of days or weeks—is determined by the fluid balance of the individual: namely, the balance between fluid intake and fluid output. By far, the most important organ in the control of body fluid volume, and hence blood pressure, is the kidney (see also

Cardiopulmonary receptors are located in the atria, ventricles, and pulmonary vessels. These baroreceptors are innervated by vagal and sympathetic afferent nerves. Cardiopulmonary reflexes are tonically active and can alter peripheral resistance in response to changes in intracardiac, venous, or pulmonary vascular pressure.

The atria contain two types of cardiopulmonary baroreceptors: those activated by the tension developed during atrial systole (type A receptors) and those activated by ofmeanarterialpressure.stretch of the atria during atrial diastole (type B receptors).

Stimulation of these atrial receptors sends impulses up vagal fibers to the vagal center in the medulla. Consequently, sympathetic activity is decreased to the kidney and increased to the sinus node. These changes in sympathetic activity increase renal blood flow, urine flow, and heart rate.

Activation of the cardiopulmonary receptors can also initiate a reflex that lowers arterial blood pressure by inhibiting the vasoconstrictor center in the cerebral medulla. Stimulation of the cardiopulmonary receptors inhibits release of angiotensin, aldosterone, and vasopressin (antidiuretic hormone); interruption of the reflex pathway has the opposite effects.

The role that activation of these baroreceptors plays in the regulation of blood volume is apparent in the body’s responses to hemorrhage. The reduction in blood volume (hypovolemia) enhances sympathetic vasoconstriction in the kidney and increases the secretion of renin, angiotensin, aldosterone, and vasopressin (see also ). The renal vasoconstriction (primarily afferent arterioles) reduces glomerular filtration and increases release of renin from the kidney. Renin acts on a plasma substrate to yield angiotensin II, which stimulates aldosterone secretion by the adrenal cortex. The enhanced release of vasopressin decreases renal water excretion, and the release of aldosterone decreases renal NaCl excretion. The kidneys retain salt and water, and hence blood volume increases. Angiotensin II (formed from angiotensin I by angiotensin-converting enzyme) also raises systemic arteriolar tone (see

Table 14.1

Insomeindividuals,thecarotidsinusisabnormallysensitivetoexternalpressure.Hence,tightcollarsorotherformsofexternalpressureovertheregionofthecarotidsinusmayelicitmarkedhypotensionandfainting.Suchhypersensitivityisknownasthecarotid sinus syndrome.

Peripheral chemoreceptors consist of small, highly vascular bodies in the region of the aortic arch (aortic bodies; see

Fig. 18.26 ) and just medial to the carotid sinuses (carotid bodies; see

Fig. 18.25 ). These vascular bodies are sensitive to changes in the PO2, PCO2, and pH of arterial blood. Although they primarily regulate respiration, they also influence the vasomotor regions. A reduction in PaO2 stimulates the chemoreceptors. The increased activity in afferent nerve fibers from the carotid and aortic bodies stimulates the vasoconstrictor regions and thereby increases the tone of resistance and capacitance vessels.

The chemoreceptors are also stimulated by increased arterial blood PCO2 (PaCO2) and by reduced pH. However, the reflex effect is small in comparison to the direct effects of hypercapnia (high PaCO2) and acidosis on the vasomotor regions in the medulla. When hypoxia and hypercapnia occur simultaneously, the effects of the chemoreceptors are greater than the sum of the effects of each of the two stimuli when they act alone.

Chemoreceptors are also located in the heart. These cardiac chemoreceptors are activated by ischemia of cardiac muscle, and they transmit the precordial pain (angina pectoris) associated with an inadequate blood supply to the myocardium.

Optimal function of the cardiovascular reflexes requires integrity of the pontine and hypothalamic structures. Furthermore, these structures are responsible for behavioral and emotional control of the cardiovascular system (see also ). Stimulation of the anterior hypothalamus produces both a fall in blood pressure and bradycardia, whereas stimulation of the posterolateral region of the hypothalamus increases both blood pressure and the heart rate. The hypothalamus also contains a temperature-regulating center that affects blood vessels in the skin. Stimulation by the application of cold to the skin or by cooling of the blood perfusing the hypothalamus results in constriction of the skin vessels and heat conservation, whereas warm stimuli to the skin result in cutaneous vasodilation and enhanced heat loss.

The cerebral cortex also affects blood flow distribution in the body. Stimulation of the motor and premotor areas affects blood pressure; usually, a pressor response occurs. However, vasodilation and depressor responses may be evoked, as in blushing or fainting, in response to an emotional stimulus.

Painful stimuli can elicit either pressor or depressor responses, depending on the magnitude and location of the stimulus. Distention of the viscera often evokes a depressor response, whereas painful stimuli to the body surface generally evoke a pressor response.

Inflation of the lungs initiates a reflex that induces systemic vasodilation and a decrease in arterial blood pressure. Conversely, collapse of the lungs evokes systemic vasoconstriction. Afferent fibers that mediate this reflex are in the vagus nerves and possibly also in the sympathetic nerves. Stimulation of these fibers by stretch of the lungs inhibits the vasomotor areas. The magnitude of the depressor response to lung inflation is directly related to the degree of inflation and to the existing level of vasoconstrictor tone (see also

Increases in PCO2 stimulate chemosensitive regions of the medulla (the central chemoreceptors), and they elicit vasoconstriction and increased peripheral resistance. A reduction in PCO2 to subnormal levels (in response to hyperventilation) decreases tonic activity in these areas in the medulla and thereby decreases peripheral resistance. The chemosensitive regions are also affected by changes in pH. Lowering of blood pH stimulates these cerebral areas, and a rise in blood pH inhibits them. These effects of changes in PCO2 and blood pH may operate through changes in cerebrospinal fluid pH, as may also the respiratory center.

PaO2 has little direct effect on the medullary vasomotor region. The primary effect of hypoxia is mediated by reflexes via the carotid and aortic chemoreceptors. A moderate reduction in PaO2 stimulates the vasomotor region, but a severe reduction depresses vasomotor activity in the same manner by which other areas of the brain are depressed by very low O2 tension.

Balance Between Extrinsic and Intrinsic Factors in Regulation of Peripheral Blood Flow

Dual control of peripheral vessels by intrinsic and extrinsic mechanisms evokes a number of important vascular adjustments. Such regulatory mechanisms enable the body to direct blood flow to areas where it is most needed and away from areas that have fewer requirements. In some tissues, the effects of the extrinsic and intrinsic mechanisms are fixed; in other tissues, the ratio is changeable and depends on the state of activity of that tissue.

Cerebralischemia,whichmayoccurbecauseofexcessivepressureexertedbyanexpandingintracranialtumor,resultsinamarkedincreaseinperipheralvasoconstriction.ThestimulationisprobablycausedbylocalaccumulationofCO2andareductioninO2andpossiblybyexcitationofintracranialbaroreceptors.Withprolonged,severeischemia,centraldepressioneventuallysupervenes,andbloodpressurefalls.

CHAPTER 18 Regulation of the Heart and Vasculature

In the brain and heart, which are vital structures with limited tolerance for a reduced blood supply, intrinsic flow-regulating mechanisms are dominant. For instance, massive discharge of the vasoconstrictor region via the sympathetic nerves, which might occur in severe, acute hemorrhage, has negligible effects on the cerebral and cardiac resistance vessels, whereas the cutaneous, renal, and splanchnic blood vessels become greatly constricted.

In the skin, extrinsic vascular control is dominant. The cutaneous vessels not only participate strongly in a general vasoconstrictor discharge but also respond selectively via hypothalamic pathways to subserve the functions of heat loss and heat conservation required for regulation of body temperature. However, intrinsic control can be elicited by local temperature changes that modify or override the central influence on resistance and capacitance vessels (see also

In skeletal muscle, the extrinsic and intrinsic mechanisms interact. In resting skeletal muscle, neural control (vasoconstrictor tone) is dominant, as can be demonstrated by the large increase in blood flow that occurs immediately after section of the sympathetic nerves to the tissue. After the onset of exercise, the intrinsic flow-regulating mechanism assumes control, and vasodilation occurs in the active muscles because of the local increase in metabolites. Vasoconstriction occurs in the inactive tissues as a manifestation of the general sympathetic discharge. However, constrictor impulses that reach the resistance vessels of the active muscles are overridden by the local metabolic effect. Operation of this dual control mechanism thus increases blood flow where it is required and shunts it away from relatively inactive areas (see also

Chapter 17).

Similar effects may be achieved in response to an increase in PCO2. Normally, the hyperventilation associated with exercise keeps PCO2 at normal levels. However, if PCO2 is increased, generalized vasoconstriction would occur because CO2 stimulates the medullary vasoconstrictor region. In active muscles, where [CO2] would be highest, the smooth muscle of the arterioles would relax in response to the local PCO2. Factors that affect and are affected by the vasomotor region are summarized in

Fig. 18.28 408 SECTION4Berne & Levy Physiology •Fig. 18.28 Schematic Diagram Illustrating Neural Input and Output of the Vasomotor Region (VR). Arrows indicatedirectionofneuralinputandoutput.IX,glossopharyngealnerve;↑PCO2,increasedpartialpressureofcarbondioxide;↓PCO2,increasedPCO2;↑PO2,increasedpartialpressureofoxygen;↓PO2,increasedPO2;SA,sinoatrial;X,vagusnerve.Aortic bodies SA node Sympathetic chain V R Carotid body X X IX ˜ PCO2 ° PO2 ˜ PCO2 ° PO2 ˜ PCO2 ° PO2 1.

Cardiac function is regulated by a number of intrinsic and extrinsic mechanisms. The principal intrinsic mechanisms that regulate myocardial contraction are the Frank-Starling mechanism and rate-induced regulation.

2.

The heart rate is regulated mainly by the autonomic nervous system. Sympathetic nervous activity increases the heart rate, whereas parasympathetic (vagal) activity decreases the heart rate. When both systems are active, the vagal effects usually dominate. The autonomic nervous system regulates myocardial performance mainly by varying the Ca++ conductance of the cell membrane via the adenylate cyclase system.

3.

The reflexes that regulate the heart rate are the baroreceptor, chemoreceptor, pulmonary inflation, atrial receptor (Bainbridge), and ventricular receptor reflexes.

4.

Certain hormones—such as epinephrine, adrenocortical steroids, thyroid hormones, insulin, glucagon, and anterior pituitary hormones—regulate myocardial performance. Changes in the arterial blood concentrations of O2, CO2, and H+ alter cardiac function directly and, through the chemoreceptors, indirectly.

5. The arterioles (resistance vessels) regulate blood flow mainly through their downstream capillaries. Smooth muscle, which makes up most of the walls of arterioles, contracts and relaxes in response to neural and humoral stimuli. Neural regulation of blood flow is almost completely accomplished by the sympathetic nervous system. Sympathetic nerves to blood vessels are tonically active; inhibition of the vasoconstrictor center in the medulla reduces peripheral vascular resistance. Stimulation of the sympathetic nerves constricts the resistance and capacitance (veins) vessels. Parasympathetic fibers innervate the head, viscera, and genitalia; they do not innervate the skin and muscle.

6.

Autoregulation of blood flow occurs in most tissues. This process is characterized by a constant blood flow in the presence of a change in perfusion pressure. Autoregulation is mediated by a myogenic mechanism whereby an increase in transmural pressure elicits a contraction of vascular smooth muscle and a decrease in transmural pressure elicits a relaxation.

7.

The striking parallelism between tissue blood flow and tissue O2 consumption indicates that blood flow is regulated largely by a metabolic mechanism. A decrease in the ratio of O2 supply to O2 demand of a tissue releases vasodilator metabolites that dilate arterioles and thereby enhance the O2 supply.

8.

The baroreceptors in the internal carotid arteries and aorta are tonically active and regulate blood pressure on a moment-to-moment basis. An increase in arterial pressure stretches these receptors to initiate a reflex that inhibits the medullary vasoconstrictor center and induces vasodilation. Conversely, a decrease in arterial pressure disinhibits the vasoconstrictor center and induces vasoconstriction. The baroreceptors in the internal carotid arteries

Fukuda K, Kanazawa H, Aizawa Y, et al. Cardiac innervation and sudden cardiac death. Circ Res. 2015;116:2005.

Iancu RV, Jones SW, Harvey RD. Compartmentation of cAMP signaling in cardiac myocytes. Biophys J. 2007;92:3317.

Liu Y, Bubolz AH, Mendoza S, et al. H2O2 is the transferrable factor mediating flow-induced dilation in human coronary arterioles. Circ Res. 2011;108:566.

predominate over those in the aorta, and they respond more vigorously to changes in pressure (stretch) than to elevated or reduced nonpulsatile pressure.

9.

Peripheral chemoreceptors (carotid and aortic bodies) and central chemoreceptors in the medulla oblongata are stimulated by a decrease in blood PaO2 and by an increase in blood PCO2. Stimulation of these chemoreceptors increases the rate and depth of respiration, but it also produces peripheral vasoconstriction. Cardiopulmonary baroreceptors are also present in the cardiac chambers and large pulmonary vessels. They have less influence on blood pressure but do participate in regulation of blood volume.

10.

Peripheral resistance and hence blood pressure are affected by stimuli that arise in the skin, viscera, lungs, and brain. The combined effect of neural and local metabolic factors distributes blood to active tissues and diverts it from inactive tissues. In vital structures, such as in the heart and brain and in contracting skeletal muscle, the metabolic factors predominate.

Llewellyn-Smith IJ, Verberne AJM, eds. Central Regulation of Autonomic Functions. New York: Oxford University Press; 2011.

Timmers HJ, Wieling W, Karemaker JM, et al. Cardiovascular responses to stress after carotid baroreceptor denervation in humans. Ann N Y Acad Sci. 2004;1018:515.

Zhang P, Mende U. Regulators of G-protein signaling in the heart and their potential as therapeutic targets. Circ Res. 2011;109: 320.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

What are the four major factors that determine cardiac output? Which two of these factors are said to be “coupling factors,” and what is the reason for that description?

2.

What is a cardiac function curve, and how is it related to the Frank-Starling mechanism?

3.

What is a vascular function curve, and how is it affected by changes in total peripheral resistance, blood volume, and venous tone?

4.

Why does the operating point of the cardiovascular system occur at the intersection of the vascular and cardiac function curves?

5.

How does evaluation of the cardiac function curve and the vascular function curve enable clinicians to determine the effect of changes in blood volume, vascular tone, and contractility on cardiac output?

6.

What mechanisms in the central nervous system, heart, and systemic vasculature allow cardiac output to increase to the necessary levels during vigorous exercise?

7.

What are the cardiovascular consequences of hemorrhage, and what are the compensatory mechanisms that tend to restore arterial pressure and cardiac output?

Regulation of Cardiac Output and Blood Pressure

Four factors control cardiac output: heart rate, myocardial contractility, preload, and afterload (

Fig. 19.1). Heart rate and myocardial contractility are strictly cardiac factors, although they are controlled by various neural and humoral mechanisms. Preload and afterload are factors that are mutually dependent on function of the heart and the vasculature and are important determinants of cardiac output. Preload and afterload are themselves determined by cardiac output and by certain vascular characteristics. Preload and afterload are called coupling factors because they constitute a functional coupling between the heart and blood vessels.

To understand regulation of cardiac output, the nature of the coupling between the heart and the vascular system must be appreciated.

In this chapter, two kinds of graphed curves are used to analyze interactions between the cardiac and vascular components of the circulatory system. The first curve, the cardiac function curve, is an expression of the well-known Frank-Starling relationship, and it illustrates the dependence of cardiac output on preload (i.e., central venous or right atrial pressure). The cardiac function curve is a characteristic of the heart itself and is usually studied in hearts completely isolated from the rest of the circulation. This curve has already been discussed in detail in . Later in this chapter, this curve is discussed in association with the other characteristic curve, the vascular function curve, to analyze interactions between the heart and the vasculature. The vascular function curve defines the dependence of central venous pressure on cardiac output. This relationship depends only on several vascular system characteristics, including peripheral vascular resistance, arterial and venous compliance, and blood volume. The vascular function curve is entirely independent of the characteristics of the heart. Because of this independence, it can be derived experimentally even if a mechanical pump replaces the heart.

The vascular function curve defines the changes in central venous pressure (Pv) that are caused by changes in cardiac output. In this curve, Pv is the dependent variable (or response), and cardiac output is the independent variable (or stimulus). These variables are opposite those of the cardiac function curve, in which Pv (or preload) is the independent variable and cardiac output is the dependent variable.

The simplified model of the circulation shown in

Fig.

19.2 helps explain how cardiac output determines the level of Pv. In this model, all essential components of the cardiovascular system have been lumped into four basic elements. The right and left sides of the heart, as well as the pulmonary vascular bed, constitute a pump-oxygenator, much like an artificial heart-lung machine used to perfuse •Fig. 19.1 Thefourfactors(inblue squares)thatdeterminecardiacoutput.

the body during open heart surgery. The high-resistance microcirculation is designated the peripheral resistance. Finally, the compliance of the system is subdivided into arterial compliance (Ca) and venous compliance (Cv). As defined in , the compliance (C) of a blood vessel is the change in volume (ΔV) that is accommodated in that vessel per unit change in transmural pressure (ΔP); that is,

Equation 19.1

Venous compliance is approximately 20 times greater than arterial compliance. In the example in

Fig. 19.2, the ratio of Cv to Ca is set at 19:1 to simplify calculations.

To show how a change in cardiac output causes an inverse change in Pv, the hypothetical model has certain characteristics that mimic those of an average adult (see

Fig. 19.2A ). The flow generated by the heart (i.e., cardiac output; Qh) is 5 L/minute; mean arterial pressure (Pa) is 102 mm Hg; and Pv is 2 mm Hg. Peripheral resistance (R) is the ratio of the arteriovenous pressure difference (Pa − Pv) to flow (Qr) through the resistance vessels; this ratio is equal to 20 mm Hg/L/minute.

An arteriovenous pressure difference of 100 mm Hg is sufficient to force a flow rate (Qr) of 5 L/minute through a peripheral resistance of 20 mm Hg/L/minute (see

Fig.

19.2A ). Under equilibrium conditions, this flow rate (Qr) is precisely equal to the flow rate (Qh) pumped by the heart. From heartbeat to heartbeat, the volume of blood in the arteries (Va) and the volume of blood in the veins (Vv) remain constant because the volume of blood transferred from the veins to the arteries by the heart is equal to the volume of blood that flows from the arteries through the resistance vessels and into the veins.

*Thus if it were necessary to add x mL of blood to the arterial system to produce a 1–mm Hg increase in arterial pressure, 19x mL of blood would need to be added to the venous system to raise venous pressure by the same amount.

Effects of Cardiac Arrest on Arterial and Venous Pressure

Fig. 19.2B depicts the circulation at the very beginning of an episode of cardiac arrest; that is, Qh = 0. In the instant immediately after arrest of the heart, the volume of blood in the arteries (Va) and veins (Vv) has not had time to change appreciably. Because arterial pressure and venous pressure depend on Va and Vv, respectively, these pressures are identical to the respective pressures in

Fig. 19.2A (i.e., Pa = 102 and Pv = 2). This arteriovenous pressure gradient of 100 mm Hg forces a flow rate (Qr) of 5 L/minute through the peripheral resistance of 20 mm Hg/L/minute. Thus although cardiac output (Qh) at that point is 0 L/ minute, the rate of flow through the microcirculation (Qt) is 5 L/minute because the potential energy stored in the arteries by the preceding pumping action of the heart causes blood to be transferred from arteries to veins. This transfer occurs initially at the control (steady-state) rate, even though the heart can no longer transfer blood from the veins to the arteries.

As cardiac arrest continues, blood flow through the resistance vessels causes the blood volume in the arteries to decrease progressively and the blood volume in the veins to increase progressively at the same absolute rate. Because the arteries and veins are elastic structures, arterial pressure falls gradually, and the venous pressure rises gradually. This process continues until arterial and venous pressures become equal (see

Fig. 19.2C ). Once this condition is reached, the rate of flow (Qr) from the arteries to the veins through the resistance vessels is 0 L/minute, as is Qh.

When the effects of cardiac arrest reach this equilibrium state (see

Fig. 19.2C ), the pressure attained in the arteries and veins depends on the relative compliance of these vessels. If arterial compliance (Ca) and venous compliance (Cv) are equal, the decline in Pa is equal to the rise in Pv because the decrease in arterial volume would be equal to the increase in venous volume (according to the principle of conservation of mass). Both Pa and Pv would attain the average of their combined values in

Fig. 19.2A ; that is, Pa = Pv = (102 + 2)/2 = 52 mm Hg. However, Ca and Cv in a living person are not equal. Veins are much more compliant than arteries; the compliance ratio (Cv/Ca) is approximately 19, the ratio assumed for the model in

Fig. 19.2 . When the effects of cardiac arrest reach equilibrium in an intact subject, the pressure in the arteries and veins is much lower than the average value of 52 mm Hg that occurs when Ca and Cv are equal. Hence, transfer of blood from arteries to veins at equilibrium induces a fall in arterial pressure 19 times greater than the concomitant rise in venous pressure. As

Fig. 19.2C shows, Pv would increase by 5 mm Hg (to 7 mm Hg), whereas Pa would fall by 95 (i.e., 19 × 5) mm Hg (to 7 mm Hg). This equilibrium pressure, which prevails in the absence of flow, is referred to as either mean circulatory pressure or static pressure. The pressure in the static system reflects the total blood volume in the system and the overall compliance of the system.

•Fig. 19.2 A toD, Simplifiedmodelofthecardiovascularsystem,consistingofapump,arterialcompliance(Ca),peripheralresistance,andvenouscompliance(Cv).

The example of cardiac arrest aids in the understanding of the vascular function curve. The clinician can now begin to assemble a vascular function curve (

Fig. 19.3 ). The independent variable (plotted along the x-axis) is cardiac output, and the dependent variable (plotted along the y-axis) is Pv. Two important points on this curve can be derived from the example in

Fig. 19.2 . One point (A in

Fig. 19.3 ) represents the control state; that is, when cardiac output is 5 L/minute, Pv is 2 mm Hg. When the heart is arrested (cardiac output = 0), Pv becomes 7 mm Hg at equilibrium (see

Fig. 19.2C ); this pressure is the mean circulatory pressure.

The inverse relationship between Pv and cardiac output simply means that when cardiac output is suddenly decreased, the rate at which blood flows from arteries to veins through the capillaries is temporarily greater than the rate at which the heart pumps blood from the veins back •Fig. 19.3 Changes in Central Venous Pressure Produced by Changes in Cardiac Output. Themeancirculatory(orstatic)pressure(Pmc)istheequilibriumpressurethroughoutthecardiovascularsystemwhencardiacoutputis0.PointsBandArepresentthevaluesofvenouspressureatcardiacoutputsof1and5L/minute,respectively.

into the arteries. During that transient period, a net volume of blood is transferred from arteries to veins; hence, Pa falls and Pv rises.

Now suppose that cardiac output suddenly increases. This example illustrates how a third point (B in

Fig. 19.3 ) on the vascular function curve is derived. Consider that the arrested heart is suddenly restarted and immediately begins pumping blood from the veins into the arteries at a rate of 1 L/minute (see

Fig. 19.2D ). When the heart first begins to beat, the arteriovenous pressure gradient is 0, and no blood is transferred from the arteries through the capillaries and into the veins. Thus when beating resumes, blood is depleted from the veins at the rate of 1 L/minute, and arterial blood volume is replenished from venous blood volume at that same absolute rate. Hence, Pv begins to fall and Pa begins to rise. Because of the difference in arterial and venous compliance, Pa rises at a rate 19 times faster than the rate at which Pv falls. The resultant arteriovenous pressure gradient causes blood to flow through the peripheral resistance vessels. If the heart maintains a constant output of 1 L/minute, Pa continues to rise and Pv continues to fall until the pressure gradient becomes 20 mm Hg. This gradient forces a rate of flow of 1 L/minute through a peripheral resistance of 20 mm Hg/L/minute. This gradient is achieved by a 19–mm Hg rise (to 26 mm Hg) in Pa and a 1–mm Hg fall (to 6 mm Hg) in Pv. This equilibrium value of Pv (6 mm Hg) for a cardiac output of 1 L/minute also appears on the vascular function curve of

Fig. 19.3 (point B). The 1–mm Hg reduction in Pv reflects a net transfer of blood from the veins to the arteries of the circuit.

The reduction in Pv that can be evoked by a sudden increase in cardiac output is limited. At some critical maximal value of cardiac output, sufficient fluid is transferred from the veins to the arteries of the circuit for Pv to fall below ambient pressure. In a system of very distensible vessels, such as the venous system, the greater external pressure causes the vessels to collapse (see ). This •Fig. 19.4 Effectsofincreasedbloodvolume(transfusion curve) anddecreasedbloodvolume(hemorrhage curve) onthevascularfunctioncurve.Similarshiftsinthevascularfunctioncurvecanbeproducedbyincreasesanddecreases,respectively,invenomotortone.

venous collapse impedes venous return to the heart. Hence, it limits the maximal value of cardiac output to 7 L/minute in this example (see

Fig. 19.3 ), regardless of the capabilities of the pump.

Factors That Influence the Vascular Function Curve

Dependence of Venous Pressure on Cardiac Output

According to experimental and clinical observations, changes in cardiac output do indeed evoke the alterations in Pa and Pv that have been predicted by the simplified model in

Fig. 19.2

The vascular function curve is affected by variations in total blood volume. During circulatory standstill (zero cardiac output), mean circulatory pressure depends only on total vascular compliance and blood volume. For a given vascular compliance, mean circulatory pressure is increased when blood volume is expanded (hypervolemia) and is decreased when blood volume is diminished (hypovolemia). This relationship is illustrated by the y-axis intercepts in

Fig.

19.4 , in which mean circulatory pressure is 5 mm Hg after hemorrhage and 9 mm Hg after transfusion, in comparison with a value of 7 mm Hg at normal blood volume (normovolemia or euvolemia).

The effects of changes in venomotor tone on the vascular function curve closely resemble those of changes in blood volume. In

Fig. 19.4 , for example, the transfusion curve could also represent increased venomotor tone, whereas the hemorrhage curve could represent decreased tone. During

Cardiacoutputmaydecreaseabruptlywhenamajorcoronaryarterysuddenlybecomesoccluded.Theacute heart failure thatoccursasaresultofmyocardial infarction (deathofmyocardialtissue)isusuallyaccompaniedbyafallinarterialbloodpressureandariseinPv.In

Fig.19.4 itisalsoapparentthatthecardiacoutputatwhichPv= 0variesdirectlywithbloodvolume.Therefore,themaximalvalueofcardiacoutputbecomesprogressivelymorelimitedasthetotalbloodvolumeisreduced.However,thePvatwhichtheveinscollapse(illustratedbythesharpchangeinslopeofthevascularfunctioncurve)isnotsignificantlyalteredbychangesinbloodvolume.Thispressuredependsonlyontheambientpressuresurroundingthecentralveins.Ambientpressureisthepleuralpressureinthethorax(see circulatory standstill, for a given blood volume, the pressure within the vascular system rises as smooth muscle tension exerted within the vascular walls increases (these contractile changes in arteriolar and venous smooth muscle are under nervous and humoral control). The fraction of the blood volume located within the arterioles is very small, whereas the blood volume in the veins is large percentage of total blood volume (see

Table 15.1 ). Thus changes in peripheral resistance (arteriolar tone) have no significant effect on mean circulatory pressure, but changes in venous tone can alter mean circulatory pressure appreciably. Hence, mean circulatory pressure rises with increased venomotor tone and falls with diminished venomotor tone.

In experiments, the mean circulatory pressure attained approximately 1 minute after abrupt circulatory standstill is usually substantially above 7 mm Hg, even when blood volume is normal. The elevation to this pressure level is attributable to the generalized venoconstriction that is caused by cerebral ischemia, activation of chemoreceptors, and reduced excitation of baroreceptors. If resuscitation fails, this reflex response subsides as central nervous activity ceases, and mean circulatory pressure then usually falls to a value close to 7 mm Hg.

Venoconstriction is considerably greater in certain regions of the body than in others. In effect, vascular beds that undergo significant venoconstriction constitute blood reservoirs. The skin’s vascular bed is one of the major blood reservoirs in humans. Blood loss evokes profound subcutaneous venoconstriction, which gives rise to the characteristically pale appearance of the skin in response to hemorrhage. Diversion of blood away from the skin frees several hundred milliliters of blood that can be perfused through more vital regions of the body. The vascular beds of the liver, lungs, and spleen are also important blood reservoirs. In humans, however, the volume changes in the spleen are considerably less extensive (see also the sections “ •Fig. 19.5 Effectsofarteriolardilationandconstrictiononthevascularfunctioncurve.

The changes in the vascular function curve induced by alterations in arteriolar tone are depicted in

Fig. 19.5 . The amount of blood in the arterioles is small; these vessels contain only approximately 3% of total blood volume (see ). Changes in the contractile state of arterioles do not significantly alter mean circulatory pressure. Thus vascular function curves that represent different peripheral resistances converge at a common point on the y-axis (see

Fig. 19.5

Pv varies inversely with total peripheral resistance (TPR) when all other factors remain constant. Physiologically, the relationship between Pv and TPR can be explained as follows: if cardiac output is held constant, a sudden increase in TPR causes a progressively greater volume of blood to be retained in the arterial system. Blood volume in the arterial system continues to increase until Pa rises sufficiently to force a flow of blood equal to cardiac output through the resistance vessels. If total blood volume does not change, this increase in arterial blood volume is accompanied by an equivalent decrease in venous blood volume. Hence, an increase in TPR diminishes Pv proportionately. This relationship between TPR and Pv, together with the inability of peripheral resistance to affect mean circulatory pressure, accounts for the clockwise rotation of the vascular function curves in response to increased arteriolar constriction (see

Fig. 19.5 ). Similarly, arteriolar dilation produces a counterclockwise rotation from the same vertical axis intercept. A higher maximal level of cardiac output is attainable when the arterioles are dilated than when they are constricted (see

Fig. 19.5).

Cardiac output and venous return are tightly linked. Except for small, transient disparities, the heart cannot pump any more blood than is delivered to it through the venous system. Similarly, because the circulatory system is a closed circuit, venous return to the heart must equal cardiac output over any appreciable time interval. The flow around the entire closed circuit depends on the capability of the pump, the characteristics of the circuit, and the total fluid volume of the system.

Thus cardiac output and venous return are simply two terms for the flow around this closed circuit. Cardiac output is the volume of blood being pumped by the heart per unit time. Venous return is the volume of blood returning to the heart per unit time. At equilibrium, these two volumes are equal. In the following section, certain techniques of circuit analysis are discussed to provide some insight into the control of flow around the circuit.

Relating the Cardiac Function Curve to the Vascular Function Curve

Coupling Between the Heart and the Vasculature

In accordance with the Frank-Starling law of the heart, cardiac output depends closely on right atrial (or central venous) pressure. Furthermore, right atrial pressure is approximately equal to right ventricular end-diastolic pressure because the normal tricuspid valve acts as a low-resistance junction between the right atrium and ventricle. Graphs of cardiac output as a function of Pv are called cardiac function curves; extrinsic regulatory influences may be expressed as shifts in such curves.

A typical cardiac function curve is plotted on the same coordinates as those for a normal vascular function curve in

Fig. 19.6 . The cardiac function curve is plotted according to the usual convention; that is, the independent variable (Pv) is plotted along the x-axis, and the dependent variable (cardiac output) is plotted along the y-axis. In accordance with the Frank-Starling mechanism, the cardiac function curve reveals that a rise in Pv increases cardiac output.

Conversely, the vascular function curve characterizes an inverse relationship between cardiac output and Pv; that is, a rise in cardiac output diminishes Pv. Pv is the dependent variable (or response) and cardiac output is the independent variable (or stimulus) for the vascular function curve. Therefore, to plot a vascular function curve in the conventional manner, Pv should be scaled along the y-axis and cardiac output along the x-axis.

To plot the cardiac and vascular function curves on the same set of axes requires a modification of the plotting convention for one of these curves. The convention for the vascular function curve is violated arbitrarily in this chapter. Note that the vascular function curve in

Fig. 19.6 is intended to reflect how Pv (scaled along the x-axis) varies in response to a change in cardiac output (scaled along the y-axis).

When the cardiovascular system is represented by a given pair of cardiac and vascular function curves, the intersection of these two curves defines the equilibrium point of that system. The coordinates of this equilibrium point represent •Fig. 19.6 Typical Vascular and Cardiac Function Curves Plotted on the Same Coordinate Axes. Toplotbothcurvesonthesamegraph,thex-axisandy-axisforthevascularfunctioncurveshadtobeswitched;comparetheassignmentofaxeswiththosein

Figs.

19.3,19.4,and19.5.

Thecoordinatesoftheequilibriumpoint,attheintersectionofthecardiacandvascularfunctioncurves,representthestablevaluesofcardiacoutputandcentralvenouspressureatwhichthesystemtendstooperate.Anyperturbation(e.g.,asuddenincreaseinvenouspressuretopointA)institutesasequenceofchangesincardiacoutputandvenouspressurethatrestorethesevariablestotheirequilibriumvalues.

the values of cardiac output and Pv at which the system tends to operate. Only transient deviations from such values of cardiac output and Pv are possible, as long as the given cardiac and vascular function curves characterize the system accurately.

The tendency to operate about this equilibrium point may best be illustrated by the response to a sudden change. Consider the changes caused by a sudden rise in Pv from the equilibrium point to point A in

Fig. 19.6 . This change in Pv might be caused by the rapid injection, during ventricular diastole, of a given volume of blood on the venous vessels of the circuit and simultaneous withdrawal of an equal volume from the arterial vessels of the circuit. Thus although Pv rises, total blood volume remains constant.

As defined by the cardiac function curve, this elevated Pv would increase cardiac output (from point A to point B in

Fig. 19.6 ) during the next ventricular systole. The increased cardiac output would then cause the transfer of a net quantity of blood from the veins to the arteries of the circuit, with a consequent reduction in Pv. In one heartbeat, the reduction in Pv would be small (from point B to point C) because the heart would transfer only a fraction of the total venous blood volume to the arteries. As a result of this reduction in Pv, cardiac output during the very next beat diminishes (from point C to point D) by an amount dictated by the cardiac function curve. Because point C is still above the intersection point, the heart pumps blood from the veins to the arteries at a rate greater than that at which blood flows across the peripheral resistance from arteries to veins. Hence, Pv continues to fall. This process continues in diminishing steps until the point of •Fig. 19.7 Enhancementofmyocardialcontractility,asbystimulationofcardiacsympatheticnerves,causestheequilibriumvaluesofcardiacoutputandcentralvenouspressure(Pv)toshiftfromtheintersection(pointA)ofthecontrolvascularandcardiacfunctioncurves(continuous curve) totheintersection(pointD)ofthesamevascularfunctioncurvewiththecardiacfunctioncurve(dashed curve) thatrepresentstheresponsetosympatheticstimulation.

intersection is reached. Only one specific combination of cardiac output and venous pressure—the equilibrium point, denoted by the coordinates of the point at which the curves intersect—satisfies the requirements of the cardiac and vascular function curves simultaneously. At the equilibrium point, cardiac output equals venous return, and the system is stable.

Combinations of cardiac and vascular function curves also help explain the effects of alterations in ventricular contractility on cardiac output and Pv. In

Fig. 19.7, the lower cardiac function curve represents the control state, whereas the upper curve reflects the influence of increased myocardial contractility. This pair of curves is analogous to the ventricular function curves shown in

Fig. 18.12.

The enhanced ventricular contractility represented by the upper curve in

Fig. 19.7 can be produced by electrical stimulation of the cardiac sympathetic nerves. When the effects of such neural stimulation are restricted to the heart, the vascular function curve is unaffected. Therefore, only one vascular function curve is needed for this hypothetical intervention (see

Fig. 19.7

During the control state of the model, the equilibrium values for cardiac output and Pv are designated by point A in

Fig. 19.7 . Cardiac sympathetic nerve stimulation abruptly raises cardiac output to point B because of the enhanced myocardial contractility. However, this high cardiac output causes an increase in the net transfer of blood from the veins to the arteries of the circuit, and as a consequence, Pv subsequently begins to fall (to point C). The reduction in Pv then leads to a small decrease in cardiac output. However, cardiac output is still sufficiently high to effect •Fig. 19.8 Duringelectricalstimulationofthecardiacsympatheticnervefibers,aorticbloodflow(cardiacoutput)increased,whereaspressuresintheleftatrium(PLA)andrightatrium(PRA)diminished.Thesedataconformtotheconclusionsderivedfrom

Fig.19.7 ,inwhichtheequilibriumvaluesofcardiacoutputandvenouspressureareobservedtoshiftfrompointAtopointD(i.e.,cardiacoutputincreased,butcentralvenouspressuredecreased)duringcardiacsympatheticnervestimulation.(RedrawnfromSarnoffSJ,etal.Circ Res. 1960;8:1108.) the net transfer of blood from the veins to the arteries of the circuit. Thus both Pv and cardiac output continue to fall gradually until a new equilibrium point (point D) is reached. This equilibrium point is located at the intersection of the vascular function curve and the new cardiac function curve. In

Fig. 19.7 , point D lies above and to the left of the control equilibrium point (point A) and indicates that sympathetic stimulation can evoke greater cardiac output despite the lower level of Pv.

The biological response to enhancement of myocardial contractility is mimicked by the hypothetical change predicted by the model in this chapter. As depicted in

Fig. 19.8 , sympathetic nerves innervating the heart are stimulated during the time denoted by the double-headed arrow. During neural stimulation, cardiac output (aortic flow) rises quickly to a peak value and then falls gradually to a steady-state value significantly higher than the control level. The increase in aortic flow is accompanied by reductions in right and left atrial pressures.

Changes in blood volume do not directly affect myocardial contractility, but they do influence the vascular function curve in the manner shown in

Fig. 19.4 . Thus to understand how changes in blood volume affect cardiac output and Pv, the appropriate cardiac function curve is plotted along with the vascular function curves that represent the control and experimental states (

Fig. 19.9 ). When blood volume is increased by a blood transfusion, the equilibrium point (point B in

Fig. 19.9 ), which denotes the values of cardiac output and Pv after transfusion, lies above and to the right of the control equilibrium point (point A). Thus transfusion increases both cardiac output and Pv. Hemorrhage causes the opposite effect. Mechanistically, the change in ventricular filling pressure (Pv) evoked by a given change in blood volume alters cardiac output by changing the sensitivity of the contractile proteins to the prevailing concentration of intracellular Ca++ (see ). For reasons explained earlier, pure increases or decreases in venomotor tone elicit responses that are like those evoked by increases or decreases, respectively, in total blood volume.

•Fig. 19.9 Afterabloodtransfusion,thevascularfunctioncurveisshiftedtotheright.Therefore,bothcardiacoutputandvenouspressureareincreased,asdenotedbytranslocationoftheequilibriumpointfrompointAtopointB.–2 A B 1086420 0 2 4 6 8 10 Central venous pressure (mm Hg) Transfusion ControlCardiac output (L/min)

Analysis of the effects of changes in peripheral resistance on cardiac output and Pv is complex because both the cardiac and vascular function curves shift. When peripheral resistance increases (

Fig. 19.11 ), the vascular function curve is rotated counterclockwise, but it converges on the same Pv axis intercept as the control curve does. Note that vasoconstriction causes a counterclockwise rotation of the vascular function curve in

Fig. 19.11

Fig. 19.5 . The direction of rotation differs because the axes for the vascular function curves were switched in these two figures, as explained earlier. The cardiac function curve in

Fig. 19.11 is also shifted downward because at any given Pv, the heart is able to pump less blood against the greater cardiac afterload imposed by the increased peripheral resistance. Because both curves in

Fig. 19.11 are displaced downward, the new equilibrium point (point B) is below the control point (point A); that is, an increase in peripheral resistance diminishes cardiac output.

Whether point B falls directly below point A or lies slightly to the right or left of it depends on the magnitude of the shift in each curve. For example, if a given increase •Fig. 19.10 Moderateorsevereheartfailureshiftsthecardiacfunctioncurvesdownwardandtotheright.Beforechangesinbloodvolume,cardiacoutputdecreasesandcentralvenouspressurerises(fromcontrolequilibriumpointAtopointBorpointC).Aftertheincreaseinbloodvolumethatusuallyoccursinheartfailure,thevascularfunctioncurveisshiftedtotheright.Hence,centralvenouspressuremaybeelevatedwithnoreductionincardiacoutput(pointD)or,insevereheartfailure,withsomereductionincardiacoutput(pointE).

Heart failure isageneraltermthatappliestoconditionsinwhichthepumpingcapabilityoftheheartisimpairedtotheextentthatthetissuesofthebodyarenotadequatelyperfused.Inheartfailure,myocardialcontractilityisimpaired.Heartfailuremaybeacuteorchronic.Consequently,inagraphofcardiacandvascularfunctioncurves,thecardiacfunctioncurveisshifteddownwardandtotheright,asdepictedin

Fig.19.10

Acuteheartfailuremaybecausedbytoxicconcentrationsofdrugsorbycertainpathologicalconditionssuchascoronaryarteryocclusion.Inacuteheartfailure,bloodvolumedoesnotchangeimmediately.In

Fig.19.10 ,therefore,theequilibriumpointshiftsfromtheintersection(pointA)ofthenormalcurvestotheintersection(pointBorpointC)ofthenormalvascularfunctioncurve.

Chronicheartfailuremayoccurinconditionssuchasessentialhypertensionorischemicheartdisease.Inchronicheartfailure,boththecardiacfunctionandvascularfunctioncurvesshift.Thevascularfunctioncurveshiftsbecauseofanincreaseinbloodvolumecausedinpartbyfluidretentionbythekidneys.ThefluidretentionisrelatedtotheconcomitantreductioninglomerularfiltrationrateandthedecreasedrenalexcretionofNaClandwater(seealso ).Theresultanthypervolemiaisreflectedbyarightwardshiftofthevascularfunctioncurve,asshownin

Fig.19.10.

Hence,withmoderatedegreesofheartfailure,Pviselevated,butcardiacoutputmaybenormal(pointD).Withmoreseveredegreesofheartfailure,Pvisstillelevated,butcardiacoutputissubnormal(pointE).

in peripheral resistance shifts the vascular function curve more than it does the cardiac function curve, equilibrium point B is below and to the left of point A; that is, both cardiac output and Pv diminish. Conversely, if the cardiac function curve is displaced more than the vascular function •Fig. 19.11 Anincreaseinperipheralresistanceshiftsthecardiacandvascularfunctioncurvesdownward.Atequilibrium,cardiacoutputisless(pointB)whenperipheralresistanceishighthanwhenperipheralresistanceisnormal(pointA).

•Fig. 19.12 Simplifiedmodelofthecardiovascularsystemthatconsistsoftheleftventricle(LV)andrightventricle(RV),systemicvascularresistance(Rs)andpulmonaryvascularresistance(Rp),systemicarterialandvenouscompliance,andpulmonaryarterialandvenouscompliance.PsaandPsvarethepressuresinthesystemicarteriesandveins,respectively;PpaandPpvarethepressuresinthepulmonaryarteriesandveins,respectively.

curve, point B falls below and to the right of point A; that is, cardiac output decreases, but Pv rises.

A More Complete Theoretical Model: The Two-Pump System

The preceding discussion shows that the interrelationships between cardiac output and Pv are complex, even in an oversimplified circulation model that includes only one pump and just the systemic circulation. In reality, the cardiovascular system includes the systemic and pulmonary circulations and two pumps: the left and right ventricles. Thus the interrelationships among ventricular output, arterial pressure, and atrial pressure are much more complex.

Fig. 19.12 depicts a more complete (but still oversimplified) cardiovascular system model that has two pumps in series (the left and right ventricles) and two vascular beds in series (the systemic and pulmonary vasculature). The series arrangement requires that the flow pumped by the two ventricles be virtually equal to each other over any substantial period; otherwise, all the blood would ultimately accumulate in one or the other of the vascular systems. Because the cardiac function curves for the two ventricles differ substantially, the filling (atrial) pressures for the two ventricles must differ appropriately to ensure equal stroke volumes (see

Fig. 18.13

Anychangeincontractilitythataffectsthetwoventriclesdifferentlyaltersthedistributionofbloodvolumeinthetwovascularsystems.Ifacoronaryarterytotheleftventriclebecomesoccluded,leftventricularcontractilityisimpaired,andacute left ventricular failure ensues.Intheinstantafterocclusion,leftatrialpressuredoesnotchange,andtheleftventriclebeginstopumpatadiminishedrateofflow.Iftherightventricleisnotaffectedbytheacutecoronaryarteryocclusion,therightventricleinitiallycontinuestopumpthenormalflow.Thedisparaterightandleftventricularoutputsresultinaprogressiveincreaseinleftatrialpressureandaprogressivedecreaseinrightatrialpressure.Therefore,leftventricularoutputincreasestowardthenormalvalue,andrightventricularoutputfallsbelowthenormalvalue.Thisprocesscontinuesuntiltheoutputsofthetwoventriclesagainbecomeequal.Atthisnewequilibrium,theoutputofthetwoventriclesissubnormal.Theelevationinleftatrialpressureisaccompaniedbyanequalelevationinpulmonaryvenouspressure,whichcanhaveseriousclinicalconsequences.Thehighpulmonaryvenouspressurecanincreaselungstiffnessandleadtorespiratorydistressbyincreasingthemechanicalworkofpulmonaryventilation(see ).Furthermore,thehighpulmonaryvenouspressurecausesanelevatethehydrostaticpressureinthepulmonarycapillariesandmayleadtothetransudationoffluidfromthepulmonarycapillariestothepulmonaryinterstitiumorintothealveoli(pulmonary edema), whichmaybelethal.

Two basic principles to remember about ventricular function are that (1) the left ventricle pumps blood through the systemic vasculature, and (2) the right ventricle pumps blood through the pulmonary vasculature. However, these principles do not necessarily imply that both ventricles are essential to perfuse the systemic and pulmonary vascular beds adequately. To better understand the relationships between the two ventricles and the two vascular beds, the right ventricular function is examined in more detail as follows.

In the circulatory system model shown in

Fig. 19.12, consider the hemodynamic consequences that would occur if the right ventricle suddenly ceased its pump function but instead served merely as a passive, low-resistance conduit between the systemic veins and the pulmonary arteries. Under these conditions, the only functional pump would be the left ventricle, which would then be required to pump blood through both the systemic and pulmonary resistances (for the purposes of this discussion, consider the resistance to the flow of blood through the inactive right ventricle to be negligible).

Normally, pulmonary vascular resistance is approximately 10% as great as systemic vascular resistance. Because the two resistances are in series with one another, total resistance would be 10% greater than systemic resistance alone (see ). In a normal cardiovascular system, a 10% (and hence left ventricular afterload) by approximately 10%. This increase would not drastically affect left ventricular function. Under certain conditions, however, this increase in Pa could significantly alter the function of the cardiovascular system. If the 10% increase in total resistance is achieved by adding a small degree of resistance (i.e., pulmonary vascular resistance) to that of the much larger systemic resistance, and if the pulmonary vascular resistance is separated from the systemic resistance by a large degree of compliance (the combined systemic venous and pulmonary arterial compliance), the 10% increase in total resistance could drastically impair operation of the cardiovascular system.

The simulated effects of inactivating the pumping action of the right ventricle in a hydraulic analogue of the circula tory system are shown in

Fig. 19.13 . In the model, the right and left ventricles generate cardiac outputs that vary directly with their respective filling pressures. Under control conditions (when the right ventricle is functioning normally), the outputs of the left and right ventricles are equal (5 L/ minute). The right ventricular pumping action causes the pressure in the pulmonary artery (not shown) to exceed the pressure in the pulmonary veins (Ppv) by an amount that forces fluid through the pulmonary vascular resistance at a rate of 5 L/minute. When the right ventricle ceases pumping (arrow 1 in

Fig. 19.13 ), the systemic venous and pulmonary arterial systems, along with the right ventricle itself, become a common passive conduit with a large compliance. When the right ventricle ceases to transfer blood actively from the systemic veins to the pulmonary arteries, pulmonary arterial pressure (Ppa) decreases rapidly (not shown) and systemic venous pressure (Psv) rises rapidly to a common value (≈5 mm Hg). At this low pressure, however, fluid flows from the pulmonary arteries to the pulmonary veins at a greatly reduced rate.

At the start of right ventricular arrest, the left ventricle is pumping fluid from the pulmonary veins to the systemic arteries at the control rate of 5 L/minute, which greatly exceeds the rate at which blood returns to the pulmonary veins once the right ventricle ceases to operate. Hence, pulmonary venous pressure (Ppv) drops sharply. Because pulmonary venous pressure is the preload for the left ventricle, left ventricular (cardiac) output drops abruptly as well and attains a steady-state value of approximately 2.5 L/minute. This effect in turn leads to a rapid reduction in systemic arterial pressure (Psa). In short, stoppage of right ventricular pumping markedly curtails cardiac output, systemic arterial pressure, and pulmonary venous •Fig. 19.13 Changesincardiacoutput,systemicarterialpressure(Psa),systemicvenouspressure(Psv),andpulmonaryvenouspressure(Ppv)evokedbysimulatedrightventricular(RV)failureandbysimulatedinfusionoffluidinthecirculatorymodelshownin

Fig.19.12 .Atarrow 1, thepumpingactionoftherightventriclewasdiscontinued(simulatedRVfailure),andtherightventricleservedonlyasalow-resistanceconduit.Atarrow 2, thefluidvolumeinthesystemwasexpanded,andtherightventriclecontinuedtoserveonlyasaconduit.(ModifiedfromFureySA,etal.Am Heart J. 1984;107:404.)

Fig. 19.13).

Most of the hemodynamic problems induced by inactivation of the right ventricle can be reversed by an increase in the fluid (blood) volume of the system (arrow 2 in

Fig.

19.13). If fluid is added until pulmonary venous pressure (left ventricular preload) is raised to its control value, cardiac output and systemic arterial pressure are restored almost to normal, but systemic venous pressure is abnormally elevated. If left ventricular function is normal, adding a normal left ventricular preload evokes normal left ventricular output. The 10% increase in peripheral resistance caused by adding the pulmonary vascular resistance to that of the systemic vascular resistance does not impose a serious burden on left ventricular pumping capacity.

When the right ventricle is inoperative, however, pulmonary blood flow is not normal unless the usual pulmonary arteriovenous pressure gradient (≈10 to 15 mm Hg) prevails. Hence, systemic venous pressure (Psv) must exceed pulmonary venous pressure (Ppv) by this amount.

Maintenance of high systemic venous pressure may lead to the accumulation of tissue fluid (edema) in dependent regions of the body, a characteristic finding in patients with right ventricular heart failure. With this information, the principal function of the right ventricle may be characterized as as follows. From the viewpoint of providing sufficient flow of blood to all tissues in the body, the left ventricle alone can carry out this function. Operation of the two ventricles in series is not essential to provide adequate blood flow to the tissues. The crucial function of the right ventricle is to prevent the rise in systemic venous (and pulmonary arterial) pressure that would be required to force the normal cardiac output through the pulmonary vascular resistance. A normal right ventricle, by preventing an abnormal rise in systemic venous pressure, prevents the development of extensive edema in dependent regions of the body.

Clinically,right ventricular heart failure maybecausedbyocclusivediseasepredominantlyofthecoronaryvesselstotherightventricle.Thesevesselsareaffectedmuchlesscommonlythanthevesselstotheleftventricle.Themajorhemodynamiceffectsofacuteright-sidedheartfailurearepronouncedreductionsincardiacoutputandarterialbloodpressure,andtheprincipaltreatmentisinfusionofbloodorplasma.Bypassoftherightventricle(byanastomosisoftherightatriumtothepulmonaryartery)maybeperformedsurgicallyinpatientswithcertaincongenital cardiac defects, suchasseverenarrowingofthetricuspidvalveormaldevelopmentoftherightventricle.Theeffectsofacuteright-sidedheartfailureorrightventricularbypassaredirectionallysimilartothosepredictedpreviouslyfromanalysisofthemodelshownin

Fig.19.13

Role of the Heart Rate in Control of Cardiac Output

Cardiac output is the product of stroke volume and heart rate. Analysis of the control of cardiac output has thus far been restricted to the control of stroke volume, and the role of heart rate has not been considered. Analysis of the effect of a change in heart rate on cardiac output is complex because a change in heart rate alters the other three factors (preload, afterload, and myocardial contractility) that determine stroke volume (see

Fig. 19.1 ). An increase in heart rate, for example, shortens the duration of diastole. Hence, ventricular filling is diminished; that is, preload is reduced. If an increase in heart rate altered cardiac output, arterial pressure would change; that is, afterload would be altered. A rise in heart rate would increase the net influx of Ca++ per minute into myocardial cells (see also ), and this influx would enhance myocardial contractility.

The effects of changes in heart rate on cardiac output have been studied extensively, and the results are similar to those shown in

Fig. 19.14 . As atrial pacing frequency is •Fig. 19.14 Changesinstrokevolume(A) andcardiacoutput (B) inducedbychangesintherateofatrialpacing.(RedrawnfromKumadaM,etal.Jpn J Physiol. 1967;17:538.) gradually increased, stroke volume progressively diminishes (see

Fig. 19.14A ). The decrease in stroke volume is caused by the reduced time for ventricular filling. The change in stroke volume is not inversely proportional to the change in heart rate because the direction of the change in cardiac output (Qh) is markedly influenced by the actual heart rate (see

Fig. 19.14B ). For example, as pacing frequency is increased from 50 to 100 beats/minute, the increase in heart rate augments Qh. Because Qh = SV × HR, the decrease in stroke volume (SV) over this frequency range must be proportionately less than the increase in heart rate (HR).

Over the frequency range from approximately 100 to 200 beats/minute, however, cardiac output is not affected significantly by changes in pacing frequency (see

Fig.

19.14B ). Hence, as pacing frequency is increased, the decrease in stroke volume must be approximately equal to the increase in heart rate. In addition, generalized vascular autoregulation tends to keep tissue blood flow constant (see also ). This adaptation leads to changes in preload and afterload that also keep cardiac output nearly constant.

Moreover, at excessively high pacing frequencies (above 200 beats/minute; see

Fig. 19.14 ), further increases in heart rate decrease cardiac output. Therefore, the induced decrease in stroke volume must have exceeded the increase in heart rate at this high range of pacing frequencies. At such high pacing frequencies, the ventricular filling time is so severely restricted that compensation is inadequate, and cardiac output decreases sharply. Although the relationship of cardiac output to heart rate is characteristically that of an inverted U in the general population, the relationship varies quantitatively among subjects and among physiological states.

Strong correlations between heart rate and cardiac output must be interpreted cautiously. In people who are exercising, for example, cardiac output and heart rate usually increase proportionately, and stroke volume may remain constant or increase only slightly (see the later section ”). It is tempting to conclude that the increase in cardiac output during exercise must be caused solely by the observed increase in heart rate. However,

Fig. 19.14 shows that over a wide range of heart rates, a change in heart rate may have little influence on cardiac output. The principal increase in cardiac output during exercise must therefore be attributed to other factors. Such ancillary factors include the pronounced reduction in peripheral vascular resistance because of the vasodilation in the active skeletal muscles and the increased contractility of cardiac muscle associated with the generalized increase in sympathetic neural activity. Nevertheless, the increase in heart rate is still an important factor. Abundant data show that if the heart rate cannot increase normally during exercise, the augmentation in cardiac output and the capacity for exercise are severely limited. Because stroke volume changes only slightly during exercise, the increase in heart rate may play an important permissive role in augmenting cardiac output during physical exercise.

Thecharacteristicrelationshipbetweencardiacoutputandheartrateexplainstheurgentneedoftreatmentbypatientswhohaveexcessivelysloworexcessivelyfastheartrates.Profoundbradycardia (slowrate)mayoccurasaresultofaveryslowsinusrhythminpatientswithsick sinus syndrome orasaresultofaslowidioventricularrhythminpatientswithcomplete atrioventricular block. Ineitherrhythmdisturbance,thecapacityoftheventriclestofillduringprolongeddiastoleislimited(oftenbythenoncompliantpericardium).Hence,cardiacoutputusuallydecreasessubstantiallybecausetheveryslowheartratecannotbecounterbalancedbyasufficientlylargestrokevolume.Asaconsequence,suchbradycardiaoftennecessitatestheimplantationofanartificialpacemaker.Inpatientswithsupraventricular orventricular tachycardia, excessivelyhighheartratesfrequentlynecessitateemergencytreatmentbecauseinsuchpatients,cardiacoutputmaybecriticallylow,andthefillingtimeissorestrictedatveryhighheartratesthatevensmalladditionalreductionsinfillingtimecausedisproportionatelyseverereductionsinfillingvolume.Slowingtheheartratetoamorenormalrhythmcangenerallybeaccomplishedpharmacologically,butelectricalcardioversionmayberequiredinemergencies(see

Ancillary Factors That Affect the Venous System and Cardiac Output

In earlier sections of this chapter, the descriptions of the interrelationships between Pv and cardiac output were simplified by restricting the discussion to the effects evoked by individual variables. However, because the cardiovascular system is regulated by so many feedback control loops, its responses are rarely simple. A change in blood volume, for example, not only affects cardiac output directly through the Frank-Starling mechanism but also triggers reflexes that alter other aspects of cardiac function (such as the heart rate, atrioventricular conduction, and myocardial contractility) and other characteristics of the vascular system (such as peripheral resistance and venomotor tone). Several other factors, especially gravity (see ) and respiration, also regulate cardiac output.

Circulatory Effects of Respiratory Activity

The normal, periodic activity of the respiratory muscles causes rhythmic variations in vena caval flow (Fig. 19.15 ). During respiration, the reduction in intrathoracic pressure is transmitted to the lumens of the thoracic blood vessels. The reduction in Pv during inspiration increases the pressure gradient between extrathoracic and intrathoracic veins. The consequent acceleration in venous return to the right atrium is shown in

Fig. 19.15 as an increase in superior vena caval blood flow from 5.2 mL/sec during expiration to 11 mL/ sec during inspiration.

The exaggerated reduction in intrathoracic pressure achieved by a strong inspiratory effort against a closed glottis (called Müller’s maneuver) does not increase venous return proportionately. The extrathoracic veins collapse near their entry into the chest when their internal pressures fall below the ambient level. As the veins collapse, flow into the chest momentarily stops. The cessation of flow causes pressure upstream to rise, which forces the collapsed segment to reopen.

During normal expiration, flow into the central veins decelerates. However, the mean rate of venous return during normal respiration exceeds the flow during a brief period of apnea (cessation of respiration). Hence, normal inspiration apparently facilitates venous return more than normal expiration impedes it. In part, venous return is facilitated by the valves in the veins of the extremities. These valves prevent any reversal of flow during expiration. Thus the respiratory muscles and venous valves constitute an auxiliary pump for venous return.

•Fig. 19.15 Duringanormalinspiration,intrathoracicpressure,rightatrialpressure(RAP),andjugularvenous(JVP)pressuredecrease,andflowinthesuperiorvenacava(SVCF)increases(from5.2to11mL/sec).AllpressuresaregiveninmmH2O.Femoralarterialpressure(notshown)didnotchangesubstantiallyduringthenormalinspiration.Jugular vein 50 5.2 mL/sec 11 mL/sec SVCF JVP RAP 0 Atrial 0 –100

Thedramaticincreaseinintrathoracicpressureinducedbycoughingconstitutesanauxiliarypumpingmechanismfortheblooddespiteitsconcurrenttendencytoimpedevenousreturn.Certaindiagnosticprocedures,suchascoronaryangiographyorelectrophysiologicaltestingofcardiacfunction,increasetheriskforventricularfibrillation;therefore,patientsundergoingsuchproceduresaretrainedtocoughrhythmicallyoncommandduringthestudy.Ifventricularfibrillationdoesoccur,eachcoughcangeneratesubstantialincreasesinarterialbloodpressure,andenoughcerebralbloodflowmaybepromotedtosustainconsciousness.Thecoughraisesintravascularpressureequallyintheintrathoracicarteriesandveins.Bloodispropelledthroughtheextrathoracictissuesbecausetheincreasedpressureistransmittedtotheextrathoracicarteriesbutnottotheextrathoracicveinsbecausethevenousvalvespreventbackwardflowfromtheintrathoracictotheextrathoracicveins.

Inmostformsofartificialrespiration(mouth-to-mouthresuscitation,mechanicalrespiration),endotrachealpressureaboveatmosphericpressureisusedtoinflatethelungs,andexpirationoccursbypassiverecoilofthethoraciccage(see ).Thuslunginflationisaccompaniedbyanappreciableriseinintrathoracicpressure.Venacavalflowdecreasessharplyduringthephaseofpositive-pressurelunginflationwhentheendotrachealpressureprogressivelyrises.Whennegativeendotrachealpressureisusedtofacilitatedeflation,venacavalflowacceleratesmorethanwhenthelungsareallowedtodeflatepassively.

Sustained expiratory efforts increase intrathoracic pressure and thus impede venous return. Straining against a closed glottis (Valsalva’s maneuver) regularly occurs during coughing, defecation, and heavy lifting. Intrathoracic pressures in excess of 100 mm Hg have been recorded in trumpet players, and pressures higher than 400 mm Hg have been observed during paroxysms of coughing. Such increases in pressure are transmitted directly to the lumens of the intrathoracic arteries. After coughing stops, arterial blood pressure may fall precipitously because of the preceding impediment to venous return.

Interplay of Central and Peripheral Factors in Control of the Circulation

The primary function of the circulatory system is to deliver the nutrients needed for tissue metabolism and growth and to remove the products of metabolism. The contributions of the components of the cardiovascular system to maintain adequate tissue perfusion under different physiological conditions were discussed previously. In this section, the interrelationships among the various components of the circulatory system are explored. The autonomic nervous system and the baroreceptors and chemoreceptors play key roles in regulating the cardiovascular system. Control of fluid balance by the kidneys, with maintenance of a constant blood volume, is also very important.

In any well-regulated system, one way to evaluate the extent and sensitivity of its regulatory mechanisms is to disturb the system and to observe how it restores the preexisting steady state. Two such disturbances, physical exercise and hemorrhage, are discussed in the following sections to illustrate operation of the various regulatory factors.

The cardiovascular adjustments that occur during exercise consist of a combination of neural and local (chemical) factors. Neural factors include (1) central command, (2) reflexes that originate in the contracting muscle, and (3) the baroreceptor reflex. Central command is the cerebrocortical activation of the sympathetic nervous system that produces cardiac acceleration, increased myocardial contractile force, and peripheral vasoconstriction. Reflexes are activated intramuscularly by stimulation of mechanoreceptors (by stretch, tension) and chemoreceptors (by metabolic products) in response to muscle contraction. Impulses from these receptors travel centrally via small myelinated (group III) and unmyelinated (group IV) afferent nerve fibers. Group IV unmyelinated fibers may represent the muscle chemoreceptors, inasmuch as no morphological chemoreceptor has been identified. The central connections of this reflex are unknown, but the efferent limb consists of sympathetic nerve fibers to the heart and peripheral blood vessels. The baroreceptor reflex is described in , and local factors that influence skeletal muscle blood flow (metabolic vasodilators) are described in . Vascular chemoreceptors are important in regulation of the cardiovascular system during exercise. Evidence for this assertion comes from the observations that the PaCO2, the PaO2, and the pH of arterial blood remain normal during exercise.

Mild to Moderate Exercise

In humans or trained animals, anticipation of physical activity inhibits vagal nerve impulses to the heart and increases sympathetic discharge. The result is an increase in heart rate and myocardial contractility. The tachycardia and enhanced contractility increase cardiac output.

When cardiac stimulation occurs, the sympathetic nervous system also changes vascular resistance in the periphery. Sympathetic nervous system–mediated vasoconstriction increases vascular resistance and thereby diverts blood away from the skin, kidneys, splanchnic regions, and inactive

Cardiac output (L/min) 22 18 14 10 6 2 0.25 1 2 3 4 O2 uptake (L/min) Skin Heart and brain Viscera Muscle Maximum Vo2 .

•Fig. 19.16 ApproximatedistributionofcardiacoutputatrestandatdifferentlevelsofexerciseuptothemaximalO2consumption(V˜O2)in 0 0 5 10 15 800 1600 0.006 0.010 0.014 60 100 140 180 5 10 15 90 100 110 60 100 140 180 Mean Systolic pressure Diastolic pressure anormalyoungman.(RedrawnfromRuchHP,PattonTC.Physiology and Biophysics. 12thed.Philadelphia:Saunders;1974.)

Fig. 19.16 ). This increase in vascular resistance persists throughout the period of exercise.

Cardiac output and blood flow to active muscles increase as the intensity of exercise increases. Blood flow to the myocardium increases, whereas flow to the brain is unchanged. Blood flow in the skin initially decreases during exercise, then increases as body temperature rises with increments in the duration and intensity of exercise, and finally decreases when the skin vessels constrict as total body O2 consumption nears its maximal value (see

Fig. 19.16

The major circulatory adjustment to prolonged exercise occurs in the vasculature of the active muscles. Local formation of vasoactive metabolites causes marked dilation of the resistance vessels. This dilation progresses with increases in the intensity of exercise. Potassium is one of the vasodilator substances released by the contracting muscle, and this ion may be partly responsible for the initial decrease in vascular resistance in the active muscles. Other contributing factors may be the release of adenosine and a decrease in tissue pH during sustained exercise. The local accumulation of metabolites causes the terminal arterioles to relax, and blood flow through the muscle may increase 15to 20-fold above the resting level. This metabolic vasodilation of the precapillary vessels in active muscles occurs very soon after the onset of exercise. The decrease in TPR enables the heart to pump more blood at a lesser load, and it pumps more efficiently than if TPR were unchanged (see •Fig. 19.17 Effectsofdifferentlevelsofexercise(i.e.,work)onseveralcardiovascularvariables.(DatafromCarlstenA,GrimbyG.The Circulatory Response to Muscular Exercise in Man. Springfield,

IL:CharlesCThomas;1966.)

Marked changes in the capillary circulation also occur during exercise. At rest, only a small percentage of the capillaries are perfused, whereas in actively contracting muscle, all or nearly all of the capillaries contain flowing blood (capillary recruitment). The surface area available for exchange of gases, water, and solutes is increased many times. Furthermore, hydrostatic pressure in the capillaries is increased because of relaxation of the resistance vessels. Hence, water and solutes move into the muscle tissue. Tissue pressure rises and remains elevated during exercise as fluid continues to move out of the capillaries; this tissue fluid is carried away by the lymphatic vessels. Lymph flow is increased as a result of the rise in capillary hydrostatic pressure and the massaging effect of the contracting muscles on the valve-containing lymphatic vessels (see

Contracting muscle avidly extracts O2 from the perfusing blood and thereby increases the arteriovenous O2 difference (

Fig. 19.17 ). This release of O2 from blood is facilitated by the shift in the oxyhemoglobin dissociation curve during exercise. During exercise, the high concentration of CO2 and the formation of lactic acid cause a reduction in tissue pH. This decrease in pH, in addition to the increase in temperature in the contracting muscle, shifts the oxyhemoglobin dissociation curve to the right (see ). Therefore, at any given PO2, less O2 is held by the hemoglobin in the red blood cells, and consequently more O2 is available for the tissues. Oxygen consumption may increase as much as 60-fold, with only a 15-fold increase in muscle blood flow. Muscle myoglobin may serve as a limited O2 store during exercise, and it can release the attached O2 at very low partial pressures. However, myoglobin can also facilitate O2 transport from capillaries to mitochondria by serving as an O2 carrier.

Because the enhanced sympathetic drive and the reduced parasympathetic inhibition of the sinoatrial node continue during exercise, tachycardia persists. If the workload is moderate and constant, the heart rate reaches a certain level and remains there throughout the period of exercise. However, if the workload increases, the heart rate increases concomitantly until a plateau of approximately 180 beats per minute is reached during strenuous exercise. In contrast to the large increase in heart rate, the increase in stroke volume is only approximately 10% to 35%, the larger values occurring in trained individuals (see

Fig. 19.17 ). In well-trained distance runners, whose cardiac output can reach six to seven times the resting level, stroke volume attains approximately twice the resting value.

Cardiacmusclesize(growth)isdirectlyrelatedtotheamountofworkthatisimposeduponit.Duringdevelopmentandinenduranceexercise,cardiacgrowthisachievedataconstantrelationbetweensystolicbloodpressureandtheratioofwallthicknesstoventricularchamberradius.Anechocardiographicmeasurementusedtodistinguishphysiologicalfrompathologicalhypertrophyisrelativewallthickness(ratioofleftventricularwallthicknesstochamberradius).Inphysiologicalhypertrophy,leftventricularmassandradiusincreaseproportionatelysothatrelativewallthicknessdoesnotchangesignificantly.Examplesofphysiologicalhypertrophyoccurinenduranceathletesandinpregnantwomen,inwhomleftventricularenlargementoccurswithvolumeoverloadatconstantrelativewallthickness.Physiologicalhypertrophyisassociatedwithanincreasedarteriolardiameterinexperimentalanimals.Also,capillarydensityincreasesinproportiontothedegreeofhypertrophy.Thisisincontrasttothesituationinpathologicalhypertrophy,inwhichareductionofcapillarydensity(rarefaction)canoccur.Neithermyocardialfibrosisnorderangementofmusclefiberorientationisdetectedinphysiologicalhypertrophy,incontrasttothefindingsinpathologicalhypertrophy.

Thus the increase in cardiac output observed during exercise is correlated principally with an increase in heart rate. If the baroreceptors are denervated, the cardiac output and heart rate responses to exercise are small in comparison with those in individuals with normally innervated baroreceptors. However, with total cardiac denervation, exercise still increases cardiac output as much as it does in normal individuals. This increase in cardiac output is achieved chiefly by means of an elevated stroke volume. However, if a β-adrenergic receptor antagonist is given to dogs with denervated hearts, exercise performance is impaired. The β-adrenergic receptor antagonist prevents the cardiac acceleration and enhanced contractility caused by increased amounts of circulating catecholamines. Therefore, the increase in cardiac output necessary for maximal exercise performance is limited.

In addition to the contribution made by sympathetically mediated constriction of the capacitance vessels in both exercising and nonexercising parts of the body, venous return is aided by the auxiliary pumping action of the working skeletal muscles and the muscles of respiration (see also ). The intermittently contracting muscles compress the veins that course through them. Because the venous valves are oriented toward the heart, the contracting muscle pumps blood back toward the right atrium (see ). In exercise, the flow of venous blood to the heart is also aided by the deeper and more frequent respirations that increase the pressure gradient between the abdominal and thoracic veins (intrathoracic pressure becomes more negative during exercise).

In humans, blood reservoirs do not contribute much to the circulating blood volume. In fact, blood volume is usually reduced slightly during exercise, as evidenced by a rise in the hematocrit ratio. This decrease in blood volume is caused by water loss externally through sweating and enhanced ventilation and by fluid movement into the contracting muscle. However, fluid loss is counteracted in several ways. Fluid loss from the vascular compartment into the contracting muscles eventually reaches a plateau as interstitial fluid pressure rises and opposes the increased hydrostatic pressure in capillaries of the active muscle. Fluid loss is partially offset by movement of fluid from the splanchnic regions and inactive muscle into the bloodstream. This influx of fluid results from (1) a decrease in hydrostatic pressure in the capillaries of these tissues and (2) an increase in plasma osmolarity because of movement of osmotically active molecules into blood from the contracting muscle. Reduction in urine formation by the kidneys also helps conserve body water.

The large volume of venous blood returning to the heart is so effectively pumped through the lungs and out into the aorta that Pv remains essentially constant. Thus the Frank-Starling mechanism of a greater initial fiber length does not account for the greater stroke volume in moderate exercise. Radiographs of individuals at rest and during exercise reveal a decrease in heart size during exercise. However, during maximal or near-maximal exercise, right atrial pressure and end-diastolic ventricular volume do increase, and the Frank-Starling mechanism contributes to the enhanced stroke volume in very vigorous exercise.

•Fig. 19.18 Cardiacandvascularfunctioncurvesaregreatlyalteredduringstrenuousexercise,whichallowscardiacoutputtoincreasefourfoldtofivefold.TheoperatingpointofthecardiovascularsystemmovesfrompointAtopointB.Thecardiacfunctioncurveduringstrenuousexerciseistheresultofincreasedheartrate,strokevolume,andcontractility.Thevascularfunctioncurvereflectsgreatlydecreasedtotalperipheralresistanceandincreasedmeancirculatorypressure.Atthenewoperatingpoint(pointB),cardiacoutputisincreasedmorethanfourfold,butfillingpressureisincreasedonlyslightly.

In an active, healthy (untrained) individual, the mechanisms previously described typically lead to a fourfold to fivefold increase in cardiac output during vigorous exercise (

Fig. 19.18 ). Increased cardiac output is the fundamental means by which more O2 is delivered to exercising muscles (see

Fig. 19.17 ). The cardiac function curve during exercise reflects increased stroke volume (up to ≈1.5-fold) and heart rate (up to ≈3-fold). The vascular function curve during dynamic exercise reflects a marked decrease in peripheral resistance (changed slope) and an increase in mean circulatory filling pressure (changed intercept), which result from increased venous constriction (tone) in the skeletal muscle “pump,” and the respiratory “pump.” In these conditions, the cardiovascular system is able to operate at a new point (point B in

Fig. 19.18 ), in which cardiac output is increased while filling pressure is little changed. The graphical analysis (see

Fig. 19.18 ) shows that without the systemic changes in vascular function, even a heart beating strongly and fast would be able to produce only a small increase in cardiac output.

If exercise involves a large proportion of the body musculature, as in running or swimming, the reduction in total vascular resistance can be considerable. Nevertheless, arterial pressure starts to rise with the onset of exercise, and the increase in blood pressure approximately parallels the severity of the exercise performed (see

Fig. 19.17 ). Therefore, the increase in cardiac output is proportionally greater than the decrease in TPR. The vasoconstriction produced in the inactive tissues by the sympathetic nervous system (and to some extent by the release of catecholamines from the adrenal medulla) is important for maintenance of normal or increased blood pressure. Sympathectomy or drug-induced blockade of the adrenergic sympathetic nerve fibers decreases arterial pressure (hypotension) during exercise.

Sympathetic neural activity also elicits vasoconstriction in active skeletal muscle when additional muscles are recruited. In experiments in which one leg is working at maximal levels and then the other leg starts to work, blood flow decreases in the first working leg. Furthermore, blood levels of norepinephrine rise significantly during exercise, and most of the norepinephrine is released from sympathetic nerves to the active muscles.

As body temperature rises during exercise, the skin vessels dilate in response to thermal stimulation of the heat-regulating center in the hypothalamus, and TPR decreases further. This reduction in TPR would reduce blood pressure were it not for the increased cardiac output and the constriction of arterioles in the renal, splanchnic, and other tissues.

In general, Pa rises during exercise as a result of the increase in cardiac output. However, the effect of enhanced cardiac output is offset by an overall decrease in TPR, and therefore mean blood pressure increases only slightly. Vasoconstriction in the inactive vascular beds helps maintain normal arterial blood pressure for adequate perfusion of the active tissues. The actual Pa attained during exercise thus represents a balance between cardiac output and TPR (see ). Systolic pressure usually increases more than diastolic pressure, which results in an increase in pulse pressure (see

Fig. 19.17 ). The larger pulse pressure is primarily attributable to a greater stroke volume, but also to more rapid ejection of blood by the left ventricle and diminished peripheral runoff during the brief ventricular ejection period (see also

During exhaustive exercise, the compensatory mechanisms begin to fail. The heart rate attains a maximal level of approximately 180 beats per minute, and stroke volume reaches a plateau. The heart rate may then decrease, which results in a fall in blood pressure. The exercising individual also frequently becomes dehydrated. Sympathetic vasoconstrictor activity supersedes the vasodilator influence on vessels of the skin so that the rate of heat loss is decreased. Body temperature is normally elevated during exercise. A reduction in heat loss through cutaneous vasoconstriction can lead to very high body temperatures and to acute distress during severe exercise. Tissue pH and blood pH decrease as a result of increased production of lactic acid and CO2. The reduced pH may be a key factor that determines •Fig. 19.19 Cardiovascularadjustmentsinexercise.Plus signs indicateincreasedactivity,andminus signs indicatedecreasedactivity.C,vasoconstrictoractivity;D,vasodilatoractivity;IX,glossopharyngeal nerve;VR,vasomotorregion;X,vagusnerve.

the maximal amount of exercise that a given individual can tolerate. Muscle pain, a subjective feeling of exhaustion, and loss of the will to continue determine exercise tolerance. A summary of the neural and local effects of exercise on the cardiovascular system is diagrammed in

Fig. 19.19

When exercise stops, the heart rate and cardiac output quickly decrease: The sympathetic drive to the heart is essentially removed. In contrast, TPR remains low for some time after the exercise is stopped, presumably because vasodilator metabolites have accumulated in the muscles during the exercise period. As a result of the reduced cardiac output and persistence of vasodilation in the muscles, arterial pressure falls, often below preexercise levels, for brief periods. Blood pressure is then stabilized at normal levels by the baroreceptor reflexes.

Limits of Exercise Performance

The two main factors that limit skeletal muscle performance in humans are the rate of O2 use by the muscles and the O2 supply to the muscles. However, O2 use by muscle is probably not a critical factor. During exercise, maximal O2 consumption (maximal V˜O2) by a large percentage of the body’s muscle mass is unchanged or increases only slightly when additional muscles are activated. In fact, during exercise of a large muscle mass, as in vigorous bicycling, the addition of bilateral arm exercise without change in the cycling effort produces only a small increase in cardiac output and maximal VO2. However, the additional arm exercise decreases blood flow to the legs. This centrally mediated (baroreceptor reflex) vasoconstriction during maximal cardiac output prevents the fall in blood pressure that would otherwise be caused by metabolically induced vasodilation in the active muscle. If use of O2 by muscles were a significant limiting factor, recruitment of more contracting muscles would entail the use of much more O2 to meet the enhanced O2 requirements.

Limitation of the O2 supply could be caused by inadequate oxygenation of blood in the lungs or limitation of the supply of O2-laden blood to the muscles. Failure by the lungs to oxygenate blood fully can be ruled out because even with the most strenuous exercise at sea level, arterial blood is fully saturated with O2. Therefore, O2 delivery to the active muscles (or blood flow because the arterial blood O2 content is normal) appears to be the limiting factor in muscle performance. This limitation could be caused by the inability to increase cardiac output beyond a critical level. In turn, this inability is caused by a limitation in stroke volume because the heart rate reaches maximal levels before maximal V˜O2 is reached. Hence, the major factor that limits muscle performance is the pumping capacity of the heart.

The response of the cardiovascular system to regular exercise is to increase its capacity to deliver O2 to the active muscles and improve the ability of the muscle to use O2. Maximal

VO2 varies with the level of physical conditioning. Train ing progressively increases maximal VO2, which reaches a plateau at the highest level of conditioning. Highly trained athletes have a lower resting heart rate, a greater stroke volume, and lower peripheral resistance than they had before training or after deconditioning. The low resting heart rate is caused by a higher vagal tone and a lower sympathetic tone. During exercise, the maximal heart rate of a trained individual is the same as that in an untrained person, but it is attained at a higher level of exercise. A trained person also exhibits low vascular resistance in the muscles. If an individual exercises one leg regularly over an extended period and does not exercise the other leg, vascular resistance is lower and maximal V˜O2 is higher in the “trained” leg than in the “untrained” leg.

Physical conditioning is also associated with greater extraction of O2 from the blood (greater arteriovenous O2 difference) by the muscles. With long-term training, capillary density in skeletal muscle increases. Also, an increase in the number of arterioles may account for the decrease in muscle vascular resistance. The number of mitochondria increases, as does the number of oxidative enzymes in mitochondria. In addition, levels of adenosine triphosphatase (ATPase) activity, myoglobin, and enzymes involved in lipid metabolism increase in response to physical conditioning.

Endurancetraining,suchasrunningorswimming,increasesleftventricularvolumewithoutincreasingleftventricularwallthickness.Incontrast,strengthexercises,suchasweightlifting,increaseleftventricularwallthickness(hypertrophy)withlittleeffectonventricularvolume.However,thisincreaseinwallthicknessissmallinrelationtothatobservedinchronichypertension,inwhichafterloadispersistentlyelevatedbecauseofhighperipheralresistance.

The cardiovascular system is the system mainly affected in an individual who has lost a large quantity of blood. Arterial systolic, diastolic, and pulse pressures decrease, and the arterial pulse is rapid and feeble. The cutaneous veins collapse, and they fill slowly when compressed centrally. The skin is pale, moist, and slightly cyanotic. Respiration is rapid but may be shallow or deep.

Course of Arterial Blood Pressure Changes

Cardiac output decreases as a result of blood loss. The amount of blood removed when it is donated is approximately 10% of total blood volume; its removal is well tolerated, and mean arterial blood pressure changes little. This is not the case when greater amounts are lost from the circulation. The changes in Pa evoked by acute hemorrhage are illustrated in

Fig. 19.19 . If sufficient blood is rapidly withdrawn to decrease Pa to 50 mm Hg, the pressure then tends to rise spontaneously toward the control level over the next 20 or 30 minutes. In some individuals (curve A in

Fig.

19.20 ), this trend continues, and normal pressure is regained within a few hours. In others (curve B in

Fig. 19.20 ), the pressure rises initially after the cessation of hemorrhage. The pressure then begins to decline, and it continues to fall at an accelerating rate until death ensues. This progressive deterioration in cardiovascular function is termed hemorrhagic shock. At some time after the hemorrhage, the deterioration in the cardiovascular system becomes irreversible. A lethal outcome in patients with hemorrhagic shock can be prevented only temporarily by any known therapy, including massive transfusions of donor blood.

•Fig. 19.20 Changes in Mean Arterial Pressure After Rapid Hem-orrhage. Attime0,rapidlossofbloodcausesareductioninthemeanarterialpressureto50mmHg.Afteraperiodinwhichthepressurereturnstowardthecontrollevel,someindividualscontinuetoimprove(curveA)untilthecontrolpressureisattained.However,inotherindividuals,thepressurebeginstodecline(curveB)untildeathensues.–2 –1 0 0 25 50 75 100 1 2 3 4 5 A B 6 7 Time (hr) Mean arterial pressure (mm Hg)

The changes in arterial pressure immediately after acute blood loss (see

Fig. 19.20 ) indicate that certain compensatory mechanisms must be operative. Any mechanism that raises arterial blood pressure toward normal in response to a reduction in pressure is designated a negative feedback mechanism. This mechanism is termed negative because the direction of the secondary change in pressure is opposite the direction of the initiating change after the acute blood loss. The following negative feedback responses are evoked: baroreceptor reflexes, (2) chemoreceptor reflexes, (3) cerebral ischemia responses, (4) reabsorption of tissue fluids, release of endogenous vasoconstrictor substances, and renal conservation of salt and water.

The reductions in Pa and pulse pressure during hemorrhage decrease stimulation of the baroreceptors in the carotid •Fig. 19.21 Changes in Mean Aortic Pressure in Response to an 8% Blood Loss in Three Conditions. A, Thecarotidsinusbaroreceptorswereintactandtheaorticreflexeswereinterrupted.B, Theaorticreflexeswereintactandthecarotidsinusreflexeswereinterrupted.C, Allsinoaorticreflexeswereabrogated.(DatafromShepherdJT.Circulation. 1974;50:418.DerivedfromthedataofEdisAJ.Am J Physiol. 1971;221:1352.) ). Several cardiovascular responses are thus evoked, all of which tend to restore arterial pressure to the normal level. Such responses include reduction of vagal tone and enhancement of sympathetic tone, increased heart rate, and enhanced myocardial contractility.

The increased sympathetic tone also produces generalized venoconstriction, which has the same hemodynamic consequences as transfusion of blood (see

Fig. 19.9). Sym pathetic activation constricts certain blood reservoirs. This vasoconstriction acts as an autotransfusion of blood into the circulation. In humans, the cutaneous, pulmonary, and hepatic branches of the vasculature constitute the principal blood reservoirs.

Generalized arteriolar constriction is a prominent response to the reduced baroreceptor stimulation during hemorrhage. The reflex increase in peripheral resistance minimizes the fall in arterial pressure caused by the reduction in cardiac output.

Fig. 19.21 shows the effect of an 8% blood loss on mean aortic pressure. When both vagus nerves were cut to eliminate the influence of the aortic arch baroreceptors and only the carotid sinus baroreceptors were operative (see

Fig. 19.21A), this hemorrhage decreased mean aortic pressure by 14%. This pressure change did not differ significantly from the decline in pressure (12%) evoked by the same hemorrhage before vagotomy (not shown). When the carotid sinuses were denervated and the aortic baroreceptor reflexes were intact, the 8% blood loss decreased mean aortic pressure by 38% (see

Fig. 19.21B).

Hence, the carotid sinus baroreceptors were more effective than the aortic baroreceptors in attenuating the fall in pressure. However, when both sets of afferent baroreceptor pathways were interrupted (see

Fig. 19.21C), an 8% blood loss reduced arterial pressure by 48%.

Arteriolar constriction is widespread during hemorrhage but it is by no means uniform. Vasoconstriction is most pronounced in the cutaneous, skeletal muscle, and splanchnic vascular beds, and it is slight or absent in the cerebral and coronary circulations in response to hemorrhage. In many instances, cerebral and coronary vascular resistance is diminished. The reduced cardiac output is redistributed to favor flow through the brain and the heart.

In the early stages of mild to moderate hemorrhage, renal resistance changes only slightly. The tendency for increased sympathetic activity to constrict the renal vessels is counteracted by autoregulatory mechanisms (see ). With more prolonged and severe hemorrhage, however, renal vasoconstriction becomes intense.

The renal and splanchnic vasoconstriction during hemorrhage is least severe in the heart and brain. However, if such constriction persists too long, it may be detrimental. Frequently, patients survive the acute hypotensive period of a prolonged, severe hemorrhage, only to die several days later from the kidney failure that results from renal ischemia. Intestinal ischemia may also have dire effects. For example, intestinal bleeding and extensive sloughing of the mucosa can occur after only a few hours of hemorrhagic hypotension. Furthermore, the diminished splanchnic flow swells the centrilobular cells in the liver. The resulting obstruction of the hepatic sinusoids causes portal venous pressure to rise, and this response intensifies intestinal blood loss.

Reductions in arterial pressure below approximately 60 mm Hg do not evoke any additional responses through the baroreceptor reflexes because this pressure level constitutes the threshold for stimulation (see ). However, low arterial pressure may stimulate peripheral chemoreceptors because inadequacy of local blood flow leads to hypoxia in the chemoreceptor tissue. Chemoreceptor excitation may then enhance the already existent peripheral vasoconstriction evoked by the baroreceptor reflexes. In addition, respiratory stimulation assists venous return by the auxiliary pumping mechanism described earlier (see also

When arterial pressure falls below approximately 40 mm Hg as a consequence of blood loss, the resulting cerebral ischemia activates the sympathoadrenal system. The discharge by sympathetic nerves is several times greater than the maximal neural activity that occurs when the baroreceptors cease to be stimulated. The vasoconstriction and increase in myocardial contractility may be pronounced. With more severe degrees of cerebral ischemia, however, the vagal centers also become activated. The resulting bradycardia aggravates the hypotension that initiated the cerebral ischemia

Reabsorption of Tissue Fluids

The arterial hypotension, arteriolar constriction, and reduced venous pressure during hemorrhagic hypotension cause the hydrostatic pressure in the capillaries to drop. The balance of these forces promotes the net reabsorption of interstitial fluid into the vascular compartment (see ). The rapidity of this response is displayed in

Fig. 19.22 . When 45% of the estimated blood volume is removed over a 30-minute period, mean arterial blood pressure declines rapidly and then is largely restored to nearly the control level. Plasma colloid osmotic pressure declines markedly during the bleeding and continues to decrease more gradually for several hours. The reduction in colloid osmotic pressure reflects dilution of the blood by tissue fluids that contain little protein.

Considerable quantities of fluid may thus be drawn into the circulation during hemorrhage. Approximately 0.25 mL of fluid per minute per kilogram of body weight may be reabsorbed by the capillaries. Thus approximately 1 L of fluid per hour might be autoinfused from the interstitial spaces into the circulatory system of an average individual after acute blood loss. Substantial quantities of fluid may shift slowly from the intracellular space to the extracellular space. This fluid exchange is probably mediated by secretion of cortisol from the adrenal cortex in response to hemorrhage. Cortisol is essential for the full restoration of plasma volume after hemorrhage.

The catecholamines epinephrine and norepinephrine are released from the adrenal medulla in response to the same stimuli that evoke widespread sympathetic nervous discharge (see

Chapter 43). Blood levels of catecholamines are high during and after hemorrhage. When blood loss is such that arterial pressure is reduced to 40 mm Hg, the level of catecholamines increases as much as 50-fold. Epinephrine comes almost exclusively from the adrenal medulla, whereas norepinephrine is derived from both the adrenal medulla and peripheral sympathetic nerve endings. These humoral substances reinforce the effects of the sympathetic nervous activity listed previously.

Vasopressin (antidiuretic hormone), a potent vasoconstrictor, is secreted by the posterior pituitary gland in response to hemorrhage (see 41).

The plasma concentration of vasopressin rises progressively as arterial blood pressure diminishes (

Fig. 19.23 ). The receptors responsible for the augmented release of vasopressin are the aortic arch and carotid sinus baroreceptors (high pressure) and stretch receptors in the left atrium (low pressure).

The diminished renal perfusion during hemorrhagic hypotension leads to the secretion of renin from the juxtaglomerular apparatus (see

Chapter 35).

This enzyme acts on a plasma protein, angiotensinogen, to form the decapeptide angiotensin I, which in turn is cleaved to the active octapeptide angiotensin II by angiotensin-converting enzyme; angiotensin II is a very powerful vasoconstrictor.

Renal Conservation of Salt and Water

Fluid and electrolytes are conserved by the kidneys during hemorrhage in response to various stimuli, including increased secretion of vasopressin, as noted previously (see

Fig. 19.23 ), and increased renal sympathetic nerve activity, which enhances NaCl reabsorption by the nephron (decreased excretion). The lower arterial pressure decreases •Fig. 19.22 Changesinarterialbloodpressureandplasmacolloid osmoticpressureinresponsetowithdrawalof45%oftheestimated•Fig. 19.23 Meanpercentagechangesinarterialbloodpressureandbloodvolumeovera30-minuteperiod,beginningattime0.(Redrawnplasmavasopressinconcentrationinresponsetobloodloss.(RedrawnfromZweifachBW.Anesthesiology. 1974;41:157.)fromShenYT,etal.Circ Res. 1991;68:1422.) the glomerular filtration rate, which also curtails the excretion of water and electrolytes. In addition, the elevated levels of angiotensin II, as described earlier, stimulate the release of aldosterone from the adrenal cortex. Aldosterone, in turn, stimulates reabsorption of NaCl by the nephrons. Thus NaCl and water excretion is decreased (see also

Chapter 35).

In contrast to negative feedback mechanisms, hemorrhage also evokes latent positive feedback mechanisms. These mechanisms exaggerate any primary change initiated by the blood loss. Specifically, positive feedback mechanisms aggravate the hypotension induced by blood loss and tend to initiate vicious cycles, which may lead to death.

Whether a positive feedback mechanism will lead to a vicious cycle depends on the gain of that mechanism. Gain is the ratio of the secondary change evoked by a given mechanism to the initiating change itself. A gain greater than 1 induces a vicious cycle; a gain of less than 1 does not. Consider a positive feedback mechanism with a gain of 2. If Pa were to decrease by 10 mm Hg, a positive feedback mechanism with a gain of 2 would then evoke a secondary reduction in pressure of 20 mm Hg, which in turn would cause a further decrease of 40 mm Hg. Thus each change would induce a subsequent one that is twice as great. Hence, Pa would decline at an ever-increasing rate until death occurred. This process is depicted in curve B in

Fig. 19.20

Conversely, a positive feedback mechanism with a gain of 0.5 also exaggerates any change in Pa, but the change would not necessarily lead to death. If arterial pressure suddenly decreased by 10 mm Hg, a positive feedback mechanism would initiate a secondary, additional fall of 5 mm Hg. This decrease, in turn, would provoke a further decrease of 2.5 mm Hg. The process would continue in ever-diminishing steps until arterial pressure approached an equilibrium value.

Some of the more important positive feedback mechanisms that are evident during hemorrhage include (1) cardiac failure, (2) acidosis, (3) central nervous system depression, (4) aberrations in blood clotting, and (5) depression of the mononuclear phagocytic system (MPS).

These mechanisms are discussed next.

Shifts to the right in ventricular function curves, particularly in the later stages of hemorrhagic shock (

Fig. 19.24 ), provide evidence of a progressive depression in myocardial contractility during hemorrhage.

*The MPS (previously called the reticuloendothelial system) consists of macrophages that are distributed throughout the body. They are derived from bone marrow and exist for a short period in circulating blood as monocytes. They then migrate into tissues, where they phagocytose foreign material and present antigens to lymphocytes to initiate the adaptive immune response. Cells of the MPS include the Kupffer cells of the liver, alveolar macrophages, microglia, and Langerhans cells.

•Fig. 19.24 Ventricular Function Curves for the Left Ventricle During the Course of Hemorrhagic Shock. Curve A representsthecontrolfunctioncurve;curves B toF representtimeafterthehemor-rhage:117minutes(curve B), 247minutes(curve C), 280minutes(curve D), 295minutes(curve E), and310minutes(curve F). (RedrawnfromCrowellJW,GuytonAC.Am J Physiol. 1962;203:248.)–2 0 2 4 6 8 10 12 0 200 400 600 800 1000 1200 1400 1600 A E F C D B Left atrial pressure (mm Hg) Cardiac output (mL/min)

The hypotension induced by hemorrhage reduces coronary blood flow and therefore depresses ventricular function. The consequent reduction in cardiac output further reduces arterial pressure, a classic example of a positive feedback mechanism. Furthermore, reduced blood flow to peripheral tissues leads to an accumulation of vasodilator metabolites that decrease peripheral resistance and therefore aggravate the fall in arterial pressure.

The inadequate blood flow during hemorrhage affects the metabolism of all cells. The decreased O2 delivery to cells accelerates tissue production of lactic acid and other acid metabolites. Moreover, impaired kidney function prevents adequate excretion of the excess H+ , and generalized metabolic acidosis ensues. The resulting depressant effect of acidosis on the heart is a further reduction in tissue perfusion, which aggravates the metabolic acidosis. Acidosis also reduces the reactivity of the heart and resistance vessels to neurally released and circulating catecholamines and thereby intensifies the hypotension.

The hypotension in shock reduces cerebral blood flow. Moderate degrees of cerebral ischemia induce pronounced sympathetic nervous stimulation of the heart, arterioles, and veins, as noted earlier. In severe hypotension, however, the cardiovascular centers in the brainstem eventually become depressed because of inadequate cerebral blood flow. The resulting loss of sympathetic tone then reduces cardiac output and peripheral resistance. The consequent reduction in Pa intensifies the inadequate cerebral perfusion.

Endogenous opioids, such as enkephalins and βendorphin, may be released into the brain substance and into the circulation in response to the same stresses that provoke circulatory shock. Opioids are stored, along with catecholamines, in secretory granules in the adrenal medulla and in sympathetic nerve terminals, and they are released together in response to stress. Similar stimuli cause the release of β-endorphin and adrenocorticotropic hormone from the anterior pituitary gland. Opioids depress the brainstem centers that mediate some of the compensatory autonomic adaptations to blood loss, endotoxemia, and other shock-provoking stress. Conversely, the opoid antagonist naloxone improves cardiovascular function and rates of survival in various forms of shock.

The alterations in blood clotting after hemorrhage are typically biphasic. An initial phase of hypercoagulability is followed by a secondary phase of hypocoagulability and fibrinolysis. In the initial phase, platelets and leukocytes adhere to the vascular endothelium, and intravascular clots, or thrombi, develop within a few minutes of the onset of severe hemorrhage. This phenomenon, called disseminated intravascular coagulation (DIC), occurs when thrombin is activated and causes widespread deposition of fibrin within narrow and medium-diameter vessels.

The initial phase is further enhanced by the release of thromboxane A2 from various ischemic tissues. Thromboxane A2 aggregates platelets. As more platelets aggregate, more thromboxane A2 is released and more platelets are trapped. This form of positive feedback intensifies and prolongs the clotting tendency. Inflammatory cytokines (interleukin-6, tumor necrosis factor) also contribute to DIC. The rate of mortality from certain standard shock-provoking procedures has been reduced considerably by the administration of anticoagulants such as heparin.

In the later stages of hemorrhagic hypotension, the clotting time is prolonged, and fibrinolysis is prominent. Fibrinolysis occurs when clotting factors and platelets are depleted.

Depression of the Mononuclear Phagocytic System

During the course of hemorrhagic hypotension, MPS function becomes depressed. The phagocytic activity of the MPS is modulated by an opsonic protein. The opsonic activity in plasma diminishes during shock, and this change may account in part for the depression in MPS function. As a result, antibacterial and antitoxin defense mechanisms are impaired. Hypoperfusion also suppresses the barrier function of the adherens junctions and tight junctions in the 1. Two important relationships between cardiac output (Qh) and central venous pressure (Pv) prevail in the cardiovascular system. With regard to the heart, Qh varies directly with Pv (or preload) over a very wide range of Pv. This relationship is represented by the cardiac function curve, and it expresses the Frank-Starling mechanism. In the vascular system, Pv varies intestinal epithelium. Endotoxins from the normal bacterial flora of the intestine constantly enter the circulation. Ordinarily, they are inactivated by the MPS, principally in the liver. Disruption of the intestinal epithelial barrier, together with depression of the MPS, allows these endotoxins to invade the general circulation. Endotoxins produce profound, generalized vasodilation, mainly by inducing the synthesis of an isoform of nitric oxide synthase in the smooth muscle of blood vessels throughout the body. The profound vasodilation aggravates the hemodynamic changes caused by blood loss.

In addition to their role in inactivating endotoxin, macrophages release many of the mediators associated with shock. These mediators include acid hydrolases, neutral proteases, oxygen free radicals, certain coagulation factors, and the following arachidonic acid derivatives: prostaglandins, thromboxanes, and leukotrienes. Macrophages also release certain monokines that modulate temperature regulation, intermediary metabolism, hormone secretion, and the immune system.

Interactions of Positive and Negative Feedback Mechanisms

Hemorrhage provokes a multitude of circulatory and metabolic derangements. Some of these changes are compensatory, and others are decompensatory. Some of these feedback mechanisms possess high gain and others possess low gain. Furthermore, the gain of any specific mechanism varies with the severity of the hemorrhage. For example, with only a slight loss of blood, Pa is maintained within the normal range and the gain of the baroreceptor reflexes is high. With greater losses of blood, when Pa is below 60 mm Hg (i.e., below the threshold for the baroreceptors), further reductions in pressure have no additional influence through the baroreceptor reflexes. Hence, below this critical pressure, the baroreceptor reflex gain is zero or near zero.

In general, with minor degrees of blood loss, the gains of negative feedback mechanisms are high, whereas those of positive feedback mechanisms are low. The opposite is true with more severe hemorrhage. The gains of the various mechanisms add algebraically. Therefore, whether a vicious cycle develops depends on whether the sum of the positive and negative gains exceeds 1. Total gains in excess of 1 are, of course, more likely to occur with severe losses of blood. Therefore, to avert a vicious cycle, serious hemorrhages must be treated quickly and intensively, preferably by whole blood transfusion, before the process becomes irreversible.

inversely with Qh. This relationship is represented by the vascular function curve, and it reflects the fact that as Qh increases, a greater fraction of the total blood volume resides in the arteries and a smaller volume resides in the veins.

2. The principal cardiac mechanisms that govern cardiac output are the changes in numbers of myocardial cross-bridges that interact and in the affinity of the contractile proteins for Ca++ . The principal factors that govern the vascular function curve are arterial and venous compliance, peripheral vascular resistance, and total blood volume.

3.

The equilibrium values of Qh and Pv that prevail under a given set of conditions are determined by the intersection of the cardiac and vascular function curves. At very low and very high heart rates, the heart is unable to produce adequate Qh. At very low heart rates, the increase in filling during diastole cannot compensate for the small number of cardiac contractions per minute. At very high heart rates, the large number of contractions per minute cannot compensate for the inadequate filling time.

4.

Gravity influences Qh because the veins are so compliant, and substantial quantities of blood tend to pool in the veins of dependent portions of the body. Respiration changes the pressure gradient between the intrathoracic and extrathoracic veins. Hence, respiration serves as an auxiliary pump, which may affect the mean level of Qh and induce rhythmic changes in stroke volume during the various phases of the respiratory cycle.

5.

In anticipation of exercise, vagus nerve impulses to the heart are inhibited and the sympathetic nervous system is activated by central command. The result is an increase in heart rate, myocardial contractile force, and regional vascular resistance. In addition, vascular resistance increases in the skin, kidneys, splanchnic regions, and inactive muscles and decreases markedly in the active muscles. The overall effect is a pronounced reduction in total peripheral resistance,

Eijsvogels TMH, Fernandez AB, Thompson PD. Are there deleterious cardiac effects of acute and chronic endurance exercise? Physiol Rev. 2016;96:99.

Fernandes T, Baraúna VG, Negrão CE, et al. Aerobic exercise training promotes physiological cardiac remodeling involving a set of microRNAs. Am J Physiol. 2015;309:H543.

Gruen RL, Brohi K, Schreiber M, et al. Haemorrhage control in severely injured patients. Lancet. 2012;380:1099.

which, along with the auxiliary pumping action of the contracting skeletal muscles, greatly increases venous return. The increases in heart rate and myocardial contractility, both induced by the activation of cardiac sympathetic nerves, enables the heart to transfer blood to the pulmonary and systemic circulations, thereby increasing cardiac output. Stroke volume increases only slightly. O2 consumption and blood O2 extraction increase, and systolic pressure and mean blood pressure increase slightly. As body temperature rises during exercise, the skin blood vessels dilate. However, when the heart rate becomes maximal during severe exercise, the skin vessels constrict. This increases the effective blood volume but causes greater increases in body temperature and a feeling of exhaustion. The limiting factor in exercise performance is delivery of blood to the active muscles.

6. Acute blood loss induces tachycardia, hypotension, generalized arteriolar constriction, and generalized venoconstriction. Acute blood loss invokes a number of negative feedback (compensatory) mechanisms, such as baroreceptor and chemoreceptor reflexes, responses to moderate cerebral ischemia, reabsorption of tissue fluids, release of endogenous vasoconstrictors, and renal conservation of water and electrolytes. Acute blood loss also invokes a number of positive feedback (decompensatory) mechanisms, such as cardiac failure, acidosis, central nervous system depression, aberrations in blood coagulation, and depression of the mononuclear phagocytic system. The outcome of acute blood loss depends on the sum of gains of the positive and negative feedback mechanisms and on the interactions between these mechanisms.

Laughlin MH, Korthuis RJ, Duncker DJ, et al. Control of blood flow to cardiac and skeletal muscles during exercise. In: Rowell LB, Shepherd JT, eds. Handbook of Physiology, Section 12: Exercise: Regulation and Integration of Multiple Systems. Bethesda, MD: Oxford University Press; 1996.

Lavie CJ, Arena R, Swift DL, et al. Exercise and the cardiovascular system: clinical science and cardiovascular outcomes. Circ Res. 2015;117:207.

Valparaiso AP, Vicente DA, Bograd BA, et al. Modeling acute traumatic injury. J Surg Res. 2015;194:220.

SECTION 5The Respiratory System MICHELLE M. CLOUTIER AND ROGER S. THRALL

Upon completion of this chapter, the student should be able to answer the following questions: 1.

Explain the anatomical structure/function relationships of the upper and lower components of the respiratory system.

2.

Compare and contrast the pulmonary and bronchial circulatory systems.

3.

Explain the relationships between innervation and muscles in the control of respiration.

4.

Compare and contrast the roles of the conducting airways and components of the respiratory unit.

5.

Compare and contrast the effects of stimulation of the parasympathetic and sympathetic nervous systems on respiratory responses.

6 Describe lung development and alveolar repair after injury to regeneration of normal architecture.

he primary function of the lungs is gas exchange, which consists of movement of oxygen (O2) into the body and removal of carbon dioxide (CO2). This chapter provides an overview of lung anatomical structure/ function relationships (i.e., upper and lower airways, the two circulatory systems, innervation, and muscles), lung development (at the embryo stage and throughout life), and lung repair. This chapter is designed to provide a broad conceptual understanding of structure/function interactions and is not intended to afford a comprehensive understanding of individual lung structures and anatomy.

The lungs are contained in a space with a volume of approximately 4 L, but they have a surface area for gas exchange that is the size of a tennis court (≈85 m2). This large surface area is composed of myriads of independently functioning respiratory units. Unlike the heart, but like the kidneys, the lungs demonstrate functional unity; that is, each unit is structurally identical and functions just like every other unit. Because the divisions of the lung and the sites of disease are designated by their anatomical locations (e.g., right upper lobe, left lower lobe), it is essential to understand pulmonary anatomy in order to comprehend respiratory physiology and pathophysiological alterations in respiratory diseases.

Upper Airways: Nose, Sinuses, and Pharynx

The respiratory system begins at the nose and ends in the most distal alveolus. Thus the nasal cavity, the posterior pharynx, the glottis and vocal cords, the trachea, and all divisions of the tracheobronchial tree are included in the respiratory system. The upper airway consists of all structures from the nose to the vocal cords, including sinuses and the larynx, whereas the lower airway consists of the trachea, airways, and alveoli. The upper airways “condition” inspired air so that by the time air reaches the trachea, inspired air is at body temperature and fully humidified. The nose also functions to filter, entrap, and clear particles larger than 10 µm in size. The interior of the nose is lined by respiratory epithelial cells interspersed with surface secretory cells. These secretory cells produce important immunoglobulins, inflammatory mediators, and interferons, which are the first line of host defense.

The paranasal sinuses (frontal, maxillary, sphenoid, and ethmoid) are lined by ciliated epithelial cells and surround the nasal passages (

Fig. 20.1A).

The cilia facilitate the movement of mucus from the upper airways and clear the main nasal passages approximately every 15 minutes. The functions of the sinuses are (1) to lessen the weight of the skull, which makes upright posture easier; (2) to offer resonance to the voice; and (3) to protect the brain from frontal trauma. The fluid covering their surfaces is continually being propelled into the nose. In some sinuses (e.g., the maxillary sinus), the opening (ostium) is at the upper edge, which makes them particularly susceptible to retention of mucus. The ostia are readily obstructed by nasal edema (swelling), and retention of secretions and secondary infection (sinusitis) can result. The volume of the nose in an adult is approximately 20 mL, but its surface area is greatly increased by the nasal turbinates, which are a series of three continuous ribbons of tissue that protrude into the nasal cavity (see

Fig. 20.1B ). The nose enables the sense of smell. Neuronal endings in the roof of the nose above the superior turbinate carry impulses through the cribriform plate to the olfactory bulb.

The pharynx is divided into three sections: the nasopharynx, oropharynx, and laryngopharynx. Important • Fig. 20.1 Illustrations of Upper Airway Anatomy.A, Anterior view of the paranasal sinuses. B, Lateral view of the nasal passage structures demonstrating the superior, middle, and inferior turbinates and the sinus ostia. C, Lateral midsagittal section view of the head and neck, showing the three divisions of the pharynx (nasopharynx, oropharynx, and laryngopharynx) and surrounding upper airway structures.

CHAPTER 20 Introduction to the Respiratory System 435 structures within these regions include the epiglottis, vocal cords, and arytenoid cartilage which is attached to the vocal cords (see

Fig. 20.1C ). The nasopharynx (2 to 3 cm wide and 3 to 4 cm long) is the most anterior and lies behind the nose. In this region, the nose and mouth are connected via an isthmus (canals) that enables both oral and nasal breathing. Also, the nasopharynx contains small masses of lymphoid tissue (adenoids), also known as pharyngeal tonsils, which fight infections. The nasopharynx is connected to the middle ear cavity via  the eustachian tubes, which aid in equalizing pressure in  the ear to atmospheric pressure; thus they represent a drainage pathway of lymphatic fluid between the throat, nose, and ears. This network of structures provides a means of fighting infections but also is a common location for infections in the head.

The soft palate separates the nasopharynx and the oropharynx, which ends at the epiglottis. The laryngopharynx begins at the epiglottis and ends at the esophagus. Its major role is to help regulate the passage of food into the esophagus and air into the lungs. With some infections, these structures can become edematous and contribute significantly to airflow resistance. The epiglottis and arytenoid cartilage (attached to the vocal cords) cover or act as a hood over the vocal cords during swallowing. Thus under normal circumstances, the epiglottis and arytenoid cartilage function to prevent aspiration of food and liquid into the lower respiratory tract. The act of swallowing food after mastication (chewing) usually occurs within 2 seconds, and it is closely synchronized with muscle reflexes that coordinate opening and closing of the airway. Hence, air is allowed to enter the lower airways, and food and liquids are kept out. Patients with some neuromuscular diseases have altered muscle reflexes and can lose this coordinated swallowing mechanism. Such patients may become susceptible to aspiration of food and liquid, which poses a risk for pneumonia.

Lower Airways: Trachea, Bronchi, Bronchioles, and Respiratory Unit

The right lung, located in the right hemithorax, is divided into three lobes (upper, middle, and lower) by two interlobular fissures (oblique, horizontal), whereas the left lung, located in the left hemithorax, is divided into two lobes (upper, including the lingula, a tongue-like projection of the anterior aspect of the upper lobe, and lower) by an oblique fissure (

Fig. 20.2 ). Both the right and left lungs are covered by a thin membrane called the visceral pleura and are encased by another membrane called the parietal pleura. The interface of these two pleurae allows for smooth gliding of the lung as it expands in the chest and produces a potential space. Air can enter between the visceral and parietal pleurae by trauma, surgery, or rupture of a group of alveoli; the resulting condition is a pneumothorax. Fluid can also enter this space and create a pleural effusion or, in the case of severe infection, an empyema.

•Fig. 20.2 Illustrations of the Topography of the Lung With Anterior and Posterior Views, Demon-strating the Lobes, Segments, and Fissures. Thefissures(orchasms)demarcatethelobesineachlung.Numbersrefertospecificbronchopulmonarysegments,asdepictedin

Fig.20.3 .SVC,superiorvenacava.Lateral 4 Anterior 3 Anterior 3 1 1 Apical 1 Apical 1 Medial 5 Inferior 5 Superior 4 Superior 6 Posterior basal 10 POSTERIOR VIEW Left Right ANTERIOR VIEW LeftRight Posterior 2 2 Lateral basal 9 Lateral basal 9 Posterior basal 10 Superior 6 8 8 8 Heart Anterior basal Lower lobe Anterior basal Oblique fissure SVC Trachea Upper lobe Middle lobe Lower lobe Anterior basal Oblique fissure Aortic arch Apical Upper lobe Lower lobe Upper lobe Lower lobe Upper lobe Posterior Apical Horizontalfissure

The trachea branches into two main stem bronchi (

Fig. 20.3 ). These main stem bronchi then divide (like the branches of a tree) into lobar bronchi (one for each lobe), which in turn divide into segmental bronchi (Fig.

20.4

Fig. 20.3 ) and then into smaller and smaller branches (bronchioles) until ending in the alveolus (

Fig.

20.5 ). Bronchi and bronchioles differ not only in size but also by the presence of cartilage, the type of epithelium, and their blood supply (

Table 20.1). Beyond the segmental bronchi, the airways divide in a dichotomous or asymmetrical branching pattern. Bronchi, distinguished by their size and the presence of cartilage, eventually become terminal bronchioles, which are the smallest airways without alveoli. Each branching of an airway results in an increase in the number of airways with smaller diameters; as a result, the total surface area for the next generation of branches increases. Terminal bronchioles terminate in an opening (duct) to a group of alveoli and are called respiratory bronchioles.

The region of the lung supplied by a segmental bronchus is called a bronchopulmonary segment and is the functional anatomical unit of the lung. Because of their structure, bronchopulmonary segments that have become irreversibly diseased can easily be removed surgically. The basic physiological unit of the lung is the gas-exchanging unit (respiratory unit), which consists of the respiratory bronchioles, the alveolar ducts, and the alveoli (see

Figs.

20.4 20.5). The bronchi, which contain cartilage, and the terminal bronchioles (i.e., lacking alveoli), in which cartilage is absent, serve to move gas from the airways to the alveoli and are referred to as the conducting airways. This area of the lung (≈150 mL in volume) does not participate in gas exchange and forms the anatomical dead space. The respiratory bronchioles with alveoli and the area beginning •Fig. 20.3 Illustration of Bronchopulmonary Segments, Anterior View. Thenumberscorrespondtothosein

Fig.20.2 :1,apical;2,posterior;3,anterior;4,lateral(superior);5,medial(inferior);6,superior;7,medialbasal;8,anteriorbasal;9,lateralbasal;10,posteriorbasal.Themedialbasalregions(7)arelocatedintheupperregionoftheposteriorbasalregions(10)in

Fig.20.2 .Upper lobe Middle lobe Lower lobe Upper lobe Lingula Thyroid cartilage Cricoid cartilage Trachea Left main stem bronchus Right main stem bronchus 12 3 4 8 9 10 5 1 2 3 4 8 77 6 6 9 10 5 •Fig. 20.4 Illustration of Conducting Airways and Alveolar Units of the Lung. Therelativesizeofthealveolarunitisgreatlyenlarged.Numbersatthebottomindicatetheapproximatenumberofgenerationsfromtracheatoalveoli,whichmayvaryfromasfewas10toasmanyas23.(FromWeibelER.Morphometry of the Human Lung. Heidelberg:Springer-Verlag;1963.) with the respiratory bronchioles to the alveoli are where all gas exchange occurs. This region is only approximately 5 mm long, but it is the largest volume of the lung, at a volume of approximately 2500 mL and with a surface area of 70 m2 when the lung and chest wall are at the resting volume (see

Table 20.1

The alveoli are polygonal in shape and approximately 250 µm in diameter. Alveolar spaces are responsible for most of the lung’s volume; these spaces are divided by tissue known collectively as the interstitium. The interstitium is composed primarily of lung collagen fibers and is a space in which fluid and cells can potentially accumulate. An adult has approximately 5 × 108 alveoli (

Fig. 20.6 ), which are composed of type I and type II epithelial cells. Under normal conditions, type I and type II cells exist in a 1 : 1 ratio. The type I cell occupies 96% to 98% of the surface area of the alveolus, and it is the primary site for gas exchange. The thin cytoplasm of type I cells is ideal for optimal gas diffusion. In addition, the basement membrane of type I cells and the capillary endothelium are fused, which minimizes the distance for gas diffusion and thereby facilitates gas exchange.

Type II cells are cuboidal and usually found in the “corners” of the alveolus, where they occupy 2% to 4% of the surface area. During embryonic development, the alveolar epithelium is composed entirely of type II cells, and only very late in gestation do they differentiate into type I cells and form the “normal” alveolar epithelium for optimal gas exchange. Also, type II cells synthesize pulmonary surfactant (see ), which reduces surface tension (nonsticking properties) in the alveolus and thereby promotes less resistance during inhalation and exhalation. Gas exchange occurs in the alveoli through a dense mesh-like network of capillaries and alveoli called the alveolar-capillary network. The barrier between gas in the alveoli and the red blood cell is only 1 to 2 µm thick and consists of type I alveolar epithelial cells, capillary endothelial cells, and their respective basement membranes. O2 and CO2 passively diffuse across this barrier into plasma and red blood cells (see

Chapter 24). Red blood cells pass through the network in less than 1 second, which is sufficient time for CO2 and O2 gas exchange.

Circulatory Systems in the Lung

The circulation to the lung is unique in its duality and ability to accommodate large volumes of blood at low pressure. The lung has two separate blood supplies, one •Fig. 20.5 The Airway From the Terminal Bronchiole to the Alveo-lus. Notetheabsenceofalveoliintheterminalbronchiole.A,alveolus;RB,respiratorybronchiole;TB,terminalbronchiole.

Theconductingairwaysareinvolvedinseveralmajorpulmonarydiseasescollectivelyreferredtoasobstructive pulmonary disease, includingasthma,bronchiolitis,chronicbronchitis,andcysticfibrosis.Obstructionofairflowthroughtheairwaysiscommonlycausedbyincreasedamountsofmucus,airwayinflammation,andsmoothmuscleconstriction.Asthma isachronicinflammatorydiseaseofthelargeandsmallairways,mediatedpredominantlybylymphocytesandeosinophils,andisassociatedwithincreasedamountsofmucusintheairwaysandwithreversibleconstrictionoftheairwaysmoothmuscle(bronchospasm).Bronchiolitis isadiseaseofthebronchiolesthatusuallyoccurinyounginfantsandiscausedbyviruses,primarilyrespiratory syncytial virus. Chronic bronchitis, adiseasetypicallyofpeoplewhosmoke,isassociatedwithamarkedincreaseinmucus-secretingcellsintheairwaysandanincreaseinmucusproduction.Cystic fibrosis isanautosomalrecessivegeneticdiseasecausedbymutationsintheCl− ionchannelofthecysticfibrosistransmembraneconductanceregulator(CFTR).MutationsintheCFTR genecauseareductioninchlorideandwatersecretionintothemucusoverlyingtheepitheliacells,whichincreasestheviscosityofmucus.Thissituationresultsinmucusaccumulationandchronicpulmonaryinfections,primarilybyPseudomonas aeruginosa.

Notallimportantobstructivelungdiseasesinvolvetheairwaysdirectly.Emphysema isanirreversible,obstructivelungdiseasecommoninpeoplewhosmoke.Thepathogenesisinvolvesaprogressivedestructionoftheelastictissuesinthelungwithalossofalveolar/capillarystructure.Themechanismsoftissuedestructionareunclearbutmayinvolveproteolyticenzymesandothertoxiccompoundsincigarettesmoke.Emphysemaalsooccursinnonsmokingindividualswiththegeneticdisorderα1-antitrypsin deficiency, causedbytheinabilitytoregulateproteolyticenzymes,particularlyelastase.

for the uptake of O2 and removal of CO2 from the body (pulmonary circulation) and the other to supply O2 to lung tissue (bronchial circulation;

Fig. 20.7

The pulmonary circulation begins in the right atrium of the heart. Deoxygenated blood from the right atrium enters the right ventricle via the tricuspid valve, and it is then pumped under low pressure (9 to 24 mm Hg) into the pulmonary artery (pulmonary trunk), which is approximately 3 cm 200 µmPVCTB PARB AD A PV PV A I A A C C C II M10 µm 200 µm PAPA A A A I I I I EL C R COL E * E Nu 1.0 µmB •Fig. 20.6 Alveoli. A. Theterminalrespiratoryunitconsistsofthealveoli(A)andthealveolarducts(AD)arisingfromarespiratorybronchiole(RB).Eachunitisapproximatelyspherical,assuggestedbythedashed outline. Pulmonaryvenousvessels(PV)haveaperipherallocation.PA,pulmonaryartery;TB,terminalbronchiole.Inset, typeIandtypeIIalveolarepithelialcells.Alargefractionofthealveolarwallconsistsofcapillaries(C)andtheircontents.B.Illustrationofacross-sectionofanalveolarwall,showingthepathofdiffusionofO2andCO2.Thethinsideofthealveolarwallbarrier(short double arrow) consistsoftypeIepithelium(I),interstitium(asterisk) formedbythefusedbasallaminaeoftheepithelialandendothelialcells,capillaryendothelium(E),plasmainthealveolarcapillary(C),andthecytoplasmoftheredbloodcell(R).Thethicksideofthegas-exchangebarrier(long double arrow) hasanaccumulationofelastin(EL),collagen(COL),andmatrixthatjointlyseparatethealveolarepitheliumfromthealveolarcapillaryendothelium.Nu,nucleusofthecapillaryendothelialcell.

in diameter, and branches quickly (5 cm from the right progressively smaller series of branching vessels—arteries ventricle) into the right and left main pulmonary arteries, (diameter, >500 µm); arterioles (diameter, 10 to 200 µm); which supply blood to the right and left lungs, respectively. and capillaries (diameter, <10 µm)—that end in a complex The arteries of the pulmonary circulation are the only arter-mesh-like network of capillaries. The sequential branching ies in the body that carry deoxygenated blood. The deoxy-pattern of the pulmonary arteries follows the pattern of genated blood in the pulmonary arteries passes through a airway branching.

•Fig. 20.7 Illustrationoftheanatomicalrelationshipofthepulmonaryartery,thebronchialartery,theairways,andthelymphaticvessels.A,alveoli;AD,alveolarducts;RB,respiratorybronchioles;TB,terminalbronchioles.AD AD AD AD RB TB Lymphatics Lymphatics Vasomotor nerves Pulmonary vein Bronchomotor nerve Pulmonary artery Bronchial artery Bronchus A A A A A

The functions of the pulmonary circulatory system are (1) to reoxygenate the blood and release CO2, (2) to aid in fluid balance in the lung, and (3) to distribute metabolic products to and from the lung. Red blood cells are oxygenated in the capillaries that surround the alveoli, where the pulmonary capillary bed and the alveoli come together in the alveolar wall in a unique configuration for optimal gas exchange. Gas exchange occurs through this alveolar-capillary network (see

The total blood volume of the pulmonary circulation is approximately 500 mL, which is approximately 10% of the circulating blood volume. Approximately 75 mL of blood is present in the alveolar-capillary network of healthy adults at any one time. The pulmonary capillary bed is the largest vascular bed in the body. It covers a surface area of 70 to 80 m2, which is nearly as large as the alveolar surface area. During exercise, the pulmonary capillary blood volume increases from 75 mL to as high as 200 mL because of the recruitment of new capillaries as the result of an increase in pressure and flow. This recruitment of new capillaries is a unique feature of the lungs, and it allows for compensation in periods of stress, as in the case of exercise.

The oxygenated blood leaves the alveolus through a network of small pulmonary venules (15 to 500 µm in diameter) and veins. These small vessels quickly coalesce to form larger pulmonary veins (>500 µm in diameter), through which the oxygenated blood returns to the left atrium of the heart. In contrast to arteries, arterioles, and capillaries, which closely follow the branching patterns of the airways, venules and veins run quite distant from the airways.

Structure of the Pulmonary Circulation

The arteries of the pulmonary circulation have thin walls, with minimal smooth muscle. They are seven times more compliant than systemic vessels, and they are easily distensible. This highly compliant state of the pulmonary arterial vessels requires lower pressure for blood flow through the pulmonary circulation than do the more muscular, noncompliant arterial walls of the systemic circulation. The vessels in the pulmonary circulation, under normal circumstances, are in a dilated state and have larger diameters than do similar arteries in the systemic system. All of these factors contribute to a very compliant, low-resistance circulatory system, which aids in the flow of blood through the pulmonary circulation via the relatively weak pumping action of the right ventricle. This low-resistance, low-work system also explains why the right ventricle is less muscular than the left ventricle. The pressure gradient differential for the pulmonary circulation from the pulmonary artery to the left atrium is only 6 mm Hg (14 mm Hg in the pulmonary artery minus 8 mm Hg in the left atrium). This pressure gradient differential is less than 7% of the pressure gradient differential of 87 mm Hg present in the systemic circulation (90 mm Hg in the aorta minus 3 mm Hg in the right atrium).

Structures of the Extra-Alveolar and Alveolar Vessels and the Pulmonary Microcirculation

Although not well defined anatomically, vessels in the pulmonary circulation can be divided into three categories (extra-alveolar, alveolar, and microcirculation) on the basis of differences in their physiological properties. The extra-alveolar vessels (arteries, arterioles, veins, and venules) are larger than their systemic counterparts. They are not influenced by alveolar pressure changes, but they are affected by intrapleural and interstitial pressure changes. Thus the caliber of extra-alveolar vessels is affected by lung volume and by lung elastin. At high lung volumes, the decrease in pleural pressure increases the caliber of extra-alveolar vessels, whereas at low lung volumes, an increase in pleural pressure decreases vessel caliber. In contrast, alveolar capillaries reside within the interalveolar septa, and they are very sensitive to changes in alveolar pressure but not to changes in pleural or interstitial pressure. Positive-pressure ventilation increases alveolar pressure and compresses these capillaries and thus blocks blood flow. The pulmonary microcirculation comprises the small vessels that participate in liquid and solute exchange in maintenance of fluid balance in the lung.

Structure of the Alveolar-Capillary Network

The sequential branching of the pulmonary arteries culminates in a dense mesh-like network of capillaries that surround alveoli. This alveolar-capillary network is composed of thin epithelial cells of the alveolus and endothelial cells of the vessels and their supportive matrix, and it has an alveolar surface area of approximately 85 m2 (the approximate size of a tennis court). The structural matrix and the tissue components of this alveolar-capillary network provide the only barrier between gas in the airway and blood in the capillary. The cells of this barrier, which is 1 to 2 µm thick, include type I alveolar epithelial cells, capillary endothelial cells, and their respective basement membranes, which are back to back (see

Fig. 20.6B ). Surrounded mostly by air, this alveolar-capillary network is an ideal environment for gas exchange. Red blood cells pass through the capillary component of this network in single file in less than 1 second, which is sufficient time for CO2 and O2 gas exchange.

In addition to gas exchange, the alveolar-capillary network regulates the amount of fluid within the lung. At the pulmonary capillary level, the balance between hydrostatic and oncotic pressure across the wall of the capillary results in a small net movement of fluid out of the vessels into the interstitial space. The fluid is then removed from the lung interstitium by the lymphatic system and enters the circulation via the vena cava in the area of the lung hilus. In normal adults, an average of 30 mL of fluid per hour is returned to the circulation via this route.

The bronchial circulation is a distinct system, separate from the pulmonary circulation in the lung, that provides systemic arterial blood to the trachea, upper airways, surface secretory cells, glands, nerves, visceral pleural surfaces, lymph nodes, pulmonary arteries, and pulmonary veins. The bronchial circulation is similar in structure to the systemic circulatory system and perfuses the upper respiratory tract; it does not reach the terminal or respiratory bronchioles or the

CHAPTER 20 Introduction to the Respiratory System alveoli. Venous blood from the capillaries of the bronchial circulation flows to the heart through either true bronchial veins or bronchopulmonary veins. True bronchial veins are present in the region of the lung hilus, and blood flows into the azygos, hemiazygos, or intercostal veins before entering the right atrium. The bronchopulmonary veins are formed through a network of tributaries from the bronchial and pulmonary circulatory vessels that anastomose and form vessels with an admixture of blood from both circulatory systems. Blood from these anastomosed vessels returns to the left atrium through pulmonary veins. Approximately two thirds of the total bronchial circulation is returned to the heart via the pulmonary veins and this anastomosis route.

The bronchial circulation receives only approximately 1% of total cardiac output; in comparison, the pulmonary circulation receives almost 100%. In the presence of diseases such as cystic fibrosis, the bronchial arteries, which normally receive only 1% to 2% of cardiac output, increase in size (hypertrophy) and receive as much as 10% to 20% of the cardiac output. The erosion of inflamed tissue into these vessels as a result of bacterial infection is responsible for the hemoptysis (coughing up blood) that can occur in this disease.

Breathing is automatic and under control of the central nervous system (CNS). The lungs are innervated by the autonomic nervous system of the peripheral nervous system (PNS), which is under CNS control (

Fig. 20.8).

The autonomic nervous system has four distinct components: parasympathetic, sympathetic, nonadrenergic noncholinergic inhibitory, and nonadrenergic noncholinergic stimulatory.

Stimulation of the parasympathetic system leads to airway smooth muscle constriction, blood vessel dilation, and increased glandular cell secretion, whereas stimulation of the sympathetic system causes relaxation of the airway smooth muscle, constriction of blood vessels, and inhibition of glandular secretion (see

Fig. 26.1 ). The functional unit of the autonomic nervous system is composed of preganglionic and postganglionic neurons in the CNS and postganglionic neurons in the ganglia of the specific organ. As with most organ systems, the CNS and PNS work in concert to maintain homeostasis. There is no voluntary motor innervation in the lung, nor are there pain fibers. Pain fibers are found only in the pleura.

The parasympathetic innervation of the lung originates from the medulla in the brainstem (cranial nerve X, the vagus nerve). Preganglionic fibers from the vagal nuclei descend in the vagus nerve to ganglia adjacent to airways and blood vessels in the lung. Postganglionic fibers from the ganglia then complete the network by innervating smooth muscle cells, blood vessels, and bronchial epithelial cells (including goblet cells and submucosal glands). In the lungs, both preganglionic and postganglionic fibers •Fig. 20.8 Innervation of the Lung. Theautonomicinnervation(motorandsensory)ofthelungandthesomatic(motor)nervesupplytotheintercostalmusclesanddiaphragmaredepicted.

contain excitatory (cholinergic) and inhibitory (nonadrenergic) motor neurons. Acetylcholine and substance P are neurotransmitters of excitatory motor neurons; dynorphin and vasoactive intestinal peptide are neurotransmitters of inhibitory motor neurons. Parasympathetic stimulation through the vagus nerve is responsible for the slightly constricted smooth muscle tone in a normal resting lung. Parasympathetic fibers also innervate the bronchial glands, and these fibers, when stimulated, increase the synthesis of mucus glycoprotein, which increases the viscosity of mucus. Parasympathetic innervation is greatest in the larger airways and most limited in the smaller conducting airways in the periphery.

Whereas the response of the parasympathetic nervous system is very specific and local, the response of the sympathetic nervous system tends to be more general. Mucous glands and blood vessels are heavily innervated by the sympathetic nervous system; however, airway smooth muscle is not. Neurotransmitters of the adrenergic nerves include norepinephrine and dopamine, although dopamine has no influence on the lung. Stimulation of the sympathetic nerves in mucous glands increases water secretion. This disrupts the balanced response of increased water and increased viscosity between the sympathetic and parasympathetic pathways. Adrenergic fibers are absent in humans. In addition to those in the sympathetic and parasympathetic systems, afferent nerve endings are present in the epithelium and in smooth muscle cells in the lung.

Central Control of Respiration

Breathing is an automatic, rhythmic, and centrally regulated process with voluntary control. The CNS, particularly the brainstem, functions as the main control center for respiration (

Fig. 20.9 ). Regulation of respiration requires (1) generation and maintenance of a respiratory rhythm; (2) modulation of this rhythm by sensory feedback loops and reflexes that allow adaptation to various conditions while minimizing energy costs; and (3) recruitment of respiratory muscles that can contract appropriately for gas exchange. Control of respiration is described in greater detail in

Cerebral cortex Respiratory center, medulla Spinal cord Respiratory muscles Lung and chest wall Alveolar-capillary barrier BloodPerfusion Mechano-receptors Chemo-receptors PCO2, PO2, pH Diffusion Ventilation Nerve impulses Nerve impulses Force displacement •Fig. 20.9 Block Diagram of the Respiratory Control System, Demonstrating Relationships Between the Respiratory Control Center and Muscles of Respiration. Therespiratorycenterneurons,dispersedintoseveralgroupsinthemedulla,demonstratespontaneouscyclicactivitybutarestronglyinfluencedbystimulidescendingfromthecerebralcortex(volitionalcontrol)andfromtwosensoryloops:mechanoreceptorandchemoreceptorpathways.Ventilationandperfusionoccurtogetherneartheendofthecycle,andtheiroutputdeterminespartialpressuresofarterialandalveolarcarbondioxide(PCO2)andoxygen(PO2)and,inpart,arterialhydrogenionconcentration(pH).Theseoutputsfeedbacktotherespiratorycenterviachemoreceptorandmechanoreceptorsensorypathways.

Muscles of Respiration

The major muscles of respiration include the diaphragm, the external intercostal muscles, and the scalene muscles, all of which are skeletal muscles. Skeletal muscles provide the driving force for ventilation; the force of contraction increases when they are stretched and decreases when they are shortened. The force of contraction of respiratory muscles increases at larger lung volumes.

The diaphragm is the major muscle of respiration, and it divides the thoracic cavity from the abdominal cavity (

Fig.

20.10). Contraction of the diaphragm forces the abdominal contents downward and forward. This increases the vertical dimension of the chest cavity and creates a pressure difference between the thorax and abdomen. In adults, the diaphragm can generate airway pressures of up to 150 to 200 cm H2O during maximal inspiratory effort. During quiet breathing (tidal breathing), the diaphragm moves approximately 1 cm; however, during deep-breathing maneuvers (vital capacity), the diaphragm can move as much as 10 cm. The diaphragm

CHAPTER 20 Introduction to the Respiratory System is innervated by the right and left phrenic nerves, whose origins are at the third to fifth cervical segments of the spinal cord (C3 to C5).

The other important muscles of inspiration are the external intercostal muscles, which pull the ribs upward and forward during inspiration (see

Fig. 20.10 ). This causes an increase in both the lateral and anteroposterior diameters of the thorax. Innervation of the external intercostal muscles originates from intercostal nerves that arise from the same level of the spinal cord (T1 and T2). Paralysis of these muscles has no significant effect on respiration because respiration is dependent primarily on the diaphragm. This is why individuals with injuries to the lower spinal cord can breathe on their own; it is only when the injury is above C3 that an individual is completely dependent on a ventilator.

Accessory muscles of inspiration (the scalene muscles, which elevate the sternocleidomastoid muscles; the alae nasi, which cause nasal flaring; and small muscles in the neck and head) do not contract during normal breathing. However, they do contract vigorously during exercise, and when airway obstruction is significant, they actively pull up the rib cage. During normal breathing, they anchor the sternum and upper ribs. Because the upper airway must remain patent during inspiration, the pharyngeal wall muscles (genioglossus and arytenoid) are also considered muscles of inspiration. All the rib cage muscles are voluntary muscles that are supplied by intercostal arteries and veins and innervated by motor and sensory intercostal nerves.

Exhalation during normal breathing is passive, but it becomes active during exercise and hyperventilation. The most important muscles of exhalation are those of the abdominal wall (rectus abdominis, internal and external oblique, and transversus abdominis) and the internal intercostal muscles, which oppose the external intercostal muscles (i.e., they pull the ribs downward and inward). The inspiratory muscles do the work of breathing. During normal breathing, this workload is low, and the inspiratory muscles have significant reserve. Respiratory muscles can be trained to do more work, but there is a limit to the work that they can perform. Respiratory muscle weakness can impair movement of the chest wall, and respiratory muscle fatigue is a major factor in the development of respiratory failure.

Lung Embryology, Development, Aging, and Repair

The epithelium of the lung arises as a pouch from the primitive foregut at approximately 22 to 26 days after fertilization of the ovum. This single lung bud branches into primitive right and left lungs. Over the next 2 to 3 weeks, further branching occurs to create the irregular dichotomous branching pattern. The pathologist Lynne Reid described “three laws of lung development”: (1) The bronchial tree has developed by week 16 of intrauterine

Diaphragm (domes descend, increasing longitudinal dimension of chest and elevating lower ribs)

A Muscles of inspiration

Internal intercostals, except parasternal intercartilaginous muscles (depress ribs)

Abdominal muscles (depress lower ribs, compress abdominal contents)

Muscles of expiration

Because respiratory muscles provide the driving force for ventilation, diseases that affect the mechanical properties of the lung affect the muscles of respiration. For example, in chronic obstructive pulmonary disease (COPD), the work of breathing is increased secondary to airflow obstruction. Exhalation no longer is passive but instead requires active, expiratory muscle contraction. In addition, total lung capacity is increased (see ). The larger total lung capacity forces the diaphragm downward, shortens the muscle fibers, life; (2) alveoli develop after birth, the number of alveoli increases until the age of 8 years, and the size of alveoli increases until growth of the chest wall is completed at adulthood; and (3) the development of preacinar vessels (arteries and veins) parallels that of the airways, whereas that of intra-acinar vessels parallels that of the alveoli.

and decreases the radius of curvature. As a result, the function and efficiency of the diaphragm are decreased. Respiratory muscles can fatigue just as other skeletal muscles do when the workload increases. Respiratory muscles can also weaken in patients with neuromuscular diseases (e.g., Guillain-Barré syndrome, myasthenia gravis). In these diseases, sufficient weakness of the respiratory muscles can impair movement of the chest wall and result in respiratory failure, even though the mechanical properties of the lung and chest wall are normal.

Thus intrauterine events that occur before 16 weeks of gestation will affect the number of airways. A condition known as congenital diaphragmatic hernia is an example of a congenital lung disease. It occurs at 6 to 8 weeks of gestation and is due to failure of the pleuroperitoneal canal to close and thereby separate the chest and abdominal cavities; the presence of the abdominal contents in the lung hemithorax results in abnormal lung growth with a decrease in the number of airways and alveoli. Before the birth of an affected infant, the alveolar epithelium is composed solely of type II epithelial cells, and it is not until birth that these cells differentiate into type I epithelial cells.

Growth of the lungs is similar and relatively proportional to growth in body length and stature. The rate of development is fastest in the neonatal and preadolescent periods (≈11 years of age), and girls’ lungs mature earlier than boys’. Although the growth rate of the lung slows after adolescence, the body and lung increase in size steadily until adulthood. Improvement in lung function occurs at all stages of growth development; however, once optimal size has been attained in early adulthood (20 to 25 years of age), lung function starts to decline with age. The decrease in lung function with age, estimated at less than 1% per year, appears to begin earlier and proceed faster in individuals who smoke or are exposed to toxic environmental factors. The major physiological insufficiencies caused by aging involve ventilatory capacity and responses, especially during exercise, and they result in abnormal ventilation with normal perfusion. In addition, gas diffusion decreases with age, probably as a result of a decrease in alveolar surface area. Age-related decreases in lung function and altered structure parallel biochemical observations of increased levels of elastin within the lung, which could explain some of the functional abnormalities.

TypeIcellslackfreeradicalscavengers(i.e.,superoxidedismutase)andaresusceptibletoinjuryanddeathinducedbytoxicO2compoundsandfreeradical(i.e.,H2O2,OH− ,andO2−).Invariousinflammatorylungdiseases,typeIcellsdie,andthealveolarepitheliumtherebybecomesdenuded,withincreasedvascularpermeabilityandensuingfluidaccumulation(impairedgasexchange).TypeIIcellshavesuperoxidedismutaseandarethusmoreresistanttothetoxicoxygenradicals.TheycansurvivetoproliferateanddifferentiateintotypeIcellstorestorethenormalalveolararchitecture.ThistypeofresponseisdependentonanintactbasementmembranetosupporttheproliferationoftypeIIcellsandisanexampleof“phylogenyrecapitulatingontogeny.”Ifthebasementmembranecannotberepopulated,thenthebody’srecourseforrepairiscollagendepositionandscarformation,whichisnotconducivetogas 1.

The lungs demonstrate anatomical and physiological unity; that is, each unit (bronchopulmonary segment) is structurally identical and functions just like every other unit.

2.

The upper airways (nose, sinuses, pharynx) condition inspired air for temperature, humidity, and atmospheric pressure, and they control, via the epiglottis, the flow of air into the lungs and food/fluids into the esophagus.

3.

Components of the lower airways (trachea, bronchi, bronchioles) are considered conducting airways in which air is transported to the gas-exchanging respiratory units composed of respiratory bronchioles, alveolar ducts, and alveoli.

4.

The lungs have unique, dual circulatory systems. The pulmonary circulatory system has the ability to accommodate large volumes of blood at low pressure and brings deoxygenated blood from the right ventricle to the gas-exchanging units in the lung. The exchange.Inlungdiseasethatinvolvesscarformation(i.e.,pulmonaryfibrosis),thetotallungvolumedecreasesasaresultofthelossofalveoliandimpairmentofO2diffusionintothecapillariesbyathickened,nonpermeablematrix.Historically,idiopathicpulmonaryfibrosishasbeenverydifficulttotreatbecauseofthelackofspecifictherapeutics,whichcaninhibitcollagendeposition.Twotherapeuticcompounds(pirfenidoneandnintedanib)havebeenshowninclinicaltrialstoslowtheprogressionofthediseaseandimproveoutcomesinpatientswithidiopathicpulmonaryfibrosis.Pirfenidoneisasmalllow-molecular-weightcompoundwithanti-inflammatoryproperties(itdecreasesprocollagentypesIandIIsynthesis),andnintedanibisatyrosinekinaseinhibitor(inhibitsvascularendothelialgrowthfactorandfibroblast-derivedgrowthfactor).

bronchial circulation arises from the aorta and provides nourishment (O2) to the lung parenchyma.

5.

Breathing is automatic; the lungs are innervated by the autonomic nervous system of the PNS while under the control of the CNS. Parasympathetic stimulation results in constriction of airway smooth muscles (airway narrowing) whereas sympathetic stimulation results in relaxation of airway smooth muscles (airway opening).

6.

Inspiration is the active phase of breathing. The diaphragm is the major muscle of respiration, and its contraction creates a pressure difference (mechanoreceptor response) between the thorax and diaphragm (negative pressure in the chest), which induces inspiration.

7.

The respiratory center is located in the medulla and regulates respiration with input from sensory (mechanoreceptor and chemoreceptor) feedback loops.

Burri PH. Structural aspects of postnatal lung development—alveolar formation and growth. Biol Neonate. 2006;89:313-322.

Hameed A, Sherkheli MA, Hussain A, Ul-haq R. Molecular and physiological determinants of pulmonary developmental biology: a review. Am J Biomed Res. 2013;1:13-24.

Harding R, Pinkertton KE, eds. The Lung: Development, Aging and the Environment. 2nd ed. London: Academic Press; 2014.

Hattrup CL, Gendler SJ. Structure and function of the cell surface (tethered) mucins. Annu Rev Physiol. 2007;70:431-457.

Reynolds HY. Lung inflammation and fibrosis: an alveolar macrophage-centered perspective from the 1970s to 1980s. Am J Respir Crit Care Med. 2005;171:98-102.

Satir P, Christensen ST. Overview of structure and function of mammalian cilia. Annu Rev Physiol. 2007;69:377-400.

Shannon JM, Hyatt BA. Epithelial cell-mesenchymal interactions in the developing lung. Annu Rev Physiol. 2004;66:625-645.

Warburton D, El-Hashash A, Carraro G, et al. Lung organogenesis. Curr Top Devel Bio. 2010;90:73-158.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

Define the different pressures in the respiratory system.

2.

Explain how a pressure gradient is created.

3.

Define the different volumes in the lung, and describe how they are measured.

4.

Explain how static lung mechanics determines lung volumes.

5.

Define lung compliance.

6.

Explain how surfactant affects lung compliance, and describe its importance in maintaining unequal alveolar volumes.

o achieve its primary function of gas exchange, air must be moved in and out of the lung. The mechanical properties of the lung and chest wall determine the ease or difficulty of this air movement. Lung mechanics is the study of the mechanical properties of the lung and chest wall (including the diaphragm, abdominal cavity, and anterior abdominal muscles). Lung mechanics is important for how the lungs work both normally and in the presence of disease, inasmuch as most lung diseases affect the mechanical properties of the lungs, chest wall, or both. In addition, death from lung disease is almost always due to respiratory muscle fatigue, which results from an inability of the respiratory muscles to overcome the altered mechanical properties of the lungs, chest wall, or both. Lung mechanics includes static mechanics (the mechanical properties of a lung whose volume is not changing with time) and dynamic mechanics (properties of a lung whose volume is changing with time). Dynamic mechanics of the lung and chest wall are described in

Chapter 22.

Pressures in the Respiratory System

In healthy people, the lungs and chest wall move together as a unit. Between these structures is the pleural space, which under normal conditions is best thought of as a potential (or virtual) space. Because the lungs and chest wall move together, changes in their respective volumes are equal during inspiration and exhalation. Volume changes in the lungs and chest wall are driven by changes in the surrounding pressure. In accordance with convention, pressures inside the lungs and chest wall are referenced in relation to atmospheric pressure, which is considered 0. Thus a negative pressure in the pleural space is a pressure that is lower than atmospheric pressure. Also in accordance with convention, pressures across surfaces such as the lungs or chest wall have been defined as the difference between the pressure inside and the pressure outside the surface. The pressure differences across the lung and across the chest wall are defined as the transmural (across a wall or surface) pressures. For the lung, this transmural pressure is called the transpulmonary (or translung) pressure (PL), and it is defined as the pressure difference between the air spaces (alveolar pressure [PA]) and the pressure surrounding the lung (pleural pressure [Ppl]):

Equation 21.1

The transmural pressure across the chest wall (Pw) is the difference between pleural (inside) pressure (Ppl) and the pressure surrounding the chest wall (Pb), which is the atmospheric pressure or body surface pressure:

Equation 21.2

The pressure across the respiratory system (Prs) is the sum of the pressure across the lung and the pressure across the chest wall:

Equation 21.3

Air flows into and out of the lungs from areas of higher pressure to areas of lower pressure. In the absence of a pressure gradient, there is no airflow. Thus at end inspiration and at the end of exhalation, which are periods of time when there is no airflow, alveolar pressure (PA) is the same as atmospheric pressure (Pb), and there is no pressure gradient (Pb − PA = 0). Pleural pressure at these same times, however, is not 0. Before inspiration begins, the pleural pressure in normal individuals is approximately −3 to −5 cm H2O. Therefore, the pressure in the pleural space is negative in relation to atmospheric pressure. This negative pressure is created by the inward elastic recoil pressure of the lung, and it acts to “pull the lung” away from the chest wall. The lung is not able, however, to pull away from the chest wall, inasmuch as the two function as a unit. Thus the inward elastic recoil pressure of the lung is balanced by the outward recoil of the chest wall.

With the onset of inspiration, the muscles of the diaphragm and chest wall contract, which causes a downward movement of the diaphragm and an outward and upward movement of the rib cage. As a result, pleural pressure decreases during inspiration. This negative pleural pressure is transmitted across the lung tissue and results in a decrease in alveolar pressure. As alveolar pressure decreases below 0 (i.e., from atmospheric pressure to a lower pressure), gas moves into the airways when the glottis is open. As gas flows into the airways to the alveoli, the pressure gradient along the airways decreases, and flow stops when there is no longer a pressure gradient from atmospheric to alveolar pressure. The decrease in pleural pressure at the start of inspiration secondary to inspiratory muscle contraction is and gas again flows from a higher (alveolar) pressure to a lower (atmospheric) pressure. In the alveoli, the driving force for exhalation is the sum of the elastic recoil of the lungs and pleural pressure (see ). This relationship between changes in pressure, changes in airflow, and changes in volume during inspiration and exhalation is displayed in

Fig. 21.2.

During tidal volume breathing in normal individuals, the decrease in alveolar pressure at the start of inspiration is small (1 to 3 cm H2O). It is much larger in individuals with airway obstruction because of the larger pressure drop that occurs across obstructed airways. Airflow stops in the absence of a pressure gradient, which occurs whenever alveolar pressure and atmospheric pressure are equal.

(Fig. 21.3 ) and the factors that determine these volumes are important components of lung mechanics greater than the transmitted fall in alveolar pressure, and, as a result, transpulmonary pressure at the start of inspiration 0.5 is positive (see

Eq. 21.1). Positive transpulmonary pressure 0.4 is necessary to increase lung volume, and lung volume increases with increasing transpulmonary pressure (

Fig.

21.1 ). Similarly, during inspiration, the chest wall expands to a larger volume. Because pleural pressure is negative in 0.1 relation to atmospheric pressure during quiet breathing, 0 the transmural pressure across the chest wall is negative

Eq. 21.2

On exhalation, the diaphragm moves higher into the chest, pleural pressure increases (i.e., becomes less nega tive), alveolar pressure becomes positive, the glottis opens, +0.5 –0.5 •Fig. 21.1 Volume of the Lung as a Function of the Transpulmonary Pressure in Health and Disease. Asthetranspulmonarypressureincreases,lungvolumeincreases.Alsoshownarethechangesinlungvolumeinthepresenceofemphysemaandpulmonaryfibrosis.Notethatforthesamechangeintranspulmonarypressure,inthepresenceofeitherofthesetypesofdiseases,thechangesinlungvolumearedifferent.TLC,totallungcapacity(thetotalvolumeofgasinthelung).

0.3 0.2 •Fig. 21.2 Changes in Alveolar and Pleural Pressure During Quiet Breathing (Tidal Volume). Inspirationisrepresentedtotheleftofthevertical dotted line, andexhalationisrepresentedtotherightofit.Positive(inrelationtoatmospheric)pressuresarerepresentedabovethehorizontal dotted line, andnegativepressuresarerepresentedbelowit.Seetextfordetails.Atpointsofnoairflow(pointsAandC),alveolarpressureis0.FRC,functionalresidualcapacity.

2 1.2 •Fig. 21.3 The Various Lung Volumes and Capacities. ERV,expiratoryreservevolume;FRC,functionalresidualcapacity;FVC,forcedvitalcapacity;IC,inspiratorycapacity;IRV,inspiratoryreservevolume;RV,residualvolume;TLC,totallungcapacity;VC,vitalcapacity;VT,tidalvolume.

and play a major role in the work of breathing (see ). All lung volumes are subdivisions of total lung capacity (TLC), the total volume of air that is contained in the lung at the point of maximal inspiration. Lung volumes are reported in liters either as volumes or as capacities. A capacity is composed of two or more volumes. Many lung volumes are measured with a spirometer. The patient is asked to first breathe normally into the spirometer, and the volume of air (the tidal volume [VT]) that is moved into and out of the lungs with each quiet breath is measured. The patient then inhales maximally and exhales forcefully and completely, and the volume of exhaled air is measured. The total volume of exhaled air, from a maximal inspiration to a maximal exhalation, is the vital capacity (VC). Residual volume (RV) is the air remaining in the lungs after a complete exhalation. TLC is the sum of VC and RV; it is the total volume of air contained in the lungs at the end of maximal inspiration, and it includes the volume of air that can be moved (VC) and the volume of air that is always present (trapped) in the lungs (RV). Functional residual capacity (FRC) is the volume of air in the lungs at the end of exhalation during quiet breathing and is also called the resting volume of the lungs. FRC is composed of RV and the expiratory reserve volume (the volume of air that can be exhaled from FRC to RV).

Measurement of Lung Volumes

RV and TLC can be measured in two ways: by helium dilution and by body plethysmography. Both methods are used clinically and provide valuable information about lung function and lung disease. The helium dilution technique is the older and simpler method, but it is often less accurate than body plethysmography, which requires sophisticated and expensive equipment.

21.1

Pulmonaryfunctiontestsareoftenusedtodiagnoseabnormalitiesinlungfunctionandtoassesstheprogressionoflungdisease.Theycandistinguishthetwomajortypesofpulmonarypathophysiologicprocesses:obstructivelungdiseasesandrestrictivelungdiseases.Forexample,innormalindividuals,theratioofRVtoTLCislessthan 0.25.Thusinahealthyindividual,approximately25%ofthetotalvolumeofairinthelungistrapped.Inobstructive pulmonary diseases, anelevationinRV/TLCratioissecondarytoanincreaseinRVoutofproportiontoanyincreaseinTLC.Incontrast,inrestrictive lung diseases, theelevationintheRV/TLCratioiscausedbyadecreaseinTLC.

In normal individuals, the FRC measured by helium dilution and the FRC measured by plethysmography are the same (

Table 21.1 ). This is not true in individuals with lung disease. The FRC measured by helium dilution is the volume of gas in the lung that communicates with the •Fig. 21.4 Measurement of Lung Volume by Helium Dilution. C1,knownconcentrationofaninertgas;C2,new(previouslyunknown)concentrationofthegas;V1,knownvolumeofabox;V2,lungvolume(initiallyunknown).

airways, whereas the FRC measured by plethysmography is the total volume of gas in the lungs at the end of a normal exhalation. If a significant amount of gas is trapped in the lungs (because of premature airway closure; see ), the FRC determined by plethysmography is considerably higher than that determined by helium dilution.

Intheheliumdilutiontechnique,aknownconcentration(C1)ofaninertgas(suchashelium)isaddedtoaboxofknownvolume(V1).Theboxisthenconnectedtoavolume(V2)thatisunknown(thelungvolumetobemeasured).Afteradequatetimefordistributionoftheinertgas,thenewconcentration(C2)oftheinertgasismeasured.Thechangeinconcentrationoftheinertgasisthenusedtodeterminethenewvolumeinwhichtheinertgashasbeendistributed(

Fig.21.4 ).Specifically,

Inbodyplethysmograph(bodybox),RobertBoyle’sgaslaw—thatpressuremultipliedbyvolumeisconstant(ataconstanttemperature)—isusedtomeasurelungvolumes.Thepatientsitsinanairtightbox(

Fig.21.5 )andbreathesthroughamouthpiecethatisconnectedtoaflowsensor(pneumotach).Thepatientthenmakesapantingrespiratoryeffortagainstaclosedmouthpiece.Duringtheexpiratoryphaseofthemaneuver,thegasinthelungbecomescompressed,lungvolumedecreases,andthepressureinsidetheboxfallsbecausethegasvolumeintheboxincreases.Oncethevolumeoftheboxandthechangeinpressureoftheboxatthemouthareknown,thechangeinvolume(ΔV)ofthelungcanbecalculated: whereP1andP2aremouthpressuresandVisFRC.FromthemeasurementofFRC,inspiratorycapacitycanberecordedasthevolumeofairinspiredaboveFRC,andexpiratoryreservevolumecanbedeterminedasthevolumeofgasexhaledfromFRC.Thesemeasurementscanthenbeusedtodeterminetheotherlungvolumes.

•Fig. 21.5 The Body Plethysmograph. Notethattheboxinwhichthepatientsitsisnotdepicted.

Determinants of Lung Volume

What determines the volume of air in the lung at TLC or at RV? The answer lies in the properties of the lung parenchyma and in the interaction between the lungs and the chest wall. The lungs and chest wall always move together as a unit in healthy individuals. The lung contains elastic fibers that (1) stretch when stress is applied, which results in an increase in lung volume, and (2) recoil passively when this stress is released, which results in a decrease in lung volume. The elastic recoil of the lung parenchyma is very high. In the absence of external forces (such as the force generated by the chest wall), the lungs become almost airless (10% of TLC). Similarly, chest wall volume can increase when the respiratory muscles are stretched and decrease when respiratory muscle length is shortened. In the theoretical absence of the lung parenchyma, the resting volume of the chest wall increases and is approximately 60% of TLC.

Lung volumes are determined by the balance between the lung’s elastic properties and the properties of the muscles of the chest wall. The maximum volume of air contained within the lung and the chest wall (i.e., TLC) is controlled by the muscles of inspiration (see ). With increasing lung volume, the chest wall muscles lengthen progressively. As these muscles lengthen, their ability to generate force decreases. TLC occurs when the inspiratory chest wall muscles are unable to generate the additional force needed to further distend the lung and chest wall. Similarly, the minimal volume of air in the –40–20 0 20 40 Pressure (cm H2O) (PL, P , or P ) •Fig. 21.6 Relaxation Pressure-Volume Curve of the Lung, Chest Wall, and Respiratory System. Thecurvefortherespiratorysystemisthesumoftheindividualcurves.Thecurveforthelungisthesameastheoneforthenormallungin

Fig.21.1 .FVC,forcedvitalcapacity;PL,transpulmonary(ortranslung)pressure(PL);Prs,thepressureacrosstherespiratorysystem;Pw,thetransmuralpressureacrossthechestwall;RV,residualvolume;TLC,totallungcapacity.

lung (i.e., RV) is controlled by the expiratory muscle force. Decreasing lung volume results in shortening of the expiratory muscles, which, in turn, results in a decrease in muscle force. The decrease in lung volume is also associated with an increase in the outward recoil pressure of the chest wall. RV occurs when expiratory muscle force is insufficient to further reduce chest wall volume.

FRC, or the volume of the lung at the end of a normal exhalation, is determined by the balance between the elastic recoil pressure generated by the lung parenchyma to become smaller (inward recoil) and the pressure generated by the chest wall to become larger (outward recoil). When the chest wall muscles are weak, FRC decreases (lung elastic recoil > chest wall muscle force). In the presence of airway obstruction, FRC increases because of premature airway closure, which traps air in the lung (see

A number of important observations can be made from an examination of the pressurevolume curves of the lung, chest wall, and respiratory system (Fig. 21.6 ). At the resting volume of the lung (FRC), the elastic recoil of the lung acts to decrease lung volume, but this inward recoil is offset by the outward recoil of the chest wall, which acts to increase lung volume. At FRC, these forces are equal and opposite, and the muscles are relaxed. As a result, the transmural pressure across the respiratory system (Prs) at FRC is 0. At TLC, both lung pressure and chest wall pressure are positive, and both require positive transmural distending pressure. The resting volume of the chest wall, in the absence of the lungs, is the volume at which the transmural pressure for the chest wall is 0, and it is approximately 60% of TLC. At volumes greater than 60% of TLC, the chest wall recoils inward and positive transmural pressure is needed, whereas at volumes below 60% of TLC, the chest wall tends to recoil outward.

The lungs alone are smallest when transpulmonary pressure is 0. The lungs, however, are not totally devoid of air when transpulmonary pressure is 0 because of the surface tension–lowering properties of surfactant (see the section “ ”). The transmural pressure for a healthy lung alone flattens at pressures higher than 20 cm H2O because the elastic limits of the lung have been reached. Thus further increases in transmural pressure produce little change in volume, and compliance (see the section ”) is low. Further distention is limited by the connective tissue (collagen, elastin) of the lung. If further pressure is applied, the alveoli near the lung surface can rupture, and air can escape into the pleural space. This is called a pneumothorax. In a pneumothorax or when the chest is opened, as during thoracic surgery, the lungs and chest wall no longer function as a single unit. The lungs recoil until transpulmonary pressure is 0; the chest wall then increases in size until trans chest wall pressure is 0.

The relationship between transpulmonary pressure and pleural, alveolar, and elastic recoil pressures is depicted in

Fig. 21.7 . Alveolar pressure is the sum of the pleural pressure and elastic recoil pressure (Pel) of the lung:

Equation 21.4

Because transpulmonary pressure (PL) = PA + Ppl,

Equation 21.5 •Fig. 21.7 Relationshipbetweentranspulmonarypressure(PL)andthepleural(Ppl),alveolar(PA),andelasticrecoil(Pel)pressuresofthelung.Alveolarpressureisthesumofpleuralpressureandelasticrecoilpressure.Transpulmonarypressureisthedifferencebetweenalveolarpressureandpleuralpressure.

In general, PL is the pressure distending the lung, whereas Pel is the pressure that tends to collapse the lung. Lung elastic recoil increases as the lung inflates.

Lung compliance (CL) is a measure of the elastic properties of the lung. It reflects how easily the lung is distended. Lung compliance is defined as the change in lung volume resulting from a 1–cm H2O change in the distending pressure of the lung. The units of compliance are in milliliters (or liters) per centimeter of water. When lung compliance is high, the lung is readily distended. When lung compliance is low (“stiff” lung), the lung is not easily distended. The compliance of the lung (CL) is expressed as

Equation 21.6 where ΔV is the change in volume and ΔP is the change in pressure. Graphically, lung compliance is the slope of the line between any two points on the deflation limb of the pressurevolume loop (

Fig. 21.8 ). The compliance of a normal human lung is approximately 0.2 L/cm H2O, but it varies with lung volume. Note that the lung is less distensible at high lung volumes. For this reason, compliance is corrected for the lung volume at which it is measured (specific compliance;

Fig. 21.9 ). Compliance is not often measured for clinical purposes because it requires placement of an esophageal balloon. The esophageal balloon, which is connected to a pressure transducer, is an excellent surrogate marker for pleural pressure, which is very difficult to measure directly. The change in pleural pressure (Ppl) is measured as a function of the change in lung volume; that is, CL =ΔV/ΔPplor .ΔPpl =ΔCL.

In addition to the elastic properties of the lungs, another major determinant of lung compliance is surfactant and its •Fig. 21.8 Deflation Pressure-Volume Curve. Thepatientinhalestototallungcapacity,andtranspulmonarypressureismeasuredwiththeuseofanesophagealballoon(whichmeasurespleuralpressure).Thepatientthenexhalesslowly,andpressureismeasuredatpointsofnoairflow,whentherespiratorymusclesarerelaxed.Thepressure-volumecurveofthelungisnotthesameininspiration(notshown)andexhalation.Thisdifferenceiscalledhysteresis, anditiscausedbytheactionofsurfactant.Inaccordancewithconvention,thedeflationpressure-volumecurveisusedformeasurements.Complianceatanypointalongthiscurveisthechangeinvolumeperchangeinpressure.Thecurvedemonstratesthatlungcompliancevarieswithlungvolume.Alinecanbedrawnbetweentwodifferentvolumesonthecurve,andtheslopeofthislinerepresentsthechangeinvolume(ΔV)forachangeinpressure(ΔP).Comparethecomplianceatline1versusline 2.Theslopeofline2islesssteepthantheslopeofline1,andsothecomplianceislessatthishigherlungvolumethanitisatthelowerlungvolume.Inaccordancewithconvention,lungcomplianceisthechangeinpressurefromfunctionalresidualcapacity(FRC)toFRC+1L.MV,minimalvolume;RV,residualvolume;TLC,totallungcapacity.

Thecomplianceofthelungsisaffectedbyseveralrespiratorydisorders.Inemphysema,anobstructivelungdiseasethatusuallyoccursinpeoplewhosmokeandisassociatedwithdestructionofthealveolarseptaandpulmonarycapillarybed,thelungismorecompliant;thatis,forevery1–cmH2Oincreaseinpressure,theincreaseinvolumeisgreaterthanthatinanormallung(see

Fig.21.1 ).Incontrast,inpulmonaryfibrosis,arestrictivelungdiseaseassociatedwithincreasedcollagenfiberdepositionintheinterstitialspace,thelungisnoncompliant;thatis,forevery1–cmH2Ochangeinpressure,thechangeinvolumeisless.Thesechangesincomplianceareofclinicalsignificancebecausealungwithlowcompliancerequireslargerchangesinpleuralpressuretoeffectchangesinlungvolumes;asresult,theworkofbreathingisincreasedforeverybreaththattheindividualtakes.

1 liter = 0.2 0.5 liter = 0.1 0.1 liter = 0.02 = 0.2 5 cm H2O 0.1 liter 0.2 = 0.2 0.1 = 0.2 0.5 liter 0.02 •Fig. 21.9 Relationship Between Compliance and Lung Volume. Imaginelungsinwhicha5–cmH2Ochangeinpressureresultsina1-Lchangeinvolume(situation1).Ifonelungisremoved(situation2),thecompliancedecreases,butwhencorrectedforvolumeofthelung,thereisnochange(specificcompliance).Evenwhentheremaininglungisreducedby90%(situation3),thespecificcomplianceisunchanged.

effect on surface tension. Surface tension is a force caused by water molecules at the airliquid interface that tends to minimize surface area, which makes inflating the lungs more difficult. The effect of surface tension on lung inflation is illustrated by a comparison of the volumepressure curves of a salinefilled lung and of an airfilled lung. Higher pressure is necessary to fully inflate the lung with air than with saline because of the higher surface tension forces in airfilled lungs than in salinefilled lungs. Surface tension is a measure of the attractive force of the surface molecules per unit length of material to which they are attached. The units of surface tension are those of a force applied per unit length. For a sphere (such as an alveolus), the relationship between the pressure within the sphere (Ps) and the tension in the wall is described by the law of Laplace:

Equation 21.7 where T is the wall tension (in dynes per centimeter) and r is the radius of the sphere.

The alveoli are lined with a predominantly lipidbased substance called surfactant. Pulmonary surfactant serves several physiological roles, including (1) reducing the work of breathing by decreasing surface tension forces; (2) preventing collapse and sticking of alveoli on exhalation; and (3) stabilizing alveoli, especially those that tend to deflate at low surface tension. In the absence of surfactant, the surface tension at the airliquid interface would remain constant, and the transalveolar pressure needed to keep it at that volume would be higher when alveolar volumes are lower (

Fig. 21.10A ). Therefore, higher transalveolar pressure would be necessary to produce a given increase in alveolar volume at lower lung volumes than at higher lung volumes. Surfactant stabilizes the inflation of alveoli because it allows the surface tension to increase as the alveoli become larger (see

Fig. 21.10B ). As a result, the transalveolar pressure necessary to keep an alveolus inflated increases as lung volume and transpulmonary pressure increases, and it decreases as lung volume decreases. In the presence of surfactant, surface tension is increased at high lung volume and decreased at low lung volume. The result is that the lungs can maintain alveoli at many different volumes. Otherwise, the gas in small alveoli would empty into large alveoli.

Pulmonary surfactant is synthesized by alveolar type II cells, stored in the cell in lamellar bodies, and secreted into the alveolar space in a precursor form (tubular myelin), from where it spreads throughout the entire alveolar surface and attains its ability to decrease surface tension. Surfactant is 85% to 90% lipids, predominantly phospholipids, and 10% to 15% proteins (

Table 21.2). The major phospholipid is phosphatidylcholine, approximately 75% of which is present as dipalmitoyl phosphatidylcholine (DPPC). DPPC decreases surface tension and is the major surfaceactive component in surfactant. The second most abundant phospholipid is phosphatidylglycerol, which accounts for •Fig. 21.10 Surface Forces in a Sphere Attempt to Reduce the Area of the Surface and Generate Pressure Within the Sphere. ByLaplace’slaw,thepressuregeneratedisinverselyproportionaltotheradiusofthesphere.A, Intheabsenceofsurfactant,surfaceforcesinthesmallerspheregeneratehigherpressure(heavier purple arrows) thandothoseinthelargersphere(lighter purple arrows). Asaresult,airmovesfromthesmallsphere(higherpressure)tothelargersphere(lowerpressure;black arrow).Thiscausesthesmallspheretocollapseandthelargespheretobecomeoverdistended.B, Surfactant(shaded layer) lowerssurfacetensionandlowersitmoreinthesmallerspherethaninthelargersphere.Thenetresultsarethatthepressuresinthesmallandlargerspheresaresimilar,andthevolumesofthespheresarestabilized.

1% to 10% of total surfactant. These lipids are important in formation of the monolayer on the alveolarair interface, and phosphatidylglycerol is important in the spreading of surfactant over a large surface area. Surfactant protein A, which is the protein most studied, is expressed in alveolar type II cells and in Clara cells in the lungs. Surfactant protein A is involved in the regulation of surfactant turnover, in immunoregulation within the lungs, and in the formation of tubular myelin.

Surfactant is secreted into the airway through exocytosis of the lamellar body by constitutive and regulated mechanisms. Numerous agents, including βadrenergic agonists, activators of protein kinase C, leukotrienes, and purinergic agonists, stimulate the exocytosis of surfactant. The major routes of clearance of pulmonary surfactant within the lung are reuptake by type II cells, absorption into the lymphatic vessels, and clearance by alveolar macrophages. Surfactant is readily inactivated by hypoxia, infection, and edema fluid, which results in a decrease in lung compliance.

In addition to surfactant, another mechanism, interdependence, contributes to stability of the alveoli. Alveoli, except for those on the pleural surface, are surrounded by other alveoli. The tendency of one alveolus to collapse is opposed by the traction exerted by the surrounding alveoli. Thus collapse of a single alveolus causes stretching and distortion of the surrounding alveoli, which in turn are connected to other alveoli. Small openings (pores of Kohn) in the alveolar walls connect adjacent alveoli, whereas the canals of Lambert connect the terminal airways to adjacent alveoli. The pores of Kohn and the canals of Lambert provide collateral ventilation and prevent alveolar collapse (atelectasis).

In1959,AveryandMeaddiscoveredthatinprematureinfantswhodiedofhyalinemembranedisease(HMD),thelungsweredeficientinsurfactant.HMD,alsoknownasinfantrespiratory distress syndrome, ischaracterizedbyprogressiveatelectasisandrespiratoryfailureinprematureinfants.Itisamajorcauseofmorbidityandmortalityintheneonatalperiod.Themajorsurfactantdeficiencyinpremature 1.

Gas flows from areas of higher pressure to areas of lower pressure. Positive transpulmonary pressure is needed to increase lung volume. The pressure across the respiratory system is 0 at points of no airflow (end inspiration and end exhalation). At functional residual capacity (FRC), the pressure difference across the respiratory system is 0, and lung elastic recoil pressure, which operates to decrease lung volume, and the pressure generated by the chest wall to become larger are equal and opposite.

2.

Pressure gradients in the respiratory system are created by the active contraction and subsequent relaxation of the muscles of respiration.

3.

Lung volumes are determined by the balance between the lung’s elastic recoil properties and the properties of the muscles of the chest wall.

Gibson GJ, Pride NB. Lung distensibility: the static pressurevolume curve of the lungs and its use in clinical assessment. Br J Dis Chest. 1976;70:143184.

Jobe AH. The alveolar lining layer: a review of studies on its role in pulmonary mechanics and in the pathogenesis of atelectasis, by Mary Ellen Avery, MD, Pediatrics, 1962:30:324330. Pediatrics. 1998;102(S1):234235.

infantsislackofphosphatidylglycerol.Ingeneral,asthelevelofphosphatidylglycerolincreasesinamnioticfluid,theinfantmortalityratedecreases.ResearchinthisfieldhasculminatedinsuccessfulattemptstotreatHMDinprematureinfantswithsurfactantreplacementtherapy.Today,surfactantreplacementtherapyisstandardcareforprematureinfants.

4.

Total lung capacity (TLC) is equal to the total volume of air that can be exhaled after a maximal inspiration (vital capacity [VC]) and the air remaining in the lung after a maximal exhalation (residual volume [RV]).

5.

Lung compliance is a measure of the elastic properties of the lung. Elastic recoil is lost in patients with emphysema, and this loss is associated with an increase in lung compliance, whereas in diseases associated with pulmonary fibrosis, lung compliance is decreased.

6.

The surface tensionreducing and antisticking properties of surfactant increase lung compliance, decrease the work of breathing, and help stabilize alveoli of different size.

Lumb AB. Nunn’s Applied Respiratory Physiology. 8th ed. St. Louis: Elsevier; 2016.

Mead J, Macklem PT, vol eds. American Physiological Society Handbook of Physiology: The Respiratory System, vol. 3: Mechanics. Bethesda, MD: American Physiological Society; 1986.

Otis AB. A perspective of respiratory mechanics. J Appl Physiol. 1983;54:11831187.

Otis AB, Fenn WO, Rahn H. Mechanics of breathing in man. J Appl Physiol. 1950;2:592607.

Upon completion of this chapter the student should be able to answer the following questions: 1.

Describe air flow in the airways.

2.

Define resistance and its affect upon airflow in the airways.

3.

List and describe two categories of factors that contribute to airway resistance.

4.

List the features of a spirogram and flow volume curve.

5.

Describe how flow limitation occurs at the equal pressure point and the role of dynamic airway compression in flow limitation.

6.

Define the components of work of breathing.

7.

Understand how dynamic compliance is different from static compliance and its contribution to work of breathing.

In this chapter, the principles that control air movement into and out of the lungs are examined. Dynamic mechanics is the study of physical systems in motion, and for the respiratory system it is the study of the properties of a lung whose volume is changing with time.

Air flows into and out of an airway when there is a pressure difference at the two ends of the airway. By way of review, during inspiration the diaphragm contracts, pleural pressure becomes more negative, and gas flows into the lung (see

Fig. 21.2 ). To meet the changing metabolic needs of the body, gas exchange depends on the speed at which fresh gas is brought to the alveoli and the rapidity with which the metabolic products of respiration (i.e., CO2) are removed. Two major factors determine the speed at which gas flows into the airways for a given pressure change: the pattern of gas flow and the resistance to airflow by the airways.

Patterns of Airflow

There are two major patterns of gas flow in the airways— laminar and turbulent. Laminar flow is parallel to the airway walls and is present at low flow rates. As the flow rate increases and particularly as the airways divide, the flow stream becomes unsteady and small eddies develop. At higher flow rates the flow stream is disorganized and turbulence occurs.

The pressure-flow characteristics of laminar flow were first described by the French physician Poiseuille and apply to both liquids and air. In straight circular tubes the flow rate (V̇ ) is defined by the following equation:

Equation 22.1 where P is the driving pressure, r is the radius of the tube, η is the viscosity of the fluid, and l is the length of the tube. It can be seen that driving pressure (P) is proportional to the flow rate (V̇ ); thus the greater the pressure, the greater the flow.

The flow resistance (R) across a set of tubes is defined as the change in driving pressure (ΔP) divided by the flow rate, or:

Equation 22.2

The units of resistance are cm H2O/L•sec. This equation is for laminar flow and demonstrates that the radius of the tube is the most important determinant of resistance. If the radius of the tube is reduced by half, the resistance will increase 16-fold. If, however, tube length is increased twofold, the resistance will increase only twofold. Thus the radius of the tube is the principal determinant of resistance. Stated another way, resistance is inversely proportional to the fourth power of the radius, and it is directly proportional to the length of the tube and to the viscosity of the gas.

In turbulent flow, gas movement occurs both parallel and perpendicular to the axis of the tube. Pressure is proportional to the flow rate squared. The viscosity of the gas increases with increasing gas density, and therefore the pressure drop increases for a given flow. Overall, gas velocity is blunted because energy is consumed in the process of generating eddies and chaotic movement. As a consequence, higher driving pressure is needed to support a given turbulent flow than to support a similar laminar flow.

Whether flow through a tube is laminar or turbulent depends on the Reynolds number. The Reynolds number (Re) is a dimensionless value that expresses the ratio of two dimensionally equivalent terms (kinematic/viscosity), as seen in the equation:

Equation 22.3 where d is the fluid density, v is the average velocity, r is the radius, and η is the viscosity. In straight tubes, turbulence occurs when the Reynolds number is greater than 2000. From this relationship it can be seen that turbulence is most likely to occur when the average velocity of the gas flow is high and the radius is large. In contrast, a low-density gas such as helium is less likely to cause turbulent flow. This is clinically relevant in states of increased airway resistance where a decrease in gas density (e.g., substituting helium for nitrogen in inspired air) can improve airflow.

Although these relationships apply well to smooth cylindrical tubes, application of these principles to a complicated system of tubes such as the airways is difficult. As a result, much of the flow in the airways demonstrates characteristics of both laminar and turbulent flow. In the trachea, for example, even during quiet breathing the Reynolds number is greater than 2000. Hence turbulent flow occurs in the trachea even during quiet breathing. Turbulence is also promoted by the glottis and vocal cords, which produce some irregularity and obstruction in the airways. As gas flows distally the total cross-sectional area increases dramatically, and gas velocities decrease significantly. As a result, gas flow becomes more laminar in the smaller airways even during maximal ventilation. Overall the gas flow in the larger airways (nose, mouth, glottis, and bronchi) is turbulent, whereas the gas flow in the smaller airways is laminar. Breath sounds heard with a stethoscope reflect turbulent airflow. Laminar flow is silent, which is why it is difficult to “hear” small airway disease with a stethoscope.

Airflow resistance is the second major factor that determines rates of airflow in the airways. Airflow resistance in the airways (Raw) differs in airways of different size. In moving from the trachea toward the alveolus, individual airways become smaller while the number of airway branches increases dramatically. Raw is equal to the sum of the resistance of each of these airways (i.e., Raw = Rlarge + Rmedium + Rsmall). From Poiseuille’s equation, one might conclude that the major site of airway resistance is in the smallest airways. In fact, however, the major site of resistance along the bronchial tree is in the first eight generations of airways.

.08 .06 Respiratory zoneConducting zone Airway generation .02 .04 0 0 5 10 15 20 Resistance (cm H2 O/L/sec) •Fig. 22.1 Airwayresistanceasafunctionoftheairwaygeneration.Inanormallung,mostoftheresistancetoairflowoccursinthefirsteightairwaygenerations.

The smallest airways contribute very little to the overall total resistance of the bronchial tree (Fig. 22.1 ). The reason for this is twofold: (1) airflow velocity decreases substantially as the effective cross-sectional area increases (i.e., flow becomes laminar), and (2) most importantly, the airway branches in each generation exist in parallel rather than in series. The resistance of airways in parallel is the inverse of the sum of the individual resistances; therefore the overall contribution to resistance of the small airways is very small. As an example, assume that each of three tubes has a resistance of 3 cm H2O. If the tubes are in series, the total resistance (Rtot) is the sum of the individual resistances:

Equation 22.4

If the tubes are in parallel (as they are in small airways), the total resistance is the sum of the inverse of the individual resistances:

Equation 22.5

This relationship is in marked contrast to the pulmonary blood vessels, in which most of the resistance is located in the small vessels (see ). Thus as airway diameter decreases, the resistance offered by each individual airway increases, but the large increase in the number of parallel pathways and cross-sectional area reduces the resistance at each generation of branching.

During normal breathing, approximately 80% of the resistance to airflow at functional residual capacity (FRC) of efferent vagal fibers, either directly or reflexively, increases ) secondary to airway constriction (recall that the vagus nerve innervates airway smooth muscle). In con trast, stimulation of sympathetic nerves and release of the postganglionic neurotransmitter norepinephrine inhibits airway constriction. Reflex stimulation of the vagus nerve by inhalation of smoke, dust, cold air, or other irritants can also result in airway constriction and coughing. Agents such as histamine, acetylcholine, thromboxane A2, prostaglandin F2, and leukotrienes (LTB4, LTC4, and LTD4) are released by resident cells (e.g., mast cells, airway epithelial cells) and recruited cells (e.g., neutrophils, eosinophils) in response to various triggers such as allergens and viral infections. These agents act directly on airway smooth muscle to cause constriction and an increase in airway resistance. Inhalation •Fig. 22.2 Airwayresistance(AWR)andconductance(Cond.)asafunctionoflungvolume.

occurs in airways with diameters greater than 2 mm. Because the small airways contribute so little to total lung resistance, measurement of airway resistance is a poor test for detecting small airway obstruction.

Factors That Contribute to Airway Resistance

In healthy individuals, airway resistance is approximately 1 cm H2O/L • sec. One of the most important factors affecting resistance is lung volume. Increasing lung volume increases the caliber of the airways because it creates a positive transairway pressure. As a result, resistance to airflow decreases with increasing lung volume and increases with decreasing lung volume. If the reciprocal of resistance (i.e., conductance) is plotted against lung volume, the relationship between lung volume and conductance is linear (Fig.

22.2 ). Other factors that increase airway resistance include airway mucus, edema, and contraction of bronchial smooth muscle, all of which decrease the caliber of the airways.

The density and viscosity of the inspired gas also affect airway resistance. When scuba diving, gas density rises and results in an increase in airway resistance; this increase can cause problems for individuals with asthma and obstructive pulmonary disease. Breathing a low-density gas such as an oxygen-helium mixture results in a decrease in airway resistance and has been exploited in the treatment of status asthmaticus, a condition associated with increased airway resistance due to a combination of bronchospasm, airway inflammation, and hypersecretion of mucus.

Neurohumoral Regulation of Airway Resistance

In addition to the effects of disease, airway resistance is regulated by various neural and humoral agents. Stimulation of methacholine, a derivative of acetylcholine, is used to diagnose airway hyperresponsiveness, which is one of the cardinal features of certain asthma phenotypes. Although everyone is capable of responding to methacholine, airway obstruction develops in individuals with asthma at much lower concentrations of inhaled methacholine.

Measurement of Expiratory Flow

Measurement of expiratory flow rates and expiratory volumes is an important clinical tool for evaluating and monitoring respiratory diseases. Commonly used clinical tests have the patient inhale maximally to total lung capacity (TLC) and then exhale as rapidly and completely as possible to residual volume (RV). The test results are displayed either as a spirogram (

Fig. 22.3A ) or as a flow-volume curve/ loop (

Fig. 22.3B ). Results from individuals with suspected lung disease are compared with results predicted from normal healthy volunteers. Predicted or normal values vary with age, sex, ethnicity, height, and to a lesser extent, weight (

Table 22.1 ). Abnormalities in values indicate abnormal pulmonary function and can be used to predict abnormalities in gas exchange. These values can detect the presence of abnormal lung function long before respiratory symptoms develop, and they can be used to determine disease severity and the response to therapy.

The Spirogram

A spirogram displays the volume of gas exhaled as a function of time (see

Fig. 22.3A ) and measures: (1) forced vital capacity (FVC), (2) forced expiratory volume in 1 second (FEV1), (3) the ratio of FEV1 to FVC (FEV1/ FVC), and (4) the average midmaximal expiratory flow (FEF25-75).

The total volume of air that is exhaled during a maximal forced exhalation from TLC to RV is called the FVC. The volume of air that is exhaled in the first second during the maneuver is called the FEV1. In normal individuals, 70% to 85% (depending on age) of the FVC can be exhaled in the first second. Thus the normal FEV1/FVC ratio is greater than 70% in healthy adults. A ratio less than 70% suggests •Fig. 22.3 Theclinicalspirogram(A)andflow-volumeloop(B).Theindividualtakesamaximalinspirationandthenexhalesasrapidly,asforcibly,andasmaximallyaspossible.Thevolumeexhaledisplottedasafunctionoftime.Inthespirogramthatisreportedinclinicalsettings,exhaledvolumeincreasesfromthebottomofthetracetothetop(A).Thisisincontrasttothephysiologist’sviewofthesamemaneuver(see

Fig.21.3 ),inwhichtheexhaledvolumeincreasesfromthetoptothebottomofthetrace.Intheflow-volumeloop(B),exhaledvolumeisplottedasafunctionoftheinstantaneousflowrate,whichismeasuredusingapneumotachometer.Themaximalexpiratoryflowrateachievedduringthemaneuveriscalledthepeak expiratory flow rate. NotethelocationsofTLCandRVonbothtracings.

difficulty exhaling because of obstruction and is a hallmark of obstructive pulmonary disease. One expiratory flow rate—the average flow rate over the middle section of the VC—can be calculated from the spirogram. This expiratory flow rate has several names, including MMEF (midmaximal expiratory flow) and FEF25-75 (forced expiratory flow from 25%–75% of VC). Although it can be calculated from the spirogram, today’s spirometers automatically calculate FEF25-75.

Another way of measuring lung function clinically is the flow-volume curve or loop. A flow-volume curve or loop is created by displaying the instantaneous flow rate during a forced maneuver as a function of the volume of gas. This instantaneous flow rate can be displayed both during exhalation (expiratory flow-volume curve) and during inspiration (inspiratory flow-volume curve) (see

Fig. 22.3B ). Expiratory flow rates are displayed above the horizontal line, and inspiratory flow rates are displayed below the horizontal line. The flow-volume loop measures: (1) the FVC, (2) the greatest flow rate achieved during the expiratory maneuver, called the peak expiratory flow rate (PEFR), and (3) multiple expiratory flow rates at various lung volumes. When the expiratory flow-volume curve is divided into quarters, the instantaneous flow rate at which 50% of the VC remains to be exhaled is called the FEF50 (also known as the V̇ max50), the instantaneous flow rate at which 75% of the VC has been exhaled is called the FEF75 (V̇ max75), and the instantaneous flow rate at which 25% of the VC has been exhaled is called the FEF25 (V̇ max25).

•Fig. 22.4 Isovolumecurves.Threesuperimposedexpiratoryflowmaneuversaremadewithincreasingeffort.Notethatpeakinspiratoryandexpiratoryflowratesaredependentoneffort,whereasexpiratoryflowrateslaterinexpirationareindependentofeffort.

Inamethacholinechallengetest,spirometrymeasurementsaremadeafterthepatientinhalesincreasingconcentrationsofthemuscarinicagonistmethacholine.ThetestisstoppedwhenFEV1fallsby20%ormoreorwhenamaximumconcentration(25mg/mL)ofmethacholinehasbeeninhaled.Theconcentrationofmethacholinethatproducesa20%decreaseinFEV1iscalledtheprovocation concentration (PC)20. ThelowerthePC20,themoresensitiveanindividualistomethacholine.MostindividualswithasthmahaveaPC20lessthan8mg/mLofmethacholine.

Determinants of Maximal Flow

The shape of the flow-volume loop reveals important information about normal lung physiology that can be altered by disease. Inspection of the flow-volume loop reveals that the maximum inspiratory flow is the same or slightly greater than the maximum expiratory flow. Three factors are responsible for the maximum inspiratory flow. First, the force generated by the inspiratory muscles decreases as lung volume increases above RV. Second, the recoil pressure of the lung increases as the lung volume increases above RV. This opposes the force generated by the inspiratory muscles and reduces maximum inspiratory flow. However, airway resistance decreases with increasing lung volume as the airway caliber increases. The combination of inspiratory muscle force, recoil of the lung, and changes in airway resistance causes maximal inspiratory flow to occur about halfway between TLC and RV.

During exhalation, maximal flow occurs early (in the first 20%) in the maneuver, and flow rates decrease progressively toward RV. Even with increasing effort, maximal flow decreases as RV is approached. This is known as expiratory flow limitation and can be demonstrated by asking an individual to perform three forced expiratory maneuvers with increasing effort.

Fig. 22.4 shows the results of these three maneuvers. As effort increases, peak expiratory flow increases. However, the flow rates at lower lung volumes converge; this indicates that with modest effort, maximal expiratory flow is achieved. No amount of effort will increase the flow rates as lung volume decreases. For this reason, expiratory flow rates at lower lung volumes are said to be effort independent and flow limited because maximal flow is achieved with modest effort, and no amount of additional effort can increase the flow rate beyond this limit. In contrast, events early in the expiratory maneuver are said to be effort dependent; that is, increasing effort generates increasing flow rates. In general the first 20% of the flow in the expiratory flow-volume loop is effort dependent.

Flow Limitation and the Equal Pressure Point

Why is expiratory flow limited and reasonably effort independent? Factors that limit expiratory flow are important because many lung diseases affect these factors and thus affect the volume and speed with which air is moved into and out of the lung. Flow limitation occurs when the airways, which are intrinsically floppy distensible tubes, become compressed. The airways become compressed when the pressure outside the airway exceeds the pressure inside the airway. How and when this occurs is important to understanding lung disease.

Fig. 22.5 shows the events that

TLC, no flow •Fig. 22.5 Flowlimitation.A,Endinspiration,beforethestartofexhalation.B,Atthestartofaforcedexhalation.C,Expiratoryflowlimitationlaterinaforcedexhalation.Expiratoryflowlimitationoccursatlocationswhereairwaydiameterisnarrowedasaresultofnegativetransmuralpressure.Seetextfordetails.

occur during expiratory flow limitation at two different lung volumes. The airways and alveoli are surrounded by the pleural space and the chest wall. The airways are shown as tapered tubes because the total or collective airway cross-sectional area decreases from the alveoli to the trachea. At the start of exhalation but before any gas flow occurs, the pressure inside the alveolus (PA) is zero (no airflow) and pleural pressure (in this example) is −30 cm H2O. Transpulmonary pressure is thus +30 cm H2O (PL = PA − Ppl). Because there is no flow, the pressure inside the airways is zero and the pressure across the airways (Pta, transairway pressure) is +30 cm H2O [Pta = Pairway − Ppl = 0 − (−30 cm H2O)]. This positive transpulmonary and transairway pressure holds the alveoli and airways open.

When an active exhalation begins and the expiratory muscles contract, pleural pressure rises to +60 cm H2O (in this example). Alveolar pressure also rises, in part because of the increase in pleural pressure (+60 cm H2O) and in part because of the elastic recoil pressure of the lung at that lung volume (which in this case is 30 cm H2O). Alveolar pressure is the sum of pleural pressure and elastic recoil pressure (i.e., PA = Pel + Ppl = 30 cm H2O + 60 cm H2O = 90 cm H2O in this example). This is the driving pressure for expiratory gas flow. Because alveolar pressure exceeds atmospheric pressure, gas begins to flow from the alveolus to the mouth when the glottis opens. As gas flows out of the alveoli, the transmural pressure across the airways decreases (i.e., the pressure head for expiratory gas flow dissipates). This occurs for three reasons: (1) there is a resistive pressure drop caused by the frictional pressure loss associated with flow (expiratory airflow resistance); (2) as the cross-sectional area of the airways decreases toward the trachea, gas velocity increases and this acceleration of gas flow further decreases the pressure; and (3) as lung volume decreases, the elastic recoil pressure decreases.

Thus as air moves out of the lung, the driving pressure for expiratory gas flow decreases. In addition, the mechanical tethering that holds the airways open at high lung volumes diminishes as lung volume decreases. There is a point between the alveoli and the mouth at which the pressure inside the airways equals the pressure that surrounds the airways. This point is called the equal pressure point. Airways toward the mouth but still inside the chest wall become compressed because the pressure outside is greater than the pressure inside (dynamic airway compression). As a consequence the transairway pressure now becomes negative [Pta = Paw − Ppl = 58 − (+60) =−2 cm H2O] just beyond the equal pressure point. No amount of effort will increase the flow further because the higher pleural pressure tends to collapse the airway at the equal pressure point, just as it also tends to increase the gradient for expiratory gas flow. Under these conditions, airflow is independent of the total driving pressure. Hence the expiratory flow is effort independent and flow limited. It is also why airway resistance is greater during exhalation than during inspiration. In the absence of lung disease, the equal pressure point occurs in airways that contain cartilage, and thus they resist collapse. The equal pressure point, however, is not static. As lung volume decreases and as elastic recoil pressure decreases, the equal pressure point moves closer to the alveoli.

One additional measurement of dynamic lung mechanics should be mentioned, and this is the measurement of dynamic compliance. A dynamic pressure-volume curve can be created by having an individual breathe over a normal lung volume range (usually from FRC to FRC +1 L). The

Whathappensinindividualswithlungdisease?Imagineanindividualwithairwayobstructionsecondarytoacombinationofmucus accumulationandairwayinflammation(Fig.

22.6A ).Atthestartofexhalationthedrivingpressureforexpiratorygasflowisthesameasinanormalindividual;thatis,thedrivingpressureisthesumoftheelasticrecoilpressureandpleuralpressure.Asexhalationproceeds,however,theresistivedropinpressureisgreaterthaninthenormalindividualbecauseofthegreaterdecreaseinairwayradiussecondarytotheaccumulationofmucusandtheinflammation.Asaresulttheequalpressurepointnowoccursinsmallairwaysthataredevoidofcartilage.Theseairwayscollapse.Thiscollapseisknownaspremature airway closure, whichresultsinaless-than-maximalexpiratoryvolumeandproducesanincreaseinlungvolumeknownasair trapping.Theincreaseinlungvolumeinitiallyhelpsoffsettheincreaseinairwayresistancecausedbytheaccumulationofmucusandinflammationbecauseitresultsinanincreaseinairwaycaliberandelasticrecoil.Asthediseaseprogresses,however,inflammationandaccumulationofmucusincreasefurther,thereisagreaterincreaseinexpiratoryresistance,andmaximalexpiratoryflowratesdecrease.

Fig.22.6B ).Atthestartofexhalationthedrivingpressureforexpiratorygasflowisreducedsecondarytoalossofelasticrecoil.Whiletheresistivedropinpressureisnormal,thesmallerinitialdrivingpressureresultsinanequalpressurepointthatoccursclosertothealveolusinairwaysthatdonotcontaincartilage.Prematureairwayclosureagainoccursbutforaverydifferentreasonthantheprematureairwayclosureobservedinindividualswithanincreaseinairwayresistance.

Individualswithprematureairwayclosurefrequentlyhavecrackles, alsosometimescalledrales, apoppingsoundusuallyheardduringinspirationonauscultation.Thesecracklesareduetotheopeningofairwaysduringinspirationthatclosed(i.e.,werecompressed)duringthepreviousexhalation.Cracklescanbeduetomucusaccumulation,airwayinflammation,fluidintheairways,oranymechanismresponsibleforairwaynarrowingorcompression.Theyarealsoheardinindividualswithemphysema,inwhichthereisadecreaseinlungelasticrecoil.Infact,acuteandchroniclungdiseasescanchangetheexpiratoryflow-volumerelationshipbychangesin(1)staticlungrecoilpressure,(2)airwayresistanceandthedistributionofresistancealongtheairways,(3)lossofmechanicaltetheringofintraparenchymalairways,(4)changesinthestiffnessormechanicalpropertiesoftheairways,and(5)differencesinseverityoftheaforementionedchangesinvariouslungregions.

mean dynamic compliance of the lung (dyn CL) is calculated as the slope of the line that joins the end-inspiratory and end-expiratory points of no flow (

Fig. 22.7

Dynamic compliance is always less than static compliance, and it increases during exercise. This is because during tidal volume breathing, a small change in alveolar surface area is insufficient to bring additional surfactant molecules to the surface, and thus the lung is less compliant. During exercise the opposite occurs; there are large changes in

Mucus, Edema, Bronchoconstriction PL = 90˜60 = 30 cm H2O

Fig. 22.6 A,Flowlimitationinthepresenceofincreasedairwayresistance.B,Flowlimitationinthepresenceofalossofelasticrecoil.

Fig. 22.7 Inflation-deflationpressure-volumecurve.Thedirectionofinspirationandexhalationisshownbythearrows.Thedifferencebetweentheinflationanddeflationpressure-volumecurvesisduetothevariationinsurfacetensionwithchangesinlungvolume.Notetheslopeofthelinejoiningpointsofnoairflow.Thisslopeislesssteepthantheslopefromthedeflationpressure-volumecurveatthesamelungvolume.

tidal volume, and more surfactant material is incorporated into the air-liquid interface. Therefore the lung is more compliant.

Sighing and yawning increase dynamic compliance by increasing tidal volume and restoring the normal surfactant layer. Both of these respiratory activities are important for D maintaining normal lung compliance. In contrast to the 1000 lung, the dynamic compliance of the chest wall is not significantly different from its static compliance.

EF Insp. Exp. B

Work of Breathing

Breathing requires the use of respiratory muscles (diaphragm, intercostals, etc.), which expends energy. Work is required to overcome the inherent mechanical properties of the lung (i.e., elastic and flow-resistive forces) and to move both the lungs and the chest wall. This work is known as the work of breathing. Changes in the mechanical properties of the lung or chest wall (or both) in the presence of disease result in an increase in the work of breathing. Respiratory muscles can perform increased work over long periods. However, the body. In the respiratory system, the work of breathing is calculated by multiplying the change in volume by the pressure exerted across the respiratory system:

Work of breathing (W) = Pressure (P) DC like other skeletal muscles they can fatigue, and respiratory failure may ensue. Respiratory muscle fatigue is the most common cause of respiratory failure, a process in which gas exchange is inadequate to meet the metabolic needs of

Although methods are not available to measure the total amount of work involved in breathing, one can estimate the mechanical work by measuring the volume and pressure changes during a respiratory cycle. Analysis of pressure-volume curves can be used to illustrate these points.

Fig.

22.8A represents a respiratory cycle of a normal lung.

The static inflation-deflation curve is represented by line ABC. The total mechanical workload is represented by the trapezoidal area OAECD.

In restrictive lung diseases, such as pulmonary fibrosis, lung compliance is decreased and the pressure-volume curve is shifted to the right. This results in a significant increase in the work of breathing (see

Fig. 22.8B ), as indicated by the increase in the trapezoidal area of OAECD. In obstructive lung diseases, such as asthma during an exacerbation or chronic bronchitis, airway resistance is elevated (see

Fig.

22.8C ) and greater negative pleural pressure is needed to maintain normal inspiratory flow rates. In addition to the increase in total inspiratory work (OAECD), individuals with obstructive lung disease have an increase in positive pleural pressure during exhalation because of the increase in resistance and the increased expiratory workload, which is visualized as area DFO. The stored elastic energy, represented by area ABCF of

Fig. 22.8A , is not sufficient, and additional energy is needed for exhalation. With time or disease progression, these respiratory muscles can fatigue and result in respiratory failure. The work of breathing is also increased when deeper breaths are taken (an increase in tidal volume requires more elastic work to overcome) and when the respiratory rate increases (an increase in minute •Fig. 22.8 Mechanicalworkdoneduringarespiratorycycleinanormallung(A),alungwithreducedcompliance(B),andalungwithincreasedairwayresistance(C).Breakdownofthetrapezoidalareasenablesonetoappreciatetheindividualaspectsofthemechanicalworkload,whichincludethefollowing:OABCD,worknecessarytoovercomeelasticresistance;AECF,worknecessarytoovercomenonelasticresistance;AECB,worknecessarytoovercomenonelasticresistanceduringinspiration;ABCF,worknecessarytoovercomenonelasticresistanceduringexhalation(representsstoredelasticenergyfrominspiration).

ventilation requires more flow resistance force to overcome) (

Fig. 22.9 ). Normal individuals and individuals with lung disease adopt respiratory patterns that minimize the work of breathing. For this reason, individuals with pulmonary fibrosis (increased elastic work) breathe more shallowly and rapidly, and those with obstructive lung disease (normal elastic work but increased resistive work) breathe more slowly and deeply.

60 50 40 Total work Elastic work Nonelastic work 20 10 30 10 20 30 40 Mechanical work (kg-m/min) Respiratory rate (no./min)

Chronic obstructive pulmonary disease (COPD)isageneraltermthatincludesdiseasessuchasemphysemaandchronicbronchitis.COPDmostcommonlyoccursinindividualswhosmoke,inwhompathologicchangesinthelungconsistentwithbothemphysemaandchronicbronchitiscancoexist.ForindividualswithCOPDinwhomemphysemaisamajorcomponent,theelastictissueinthealveolarandcapillarywallsisprogressivelydestroyed,whichresultsinincreasedlungcomplianceanddecreasedelasticrecoil.Thedecreaseinelasticrecoilresultsinmovementoftheequalpressurepointtowardthealveolusandprematureairwayclosure.ThisproducesairtrappingandincreasesinRV,FRC,andTLC.Airwayresistanceisalsoincreased.Theseincreasesinlungvolumesincreasetheworkofbreathingbystretchingtherespiratorymusclesanddecreasingtheirefficiency.

Inchronicbronchitis,accumulationofmucusandairwayinflammationcausetheequalpressurepointtomovetowardthealveolus,whichleadstoprematureairwayclosureandincreasesinRV,FRC,andTLC.Airwayresistanceandtheworkofbreathingareincreased,butlungcomplianceisnormal.

Inrestrictivelungdiseasessuchaspulmonaryfibrosis,lungcomplianceisdecreased.Lungvolumesaredecreased,butflowratesarereasonablynormal.Someofthechangesinpulmonaryfunctionvaluesinobstructiveandrestrictivepulmonarydiseasesareshownin

Table22.1

Inthethirdtrimesterofpregnancy,theenlargeduterusincreasesintraabdominalpressureandrestrictsmovementofthediaphragm.TheFRC,asaresult,decreases.Thischangeinlungvolumeresultsindecreasedlungcomplianceandincreasedairwayresistanceinotherwisehealthywomen.

•Fig. 22.9 Effectoftherespiratoryrateontheelastic,nonelastic,andtotalmechanicalworkofbreathingatagivenlevelofalveolarventilation.Individualstendtoadopttherespiratoryrateatwhichthetotalworkofbreathingisminimal(arrow) forthosewithoutlungdisease.

1.

There are two major patterns of gas flow in the airways: turbulent and laminar.

2.

Resistance to airflow is the change in pressure per unit of flow. Airway resistance varies with the inverse of the fourth power of the radius and is higher in turbulent than in laminar flow. The major site of airway resistance is the first eight airway generations. Airway resistance decreases with increases in lung volume and with decreases in gas density. Airways resistance is also regulated by neural and humoral agents.

3.

Pulmonary function tests (spirometry, flow-volume loop, body plethysmography) can detect abnormalities in lung function before individuals become symptomatic. Test results are compared with results obtained in normal individuals and vary with sex, ethnicity, age, and height. COPD is characterized by increases in lung volumes and airway resistance and by decreases in expiratory flow rates. Emphysema, a specific type of COPD, is further characterized by increased lung compliance. Restrictive lung diseases are characterized by decreases in lung volume, normal expiratory flow rates and resistance, and a marked decrease in lung compliance.

4.

The equal pressure point is the point at which the pressure inside and surrounding the airway is the same. The location of the equal pressure point is dynamic. Specifically, as lung volume and elastic recoil decrease, the equal pressure point moves toward the alveolus in normal individuals. In individuals with chronic obstructive pulmonary disease (COPD), the equal pressure point at any lung volume is closer to the alveolus. Expiratory flow limitation occurs at the equal pressure point.

5.

Energy is expended during breathing to overcome the inherent mechanical properties of the lung. Respiratory muscle fatigue is the most common cause of respiratory failure. Individuals breathe at a respiratory rate to minimize work. For individuals with increased airway resistance, work is minimized by breathing at lower frequencies. For individuals with restrictive lung diseases, work is minimized by shallow breathing at high frequencies.

6.

The dynamic compliance of the lung is always less than the static compliance and increases during exercise, sighing, and yawning.

Calverley PMA, Koulouris NG. Flow limitation and dynamic hyperinflation: key concepts in modern respiratory physiology. Eur Respir J. 2005;25:186-199.

Crapo RO, et al. Reference spirometric values using techniques and equipment that meet ATS recommendations. Am Rev Respir Dis. 1981;123:659-664.

Otis AB. A perspective of respiratory mechanics. J Appl Physiol. 1983;54:1183-1187.

Otis AB, et al. Mechanics of breathing in man. J Appl Physiol. 1950;2:592-607.

Leff AR, Schumacker PT. Respiratory Physiology: Basics and Applications. Philadelphia: Saunders; 1993. Lumb AB. Nunn’s Applied Respiratory Physiology. 8th ed. St. Louis: Elsevier; 2016.

Mead J, MacKlem PT. Mechanics of breathing. In: Handbook of Physiology. Section 3, The Respiratory System. Bethesda, MD: American Physiological Society; 1986. Wiley Online Library doi:10.1002/cphys.cp0303fmo1.

Ventilation, Perfusion, and Ventilation/ Perfusion Relationships

Upon completion of this chapter, the student should be able to answer the following questions: 1.

Define two types of dead space ventilation, and describe how dead space ventilation changes with tidal volume.

2.

Describe the composition of gas in ambient air, the trachea, and the alveolus, and understand how this composition changes with changes in oxygen fraction and barometric pressure.

3.

Use the alveolar air equation to calculate the alveolar-arterial difference for oxygen (AaDO2).

4.

Understand the alveolar carbon dioxide equation and identify how it changes with alterations in alveolar ventilation.

5.

Compare the distribution of pulmonary blood flow to the distribution of ventilation.

6.

List and define the four categories of hypoxia and the six causes of hypoxic hypoxia.

7.

Distinguish the causes of hypoxic hypoxia on the basis of the response to 100% O2.

8.

Describe the two causes of hypercapnia.

he major determinant of normal gas exchange and thus the level of PO2 and PCO2 in blood is the relationship between ventilation (V̇ ) and perfusion (Q̇ ). This relationship is called the ventilation/perfusion (V̇ /Q̇ ) ratio.

Ventilation is the process by which air moves in and out of the lungs. The incoming air is composed of a volume that fills the conducting airways (dead space ventilation) and a portion that fills the alveoli (alveolar ventilation). Minute (or total) ventilation (V̇ E) is the volume of air that enters or leaves the lung per minute:

Equation 23.1 where f is the frequency or number of breaths per minute and VT (also known as TV) is the tidal volume, or volume of air inspired (or exhaled) per breath. Tidal volume varies with age, sex, body position, and metabolic activity. In an average-sized adult at rest, tidal volume is 500 mL. In children, it is 3 to 5 mL/kg.

Dead Space Ventilation: Anatomical and Physiological

Dead space ventilation is ventilation to airways that do not participate in gas exchange. There are two types of dead space: anatomical dead space and physiological dead space. Anatomical dead space (VD) is composed of the volume of gas that fills the conducting airways:

Equation 23.2 where V refers to volume and the subscripts T, D, and A refer to tidal, dead space, and alveolar. A “dot” above V denotes a volume per unit of time (n):

Equation 23.3

Equation 23.4 where V̇ E is the total volume of gas in liters expelled from the lungs per minute (also called exhaled minute volume), V̇ D is the dead space ventilation per minute, and V̇ A is alveolar ventilation per minute.

In a healthy adult, the volume of gas contained in the conducting airways at functional residual capacity (FRC) is approximately 100 to 200 mL, in comparison with the 3 L of gas in an entire lung. The ratio of the volume of the conducting airways (dead space) to tidal volume represents the fraction of each breath that is “wasted” in filling the conducting airways. This volume is related to tidal volume (VT) and to exhaled minute ventilation (V̇ E) in the following way:

Equation 23.5

CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships

If the dead space volume is 150 mL and tidal volume increases from 500 to 600 mL for the same exhaled minute ventilation, what is the effect on dead space ventilation?

= 0 3. × VE and, similarly,

Increasing tidal volume is an effective way to increase alveolar ventilation (and thus normal blood gas values), as might occur during exercise or periods of stress. As tidal volume increases, the fraction of the dead space ventilation decreases for the same exhaled minute ventilation.

Dead space ventilation (VD) varies inversely with tidal volume (VT). The larger the tidal volume, the smaller the proportion of dead space ventilation. Normally, VD/VT is 20% to 30% of exhaled minute ventilation. Changes in dead space are important contributors to work of breathing. If the dead space increases, the individual must inspire a larger tidal volume to maintain normal levels of blood gases. This adds to the work of breathing and can contribute to respiratory muscle fatigue and respiratory failure. If metabolic demands increase (e.g., during exercise or with fever), individuals with lung disease may not be able to increase tidal volume sufficiently.

The second type of dead space is physiological dead space. Often in diseased lungs, some alveoli are perfused but not ventilated. The total volume of gas in each breath that does not participate in gas exchange is called the physiological dead space. This volume includes the anatomical dead space and the dead space secondary to perfused but unventilated alveoli. The physiological dead space is always at least as large as the anatomical dead space, and in the presence of disease, it may be considerably larger.

Both anatomical and physiological dead space can be measured, but they are not measured routinely in the course of patient care.

Composition of Air

Inspiration brings ambient or atmospheric air to the alveoli, where O2 is taken up and CO2 is excreted. Ambient air is a

In individuals with certain types of chronic obstructive pulmonary disease (COPD), such as emphysema, physiological dead space is increased. If dead space doubles, tidal volume must increase in order to maintain the same level of alveolar ventilation. If tidal volume is 500 mL and VD/VT is 0.25, then

If VD increases to 250 mL in this example, tidal volume (VT) must increase to 625 mL to maintain a normal alveolar ventilation (i.e., VA = 375 mL): gas mixture composed of N2 and O2, with minute quantities of CO2, argon, and inert gases. The composition of this gas mixture can be described in terms of either gas fractions or the corresponding partial pressure.

Because ambient air is a gas, the gas laws can be applied, from which two important principles arise. The first is that when the components are viewed in terms of gas fractions (F), the sum of the individual gas fractions must equal one:

Equation 23.6 1 0. = FN2 + FO2 + Fargon and other gases

It follows, then, that the sum of the partial pressures (in millimeters of mercury) of a gas, also known as the gas tension (in torr), must be equal to the total pressure. Thus at sea level, where atmospheric pressure (also known as barometric pressure [Pb]) is 760 mm Hg, the partial pressures of the gases in air are as follows:

Equation 23.7

Three important gas laws govern ambient air and alveolar ventilation. According to Boyle’s law, when temperature is constant, pressure (P) and volume (V) are inversely related; that is,

Boyle’s law is used in the measurement of lung volumes (see

Fig. 21.4 ). Dalton’s law is that the partial pressure of a gas in a gas mixture is the pressure that the gas would exert if it occupied the total volume of the mixture in the absence of the other components.

Eq. 23.7 is an example of how Dalton’s law is used in the lung. According to Henry’s law, the concentration of a gas dissolved in a liquid is proportional to its partial pressure.

The second important principle is that the partial pressure of a gas (Pgas) is equal to the fraction of that gas in the gas mixture (Fgas) multiplied by the atmospheric (barometric) pressure:

Equation 23.8

Ambient air is composed of approximately 21% O2 and 79% N2. Therefore, the partial pressure of O2 in inspired ambient air (PO2) is calculated as follows:

Equation 23.9 where (FiO2) is the fraction of oxygen in inspired air. The partial pressure of O2, or oxygen tension, in ambient air at the mouth at the start of inspiration is therefore 159 mm Hg, or 159 torr. The O2 tension at the mouth can be altered in one of two ways: by changing the fraction of O2 in inspired air (FiO2) or by changing barometric pressure. Thus ambient O2 tension can be increased through the administration of supplemental O2 and is decreased at high altitude.

The partial pressure of O2 in ambient air varies with altitude. The highest and lowest points in the contiguous United States are Mount Whitney in Sequoia National Park/ Inyo National Forest (14,505 feet; barometric pressure, 437 mm Hg) and Badwater Basin in Death Valley National Park (282 feet; barometric pressure, 768 mm Hg). On Mount Whitney, the partial pressure of O2 in ambient air is calculated as follows: . = 92 mm Hg whereas in Death Valley Badwater Basin, the partial pressure of oxygen is calculated as follows: . = 161mm Hg

Note that the FiO2 does not vary at different altitudes; only the barometric pressure varies. These differences in oxygen tension have profound effects on arterial blood gas values.

As inspiration begins, ambient air is brought into the nasopharynx and laryngopharynx, where it becomes warmed to body temperature and humidified. Inspired air becomes saturated with water vapor by the time it reaches the glottis. Water vapor exerts a partial pressure and dilutes the total pressure in which the other gases are distributed. Water vapor pressure at body temperature is 47 mm Hg. To calculate the partial pressures of O2 and N2 in a humidified mixture, the water vapor partial pressure must be subtracted from the total barometric pressure. Thus in the conducting airways, which begin in the trachea, the partial pressure of O2 is calculated as follows:

Equation 23.10 = . = 150 mm Hg and the partial pressure of N2 is calculated similarly:

Equation 23.11 = 563 mm Hg Note that the total pressure remains constant at 760 mm Hg (150 + 563 + 47 mm Hg) and that the fractions of O2 and N2 are unchanged. Water vapor pressure, however, reduces the partial pressures of O2 and N2. Note also that in the calculation of the partial pressure of ambient air (

Eq. 23.9), water vapor is ignored, and ambient air is considered “dry.” The conducting airways do not participate in gas exchange. Therefore, the partial pressures of O2, N2, and water vapor remain unchanged in the airways until the air reaches the alveolus.

When the inspired air reaches the alveolus, O2 is transported across the alveolar membrane into the capillary bed, and CO2 moves from the capillary bed into the alveolus. The process by which this occurs is described in . At the end of inspiration and with the glottis open, the total pressure in the alveolus is atmospheric; thus, the partial pressures of the gases in the alveolus must equal the total pressure, which in this case is atmospheric. The composition of the gas mixture, however, is changed and can be described as follows:

Equation 23.12 1 0. = FO2 + FN2 + FH2O + FCO2 + Fargon and other gases where N2 and argon are inert gases, and therefore the fraction of these gases in the alveolus does not change from ambient fractions. The fraction of water vapor also does not change because the inspired gas is already fully saturated with water vapor and is at body temperature. As a consequence of gas exchange, however, the fraction of O2 in the alveolus decreases, and the fraction of CO2 in the alveolus increases. Because of changes in the fractions of O2 and CO2, the partial pressures exerted by these gases also change. The partial pressure of O2 in the alveolus (PAO2) is given by the alveolar gas equation, which is also called the ideal alveolar oxygen equation:

Equation 23.13

CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships

PO2 159 150 102 90 40 PCO2 0 040 40 46 PH2O, 37°C 0 47 47 47 47 PN2 601 563 571

PCO2, partial pressure of carbon dioxide; PH2O, partial pressure of water; PN2, partial pressure of nitrogen; PO2, partial pressure of oxygen; PTOTAL, partial pressure of all parameters; R, respiratory quotient. *PN2 is increased in alveolar gas by 1% because R is normally less than 1.

†Ptotal is less in venous than in arterial blood because PO2 has decreased more than PCO2 has increased.

where PiO2 is the partial pressure of inspired O2, which is equal to the fraction of O2 (FiO2) multiplied by the barometric pressure (Pb) minus water vapor pressure (PH2O); PACO2 is the partial pressure of alveolar CO2; and R is the respiratory exchange ratio, or respiratory quotient. The respiratory quotient is the ratio of the amount of CO2 excreted (V̇ CO2) to the amount of O2 taken up (V̇ O2) by the lungs. This quotient is the amount of CO2 produced in relation to the amount of O2 consumed by metabolism and is dependent on caloric intake. The respiratory quotient varies between 0.7 and 1.0; it is 0.7 in states of exclusive fatty acid metabolism and 1.0 in states of exclusive carbohydrate metabolism. Under normal dietary conditions, the respiratory quotient is assumed to be 0.8. Thus the quantity of O2 taken up exceeds the quantity of CO2 that is released in the alveoli. The partial pressures of O2, CO2, and N2 from ambient air to the alveolus at sea level are shown in

Table 23.1

A similar approach can be used to calculate the estimated PACO2. The fraction of CO2 in the alveolus is a function of the rate of CO2 production by the cells during metabolism and the rate at which the CO2 is eliminated from the alveolus. This process of elimination of CO2 is known as alveolar ventilation. The relationship between CO2 production and alveolar ventilation is defined by the alveolar carbon dioxide equation:

Equation 23.14 where V̇ CO2 is the rate of CO2 production by the body, V̇ A is alveolar ventilation per minute, and FACO2 is the fraction of CO2 in dry alveolar gas. This relationship demonstrates that the rate of elimination of CO2 from the alveolus is related to alveolar ventilation and to the fraction of CO2 in the alveolus. Like the partial pressure of any other gas (see

Eq. 23.8 ), PACO2 is defined by the following:

Equation 23.15

Substituting for FACO2 in the previous equation yields the following relationship:

Equation 23.16

This equation demonstrates several important relationships. First, there is an inverse relationship between the partial pressure of CO2 in the alveolus (PACO2) and alveolar ventilation per minute (V̇ A), regardless of the exhaled CO2. Specifically, if ventilation is doubled, PACO2 decreases by 50%. Conversely, if ventilation is decreased by half, the PACO2 doubles. Second, at a constant alveolar ventilation per minute (V̇ A), doubling of the metabolic production of CO2 (V̇ CO2) causes the PACO2 to double. The relationship between V̇ A and PACO2 is depicted in

Fig. 23.1

In normal lungs, PaCO2 is tightly regulated and maintained at 40 ± 2 mm Hg. Increases or decreases in PaCO2, particularly when associated with changes in arterial pH, have profound effects on cell function, including enzyme and protein activity. Specialized chemoreceptors monitor PaCO2 in the brainstem ( ), and exhaled minute ventilation (

Eq. 23.1 ) varies in accordance with the level of PaCO2.

An acute increase in PaCO2 results in respiratory acidosis (pH < 7.35), whereas an acute decrease in PaCO2 results in respiratory alkalosis (pH > 7.45). Hypercapnia is defined as an elevation in PaCO2, and it occurs when CO2 production exceeds alveolar ventilation (hypoventilation). Conversely, hyperventilation occurs when alveolar ventilation exceeds CO2 production, and it decreases PaCO2 (hypocapnia).

• Fig. 23.1 The Alveolar Partial Pressure of Carbon Dioxide (PCO2; y-axis) as a Function of Alveolar Ventilation per Minute (VA; x-axis) • Fig. 23.2 Regional Distribution of Lung Volume, Including in the Lung. Each line corresponds to a given metabolic rate asso-

Alveolar Size (Circles) and Location on the Pressure-Volume ciated with a constant production of CO2 CO2 isometabolic line).

Curve of the Lung at Different Lung Volumes. Because the lungs are suspended in the upright position, the pleural pressure (Ppl) and translung pressure (PL) of lung units at the apex are greater than those at the base. These lung units are larger at any lung volume than are those at the base. The effect is greatest at residual volume (RV), less so at functional residual capacity (FRC), and absent at total lung capacity (TLC). Note also that because of their “location” on the pressure-volume curve, inspired air is differentially distributed to these lung units; those at the apex are less compliant and receive a smaller Normally, alveolar ventilation is controlled to maintain an alveolar PCO2 of approximately 40 mm Hg. Thus at rest, when VCO2 is approximately 250 mL/minute, alveolar ventilation of 5 L/minute results in an alveolar

PCO2 of 40 mm Hg. A 50% decrease in ventilation at rest (i.e., from 5 to 2.5 L/minute) results in doubling of alveolar PCO2. During exercise,

CO2 production is increased (V̇ CO2 = 750 mL/min), and to maintain normal alveolar PCO2, ventilation must increase (in this case, to 15 L/ minute). Again, however, a 50% reduction in ventilation (from 15 to 7.5 L/minute) results in doubling of the alveolar PCO2.

proportion of the inspired air than do the lung units at the base, which are more compliant (i.e., are represented at a steeper part of the

Distribution of Ventilation

Ventilation is not uniformly distributed in the lung, largely because of the effects of gravity. In the upright position, at most lung volumes, alveoli near the apex of the lung are more expanded than are alveoli at the base. Gravity pulls the lung downward and away from the chest wall. As a result, pleural pressure is lower (i.e., more negative) at the apex than at the base of the lung, and static translung pressure (PL = PA − Ppl) is increased; this results in an increase in alveolar volume at the apex. Because of the difference in alveolar volume at the apex and at the base of the lung (Fig.

23.2 ), alveoli at the lung base are represented along the steep portion of the pressure-volume curve, and they receive more of the ventilation (i.e., they have greater compliance). In contrast, the alveoli at the apex are represented closer to the top or flat portion of the pressure-volume curve. They have lower compliance and thus receive proportionately less of the tidal volume. The effect of gravity is less pronounced when a person is supine rather than upright, and it is less pressure-volume curve).

Alveolar units with long time constants fill and empty slowly. Thus an alveolar unit with increased airway resistance or increased compliance takes longer to fill and longer to empty. In adults, the normal respiratory rate is approximately 12 breaths per minute, the inspiratory time is approximately 2 seconds, and the expiratory time is approximately 3 seconds. In normal lungs, this time is sufficient to approach volume equilibrium (

Fig. 23.3 ). In the presence of increased resistance or increased compliance, however, volume equilibrium is not reached.

Adults with COPD have a very long time constant as a result when a person is supine rather than prone. This is because the diaphragm is pushed in a cephalad direction when a person is supine, and it affects the size of all of the alveoli.

In addition to gravitational effects on the distribution of ventilation, ventilation in alveoli is not uniform. The reason for this is variable airway resistance (R) or compliance (C), and it is described quantitatively by the time constant (τ):

Equation 23.17 of an increase in resistance and, in the case of individuals with emphysema, an increase in compliance. As a result, such affected adults tend to breathe at a low respiratory rate. Imagine now what happens when individuals with COPD climb a flight of stairs. The increase in respiratory rate does not allow sufficient time for a full exhalation, and a process called dynamic hyperinflation occurs (

Fig. 23.4 ); lung volumes, which are already increased, increase further, the lung becomes less compliant, and the work of breathing is very high.

CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships ˛=0.28 ˛=0.56 C=0.4 Decreased compliance C=0.8 Increased resistance R=0.7 R=1.4 R=0.7 ˛=1.12 • Fig. 23.4 Dynamic Hyperinflation. The total time for respiration (Ttot) is composed of the time for inspiration (Ti) and the time for exhalation (Te). When the respiratory rate increases (e.g., during exercise), Ttot decreases. In individuals with chronic obstructive pulmonary disease

C=0.8 Normal (COPD), the effect of the increase in Ttot on Te may not allow for complete emptying of the alveoli with a long time constant, and with each succeeding breath, there is an increase in the lung volume (air trapping). This increase in lung volume eventually results in such a degree of hyperinflation that the affected person is no longer able to do the work needed to overcome the decreased compliance of the lung at this high lung volume. In such individuals, it is a major cause of shortness of breath with activity. FRC, functional residual capacity.

• Fig. 23.3 Examples of Local Regulation of Ventilation as a Result of Variation in the Resistance (R) or Compliance (C) of Individual Lung Units. Top, The individual resistance and compliance values of three different lung units are illustrated. Bottom, The graph illustrates the volume of these three lung units as a function of time. In the upper schema, the normal lung has a time constant (τ) of 0.56 second. This lung unit reaches 97% of final volume equilibrium in 2 seconds, which is the normal inspiratory time. The lung unit at the right has a twofold increase in resistance; hence its time constant is doubled. That lung unit fills more slowly and reaches only 80% volume equilibrium during a normal inspiratory time (see graph); thus this lung unit is underventilated. The lung unit on the left has decreased compliance (is “stiff”), which acts to reduce its time constant. This lung unit fills quickly, reaching its maximum volume within 1 second, but receives only half the ventilation of a normal lung unit.

Blood flow in the pulmonary circulation is pulsatile and influenced by pulmonary vascular resistance (PVR), gravity, alveolar pressure, and the arterial-to-venous pressure gradient. PVR is calculated as the change in pressure from the pulmonary artery (PPA) to the left atrium (PLA), divided by the flow (QT), which is cardiac output:

Equation 23.18

Under normal circumstances,

Equation 23.19

This resistance is about 10 times less than that in the systemic circulation. The pulmonary circulation has two unique features that allow increased blood flow on demand without an increase in pressure: (1) With increased demand, as during exertion or exercise, pulmonary vessels that are normally closed are recruited; and (2) the blood vessels in the pulmonary circulation are highly distensible, and their diameter increases with only a minimal increase in pulmonary arterial pressure.

Fig. 23.5). At end inspiration, the air-filled alveoli compress the alveolar capillaries and increase PVR. In contrast to the capillary beds in the systemic circulation, the capillary beds in the lungs account for approximately 40% of PVR. The diameters of the larger extra-alveolar vessels increase at end inspiration because of radial traction and elastic recoil, and their PVR is lower at higher lung volume. During exhalation, the deflated alveoli apply the least resistance to the alveolar capillaries and their PVR is diminished, whereas the higher pleural pressure during exhalation increases the PVR of extra-alveolar vessels. As a result of these opposite effects of lung volume on PVR, total PVR in the lung is lowest at FRC.

• Fig. 23.5 Schematic Representation of the Effects of Changes in Vital Capacity on Total Pulmonary Vascular Resistance and the Contributions to the Total Afforded by Alveolar and Extra-Alveolar Vessels. During inflation from residual volume (RV) to total lung capacity (TLC), resistance to blood flow through alveolar vessels increases, whereas resistance through extra-alveolar vessels decreases. Thus changes in total pulmonary vascular resistance are plotted as a U-shaped curve during lung inflation, with the nadir at functional residual capacity (FRC).

Distribution of Pulmonary Blood Flow

Because the pulmonary circulation is a low-pressure/lowresistance system, it is influenced by gravity much more dramatically than is the systemic circulation. This gravitational effect contributes to an uneven distribution of blood flow in the lungs. In normal upright persons at rest, the volume of blood flow increases from the apex of the lung to the base of the lung, where it is greatest. Similarly, in a supine individual, blood flow is least in the uppermost (anterior) regions and greatest in the lower (posterior) regions. Under conditions of stress, such as exercise, the difference in blood flow in the apex and base of the lung in upright persons becomes less, mainly because of the increase in arterial pressure.

On leaving the pulmonary artery, blood must travel against gravity to the apex of the lung in upright people. For every 1-cm increase in location of a pulmonary artery segment above the heart, there is a corresponding decrease in hydrostatic pressure equal to 0.74 mm Hg. Thus the pressure in a pulmonary artery segment that is 10 cm above the heart is 7.4 mm Hg less than the pressure in a segment at the level of the heart. Conversely, a pulmonary artery segment 5 cm below the heart has a 3.7–mm Hg increase in pulmonary arterial pressure. This effect of gravity on blood flow affects arteries and veins equally and results in wide variations in arterial and venous pressure from the apex to • Fig. 23.6 Model to Explain the Uneven Distribution of Blood Flow in the Lung According to the Pressures Affecting the Capillaries.

PA, pulmonary alveolar pressure; Pa, pulmonary arterial pressure; Pv, pulmonary venous pressure. (From West JB, et al. J Appl Physiol. 1964;19:713.) the base of the lung. These variations influence both flow and ventilation/perfusion relationships.

In addition to the pulmonary arterial pressure (Pa) to pulmonary venous pressure (Pv) gradients, differences in pulmonary alveolar pressure (PA) also influence blood flow in the lung. Classically, the lung has been thought to be divided into three functional zones (

Fig. 23.6 ). Zone 1 represents the lung apex, where Pa is so low that it can be exceeded by PA. The capillaries collapse because of the greater external PA, and blood flow ceases. Under normal conditions, this zone does not exist; however, this state could be reached during positive-pressure mechanical ventilation or if Pa decreases sufficiently (such as might occur with a marked decrease in blood volume). In zone 2, or the upper third of the lung, Pa is greater than PA, which is in turn is greater than Pv. Because PA is greater than Pv, the greater external PA partially collapses the capillaries and causes a “damming” effect. This phenomenon is often referred to as the waterfall effect. In zone 3, Pa is greater than Pv, which is greater than PA, and blood flows in this area in accordance with the pressure gradients. Thus, pulmonary blood flow is greater in the base of the lung because the increased trans-mural pressure distends the vessels and lowers the resistance.

Active Regulation of Blood Flow

Blood flow in the lung is regulated primarily by the passive mechanisms described previously. There are, however, several active mechanisms that regulate blood flow. Although the smooth muscle around pulmonary vessels is much thinner than that around systemic vessels, it is sufficient to affect vessel caliber and thus PVR. Oxygen levels have a major effect on blood flow. Hypoxic vasoconstriction occurs in arterioles in response to decreased PAO2. The response is local, and the result is the shifting of blood flow from

CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships

AT THE CELLULAR LEVEL • BOX 23.1 Factors and Hormones That Regulate Pulmonary Blood Flow hypoxic areas to well-perfused areas in an effort to enhance gas exchange. Isolated, local hypoxia does not alter PVR; approximately 20% of the vessels must be hypoxic before a change in PVR can be measured. Low inspired O2 levels as a result of high altitude have a greater effect on PVR because all vessels are affected. High levels of inspired O2 can dilate pulmonary vessels and decrease PVR. Other factors and some hormones (

Box 23.1 ) can also influence vessel caliber, but their effects are usually local, brief, and important only in pathological conditions. Pulmonary capillaries lack smooth muscle and are thus not affected by these mechanisms. In some individuals, as a consequence of chronic hypoxia or collagen vascular disease, or for no apparent reason, pulmonary artery vascular resistance and subsequently pulmonary artery pressures rise (pulmonary artery hypertension).

Both ventilation (V̇ ) and lung perfusion (Q̇ ) are essential components of normal gas exchange, but a normal relationship between the two components is insufficient to ensure normal gas exchange. The ventilation/perfusion ratio (also referred to as the V̇ /Q̇ ratio) is defined as the ratio of ventilation to blood flow. This ratio can be defined for a single alveolus, for a group of alveoli, or for the entire lung. At the level of a single alveolus, the ratio is defined as alveolar ventilation per minute (V̇ A) divided by capillary flow (Q̇ c). At the level of the lung, the ratio is defined as total alveolar ventilation divided by cardiac output. In normal lungs, alveolar ventilation is approximately 4.0 L/min, whereas pulmonary blood flow is approximately 5.0 L/min. Thus

Endothelin-1 is an amino acid peptide that is produced by the vascular endothelium. Endothelin regulates the tone of pulmonary arteries, and increased expression of endothelin-1 has been found in individuals with pulmonary artery hypertension. Endothelin-1 also decreases endothelial expression of nitric oxide synthase, which reduces levels of nitric oxide, an endothelial vasodilator. Endothelin-1 antagonists (e.g., bosentan, sitaxentan) have been produced and are important drugs in the treatment of pulmonary arterial hypertension.

in a normal lung, the overall ventilation/perfusion ratio is approximately 0.8, but the range of V̇ /Q̇ ratios varies widely in different lung units. When ventilation exceeds perfusion, the ventilation/perfusion ratio is greater than 1 (V̇ /Q̇ > 1), and when perfusion exceeds ventilation, the ventilation/ perfusion ratio is less than 1 (V̇ /Q̇ < 1). Mismatching of pulmonary blood flow and ventilation results in impaired O2 and CO2 transfer. In individuals with cardiopulmonary disease, mismatching of pulmonary blood flow and alveolar ventilation is the most frequent cause of systemic arterial hypoxemia (reduced PaO2). In general, V̇ /Q̇ ratios greater than 1 are not associated with hypoxemia.

A normal ventilation/perfusion ratio does not mean that ventilation and perfusion of that lung unit are normal; it simply means that the relationship between ventilation and perfusion is normal. For example, in lobar pneumonia, ventilation to the affected lobe is decreased. If perfusion to this area remains unchanged, perfusion would exceed ventilation; that is, the ventilation/perfusion ratio would be less than 1 (V̇ /Q̇ < 1). However, the decrease in ventilation to this area produces hypoxic vasoconstriction in the pulmonary arterial bed supplying this lobe. This results in a decrease in perfusion to the affected area and a more “normal” ventilation/perfusion ratio. Nonetheless, neither the ventilation nor the perfusion to this area is normal (both are decreased), but the relationship between the two could approach the normal range.

The ventilation/perfusion ratio varies in different areas of the lung. In an upright individual, although both ventilation and perfusion increase from the apex to the base of the lung, the increase in ventilation is less than the increase in blood flow. As a result, the normal V̇ /Q̇ ratio at the apex of the lung is much greater than 1 (ventilation exceeds perfusion), whereas the V̇ /Q̇ ratio at the base of the lung is much less than 1 (perfusion exceeds ventilation). The relationship between ventilation and perfusion from the apex to the base of the lung is depicted in

Fig. 23.7 • Fig. 23.7 Ventilation/Perfusion Relationships in a Normal Lung in the Upright Position. Only the apical and basal values are shown for clarity. In each column, the number on top represents values at the apex of the lung, and the number on the bottom represents values at the base. PCO2, partial pressure of carbon dioxide; PN2, partial pressure of nitrogen; PO2, partial pressure of oxygen; Q̇ , perfusion per minute; VA, alveolar ventilation per minute.

PACO2 and PaCO2 are equal because of the solubility properties of CO2 (see ). The same is not true for alveolar and arterial O2. Even in individuals with normal lungs, PAO2 is slightly greater than PaO2. The difference between PAO2 and PaO2 is called the alveolar-arterial difference for oxygen (AaDO2). An increase in the AaDO2 is a hallmark of abnormal O2 exchange. This small difference in healthy individuals is not caused by “imperfect” gas exchange, but by the small number of veins that bypass the lung and empty directly into the arterial circulation. The thebesian vessels of the left ventricular myocardium drain directly into the left ventricle (rather than into the coronary sinus in the right atrium), and some bronchial and mediastinal veins drain into the pulmonary veins. This results in venous admixture and a decrease in PaO2. (This is an example of an anatomical shunt; see the section .”) Approximately 2% to 3% of the cardiac output is shunted in this way.

To measure the clinical effectiveness of gas exchange in the lung, PaO2 and PaCO2 are measured. PAO2 is calculated from the alveolar air equation (

Eq. 23.13 ). The difference between the calculated PAO2 and the measured PaO2 is the AaDO2. In individuals with normal lungs who are breathing room air, the AaDO2 is less than 15 mm Hg. The mean value rises approximately 3 mm Hg per decade of life after 30 years of age. Hence, an AaDO2 lower than 25 mm Hg is considered the upper limit of normal.

Assume that an individual with pneumonia is receiving 30% supplemental O2 by face mask. Arterial blood gas pH is 7.40, PaCO2 is 44 mm Hg, and PaO2 is 70 mm Hg. What is the patient’s AaDO2? (Assume that the patient is at sea level and the patient’s respiratory quotient is 0.8.) According to the alveolar air equation (

Eq. 23.13

R 44PAO2 = [0 3. × (760 − 47)] − 0 8. = 159 mm Hg

Therefore,

This high AaDO2 suggests that the patient has lung disease (in this case, pneumonia).

Abnormalities in PaO2 can occur with or without an elevation in AaDO2. Hence, the relationship between PaO2 and AaDO2 is useful in determining the cause of an abnormal PaO2 and in predicting the response to therapy (particularly to supplemental O2 administration). Causes of a reduction in PaO2 (arterial hypoxemia) and their effect on AaDO2 are listed in

Table 23.2 . Each of these causes is discussed in greater detail in the following sections.

Arterial Blood Hypoxemia, Hypoxia, and Hypercarbia

Arterial hypoxemia is defined as a PaO2 lower than 80 mm Hg in an adult who is breathing room air at sea level. Hypoxia is defined as insufficient O2 to carry out normal metabolic functions; hypoxia often occurs when the PaO2 is less than 60 mm Hg. There are four major categories of hypoxia. The first, hypoxic hypoxia, is the most common. The six main pulmonary conditions associated with hypoxic hypoxia—anatomical shunt, physiological shunt, decreased FiO2, V̇ /Q̇ mismatching, diffusion abnormalities, and hypoventilation—are described in the following sections and in

Table 23.2 . A second category is anemic hypoxia, which is caused by a decrease in the amount of functioning hemoglobin as a result of too little hemoglobin, abnormal hemoglobin, or interference with the chemical combination of oxygen and hemoglobin (e.g., carbon monoxide poisoning; see the following “

In the Clinic” box). The third category is hypoperfusion hypoxia, which results from low

CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships

AaDO2, alveolar-arterial difference for oxygen; FiO2, fraction of inspired oxygen; PaO2, partial pressure of arterial oxygen.

blood flow (e.g., decreased cardiac output) and reduced oxygen delivery to the tissues. Histotoxic hypoxia, the fourth category of hypoxia, occurs when the cellular machinery that uses oxygen to produce energy is poisoned, as in cyanide poisoning. In this situation, arterial and venous PO2 are normal or increased because oxygen is not being utilized.

Carbon monoxide can be generated from a malfunctioning space heater, from car exhaust, or from a burning building. Individuals exposed to carbon monoxide experience headache, nausea, and dizziness, and if it is not recognized, such individuals may die. They often have a cherry-red appearance, and oxygen saturation as measured with an oximeter is high (approaching 100%). Even on an arterial blood gas, the PAO2 may be normal. Nevertheless, the tissues are depleted of O2. Thus it is imperative that the clinician recognize a potential case of carbon monoxide poisoning and order an oxygen saturation measurement with the use of a carbon monoxide oximeter. If a patient has carbon monoxide poisoning, there will be a marked difference between the measurement of oxygen saturation by oximetry and that measured with a carbon monoxide oximeter.

A useful way to examine the relationship between ventila tion and perfusion is with the two–lung unit model (Fig.

23.8 ). Two alveoli are ventilated, each of which is supplied by blood from the heart. When ventilation is uniform, half the inspired gas goes to each alveolus, and when perfusion is uniform, half the cardiac output goes to each alveolus. In this normal unit, the ventilation/perfusion ratio in each of the alveoli is the same and is equal to 1. The alveoli are perfused by mixed venous blood that is deoxygenated and contains increased PaCO2. PAO2 is higher than mixed venous O2, and this provides a gradient for movement of O2 into blood. In contrast, mixed venous CO2 is greater than PACO2, and this provides a gradient for movement • Fig. 23.8 Simplified Lung Model of Two Normal Parallel Lung Units. Both units receive equal volumes of air and blood flow for their size. The blood and alveolar gas partial pressures are normal values in a resting person at sea level. PACO2, partial pressure of alveolar carbon dioxide; PAO2, partial pressure of alveolar oxygen; PiCO2, partial pressure of inspired carbon dioxide; PiO2, partial pressure of inspired oxygen; PpvCO2, partial pressure of carbon dioxide in portal venous blood; PpvO2, partial pressure of oxygen in portal venous blood; PvCO2, partial pressure of carbon dioxide in mixed venous blood; PvO2, partial pressure of oxygen in mixed venous blood.

of CO2 into the alveolus. Note that in this ideal model, alveolar-arterial O2 values do not differ.

An anatomical shunt occurs when mixed venous blood bypasses the gas-exchange unit and goes directly into the arterial circulation (

Fig. 23.9 ). Alveolar ventilation, the distribution of alveolar gas, and the composition of alveolar gas are normal, but the distribution of cardiac output is changed. Some of the cardiac output goes through the pulmonary capillary bed that supplies the gas-exchange units, but the rest of it bypasses the gas-exchange units and goes directly into the arterial circulation. The blood that bypasses the gas-exchange unit is thus shunted, and because the blood is deoxygenated, this type of bypass is called a right-to-left shunt. Most anatomical shunts develop within the heart, and they develop when deoxygenated blood from the right atrium or ventricle crosses the septum and mixes with blood from the left atrium or ventricle. The effect of this right-to-left shunt is to mix deoxygenated blood with oxygenated blood, and it results in varying degrees of arterial hypoxemia.

• Fig. 23.9 Right-to-Left Shunt. Alveolar ventilation is normal, but a portion of the cardiac output bypasses the lung and mixes with oxygenated blood. PaO2 varies according to the size of the shunt. PACO2, partial pressure of alveolar carbon dioxide; PAO2, partial pressure of alveolar oxygen; PiCO2, partial pressure of inspired carbon dioxide; PiO2, partial pressure of inspired oxygen; PpvCO2, partial pressure of carbon dioxide in portal venous blood; PpvO2, partial pressure of oxygen in portal venous blood; PvCO2, partial pressure of carbon dioxide in mixed venous blood; PvO2, partial pressure of oxygen in mixed venous blood.

An important feature of an anatomical shunt is that if an affected individual is given 100% O2 to breathe, the response is blunted severely. The blood that bypasses the gas-exchanging units is never exposed to the enriched O2, and thus it continues to be deoxygenated. The PO2 in the blood that is not being shunted increases and it mixes with the deoxygenated blood. Thus the degree of persistent hypoxemia in response to 100% O2 varies with the volume of the shunted blood. Normally, the hemoglobin in the blood that perfuses the ventilated alveoli is almost fully saturated. Therefore, most of the added O2 is in the form of dissolved O2 (see

The PaCO2 in an anatomical shunt is not usually increased even though the shunted blood has an elevated level of CO2. The reason for this is that the central chemoreceptors (see ) respond to any elevation in CO2 with an increase in ventilation and reduce PaCO2 to the normal range. If the hypoxemia is severe, the increased respiratory drive secondary to the hypoxemia increases the ventilation and can decrease PaCO2 to below the normal range.

A physiological shunt (also known as venous admixture) can develop when ventilation to lung units is absent in the presence of continuing perfusion (

Fig. 23.10 ). In this situation, in the two–lung unit model, all the ventilation goes to the other lung unit, whereas perfusion is equally distributed between both lung units. The lung unit without ventilation but with perfusion has a V̇ /Q̇ ratio of 0. The blood perfusing this unit is mixed venous blood; because • Fig. 23.10 Schema of a Physiological Shunt (Venous Admixture). Notice the marked decrease in PaO2 in comparison to PCO2. The alveolar-arterial difference for oxygen (AaDO2) in this example is 85 mm Hg. PACO2, partial pressure of alveolar carbon dioxide; PAO2, partial pressure of alveolar oxygen; PiCO2, partial pressure of inspired carbon dioxide; PiO2, partial pressure of inspired oxygen; PpvCO2, partial pressure of carbon dioxide in portal venous blood; PpvO2, partial pressure of oxygen in portal venous blood; PvCO2, partial pressure of carbon dioxide in mixed venous blood; PvO2, partial pressure of oxygen in mixed venous blood.

there is no ventilation, no gas is exchanged in the unit, and the blood leaving this unit continues to resemble mixed venous blood. The effect of a physiological shunt on oxygenation is similar to the effect of an anatomical shunt; that is, deoxygenated blood bypasses a gas-exchanging unit and admixes with arterial blood. Clinically, atelectasis (which is obstruction to ventilation of a gas-exchanging unit with subsequent loss of volume) is an example of a situation in which the lung region has a V̇ /Q̇ of 0. Causes of atelectasis include mucous plugs, airway edema, foreign bodies, and tumors in the airway.

Mismatching between ventilation and perfusion is the most frequent cause of arterial hypoxemia in individuals with respiratory disorders. In the most common example, the composition of mixed venous blood, total blood flow (cardiac output), and the distribution of blood flow are normal. However, when alveolar ventilation is distributed unevenly between the two gas-exchange units (

Fig. 23.11 ) and blood flow is equally distributed, the unit with decreased ventilation has a V̇ /Q̇ ratio of less than 1, whereas the unit with the increased ventilation has a V̇ /Q̇ of greater than 1. This causes the alveolar and end-capillary gas compositions to vary. Both the arterial O2 content and CO2 content are abnormal in the blood that has come from the unit with the decreased ventilation (V̇ /Q̇ , <1). The unit with the increased ventilation (V̇ /Q̇ , >1) has a lower CO2 content and a higher O2 content because it is being overventilated. The actual PaO2 and PaCO2 vary, depending on the relative contribution of each of these units to arterial blood. The alveolar-arterial O2 gradient (AaDO2) is increased because the relative overventilation of one unit does not fully

PlO2 = 150 PlCO2 = 0 PAO2 = 77 PACO2 = 45 PCO2= 45PO2= 77 PAO2 = 105 PACO2 = 36 PpvO2 = 89 PpvCO2 = 40.5 Pulmonary vein Pulmonary artery PvO2 = 40 PvCO2 = 46 – – Anatomic dead space • Fig. 23.11 Effects of Ventilation/Perfusion Mismatching on Gas Exchange. The decrease in ventilation to the one lung unit could be due to mucus obstruction, airway edema, bronchospasm, a foreign body, or a tumor. PACO2, partial pressure of alveolar carbon dioxide; PAO2, partial pressure of alveolar oxygen; PiCO2, partial pressure of inspired carbon dioxide; PiO2, partial pressure of inspired oxygen; PpvCO2, partial pressure of carbon dioxide in portal venous blood; PpvO2, partial pressure of oxygen in portal venous blood; PvCO2, partial pressure of carbon dioxide in mixed venous blood; PvO2, partial pressure of oxygen in mixed venous blood.

compensate (either by the addition of extra O2 or by the removal of extra CO2) for underventilation of the other unit. The failure to compensate is greater for O2 than for CO2, as indicated by the flatness of the upper part of the oxyhemoglobin dissociation curve, in contrast to the slope of the CO2 dissociation curve (see

Chapter 24).

In other words, increased ventilation increases PAO2, but it adds little extra O2 content to the blood because hemoglobin is close to being 100% saturated in the overventilated areas. This is not the case for CO2, for which the steeper slope of the CO2 curve indicates removal of more CO2 when ventilation increases. Thus inasmuch as CO2 moves by diffusion, then as long as a CO2 gradient is maintained, CO2 diffusion will occur.

The PAO2 is determined by a balance between the rate of O2 uptake and the rate of O2 replenishment by ventilation. Oxygen uptake depends on blood flow through the lung and the metabolic demands of the tissues. If ventilation decreases, PAO2 decreases, and PaO2 subsequently decreases. In addition, VA and PACO2 are directly but inversely related. When ventilation is halved, the PACO2 doubles and thus so does the PaCO2 (see

Eq. 23.16 ). Ventilation insufficient to maintain normal levels of CO2 is called hypoventilation. Hypoventilation always decreases PaO2 and increases PaCO2.

One of the hallmarks of hypoventilation is a normal AaDO2. Hypoventilation reduces PAO2, which in turn results in a decrease in PaO2. Because gas exchange is normal, the AaDO2 remains normal. Hypoventilation accompanies diseases associated with muscle weakness and is associated with drugs that reduce the respiratory drive. In the presence of hypoventilation, however, areas of atelectasis develop

Ventilation, Perfusion, and Ventilation/Perfusion Relationships rapidly; atelectasis creates regions with V̇ /Q̇ ratios of 0, and when this occurs, the AaDO2 rises.

Abnormalities in diffusion of O2 across the alveolar-capillary barrier could potentially result in arterial hypoxia. Equilibration between alveolar and capillary O2 and CO2 content occurs rapidly: in a fraction of the time that it takes for red blood cells to transit the pulmonary capillary network. Hence, diffusion equilibrium almost always occurs in normal people, even during exercise, when the transit time of red blood cells through the lung increases significantly. An increased AaDO2 attributable to incomplete diffusion (diffusion disequilibrium) has been observed in normal persons only during exercise at high altitude (≥10,000 feet). Even in individuals with an abnormal diffusion capacity, diffusion disequilibrium at rest is unusual but can occur during exercise and at altitude. Alveolar capillary block, or thickening of the air-blood barrier, is an uncommon cause of hypoxemia. Even when the alveolar wall is thickened, there is usually sufficient time for gas diffusion unless the red blood cell transit time is increased.

Mechanisms of Hypercapnia

Two major mechanisms account for the development of hypercapnia (elevated PCO2): hypoventilation and wasted, or increased, dead space ventilation. As noted previously, alveolar ventilation and alveolar CO2 are inversely related. When ventilation is halved, PACO2 and PaCO2 double. Hypoventilation always decreases PaO2 and increases PaCO2 and thereby results in a hypoxemia that responds to an enriched source of O2. Dead space ventilation is wasted, or increased, when pulmonary blood flow is interrupted in the presence of normal ventilation. This is most often caused by a pulmonary embolus that obstructs blood flow. The embolus halts blood flow to pulmonary areas with normal ventilation (V̇ /Q̇ =∞). In this situation, the ventilation is wasted because it fails to oxygenate any of the mixed venous blood. The ventilation to the perfused regions of the lung is less than ideal (i.e., there is relative “hypoventilation” to this area because in this situation, it receives all the pulmonary blood flow with “normal” ventilation). If compensation does not occur, PaCO2 increases and PaO2 decreases. Compensation after a pulmonary embolus, however, begins almost immediately; local bronchoconstriction occurs, and the distribution of ventilation shifts to the areas being perfused. As a result, changes in arterial CO2 and O2 content are minimized.

Effect of 100% Oxygen on Arterial Blood Gas Abnormalities

One of the ways that a right-to-left shunt can be distinguished from other causes of hypoxemia is for the individual to breathe 100% O2 through a non-rebreathing face mask for approximately 15 minutes. When the individual breathes 100% O2, all of the N2 in the alveolus is replaced by O2. Thus the PAO2, according to the alveolar air equation (

Eq.

23.13), is calculated as follows:

Equation 23.20

PAO2 =[1 0. × (Pb − PH2O)]− PaCO2 0 8. =[1 0 × (760 4 . − 47)]− 40 08

In a normal lung, the PAO2 rapidly increases, and it provides the gradient for transfer of O2 into capillary blood. This is associated with a marked increase in PaO2 (see 23.2 ). Similarly, over the 15-minute period of breathing enriched with O2, even areas with very low V̇ /Q̇ ratios develop high alveolar O2 pressure as the N2 is replaced by O2. In the presence of normal perfusion to these areas, there is a gradient for gas exchange, and the end-capillary blood is highly enriched with O2. In contrast, in the presence of a right-to-left shunt, oxygenation is not corrected because mixed venous blood continues to flow through the shunt and mix with blood that has perfused normal units. The poorly oxygenated blood from the shunt lowers the arterial O2 content and maintains the AaDO2. An elevated AaDO2 during a properly conducted study with 100% O2 signifies the presence of a shunt (anatomical or physiological); the magnitude of the AaDO2 can be used to quantify the proportion of the cardiac output that is being shunted.

The regional differences in ventilation and perfusion and the relationship between ventilation and perfusion were discussed earlier in this chapter. The effects of various physiological abnormalities (e.g., shunt, V̇ /Q̇ mismatch, and hypoventilation) on arterial O2 and CO2 levels were also described. In addition, however, it should be noted that because the V̇ /Q̇ ratio varies in different regions of the lung, the end-capillary blood coming from these regions has different O2 and CO2 levels. These differences are shown in

Fig. 23.7 , and they demonstrate the complexity of the lung. First, recall that the volume of the lung at the apex is less than the volume at the base. As previously described, ventilation and perfusion are less at the apex than at the base, but the differences in perfusion are greater than the differences in ventilation. Thus the V̇ /Q̇ ratio is high at the apex and low at the base. This difference in ventilation/perfusion ratios is associated with a difference in alveolar O2 and CO2 content between the apex and the base. The PAO2 is higher and the PACO2 is lower in the apex than in the base. This results in differences in end-capillary contents for these gases. End-capillary PO2 is lower, and, as a consequence, the O2 content is lower in end-capillary blood at the lung base than at the apex. In addition, there is significant variation in blood pH in the end capillaries in these regions because of the variation in CO2 content. During exercise, blood flow to the apex increases and becomes more uniform in the lung; as a result, the difference between the content of gases in the apex and in the base of the lung diminishes with exercise.

1.

The volume of air in the conducting airways is called the anatomical dead space. Dead space ventilation varies inversely with tidal volume. The total volume of gas in each breath that does not participate in gas exchange is called the physiological dead space. It includes the anatomical dead space and the dead space secondary to ventilated but unperfused alveoli.

2.

The sum of the partial pressures of a gas is equal to the total pressure. The partial pressure of a gas (Pgas) is equal to the fraction of the gas in the gas mixture (Fgas) multiplied by the total pressure (Ptotal). The conducting airways do not participate in gas exchange. Therefore, the partial pressures of O2, N2, and water vapor in humidified air remain unchanged in the airways until the gas reaches the alveolus.

3.

The partial pressure of O2 in the alveolus is given by the alveolar air equation (

Eq. 23.13 ). This equation is used to calculate the AaDO2, a useful measurement of abnormal arterial O2.

4.

The relationship between CO2 production and alveolar ventilation is defined by the alveolar carbon

Eq. 23.14 ). There is an inverse relationship between the PACO2 and VA, regardless of the exhaled quantity of CO2. In normal lungs, PaCO2 is tightly regulated to remain constant at around 40 mm Hg.

5. Because of the effects of gravity, there are regional differences in ventilation and perfusion. The ventilation/perfusion (V̇ /Q̇ ) ratio is defined as the ratio of ventilation to blood flow. In a normal lung, the overall ventilation/perfusion ratio is approximately 0.8. When ventilation exceeds perfusion, the ventilation/perfusion ratio is greater than 1 (V̇ /Q̇ > 1), and when perfusion exceeds ventilation, the ventilation/perfusion ratio is less than 1 (V̇ /Q̇ < 1). The V̇ /Q̇ ratio at the apex of the lung is high (ventilation is increased in relation to very little blood flow), whereas the V̇ /Q̇ ratio at the base of the lung is low. In individuals with normal lungs who are breathing room air, the AaDO2 is less than 15 mm Hg; the upper limit of normal is 25 mm Hg.

CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships 6. The pulmonary circulation is a low-pressure, low-resistance system. Recruitment of new capillaries and dilation of arterioles without an increase in pressure are unique features of the lung and allow for adjustments during stress, as in the case of exercise. Pulmonary vascular resistance is the change in pressure from the pulmonary artery (PPA) to the left atrium (PLA), divided by cardiac output (QT). This resistance is about 10 times less than in the systemic circulation.

Leff AR, Schumacker PT. Respiratory Physiology: Basics and Applications. Philadelphia: WB Saunders; 1993.

Lumb AB. Nunn’s Applied Respiratory Physiology. 8th ed. St. Louis: Elsevier; 2016.

Mead J, Macklem PT, vol eds. American Physiological Society Handbook of Physiology: The Respiratory System. Vol. 3. Mechanics. Bethesda, MD: American Physiological Society; 1986.

7.

There are four categories of hypoxia (hypoxic hypoxia, anemic hypoxia, diffusion hypoxia, and histotoxic hypoxia) and six mechanisms of hypoxic hypoxia and hypoxemia: anatomical shunt, physiological shunt, decreased FiO2, V̇ /Q̇ mismatching, diffusion abnormalities, and hypoventilation.

8.

There are two mechanisms of the development of hypercapnia: increase in dead space ventilation and hypoventilation.

Wasserman K, Beaver WL, Whipp BI. Gas exchange theory and the lactic acidosis (anaerobic) threshold. Circulation. 1990;81(1 suppl):1114-1130.

West JB. Ventilation/Blood Flow and Gas Exchange. 5th ed. New York: Blackwell Scientific; 1991.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

Describe the basic gas diffusion principles and how they affect O2 and CO2 absorption and expiration.

2.

Compare and contrast the chemical transport mechanisms of O2 and CO2 in blood.

3.

Describe the basic principles and clinical significances of the O2 and CO2 dissociation curves.

4.

Describe the chemical synthesis of H+ ions and their role in the regulation of acid-base balance.

5.

Explain why the diffusion of O2 and that of CO2 are considered perfusion limited and that of CO is considered diffusion limited.

6.

Compare and contrast factors that shift the oxyhemoglobin dissociation curve.

7.

Explain the clinical significance of the differences in the oxyhemoglobin and carboxyhemoglobin dissociation curves.

8.

Compare and contrast the chloride shift and the Haldane effect on CO2 transport.

he respiratory and circulatory systems function together to transport oxygen (O2) from the lungs to the tissues to sustain normal cellular activity and to transport carbon dioxide (CO2) from the tissues to the lungs for expiration. CO2, a product of active cellular metabolism, is transported from the tissues via systemic veins to the lungs, where it is expired (Fig. 24.1 ). To enhance uptake and transport of these gases between the lungs and tissues, specialized mechanisms (e.g., binding of O2 and hemoglobin and HCO3 − transport of CO2) have evolved that enable O2 uptake and CO2 expiration to occur simultaneously. Moreover, these specialized mechanisms facilitate uptake of O2 and expiration of CO2. To understand the mechanisms involved in the transport of these gases, gas diffusion properties, as well as transport and delivery mechanisms, must be considered.

Gas movement throughout the respiratory system occurs predominantly via diffusion. The respiratory and circulatory systems contain several unique anatomical and physiological features to facilitate gas diffusion: (1) large surface areas for gas exchange (alveolar to capillary and capillary to tissue membrane) with short distances to travel, (2) substantial partial pressure gradient differences, and (3) gases with advantageous diffusion properties. Transport and delivery of O2 from the lungs to the tissue and vice versa for CO2 are dependent on basic gas diffusion laws.

Diffusion of Gases From Regions of Higher to Lower Partial Pressure in the Lungs

The process of gas diffusion is passive and similar whether diffusion occurs in a gaseous or liquid state. The rate of diffusion of a gas through a liquid is described by Graham’s law, which states that the rate is directly proportional to the solubility coefficient of the gas and inversely proportional to the square root of its molecular weight. Calculation of the diffusion properties for O2 and CO2 reveals that CO2 diffuses approximately 20 times faster than O2. Rates of O2 diffusion from the lungs into blood and from blood into tissue, and vice versa for CO2, are predicted by Fick’s law of gas diffusion (

Fig. 24.2). The ratio AD:T represents the con ductance of a gas from the alveolus to blood. The diffusing capacity of the lung (DL) is its conductance (A•D/T) when considered for the entire lung; thus, with Fick’s equation, DL can be calculated as follows:

Equation 24.1 where V̇ gas = gas diffusion.

Fick’s law of diffusion could be used to assess the diffusion properties of O2 in the lungs, except that the capillary partial pressure of oxygen cannot be measured. This limitation can be overcome with the use of carbon monoxide (CO) rather than O2. Because CO has low solubility in the capillary membrane, the rate of CO equilibrium across the capillary is slow, and the partial pressure of CO in capillary blood remains close to 0. In contrast, the solubility of CO in blood is high. Thus the only limitation for diffusion of CO is the alveolar-capillary membrane, and thus CO is a •Fig. 24.1 Oxygen (O2) and Carbon Dioxide (CO2) Transport in Arterial and Venous Blood. Oxygeninarterialbloodistransferredfromarterialcapillariestotissues.TheflowratesforO2andCO2areshownfor1Lofblood.

•Fig. 24.2 AccordingtoFick’slaw,diffusionofagasacrossasheetoftissue(Vgas)isdirectlyrelatedtothesurfacearea(A)ofthetissue, thediffusionconstant(D)ofthespecificgas,andthepartialpressuredifference(P1− P2)ofthegasoneachsideofthetissue,anditisinverselyrelatedtotissuethickness(T).

useful gas for calculating DL. The capillary partial pressure (P2 in

Eq. 24.1 ) is essentially 0 for CO, and therefore DL can be measured from the diffusion of carbon monoxide (V̇ CO) and the average partial pressure of CO in the alveolus; that is,

Equation 24.2 where DLCO = diffusion capacity of the lung for carbon monoxide.

Assessment of DLCO has become a classic measurement of the diffusion barrier of the alveolar-capillary membrane. It is useful in the differential diagnosis of certain obstructive lung diseases, such as emphysema.

Apatientwithinterstitialpulmonaryfibrosis(arestrictivelungdisease)inhalesasinglebreathof0.3%COfromresidualvolumetototallungcapacity.Heholdshisbreathfor10secondsandthenexhales.Afterdiscardingtheexhaledgasfromthedeadspace,arepresentativesampleofalveolargasfromlateinexhalationiscollected.TheaveragealveolarCOpressureis0.1mmHg,and0.25mLofCOhasbeentakenup.ThediffusioncapacityforCOinthispatientis . mL/10 seconds × 0 1. mm Hg = 15 mL/minute/mm Hg

ThenormalrangeforDLCOis20to30mL/minute/mmHg.Patientswithinterstitialpulmonaryfibrosishaveaninitialalveolarinflammatoryresponsewithsubsequentscarformationwithintheinterstitialspace.Theinflammationandscarreplacethealveolianddecreasethesurfaceareaforgasdiffusiontooccur,whichresultsindecreasedDLCO.Thisisaclassiccharacteristicofcertaintypesofrestrictivelungdisease.

Oxygen and Carbon Dioxide Exchange in the Lung Is Perfusion Limited

Different gases have different solubility factors. Gases that are insoluble in blood (i.e., anesthetic gases such as nitrous oxide and ether) do not chemically combine with proteins in blood and equilibrate rapidly between alveolar gas and blood. The equilibration occurs in less time than the 0.75 seconds that the red blood cell spends in the capillary bed (capillary transit time). The diffusion of insoluble gases between alveolar gas and blood is considered perfusion limited because the partial pressure of gas in the blood leaving the capillary has reached equilibrium with alveolar gas and is limited only by the amount of blood perfusing the alveolus. In contrast, a gas that is diffusion limited, such as CO, has low solubility in the alveolar-capillary membrane but high solubility in blood because of its high affinity for hemoglobin (Hgb). These features prevent the equilibration of CO between alveolar gas and blood during the red blood cell transit time.

The high affinity of CO for Hgb enables large amounts of CO to be taken up in blood with little or no appreciable increase in its partial pressure. Gases that are chemically bound to Hgb do not exert a partial pressure in blood. Like CO, both CO2 and O2 have relatively low solubility in the alveolar-capillary membrane but high solubility in blood because of their ability to bind to Hgb. However, their rate of equilibration is sufficiently rapid for complete equilibration to occur during the transit time of the red blood cell within the capillary. Equilibration for O2 and CO2 usually occurs within 0.25 seconds. Thus O2 and CO2 transfer is normally perfusion limited. The partial pressure of a gas that is diffusion limited (i.e., CO) does not reach equilibrium with the alveolar pressure over the time that it spends in the capillary (

Fig. 24.3 ). Although CO2 has a greater rate of diffusion in blood than O2 does, it has a lower membrane-blood solubility ratio and consequently takes approximately the same amount of time to reach equilibration in blood.

Start of

End of 0 .25 .50 .75 Time in capillary (sec) •Fig. 24.3 Uptake of Nitrous Oxide (N2O), Carbon Monoxide (CO), and O2in Blood in Relation to Their Partial Pressures and the Transit Time of the Red Blood Cell in the Capillary. Forgasesthatareperfusionlimited(N2OandO2),theirpartialpressureshaveequilibratedwithalveolarpressurebeforeexitingthecapillary.Incontrast,thepartialpressureofCO,agasthatisdiffusionlimited,doesnotreachequilibriumwithalveolarpressure.Inrareconditions,O2uptakecanbecomediffusionlimited.

Diffusion limitation for O2 and CO2 would occur if red blood cells spent less than 0.25 seconds in the capillary bed. This is occasionally the case in very fit athletes during vigorous exercise and in healthy subjects who exercise at high altitude.

Oxygen is carried in blood in two forms: dissolved O2 and O2 bound to Hgb. The dissolved form is measured clinically in an arterial blood gas sample as the partial pressure of arterial oxygen (PaO2). Only a small percentage of O2 in blood is in the dissolved form, and its contribution to O2 transport under normal conditions is almost negligible. However, dissolved O2 can become a significant factor in conditions of severe hypoxemia. Binding of O2 to Hgb to form oxyhemoglobin within red blood cells is the primary transport mechanism of O2. Hgb not bound to O2 is referred to as deoxyhemoglobin or reduced Hgb. The O2-carrying capacity of blood is enhanced about 65 times by its ability to bind to Hgb.

Hgb is the major transport molecule for O2. The Hgb molecule is a protein with two major components: four nonprotein heme groups, each containing iron in the reduced ferric (Fe+++) form, which is the site of O2 binding, and a globin portion consisting of four polypeptide chains. Normal adults have two α-globin chains and two β-globin chains (HgbA), whereas children younger than 6 months of age have predominantly fetal Hgb (HgbF), which consists of two α chains and two γ chains. This difference in the structure of HgbF increases its affinity for O2 and aids in the transport of O2 across the placenta. In addition, HgbF is not inhibited by 2,3-diphosphoglycerate (2,3DPG), a product of glycolysis; thus O2 uptake is further enhanced.

Binding of O2 to Hgb alters the ability of Hgb to absorb light. This effect of O2 on Hgb is responsible for the change in color between oxygenated arterial blood (bright red) and deoxygenated venous blood (dark red-bluish). Binding and dissociation of O2 with Hgb occur in milliseconds, thus facilitating O2 transport because red blood cells spend only 0.75 seconds in the capillaries. There are approximately 280 million Hgb molecules per red blood cell, which provides an efficient mechanism to transport O2. Myoglobin, a protein similar in structure and function to Hgb, has only one subunit of the Hgb molecule. It aids in the transfer of O2 from blood to muscle cells and in the storage of O2, which is especially critical in O2-deprived conditions.

Abnormalities of the Hgb molecule occur with mutations in the amino acid sequence (i.e., sickle cell disease) or in the spatial arrangement of the globin polypeptide chains and result in abnormal function. Compounds such as CO, nitrites (nitric oxide), and cyanides can oxidize the •Fig. 24.4 Oxyhemoglobin Dissociation Curve Showing the Relationship Between the Partial Pressure of Oxygen (Po2) in Blood and the Percentage of Hemoglobin (Hgb) Binding Sites That Are Occupied by Oxygen Molecules (Percentage Saturation). Adulthemoglobin(HgbA)isabout50%saturatedwithoxygenataPO2of27mmHg,90%saturatedat60mmHg,andabout98%saturatedat100mmHg.TheP50isthepartialpressureatwhichHgbis50%saturatedwithO2.WhentheO2dissociationcurveshiftstotheright,P50increases.Whenthecurveshiftstotheleft,P50decreases.

iron molecule in the heme group and change it from the reduced ferrous state (Fe++) to the ferric state (Fe+++), which reduces the ability of O2 to bind to Hgb.

In the alveoli, the majority of O2 in plasma quickly diffuses into red blood cells and chemically binds to Hgb. This process is reversible, so that Hgb quickly gives up its O2 to tissue through passive diffusion (the concentration of O2 in Hgb decreases). The oxyhemoglobin dissociation curve illustrates the relationship between PO2 in blood and the number of O2 molecules bound to

Hgb (Fig. 24.4 ). The S shape of the curve demonstrates the dependence of Hgb saturation on PO2, especially at partial pressures lower than 60 mm Hg. The clinical significance of the flat portion of the oxyhemoglobin dissociation curve (>60 mm Hg) is that a drop in PO2 over a wide range of partial pressures (100 to 60 mm Hg) has a minimal effect on Hgb saturation, which remains at 90% to 100%, a level sufficient for normal O2 transport and delivery. The clinical significance of the steep portion (<60 mm Hg) of the curve is that a large amount of O2 is released from Hgb with only a small change in PO2, which facilitates the release and diffusion of O2 into tissue. The point on the curve at which Hgb is 50% saturated with O2 is called the P50, and it is 27 mm Hg in normal adults.

˜˜˜˜°°°°Temperature PCO2 2,3-DPG pH Decreased P50 (increased affinity) Temperature PCO2 2,3-DPG pH Increased P50 (decreased affinity) •Fig. 24.5 Factorsthatshifttheoxyhemoglobindissociationcurvetotheright(decreasedaffinityofHgbforO2)ortotheleft(increasedaffinity).2,3-DPG,2,3-diphosphoglycerate;PCO2,partialpressureofcarbondioxide.

Physiological Factors That Shift the Oxyhemoglobin Dissociation Curve

The oxyhemoglobin dissociation curve can shift in numerous clinical conditions, either to the right or to the left (

Fig.

24.5 ). The curve is shifted to the right when the affinity of

Intheinheritedhomozygousconditionknownassickle cell disease, affectedindividualshaveanaminoacidsubstitution(valineforglutamicacid)ontheβ chainoftheHgbmolecule.ThiscreatesasicklecellHgb(HgbS),which,whennotboundtooxygen(deoxyhemoglobinordesaturatedHgb),cantransformintoastiffgelatinousmaterialthatdistortsthenormalbiconcaveshapeoftheredbloodcelltoacrescent,orsickle-shaped,form.Thischangeinappearancefromsphericaltoasickleshapeincreasesthetendencyoftheredbloodcelltoformthrombiorclotsthatobstructsmallvesselsandcreatesaclinicalconditionknownasacute sickle cell episode. Thesymptomsofsuchanepisodevary,dependingonthesiteoftheobstruction(e.g.,inthebrain,stroke;inthelungs,pulmonaryinfarction)andarecommonlyassociatedwithintensepain.Thespleenisacommonsiteofobstruction/infarction,andtheensuingtissuedamagecompromisestheimmunecapabilitiesofaffectedindividualsandrendersthemsusceptibletorecurrentinfections.Inthehomozygousform,thisconditionislife-shortening;however,individualswiththeheterozygousformareresistanttomalaria.Thusanindividualwithheterozygousalleleshasasurvivaladvantageinregionsoftheworldwheremalariaisprevalent,whichmayexplainwhythesicklecellmutationhasbeenpreservedthroughevolution.TheincreasedaffinityofHgbFforO2confersadvantagestoindividualswithsicklecelldiseaseinthatthecellsdonotdesaturateasmuchwhenO2isreleasedfromHgbtothetissueandthusarelesslikelytobecomedeformedinthesickleshape.SicklecelldiseaseismostprevalentamongindividualsofAfricanAmericandescent,butitisalsoobservedinHispanic,Turkish,Asian,andotherethnicgroups.

Hgb for O2 decreases, which enhances O2 dissociation. This results in decreased Hgb binding to O2 at a given PO2, which causes the P50 to increase. When the affinity of Hgb for O2 increases, the curve is shifted to the left, which causes the P50 to decrease. In this state, O2 dissociation and delivery to tissue are inhibited. Shifts to the right or left of the dissociation curve have little effect when they occur at O2 partial pressures within the normal range (80 to 100 mm Hg). However, at O2 partial pressures below 60 mm Hg (steep part of the curve), shifts in the oxyhemoglobin dissociation curve can dramatically influence O2 transport.

Changes in blood hydrogen ion concentration (pH) shift the oxyhemoglobin dissociation curve. An increase in CO2 production by tissue and its release into blood results in the generation of hydrogen ions (H+) and a decrease in pH. This shifts the dissociation curve to the right, which has a beneficial effect by aiding in the release of O2 from Hgb for diffusion into tissues. The shift to the right in the dissociation curve is due to the decrease in pH and to a direct effect of CO2 on Hgb. Conversely, as blood passes through the lungs, CO2 is exhaled, which results in an increase in pH, which in turn causes the oxyhemoglobin dissociation curve to shift to the left. This effect of CO2 on the affinity of Hgb for O2 is known as the Bohr effect, and it serves to enhance O2 uptake in the lungs and delivery of O2 to tissues. An increase in body temperature, as occurs during exercise, shifts the oxyhemoglobin dissociation curve to the right and enables more O2 to be released to tissues, where it is needed because the demand increases. During cold weather, a decrease in body temperature, especially in the extremities (lips, fingers, toes, and ears), shifts the O2 dissociation curve to the left (higher Hgb affinity). In this instance, PaO2 may be normal, but release of O2 in these extremities is not facilitated.

2,3-Diphosphoglycerate

Mature red blood cells do not have mitochondria, and therefore they depend on anaerobic glycolysis. Large quantities of a metabolic intermediary, 2,3-DPG, are formed in red blood cells during glycolysis, and the affinity of Hgb for O2 decreases as 2,3-DPG levels increase. Thus the oxyhemoglobin dissociation curve shifts to the right. Although the binding sites of 2,3-DPG and O2 differ on the Hgb molecule, binding of 2,3-DPG creates an allosteric effect that inhibits the binding of O2. Conditions that increase 2,3-DPG include hypoxia, decreased Hgb, and increased pH. Decreased levels of 2,3-DPG are observed in stored blood samples and thus may present a problem to transfusion recipients because of the greater affinity of Hgb for O2, which inhibits the unloading of O2 to tissues.

As discussed previously, HgbF has a greater affinity for O2 than does adult Hgb, and the oxyhemoglobin dissociation curve thus shifts to the left.

•Fig. 24.6 Theoxyhemoglobinandcarboxyhemoglobindissociationcurvesclearlyillustratetheincreasedaffinitythatcarbonmonoxide(CO)hasforHgb,incomparisonwithO2.100 80 60 40 20 0 0 20 40 60 PO2 (mm Hg) Hemoglobin saturation (%) 80 100 O2 + hemoglobin CO + hemoglobin

CO binds to the heme group of the Hgb molecule at the same site as O2 and forms carboxyhemoglobin (HgbCO). A major difference between the ability of CO and that of O2 to bind to Hgb is illustrated by a comparison of the oxyhemoglobin and carboxyhemoglobin dissociation curves. The affinity of CO for Hgb is about 200 times greater than it is for O2 (

Fig. 24.6 ). Thus small amounts of CO can greatly influence the binding of O2 to Hgb. In the presence of CO, the affinity of Hgb for O2 is enhanced. This causes the dissociation curve to shift to the left, which further prevents the unloading and delivery of O2 to tissues. As the PCO of blood approaches 1.0 mm Hg, all of the Hgb binding sites are occupied by CO, and Hgb is unable to bind to O2. This situation is not compatible with life and is the cause of death in cases of CO poisoning. In healthy individuals, HgbCO occupies about 1% to 2% of the Hgb binding sites; however, in cigarette smokers and in individuals who reside in high-density urban traffic areas, occupation of Hgb binding sites can be increased to 10%. Levels above 5% to 7% are considered hazardous. Treatment of individuals with high levels of CO, such as those who have inhaled car exhaust or smoke from a burning building, consists of administering high concentrations of O2 to displace CO from Hgb. Increasing the ambient pressure above atmospheric pressure, through the use of a barometric chamber, substantially increases the O2 tension, which promotes the dissociation of CO from Hgb. Another gas, nitric oxide, has great affinity (200,000 times greater than O2) for Hgb, and it binds irreversibly to Hgb at the same site that O2 does. Endothelial cells synthesize nitric oxide, which has vasodilation properties. Thus nitric oxide is used therapeutically as an inhalant in patients with pulmonary hypertension to reduce pressure. Although nitric oxide poisoning is not common, the clinician should be cautious when administering nitric oxide therapy for long periods. Hgb-bound CO and nitric oxide is referred to as methemoglobin. Under normal conditions, about 1% to 2% of Hgb is bound to CO and nitric oxide.

Oxygen Saturation, Content, and Delivery

Each Hgb molecule can bind up to four O2 atoms, and each gram of Hgb can bind up to 1.34 mL of O2. The term O2 saturation (SO2) refers to the amount of O2 bound to Hgb in relation to the maximal amount of O2 (100% O2 capacity) that can bind Hgb. At 100% O2 capacity, the heme groups of the Hgb molecules are fully saturated with O2, and at 75% O2 capacity, three of the four heme groups are occupied. Binding of O2 to each heme group increases the affinity of the Hgb molecule to bind additional O2. The O2 content in blood is the sum of the O2 bound to Hgb and the dissolved O2. Oxygen content is decreased in the presence of increased CO2 and CO and in individuals with anemia (

Fig. 24.7

Oxygen delivery from the lungs to tissues is dependent on several factors, including cardiac output, the Hgb content of blood, and the ability of the lung to oxygenate the blood. Not all of the O2 carried in blood is unloaded at the tissue level. The actual O2 extracted from blood by the tissue is the difference between the arterial O2 content and the venous O2 content, multiplied by cardiac output. Under normal conditions, Hgb leaves the tissue 75% saturated with O2, and only about 25% is actually used by tissues. Hypothermia, relaxation of skeletal muscles, and an increase in cardiac output reduce O2 extraction. Conversely, a decrease in cardiac output, anemia, hyperthermia, and exercise increase O2 extraction.

•Fig. 24.7 AcomparisonofO2contentcurvesunderthreeconditionsshowswhycarboxyhemoglobin(HgbCO)dramaticallyreducestheO2transportsystem.FiftypercentHgbCOrepresentsthebindingofhalfthecirculatingHgbwithcarbonmonoxide(CO).The50%hemoglobinand50%HgbCOcurvesshowthesamedecreasedO2contentinarterialblood.However,COhasaprofoundeffectinloweringvenouspartialpressureofoxygen.Thearterial(a)andmixedvenous(v)pointsofconstantcardiacoutputareindicated.

Tissue hypoxia is a condition in which O2 available to cells is insufficient for maintaining adequate aerobic metabolism. Thus anaerobic metabolism is stimulated and results in the increases in levels of lactate and H+ and the subsequent formation of lactic acid. The net result can lead to a significant decrease in blood pH. In cases of severe hypoxia, the extremities, toes, and fingertips may appear blue-gray (cyanotic) because of lack of O2 and increased deoxyhemoglobin levels. There are four major types of tissue hypoxia (hypoxic hypoxia, circulatory hypoxia, anemic hypoxia, histotoxic hypoxia), discussed in detail in

Chapter 23.

Tissue oxygenation depends on the concentration of Hgb and thus on the number of red blood cells available in the circulation. Red blood cell production (erythropoiesis) in the bone marrow is controlled by the hormone erythropoietin, which is synthesized in the kidneys by cortical interstitial cells. Although Hgb levels are normally very stable, decreased O2 delivery, low Hgb concentration, and low PaO2 stimulate the secretion of erythropoietin. This increases the production of red blood cells. Chronic renal disease damages the cortical interstitial cells and thereby suppresses their ability to synthesize erythropoietin. This causes anemia, along with decreased Hgb because of the lack of erythropoietin. Erythropoietin replacement therapy using epoetin alfa (Epogen, Procrit) or darbepoetin alfa (Aranesp) effectively increases red blood cell production.

CO2 is produced at a rate of approximately 200 mL/minute under healthy conditions, and typically, 80 molecules of CO2 are expired by the lung for every 100 molecules of O2 that enter the capillary bed. The ratio of expired CO2 to O2 uptake is referred to as the respiratory exchange ratio and, under normal conditions, is 0.8 (80 molecules of CO2 to 100 molecules of O2). This ratio is similar at the tissue to blood compartment, where it is referred to as the respiratory quotient.

The body has enhanced storage capabilities for CO2, in comparison with O2, and hence PaO2 is much more sensitive to changes in ventilation than is PaCO2. Whereas PaO2 is dependent on several factors, in addition to alveolar ventilation, arterial PaCO2 is solely dependent on alveolar ventilation and CO2 production. There is an inverse relationship between alveolar ventilation and PaCO2.

In blood, CO2 is transported in red blood cells primarily as bicarbonate (HCO3 −) but also as dissolved CO2 and as

H2CO3 = PCO2 ˜ 0.0301 •Fig. 24.8 Mechanisms of CO2Transport in Blood. v. ThepredominantmechanismbywhichCO2istransportedfromtissuecellstothelungisintheformofbicarbonateanion(HCO3−).H2CO3,carbonicacid;HgbO,oxyhemoglobin;NaHCO3,sodiumbicarbonate;RBC,redbloodcell.

carbamino protein complexes (i.e., CO2 binds to plasma proteins and to Hgb;

Fig. 24.8 ). Once CO2 diffuses through the tissue and enters plasma, it quickly dissolves. The reaction of CO2 with H2O to form carbonic acid (H2CO3) is the major mechanism for the generation of HCO3 − in red blood cells:

Equation 24.3

The reaction normally proceeds quite slowly; however, it is catalyzed within red blood cells by the enzyme carbonic anhydrase. The HCO3 − diffuses out of the red blood cell in exchange for Cl−, in a process known as the chloride shift, which helps the cell maintain its osmotic equilibrium. This chemical reaction (see

Fig. 24.8 ) is reversible and can be shifted to the right to generate more HCO3 − in response to more CO2 entering the blood from tissues, or it can be shifted to the left as CO2 is exhaled in the lungs, which reduces HCO3 − levels. The free H+ is quickly buffered within the red blood cell by binding to Hgb. Buffering of H+ is critical for keeping the reaction moving toward the synthesis of HCO3; high levels of free H+ (low pH) cause the reaction to shift to the left.

Regulation of Hydrogen Ion Concentration and Acid-Base Balance

The H+ concentration (pH) has a dramatic effect on many metabolic processes within cells, and regulation of pH is essential for normal homeostasis. In the clinical setting, blood pH is measured to assess the concentration of H+ . The normal pH range for adults is 7.35 to 7.45 and is maintained by the lungs, kidneys, and chemical buffer systems. In the respiratory system, conversion of CO2 to HCO3 − , illustrated as follows, is a major mechanism of buffering and regulating the H+ concentration (pH):

Equation 24.4

As PACO2 changes, so does the concentration of HCO3 − and H2CO3, as well as PaCO2. The Henderson-Hasselbalch equation is used to calculate how changes in CO2 and HCO3 − affect pH:

Equation 24.5 where α= solubility. Thus

Equation 24.6 log[HCO3 − ]pH = 6 1. + 0 03 × PCO2

In these equations, the amount of CO2 is determined from the PCO2 and its solubility (α) in solution. For plasma at 37°C, α has a value of 0.03. Also, pK′ is the negative logarithm of the overall dissociation constant for the reaction and has a logarithmic value of 6.1 for plasma at 37°C.

Acute hyperventilation secondary to exercise or anxiety reduces PCO2 and thereby causes an increase in pH (respiratory alkalosis). Conversely, if PCO2 increases because of hypoventilation secondary to an overdose of a respiratory depressant, the pH decreases (respiratory acidosis). Acid-base imbalances are also caused by metabolic disorders such as metabolic acidosis (e.g., lactic acidosis, ketoacidosis, and renal failure) and metabolic alkalosis (e.g., hypokalemia, hypochloremia, vomiting, high doses of steroids).

In contrast to O2, the dissociation curve for CO2 in blood is linear and directly related to PCO2 (

Fig. 24.9). The degree of Hgb saturation with O2 has a major effect on the CO2 dissociation curve. Although O2 and CO2 bind to Hgb 1.

Gases (nitrous oxide, ether, helium) that have a rapid rate of air-to-blood equilibration are perfusion limited. Gases (CO) that have a slow air-to-blood equilibration rate are diffusion limited. Under normal conditions, O2 and CO2 exchange are perfusion limited but can be diffusion limited in some situations.

2.

The major transport mechanism of O2 in blood is within the red blood cell bound to Hgb, and for CO2, it is within red blood cells in the form of HCO3 − .

3.

The reversible reaction of CO2 with H2O to form H2CO3, with its subsequent dissociation to HCO3 − and H+ , is catalyzed by the enzyme carbonic anhydrase within red blood cells and is the major mechanism for generation of HCO3 − .

4.

The O2 dissociation curve is S shaped. In the plateau area (>60 mm Hg), increasing or decreasing PO2 has only a minimal effect on Hgb saturation from 100% •Fig. 24.9 Equilibrium Curves for CO2in Arterial and Venous Blood. VenousbloodcantransportmoreCO2thanarterialbloodcanatanygivenPCO2.IncomparisonwiththeHgbO2equilibriumcurve,theCO2curvesareessentiallystraightlinesbetweenaPCO2of20andaPCO2of80mmHg.Long dashes representthearterialequilibriumcurve;short dashes representthevenousequilibriumcurve.

at different sites, deoxygenated Hgb has greater affinity for CO2 than oxygenated Hgb. Thus deoxygenated blood (venous blood) freely takes up and transports more CO2 than oxygenated arterial blood does. The deoxygenated Hgb more readily forms carbamino compounds and also more readily binds free H+ released during the formation of HCO3 − . The effect of changes in oxyhemoglobin saturation on the relationship of CO2 content to PCO2 is referred to as the Haldane effect and is reversed in the lungs when O2 is transported from the alveoli to red blood cells. This effect is illustrated by a shift to the left in the CO2 dissociation curve in venous blood in comparison with arterial blood.

to 90%. This ensures adequate Hgb saturation over a large range of PO2 values.

5.

The CO2 dissociation curve is linear and directly related to PCO2. PCO2 is solely dependent on alveolar ventilation and CO2 production.

6.

The CO2 to HCO3 − pathway plays a critical role in the regulation of H+ ions and in maintaining acid-base balance in the body.

7.

Tissue oxygenation is dependent on Hgb within red blood cells and subsequently the number (and production) of red blood cells, which is controlled by the hormone erythropoietin. Low O2 delivery, low Hgb concentration, and low PaO2 stimulate the secretion of erythropoietin in the kidneys.

8.

Tissue hypoxia occurs when insufficient amounts of O2 are supplied to the tissue to conduct normal levels of aerobic metabolism.

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Calverley PMA, Koulouris NG. Flow limitation and dynamic hyperinflation: key concepts in modern respiratory physiology. Eur Respir J. 2005;25:186-199.

Hillman SS, Hancock TV, Hedrick MS. A comparative meta-analysis of maximal aerobic metabolism of vertebrates: implications for respiratory and cardiovascular limits to gas exchange. J Comp Physiol [B]. 2013;183:167-179.

Hughes JM. Assessing gas exchange. Chron Respir Dis. 2007;4:205-214.

Petersson J, Glenny RW. Gas exchange and ventilation-perfusion relationships in the lung. Eur Respir J. 2014;44:1023-1041.

Sheel AW, Romer LM. Ventilation and respiratory mechanics. Compr Physiol. 2012;2:1093-1142.

Stickland MK, Lindinger MI, Olfert IM, et al. Pulmonary gas exchange and acid-base balance during exercise. Compr Physiol. 2013;3:693-739.

Whipp BJ. Physiological mechanisms dissociating pulmonary CO2 and O2 exchange dynamics during exercise in humans. Exp Physiol. 2007;92:347-355.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

Describe the central organization of breathing.

2.

Explain the role of central and peripheral chemoreceptors in regulating respiration.

3.

Compare and contrast the roles of chemoreceptors and pulmonary mechanoreceptors in regulating respiration.

4.

Describe ventilatory control during special circumstances (e.g., exercise and high altitude).

5.

Describe the effects of abnormalities in ventilatory control.

eople breathe without thinking, and they can willingly modify their breathing pattern and even hold their breath. Control of ventilation includes the generation and regulation of rhythmic breathing by the respiratory center in the brainstem and its modification by the input of information from higher brain centers and from systemic receptors. The goals of breathing are, from a mechanical perspective, to minimize work and, from a physiological perspective, to maintain and regulate positive pressures of arterial blood O2 (PaO2) and CO2 (PaCO2). Another goal of breathing is to maintain acid-base balance in the brain by regulating PaCO2. Automatic respiration begins at birth. In utero, the placenta, not the lung, is the organ of gas exchange in the fetus. Its microvilli interdigitate with the maternal uterine circulation, and PaO2 transport and PaCO2 removal from the fetus occur by passive diffusion across the maternal circulation.

Ventilatory Control: An Overview

There are four major sites of ventilatory control: (1) the respiratory control center, (2) central chemoreceptors, (3) peripheral chemoreceptors, and (4) pulmonary mechanoreceptors/sensory nerves. The respiratory control center is located in the medulla oblongata of the brainstem and is composed of multiple nuclei that generate and modify the basic ventilatory rhythm. This center consists of two main parts: (1) a ventilatory pattern generator, which sets the rhythmic pattern, and (2) an integrator, which controls generation of the pattern, processes input from higher brain centers and chemoreceptors, and controls the rate and amplitude of the ventilatory pattern. Input to the integrator arises from higher brain centers, including the cerebral cortex, hypothalamus, limbic system including the amygdalae, and cerebellum.

Central chemoreceptors are located in the central nervous system just below the ventrolateral surface of the medulla. These central chemoreceptors detect changes in the PaCO2 and pH of interstitial fluid in the brainstem, and they modulate ventilation. Peripheral chemoreceptors are located on specialized cells in the aortic arch (aortic bodies) and at the bifurcation of the internal and external carotid arteries (carotid bodies) in the neck. These peripheral chemoreceptors sense the PaO2, PaCO2, and pH of arterial blood, and they feed information back to the integrator nuclei in the medulla through the vagus nerves and carotid sinus nerves, which are branches of the glossopharyngeal nerves. Pulmonary mechanoreceptors and sensory nerve stimulation, in response to lung inflation or to stimulation by irritants or release of local mediators in the airways, modify the ventilatory pattern.

The collective output of the respiratory control center to motor neurons located in the anterior horn of the spinal column controls the muscles of respiration, and this output determines the automatic rhythmic pattern of respiration. Motor neurons located in the cervical region of the spinal column control the activity of the diaphragm through the phrenic nerves, whereas other motor neurons located in the thoracic region of the spine control the intercostal muscles and the accessory muscles of respiration.

In contrast to automatic respiration, voluntary respiration bypasses the respiratory control center in the medulla. The neural activity controlling voluntary respiration originates in the motor cortex, and signaling passes directly to motor neurons in the spine through the corticospinal tracts. The motor neurons to the respiratory muscles act as the final site of integration of the voluntary (corticospinal tract) and automatic (ventrolateral tracts) control of ventilation. Voluntary control of these muscles competes with automatic influences at the level of the spinal motor neurons, and this competition can be demonstrated by breath holding. At the start of the breath hold, voluntary control dominates the spinal motor neurons. However, as the breath hold continues, the automatic ventilatory control eventually overpowers the voluntary effort and limits the duration of the breath hold. Motor neurons also innervate muscles 490 SECTION5Berne & Levy Physiology 15 10 5 0 25 35 45 55 65 PaCO2 (mm Hg) Alveolar ventilation (L/min) Metabolic acidosis Awake normal Sleep Morphine, barbiturates COPD Anesthetic agents •Fig. 25.1 Relationship between Partial Pressure of Arterial Carbon Dioxide (PaCO2) and Alveolar Ventilation in Awake Normal States, during Sleep, after Narcotic Ingestion and Deep Anesthesia, and in the Presence of Metabolic Acidosis. Boththeslopesoftheresponse(sensitivity)andthepositionoftheresponsecurves(threshold,thepointatwhichthecurvecrossesthex-axis[notshown])arechanged,whichindicatesdifferencesinventilatoryresponsesandresponsethresholds.COPD,chronicobstructivepulmonarydisease.

of the upper airway. These neurons are located within the medulla near the respiratory control center. They innervate muscles in the upper airways through the cranial nerves. When activated, they dilate the pharynx and large airways at the initiation of inspiration.

Response to Carbon Dioxide

Ventilation is regulated by PaCO2, PaO2, and pH in arterial blood. PaCO2 is the most important of these regulators. Both the rate and depth of breathing are controlled to maintain PaCO2 close to 40 mm Hg. In a normal awake individual, there is a linear rise in ventilation as PaCO2 reaches and exceeds 40 mm

Hg (Fig. 25.1 ). The ventilatory drive or response to changes in PaCO2 can be reduced by hyperventilation and by drugs that depress the respiratory center and decrease the ventilatory response to both CO2 and O2, such as morphine, barbiturates, and anesthetic agents. In these instances, the stimulus is inadequate to stimulate the motor neurons that innervate the muscles of respiration. It is also depressed during sleep. In addition, the ventilatory response to changes in PaCO2 is reduced if the work of breathing is increased, which can occur in individuals with chronic obstructive pulmonary disease (COPD). This effect occurs primarily because the neural output of the respiratory center is less effective in promoting ventilation as a result of the mechanical limitation to ventilation.

Changes in PaCO2 are sensed by central and peripheral chemoreceptors, and they transmit this information to the medullary respiratory centers. The respiratory control center then regulates minute ventilation and thereby maintains PaCO2 within the normal range. In the presence of a normal PaO2, ventilation increases by approximately 3 L/minute for each 1 mm Hg rise in PaCO2. The response to an increase in PaCO2 is further increased when the PaO2 is low (

Fig. 25.2, ). With a low PaO2, ventilation is greater for any given PaCO2, and the increase in ventilation for a given increment in PaCO2 is enhanced. The slope of the minute ventilation response as a function of the inspired CO2 is termed the ventilatory response to CO2 and is a test of CO2 sensitivity. It is important to recognize that this relationship is amplified by low O2 (see

Fig. 25.2, B ). The responsiveness to low O2 is enhanced because different mechanisms are responsible for sensing PaO2 and PaCO2 in the peripheral chemoreceptors. Thus the presence of both hypercapnia-elevated CO2 and hypoxemia-low O2 (often called asphyxia when both changes are present) has an additive effect on chemoreceptor output and on the resulting ventilatory stimulation.

Control of Ventilation: The Details

The Respiratory Control Center

When the brain is transected experimentally between the medulla and the pons, periodic breathing is maintained, thus demonstrating that the inherent rhythmicity of breathing originates in the medulla. Although no single group of neurons in the medulla has been found to be the breathing “pacemaker,” two distinct nuclei within the medulla are involved in generation of the respiratory pattern (

Fig. 25.3 ). One nucleus is the dorsal respiratory group (DRG), which is composed of cells in the nucleus tractus solitarius and is located in the dorsomedial region of the medulla. Cells in the DRG receive afferent input from cranial nerves IX •Fig. 25.2 The Effects of Hypoxia and Hypercapnia on Ventilation as the Other Respiratory Gas Partial Pressures Are Varied. A, Atagivenpartialpressureofarterialcarbondioxide(PaCO2),ventilationincreasesmoreandmoreaspartialpressureofarterialoxygen(PaO2)decreases.WhenPaCO2isallowedtodecrease(thenormalcondition)duringhypoxia,thereislittlestimulationofbreathinguntilPaO2fallsbelow60mmHg.Thehypoxicresponseismediatedthroughthecarotidbodychemoreceptors.B, ThesensitivityoftheventilatoryresponsetoCO2isenhancedbyhypoxia.

and X, which originate from airways and the lungs and constitute the initial intracranial processing station for this afferent input. The second group of medullary cells is the ventral respiratory group (VRG), located in the ventrolateral region of the medulla. The VRG is composed of three cell groups: the rostral nucleus retrofacialis, the caudal nucleus retroambiguus, and the nucleus para-ambiguus. The VRG contains both inspiratory and expiratory neurons. The nucleus retrofacialis and the caudally located cells of the nucleus retroambiguus are active during exhalation, whereas the rostrally located cells of the nucleus retroambiguus are active during inspiration. The nucleus para-ambiguus has inspiratory and expiratory neurons that travel in the vagus nerve to the laryngeal and pharyngeal muscles. Discharges •Fig. 25.3 The Respiratory Control Center is Located in the Medulla, the Most Primitive Portion of the Brain. Theneuronsaremainlyintwoareas:thedorsalrespiratorygroup(DRG),whichconsistsofthenucleustractussolitarius,andtheventralrespiratorygroup(VRG),whichconsistsoftherostralnucleusretrofacialis,nucleusparaambiguus,andthecaudalnucleusretroambiguus.C1referstothefirstcervicalsignalsegmenttothecaudalborderofthepons.Thefourthventricleofthebrainislocatedbelowthecerebellumandabove,andbetween,theponsandthemedulla.

•Fig. 25.4 Diagram of the Basic Wiring of the Brainstem Ventilatory Controller. Thesignsofthemainoutput(arrows) oftheneuronpoolsindicatewhethertheoutputisexcitatory(+)orinhibitory(−).PoolAprovidestonicinspiratorystimulitothemusclesofbreathing.PoolBisstimulatedbypoolAandprovidesadditionalstimulationtothemusclesofbreathing,andpoolBstimulatespoolC.OtherbraincentersfeedintopoolC(inspiratorycutoffswitch),whichsendsinhibitoryimpulsestopoolA.Afferentinformation(feedback)fromvarioussensorsactsatdifferentlocations:ChemoreceptorsactonpoolA,andintrapulmonarysensoryfibersactviathevagusnervesonpoolB.Apneumotaxiccenterintheanteriorponsreceivesinputfromthecerebralcortex,anditactsonpoolC.

from cells in these areas excite some cells and inhibit other cells.

At the level of the respiratory control center, inspiration and exhalation involve three phases: one inspiratory and two expiratory (

Fig. 25.4 ). Inspiration begins with an abrupt increase in discharge from cells in the nucleus tractus solitarius, the nucleus retroambiguus, and the nucleus para-ambiguus, followed by a steady ramp-like increase in firing rate throughout inspiration. This leads to progressive contraction of the respiratory muscles during automatic breathing. At the end of inspiration, an “off-switch” event causes neuron firing to decrease markedly, at which point exhalation begins. At the start of exhalation (phase I of expiration), a paradoxical increase in inspiratory neuron firing slows the expiratory phase down by increasing inspiratory muscle tone and expiratory neuron firing. This inspiratory neuron firing decreases and stops during phase II of exhalation. Although many different neurons in the DRG and VRG are involved in ventilation, each cell type appears to have a specific function. For example, the Hering-Breuer reflex is an inspiratory-inhibitory reflex that arises from afferent stretch receptors located in the smooth muscles of the airways. Increasing lung inflation stimulates these stretch receptors and results in early exhalation by stimulating the neurons associated with the off-switch phase of inspiratory muscle control. Thus rhythmic breathing depends on a continuous (tonic) inspiratory drive from the DRG and an intermittent (phasic) expiratory drive from the cerebrum, thalamus, cranial nerves, and ascending sensory tracts in the spinal cord.

A chemoreceptor is a receptor that responds to a change in the chemical composition of blood or other fluid around it. Central chemoreceptors are specialized cells on the ventrolateral surface of the medulla. Chemoreceptors are sensitive to the pH of the surrounding extracellular fluid. Because this extracellular fluid is in contact with cerebrospinal fluid (CSF), changes in the pH of CSF affect ventilation by acting on these chemoreceptors

CSF is an ultrafiltrate of plasma that is secreted continuously by the choroid plexus and is reabsorbed by the arachnoid villi. Because it is in contact with the extracellular fluid in the brain, the composition of CSF is influenced by the metabolic activity of the cells in the surrounding area and the composition of the blood. Although the origin of CSF is plasma, the composition of CSF is not the same as that of plasma because the blood-brain barrier exists between the two sites (

Fig. 25.5 ). The blood-brain barrier is composed of endothelial cells, smooth muscle, and the pial and arachnoid membranes, and it regulates the movement of ions between blood and CSF. In addition, the choroid plexus also determines the ionic composition of CSF by transporting ions into and out of CSF. The blood-brain barrier is relatively impermeable by H+ and HCO3 − ions, but it is very permeable by CO2. Thus the PCO2 in CSF parallels the arterial PCO2 tension. CO2 is also produced by cells of the brain as a product of metabolism. As a consequence, the PCO2 in CSF is usually a few millimeters of mercury higher than that in arterial blood, and so the pH is slightly more acidic (7.33) in CSF than in plasma (

Table 25.1 •Fig. 25.5 Carbon Dioxide and the Blood-Brain Barrier. PaCO2crossestheblood-brainbarrierandrapidlyequilibrateswithCO2incerebrospinalfluid(CSF).H+ andHCO3− ionscrossthebarrierslowly.Thepartialpressureofarterialcarbondioxide(PaCO2)combineswithCO2generatedbymetabolismtodilatethesmoothmuscle.Incomparisonwitharterialblood,thepHofCSFislowerandthePCO2ishigher,withlittleproteinbuffering.

PCO2,partialpressureofcarbondioxide.

The carotid and aortic bodies are peripheral chemoreceptors that respond to changes in PaO2 (not the O2 content), PaCO2, and pH, and they transmit afferent information to the central respiratory control center. The peripheral chemoreceptors are the only chemoreceptors that respond to changes in PaO2. The peripheral chemoreceptors are also responsible for approximately 40% of the ventilatory response to PaCO2. These chemoreceptors are small, highly vascularized structures. They consist of type I (glomus) cells that are rich in mitochondria and endoplasmic reticulum. They also have several types of cytoplasmic granules (synaptic vesicles) that contain various neurotransmitters, including dopamine, acetylcholine, norepinephrine, and neuropeptides. Afferent nerve fibers synapse with type I cells, and they transmit information to the brainstem through the carotid sinus nerve (carotid body) and vagus

TheHenderson-Hasselbalch equation relatesthepHofCSFtotheconcentrationofbicarbonate([HCO3−]): whereα isthesolubilitycoefficient(0.03mmol/L/mmHg)andpKisthenegativelogarithmofthedissociationconstantforcarbonicacid(6.1).TheHenderson-HasselbalchequationdemonstratesthatanincreaseinCSFPCO2causesthepHofCSFtodecreaseatanygiven[HCO3−].ThedecreaseinpHstimulatesthecentralchemoreceptorsandtherebyincreasesventilation.ThusCO2inbloodregulatesventilationbyitseffectonthepHofCSF.TheresultinghyperventilationreducesthePaCO2,andthereforethePCO2ofCSF,andreturnsthepHofCSFtowardanormalvalue.Furthermore,cerebralvasodilationaccompaniesanincreaseinPaCO2,andthisenhancesthediffusionofCO2intoCSF.Incontrast,anincreaseinCSF[HCO3−]causesanincreaseinthepHofCSFatanygivenPaCO2.

ChangesinPaCO2thatresultfromalterationsinpHactivatehomeostaticmechanismsthatreturnthepHbacktowardanormalvalue.Theblood-brainbarrierregulatesthepHofCSFbyadjustingtheioniccompositionand[HCO3 −]ofCSF.ThesechangesinCSF[HCO3−],however,occurslowly,overaperiodofseveralhours,whereaschangesinCSFPCO2canoccurwithinminutes.ThuscompensationforchangesinthepHofCSFrequireshourstodevelopfully.

Several reflexes that arise from the chest wall and lungs affect ventilation and ventilatory patterns (

Table 25.2 ). The Hering-Breuer inspiratory-inhibitory reflex is stimulated by increases in lung volume, especially those associated with an increase in both ventilatory rate and tidal volume. This stretch reflex is mediated by vagal fibers, and when nerve (aortic body). Type I cells are the cells primarily responsible for sensing PaO2, PaCO2, and pH. In response to even small decreases in PaO2, there is an increase in chemoreceptor discharge, which enhances respiration. The response is robust when PaO2 decreases below 75 mm Hg. Thus ventilation is regulated by changes in arterial and CSF pH through effects on peripheral and central chemoreceptors (

Fig. 25.6 •Fig. 25.6 The Ventilatory Response to Partial Pressure of Arterial Carbon Dioxide (PaCO2) is Affected by the Concentration of Hydrogen ([H+]) in Cerebrospinal Fluid (CSF) and Brainstem Interstitial Fluid. Duringchronicmetabolicacidosis(e.g.,diabeticketoacidosis),the[H+]inCSFisincreased,andtheventilatoryresponsetoinspiredPaCO2isincreased(steeperslope).Conversely,duringchronicmetabolicalkalosis(arelativelyuncommoncondition),the[H+]inCSFisdecreasedandtheventilatoryresponsetoinspiredPaCO2isdecreased(reducedslope).Thepositionsoftheresponselinesarealsoshifted,whichindicatesalteredthresholds.

ImagineflyingfromNewYorkCitytoDenver.ThebarometricpressureinNewYorkisapproximately760mmHg,whereasinthemountainssurroundingDenver,Colorado,itis600mmHg.Atsealevel,thePaO2isapproximately95mmHgandPAO2= [(760− 47)× 0.21]− [40/0.8]= 100mmHg(accordingtothealveolarairequation;see ).Ifthealveolar-arterialPO2difference[AaDO2]is5mmHg,PaO2= 100mmHg− 5mmHg= 95mmHg.IntheCSF,pHwouldbeapproximately7.33,PaCO2wouldbe44mmHg(PaCO2+ CO2producedbymetabolismofbraincells),andHCO3− wouldbeapproximately22mEq/L.

WhenyouarriveinDenver,thereisanabruptdecreaseinthepartialpressureofinspiredO2(PiO2):PiO2= (600− 47)× 0.21= 116mmHg;therearealsodecreasesinthepartialpressuresofalveolarandarterialO2:PAO2= 116− (40/0.8)= 66mmHg,andPaO2= 61mmHgifthereisnochangeinAaDO2).ThisdecreaseinarterialO2stimulatestheperipheralchemoreceptorsandtherebyincreasesventilation.TheincreaseinventilationdecreasesPaCO2andelevatesarterialpH.TheresultofthisincreaseinventilationistominimizethehypoxemiabyincreasingPAO2.Forexample,assumethatPACO2decreasesto30mmHg.ThenPAO2= [(600− 47)× 0.21]− [30/0.8]= 78mmHg,a12–mmHgincreaseinPAO2.

ThedecreaseinPaCO2alsocausesadecreaseinthePCO2ofCSF.Because[HCO3−]isunchanged,thepHofCSFincreases.ThisincreaseinthepHofCSFattenuatestherateofdischargeofthecentralchemoreceptorsanddecreasestheircontributiontotheventilatorydrive.Overthenext12to36hours,[HCO3−]inCSFdecreasesasacid-basetransporterproteinsintheblood-brainbarrierreduce[HCO3−].Asaconsequence,thepHofCSFreturnstowardnormal.Centralchemoreceptordischargeincreases,andminuteventilationisfurtherincreased.Atthesametimethat[HCO3−]inCSFdecreases,HCO3− isgraduallyexcretedfromplasmabythekidneys.ThisresultsinagradualreturnofarterialpHtowardnormalvalues.PeripheralchemoreceptorstimulationincreasesfurtherasarterialpHbecomesnormal(peripheralchemoreceptorsareinhibitedbytheelevatedarterialpH).Finally,within36hoursofarrivingathighaltitude,minuteventilationincreasessignificantly.Thisdelayedresponseisgreaterthantheimmediateeffectofthehypoxemiaonventilation.Thisfurtherincreaseinventilationisduetobothcentralandperipheralchemoreceptorstimulation.Thusafter36hours,botharterialpHandCSFpHareapproachingnormalvalues;minuteventilationisincreased,PaO2isdecreased,andPaCO2isdecreased.

Younowreturnhome.WhenyoulandinNewYork,thePiO2returnstoanormalvalue,andthehypoxicstimulustoventilationisremoved.PaO2returnstoanormalvalue,andtheperipheralchemoreceptorstimulationtoventilationdecreases.Thiscausesanincreaseinarterial[CO2]towardnormalvalues,whichinturncausesanincreaseinCSF[CO2].ThisincreaseisassociatedwithadecreaseinthepHofCSFas[HCO3−]inCSFisreducedandventilationisaugmented.Overthenext12to36hours,theacid-basetransportersintheblood-brainbarriertransportHCO3− backintoCSF,andthepHofCSFgraduallyreturnstowardnormalvalues.Similarly,thepHofblooddecreasesasPaCO2risesbecausearterial[HCO3−]decreases.Thisstimulatestheperipheralchemoreceptors,andminuteventilationremainsaugmented.Overthenext12to36hours,blood[HCO3−]increasesinthekidneys(see ),arterialpHreturnstoanormalvalue,andminuteventilationreturnstoanormallevel.

elicited, it results in cessation of inspiration by stimulating the off-switch neurons in the medulla. This reflex is inactive during quiet breathing and appears to be most important in newborns. Stimulation of nasal or facial receptors with cold water initiates the diving reflex. When this reflex is elicited, apnea, or cessation of breathing, and bradycardia occur. This reflex protects individuals from aspirating water in the initial stages of drowning. Activation of receptors in the nose is responsible for the sneeze reflex.

The aspiration or sniff reflex can be elicited by stimulation of mechanical receptors in the nasopharynx and pharynx. This is a strong, short-duration inspiratory effort that brings material from the nasopharynx to the pharynx, where it can be swallowed or expectorated. The mechanical receptors responsible for the sniff reflex are also important in swallowing by inhibiting respiration and causing laryngeal closure. For anatomical reasons, only newborns can breathe and swallow simultaneously, which allows more rapid ingestion of nutrients.

The larynx contains both superficial and deep receptors. Activation of the superficial receptors results in apnea, cough, and expiratory movements that protect the lower respiratory tract from aspirating foreign material. The deep receptors are located in the skeletal muscles of the larynx, and they control muscle fiber activation, as in other skeletal muscles.

Three major types of sensory receptors located in the tracheobronchial tree respond to a variety of different stimuli, and those responses result in changes in the lung’s mechanical properties, alterations in the respiratory pattern, and the development of respiratory symptoms. Inhaled dust, noxious gases, and cigarette smoke stimulate irritant receptors in the trachea and large airways that transmit information through myelinated vagal afferent fibers. Stimulation of these receptors results in an increase in airway resistance, reflex apnea, and coughing. These receptors are also known as rapidly adapting pulmonary stretch receptors. Slowly adapting pulmonary stretch receptors respond to mechanical stimulation, and they are activated by lung inflation. They also transmit information through myelinated, vagal afferent fibers. The increase in lung volume in people with COPD stimulates these pulmonary stretch receptors and delays the onset of the next inspiratory effort. This explains the long, slow expiratory effort in affected individuals, and it is essential to minimize dynamic, expiratory airway compression.

In addition, specialized sensory receptors located in the lung parenchyma respond to chemical or mechanical stimulation in the lung interstitium. These receptors are called juxta-alveolar, (or J) receptors. They transmit their afferent input through unmyelinated, vagal C fibers. They may be responsible for the sensation of dyspnea (abnormal shortness of breath) and the rapid, shallow ventilatory patterns that occur in interstitial lung edema and some inflammatory lung states.

VO2 (L/min) . 1 1.5 2 VCO2 . PaCO2 pH Ventilation PaO2 Lactate •Fig. 25.8 The Two Main Categories of Sleep Apnea. A, Obstruc-tivesleepapnea,thepleuralpressureoscillationsincreaseasCO2levelrises.Thisindicatesthatresistancetoairflowisveryhighasaresultofupperairwayobstruction.B, Centralsleepapneaischaracterizedbynoattempttobreathe,asdemonstratedbynooscillationsinpleuralpressure.

•Fig. 25.7 Oxygen Consumption (V̇ O2) as a Function of the Metabolic Changes That Occur During Exercise. Theanaerobicthreshold(arrow) isthepointatwhichtheillustratedvariableschangeandisduetolacticacidosis.PaCO2,partialpressureofarterialcarbondioxide;PaO2,partialpressureofarterialoxygen;VCO2,carbondioxide consumption.

Somatic receptors are also located in the intercostal muscles, rib joints, accessory muscles of respiration, and tendons, and they respond to changes in the length and tension of the respiratory muscles. Although they do not directly control respiration, they do provide information about lung volume and play a role in terminating inspiration. They are especially important in individuals with increased airway resistance and decreased pulmonary compliance because they can augment muscle force within the same breath. Somatic receptors also help minimize the chest wall distortion during inspiration in newborns, who have very compliant rib cages.

The ability to exercise depends on the capacity of the cardiac and respiratory systems to increase delivery of O2 to tissues and remove CO2 from the body. Ventilation increases immediately when exercise begins, and this increase in minute ventilation closely matches the increases in O2 consumption and CO2 production that accompany exercise (

Fig. 25.7 ). Ventilation is linearly related to both CO2 production and O2 consumption at low to moderate levels (see

Fig. 25.7 ). During maximal exercise, a physically fit individual can achieve an O2 consumption of 4 L/minute with a minute ventilation volume of 120 L/minute, which is almost 15 times the resting level.

Exercise is remarkable because of the lack of significant changes in blood gases. Except at maximal exertion, changes in PaCO2 and PaO2 are minimal during exercise. Arterial pH remains within normal values during moderate exercise. During strenuous exercise, arterial pH begins to fall as lactic acid is liberated from muscles during anaerobic metabolism. This decrease in arterial pH stimulates ventilation that is out of proportion to the level of exercise. The level of exercise at which sustained metabolic (lactic) acidosis begins is called the anaerobic threshold (see

Fig. 25.7

Abnormalities in the Control of Breathing

Changes in the ventilatory pattern can occur for both primary and secondary reasons. During sleep, approximately one third of normal individuals have brief episodes of apnea or hypoventilation that have no significant effects on PaO2 or PaCO2. The apnea usually lasts less than 10 seconds, and it occurs in the lighter stages of slow-wave and rapid eye movement (REM) sleep. In sleep apnea syndromes, the duration of apnea is abnormally prolonged, and it changes PaO2 and PaCO2. There are two major categories of sleep apnea (

Fig. 25.8 ). The first, obstructive sleep apnea (OSA), is the most common of the sleep apnea syndromes, and it occurs when the upper airway (generally the hypopharynx) closes during inspiration. Although the process is similar to what happens during snoring, it is more severe, inasmuch as it obstructs the airway and causes cessation of airflow.

The second sleep apnea syndrome is central sleep apnea. This variant of apnea occurs when the ventilatory drive to

TheclinicalhistoriesofindividualswithOSAareverysimilar.Aspouseusuallyreportsthattheaffectedindividualsnores.Thesnoringbecomeslouderandlouderandthenstopswhiletheindividualcontinuestomakevigorousrespiratoryefforts(see

Fig.25.8 ).Theindividualthenawakens,fallsbacktosleep,andcontinuesthesameprocessrepetitivelythroughoutthenight.IndividualswithOSAawakenwhenthearterialhypoxemiaandhypercapniastimulatebothperipheralandcentralchemoreceptors.Respirationisrestoredbrieflybeforethenextapneiceventoccurs.IndividualswithOSAcanhavehundredsoftheseeventseachnightthatinterruptsleep.ComplicationsofOSAincludesleepdeprivation,polycythemia,right-sidedcardiacfailure(corpulmonale),andpulmonaryhypertensionsecondarytotherecurrent,hypoxicevents.OSAiscommoninindividualswithobesityandinthosewithexcessivecomplianceofthehypopharynx,upperairwayedema,andstructuralabnormalitiesoftheupperairway.

the respiratory motor neurons decreases. Individuals with central sleep apnea have repeated episodes of apnea, during which time they make no respiratory effort, every night (see

Fig. 25.8 ). The degree of hypercapnia and hypoxemia in individuals with central sleep apnea is less than that in individuals with OSA, but the same complications (e.g., polycythemia) can occur when central sleep apnea is recurrent and severe.

Cheyne-Stokes ventilation is another abnormality of breathing that is characterized by varying tidal volume and ventilatory frequency (

Fig. 25.9 ). After a period of apnea, tidal volume and respiratory frequency increase progressively over several breaths, and then they progressively decrease until apnea recurs. This irregular breathing pattern is seen in some individuals with central nervous system diseases, head trauma, and increased intracranial pressure. It is also present on occasion in normal individuals during sleep at high altitude. The mechanism underlying Cheyne-Stokes respiration is not known. In some individuals, it appears to be due to slow blood flow in the brain in association with periods of overshooting and undershooting ventilatory effort in response to changes in PCO2.

Central alveolar hypoventilation, alsoknownasOndine’s curse, isararediseaseinwhichvoluntarybreathingisintactbutabnormalitiesinautomaticityexist.Itisthemostsevereofthecentralsleepapneasyndromes.Asaresult,peoplewithcentralalveolarhypoventilationcanbreatheaslongastheydonotfallasleep.Fortheseindividuals,mechanicalventilationor,morerecently,bilateraldiaphragmaticpacing(similartoacardiacpacemaker)canbelifesaving.

Apneustic breathing is another abnormal breathing pattern that is characterized by sustained periods of inspiration separated by brief periods of exhalation (

Fig. 25.10, C •Fig. 25.9 In Cheyne-Stokes Breathing, Tidal Volume and, as a Consequence, Arterial Blood Gas Levels Wax and Wane. Ingeneral,Cheyne-Stokesbreathingisasignofvasomotorinstability,particularlylowcardiacoutput.PaCO2,partialpressureofarterialcarbondioxide;PaO2,partialpressureofarterialoxygen.

Lung volumeRemove input from cortex, thalamus, and vagus nerves A B Remove sensory input from lungs •Fig. 25.10 Some Patterns of Breathing. A, Normalrateofbreathingisintherangeof12to20breathsperminute.B, Whensensoryinputisremovedfromvariouslungreceptors(mainlystretch),eachbreathingcycleislengthenedandtidalvolumeisincreased,sothatalveolarventilationisnotsignificantlyaffected.C, Wheninputfromthecerebralcortexandthalamusisalsoeliminated,togetherwithvagalblockade,theresultisprolongedinspiratoryactivitybrokenafterseveralsecondsbybriefexpirations(apneusis).

The mechanism underlying this ventilatory pattern appears to be a loss of inspiratory-inhibitory activities that results in augmentation of the inspiratory drive. The pattern sometimes occurs in individuals with central nervous system injury.

Sudden infant death syndrome (SIDS)isthemostcommoncauseofdeathininfantsinthefirstyearoflifeaftertheperinatalperiod.AlthoughthecauseofSIDSisnotknown,abnormalitiesinventilatorycontrol,particularlyin 1.

Ventilatory control is composed of the respiratory control center, central chemoreceptors, peripheral chemoreceptors, and pulmonary mechanoreceptors/ sensory nerves. PaCO2 is the major factor that influences ventilation.

2.

The respiratory control center is composed of the dorsal respiratory group and the ventral respiratory group. Rhythmic breathing depends on a continuous (tonic) inspiratory drive from the dorsal respiratory group and on intermittent (phasic) expiratory input from the cerebrum, thalamus, cranial nerves, and ascending spinal cord sensory tracts. The peripheral and central chemoreceptors respond to changes in PaCO2 and pH. The peripheral chemoreceptors (carotid and aortic bodies) are the only chemoreceptors that respond to changes in PaO2.

Calverley PMA, Koulouris NG. Flow limitation and dynamic hyperinflation: key concepts in modern respiratory physiology. Eur Respir J. 2005;25:186-199.

Canning BJ. Afferent nerves regulating the cough reflex: mechanisms and mediators of cough in disease. Otolaryngol Clin N Am. 2010;43:15-25.

Leff AR, Schumacker PT. Respiratory Physiology: Basics and Applications. Philadelphia: WB Saunders; 1993. Nishino T. Dyspnoea underlying mechanisms and treatment. Br J Anaesth. 2011;106:463-474.

CO2responsiveness,havebeenimplicated.Placinginfantsontheirbackstosleep(whichreducesthepotentialforCO2rebreathing)hasdramaticallydecreased(butnoteliminated)therateofdeathfromthissyndrome.

3.

Acute hypoxia and chronic hypoxia affect breathing differently because the slow adjustments in CSF [H+] in chronic hypoxia alter sensitivity to CO2.

4.

Irritant receptors protect the lower respiratory tract from particles, chemical vapors, and physical factors, primarily by inducing cough. C fiber J receptors in the terminal respiratory units are stimulated by distortion of the alveolar walls (by lung congestion or edema).

5.

The two most important clinical abnormalities of breathing are obstructive and central sleep apnea.

6.

PaO2, PaCO2, and pH remain within normal limits during moderate exercise; however, during strenuous exercise, pH falls, which stimulates ventilation, whereas PaO2 and PaCO2 remain relatively normal.

Otis AB. A perspective of respiratory mechanics. J Appl Physiol. 1983;54:1183-1187.

Sheel AW, Romer LM. Ventilation and respiratory mechanics. Compr Physiol. 2012;2:1093-1142.

Wasserman K, Beaver WL, Whipp BI. Gas exchange theory and the lactic acidosis (anaerobic) threshold. Circulation. 1990;81(1 suppl):1114-1130.

Wine JJ. Parasympathetic control of airway submucosal glands: central reflexes and the airway intrinsic nervous system. Auton Neurosci. 2007;133:35-54.

Upon completion of this chapter, the student should be able to answer the following questions: 1.

Describe the components of the mucociliary clearance system and their role in the removal of xenobiotic substances and particulates.

2.

Explain how particle characteristics and properties influence their deposition and clearance.

3.

Compare and contrast the major mechanisms of particle deposition.

4.

Compare and contrast the mucosal and systemic immune responses.

5.

Describe unique features of immunoglobulin A and why it is well suited for the mucosal environment and protection.

6.

Compare and contrast adaptive and innate immune cells in the respiratory system.

7.

Describe the various metabolic processes in the lung.

n addition to their primary function of gas exchange, the lungs act as a primary barrier between the outside world and the inside of the body, with host defense functions. They are also active organs in the metabolism of xenobiotic and endogenous compounds.

To cope with the inhalation of foreign substances, the respiratory system and, in particular, the conducting airways have developed unique structural features: the mucociliary clearance system and specialized adaptive and innate immune response mechanisms.

The mucociliary clearance system protects the conducting airways by trapping and removing inhaled pathogenic viruses and bacteria, in addition to nontoxic and toxic particulates (e.g., pollen, ash, mineral dust, mold spores, and organic particles), from the lungs. These particulates are inhaled with each breath and must be removed. The three major components of the mucociliary clearance system are two fluid layers, referred to as the sol (periciliary fluid) and gel (mucus) layers, and cilia, which are positioned on the surface of bronchial epithelial cells (

Fig. 26.1 ). Inhaled material is trapped on the viscoelastic (sticky) mucus layer, whereas the watery periciliary fluid allows the cilia to move freely and establish an upward flow to clear particulates from the lung. Effective clearance requires both ciliary activity and the appropriate balance of periciliary fluid and mucus.

The periciliary fluid layer is composed of nonviscous serous fluid, which is produced by the pseudostratified ciliated columnar epithelial cells that line the airways. These cells have the ability to either secrete fluid, a process that is mediated by activation of cystic fibrosis transmembrane regulator (CFTR) chloride (Cl−) ion channels (Na+ secretion follows passively between cells across the tight junctions) or reabsorb fluid, a process that is mediated by activation of epithelial sodium channels (ENaC; Cl− absorption follows passively between cells across the tight junctions). NaCl secretion or reabsorption temporarily establishes an osmotic gradient across the pseudostratified epithelium, which provides the driving force for passive water movement. The balance between CFTR-mediated Cl− secretion and ENaC-mediated Na+ absorption is regulated by a variety of hormones and determines the volume of the periciliary fluid, which in the healthy lung is 5 to 6 µm deep, a level that is optimal for rhythmic beating of the cilia and mucociliary clearance.

The mucus layer lies on top of the periciliary fluid layer and is composed of a complex mixture of macromolecules and electrolytes. Because the mucus layer is in direct contact with air, it entraps inhaled substances, including pathogens. The mucus layer is predominantly water (95% to 97%), 5 to 10 µm thick and exists as a discontinuous blanket (i.e., islands of mucus). Mucus has low viscosity and high elasticity and is composed of glycoproteins with groups of oligosaccharides attached to a protein backbone. Healthy •Fig. 26.1 Overview of the Epithelial Lining and Innervation of the Tracheobronchial Tree. Theciliaoftheepithelialcellresideinthepericiliaryfluidlayer,andthemucuslayerisontop.Interspersedbetweentheciliatedepithelialcellsaresurfacesecretory(goblet)cellsandsubmucosalglands.Sympatheticandparasympatheticnervefibersdescendintothesubmucosalglandsandsmoothmuscles.

Cystic fibrosis (CF)isthemostcommonlethalinheriteddiseaseamongwhitepeople.ItisanautosomalrecessivediseasecausedbymutationsintheCFTR gene.Itischaracterizedbychronicbacteriallunginfection,progressivedeclineinlungfunction,andprematuredeathatanaverageageof38years.Morethan1000mutationsoftheCFTR genehavebeendescribed,but70%ofaffectedindividualshaveadeletionofphenylalanineatcodon508(ΔF508-CFTR)inatleastoneallele.ThismutationresultsinalackofCl− secretionandanincreaseinENaC-mediatedNa+ reabsorption,whichinturnresultsinareductioninthevolumeofthepericiliaryfluid.

DetailedstudyofthedifferentCFTR mutationshasresultedinanunderstandingofvariousdisease-relatedphenotypes,someofwhichareassociatedwithmilderdiseaseandsomewithmoreseveredisease.Sincethe1980s,researchfindingshaveelucidatedhowmanyofthemostcommonmutationsintheCFTR genecauseCF,andthishasledtothedevelopmentofdrugsthattargetspecificmutationsandreversethe individuals produce approximately 100 mL of mucus each day. Four cell types contribute to the quantity and composition of the mucus layer: goblet cells and Clara cells within the tracheobronchial epithelium, and mucous cells and serous cells within the tracheobronchial submucosal glands. Goblet cells, also referred to as surface secretory cells, represent approximately 15% to 20% of the tracheobronchial epithelium, and are found in the tracheobronchial tree up to the 12th division. In many respiratory diseases, goblet cells appear further down the tracheobronchial tree; thus the smaller airways are more susceptible to obstruction progressivereductioninlungfunction.Forexample,inonemutation(G551D-CFTR,whichaffects≈5%ofpatientswithCF),theCFTRCl− channelreachestheplasmamembraneofairwayepithelialcellsbutdoesnotsecreteCl− .Throughprecisionmedicine,adrug,ivacaftor(Kalydeco),hasbeenfoundtostimulateCl− secretionviatheG551D-CFTR,therebyimprovinglungfunctionanddecreasingtherateofdiseaseprogression.WhentheallelesarehomozygousfortheΔF508CFTRmutation(whichaffects≈50%ofpatientswithCF),theCFTRCl− channeldoesnotreachtheplasmamembraneofairwayepithelialcells.

In2015,theU.S.FoodandDrugAdministrationapprovedacombinationdrugtherapy,lumacaftor/ivacaftor(Orkambi),thathasbeenshowntocorrectthegenedefect,increasetheamountofΔF508-CFTRintheplasmamembrane,andimproveCl− transport.Clinically,bothivacaftorandlumacaftor/ivacaftorhavebeenshowntoimprovelungfunctionsignificantlyandtodecreasetherateofdeclineinlungfunction.

by mucus plugging. Goblet cells secrete neutral and acidic glycoproteins rich in sialic acid in response to chemical stimuli. In the presence of infection or cigarette smoke or in patients with chronic bronchitis, goblet cells can increase in size and number, extend above the 12th division of the tracheobronchial tree, and secrete copious amounts of mucus. Injury and infection increase the viscosity of the mucus secreted by goblet cells, which reduces mucociliary clearance of inhaled particles and pathogens.

Submucosal tracheobronchial glands are present wherever there is cartilage in the upper regions of the conducting airways, and they secrete water, ions, and mucus into the airway lumen through a ciliated duct (see

Fig. 26.1 ). Although both mucous and serous cells secret mucus, their cellular structure and mucus composition are distinctly different (

Table 26.1 ). In several lung diseases, including chronic bronchitis, the number and size of submucosal glands are increased, which leads to increases in mucus production, alterations in chemical composition of mucus (i.e., increased viscosity and decreased elasticity), and the formation of mucus plugs that cause airway obstruction. Mucus secretion from submucosal tracheobronchial glands is stimulated by parasympathetic (cholinergic) compounds such as acetylcholine and substance P and inhibited by sympathetic (adrenergic) compounds such as norepinephrine and vasoactive intestinal polypeptide. Local inflammatory mediators such as histamine and arachidonic acid metabolites also stimulate mucus production.

Clara cells, located in the epithelium of bronchioles, also contribute to the composition of mucus through secretion of a nonmucinous material containing carbohydrates and proteins. These cells play a role in bronchial regeneration after injury.

As noted previously, the respiratory tract to the level of the bronchioles is lined by a pseudostratified, ciliated columnar epithelium (see

Fig. 26.1 ). These cells maintain the level of the periciliary fluid in which cilia and the mucociliary transport system function. Mucus and inhaled particles are removed from the airways by the rhythmic beating of the cilia. There are approximately 250 cilia per airway epithelial cell, and each is 2 to 5 µm in length. Cilia are composed of nine microtubular doublets that surround two central microtubules held together by dynein arms, nexin links, and spokes. The central microtubule doublet contains an adenosine triposphatase (ATPase) that is responsible for the contractile beat of the cilium. Cilia beat with a coordinated oscillation in a characteristic, biphasic, and wave-like rhythm called metachronism. They beat at approximately

Sputumisexpectoratedmucus.However,inadditiontomucus,sputumcontainsserumproteins,lipids,electrolytes,Ca++ ,DNAfromdegeneratedwhitebloodcells(collectivelyknownasbronchial secretions),andextrabronchialsecretions;includingnasal,oral,lingual,pharyngeal,andsalivarysecretions.Thecolorofsputumismorecloselycorrelatedwiththeamountoftimethatithasbeenpresentinthelowerrespiratorytractthanwiththepresenceofinfection.Althoughnotpreciselyidentifiablewithdiseasediagnosis,thecolorofsputumcanbeinformativeinhelpingleadtoadiagnosisandstageofdisease.Mucushasmanycolors:white,yellow,green,red,pink,brown,gray,andblack.Thecolorationiscommonlyduetothetypeofcellpresentintheairways(inflammatorycells,suchasneutrophilsoreosinophils,orredbloodcells)andhowlongtheyhavebeenthere.Clearorcloudywhite thinmucusisconsiderednormal;however,ifamountsandthicknessareincreased,itmayrepresentanearlysignofinfection.Thickwhitemucuscanbetheonlyidentifiablefeatureofgastroesophagealrefluxdiseasecausedbygastricacidrefluxintotheairways.Yellow andgreen colorationofmucusisduetothepresenceandbreakdownofneutrophilsandeosinophilsininfectiousandallergicdiseases.Yellowistypicallyassociatedwithmoreacutedisease(infection,allergy)andgreenusuallyindicatesamorechronicstagewiththepresenceofbacteria(chronicbronchitis,bronchiectasis,cysticfibrosis,andlungabscess).Red mucusindicatesthepresenceofredbloodcellsintheairwaysandisassociatedwithpneumococcalpneumonia,lungcancer,tuberculosis,andpulmonaryemboli.Pink mucusistypicallyassociatedwiththebreakdownofeosinophilsinindividualswithallergies.Gray,brown,andblackmucusisoftenassociatedwithcigaretteormarijuanasmoking,cocaineuse,airpollution(workplaceenvironment,suchascoalmines),andoldblood.

1000 strokes per minute, with a power forward stroke and a slow return or recovery stroke. During their power forward stroke, the tips of the cilia extend upward into the viscous mucus layer and thereby move it and the entrapped particles. On the reverse beat, the cilia release the mucus and withdraw completely into the sol layer. Cilia in the nasopharynx beat in the direction that propels the mucus into the pharynx, whereas cilia in the trachea propel mucus upward toward the pharynx, where it is swallowed.

In general, deposition of particles in the lung depends on the particle’s size, density, and shape; the distance over which it has to travel; airflow speed; and the relative humidity of the air. The four major mechanisms for deposition are impaction, sedimentation, interception, and brownian movement. Particle characteristics and properties, which influence the mechanism of deposition, are listed in 26.2 . In general, particles larger than 10 µm are deposited by impaction in the nasal passages and do not penetrate into the lower respiratory tract. Particles 2 to 10 µm in size

Sedimentation0.2to2.0InterceptionNABrownianmovement<0.2

NA,notapplicable.

are deposited in the lower respiratory tract predominantly by inertial impaction at points of turbulent airflow (i.e., nasopharynx, trachea, and bronchi) and at airway bifurcations because their tendency to move in a straight direction prevents them from changing directions rapidly. In more distal areas, where airflow is slower, smaller particles (0.2 to 2 µm) are deposited on the surface by sedimentation as a result of gravity. For substances with elongated shapes (i.e., asbestos, silica), the mechanism of deposition is interception. The elongated particle’s center of gravity is compatible with the flow of air; however, when the distal tip of the particulate comes in contact with a cell or mucus layer, deposition is facilitated. Particles smaller than 0.2 µm are deposited in the smaller airways and alveoli and are influenced mainly by their diffusion coefficient and brownian motion. Unlike the deposition of larger particles in the upper airways, particle density does not influence diffusion of these smaller particles, and deposition is enhanced with decreased size. These smaller particles come in contact with the alveolar epithelium, where cilia and the mucociliary transport system do not exist; thus they are removed by the phagocytic activity of alveolar macrophages or absorption into the interstitium with subsequent clearance by lymphatic drainage. Although most alveolar macrophages are adjacent to the epithelium of the alveolus, some are located in the terminal airways and interstitial space.

In the conducting airways, the mucociliary clearance system transports deposited particles from the terminal bronchioles to the major airways, where they are coughed up and either expectorated or swallowed. Deposited particles can be removed in a matter of minutes to hours. In the trachea and main bronchi, the rate of particle clearance is 5 to 20 µm/minute, but it is slower in the bronchioles (0.5 to 1 µm/minute). In general, the longer an inhaled material remains in the airways, the greater is the probability that the material will cause lung damage. The region from the terminal bronchioles to the alveoli is devoid of ciliated cells and is considered the “Achilles heel” in what is otherwise a highly effective system. The relatively slow rate of particle clearance in this area, which is mediated by macrophages, renders it the most common location for many occupational lung diseases.

Insomelungdiseases—forexample,thosecausedbyinhalationofsilicaparticles(silicosis) orcoaldustparticles(pneumoconiosis, the“blacklung”diseaseofcoalminers)—alveolarmacrophagesphagocytizetheparticlesbutareunabletodestroythem,andthemacrophageseventuallydie.Alveolarmacrophageshavelocalizedandconcentratedtheparticlesinthe“Achillesheel”regionofthelung.Theseparticlesarenotremovedviamucociliaryclearanceandeventuallyenterthelunginterstitium.Theensuinginflammatoryresponseleadstoagranulomatouslikelesionwithfibrosis,arestrictivelungdisease.Silicosisandpneumoconiosisareclassicalexamplesofdiseasesoriginatingthroughenvironmentalworkplaceexposure.Increasedawarenessofthecauseofthesediseasesandimprovedworkplaceenvironmentshaveledtoreductionintheincidenceofthesetypesoflungdiseases.

Mucosal Immune System: Adaptive and Innate Immunity

In nonmucosal tissues (e.g., spleen, liver, kidney), the body’s primary defense is the classical proinflammatory adaptive, antigen-specific immune response orchestrated in the local draining lymph nodes with afferent and efferent lymph flow. The major adaptive immune cells in the systemic immune system are T lymphocytes with αβ T cell receptors (TCRαβ T cells) for specific antigen recognition and plasma B cells that synthesize immunoglobulin M (IgM) and immunoglobulin G (IgG) complement–binding antibodies, which can induce inflammation. However, mucosal tissues (i.e., those of the respiratory, gastrointestinal, and urinary systems, as well as the eyes, nose, throat, and mouth) must constantly discriminate between what is harmful and what is not, and although inflammation is protective, it usually disrupts the normal physiologic processes and is not desirable unless absolutely necessary. Accordingly, mucosal tissues have developed specialized “noninflammatory” defense mechanisms, which form the basis of the mucosal immune system and can function independently of the systemic immune system. The mucosal immune system contains both specialized innate lymphoid cells (macrophages, natural killer cells, dendritic cells [DCs]) and adaptive T lymphocytes with γδ T cell receptors (TCRγδ T cells) and plasma B cells that synthesize immunoglobulin A (IgA), a nonclassical complement-binding antibody. These innate and unique adaptive responses can prevent or limit responses to foreign nonpathological agents while eliminating pathological agents/substances with little or no inflammation. In addition, if this front-line defense system fails or is bypassed, the lungs do have a classical adaptive immune response system in which lymphatic drainage is via the mediastinal lymph node located in the upper region of the thoracic cavity adjacent to the main left-right lung bifurcation.

A distinctive feature of the mucosal immune system is that antigens are processed through lymphoid aggregates rather than through a true lymph node. Unlike a true lymph node, which has afferent and efferent lymph flow, lymphoid aggregates have only afferent drainage of material into the aggregate without efferent flow. This lymphoid network is commonly referred to as mucosa-associated lymphoid tissue (MALT); in the gastrointestinal tract, it is referred to as gut-associated lymphoid tissue (GALT); and in the lungs, it is known as bronchus-associated lymphoid tissue (BALT;

Fig. 26.2 ). BALT is present in the conducting airways, where the epithelium is composed mainly of ciliated cells with consistent mucus flow. However, an interesting feature of BALT is that its airway epithelium is not ciliated; it is referred to as a lymphoepithelium, which creates a break in the mucus flow (like a drain) and allows the substances/ particulates to be processed in the lymphoid aggregate (or follicle).

It appears that there is communication between mucosal tissues and that sensitization via one organ is transposed to all MALT/BALT tissues via a lymphatic-like drainage network. The systemic immune system and MALT/BALT may work independently of each other, and the fact that one is sensitized may not be true of the other. This may serve as a defense mechanism in limiting sensitization only to mucosal tissue. Lymphocytes also cluster in smaller numbers and density in what is referred to as tertiary ectopic lymphoid tissue (TELT), in which they can also process antigens (see

Fig. 26.2, A ). Another prominent feature is a diffuse submucosal and intraepithelial network of solitary lymphocytes and innate lymphoid cells scattered throughout the respiratory tract. Because inhaled particles are broadly dispersed throughout the respiratory tract, each type of lymphoid cell and tissue (BALT, TELT, and solitary lymphocytes) play important and unique roles in the overall defense of the lungs.

One of the specialized features of MALT, GALT, and BALT is a unique antibody system in which specialized features • Fig. 26.2 Representation of Bronchus-Associated Lymphoid Tissue (BALT)/Tertiary Ectopic Lymphoid Tissue (TELT), M Cells, and Immunoglobulin a (IgA) Synthesis.A, M cells located in mucosal epithelium endocytose antigen in the lumen and transport it for processing to loosely organized submucosal pockets of lymphoid cells, predominantly lymphocytes and macrophages (TELT). B, Diagram of a mucous membrane, showing secretion of IgA antibodies in response to antigen endocytosed by M cells. Activated B cells differentiate into IgA-producing plasma cells (lymphocytes) and migrate from the densely organized lymphoid follicle (in bronchus-associated lymphoid tissue [BALT]) to the nearby submucosa, where they secret IgA.

of the IgA antibody (non–complement-fixing, J chain for transport, and dimeric structure for stability in the airway lumen) are used. In submucosal areas, plasma cells synthesize and secrete IgA, which migrates to the submucosal surface of epithelial cells, where it binds to a surface protein •Fig. 26.3 Structure and Formation of Secretory Immunoglobulin a (IgA). A, SecretoryIgAconsistsofatleasttwoIgAmoleculesthatarecovalentlylinkedviaJchainandcovalentlyassociatedwiththesecretorycomponent.Thesecretorycomponentcontainsfiveimmunoglobulin-likedomainsandislinkedtodimericIgAthroughbindingtoanIgAheavychain.B, SecretoryIgAisformedduringtransportthroughepithelialcells.Poly-Ig,polymericimmunoglobulin.

receptor, polymeric immunoglobulin (poly-Ig;

Fig. 26.3 ). The poly-Ig receptor aids in the pinocytosis of IgA into the epithelial cell and its eventual secretion into the airway lumen. During exocytosis of the IgA complex, the poly-Ig is enzymatically cleaved, and a portion of it, the secretory piece, remains associated with the complex. The secretory piece stays attached to the IgA complex in the airway and helps protect it from proteolytic cleavage. The IgA-antigen immune complex does not bind complement in the same classical manner as do other immune complexes, and thus its proinflammatory properties are limited. The IgA-antibody system is very effective in binding particulates and viruses to form a large complex, which promotes its removal via the mucociliary clearance system, before they invade epithelial cells.

Most classical adaptive immune T lymphocytes are CD3+ cells with TCRs that are composed of α and β chains (TCRαβ T cells). These cells mature in the thymus and egress mostly to lymph nodes and the spleen. The classical activation of TCRαβ cells requires antigen processing/ presentation, typically via the major histocompatibility complex, in a DC, usually to induce an inflammatory response. CD3+ T lymphocytes with TCRs expressing γ and δ chains (TCRγδ T cells) also mature in the thymus, but the majority of these cells egress to mucosal tissues (i.e., lung, intestines, and skin) and represent only a minority of T cells in the peripheral blood and systemic lymphoid tissues. TCRγδ T cells, often referred to as intraepithelial lymphocytes, preferentially localize to submucosal sites and

BALT,bronchus-associatedlymphoidtissue;IFN-γ,interferonγ;IgA,IgE,IgG,andIgM,immunoglobulinsA,E,G,andM;IL-10,interleukin10;iNKT,invariantnaturalkillerT;NK,naturalkiller;TCR,Tcellreceptor;TELT,tertiaryectopiclymphoidtissue;Th1andTh2,Thelper1andThelper2.

epithelium and are considered a first line of defense of epithelial surfaces.

In contrast to TCRαβ T cells, the classical antigen activation of TCRγδ T cells does not require antigen processing or presentation by DCs. Of interest is that TCRγδ T cells have been shown to be capable of responding to antigen either in the manner of an innate cellular response, via pathogen-associated molecular patterns (PAMPs, described later in this chapter), or, in some circumstances, in the manner of a classical TCRαβ T cell response. Whether this variation in responsiveness is due to the presence of subpopulations of TCRγδ T cells or to plasticity (alterations) of the same cell is not clear. Because these cells express this unique duality, they are considered to be a link between adaptive and innate immune responses. Furthermore, TCRγδ cells can respond immediately to antigen and generally do not demonstrate memory. In contrast to the typical TCRαβ T cell proinflammatory response, TCRγδ T cells can provide protection without inducing inflammation. TCRγδ T cells have been shown to expand in response to viral and bacterial infections and synthesize either proinflammatory cytokines (interleukin [IL]–17, interferon [IFN]–γ) or regulatory/ suppressive cytokines (transforming growth factor β [TGFβ], lymphocyte-activation gene 3). TCRγδ T cells also suppress the immunoglobulin E (IgE) response to inhaled antigen, further preventing allergen-induced inflammation.

Innate lymphoid cells (ILCs) generally fall into four categories, TCRγδ T cells, dendritic cells (DCs), macrophages, natural killer (NK) cells, and subpopulations of each ( 26.3 ). ILCs are distinctively different from adaptive immune cells by their lack of “antigen” specificity for the offending agents and their ability to distinguish self from non-self through recognition of PAMPs present on the pathogens. Since most inhaled substances are nonpathogenic, the body has developed a specialized recognition system to identify harmful pathogenic substances and organisms. Rather than specific antigen recognition, this system enables discrimination of self from non-self material through the recognition of PAMPs on the pathogenic organisms/substances, which are then recognized by a family of receptors on host defense cells (i.e., ILCs) called pattern recognition receptors (PRRs). PAMPs are common distinguishing features on many pathogens and are composed of peptidoglycans, lipopolysaccharides, and unmethylated 5′-C-phosphate-G-3′ (CpG) DNA, which bind to their corresponding PPRs on ILCs. There are two major classifications for PRRs: toll-like receptors (TLRs), which are expressed mainly on cell surfaces, and nucleotide-oligomerization domain (NOD) receptors, which are expressed intracellularly in the cytoplasma of ILCs. Both of these pathways activate nuclear factor Kβ (NFKβ), a transcription factor, which stimulates release of proinflammatory cytokines, as well the antiviral type I interferons (IFN-α and IFN-β). The TLRs are a family of transmembrane proteins with different specificities for various pathogens. TLR-2 is specific for lipoteichoic acids associated with gram-positive bacteria, whereas TLR-4 is specific for lipopolysaccharide (endotoxin), a product of gram-negative bacteria. In the lung, bronchial epithelial cells, macrophages, DCs, mast cells, eosinophils, and alveolar type II epithelial cells express both TLR-2 and TLR-4. Other TLRs are specific for viruses: TLR-3 binds to double-stranded RNA viruses, and TLR-7 and TLR-8 bind to single-stranded RNA viruses. NK cells express TLR-3, TLR-7, and TLR-8 and plasmacytoid DCs, eosinophils, and B cells express TLR-7. A variety of phagocytic cells and DCs in the lung and other mucosal tissues also express TLRs. Thus in addition to classical phagocytic cells, bronchial and alveolar epithelial cells play active roles in host defense by means of the PAMP-PRR nexus.

Perhaps the most unique and intriguing aspect of this specialized mucosal defense system is the ability of ILCs to respond to pathogens immediately, as opposed to the days or weeks it takes to mount a classical TCRαβ T cell adaptive immune response with clonal expansion and memory. The fast response, and the lack thereof for innocuous substances, are highly advantageous in the mucosal tissues, which are common sites for parasitic invasion and toxic chemical exposure. In addition, many of the ILCs demonstrate plasticity, meaning that they can be induced by the environment (i.e., cytokines) to alter their functionality to either a proinflammatory or regulatory (suppressive) phentoype.

Both macrophages and DCs originate in the bone marrow but vary somewhat in their differentiation lineage: Macrophages develop through the common myeloid-granulocyte/ macrophage progenitor linage with other granulocytic cells (basophils, mast cells, eosinophils, neutrophils), and DCs develop through either the common lymphoid progenitor or the common myeloid progenitor lineages. Macrophages and DCs are the first nonepithelial cells to contact and respond to a foreign substance. In the lungs, there are three major types of macrophages (alveolar, M-1 [proinflammatory], and M-2 [regulatory]) and two types of DCs (conventional and plasmacytoid).

If the inhaled foreign material stays within the airspace in the lower respiratory system (alveolar ducts and alveoli), it will probably be phagocytized by alveolar macrophages. However, if the foreign material/organism penetrates and reaches submucosal areas, it will come into contact with M-1 or M-2 macrophages and conventional or plasmacytoid DCs. Alveolar macrophages are found mostly in the alveolus adjacent to the epithelium and less frequently in the terminal airways and interstitial space. They migrate freely throughout the alveolar spaces and serve as a first line of defense in the terminal bronchioles and alveoli. They phagocytize foreign particles and substances, as well as surfactant and cellular debris from dead cells. For a particle/organism that is phagocytized by a macrophage, the major mechanisms of destruction include formation of O2 radicals, enzyme activity, and halogen derivatives within lysosomes.

The phagocytic activity of the macrophage inhibits the binding of particulates to the alveolar epithelium and their subsequent penetration into the interstitium. The alveolar macrophage also transports engulfed particles to ciliated regions of the mucociliary transport system for elimination and thus provides an important link between the alveolar spaces, the postterminal bronchiole “Achilles heel” region, and the mucociliary clearance system.

In addition, alveolar macrophages and M-2 macrophages present in the submucosa can also suppress T cell activity by direct contact or by the secretion of soluble factors such as nitric acid, prostaglandin E2, and the immunosuppressive cytokines (IL-10 and TGF-β). M-1 macrophages are also located in submucosal sites and represent the classical “proinflammatory” phagocytic cell, with similar killing capabilities as those of alveolar macrophages. In addition, the T helper 2 (Th2) proinflammatory (and proasthma) cytokines IL-4 and IL-13 have been shown to promote the differentiation of M-1 macrophages into regulatory/suppressive M-2 macrophages, which demonstrates the plasticity of these cells. The ability of these macrophage populations to demonstrate plasticity and dispose of foreign material rapidly with either a proinflammatory or regulatory response considerably enhances the lung defense system and is a unique contributor to the overall mucosal defense system.

Conventional DCs reside in the submucosa of lung and other mucosa tissues and are considered the major antigen-presenting cells. They are usually in a resting immature state in which they function as sentinels to capture and process antigen, after which they mature and migrate to the local draining lymph node (mediastinal node for the lungs). At this node, they present antigen to T cells, which initiates an adaptive immune response, either proinflammatory or suppressive, depending on the antigen and DC. CD103+ DCs have been shown to induce regulatory/suppressor T cells by means of their synthesis and secretion of TGF-β and indoleamine dioxygenase. Plasmacytoid DCs are located in similar submucosal areas but function more in an antiviral role, as opposed to antigen presentation; upon viral activation, they rapidly secrete the antiviral cytokine IFN-γ. Macrophages and DCs commonly synthesize and secrete many similar cytokines, depending on the stimulus; such cytokines include IL-1β (which activates vascular endothelium and lymphocytes), IL-6 (which activates lymphocytes and enhances antibody production), IL-12 (which activates NK cells, CD4 T cells to Th1 T cells), tumor necrosis factor α (which activates vascular endothelium and increases permeability), IL-8 (which recruits neutrophis, basophils, and T cells), and IFN-γ (which has antiviral effects).

NK cells originate in the bone marrow and differentiate through the common lymphoid progenitor lineage . They are neither T or B cells, and resident populations of functionally active NK and invariant natural killer T (iNKT) cells are present in mucosal sites and the lung interstitium. NK cells are a major component of the body’s innate immune system of defense against invading pathogens such as herpesviruses, various bacterial infections, and tumor cells. NK cells can be activated by interferons (IFN-α and IFN-β) and other macrophage-derived cytokines (IL-12 and IL-18), which enhances their killing capabilities. The mechanism of killing is through the release of granular enzymes (granzymes, perforins, and serine esterases), which create holes or pores within the target cell membranes and thereby cause cell death. In addition, NK cells can synthesize IFN-γ, which is capable of inhibiting viral infections and stimulating CD4 and CD8 T cell responses.

NK cells have a complicated set of receptors to recognize a wide array of infectious bacteria and viruses. Individuals who lack NK cells are very prone to herpesvirus infections and often have recurrent infection. The iNKT cells belong to a category of lymphoid cells termed innate-like lymphocytes, reside together with TCRγδ cells in mucosal tissues, and have antigen receptors with limited diversity, unlike the adaptive immune cells. These types of cells are thought to be links between innate and adaptive immunity. A critical aspect of the link is that these cells can respond must faster than cells of the classical adaptive immune response, limiting early damage until the adaptive response can mobilize a stronger defense. The iNKT cells have an invariant T cell receptor α chain and, on activation, secrete the regulatory cytokines IL-10 and IFN-γ.

Epithelial Cells and Commensal Microbiota Protect the Lumen of the Airways

Airway epithelial cells can produce and secret both antibacterial enzymes and amphipathic (hydrophilic–positively charged) antimicrobial peptides, which generally target and disrupt cell wall components of the bacteria, which leads to cell death. Lysozyme, elastase, hydrolases, and secretory phospholipase A2 are examples of antibacterial enzymes targeted at various pathogens. Antimicrobial peptides are typically cationic peptides synthesized as propeptides and activated through cleavage of an anionic propiece. Peptides secreted from epithelial cells and alveolar macrophages usually enter the mucosal fluid, whereas peptides secreted from submucosal macrophages typically stay within the local tissue.

There are three classes of antimicrobial peptides: defensins, cathelicidins, and histatins. Defensins, which are categorized as α and β can disrupt cell membranes within minutes and are very effective on bacteria, fungi, and viruses. β–Defensins are synthesized in alveolar type II cells, are stored in lamellar bodies, and are a component of surfactant. α–Defensins are synthesized and stored within epithelial cells, macrophages, and neutrophils. Cathelicidins are stored in secondary granules and activated intracellularly by fusion of the phagosomes with the granule or also directly by elastase. In the lungs, cathelicidins are secreted from lung type II epithelial cells, as well as from neutrophils and macrophages. Histatins are present mainly in the oral cavity and are more specific for pathogenic fungi. Although it is not clear whether they exist in the respiratory tract, the carbodydrate-binding proteins (lectins) in the gut can directly kill Gram-positive bacteria by binding to their cell walls. Furthermore, lactoferrins and lysozyme present in epithelial cells have been shown to have bacteriostatic and bactericidal effects; the precise mechanism by which this occurs is not clearly understood, although it is postulated that they may work synergistically with iron and calcium.

The human microbiome, especially the gut, has received a lot of attention, and exploration into the role of these commensal microorganisms in health and disease is rapidly developing. Although much is known about the gut microbiome, the lung microbiome has been difficult to explore. Once thought to be a sterile environment, the lung remains a difficult site to sample for a variety of reasons, including subject safety during sampling (invasive procedures), complexity and variety of epithelial surfaces throughout the respiratory tract, and the simple fact that far fewer bacteria are available to sample. Furthermore, the lumen ecosystem and epithelium of the respiratory tract differ considerably along the respiratory tract, from the upper airways to the alveoli; it is likely that the density and types of bacteria residing in these various regions also differ. It is clear, however, that the lung does have a microbiome, and three primary phyla appear to be represented: Bacteroidetes (Prevotella, Bacteroides), Firmicutes (Veillonella, Streptococcus, Staphylococcus), and Proteobacteria (Pseudomonas, Haemophilus, Moraxella, Neisseria, Acinetobacter).

Alterations in the lung microbiome have been shown to be associated with several pulmonary conditions (including asthma, cystic fibrosis, and chronic obstructive pulmonary disease) and with lung transplantation. The increased rate of asthma in children has been linked to the increased rate of antibiotic use, which has a vast effect on the microbiome. Organisms in the Proteobacteria (Haemophilus, Moraxella) and Firmicutes (Streptococcus) phyla have been shown to correlate with asthma. An experimental study in mice showed an improvement in asthma when Proteobacteria organisms were replaced with Bacteriodetes organisms, which coincided with an increase in regulatory T cells and suppression of the Th2 proinflammatory cytokines prominent in the pathogenesis of asthma.

From a therapeutic standpoint, the use of probiotics to maintain a “normal” microbiome has proved useful in treating infectious diseases in the gut, and the use of probiotic therapy in patients with chronic lung infections (e.g., cystic fibrosis, upper respiratory tract infections) have been promising. The precise mechanism or mechanisms by which the bacteria may “protect” the lungs is still poorly understood but probably varies considerably according to the microorganism.

It has been shown that Bifidobacterium can activate DCs and TLRs to secrete IL-10 and retinoic acid, mediators capable of inducing regulatory T cells, which downregulate the Th2 inflammatory response. From a causative perspective, histamine, a major component of mast cell granules and secreted from Lactobacillus organisms, has been shown to have an association with priming DCs to stimulate Th2 inflammatory responses, as seen in individuals with asthma. In addition, it has been shown that ILCs can play a role in influencing the microbiome through their ability to bind to receptors on epithelial cells, thus altering the interaction of the epithelium with the microorganisms and possibly promoting colonization of either pathogenic or nonpathogenic microorganisms. Much is still to be learned and absorbed from future studies of the lung microbiome.

By far the most common pathological conditions associated with mucosal tissue are allergic responses (e.g., allergic asthma, allergic rhinitis, and food and skin allergies). Some common human allergens include components of inhaled house dust mites, cockroaches, and cat dander, as well as bee stings and ingestion of peanuts. One of the most common drug allergens is penicillin, which can bind to many endogenous proteins and also alter their antigenicity.

As previously described, the predominant antibody response in MALT is IgA; however, in an allergic response, IgE is the predominant antibody. It is generated by a switchover mechanism induced by the synthesis of IL-4 from Th2-primed CD4 T cells. The IL-4 induces the antibody-producing B cells to switch over from synthesizing IgG antibodies to IgE. IgE binds to the surface of mast cells in the submucosa through its crystallizable fragment (constant) region of the antibody molecule. Upon reexposure to the inhaled antigen and its subsequent migration from the airway lumen to the submucosa, the allergen then binds to the antigen-binding fragment region of the IgE molecule, which forms an immune complex (IgE-Ag) on the surface of the mast cell.

The final step is that IgE-Ag complexes must crosslink with other IgE-Ag complexes on the surface of the mast cell, which induce intracellular signaling pathways to initiate degranulation and immediate release of preformed Th2 mediators (histamine, heparin, prostaglandins, leukotrienes, IL-4, IL-5, IL-13, proteases). These mediators induce the classical signs of asthma: smooth muscle constriction

The inflammatory response can resolve spontaneously or as a result of therapy (bronchodilator or anti-inflammatory drugs, such as corticosteroids). Low-grade inflammation may persist and result in a process called airway remodeling, manifested by permanent, irreversible structural changes such as submucosal fibrosis and airway smooth muscle hypertrophy. The mechanisms responsible for airway remodeling in allergic diseases are not clearly understood, but chemokines and cytokines such as TGF-β, a potent profibrotic cytokine, play important roles. Thus a very elegant, highly effective defensive system against infectious and parasitic organisms is “tricked” into responding to an innocuous substance, an allergen, as if it were harmful and initiates its defenses; the result is allergic airway disease.

Metabolic Functions of the Lung

The lungs are exposed to and metabolize a wide variety of xenobiotic substances. The endothelial cells within the lung capillary bed have a large surface area that receives very high blood flow, and lung endothelial cells have developed various mechanisms and cell surface receptors to metabolize xenobiotics. Most of the metabolic processing of inhaled or ingested xenobiotic compounds occurs enzymatically within the liver and intestinal tract with members of the cytochrome P-450 (CYP) enzyme families (e.g., CYP1, CYP2, CYP3). The lungs and other organs also selectively participate in the processing of xenobiotics and typically have lower levels of cytochrome P-450 enzymes. Prominent cytochrome P-450 enzymes in the lung include CYP1B1, CYP2B6, CYP2E1, CYP2J2, CYP3A5, and CYP1A1, the last of which is present in high levels in people who smoke cigarettes.

Drugs for the treatment of asthma and chronic obstructive pulmonary disease—such as corticosteroids, long-acting β2 receptor agonists, leukotriene receptor antagonists, and methylxanthines—are degraded enzymatically in the lungs. In addition, a wide array of endogenous substances are metabolized by endothelial cells within the pulmonary capillary bed, including vasoactive amines, cytokines, lipid mediators, and proteins.

Box 26.1 provides a list of compounds metabolized in the lung. Metabolism can occur • BOX 26.1 Compounds Metabolized in the Lungs (bronchoconstriction), eosinophil recruitment and activa-Vasoactiveaminestion (inflammation), and connective tissue remodeling. Cytokines

Symptoms of wheezing, coughing, and shortness of breath occur within minutes, followed by a late response of eosinophilia and airway inflammation.

through either intracellular or extracellular processing of endogenous substances that pass though the capillaries or by direct synthesis and secretion by endothelial cells. For example, angiotensin I is activated by angiotensinconverting enzyme, which is on the surface of endothelial cells. Serotonin, a vasoconstrictor, binds to a specific receptor on the surface of endothelial cells and is internalized and metabolized inside cells. Approximately 80% of the serotonin entering the lungs is metabolized in a single pass through the pulmonary capillary bed.

Endothelial cells also have surface receptors for bradykinin, tumor necrosis factor, components of the complement system, immunoglobulin crystallizable fragments, and adhesion molecules. In addition, vascular endothelial cells synthesize and secrete prostacyclin, endothelin, clotting factors, nitric oxide, prostaglandins, and cytokines. Vascular endothelial cells, however, lack 5-lipoxygenase and are not able to synthesize leukotrienes (smooth muscle constrictors). Compounds not metabolized by the pulmonary capillary bed include epinephrine, dopamine, histamine, isoproterenol, angiotensin II, and substance P.

Angiotensin-convertingenzyme(ACE)ispresentinsmallindentations(caveolae)onthesurfaceofpulmonaryendothelialcellsandcatalyzestheconversionofthephysiologicalinactiveangiotensinItotheactiveangiotensinII,apotentvasoconstrictor.Thisisamajormechanisminthebody’sabilitytosupplysystemiclevelsofangiotensinIIandthusinfluencebloodpressure(see

Chapter36).

ThetherapeuticuseofACEinhibitorsisimportantinthemanagementofpatientswithhighbloodpressure.

Thelungsplayakeyroleinthemetabolismofmanyprodrugs,whichareinactive,intoactivedrugs.Administrationofprodrugsimprovestheamountofactivedrugdeliveredtotheirtargetsinthebody.Inmanycases,inactiveprecursorprodrugsaredeliveredsystemicallyorlocally(throughinhalation)tothelungs,wheretheyareactivatedinsitu.Anexampleofthisisbeclomethasonedipropionate(Qvar,BeconaseAQ),amedicationinhaledbypatientswithasthma,whichisactivatedbyesteraseswithinthelungtotheactiveform17-beclamethasonemonopropionate.

1.

The respiratory system has developed unique structural (mucociliary transport system) and immunological (mucosal immune system) features to cope with the constant environmental exposure to foreign substances; these features limit or inhibit inflammation.

2.

The three components of the mucociliary transport system are the sol phase (periciliary fluid), the gel phase (mucus), and cilia.

3.

The depth of the periciliary fluid layer is maintained by the balance between Cl− secretion and Na+ absorption and is essential to normal ciliary beating.

4.

Mucus is a complex macromolecule composed of glycoproteins, proteins, electrolytes, and water. It has low viscosity and high elastic mechanical properties.

5.

Goblet cells, Clara cells, and the mucous and serous cells residing in the tracheobronchial glands produce mucus.

6.

Particle deposition in the lung is dependent on their size, density, and shape; the distance traveled; airflow speed; and relative humidity. The major mechanisms for particle deposition are impaction (particles larger than 10 µm, in nasal passages, and particles 2 to 10 µm, in the nasopharynx, trachea, and bronchi), sedimentation (particles 0.2 to 2 µm in size, in distal airways), interception (particles with elongated shape, in the lower airways), and brownian movement (particles smaller than 0.2 µm, in the alveoli).

7.

The respiratory system is part of the mucosal immune system, which is composed of the intestinal (GALT), the respiratory (BALT), and urinary tract systems. These systems do not contain true lymph nodes with afferent and efferent lymph flow; they are composed mainly of nonencapsulated lymph nodules without true lymphatic drainage.

8.

The nonciliated lymphoepithelium of BALT establishes a break in the mucociliary blanket that acts as a drain to facilitate the collection and immune processing of foreign particulates throughout the conducting airways.

9.

TCRγδ T cells, IgA synthesizing plasma cells, NK cells, and alveolar macrophages are highly specialized innate and adaptive immune cells unique to the anti-inflammatory defense system in the lung and other mucosal tissues.

Cua DJ, Tato CM. Innate IL-17–producing cells: the sentinels of the immune system. Nat Rev Immunol. 2010;10:479-489.

Dickson RP, Erb-Downward JR, Martinez FJ, Huffnagle GB. The microbiome and the respiratory tract. Annu Rev Physiol. 2016;78:481-504.

Martin TR, Frevert CW. Innate immunity in the lungs. Proc Am Thorac Soc. 2005;2:403-411.

McKenize AN, Spits H, Eberl G. Innate lymphoid cells in inflammation and immunity. Immunity. 2014;41:366-374.

Monticelli LA, Sonnenberg GF, Abt MC, et al. Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus. Nat Immunol. 2011;12:1045-1054.

Olsson B, Bondesson E, Borgström L, et al. Pulmonary drug metabolism, clearance, and adsorption. In: Smyth HDC, Hickey AJ, eds. Controlled Pulmonary Drug Delivery. New York: Springer; 2011:21-51.

Spits H, Artis D, Colonna M, et al. Innate lymphoid cells—a proposal for uniform nomenclature. Nat Rev Immunol. 2013;13: 145-149.

Vantourout P, Hayday A. The inbetweeners: innate-like lymphocytes. Six of the best: unique contributions of γδ T cells to immunology. Nat Rev Immunol. 2014;13:88-100.

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SECTION 6Gastrointestinal Physiology KIM E. BARRETT AND HELEN E. RAYBOULD

Chapter 27 Functional Anatomy and General Principles of Regulation in the Gastrointestinal Tract

Chapter 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal

Chapter 29 The Gastric Phase of the Integrated Response to a Meal

Chapter 30 The Small Intestinal Phase of the Integrated Response to a Meal

Chapter 31 The Colonic Phase of the Integrated Response to a Meal

Chapter 32 Transport and Metabolic Functions of the Liver

Upon completion of this chapter the student should be able to answer the following questions: 1.

What is the neural innervation of the GI tract, and how is GI function regulated?

2.

What are some examples of neural, paracrine, and humoral regulation of GI function?

he gastrointestinal (GI) tract consists of the alimentary tract from the mouth to the anus and includes the associated glandular organs that empty their contents into the tract. The overall function of the GI tract is to absorb nutrients and water into the circulation and eliminate waste products. The major physiological processes that occur in the GI tract are motility, secretion, digestion, and absorption. Most of the nutrients in the diet of mammals are taken in as solids and as macromolecules that are not readily transported across cell membranes to enter the circulation. Thus digestion consists of physical and chemical modification of food such that absorption can occur across intestinal epithelial cells. Digestion and absorption require motility of the muscular wall of the GI tract to move the contents along the tract and to mix the food with secretions. Secretions from the GI tract and associated organs consist of enzymes, biological detergents, and ions that provide an intraluminal environment optimized for digestion and absorption. These physiological processes are highly regulated to maximize digestion and absorption, and the GI tract is endowed with complex regulatory systems to ensure this occurs. In addition the GI tract absorbs drugs administered by the oral or rectal routes.

The GI tract also serves as an important organ for excretion of substances. It stores and excretes waste substances from ingested food materials and excretes products from the liver such as cholesterol, steroids, and drug metabolites (all sharing the common property of being lipid-soluble molecules).

When considering the physiology of the GI tract, it is important to remember that it is a long tube that is in contact with the body’s external environment. As such, it is vulnerable to infectious microorganisms that can enter along with food and water. To protect itself the GI tract possesses a complex system of defenses consisting of immune cells and other nonspecific defense mechanisms. In fact the GI tract represents the largest immune organ of the body. This chapter provides an overview of the functional anatomy and general principles of regulation in the GI system.

The structure of the GI tract varies greatly from region to region, but there are common features in the overall organization of the tissue. Essentially the GI tract is a hollow tube divided into major functional segments; the major structures along the tube are the mouth, pharynx, esophagus, stomach, duodenum, jejunum, ileum, colon, rectum, and anus (

Fig. 27.1 ). Together the duodenum, jejunum, and ileum make up the small intestine, and the colon is sometimes referred to as the large intestine. Associated with the tube are blind-ending glandular structures that are invaginations of the lining of the tube; these glands empty their secretions into the gut lumen (e.g., Brunner’s glands in the duodenum, which secrete copious amounts of HCO3 −). Additionally there are glandular organs attached to the tube via ducts through which secretions empty into the gut lumen—for example, the salivary glands and pancreas.

The major structures along the GI tract have many functions. One important function is storage; the stomach and colon are important storage organs for processed food (also referred to as chyme) and exhibit specialization in terms of both their functional anatomy (e.g., shape and size) and control mechanisms (characteristics of smooth muscle to produce tonic contractions) that enable them to perform this function efficiently. The predominant function of the small intestine is digestion and absorption; the major specialization of this region of the GI tract is a large surface area over which absorption can occur. The colon reabsorbs water and ions to ensure they do not get eliminated from the body. Ingested food is moved along the GI tract by the action of muscle in its walls. Separating the regions  of the GI tract are also specialized muscle structures called sphincters. These function to isolate one region from the next and provide selective retention of contents or prevent backflow, or both.

The blood supply to the intestine is important for carrying absorbed nutrients to the rest of the body. Unlike other organ systems of the body, venous drainage from the GI tract does not return directly to the heart but first enters • Fig. 27.1 General anatomy of the GI system and its division into functional segments.

the portal circulation leading to the liver. Thus the liver is unusual in receiving a considerable part of its blood supply from other than the arterial circulation. GI blood flow is also notable for its dynamic regulation. Splanchnic blood flow receives about 25% of cardiac output, an amount disproportionate to the mass of the GI tract it supplies. After a meal, blood can also be diverted from muscle to the GI tract to subserve the metabolic needs of the gut wall and also to remove absorbed nutrients.

The lymphatic drainage of the GI tract is important for the transport of lipid-soluble substances that are absorbed across the GI tract wall. As we will see later, lipids and other lipid-soluble molecules (including some vitamins and drugs) are packaged into particles that are too large to pass into the capillaries and instead pass into lymph vessels in the intestinal wall. These lymph vessels drain into larger lymph ducts, which finally drain into the thoracic duct and thus into the systemic circulation on the arterial side. This has major physiological implications in lipid metabolism and also in the ability of drugs to be delivered straight into the systemic circulation.

The wall of the tubular gut is made up of layers consisting of specialized cells (

Fig. 27.2

The mucosa is the innermost layer of the GI tract. It consists of the epithelium, the lamina propria, and the muscularis mucosae. The epithelium is a single layer of specialized cells that line the lumen of the GI tract. It forms a continuous layer along the tube and with the glands and organs that drain into the lumen of the tube. Within this cell layer are • Fig. 27.2 General organization of the layers composing the wall of the GI tract.

a number of specialized epithelial cells; the most abundant are cells termed absorptive enterocytes, which express many proteins important for digestion and absorption of macronutrients. Enteroendocrine cells contain secretory granules that release regulatory peptides and amines to help regulate GI function. In addition, cells in the gastric mucosa are specialized for production of protons, and mucin-producing cells throughout the GI tract produce a glycoprotein (mucin) that helps protect the GI tract and lubricate the luminal contents.

The columnar epithelial cells are linked together by intercellular connections called tight junctions. These junctions are complexes of intracellular and transmembrane proteins, and the tightness of these junctions is regulated throughout the postprandial period. The nature of the epithelium varies greatly from one part of the digestive tract to another, depending on the predominant function of that region. For example, the intestinal epithelium is designed for absorption; these cells mediate selective uptake of nutrients, ions, and water. In contrast, the esophagus has a squamous epithelium that has no absorptive role. It is a conduit for transportation of swallowed food and thus needs some protection, provided by the squamous epithelium, from rough food such as fiber.

The surface area of the epithelium is arranged into villi and crypts (Fig. 27.3 ). Villi are finger-like projections that serve to increase the surface area of the mucosa. Crypts are invaginations or folds in the epithelium. The epithelium lining the GI tract is continuously renewed and replaced by dividing cells; in humans this process takes about 3 days. These proliferating cells are localized to the crypts, where there is a proliferative zone of intestinal stem cells.

•Fig. 27.3 Comparisonofthemorphologyoftheepitheliumofthesmallintestineandcolon.

The lamina propria immediately below the epithelium consists largely of loose connective tissue that contains collagen and elastin fibrils (see

Fig. 27.2 ). The lamina propria is rich in several types of glands and contains lymph vessels and nodules, capillaries, and nerve fibers. The muscularis mucosae is the thin innermost layer of intestinal smooth muscle. When seen through an endoscope, the mucosa has folds and ridges that are caused by contractions of the muscularis mucosae.

The next layer is the submucosa (see

Fig. 27.2 ), which consists largely of loose connective tissue with collagen and elastin fibrils. In some regions of the GI tract, glands (invaginations or folds of the mucosa) are present in the submucosa. The larger nerve trunks, blood vessels, and lymph vessels of the intestinal wall lie in the submucosa, together with one of the plexuses of the enteric nervous system (ENS), the submucosal plexus.

The muscularis externa, or muscularis propria, typically consists of two substantial layers of smooth muscle cells: an inner circular layer and an outer longitudinal layer (see

Fig. 27.2 ). Muscle fibers in the circular muscle layer are oriented circumferentially, whereas muscle fibers in the longitudinal muscle layer are oriented along the longitudinal axis of the tube. In humans and most mammals, the circular muscle layer of the small intestine is subdivided into an inner dense circular layer that consists of smaller, more closely packed cells, and an outer circular layer. Between the circular and longitudinal layers of muscle lies the other plexus of the ENS, the myenteric plexus. Contractions of the muscularis externa mix and circulate the contents of the lumen and propel them along the GI tract.

The wall of the GI tract contains many interconnected neurons. The submucosa contains a dense network of nerve cells called the submucosal plexus (also referred to as Meissner’s plexus). The prominent myenteric plexus (Auerbach’s plexus) is located between the circular and longitudinal smooth muscle layers. These intramural plexuses constitute the ENS. The ENS helps integrate the motor and secretory activities of the GI system. If the sympathetic and parasympathetic nerves to the gut are cut, many motor and secretory activities continue because the ENS directly controls these processes.

The serosa, or adventitia, is the outermost layer of the GI tract and consists of a layer of squamous mesothelial cells (see

Fig. 27.2 ). It is part of the mesentery that lines the surface of the abdominal wall and suspends the organs within the abdominal cavity. The mesenteric membranes secrete a thin viscous fluid that helps lubricate the abdominal organs so movement of the organs can occur as the muscle layers contract and relax.

Regulatory Mechanisms in the Gastrointestinal Tract

Unlike the cardiovascular or respiratory systems, the GI tract undergoes periods of relative quiescence (intermeal period) and periods of intense activity after the intake of food (postprandial period). Consequently the GI tract has to detect and respond appropriately to food intake. In addition the macronutrient content of a meal can vary considerably, and there have to be mechanisms that can detect this and mount appropriate physiological responses. Thus the GI tract has to communicate with associated organs such as the pancreas. Finally, because the GI tract is essentially a long tube, there have to be mechanisms by which events occurring in the proximal portion of the GI tract are signaled to the more distal parts, and vice versa.

There are three principal control mechanisms involved in the regulation of GI function: endocrine, paracrine, and neurocrine (Fig. 27.4

Endocrine regulation describes the process whereby the sensing cell in the GI tract, an enteroendocrine cell (EEC), responds to a stimulus by secreting a regulatory peptide or hormone that travels via the bloodstream to target cells removed from the point of secretion. Cells responding to a GI hormone express specific receptors for the hormone. Hormones released from the GI tract have effects on cells located in other regions of the GI tract and also on glandular structures associated with the GI tract, such as the pancreas. In addition, GI hormones have effects on other tissues that have no direct role in digestion and absorption, including endocrine cells in liver and brain.

EECs are packed with secretory granules, the products of which are released from the cell in response to chemical and mechanical stimuli to the wall of the GI tract (Fig.

27.5 ). In addition, EECs can be stimulated by neural input or other factors not associated with a meal. The most common EECs in the gut wall are referred to as the “open” type; these cells have an apical membrane that is in contact with the lumen of the GI tract (generally regarded as the location where sensing occurs) and a basolateral membrane through which secretion occurs. There are also “closed”-type EECs that do not have part of their membrane in contact with the luminal surface of the gut; an example is the enterochromaffin-like (ECL) cell in the gastric epithelium, which secretes histamine.

There are many examples of hormones secreted by the GI tract (see

Table 27.1 ); it is worth remembering that the first hormone ever identified was the GI hormone secretin. One of the most well-characterized GI hormones is gastrin, which is released from endocrine cells located in the wall of the distal part of the stomach. Release of gastrin is stimulated by activation of parasympathetic outflow to the GI tract, and gastrin potently stimulates gastric acid secretion in the postprandial period.

•Fig. 27.4 ThethreemechanismsbywhichfunctionintheGItractisregulatedintheintegratedresponsetoameal.

Paracrine regulation describes the process whereby a chemical messenger or regulatory peptide is released from a sensing cell (often an EEC) in the intestinal wall that acts on a nearby target cell by diffusion through the interstitial space. Paracrine agents exert their actions on several different cell types in the wall of the GI tract, including smooth muscle cells, absorptive enterocytes, secretory cells in glands, and even on other EECs. There are several important paracrine agents, and they are listed in

Table 27.1 along with their site of production, site of action, and function. An important paracrine mediator in the gut wall is histamine. In the stomach, histamine is stored and released by ECL cells located in the gastric glands. Histamine diffuses through the interstitial space in the lamina propria to neighboring parietal cells and stimulates production of acid. Serotonin (5-hydroxytryptamine [5-HT]), released from enteric neurons, mucosal mast cells, and specialized EECs called enterochromaffin cells, regulates smooth muscle function and water absorption across the intestinal wall. There are other paracrine mediators in the gut wall, including prostaglandins, adenosine, and nitric oxide (NO); the functions of these mediators are not well described, but they are capable of producing changes in GI function.

Fig. 27.5 Electronmicrographofanopen-typeendocrinecellintheGItract.Notethemicrovilliattheapicalprojectionandthesecretorygranulesinthebasolateralportionofthecell.(FromBarrett

K.

Gastrointestinal Physiology [Lange Physiology Series]. NewYork:McGraw-Hill;2005.)(CourtesyofLeonardR.Johnson,Ph.D.)

Many substances can be both paracrine and endocrine regulators of GI function. For example, cholecystokinin, which is released from the duodenum in response to dietary protein and lipid, acts locally on nerve terminals in a paracrine fashion and also affects the pancreas. This will be discussed in more detail in

Neural Regulation of Gastrointestinal Function

Nerves and neurotransmitters play an important role in regulating the function of the GI tract. In its simplest form, neural regulation occurs when a neurotransmitter is released from a nerve terminal located in the GI tract and the neurotransmitter has an effect on the cell that is innervated. However, in some cases there are no synapses between motor nerves and effector cells in the GI tract. Neural regulation of GI function is very important within an organ, as well as between distant parts of the GI tract.

Therearemultiplereceptorsubtypesfortheregulatorypeptidehormonesreleasedfromendocrinecellsinthewallofthegut.Theselectivityofreceptorstopeptidehormonesisdeterminedbyposttranslationalmodifications,whichthenconfersreceptorselectivity.AnexampleofthisispeptideYY(PYY).TherearemultiplereceptorsubtypesforPYY,classifiedasY1toY7.PYYisreleasedfromendocrinecellsinthewallofthegut,mainlyinresponsetofattyacids.Itisreleasedasa36–aminoacidpeptideandbindstotheY1,Y2,andY5receptors;however,itcanbecleavedtoPYY3-36bytheenzymedipeptidylpeptidaseIV,amembranepeptidase.ThisformofthepeptideismoreselectivefortheY2receptor.ThusthepresenceoftheenzymethatcleavesthepeptidecanalterthebiologicalresponsetoPYYsecretion.

Glucagon-likepeptide1(GLP-1)isaregulatorypeptidereleasedfromEECscellsinthegutwallinresponsetothepresenceofluminalcarbohydrateandlipids.GLP-1arisesfromdifferentialprocessingoftheglucagongene,thesamegenethatisexpressedinthepancreasandthatgivesrisetoglucagon.GLP-1isinvolvedinregulationofthebloodglucoselevelviastimulationofinsulinsecretionandalsoinsulinbiosynthesis.AgonistsoftheGLP-1receptorimproveinsulinsensitivityindiabeticanimalmodelsandhumansubjects.AdministrationofGLP-1alsoreducesappetiteandfoodintakeanddelaysgastricemptying,responsesthatmaycontributetoimprovingglucosetolerance.Long-actingagonistsfortheGLP-1receptor(e.g.,exenatide)havebeenapprovedforthetreatmentoftype2diabetes.

Neural regulation of the GI tract is surprisingly complex. The gut is innervated by two sets of nerves, the extrinsic and intrinsic nervous systems. The extrinsic nervous system is defined as nerves that innervate the gut, with cell bodies located outside the gut wall; these extrinsic nerves are part of the autonomic nervous system (ANS). The intrinsic nervous system, also referred to as the enteric nervous system, has cell bodies that are contained within the wall of the gut (submucosal and myenteric plexuses). Some GI functions are highly dependent on the extrinsic nervous system, yet others can take place independently of the extrinsic nervous system and are mediated entirely by the ENS. However, extrinsic nerves can often modulate intrinsic nervous system function (

Fig. 27.6 •Fig. 27.6 HierarchicalneuralcontrolofGIfunction.StimulitotheGItractfromameal(e.g.,chemical,mechanical,osmotic)willactivateboththeintrinsicandextrinsicsensory(afferent)pathways,whichinturnwillactivatetheextrinsicandintrinsicneuralreflexpathways.

Extrinsic neural innervation to the gut is via the two major subdivisions of the ANS, namely, parasympathetic and sympathetic innervation (

Fig. 27.7 ). Parasympathetic innervation to the gut is via the vagus and pelvic nerves. The vagus nerve, the 10th cranial nerve, innervates the esophagus, stomach, gallbladder, pancreas, first part of the intestine, cecum, and the proximal part of the colon. The pelvic nerves innervate the distal part of the colon and the anorectal region, in addition to the other pelvic organs that are not part of the GI tract.

Consistent with the typical organization of the parasympathetic nervous system, the preganglionic nerve cell bodies lie in the brainstem (vagus) or the sacral spinal cord (pelvic). Axons from these neurons run in the nerves to the gut (vagus and pelvic nerves, respectively), where they synapse with postganglionic neurons in the wall of the organ, which in this case are enteric neurons in the gut wall. There is no direct innervation of these efferent nerves to effector cells within the wall of the gut; the transmission pathway is always via a neuron in the ENS.

Consistent with transmission in the ANS, the synapse between preganglionic and postganglionic neurons is an obligatory nicotinic synapse. That is, the synapse between preganglionic and postganglionic neurons is mediated via acetylcholine released from the nerve terminal and acting at nicotinic receptors localized on the postganglionic neuron, which in this case is an intrinsic neuron.

Sympathetic innervation is supplied by cell bodies in the spinal cord and fibers that terminate in the prevertebral ganglia (celiac, superior, and inferior mesenteric ganglia); these are the preganglionic neurons. These nerve fibers synapse with postganglionic neurons in the ganglia, and the fibers leave the ganglia and reach the end organ along the major blood vessels and their branches. Rarely there is a synapse in the paravertebral (chain) ganglia, as seen with sympathetic innervation of other organ systems. Some vasoconstrictor sympathetic fibers directly innervate blood vessels of the GI tract, and other sympathetic fibers innervate glandular structures in the wall of the gut.

The ANS, both parasympathetic and sympathetic, also carries the fibers of afferent (toward the central nervous system [CNS]) neurons; these are sensory in nature. The cell bodies for the vagal afferents are in the nodose

SuperiorcervicalganglionPrevertebral ganglia1. Celiac2. Superior mesenteric3. Inferior mesenteric 123 •Fig. 27.7 ExtrinsicinnervationoftheGItract,consistingoftheparasympathetic(A)andsympathetic(B)subdivisionsoftheautonomicnervoussystem.

ganglion. These neurons have a central projection terminating in the nucleus of the tractus solitarius in the brainstem and the other terminal in the gut wall. The cell bodies of the spinal afferent neurons that run with the sympathetic pathway are segmentally organized and are found in the dorsal root ganglia. Peripheral terminals of the spinal and vagal afferents are located in all layers of the gut wall, where they detect information about the state of the gut. Afferent neurons send this information to the CNS. Information sent to the CNS relays the nature of the luminal contents (e.g., acidity, nutrient content, osmolality of the luminal contents), as well as the degree of stretch or contraction in smooth muscle. Afferent innervation is also responsible for transmitting painful stimuli to the CNS.

The components of a reflex pathway—afferents, inter-neurons, and efferent neurons—exist within the extrinsic innervation to the GI tract. These reflexes can be mediated entirely via the vagus nerve (termed a vagovagal reflex), which has both afferent and efferent fibers. The vagal afferents send sensory information to the CNS, where they synapse with an interneuron, which then drives activity in the efferent motor neuron. These extrinsic reflexes are very important in regulating GI function after ingestion of a meal. An example of an important vagovagal reflex is the gastric receptive relaxation reflex, in which distention of the stomach results in relaxation of the smooth muscle in the stomach; this allows filling of the stomach to occur without an increase in intraluminal pressure.

In general, as with other visceral organ systems, the parasympathetic and sympathetic nervous systems tend to work in opposition. However, this is not as simple as in the cardiovascular system, for example. Activation of the parasympathetic nervous system is important in the integrative response to a meal and is discussed in the following chapters. The parasympathetic nervous system generally results in activation of physiological processes in the gut wall, although there are notable exceptions. In contrast, the sympathetic nervous system tends to be inhibitory to GI function and is more frequently activated in pathophysiological circumstances. Overall, sympathetic activation inhibits smooth muscle function. The exception to this is the sympathetic innervation of GI sphincters, in which sympathetic activation tends to induce contraction of smooth muscle. Moreover, the sympathetic nervous system is notably important in regulation of blood flow in the GI tract.

The ENS is made up of two major plexuses, which are collections of nerve cell bodies (ganglia) and their fibers, all originating in the wall of the gut (

Fig. 27.8). The myenteric plexus lies between the longitudinal and circular muscle layers, and the submucosal plexus lies in the submucosa. Interganglionic strands link neurons in the two plexuses.

Neurons in the ENS are characterized functionally as afferent neurons, interneurons, or efferent neurons, similar to neurons in the extrinsic part of the ANS. Thus all components of a reflex pathway can be contained within the ENS. Stimuli in the wall of the gut are detected by afferent neurons, which activate interneurons and then efferent neurons to alter function. In this way the ENS can act autonomously from extrinsic innervation. However, neurons in the ENS, as we have already seen, are innervated by extrinsic neurons, and thus the function of these reflex pathways can be modulated by the extrinsic nervous system. Because the ENS is capable of performing its own integrative functions and complex reflex pathways, it is sometimes referred to as the “little brain in the gut” as a result of its importance and complexity. It is estimated that there are as many neurons in the ENS as in the spinal cord. In addition, many GI hormones also act as neurotransmitters in the ENS and in the brain in regions involved in autonomic outflow. These mediators and regulatory peptides are thus • Fig. 27.8 The enteric nervous system in the wall of the GI tract.

referred to as brain-gut peptides, and the extrinsic and intrinsic components innervating the gut are sometimes referred to as the brain-gut axis.

Response of the GI Tract to a Meal

This introductory chapter provides a broad overview of the anatomy and regulatory mechanisms in the GI tract. In the following chapters there will be discussion of the integrated response to a meal to provide the details of GI physiology. The response to a meal is classically divided into phases: cephalic, oral, esophageal, gastric, duodenal, and intestinal. In each phase the meal presents certain stimuli (e.g., chemical, mechanical, and osmotic) that activate different pathways (e.g., neural, paracrine, and humoral reflexes) that result in changes in effector function (e.g., secretion and motility). There is considerable crosstalk between the regulatory mechanisms that have been outlined, and this will be discussed in the next chapters. As with maintenance of homeostasis in other systems of the body, control of GI function requires complex regulatory mechanisms to sense and act in a dynamic fashion.

1.

The GI tract is a tube subdivided into regions that subserve different functions associated with digestion and absorption.

2.

The lining of the GI tract is subdivided into layers— the mucosal, submucosal, and muscle layers.

3.

There are three major control mechanisms: hormonal, paracrine, and neurocrine.

4.

The innervation of the GI tract is particularly interesting because it consists of two interacting components, extrinsic and intrinsic.

5.

Extrinsic innervation (cell bodies outside the wall of the GI tract) consists of the two subdivisions of the

Baldwin GS. Posttranslational modification of gastrointestinal peptides. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012.

Brierley SM, et al. Innervation of the gastrointestinal tract by spinal and vagal afferent nerves. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012.

Brookes SJ, et al. Extrinsic primary afferent signalling in the gut. Nat Rev Gastroenterol Hepatol. 2013;10:286-296.

Chao C, Hellmich MR. Gastrointestinal peptides: gastrin, cholecystokinin, somatostatin and ghrelin. In: Johnson LR, ed.

ANS: parasympathetic and sympathetic. Both have an important sensory (afferent) component.

6.

The intrinsic or enteric nervous system (cell bodies in the wall of the GI tract) can act independently of extrinsic neural innervation.

7.

When a meal is in different regions of the tract, sensory mechanisms detect the presence of the nutrients and mount appropriate physiological responses in that region of the tract, as well as in more distal regions. These responses are mediated by endocrine, paracrine, and neurocrine pathways.

Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012.

Furness JB. The enteric nervous system and neurogastroenterology. Nat Rev Gastroenterol Hepatol. 2012;9:286-294.

Gomez GA, et al. Postpyloric gastrointestinal peptides. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012.

Parker HE, et al. The role of gut endocrine cells in control of metabolism and appetite. Exp Physiol. 2014;99:1116-1120.

Sandoval DA, D’Alessio DA. Physiology of proglucagon peptides: role of glucagon and GLP-1 in health and disease. Physiol Rev. 2015;95:513-548.

28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal

Upon completion of this chapter the student should be able to answer the following questions: 1.

What are the structures of the functional anatomy of salivary glands, including their secretory elements?

2.

What are the cephalic and oral phases (what, why, how it happens) of the response to a meal?

3.

What are the general principles of secretion along the gastrointestinal (GI) tract (where do secretions come from, what are the components)?

4.

How do the components of secretion vary with the gland or region of the GI tract?

5.

What is the correlation between the composition and functions of salivary secretion?

6.

How are primary and secondary secretion within salivary glands generated and regulated?

7.

What is the sequence of events in swallowing?

8.

What are the stimulus and neural pathways generating primary and secondary esophageal peristalsis?

9.

What changes in gastric motility take place during swallowing, and what is the significance?

10.

What are the major functions of the esophagus and associated structures in terms of protection and propulsion?

his chapter will describe the processes that occur in the gastrointestinal (GI) tract in the early stages of the integrated response to a meal. There are changes in GI tract physiology (1) before food is ingested (the cephalic phase), (2) when ingested food is in the mouth (the oral phase), and (3) when food is transferred from the mouth to the esophagus (the esophageal phase). The responses of the GI tract to the presence of food are mainly associated with preparing the GI tract for digestion and absorption.

The main feature of the cephalic phase is activation of the GI tract in readiness for the meal. The stimuli involved are cognitive and include anticipation or thinking about the consumption of food, olfactory input, visual input (seeing or smelling appetizing food when hungry), and auditory input. The latter may be an unexpected link but was clearly demonstrated in the classic conditioning experiments of Pavlov, in which he paired an auditory stimulus to the presentation of food to dogs; eventually the auditory stimulus alone could stimulate secretion. A real-life analogy is presumably being told that dinner is ready. All these stimuli result in an increase in excitatory parasympathetic neural outflow to the gut. Sensory input (e.g., smell) stimulates sensory nerves that activate parasympathetic outflow from the brainstem. Higher brain sites (e.g., limbic system, hypothalamus, cortex) are also involved in the cognitive components of this response. The response can be both positive and negative; thus anticipation of food and a person’s psychological status, such as anxiety, can alter the cognitive response to a meal. However, the final common pathway is activation of the dorsal motor nucleus in the brainstem, the region where the cell bodies of the vagal preganglionic neurons arise. Activation of the nucleus leads to increased activity in efferent fibers passing to the GI tract in the vagus nerve. In turn the efferent fibers activate the postganglionic motor neurons (referred to as motor because their activation results in change of function of an effector cell). Increased parasympathetic outflow enhances salivary secretion, gastric acid secretion, pancreatic enzyme secretion, gallbladder contraction, and relaxation of the sphincter of Oddi (the sphincter between the common bile duct and duodenum). All these responses enhance the ability of the GI tract to receive and digest the incoming food. The salivary response is mediated via the ninth cranial nerve; the remaining responses are mediated via the vagus nerve.

Many of the features of the oral phase are indistinguishable from the cephalic phase. The only difference is that food is in contact with the surface of the GI tract. Thus there are additional stimuli generated from the mouth, both mechanical and chemical (taste). However, many of

CHAPTER 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal the responses initiated by the presence of food in the oral cavity are identical to those initiated in the cephalic phase, because the efferent pathway is the same. The responses specifically initiated in the mouth, which consist mainly of the stimulation of salivary secretion, will be discussed next.

The mouth is important for the mechanical disruption of food and for initiation of digestion. Chewing subdivides and mixes the food with the enzymes salivary amylase and lingual lipase and with the glycoprotein mucin, which lubricates food for chewing and swallowing. Minimal absorption occurs in the mouth, although alcohol and some drugs are absorbed from the oral cavity, and this can be clinically important. However, as with the cephalic phase, it is important to realize that stimulation of the oral cavity initiates responses in the more distal GI tract, including increased gastric acid secretion, increased pancreatic enzyme secretion, gallbladder contraction, and relaxation of the sphincter of Oddi, mediated via the efferent vagal pathway.

Properties of Secretion

Secretions in the GI tract come from glands associated with the tract (salivary glands, pancreas, and liver), from glands formed by the gut wall itself (e.g., submucosal glands in esophagus and duodenum), and from the intestinal mucosa itself. The exact nature of the secretory products can vary tremendously, depending on the function of that region of the GI tract. However, these secretions have several characteristics in common. Secretions from the GI tract and associated glands include water, electrolytes, protein, and humoral agents. Water is essential for generating an aqueous environment for efficient enzyme action. Secretion of electrolytes is important for generation of osmotic gradients to drive the movement of water. Digestive enzymes in secreted fluid catalyze the breakdown of macronutrients in ingested food. Moreover, many additional proteins secreted along the GI tract have specialized functions, some of which are fairly well understood, such as those of mucin and immunoglobulins, and others that are only just beginning to be understood, such as those of trefoil peptides.

Secretion is initiated by multiple signals associated with the meal, including chemical, osmotic, and mechanical components. Secretion is elicited by the action of specific effector substances called secretagogues acting on secretory cells. Secretagogues work in one of the three ways that have already been described in

Chapter 27—endocrine, paracrine, and neurocrine.

Constituents of Secretions

Inorganic secretory components are region or gland specific, depending on the particular conditions required in that part of the GI tract. The inorganic components are electrolytes, including H+ and HCO3 − . Two examples of different secretions include acid (HCl) in the stomach, which is important to activate pepsin and start protein digestion, and HCO3 − in the duodenum, which neutralizes gastric acid and provides optimal conditions for the action of digestive enzymes in the small intestine.

Organic secretory components are also gland or organ specific and depend on the function of that region of the gut. The organic constituents are enzymes (for digestion), mucin (for lubrication and mucosal protection), and other factors such as growth factors, immunoglobulins, bile acids, and absorptive factors.

During the cephalic and oral phases of the meal, considerable stimulation of salivary secretion takes place. Saliva has a variety of functions, including those important for the integrative responses to a meal and for other physiological processes (

Box 28.1 ). The main functions of saliva in digestion include lubrication and moistening of food for swallowing, solubilization of material for taste, initiation of carbohydrate digestion, and clearance and neutralization of refluxed gastric secretions in the esophagus. Saliva also has antibacterial actions that are important for overall health of the oral cavity and teeth.

Functional Anatomy of the Salivary Glands

There are three pairs of major salivary glands: parotid, submandibular, and sublingual. In addition, many smaller glands are found on the tongue, lips, and palate. These glands are the typical tubuloalveolar structures of glands located in the GI tract (

Fig. 28.1 ). The acinar portion of the gland is classified according to its major secretion: serous (“watery”), mucous, or mixed. The parotid gland produces mainly serous secretion, the sublingual gland secretes mainly mucus, and the submandibular gland produces a mixed secretion.

Cells in the secretory end pieces, or acini, are called acinar cells and are characterized by basally located nuclei, abundant rough endoplasmic reticulum, and apically located secretory granules that contain the enzyme amylase and other secreted proteins. There are also mucous cells in the acinus; the granules in these cells are larger and contain the specialized glycoprotein mucin. There are three kinds of ducts in the gland that transport secretions from the acinus to the opening in the mouth and also modify the secretion: intercalated ducts drain acinar fluid into larger •BOX 28.1FunctionsofSalivaandChewing

Disruption of food to produce smaller particles Formation of a bolus for swallowing Initiation of starch and lipid digestion Facilitation of taste Production of intraluminal stimuli in the stomach Regulation of food intake and ingestive behavior Cleansing of the mouth and selective antibacterial action Neutralization of refluxed gastric contents Mucosal growth and protection in the rest of the GI tract Aid in speech 522 SECTION6Berne & Levy Physiology • Fig.28.1General structure of tubuloalveolar secretory glands (e.g., salivary glands, pancreas) associ-ated with the digestive tract. Demilune of serous cells Serous cell Basement membrane Intercalated duct Mucous cell Salivary duct (secretory) ducts, the striated ducts, which then empty into even larger excretory ducts. In addition, a single large duct from each gland drains saliva to the mouth. The ductal cells lining the striated ducts, in particular, modify the ionic composition and osmolarity of saliva.

Composition of Saliva

The important properties of saliva are a large flow rate relative to the mass of gland, low osmolarity, high K+ concentration, and organic constituents, including enzymes (amylase, lipase), mucin, and growth factors. The latter are not important in the integrated response to a meal but are essential for long-term maintenance of the lining of the GI tract.

The inorganic composition is entirely dependent on the stimulus and the rate of salivary flow. In humans, salivary secretion is always hypotonic. The major components are − , Ca++ , Mg++

Na+ , K+ , HCO3 , and Cl− . Fluoride can be secreted in saliva, and fluoride secretion forms the basis of oral fluoride treatment for prevention of dental caries. The concentration of ions varies with the rate of secretion; the flow rate of salivary secretion is stimulated during the postprandial period.

The primary secretion is produced by acinar cells in the secretory end pieces (or acini) and is modified by duct cells as saliva passes through the ducts. The primary secretion is isotonic, and the concentration of the major ions is similar to that in plasma. Secretion is driven predominantly by Ca++-dependent signaling, which opens apical Cl− channels in the acinar cells. Cl− therefore flows out into the duct lumen and establishes an osmotic and electrical gradient. Because the epithelium of the acinus is relatively leaky, Na+ and water then follow across the epithelium via the tight junctions (i.e., via paracellular transport). Transcellular water movement may also occur, mediated by aquaporin 5 water channels. The amylase content and rate of fluid secretion vary with the type and level of stimulus. As the fluid passes along the ducts, the excretory and striated duct cells modify the ionic composition of the primary secretion to produce the secondary secretion. The duct cells reabsorb Na+ and Cl− , and secrete K+ and HCO3 − into the lumen. Na+ is exchanged for protons, but some of the secreted protons are then reabsorbed in exchange for K+ . HCO3 − on the other hand is secreted only in exchange for Cl−, thereby alkalinizing salivary secretion.

At rest, final salivary secretion is hypotonic and slightly alkaline. The alkalinity of saliva is important in restricting microbial growth in the mouth, as well as in neutralizing refluxed gastric acid once the saliva is swallowed. When salivary secretion is stimulated, there is a small decrease in the K+ concentration (but it always remains above plasma concentrations), the Na+ concentration increases toward plasma levels, and Cl− and HCO3 − concentrations increase, thus the secreted fluid becomes even more alkaline (

Fig. 28.2 ). Note that HCO3 − secretion can be directly stimulated by the action of secretagogues on duct cells. The duct epithelium is relatively tight and lacks expression of aquaporin, and therefore water cannot follow the ions rapidly enough to maintain isotonicity at moderate or high flow rates during stimulated salivary secretion. Thus with

CHAPTER 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal 1.0 2.0 3.0 4.0 Flow of saliva (mL/min) Na+ Saliva Plasma Cl– K+ Cl– Na+ K+ HCO– 3 HCO– 3

Amylase-containing PRIMARY SECRETION (nearly isotonic; levels of Na+, K+, Cl–, and probably HCO3 similar to plasma)

Modification of ionic content • Fig. 28.2 A, The composition of salivary secretion as a function of the salivary flow rate compared with the concentration of ions in plasma. Saliva is hypotonic to plasma at all flow rates. [HCO3 −] in saliva exceeds that in plasma except at very low flow rates. B, Schematic representation of the two-stage model of salivary secretion. The primary secretion containing amylase and electrolytes is produced in the acinar cell. The concentration of electrolytes in plasma is similar to that in the primary secretion, but it is modified as it passes through ducts that absorb Na+ and Cl− and secrete K+ and HCO3 − .

an increase in secretion rate, there is less time for ionic modification by the duct cells, and the resulting saliva more closely resembles the primary secretion and therefore plasma. However, [HCO3 −] remains high because secretion from duct cells and possibly acinar cells is stimulated (see

Fig. 28.2).

The organic constituents of saliva—proteins and glycoproteins—are synthesized, stored, and secreted by the acinar cells. The major products are amylase (an enzyme that initiates starch digestion), lipase (important for lipid digestion), glycoprotein (mucin, which forms mucus when hydrated), and lysozyme (attacks bacterial cell walls to limit colonization of bacteria in the mouth). Although salivary amylase begins the process of digestion of carbohydrates, it is not required in healthy adults because of the excess of pancreatic amylase. Similarly the importance of lingual lipase is unclear.

Metabolism and Blood Flow of Salivary Glands

The salivary glands produce a prodigious flow of saliva. The maximal rate of saliva production in humans is about 1 mL/ min/g of gland; thus at this rate, the glands are producing their own weight in saliva each minute. Salivary glands have a high rate of metabolism and high blood flow; both are proportional to the rate of saliva formation. Blood flow to maximally secreting salivary glands is approximately 10 times that of an equal mass of actively contracting skeletal muscle. Stimulation of the parasympathetic nerves to salivary glands increases blood flow by dilating the vasculature of the glands. Vasoactive intestinal polypeptide (VIP) and acetylcholine are released from parasympathetic nerve terminals in the salivary glands and are vasodilatory during secretion.

Regulation of Salivary Secretion

Control of salivary secretion is exclusively neural. In contrast, control of most other GI secretions is primarily hormonal. Salivary secretion is stimulated by both the sympathetic and parasympathetic subdivisions of the autonomic nervous system. Excitation of either sympathetic or parasympathetic nerves to the salivary glands stimulates salivary secretion. Primary physiological control of the salivary glands during the response to a meal is by the parasympathetic nervous system. If the parasympathetic supply is interrupted, salivation is severely impaired and the salivary glands atrophy.

Sympathetic fibers to the salivary glands stem from the superior cervical ganglion. Preganglionic parasympathetic fibers travel via branches of the facial and glossopharyngeal nerves (cranial nerves VII and IX, respectively). These fibers form synapses with postganglionic neurons in ganglia in or near the salivary glands. The acinar cells and ducts are supplied with parasympathetic nerve endings.

Parasympathetic stimulation increases synthesis and secretion of salivary amylase and mucins, enhances the transport activities of the ductular epithelium, greatly increases blood flow to the glands, and stimulates glandular metabolism and growth.

Ionic Mechanisms of Salivary Secretion

Fig. 28.3 shows a simplified view of the mechanisms of ion secretion by serous acinar cells. The basolateral membrane of the cell contains Na+,K+-ATPase and an Na+-K+-2Cl− symporter. The concentration gradient for Na+ across the basolateral membrane, which is dependent on Na+,K+-ATPase, provides the driving force for entry of Na+ , K+ , and Cl− into the cell. Cl− and HCO3 − leave the acinar cell and enter the lumen via an anion channel located in the apical membrane of the acinar cell. This secretion of anions drives the entry of Na+ and thus water into the acinar lumen across the relatively leaky tight junctions.

Acinar cell fluid secretion is strongly enhanced in response to elevations in intracellular [Ca++] as a result of activation of the muscarinic receptor for acetylcholine.

Fig. 28.4 shows a simplified model of ion transport processes in epithelial cells of the excretory and striated ducts. Na+,K+-ATPase located in the basolateral membrane maintains the electrochemical gradients for Na+ and K+ that drive most of the other ionic transport processes of the cell. In the apical membrane the parallel operation of the Na+/ H+ antiporter, the Cl−/HCO3 − antiporter, and the H+/K+ antiporter results in absorption of Na+ and Cl− from the lumen and secretion of K+ and HCO3 − into the lumen. The relative impermeability of the ductular epithelium to water prevents the ducts from absorbing too much water by osmosis.

• Fig.28.3Ionic transport mechanism involved in the secretion of amylase and electrolytes in salivary acinar cells. ATP2Cl–HCO3LumenofacinusNa˜Na˜Na˜Cl°Na˜K˜K˜K˜K˜°H˜

Swallowing can be initiated voluntarily, but thereafter it is almost entirely under reflex control. The swallowing reflex is a rigidly ordered sequence of events that propel food from the mouth to the pharynx and from there to the stomach. This reflex also inhibits respiration and prevents entrance of food into the trachea during swallowing. The afferent limb of the swallowing reflex begins when touch receptors, most notably those near the opening of the pharynx, are stimulated. Sensory impulses from these receptors are transmitted to an area in the medulla and lower pons called the swallowing center. Motor impulses travel from the swallowing center to the musculature of the pharynx and upper esophagus via various cranial nerves and to the remainder of the esophagus by vagal motor neurons.

The timing of events in swallowing is shown in

Fig. 28.5 . The voluntary phase of swallowing is initiated when the tip of the tongue separates a bolus of food from the mass of food in the mouth. First the tip of the tongue and later the more posterior portions of the tongue press against the hard palate. The action of the tongue moves the bolus upward and then backward into the mouth. The bolus is forced into the pharynx, where it stimulates the touch receptors

CHAPTER 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal • Fig.28.4Ionic transport mechanism involved in secretion and absorption in epithelial cells of the striated and excretory duct of the salivary gland. ATPHCO3LumenofductNa˜Na˜Na˜Na˜Cl°Cl°K˜K˜K˜°H˜H˜H˜

The acinar cells and duct cells of the salivary glands respond to both cholinergic and adrenergic agonists. Nerves stimulate the release of acetylcholine, norepinephrine, substance P, and VIP by salivary glands, and these hormones increase the secretion of amylase and the flow of saliva. These neurotransmitters act mainly by elevating the intracellular concentration of cyclic adenosine monophosphate (cAMP) and by increasing the concentration of Ca++ in the cytosol. Acetylcholine and substance P, acting on muscarinic and tachykinin receptors, respectively, increase the cytosolic concentration of Ca++ in serous acinar cells. In contrast, norepinephrine, acting on β receptors, and VIP, binding to its receptor, elevate the cAMP concentration in acinar cells. Agonists that elevate the cAMP concentration in serous acinar cells elicit a secretion that is rich in amylase; agonists that mobilize Ca++ elicit a secretion that is more voluminous but has a lower concentration of amylase. Ca++-mobilizing agonists may also elevate the concentration of cyclic guanosine monophosphate (cGMP), which may mediate the trophic effects evoked by these agonists.

that initiate the swallowing reflex. The pharyngeal phase of swallowing involves the following sequence of events, which occur in less than 1 second: 1. The soft palate is pulled upward and the palatopharyngeal folds move inward toward one another; these movements 0.2 0.4 0.6 0.8 1.0 1.2 Time (sec) Bolus in mouth Bolus moves through pharynx and UES Bolus enters esophagus Tongue thrust up and back Nasopharynx closed Larynx elevated Airway closed UES open Pharynx contracts • Fig. 28.5 Timing of motor events in the pharynx and UES during a swallow.

Xerostomia, or dry mouth, is caused by impaired salivary secretion. It can be congenital or develop as part of an autoimmune process. The decrease in secretion reduces the pH in the oral cavity, which causes tooth decay and is associated with esophageal erosions. Reduced secretion also causes difficulty swallowing.

The ability to measure and monitor a wide range of molecular components that are indicative of overall health is useful in diagnosis and monitoring. Saliva is easy to access, and collection of it is noninvasive. It is used to identify individuals with disease (presence of biomarkers) and to monitor the progress of affected individuals under treatment. In endocrinology, levels of steroids can be measured in the free form rather than as the free and bound form, as in plasma (e.g., the stress hormone cortisol and the sex hormones estradiol, progesterone, and testosterone). Viral infections such as human immunodeficiency virus (HIV), herpes, hepatitis C, and Epstein-Barr virus infection can be detected by polymerase chain reaction (PCR) techniques. Bacterial infections, such as Helicobacter pylori, can likewise be detected in saliva, and saliva is also used for monitoring drug levels.

prevent reflux of food into the nasopharynx and open a narrow passage through which food moves into the pharynx.

2. The vocal cords are pulled together and the larynx is moved forward and upward against the epiglottis; these actions prevent food from entering the trachea and help open the upper esophageal sphincter (UES).

3.

The UES relaxes to receive the bolus of food.

4.

The superior constrictor muscles of the pharynx then contract strongly to force the bolus deeply into the pharynx. A peristaltic wave is initiated with contraction of the pharyngeal superior constrictor muscles, and the wave moves toward the esophagus. This wave forces the bolus of food through the relaxed UES. During the pharyngeal stage of swallowing, respiration is also reflexively inhibited. After the bolus of food passes the UES, a reflex action causes the sphincter to constrict.

Gastroesophageal reflux disease (GERD) is commonly referred to as heartburn or indigestion. It occurs when the lower esophageal sphincter allows the acidic contents of the stomach to reflux back into the distal part of the esophagus. This region of the esophagus, unlike the stomach, does not have a robust system to protect the mucosal lining. Thus the acid will activate pain fibers and thereby result in discomfort and pain. This is not an uncommon phenomenon, even in healthy individuals. In the long term, continual reflux can result in damage to the esophageal mucosa. In this case, this condition is classed as GERD and can be treated by H2 receptor antagonists that reduce gastric acid secretion (e.g., ranitidine [Zantac]) or by proton pump inhibitors (e.g., omeprazole [Prilosec]).

The esophagus, the UES, and the lower esophageal sphincter (LES) serve two main functions (

Fig. 28.6 ). First, they propel food from the mouth to the stomach. Second, the sphincters protect the airway during swallowing and protect the esophagus from acidic gastric secretions.

The stimuli that initiate the changes in smooth muscle activity that result in these propulsive and protective functions are mechanical and consist of pharyngeal stimulation during swallowing and distention of the esophageal wall itself. The pathways are exclusively neural and involve both extrinsic and intrinsic reflexes. Mechanosensitive afferents in both the extrinsic (vagus) nerves and intrinsic neural pathways respond to esophageal distention. These pathways include activated reflex pathways via the brain-stem (extrinsic, vagus) or solely intrinsic pathways. The striated muscle is regulated from the nucleus ambiguus in the brainstem, and the smooth muscle is regulated by parasympathetic outflow via the vagus nerve. The changes in function resulting from mechanosensitive stimuli and activation of reflex pathways are peristalsis of striated and smooth muscle, relaxation of the LES, and relaxation of the proximal portion of the stomach.

Functional Anatomy of the Esophagus and Associated Structures

The esophagus, like the rest of the GI tract, has two muscle layers—circular and longitudinal—but the esophagus is one of two places in the gut where striated muscle occurs, the other being the external anal sphincter. The type of muscle (striated or smooth) in the esophagus varies along its length. The UES and LES are formed by thickening of striated or circular smooth muscle, respectively.

Motor Activity During the Esophageal Phase

The UES, esophagus, and LES act in a coordinated manner to propel material from the pharynx to the stomach. At the end of a swallow, a bolus passes through the UES, and the presence of the bolus, via stimulation of mechanoreceptors and reflex pathways, initiates a peristaltic wave (alternating contraction and relaxation of the muscle) along the esophagus that is called primary peristalsis (

Fig. 28.7 ). This wave moves down the esophagus slowly (3–5 cm/s). Distention of the esophagus by the moving bolus initiates another • Fig.28.6The esophagus and associated sphincters have multiple functions involved in movement of food from the mouth to the stomach and also in protection of the airway and esophagus. PropulsivefunctionsProtectiveeffectsFood transfer to esophagus Allows entry of food into esophagus Transports bolus from pharynx to stomach Allows entry of food into stomach Protects airway from swallowed material Protects airway from gastric reflux Clears material refluxed from stomach Protects esophagus from gastric reflux LES Esophagus UES Pharynx

CHAPTER 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal • Fig. 28.7 Changes in pressure in the different regions of the pharynx, esophagus, and associated sphincters initiated during a swallow. The pressure trace is a diagrammatic representation from that obtained during manometry in an awake human. Stimulation of the pharynx by the presence of a bolus initiates a decrease in pressure (= opening) of the UES and a peristaltic wave of contraction along the esophagus. Stimulation of the pharynx also relaxes the smooth muscle of the LES to prepare for entry of food.

• Fig. 28.8 Swallowing in the form of pharyngeal stimulation induces neural reflex relaxation of the LES and the proximal part of the stomach to allow entry of food.

wave called secondary peristalsis. Frequently, repetitive secondary peristalsis is required to clear the esophagus of the bolus. Stimulation of the pharynx by the swallowed bolus also produces reflex relaxation of the LES and the most proximal region of the stomach. Thus when the bolus reaches the LES, it is already relaxed to allow passage of the bolus into the stomach. Similarly the portion of the stomach that receives the bolus is relaxed. In addition, esophageal distention produces further receptive relaxation of the stomach. The proximal part of the stomach relaxes at the same time as the LES; this occurs with each swallow, and its function is to allow the stomach to accommodate large volumes with a minimal rise in intragastric pressure. This process is called receptive relaxation (

Fig. 28.8

The LES also has important protective functions. It is involved in preventing acid reflux from the stomach back into the esophagus. An insufficient tonic contraction of the LES is associated with reflux disease, a gradual erosion of the esophageal mucosa, which is not as well protected as the gastric and duodenal mucosa. There is also some evidence that peristalsis in the absence of swallowing (secondary peristalsis) is important for clearing refluxed gastric contents.

1.

The cephalic and oral phases of the meal share many characteristics and prepare the remainder of the GI tract for the meal; these responses are neurally mediated, predominantly by the efferent vagus nerve.

2.

Salivary secretion has important functions and, together with chewing of the food, allows the formation of a bolus that can be swallowed and passed along the esophagus to the stomach.

3.

The ionic composition of salivary secretion varies with the flow rate, which is stimulated during a meal. The primary secretion comes from cells in the acini and is modified by epithelial cells as it passes through the ducts.

4.

Regulation of salivary secretion is exclusively neural; parasympathetic innervation is most important in the response to food.

Catalan MA, et al. Salivary gland secretion. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012.

5.

The swallowing reflex is a rigidly ordered sequence of events that propel food from the mouth to the pharynx and from there to the stomach.

6.

The major function of the esophagus is to propel food from the mouth to the stomach. The esophagus has sphincters at either end that are involved in protective functions important in swallowing and preserving the integrity of the esophageal mucosa.

7.

Esophageal peristalsis (primary) is stimulated by mechanical stimulation of the pharynx, and secondary peristalsis is stimulated by distention of the esophageal wall.

8.

Esophageal function and the associated sphincters are regulated by extrinsic and intrinsic neural pathways.

Lee MG, et al. Molecular mechanism of pancreatic and salivary gland fluid and HCO3 secretion. Physiol Rev. 2012;92:39-74.

Mittal RK. Motor Function of the Pharynx, Esophagus, and its Sphincters. San Rafael, CA: Morgan & Claypool Life Sciences; 2011.

Upon completion of this chapter the student should be able to answer the following questions: 1.

What are the major functions of the monogastric stomach?

2.

What are the gross functional regions of the stomach?

3.

What is the role of the gastric epithelium in digestion and absorption?

4.

What is the role of the proton pump in parietal cell function?

5.

What are some examples of how gastric acid secretion is regulated during the postprandial period?

6.

What are the differences between gastric mucosal protection and defense?

7.

What are the components of the functional anatomy of GI smooth muscle?

8.

What is the significance of gap junctions, interstitial cells of Cajal, and pacemaker cells in the functioning of GI smooth muscle?

9.

How is the basic electrical rhythm (slow wave) generated, how is it regulated by chemical messengers (hormones, paracrine, neurotransmitters), and what causes contractions associated with the slow wave to occur?

10.

What physiological events in gastric motility occur in the gastric phase?

n this chapter, gastrointestinal (GI) tract physiology when food is in the stomach (i.e., the gastric phase of digestion) will be discussed. This includes gastric function and its regulation, in addition to changes in function that occur in more distal regions of the GI tract. The main functions of the stomach are to act as a temporary reservoir for the meal and to initiate protein digestion through secretion of acid and the enzyme precursor pepsinogen. Other functions are listed in

Box 29.1

Food entering the stomach from the esophagus causes mechanical stimulation of the gastric wall via distention and stretching of smooth muscle. Food—predominantly oligopeptides and amino acids—also provides chemical stimulation when present in the gastric lumen. Regulation of gastric function during the gastric phase is dependent on endocrine, paracrine, and neural pathways. These pathways are activated by mechanical and chemical stimuli, which result in intrinsic and extrinsic neural reflex pathways that are important for regulation of gastric function. Afferent neurons that pass from the GI tract to the central nervous system (and to a lesser extent to the spinal cord) via the vagus nerve respond to these mechanical and chemical stimuli and activate parasympathetic outflow.

The endocrine pathways include the release of gastrin, which stimulates gastric acid secretion, and the release of somatostatin, which inhibits gastric secretion. Important paracrine pathways include histamine release, which stimulates gastric acid secretion. The responses elicited by activation of these pathways include both secretory and motor responses; secretory responses include secretion of acid, pepsinogen, mucus, intrinsic factor, gastrin, lipase, and HCO3 − . Overall these secretions initiate protein digestion and protect the gastric mucosa. Motor responses (changes in activity of smooth muscle) include inhibition of motility of the proximal part of the stomach (receptive relaxation) and stimulation of motility of the distal part of the stomach, which causes antral peristalsis. These changes in motility play important roles in storage and mixing of the meal with secretions and are also involved in regulating the flow of contents out of the stomach.

Functional Anatomy of the Stomach

The stomach is divided into three regions: the cardia, the corpus (also referred to as the fundus or body), and the antrum (

Fig. 29.1 ). However, when discussing the physiology of the stomach, it is helpful to think of it as subdivided into two functional regions: the proximal and distal parts of the stomach. The proximal portion of the stomach (called proximal because it is the most cranial) and the distal portion of the stomach (furthest away from the mouth) have quite different functions in the postprandial response to a meal, which will be discussed later.

The lining of the stomach is covered with a columnar epithelium folded into gastric pits; each pit is the opening of a duct into which one or more gastric glands empty (Fig.

29.2 ). The gastric pits account for a significant fraction of the total surface area of the gastric mucosa. The gastric mucosa is divided into three distinct regions based on the structure of the glands. The small cardiac glandular region, located just below the lower esophageal sphincter (LES), primarily contains mucus-secreting gland cells. The remainder of the gastric mucosa is divided into the oxyntic or parietal (acid-secreting) gland region, located above the gastric notch (equivalent to the proximal part of the stomach), and the pyloric gland region, located below the notch (equivalent to the distal part of the stomach).

The structure of a gastric gland from the oxyntic glandular region is illustrated in

Fig. 29.2 . Surface epithelial cells extend slightly into the duct opening. The opening of the gland is called the isthmus and is lined with surface mucous cells and a few parietal cells. Mucous neck cells are located in the narrow neck of the gland. Parietal or oxyntic cells, which secrete HCl and intrinsic factor (involved in absorption of vitamin B12), and chief or peptic cells, which secrete pepsinogens, are located deeper in the gland. Oxyntic glands also contain enterochromaffin-like (ECL) cells that secrete histamine, and D cells that secrete somatostatin. Parietal cells are particularly numerous in glands in the fundus, whereas mucus-secreting cells are more numerous in glands •BOX 29.1FunctionsoftheStomach

Storage—acts as temporary reservoir for the meal

Secretion of H+ to kill microorganisms and convert pepsinogen to its active form

Secretion of intrinsic factor to absorb vitamin B12 (cobalamin)

Secretion of mucus and HCO3 − to protect the gastric mucosa

Secretion of water for lubrication and to provide aqueous suspension of nutrients

Motor activity for mixing secretions (H+ and pepsin) with ingested food

Coordinated motor activity to regulate the emptying of contents into the duodenum of the pyloric (antral) glandular region. In addition the pyloric glands contain G cells that secrete the hormone gastrin. The parietal glands are also divided into regions: the neck (mucous neck cells and parietal cells) and the base (peptic/chief and parietal cells). Endocrine cells are scattered throughout the glands.

The fluid secreted into the stomach is called gastric juice. Gastric juice is a mixture of the secretions of the surface epithelial cells and the secretions of gastric glands. One of the most important components of gastric juice is H+, which is secreted against a very large concentration gradient. Thus H+ secretion by the parietal mucosa is an energy-intensive process. The cytoplasm of the parietal cell is densely packed with mitochondria, which have been estimated to fill 30% to 40% of the cell’s volume. One major function of H+ is conversion of inactive pepsinogen (the major enzyme product of the stomach) to pepsins, which initiate protein digestion in the stomach. Additionally, H+ ions are important for preventing invasion and colonization of the gut by bacteria and other pathogens that may be ingested with food. The stomach also secretes significant amounts of HCO3 − and mucus, which are important for protection of the gastric mucosa against the acidic and peptic luminal environment. The gastric epithelium also secretes intrinsic factor, which is necessary for absorption of vitamin B12 (cobalamin). The functions of other components of gastric juice are redundant with secretions provided more distally in the GI tract.

Composition of Gastric Secretions

Like other GI secretions, gastric juice consists of inorganic and organic constituents together with water. Among the important components of gastric juice are HCl, salts, • Fig.29.1The three functional regions of the stomach. The regions have different luminal secretions and patterns of smooth muscle activity indicative of their unique functions in response to food.

CHAPTER 29 The Gastric Phase of the Integrated Response to a Meal 531

Opening of gastric pit

Opening of gastric pit • Fig. 29.2 Representation of the structure of the gastric mucosa showing a section through the wall of the stomach (A) and detail of the structure of gastric glands and cell types in the mucosa (B).

pepsins, intrinsic factor, mucus, and HCO3 − . Secretion of all these components increases after a meal.

Inorganic Constituents of Gastric Juice

The ionic composition of gastric juice depends on the rate of secretion. The higher the secretory rate, the higher the concentration of H+ ions. At lower secretory rates, [H+] decreases and [Na+] increases. [K+] is always higher in gastric juice than in plasma. Consequently, prolonged vomiting may lead to hypokalemia. At all rates of secretion, Cl− is the major anion of gastric juice. Gastric HCl converts pepsinogens to active pepsins and provides the acid pH at which pepsins are active.

The rate of gastric H+ secretion varies considerably among individuals. In humans, basal (unstimulated) rates of gastric H+ production typically range from about 1 to 5 mEq/hr. During maximal stimulation, HCl production rises to 6 to 40 mEq/hr. The basal rate is greater at night and lowest in the early morning. The total number of parietal cells in the stomach of normal individuals varies greatly, and this variation is partly responsible for the wide range in basal and stimulated rates of HCl secretion.

Organic Constituents of Gastric Juice

The predominant organic constituent of gastric juice is pepsinogen, the inactive proenzyme of pepsin. Pepsins, often collectively called “pepsin,” are a group of proteases secreted by the chief cells of the gastric glands. Pepsinogens are contained in membrane-bound zymogen granules in the chief cells. Zymogen granules release their contents by exocytosis when chief cells are stimulated to secrete 29.1 Response to a Meal

Table 29.1 ). Pepsinogens are converted to active pepsins by the cleavage of acid-labile linkages. The lower the pH, the more rapid the conversion. Pepsins also act proteolytically on pepsinogens to form more pepsin. Pepsins are most proteolytically active at pH 3 and below. Pepsins may digest as much as 20% of the protein in a typical meal but are not required for digestion, because their function can be replaced by that of pancreatic proteases. When the pH of the duodenal lumen is neutralized, pepsins are inactivated by the neutral pH.

Intrinsic factor, a glycoprotein secreted by parietal cells of the stomach, is required for normal absorption of vitamin B12. Intrinsic factor is released in response to the same stimuli that elicit secretion of HCl by parietal cells.

Cellular Mechanisms of Gastric Acid Secretion

Fig. 29.3 ). Branching secretory canaliculi course through the cytoplasm • Fig. 29.3

Parietal cell ultrastructure. A, A resting parietal cell showing the tubulovesicular apparatus in the cytoplasm and the intracellular canaliculus. B, An activated parietal cell that is secreting acid. The tubulovesicles have fused with the membranes of the intracellular canaliculus, which is now open to the lumen of the gland and lined with abundant long microvilli.

• Fig. 29.4

Mechanism of H+ and Cl− secretion by an activated parietal cell in the gastric mucosa. ATP, adenosine triphosphate.

and are connected by a common outlet to the cell’s luminal surface. Microvilli line the surfaces of the secretory canaliculi. The cytoplasm of unstimulated parietal cells contains numerous tubules and vesicles called the tubulovesicular system. The membranes of tubulovesicles contain the transport proteins responsible for secretion of H+ and Cl− into the lumen of the gland. When parietal cells are stimulated to secrete HCl (see

Fig. 29.3 ), tubulovesicular membranes fuse with the plasma membrane of the secretory canaliculi. This extensive membrane fusion greatly increases the number of H+/K+ antiporters in the plasma membrane of the secretory canaliculi. When parietal cells secrete gastric acid at the maximal rate, H+ is pumped against a concentration gradient that is about 1 million–fold. Thus the pH is 7 in the parietal cell cytosol and 1 in the lumen of the gastric gland.

The cellular mechanism of H+ secretion by the parietal cell is depicted in

Fig. 29.4 . Cl− enters the cell across the basolateral membrane in exchange for HCO3 − generated in the cell by the action of carbonic anhydrase, which produces HCO3 − and H+ . H+ is secreted across the luminal membrane by H+,K+-ATPase in exchange for K+ . K+ recycles across the luminal membrane via a K+ channel. Cl− enters the lumen via an ion channel (a CLC family Cl− channel) located in the luminal membrane. Increased intracellular Ca++ and cyclic adenosine monophosphate (cAMP) stimulate luminal membrane conduction of Cl− and K+ . Increased K+ conductance hyperpolarizes the luminal membrane

CHAPTER 29 The Gastric Phase of the Integrated Response to a Meal 533 potential, which increases the driving force for efflux of Cl− across the luminal membrane. The K+ channel in the basolateral membrane also mediates the efflux of K+ that accumulates in the parietal cell via the activity of H+,K+-ATPase. In addition, cAMP and Ca++ promote trafficking of Cl− channels into the luminal membrane, as well as fusion of cytosolic tubulovesicles containing H+,K+-ATPase with the membrane of the secretory canaliculi (see

Figs. 29.3 29.4 ). Parietal cell secretion of H+ is also accompanied by transport of HCO3 − into the bloodstream to maintain intracellular pH.

Secretion of HCO3 −

The surface epithelial cells also secrete a watery fluid that contains Na+ and Cl− in concentrations similar to those in plasma but with higher K+ and HCO3 − concentrations. HCO3 − is entrapped by the viscous mucus that coats the surface of the stomach; thus the mucus secreted by the resting mucosa lines the stomach with a sticky alkaline coat. When food is eaten, rates of secretion of both mucus and HCO3 − increase.

Secretion of Mucus

Secretions that contain mucins are viscous and sticky and are collectively termed mucus. Mucins are secreted by mucous neck cells located in the necks of gastric glands and by the surface epithelial cells of the stomach. Mucus is stored in large granules in the apical cytoplasm of mucous neck cells and surface epithelial cells and is released by exocytosis.

Gastric mucins are about 80% carbohydrate by weight and consist of four similar monomers of about 500,000 Da each that are linked together by disulfide bonds (Fig. 29.5

These tetrameric mucins form a sticky gel that adheres to the surface of the stomach. This gel is subject to proteolysis by pepsins that cleave disulfide bonds near the center of the tetramers. Proteolysis releases fragments that do not form gels and thus dissolves the protective mucous layer. Maintenance of the protective mucous layer requires continuous synthesis of new tetrameric mucins to replace the mucins cleaved by pepsins.

Mucus is secreted at a significant rate in the resting stomach. Secretion of mucus is stimulated by some of the same stimuli that enhance acid and pepsinogen secretion, especially acetylcholine released from parasympathetic nerve endings near the gastric glands. If the gastric mucosa is mechanically deformed, neural reflexes are evoked to enhance mucus secretion.

Regulation of Gastric Secretion

Parasympathetic innervation via the vagus nerve is the strongest stimulant of gastric H+ secretion. Extrinsic efferent fibers terminate on intrinsic neurons that innervate parietal cells, ECL cells that secrete the paracrine mediator histamine, and endocrine cells that secrete the hormone gastrin. In addition, vagal stimulation results in secretion of pepsinogen, mucus, HCO3 − , and intrinsic factor. Stimulation of the parasympathetic nervous system also occurs during the cephalic and oral phase of the meal. However, the gastric phase produces the largest stimulation of gastric secretion of the postprandial period (

Fig. 29.6

Stimulation of gastric acid secretion is an excellent example of a “feed-forward” (or cascade) response that uses endocrine, paracrine, and neural pathways. Activation of intrinsic neurons by vagal efferent activity results in release

Protein core: protected from further proteolysisGlycosylated part of by carbohydrate chains peptide cores (resistant to

Sheath of branched proteolysis) carbohydrate chains with average of 15 Nonglycosylated part of sugars per chain peptide cores with disulfide bridges joining subunits (site of proteolysis) • Fig.29.5Schematic representation of the structure of gastric mucins before and after hydrolysis by pepsin. Intact mucins are tetramers of four similar monomers of about 500,000 Da. Each monomer is largely covered by carbohydrate side chains that protect it from proteolytic degradation. The central portion of the mucin tetramer, near the disulfide cross-links, is more susceptible to proteolytic digestion. Pepsins cleave bonds near the center of the tetramers to release fragments about the size of monomers.

• Fig.29.6Neural regulation of gastric acid secretion in the gastric phase of the meal is mediated by the vagus nerve. The stimulation that occurs in the cephalic and oral phases (before food reaches the stomach) results in stimulation of parietal cells to secrete acid and chief cells to secrete pepsinogen. Thus when food reaches the stomach, protein digestion is initiated by generating protein hydrolysate, which further stimulates secretion of gastrin from the mucosa of the gastric antrum. In addition, gastric distention activates a vagovagal reflex that further stimulates gastric acid and pepsinogen secretion. ˜ H+ ˜ Pepsinogen Endocrine (gastrin) Oligopeptides Protein Pepsin Pepsin pH 2.0 Pepsinogen ˜ H+ ˜ Pepsinogen ˜ Gastrin • Intrinsic neural pathways are also activated by distention Distention Dorsal vagal complex Neural (vagovagal) of acetylcholine from nerve terminals, which activates cells in the gastric epithelium. Parietal cells express muscarinic receptors and are activated to secrete H+ in response to vagal efferent nerve activity. In addition, parasympathetic activation, via gastrin-releasing peptide from intrinsic neurons, releases gastrin from G cells located in the gastric glands in the gastric antrum (see

Fig. 29.6 ). Gastrin enters the bloodstream and, via an endocrine mechanism, further stimulates the parietal cell to secrete H+ . Parietal cells express cholecystokinin type 2 (CCK2) receptors for gastrin. Histamine is also secreted in response to vagal nerve stimulation, and ECL cells express muscarinic and gastrin receptors. Thus gastrin and vagal efferent activity induce release of histamine, which potentiates the effects of both gastrin and acetylcholine on the parietal cell. Hence activation of parasympathetic (vagal) outflow to the stomach is very efficient at stimulating the parietal cell to secrete acid (

Fig. 29.7

In the gastric phase the presence of food in the stomach is detected and activates vagovagal reflexes to stimulate secretion. Food in the stomach results in distention and stretch, which are detected by afferent (or sensory) nerve endings in the gastric wall. These are the peripheral terminals of vagal afferent nerves that transmit information to the brainstem and thereby drive activity in vagal efferent fibers, a vagovagal reflex (see

Fig. 29.6 ). In addition, digestion of proteins increases the concentration of oligopeptides and free amino acids in the lumen, which are detected by chemosensors in the gastric mucosa. Oligopeptides and amino acids also stimulate vagal afferent activity. The exact nature of the

ACETYLCHOLINE, GASTRIN, AND HISTAMINE STIMULATE THE PARIETAL CELL • Fig.29.7The parietal cell is regulated by neural, hormonal, and paracrine pathways. Activation of vagal parasympathetic preganglionic outflow to the stomach acts in three ways to stimulate gastric acid secretion. There is direct neural innervation and activation of the parietal cell via release of acetylcholine (A) from enteric neurons, which acts on the parietal cell via muscarinic receptors. In addition, neural activation of the ECL cell stimulates release of histamine (H), which acts via a paracrine pathway to stimulate the parietal cell. Finally, G cells located in gastric glands in the gastric antrum are activated by release of gastrin-releasing peptide (GRP) from enteric neurons, which acts on the G cell to stimulate release of gastrin (G). Gastrin thereafter acts via a humoral pathway to stimulate the parietal cell.

chemosensors is not clear but may involve endocrine cells that release their contents to activate nerve endings. This topic will be discussed in more detail in

Chapter 30.

There is also an important negative feedback mechanism whereby the presence of acid in the distal part of the stomach (antrum) induces a feedback loop to inhibit the parietal cell such that meal-stimulated H+ secretion does not go unchecked. When the concentration of H+ in the lumen reaches a certain threshold (<pH 3), somatostatin is released from endocrine cells in the antral mucosa. Somatostatin has a paracrine action on neighboring G cells to decrease the release of gastrin and thereby decrease gastric acid secretion (Fig. 29.8

The receptors on the parietal cell membrane for acetylcholine, gastrin, and histamine, as well as the intracellular second messengers by which these secretagogues act, are shown in

Fig. 29.9 . Histamine is the strongest agonist of H+ secretion, whereas gastrin and acetylcholine are much • Fig.29.8Feedback regulation of gastric acid secretion by release of somatostatin and its action on G cells in the gastric antrum. Endocrine cells in the mucosa of the gastric antrum sense the presence of H+and secrete somatostatin. This in turn acts on specific receptors on G cells to inhibit release of gastrin and thus bring about inhibition of gastric acid secretion. Circulation G cell – + D cell Antral lumen GastrinSomatostatin weaker agonists. However, histamine, acetylcholine, and gastrin potentiate one another’s actions on the parietal cell. Antagonists of H2 histamine receptors (e.g., cimetidine [Tagamet]) block secretagogue-stimulated acid secretion. Thus much of the response to gastrin results from gastrinstimulated release of histamine. Gastrin also has important trophic effects; elevation of gastrin levels causes ECL cells to increase in size and number. Binding of histamine to H2 receptors on parietal cell plasma membranes activates adenylyl cyclase and elevates the cytosolic concentration of cAMP. These events stimulate H+ secretion by activating basolateral K+ channels and apical Cl− channels and by causing more H+,K+-ATPase molecules and Cl− channels to be inserted into the apical plasma membrane (see

Fig. 29.4 ). Acetylcholine binds to M3 muscarinic receptors and opens Ca++ channels in the apical plasma membrane. Acetylcholine also elevates intracellular [Ca++] by promoting release of Ca++ from intracellular stores, which enhances H+ secretion by activating basolateral K+ channels and causing more H+,K+-ATPase molecules and Cl− channels to be inserted into the apical plasma membrane. Gastrin enhances acid secretion by binding to CCK2 receptors (Fig. 29.10

Digestion in the Stomach

Some digestion of nutrients occurs in the stomach. However, this is not required for full digestion of a meal; intestinal digestion is sufficient. Some amylase-mediated digestion of carbohydrates occurs in the stomach. Amylase is sensitive to pH and inactivated at low pH; however, some amylase is active even in the acidic gastric environment of the stomach because of substrate protection. Thus when carbohydrate occupies the active site of amylase, it protects the enzyme from degradation.

• Fig.29.9Vagal parasympathetic stimulation of gastric secretions via enteric neurons. Vagal preganglionic neurons innervate the myenteric and submucosal plexus. The terminals of the vagal preganglionic neurons innervate many enteric neurons and thus bring about changes in function as described in

Fig.

29.7 . ACh, acetylcholine; GRP, gastrin-releasing peptide.

• Fig.29.10Signal transduction mechanisms showing the mechanism of action of agonists (secretagogues) and antagonists that regulate secretion in parietal cells. Acetylcholine (ACh) binds to muscarinic M3 receptors. Histamine acts via the H2 receptor. Gastrin binds to the cholecystokinin type 2 (CCK2) receptor. Activation of M3 and CCK2 receptors results in opening of Ca++ channels and release of Ca++ from intracellular stores and thus an increase in cytosolic [Ca++]. Activation of H2 receptors activates adenylyl cyclase to increase intracellular levels of cAMP. Ac, adenylyl cyclase; ACh, acetylcholine; CCK, cholecystokinin; DAG, diacylglycerol; EGF, epidermal growth factor; IP3, inositol triphosphate; PGE2, prostaglandin E2; PIP2, phosphatidylinositol 4,5-diphosphate; PKC, protein kinase C; PLC, protein lipase C; TGF-α, transforming growth factor α.

Digestion of lipids also starts in the stomach. The mixing patterns of gastric motility result in formation of an emulsion of lipids and gastric lipase, which attaches to the surface of lipid droplets in the emulsion and generates free fatty acids and monoglyceride from dietary triglyceride. However, the extent of hydrolysis of triglyceride is approximately 10%, and hydrolysis is not essential for normal digestion and absorption of dietary lipids. Moreover, as discussed in the next chapter, the products of lipolysis are not available for absorption in the stomach because of its low luminal pH.

Mucus and HCO3 − protect the surface of the stomach from the effects of H+ and pepsins. The protective mucus gel that forms on the luminal surface of the stomach, as well as alkaline secretions entrapped within it, constitute a gastric mucosal barrier that prevents damage to the mucosa by gastric contents (

Fig. 29.11 ). The mucus gel layer, which is about 0.2 mm thick, effectively separates the HCO3 −-rich secretions of the surface epithelial cells from the acidic contents of the gastric lumen. The mucus allows the pH of epithelial cells to be maintained at nearly neutral despite a luminal pH of about 2. Mucus also slows the diffusion of acid and pepsins to the epithelial cell surface. Protection of the gastric epithelium depends on both mucus and HCO3 − secretion.

To understand GI motility it is necessary to review some properties of smooth muscle function. The motion of the gut wall governs the flow of the luminal contents along its length; the main patterns of motility are mixing (segmentation) and propulsion (peristalsis). In addition, smooth muscle activity in the stomach and colon subserves a storage function.

• Fig.29.11The surface of the stomach is protected by the gastric mucosal barrier. Buffering by the HCO3 −-rich secretions and the high viscosity of the layer of mucus allow the pH at the cell surface to remain near 7, whereas the pH in the gastric juice in the lumen is 2.

Functional Anatomy of Gastrointestinal Smooth Muscle

The smooth muscle in the GI tract is similar in structure to other smooth muscle found in the body. Fusiform cells are packed together in bundles surrounded by a connective tissue sheath. Gap junctions functionally couple the smooth muscle cells so that contraction of bundles occurs synchronously. The interstitial cells of Cajal (ICCs) are a specialized group of cells in the intestinal wall that are involved in transmission of information from enteric neurons to smooth muscle cells (

Fig. 29.12 ). It is also thought that ICCs are “pacemaker” cells that have the capacity to generate the basic electrical rhythm, or slow-wave activity, that is a consistent feature of GI smooth muscle (

Fig. 29.13).

Electrophysiology of Gastrointestinal Smooth Muscle

The cyclic variation in resting membrane potential of GI smooth muscle is called the basic electrical rhythm or slow wave. The frequency of slow waves is 3 to 5 per minute in the stomach and about 12 to 20 per minute in the small intestine; it decreases to 6 to 8 per minute in the colon. The frequency of the slow wave is set by a pacemaker region in the different regions of the GI tract (see

Fig. 29.13 ). Slow waves are thought to be generated by ICCs. These cells are located in a thin layer between the longitudinal and

Slow waves are generated in interstitial cells of Cajal

Slow wave conducted to smooth muscle cells • Fig.29.12Diagrammatic representation of the interstitial cells of Cajal network in the smooth muscle wall of the GI tract.

circular layers of the muscularis externa and in other places in the wall of the GI tract. Interstitial cells have properties of both fibroblasts and smooth muscle cells. Their long processes form gap junctions with the longitudinal and circular smooth muscle cells; the gap junctions enable the slow waves to be conducted rapidly to both muscle layers. Because gap junctions electrically and chemically couple the smooth muscle cells of both longitudinal and circular layers, the slow wave spreads throughout the smooth muscle of each segment of the GI tract.

538 SECTION6Berne & Levy Physiology • Fig.29.13Amplitude of slow wave determines the strength of muscle contraction. The slow wave will initiate a contraction in smooth muscle when it reaches a threshold amplitude. The amplitude of the slow wave is altered by release of neurotransmitters from enteric neurons. At rest Stimulated Inhibited Membrane potential Muscle tone Membrane potential Muscle tone Membrane potential Muscle tone

There are times when the gastric mucosal barrier fails. Superficial breakdowns of the GI lining not involving the submucosa are called erosions. They generally heal without intervention. In contrast, breakdowns of the GI lining involving the muscularis and deeper layers are called ulcers. Gastric and duodenal erosions and ulcers occur as a result of an imbalance between the mechanisms that protect the mucosa and aggressive factors that can break it down. A healthy stomach/duodenum has ample natural protection against the destructive effects of H+ . Factors that magnify the harmful effect of H+ on the stomach/duodenum or act separately from H+ include pepsin, bile, the bacterium Helicobacter pylori, and the class of drugs known as nonsteroidal antiinflammatory drugs (NSAIDs). Indeed, ulcer disease is becoming more common as the population ages and has more need of NSAIDs for non-GI complaints such as arthritis. Alcohol, tobacco, and caffeine are also risk factors for ulcers. Infectious agents can also cause gastritis (inflammation of the gastric epithelium). H. pylori is a spiral bacterium that has now become widely recognized as one factor that can lead to gastritis, ulcer formation, and in humans, gastric carcinoma. H. pylori exists in the stomach because it secretes an enzyme, urease, that converts urea to NH3, which is used to buffer H+ by forming NH4 + . An aggressive regimen of antibiotic treatment, sometimes in combination with an H+,K+-ATPase inhibitor, can often eliminate the infection, after which the gastritis and ulcer symptoms improve.

The amplitude and, to a lesser extent, the frequency of the slow wave can be modulated by the activity of intrinsic and extrinsic nerves and by hormones and paracrine substances. If the depolarization of the slow wave exceeds the threshold, a train of action potentials may be triggered during the peak of the slow wave.

Action potentials in GI smooth muscle are more prolonged (10–20 msec) than those in skeletal muscle and have little or no overshoot. The rising phase of the action potential is caused by flow of ions through channels that conduct both Ca++ and Na+ and are relatively slow to open. The Ca++ that enters the cell during the action potential helps initiate contraction. The extent of depolarization of the cells and the frequency of action potentials are enhanced by some hormones and paracrine agonists and by neurotransmitters from excitatory enteric nerve endings (e.g., acetylcholine and substance P). Inhibitory hormones and neuroeffector substances (e.g., vasoactive intestinal polypeptide and nitric oxide) hyperpolarize the smooth muscle cells and may diminish or abolish action potential spikes.

Slow waves that are not accompanied by action potentials elicit little or no contraction of the smooth muscle cells. Much stronger contractions are evoked by the presence of action potentials. The greater the number of action potentials that occur at the peak of a slow wave, the more intense the contraction of the smooth muscle. Because smooth muscle cells contract rather slowly (about a 10th as fast as skeletal muscle cells), the individual contractions caused by each action potential in a train do not cause distinct twitches; rather they sum temporally to produce a smoothly increasing level of tension.

Between the trains of action potentials, the tension developed by GI smooth muscle falls, but not to zero. This nonzero resting, or baseline, tension of smooth muscle is called tone. The tone of GI smooth muscle is altered by neuroeffectors, hormones, paracrine substances, and drugs and is important in the sphincters and also in regions where storage of contents is important, such as the stomach and colon.

Specialized Patterns of Motility

Peristalsis is a moving ring of contraction that propels material along the GI tract. It involves neurally mediated contraction and relaxation of both muscle layers. Peristalsis occurs in the pharynx, esophagus, gastric antrum, and the small and large intestine.

Segmental contractions produce narrow areas of contracted segments between relaxed segments. These movements allow mixing of the luminal contents with GI tract secretions and increase exposure to the mucosal surfaces where absorption occurs. Segmentation occurs predominantly in the small and large intestine.

There are also characteristic pathological patterns of motility. During spasm, maximal contractile activity occurs continuously in a dysregulated manner. In ileus, contractile activity is markedly decreased or absent; it often results from irritation of the peritoneum, such as occurs in surgery, peritonitis, and pancreatitis.

Functional Anatomy of the Stomach

As discussed, the stomach is divided into two functional regions—proximal and distal, with sphincters at either end. The LES and cardia (defined as the region of the stomach immediately surrounding the LES) have important functions. Relaxation of the LES and cardia allows entry of food from the esophagus into the stomach and the release of gas, called belching. By maintaining tone, reflux of contents from the stomach back into the esophagus is prevented.

The proximal part of the stomach (the fundus together with the corpus or body) produces slow changes in tone compatible with its reservoir function. It is important for receiving and storing food and for mixing the contents with gastric juice (

Table 29.2 ). Generation of tone in the proximal portion of the stomach is also an important driving force in the regulation of gastric emptying. Low tone and consequently low intragastric pressure are associated with delayed or slow gastric emptying, and an increase in tone in this region is required for gastric emptying to occur.

The distal part of the stomach is important in the mixing of gastric contents and for propulsion through the pylorus and into the duodenum. The muscle layers in the region of the gastric antrum are much thicker than in the more proximal regions of the stomach, and thus the antrum is capable of producing strong phasic contractions. Contractions initiated by the slow wave begin in the midportion of the stomach and move toward the pylorus. The strength of these contractions varies during the postprandial period. In the gastric phase of the meal the pylorus is usually closed, and these antral contractions serve to mix the gastric contents and reduce the size of solid particles (grinding). However, eventually these antral contractions are also important in emptying the stomach of its contents.

The pyloric sphincter is the gastroduodenal junction and is defined as an area of thickened circular muscle. This is a region of high pressure generated by tonic smooth muscle contraction. It is important in regulating gastric emptying.

Control of Gastric Motility in the Gastric Phase

Gastric motility is highly regulated and coordinated to perform the functions of storage and mixing. Regulation of emptying of contents into the small intestine, an important part of gastric motor function, will be considered in detail in the discussion of the duodenal phase of the meal, because the controls are generated in the duodenum.

The stimuli regulating gastric motor function that result from the presence of the meal in the stomach are both mechanical and chemical and include distention and the presence of products of protein digestion (amino acids and small peptides). The pathways regulating these processes are predominantly neural and consist of vagovagal reflexes initiated by extrinsic vagal afferent fibers that terminate in the muscle and mucosa. Mucosal afferents respond to chemical stimuli, and mechanosensitive afferents respond to distention and contraction of smooth muscle. This afferent stimulation results in reflex activation of vagal efferent (parasympathetic) outflow and activation of enteric neurons that innervate the smooth muscle. Activation of enteric neurons produces both inhibitory and excitatory effects on gastric smooth muscle; these effects vary depending on the region of the stomach. Thus distention of the gastric wall results in inhibition of smooth muscle in the proximal portion of the stomach and subsequent reflex accommodation, which allows entry and storage of the meal to occur with minimal increase in intragastric pressure.

In contrast, the predominant motor pattern of the distal part of the stomach in the gastric phase of the meal is activation of smooth muscle to produce and strengthen the antral contractions. The rate of antral contractions is set by the gastric pacemaker; however, the magnitude of the contractions is regulated by release of neurotransmitters from enteric neurons, including substance P and acetylcholine, which increase the level of depolarization of the smooth muscle and therefore produce stronger contractions. In this phase of the meal the pylorus is mostly closed. Thus antral contractions will tend to move the contents toward the pylorus; however, because the pylorus is closed, the contents will be returned to the more proximal part of the stomach. In this way the gastric contents will be mixed. In addition, antral contractions can occlude the lumen, and thus larger particles will be dispersed, a process referred to as grinding (

Fig. 29.14

Onset of terminal Complete terminal antral contraction antral contraction • Force for retropulsion is increased pressure in terminal antrum as the antral contraction approaches the closed pylorus.

•Fig.29.14Coordinated activity in the smooth muscle of the proximal and distal portions of the stomach and the pyloric sphincter results in mixing and grinding in the gastric antrum. The peristaltic wave moves down the gastric body and antrum toward the pylorus. If the pylorus is closed, the contents of the gastric antrum are retropulsed back into the more proximal part of the stomach. This pattern of motility results in grinding and mixing of the food with secretions from the gastric wall and eventually leads to a reduction in particle size and the presence of digestive products that will empty into the duodenum.

1.

The main functions of the stomach are storage and initiation of protein digestion.

2.

Regulation of gastric function is driven by extrinsic and intrinsic neural pathways together with key humoral (gastrin) and paracrine (histamine) mediators.

3.

The key secretions from the stomach are acid and pepsinogen, which together begin protein digestion.

4.

H+ is secreted across the apical plasma membrane of parietal cells via H+,K+-ATPase.

5.

The stomach also secretes intrinsic factor, which is involved in absorption of vitamin B12.

Huizinga JD, Chen JH. Interstitial cells of Cajal: update on basic and clinical science. Curr Gastroenterol Rep. 2014;16:363.

Hunt RH, et al. The stomach in health and disease. Gut. 2015;64:1650-1668.

Said H, et al. Gastroduodenal mucosal defense mechanisms. Curr Opin Gastroenterol. 2015;31:486-491.

6.

The gastric epithelium secretes HCO3 − and mucus to form a gel-like mucosal barrier that protects it against the acidic and peptic luminal contents.

7.

The smooth muscle of the gut wall undergoes cyclic changes in membrane potential, termed the basic electrical rhythm or the slow wave.

8.

The interstitial cells of Cajal are pacemakers in the gut wall, and they set the frequency of the slow wave.

9.

The proximal part of the stomach undergoes a slow change in tone compatible with its storage function.

10.

The distal part of the stomach undergoes phasic contractions that can vary considerably in strength.

11.

Gastric emptying is regulated by vagovagal reflexes.

Sanders KM, et al. Regulation of gastrointestinal smooth muscle function by interstitial cells. Physiology (Bethesda). 2016;31:316-326.

Schubert ML. Functional anatomy and physiology of gastric secretion. Curr Opin Gastroenterol. 2015;31:479-485.

Schubert ML. Regulation of gastric acid secretion. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012.

Upon completion of this chapter the student should be able to answer the following questions: 1.

How are the various components of a mixed meal digested and absorbed in the small intestine?

2.

What are the constituents and functions of pancreatic juice, and how is their secretion controlled?

3.

How do bile acids assist with digestion and assimilation of lipids?

4.

What are the mechanisms that provide for appropriate levels of fluidity of the intestinal contents?

5.

What are the motor patterns of the small intestine in the postprandial period as well as during fasting, and what functions do these patterns subserve?

he small intestine is the critical portion of the intestinal tract for assimilation of nutrients. In this site the meal is mixed with a variety of secretions that permit its digestion and absorption, and motility functions ensure adequate mixing and exposure of the intestinal contents (chyme) to the absorptive surface. The small intestine has many specializations that enable it to perform its functions efficiently. One of the most obvious specializations is the substantial surface area of the mucosa; this is achieved in a number of different ways. The small intestine is essentially a long tube that is coiled inside the abdominal cavity, there are folds of the full thickness of the mucosa and submucosa, the mucosa has finger-like projections called villi, and finally, each epithelial cell has microvilli on its apical surface. Thus a large surface area exists over which digestion and absorption occur.

The main characteristic of the small intestinal phase of the response to a meal is controlled delivery of chyme from the stomach to match the digestive and absorptive capacity of the intestine. In addition there is further stimulation of pancreatic and biliary secretion and emptying of these secretions into the small intestine. Therefore the function of this region is highly regulated by feedback mechanisms that involve hormonal, paracrine, and neural pathways.

The stimuli that regulate these processes are both mechanical and chemical and include distention of the intestinal wall and the presence of increased [H+], high osmolarity, and nutrients in the intestinal lumen. These stimuli result in a set of changes that represent the intestinal phase of the response to the meal: (1) increased pancreatic secretion, (2) increased gallbladder contraction, (3) relaxation of the sphincter of Oddi, (4) regulation of gastric emptying, (5) inhibition of gastric acid secretion, and (6) interruption of the migrating motor complex (MMC). The goal of this chapter is to discuss how such changes are brought about and how they result ultimately in assimilation of nutrients. Changes in small intestinal function that occur after the meal has passed through will also be addressed.

Gastric Emptying in the Small Intestinal Phase

Immediately after a meal the stomach may contain up to a liter of material that will empty slowly into the small intestine. The rate of gastric emptying is dependent on the macronutrient content of the meal and the amount of solids it contains. Thus solids and liquids of similar nutritional composition will empty at different rates. Liquids empty rapidly, but solids do so only after a lag phase, which means that after a solid meal, there is a period of time during which little or no emptying occurs (Fig. 30.1

Regulation of gastric emptying is achieved by alterations in motility of the proximal part of the stomach (fundus and corpus) and distal part of the stomach (antrum and pylorus) as well as in the duodenum. Motor function in these regions is highly coordinated. Recall that during the esophageal and gastric phase of the meal, the predominant reflex response is receptive relaxation. At the same time, peristaltic movements in the more distal part of the stomach (antrum) mix the gastric contents with gastric secretions. The pyloric sphincter is largely closed. Even if it opens periodically, little emptying will occur, because the proximal portion of the stomach is relaxed and the antral pump (antral contractions) is not very strong. Subsequently, gastric emptying is brought about by an increase in tone (intraluminal pressure) in the proximal portion of the stomach, increased strength of antral contractions (increased strength of the •Fig. 30.1 Ratesofemptyingofdifferentmealsfromadog’sstomach.Asolution(1%glucose)isemptiedfasterthanadigestiblesolid(cubedliver).Notethelagphaseforemptyingofthesolids,whichisrelatedtothetimeneededtoreduceparticlesbelow2mminsize.(AdaptedfromHinderRA,KellyKA.Am J Physiol 1977;233:E335.)100 80 60 40 20 21 3 4 % emptied Hours400 mL 1% glucose 50 g cubed liver Protein Glucose

Thegastrointestinal(GI)tractplaysamajorroleinthesensingandsignalingofingestednutrientsbyactivatingneuralandendocrinepathwaysthatconnectwithothersignals,suchasfatenergystorageandutilization,whichtogetherregulateenergyhomeostasis.SatietysignalsfromtheGItractaregenerallyinvolvedinshort-termregulationoffoodintake,suchasindividualmealsizeandmealduration.Forexample,theluminalcontentsactivatevagalafferentpathways,leadingtosuppressionofmealsize.Inaddition,severalGIhormonesreleasedbynutrientsalsoinfluencefoodintake.Cholecystokinin(CCK)isawell-describedsatietyhormone;itisreleasedbynutrientsanddecreasesfoodintakeafterexogenousadministration.OtherGIhormonesinthisclassincludeglucagon-likepeptide1(GLP-1)andpeptideYY(PYY).Inbothleanandobesehumans,injectionofexogenousPYYinhibitsfoodintake.Along-actinganalogueofGLP-1,exendin-4,iscurrentlybeingusedasanagentforweightcontrolinhumans.

antral pump), opening of the pylorus to allow the contents to pass, and simultaneous inhibition of duodenal segmental contractions. Liquids and the semiliquid chyme flow down the pressure gradient from the stomach to the duodenum.

As the meal enters the small intestine, it feeds back via both neural and hormonal pathways to regulate the rate of gastric emptying based on the chemical and physical composition of the chyme. Afferent neurons, predominantly of vagal origin, respond to nutrients, [H+], and the hyperosmotic content of chyme as it enters the duodenum. Reflex activation of vagal efferent outflow decreases the strength of antral contractions, contracts the pylorus, and decreases proximal gastric motility (with a decrease in intragastric pressure), thereby resulting in inhibition (slowing) of gastric emptying. This same pathway is responsible for the inhibition of gastric acid secretion that occurs when nutrients are in the duodenal lumen. Cholecystokinin (CCK) is released from endocrine cells in the duodenal mucosa in response to such nutrients. This hormone is physiologically important, in addition to its role in neural pathways, in the regulation of gastric emptying, gallbladder contraction, relaxation of the sphincter of Oddi, and pancreatic secretion. Recent evidence suggests that CCK acts both directly (inhibits gastric emptying) and indirectly (stimulates vagal afferent fiber discharge to produce an indirect vagovagal reflex–mediated decrease in gastric emptying).

How then can gastric emptying proceed in the face of these inhibitory pathways? The amount of chyme in the duodenum decreases as it passes further down the small intestine into the jejunum; thus the strength of intestinal feedback inhibition fades as there is less activation of duodenal sensory mechanisms by nutrients. At this time, intragastric pressure in the proximal portion of the stomach increases, thereby moving material into the antrum and toward the antral pump. Antral peristaltic contractions again deepen and culminate in opening of the pylorus and release of gastric contents into the duodenum.

Surgicaltreatmentofobesity,so-calledbariatricsurgery,canachievesubstantialandlastingweightlossandalsohelpameliorateassociatedhealthproblemssuchasinsulinresistance,hyperlipidemia,andelevatedbloodpressure.Initially,surgeryinvolvedjejunoileal bypass, theremovalofasubstantialpartoftheabsorptivesmallintestine,butthisprocedureisassociatedwithmalabsorptionandsubsequentundesirablesequelaesuchasdiarrhea.Avarietyofrevisedsurgicalapproachestoobesityhavebeendevised,includingRoux-en-Ygastricbypassandverticalsleevegastrectomy.Themechanismsbywhichtheseproceduresarethoughttobesuccessfullieinthesmallsizeoftheresidualgastricpouch,wherebymealsizeisdecreasedbecauseofearlysatiety,andabeneficialeffectofthebypassontheprofilesofGIhormones.Recentdataimplythateffectsofsurgeryonbileacidsandthemicrobiomemayalsocontributetobothweightlossandmetabolicbenefits.

Most of the nutrients ingested by humans are in the chemical form of macromolecules. However, such molecules are too large to be assimilated across the epithelial cells that line the intestinal tract and must therefore be broken down into their smaller constituents by processes of chemical and enzymatic digestion. Secretions arising from the pancreas are quantitatively the largest contributors to enzymatic digestion of the meal. The pancreas also provides additional important secretory products that are vital for normal digestive function. Such products include substances that regulate the function or secretion (or both) of other pancreatic products, as well as water and bicarbonate ions. The latter are involved in neutralizing gastric acid so that the small intestinal lumen has a pH approaching 7.0. This is important because pancreatic enzymes are inactivated by high levels of acidity and also because neutralization of gastric acid reduces the likelihood that the small intestinal mucosa will be injured by such acid acting in combination with pepsin. Quantitatively the pancreas is the largest contributor to the supply of bicarbonate ions needed to neutralize the gastric acid load, although the biliary ductules and duodenal epithelial cells also contribute.

As in the salivary glands, the pancreas has a structure that consists of ducts and acini. The pancreatic acinar cells line the blind ends of a branching ductular system that eventually empties into the main pancreatic duct and from there into the small intestine under control of the sphincter of Oddi. Also in common with salivary glands, a primary secretion arises in the acini, which is subsequently modified as it passes through the pancreatic ducts. In general the acinar cells supply the organic constituents of the pancreatic juice in a primary secretion whose ionic composition is comparable to that of plasma, whereas the ducts dilute and alkalinize the pancreatic juice while reabsorbing chloride ions (Fig. 30.2 ). The major constituents of pancreatic juice, which amounts to approximately 1.5 L/day in adult humans, are listed in

Box 30.1 . This list also outlines the functions of pancreatic secretory products. Many of the digestive enzymes produced by the pancreas, particularly the proteolytic enzymes, are produced as inactive precursor forms. Storage in these inactive forms is critically important in preventing the pancreas from digesting itself.

Pancreatitis canresultwhenenzymessecretedbypancreaticacinarcellsbecomeproteolyticallyactivatedbeforetheyhavereachedtheirappropriatesiteofactioninthesmallintestinallumen.Indeed,pancreaticjuicecontainsavarietyoftrypsininhibitorstoreducetheriskofprematureactivation,becausetrypsinistheactivatorofotherpro-formsofenzymessecretedinpancreaticjuice.Asecondlevelofprotectionliesinthefactthattrypsincanbedegradedbyothertrypsinmolecules.Despitethesedefenses,someindividualsaresusceptibletohereditarypancreatitisthatoccursspontaneouslyintheabsenceofknownriskfactors.Insomeofthesepatientsthereisamutationintrypsinthatrendersitresistanttodegradationbyothertrypsinmolecules.Othersharbormutationsintrypsininhibitors,renderingtheinhibitorsinactive.Inanyevent,ifotherdefenseshavebeenbreachedandtrypsinbecomesactiveprematurely,aviciouscycleofenzymeactivationensuesandboutsofpancreatitisfollow.

Characteristics and Control of Ductular Secretion

In this section we consider how the pancreatic ductular cells contribute to the flow and composition of pancreatic juice in the postprandial period. The ducts of the pancreas can be considered the effector arm of a pH regulatory

Lobules of acinar and ductal parenchyma

Secretion of H2O and –

Extralobular HCO3 in response to ductal system absorption of Cl˜ Cl˜

Direction of fluid movement •Fig. 30.2 Locationsofimportanttransportprocessesinvolvedintheelaborationofpancreaticjuice.AcinarfluidisisotonicandresemblesplasmainitsconcentrationsofNa+ ,K+ ,Cl− ,andHCO3− .SecretionofacinarfluidandtheproteinsitcontainsisstimulatedprimarilybyCCK.Thehormonesecretinstimulatessecretionofwaterandelectrolytesfromthecellsthatlinetheextralobularducts.Thesecretin-stimulatedsecretionisricherinHCO3− thantheacinarsecretionbecauseofCl−/HCO3− exchange.(AdaptedfromSwansonCH,SolomonAK.J Gen Physiol 1973;62:407.) system designed to respond to luminal acid in the small intestine and secrete just enough bicarbonate to restore pH to neutrality (

Fig. 30.3 ). This regulatory function also requires mechanisms to sense luminal pH and convey this information to the pancreas as well as other epithelia (e.g., biliary ductules and the duodenal epithelium itself) capable of secreting bicarbonate. The pH-sensing mechanism is embodied in specialized endocrine cells known as S cells, localized within the small intestinal epithelium. When luminal pH falls below approximately 4.5, S cells are triggered to release secretin in response to the increase in [H+]. The components of this regulatory loop constitute a self-limited system. Thus as secretin evokes secretion of bicarbonate, pH in the small intestinal lumen will rise and the signal for release of secretin from S cells will be terminated.

At the cellular level, secretin stimulates epithelial cells to secrete bicarbonate into the ductular lumen, with water following via the paracellular route to maintain osmotic equilibrium. Secretin increases cyclic adenosine monophosphate (cAMP) in the ductular cells and thereby opens cystic fibrosis transmembrane conductance regulator (CFTR) • BOX 30.1 Products of Pancreatic Acinar Cells

Precursors of Proteases channels (Fig. 30.4 ) and causes an outflow of Cl− into the duct lumen. This secondarily drives the activity of an adjacent antiporter that exchanges the chloride ions for bicarbonate. CFTR is also permeable to bicarbonate. Thus the bicarbonate secretory process is dependent on CFTR, which provides an explanation for the defects in pancreatic function seen in the disease cystic fibrosis, in which CFTR is mutated. The bicarbonate needed for this secretory process is derived from two sources. Some is taken up across the basolateral membrane of the ductular epithelial cells via the sodium-bicarbonate cotransporter type 1 (NBC-1) symporter. Recall that the process of gastric acid secretion results in an increase in circulating bicarbonate ions, which can serve as a source of bicarbonate to be secreted by the pancreas. However, bicarbonate can also be generated intracellularly via the activity of the enzyme carbonic anhydrase. The net effect is to move HCO3 − into the lumen and thereby increase the pH and volume of pancreatic juice.

•Fig. 30.3 ParticipationofsecretinandHCO3−secretioninaclassicnegative-feedbackloopthatrespondstoafallinluminalpHintheduodenum.˜ pH in duodenum S cells Ductular bicarbonate secretion Secretin release ˜°°° •Fig. 30.4 Iontransportpathwaysinpancreaticductcells.CA,carbonicanhydrase;CFTR,cysticfibrosistransmembraneconductanceregulator;NBC-1,sodium/bicarbonatecotransporter(symporter)type1;NHE-1,sodium-hydrogenexchanger(antiporter)type1.NBC-1 NHE-1 Duct cell 2K˜Cl °HCO3 Duct lumen CFTR Na˜Na˜3Na˜2HCO3 H2CO3 CO2 ˜ H2O H2O ˜CO2 ˜H˜H2CO3 CA Cl °Na˜K˜H2O °2HCO3 °H˜˜ HCO3 °°HCO3°

Characteristics and Control of Acinar Secretion

In contrast to the pancreatic ductules, where secretin is the most important physiological agonist, CCK plays the predominant role at the level of the acinar cells. Thus it is important to understand how release of CCK is controlled during the small intestinal phase of the response to a meal.

Cysticfibrosis(CF)isageneticdiseasethataffectsthefunctionofavarietyofepithelialorgans,includingthelung,intestine,biliarysystem,andpancreas.Previouslythediseasewasalmostuniformlyfatalduringadolescenceasaresultofsevererespiratoryinfections.However,improvedantibiotics,drugsthatimproveclearanceofmucusfromthelungs,correctionofpancreaticinsufficiencyandundernutrition,andrecentUSFoodandDrugAdministration(FDA)approvalofdrugssuchasivacaftor(Kalydeco)andlumacaftor/ivacaftor(Orkambi)nowextendlifeintothefifthdecadeorlaterinsomepatients.CFiscausedbyamutationinCFTR, whichinthegutimpairstheabilitytohydrateandalkalinizetheluminalcontents.IntheGIsystemspecifically,thiscanresultinintestinalobstruction,duodenalmucosalinjury,anddamagetotheliverandbiliarysystemaswellasthepancreas.InmanyCFpatientstheendocrinepancreasisdysfunctional,andtheymustbegivendigestiveenzymesupplementstomaintainadequatenutrientdigestion.Inotherpatientswithmildermutations,pancreatitismaydeveloplaterinlifeintheabsenceofotherclassicCFsymptoms,presumablybecauseofretentionofdigestiveenzymesinthepancreas.Ineithercase,improvedrecognitionandtreatmentofthepulmonarycomplicationsofCFmeanthatGIsymptomssuchasliverfailure,reducedbileflow,pancreatitis,obstruction,andmaldigestion/malabsorptionofnutrientsareacquiringincreasedimportanceasfacetsofthediseasethatmustbemanagedinadults,oftenbymultidisciplinaryteamsofphysiciansandotherhealthcareprofessionals.

CCK is the product of I cells, which are also localized in the small intestinal epithelium. These classic enteroendocrine cells release CCK into the interstitial space when specific food components are present in the lumen, particularly free fatty acids and certain amino acids. Release of CCK may occur following direct interaction of fatty acids or amino acids, or both, with the I cells. Release of CCK is also regulated by two luminally acting releasing factors that can stimulate the I cell. The first of these, CCK-releasing peptide, is secreted by paracrine cells within the epithelium into the small intestinal lumen in response to products of fat •Fig. 30.5 MechanismsresponsibleforcontrollingreleaseofCCKfromduodenalIcells.ACh,acetylcholine;CCK-RP,CCK-releasingpeptide;GRP,gastrin-releasingpeptide.Solidarrowsrepresentstimulatoryeffects,whereasdashedarrowsindicateinhibition.(RedrawnfromBarrettKE.Gastrointestinal Physiology. NewYork:McGraw-Hill;2006.) and protein digestion. The second releasing factor, monitor peptide, is released by pancreatic acinar cells into pancreatic juice. Both CCK-releasing peptide and monitor peptide can also be released in response to neural input, which is particularly important in initiating pancreatic secretion during the cephalic and gastric phases, thereby preparing the system to digest the meal as soon as it enters the small intestine.

What is the significance of these CCK-releasing factors? Their primary role is to match CCK release, as well as the resulting availability of pancreatic enzymes, to the need for these enzymes to digest the meal in the small intestinal lumen (

Fig. 30.5 ). Because the releasing factors are peptides, they will be subject to proteolytic degradation by enzymes such as pancreatic trypsin in exactly the same way as dietary protein. However, when dietary protein is ingested, it is present in much greater amounts in the lumen than the releasing factors and thus “competes” with the releasing factors for proteolytic degradation. The net effect is that the releasing factors will be protected from breakdown while the meal is in the small intestine and are therefore available to continue stimulation of CCK release from I cells. However, once the meal has been digested and absorbed, the releasing factors are degraded and the signal for release of CCK is shut off.

CCK evokes secretion by pancreatic acinar cells in two ways. First, it is a classic hormone that travels through the bloodstream to encounter acinar cell CCK1 receptors. However, CCK also stimulates neural reflex pathways that impinge on the pancreas. Vagal afferent nerve endings in •Fig. 30.6 Receptorsofthepancreaticacinarcellandregulationofsecretion.ThethickblackarrowindicatesthatCa++-dependentsignalingpathwaysplaythemostprominentrole.ACh,acetylcholine;CCK,cholecystokinin;GRP,gastrin-releasingpeptide;VIP,vasoactiveintestinalpolypeptide;M3,M3muscarinicreceptor;CCK-1;CCKreceptortype1.(RedrawnfromBarrettKE.Gastrointestinal Physiology. NewYork:McGraw-Hill;2006.) the wall of the small intestine are responsive to CCK by virtue of their expression of CCK1 receptors. As described earlier for the effect of CCK on gastric emptying, binding of CCK activates a vagovagal reflex that can further enhance acinar cell secretion via activation of pancreatic enteric neurons and release of a series of neurotransmitters such as acetylcholine, gastrin-releasing peptide, and vasoactive intestinal polypeptide (VIP).

The secretory products of pancreatic acinar cells are largely presynthesized and stored in granules that cluster toward the apical pole of acinar cells (

Fig. 30.6 ). The most potent stimuli of acinar cell secretion, including CCK, acetylcholine, and gastrin-releasing peptide (GRP), act by mobilizing intracellular Ca++ . Stimulation of acinar cells results in phosphorylation of a series of regulatory and structural proteins within the cell cytosol that move the granules closer to the apical membrane, where the granules fuse with the plasma membrane. The contents of the granule are then discharged into the acinar lumen and washed out by an exudate of plasma crossing the tight junctions, linking the acinar cells together, and subsequently by ductular secretions. In the period between meals the granule constituents are resynthesized by the acinar cells and then stored until needed to digest the next meal. Resynthesis may be stimulated by the same agonists that evoke the initial secretory response.

Another important digestive juice that is mixed with the meal in the small intestinal lumen is bile. Bile is produced by the liver, and the mechanisms that are involved, as well as the specific constituents, will be discussed in greater detail in when we address the transport and metabolic functions of the liver. However, for purposes of the current discussion, bile is a secretion that serves to aid in digestion and absorption of lipids. Bile flowing out of the liver is stored and concentrated in the gallbladder until it is released in response to ingestion of a meal. Contraction of the gallbladder, as well as relaxation of the sphincter of Oddi, are evoked predominantly by CCK.

When considering the small intestinal phase of meal assimilation, the bile constituents we are most concerned with are the bile acids. These form structures known as micelles that serve to shield the hydrophobic products of lipid digestion from the aqueous environment of the lumen. Bile acids are in essence biological detergents, and large quantities are needed on a daily basis for optimal lipid absorption—as much as 1 to 2 g/day. The majority of the bile acid pool is recycled from the intestine back to the liver after each meal via the enterohepatic circulation (Fig.

30.7 ). Thus bile acids are synthesized in a conjugated form that limits their ability to passively cross the epithelium •Fig. 30.7 Enterohepaticcirculationofbileacids.Activeuptakeofconjugatedbileacidsoccursviatheapicalsodium-dependentbileacidtransporter(asbt).

lining the intestine so that they are retained in the lumen to participate in lipid assimilation (discussed in detail under that heading). However, when the meal contents reach the terminal ileum, after lipid absorption has been completed, the conjugated bile acids are reabsorbed by a symporter, the apical Na+-dependent bile acid transporter (asbt), that specifically takes up conjugated bile acids in association with sodium ions. Only a minor portion of the bile acid pool is left to spill over into the colon in health, and here bile acids become deconjugated and subject to passive reabsorption (see

Fig. 30.7).

The net effect is to cycle the majority of the bile acid pool between the liver and intestine on a daily basis, coincident with signals arising in the postprandial period. Bile acids also exert biological actions beyond their role as detergents by binding to both cell surface and nuclear receptors in a variety of cell types throughout the body. In this way they regulate their own synthesis as well as other metabolic processes.

The most important physiological function of the small intestine is to take up the products of digestion of ingested nutrients. Quantitatively the most significant nutrients (macronutrients) fall into three classes: carbohydrates, proteins, and lipids. The small intestine is critical not only

The Small Intestinal Phase of the Integrated Response to a Meal for absorption of nutrients into the body but also for the final stages of their digestion into molecules that are simple enough to be transported across the intestinal epithelium. We will consider the processes involved in assimilation of each of these nutrients in turn, beginning with carbohydrates. Carbohydrate digestion occurs in two phases: in the lumen of the intestine and then on the surface of enterocytes in a process known as brush border digestion. The latter is important in generating simple absorbable sugars only at the point where they can be absorbed. This may therefore limit their exposure to the small number of bacteria present in the small intestinal lumen that might otherwise use these sugars as nutrients.

Digestion of Carbohydrates

Dietary carbohydrates are composed of several different molecular classes. Starch, the first of these, is a mixture of both straightand branched-chain polymers of glucose. The straight-chain polymers are called amylose and the branched-chain molecules are called amylopectin (Fig.

30.8 ). Starch is a particularly important source of calories, especially in developing countries, and is found predominantly in cereal products. Disaccharides are a second class of carbohydrate nutrients that includes sucrose (consisting of glucose and fructose) and lactose (consisting of glucose and galactose), the latter being an important caloric source in infants. It is, however, a key principle that the intestine can absorb only monosaccharides and not larger carbohydrates. Finally, many food items of vegetable origin contain dietary fiber, which consists of carbohydrate polymers that cannot be digested by human enzymes. These polymers are instead digested by bacteria present largely in the colonic lumen (see ), thereby allowing salvage of their caloric value.

Dietary disaccharides are hydrolyzed to their component monomers directly on the surface of small intestinal epithelial cells by brush border digestion, mediated by a family of membrane-bound, heavily glycosylated hydrolytic enzymes synthesized by small intestinal epithelial cells. Brush border hydrolases critical to the digestion of dietary carbohydrates include sucrase, isomaltase, glucoamylase, and lactase (

Table 30.1). Glycosylation of these hydrolases is believed to protect them to some extent from degradation by luminal pancreatic proteases. However, between meals the hydrolases are degraded and must therefore be resynthesized by the enterocyte to participate in digesting the next carbohydrate meal. Sucrase/isomaltase and glucoamylase are synthesized in quantities that are in excess of requirements, and assimilation of their products into the body is limited by the availability of specific membrane transporters for these monosaccharides (see

Uptake of Carbohydrates). Lactase, in contrast, shows a developmental decline in expression after weaning. The relative paucity of lactase means that digestion of lactose, rather than uptake of the resulting products, is rate limiting for assimilation. If lactase levels fall below a certain threshold, the disease of lactose intolerance results.

•Fig. 30.8 Structureofamylopectinandtheactionofamylase.Thebluecirclesrepresentglucosemonomerslinkedbyα-1,4bonds.Theblackcirclesrepresentglucoseunitslinkedbyα-1,6bondsatthebranchpoints.

Lactose intolerance isrelativelycommoninadultsfromspecificethnicgroups,suchasAsians,AfricanAmericans,andHispanics.Thedisorderreflectsanormaldevelopmentaldeclineintheexpressionoflactasebyenterocytes,particularlywhenlactoseisnotaconsistentcomponentofthediet.Insuchindividuals,consumptionoffoodscontaininglargequantitiesoflactose(e.g.,milk,icecream)canresultinabdominalcramping,gas,anddiarrhea.Thesesymptomsreflectarelativeinabilitytodigestlactose;thusitremainsinthelumen,andwaterisretained.Somelactose-intolerantpatientsbenefitfromoraladministrationofabacteriallyderivedlactaseenzymebeforeingestingdairyproducts.

Digestion of starch occurs in two phases. The first takes place in the lumen and is actually initiated in the oral cavity via the activity of salivary amylase, as discussed in . Salivary amylase, however, is not essential for starch digestion, although it may assume greater importance in neonates or patients in whom the output of pancreatic enzymes is impaired by disease. Quantitatively the most significant contributor to the luminal digestion of starch is pancreatic amylase. Both enzymes hydrolyze internal α-1,4 bonds in both amylose and amylopectin, but not external bonds nor the α-1,6 bonds that form the branch points in the amylopectin molecule (see

Fig. 30.8 ). Thus digestion of starch by amylase is of necessity incomplete and results in short oligomers of glucose, including dimers (maltose) and trimers (maltotriose), as well as the simplest branching structures, which are called α-limit dextrins. Thus to allow absorption of its constituent monosaccharides, starch must also undergo brush border digestion.

At the brush border, straight-chain glucose oligomers can be digested by the hydrolases glucoamylase, sucrase, or isomaltase (see

Table 30.1 ). All yield free glucose monomers, which can then be absorbed by the mechanisms discussed later. For α-limit dextrins, on the other hand, isomaltase activity is critical because it is the only enzyme that can cleave α-1,6 bonds that make up the branch points as well as α-1,4 bonds.

Uptake of Carbohydrates

Water-soluble monosaccharides resulting from digestion must next be transported across the hydrophobic plasma membrane of the enterocyte. The sodium/glucose transporter 1 (SGLT1) is a symporter that takes up glucose (and galactose) against its concentration gradient by coupling its transport to that of Na+ (

Fig. 30.9 ). Once inside the cytosol, glucose and galactose can be retained for the epithelium’s metabolic needs or can exit the cell across its basolateral pole via a transporter known as GLUT2. Fructose, in contrast, is taken up across the apical membrane by GLUT5. However, •Fig. 30.9 Absorptionofglucose,galactose,andfructoseinthesmallintestine.GLUT,glucosetransporter;SGLT1,sodium/glucosetransporter1.GlucoseGalactoseFructoseFructoseGLUT2GLUT5 GLTI?K+ATP because fructose transport is not coupled to that of Na+, its uptake is relatively inefficient and can easily be overwhelmed if large quantities of food containing this sugar are ingested. The symptoms that occur from this malabsorption are similar to those experienced by a lactose-intolerant patient who consumes lactose.

Proteins are also water-soluble polymers that must be digested into their smaller constituents before absorption is possible. Their absorption is more complicated than that of carbohydrates because they contain 20 different amino acids and short oligomers of these amino acids (dipeptides, tripeptides, and perhaps even tetrapeptides) can also be transported by enterocytes. The body, particularly the liver (see ), has substantial ability to interconvert various amino acids subject to the body’s needs. However, some amino acids, termed the essential amino acids, cannot be synthesized by the body either de novo or from other amino acids and thus must be obtained from the diet. The amino acids that must be obtained in this way in humans are shown in

Fig. 30.10

Araregeneticdisorderresultsinaninabilityoftheintestinetoabsorbglucoseorgalactose.ThisdiseasehasbeenmappedtoavarietyofmutationsintheSGLT1 genethatresultinafaultyorunexpressedproteinor,morecommonly,failureoftheproteintotrafficappropriatelytotheapicalmembraneofenterocytes.Inpatientscarryingsuchmutations,malabsorbedglucosecontributestodiarrhealandothersymptoms,asdiscussedearlierforlactoseintolerance.Despitetherarityofthedisease,itisimportantintermsoftheinsightitprovidedintoacriticalprocessofintestinalepithelialtransport.

•Fig. 30.10 Naturallyoccurringdietaryaminoacids.Thoseinboxesareessentialaminoacidsthatcannotbesynthesizedbyhumansandthusmustbeobtainedfromthediet.(RedrawnfromBarrettKE.Gastrointestinal Physiology. NewYork:McGraw-Hill;2006.)Amino acids NeutralBasicArg HisLys AcidicGlu, Gln Asp, Asn AllphaticGly, Ala AromaticTyr Phe Try HydroxylSer Thr SulfurCys Met IminoPro HydroxyproLeu lleVal , , , ,

Digestion of Proteins

Proteins can be hydrolyzed to long peptides simply by virtue of the acidic pH that exists in the gastric lumen. However, for assimilation of proteins into the body, three phases of enzymatically mediated digestion are required (

Fig. 30.11 ). Like acid hydrolysis, the first of these phases takes place in the gastric lumen and is mediated by pepsin, the product of chief cells localized to the gastric glands. When gastric secretion is activated by signals coincident with ingestion of a meal, pepsin is released from the chief cells as the inactive precursor pepsinogen. At acidic pH, this precursor is autocatalytically cleaved to yield the active enzyme. Pepsin is highly specialized to act in the stomach, since it is activated by low pH. The enzyme cleaves proteins at sites of neutral amino acids, with a preference for aromatic or large aliphatic side chains. Because such amino acids occur only relatively infrequently in a given protein, pepsin is not capable of digesting protein fully into a form that can be absorbed by the intestine. Instead it yields a mixture of intact protein, large peptides (the majority), and a limited number of free amino acids.

On moving into the small intestine, the partially digested protein encounters the proteases provided in pancreatic juice. Recall that these enzymes are secreted in inactive forms. How then are they activated to begin the process of protein digestion? In fact, activation of proteases is delayed until these enzymes are in the lumen by virtue of the localized presence of an activating enzyme, enterokinase, only on the brush border of small intestinal epithelial cells (

Fig. 30.12 ). Enterokinase cleaves trypsinogen to yield active trypsin. Trypsin in turn cleaves all the other protease precursors secreted by the pancreas, thereby resulting in a mixture of enzymes that can almost completely digest the vast majority of dietary proteins. Trypsin is an endopeptidase that cleaves proteins only at internal bonds within the peptide chain rather than releasing individual amino acids from the end of the chain. Trypsin is specific for cleavage at basic amino acids, and such cleavage results in a set of shorter peptides with a basic amino acid at their C-terminus. The two other pancreatic endopeptidases, chymotrypsin and elastase, have a similar mechanism of action but cleave at sites of neutral •Fig. 30.11 Hierarchyofproteasesandpeptidasesthatfunctioninthestomachandsmallintestinetodigestdietaryprotein.Proteinsareabsorbedaseithersingleaminoacids(70%)orshortpeptides(30%).(AdaptedfromVanDykeRW.In:SleisengerMH,FordtranJS[eds].Gastrointestinal Disease. 4thed.

Philadelphia:Saunders;1989.) •Fig. 30.12 Conversionoftheinactiveproenzymesofpancreaticjuicetoactiveenzymesbytheactionoftrypsin.Trypsinogeninpancre-aticjuiceisproteolyticallyconvertedtoactivetrypsinbyenterokinaseexpressedonthesurfaceofepithelialcellsoftheduodenumandjejunum.Trypsinthenactivatestheotherproenzymesasshown.

amino acids. The peptides that result from endopeptidase activity are then acted on by pancreatic ectopeptidases. These enzymes cleave single amino acids from the end of a peptide chain, and those present in pancreatic juice are specific for either neutral (carboxypeptidase A) or basic (carboxypeptidase B) amino acids situated at the C-terminus. Thus the products that result after digestion of a protein meal by gastric and pancreatic secretions include neutral and basic amino acids, as well as short peptides that have acidic amino acids at their C-termini and thus are resistant to carboxypeptidase A or

B (Fig. 30.13

The final phase of protein digestion takes place at the brush border. Mature enterocytes express a variety of peptidases on their brush borders, including both amino-peptidases and carboxypeptidases that generate products suitable for uptake across the apical membrane (see

Fig.

30.11 ). However, it should be noted that even with the substantial complement of active proteolytic enzymes, some dietary peptides are either relatively or totally resistant to hydrolysis. In particular, peptides containing either proline or glycine are digested very slowly. Fortunately the intestine can take up short peptides in addition to single amino acids. The majority of peptides taken up into the enterocyte in their intact form are then subjected to a final stage of digestion in the cytosol of the enterocyte to liberate their constituent amino acids for use in the cell or elsewhere in the body (Fig. 30.14 ). However, some di-and tripeptides may also be transported into the blood in their intact form.

•Fig. 30.13 Luminaldigestionofpeptidesresultingfrompartialproteolysisinthestomach.AA,aminoacid.(RedrawnfromBarrettKE.Gastrointestinal Physiology. NewYork:McGraw-Hill;2006.)Peptide with C-terminal neutral AA Carboxypeptidase AChymotrypsin Elastase Large peptides Trypsin Peptide with C-terminal basic AA Short peptides free neutral and basic AAs SerArg Carboxypeptidase B Ser Arg •Fig. 30.14 Awidevarietyofdipeptidesandtripeptidesaretakenupacrossthebrushbordermembranebytheproton-coupledsymporterknownaspeptide transporter 1 (PepT1).Theprotongradientiscreatedbytheactionofsodium/hydrogenexchangers(NHEs)intheapicalmembrane.Peptidesarelargelydigestedinthecytosoltotheirconstituentaminoacidsforexporttothebody,butasmallproportionmaybeexportedintact.

Uptake of Peptides and Amino Acids

The body is also endowed with a series of plasma membrane transporters capable of promoting uptake of the water-soluble products of protein digestion. Given the large number of amino acids, there is a relatively large number of specific transporters (see

Figs. 30.11 30.14 ). Amino acid transporters are of clinical interest because their absence in a variety of genetic disorders results in diminished ability to transport the relevant amino acid or acids. However, such mutations are often clinically silent, at least from a nutritional standpoint, because the amino acid in question can be assimilated by other transporters with overlapping specificity or in the form of peptides. This does not rule out the possibility of pathology in other organ systems in

The Small Intestinal Phase of the Integrated Response to a Meal which the transporter of interest may normally be expressed (e.g., cystinuria). In general, amino acid transporters have reasonably broad specificity and usually transport a subset of amino acids (e.g., neutral, anionic, or cationic) but with some overlap in their affinity for particular amino acids. Furthermore, some (but not all) of the amino acid transporters are symporters that carry their substrate amino acids in conjunction with obligatory uptake of Na+ .

The small intestine is also notable for its ability to take up short peptides (see

Fig. 30.14 ). The primary transporter responsible for such uptake is called peptide transporter 1 (PepT1) and is a symporter that transports peptides in conjunction with protons. Amino acids liberated from these peptides that are not required by the enterocyte are in turn exported across the basolateral membrane and enter blood capillaries to be transported to the liver via the portal vein. PepT1 is also of clinical interest because it can mediate the uptake of so-called peptidomimetic drugs, which include a variety of antibiotics as well as cancer chemotherapeutic agents. The mechanisms by which amino acids and peptidomimetic drugs exit the enterocyte are not fully understood but are presumed to involve additional transport proteins.

Theredundancyinuptakemechanismsfortheproductsofproteindigestionunderscorestheimportanceofthisprocessandalsomeansthatdeficienciesinspecificaminoacidassimilationacrosstheintestinearerelativelyrare.However,undercertaincircumstances,mutationsinproteinsresponsibleforspecificaminoacidtransportcanleadtopathologyinotherorgans.Oneexampleisthediseaseofcysteinuria,whichisamolecularlyheterogeneousdiseaseinvolvingmutationsinavarietyofaminoacidtransporterscapableoftransportingcysteine.Becausecysteinecanalsobeassimilatedacrossthegutintheformofpeptides,nutritionaldeficienciesdonotoccurdespitealackofintestinaluptakemechanismsforthisparticularaminoacid.Incontrast,cysteinecanonlybepoorlyreabsorbedfromtheurineofpatientssufferingfromcystinuria,andkidneystonescanformbecausethisaminoacidisrelativelyinsoluble.PathophysiologycanalsoarisesecondarytomutationsinSLC6A19, aNa+-independenttransporterofneutralaminoacids,andresultinaconditionknownasHartnup disease, arelativelyrareinbornerrorofmetabolismthatcauseslossofneutralaminoacidsinurine,resultinginpsychiatricissues,intellectualdisability,shortstature,headaches,andunsteadygait,althoughnotnutritionaldeficienciesperse.

Lipids, defined as substances that are more soluble in organic solvents than in water, are the third major class of macronutrients making up the human diet. Lipids supply more significant calories on a per-gram basis than proteins or carbohydrates do and are thus of major nutritional significance; they also have a propensity to contribute to obesity if consumed in excessive amounts. Lipids also dissolve volatile compounds that contribute to food’s taste and aroma.

The predominant form of lipid in the human diet is triglyceride, found in oils and other fats. The majority of these triglycerides have long-chain fatty acids (carbon chains > 12 carbons) esterified to the glycerol backbone. Additional lipid is supplied in the form of phospholipids and cholesterol, mostly arising from cell membranes. It is also important to consider that the intestine is presented daily not only with dietary lipid but also with lipid originating from the liver in biliary secretions, as described in more detail in . Indeed, the cholesterol supplied in bile exceeds that provided in the diet on a daily basis in all but the most egg-loving individuals. Finally, though present in only trace amounts, the fat-soluble vitamins (A, D, E, K) are essential nutrients that should be supplied in the diet to avoid disease. These substances are almost entirely insoluble in water and thus require special handling to promote their uptake into the body.

Emulsification and Solubilization of Lipids

When a fatty meal is ingested, the lipid becomes liquefied at body temperature and floats on the surface of the gastric contents. This would limit the area of the interface between the aqueous and lipid phases of the gastric contents and thus restrict access of enzymes capable of breaking down the lipid to forms that can be absorbed. This is because the lipolytic enzymes, as proteins, reside in the aqueous phase. Therefore an early stage in the assimilation of lipid is its emulsification. The mixing action of the stomach churns the dietary lipid into a suspension of fine droplets, which vastly increases the surface area of the lipid phase.

Lipid absorption is also facilitated by formation of a micellar solution with the aid of bile acids supplied in biliary secretions. Details of this process will be discussed subsequently.

Digestion of Lipids

Lipid digestion begins in the stomach. Gastric lipase is released in large quantities from gastric chief cells; it adsorbs to the surface of fat droplets dispersed in the gastric contents and hydrolyzes component triglycerides to diglycerides and free fatty acids. However, little lipid assimilation can take place in the stomach because of the acidic pH of the lumen, which results in protonation of the free fatty acids released by gastric lipase. Lipolysis is also incomplete in the stomach because gastric lipase, despite its optimum catalytic activity at acidic pH, is not capable of hydrolyzing the second position of the triglyceride ester, which means that the molecule cannot be fully broken down into components that can be absorbed into the body. There is also little if any breakdown of cholesterol esters or the esters of fat-soluble vitamins. Indeed, gastric lipolysis is dispensable in healthy individuals because of the marked excess of pancreatic enzymes.

The majority of lipolysis takes place in the small intestine in health. Pancreatic juice contains three important lipolytic enzymes that are optimized for activity at neutral pH. The first of these is pancreatic lipase. This enzyme differs from the stomach enzyme in that it is capable of hydrolyzing both the 1 and 2 positions of triglyceride to yield a large quantity of free fatty acids and monoglycerides. At neutral pH, the head groups of the free fatty acids are charged, and thus these molecules migrate to the surface of the oil droplets. Lipase also displays an apparent paradox in that it is inhibited by bile acids, which also form part of the small intestinal contents. Bile acids adsorb to the surface of the oil droplets and cause lipase to dissociate. However, lipase activity is sustained by an important cofactor, colipase, which is also supplied in pancreatic juice. Colipase is a bridging molecule that binds to bile acids and to lipase; it anchors lipase to the oil droplet even in the presence of bile acids.

Pancreatic juice also contains two additional enzymes that are important in fat digestion. The first of these is phospholipase A2, which hydrolyzes phospholipids such as those present in cell membranes. Predictably this enzyme would be quite toxic in the absence of dietary substrates, and thus it is secreted as an inactive pro-form that is activated only when it reaches the small intestine. Furthermore, pancreatic juice contains a relatively nonspecific cholesterol esterase that can break down esters of cholesterol, as its name implies, as well as esters of fat-soluble vitamins and even triglycerides. Interestingly this enzyme requires bile acids for activity (contrast with lipase, discussed earlier), and it is related to an enzyme produced in breast milk that plays an important role in lipolysis in neonates.

As lipolysis proceeds, the products are abstracted from the lipid droplet, first into a lamellar (membrane) phase and subsequently into mixed micelles composed of lipolytic products as well as bile acids. The amphipathic (meaning they have both a hydrophobic and hydrophilic face) bile acids serve to shield the hydrophobic regions of lipolytic products from water while presenting their own hydrophilic faces to the aqueous environment (

Fig. 30.15 ). Micelles are truly in solution and thus markedly increase lipid solubility in the intestinal contents. This increases the rate at which molecules such as fatty acids diffuse to the absorptive epithelial surface. Nevertheless, given the very large surface area of the small intestine and the appreciable molecular solubility of the products of triglyceride hydrolysis, micelles are not essential for the absorption of triglyceride. Thus patients who have insufficient output of bile acids (caused, for example, by a gallstone that obstructs bile output) do not normally show fat malabsorption. On the other hand, cholesterol and the fat-soluble vitamins are almost totally insoluble in water and accordingly require micelles to be absorbed even after they have been digested. Thus if luminal bile acid concentrations fall below the critical micellar concentration, patients can become deficient in fat-soluble vitamins.

•Fig. 30.15 Schematicdepictionofbileacids(A)andmixedmicelles(B).Bileacidsinsolutionareamphipathic.Mixedmicellesarecylindricalassemblagesofbileacidswithotherdietarylipids.

Uptake of Lipids and Subsequent Handling

The products of fat digestion are believed to be capable of crossing cell membranes readily because of their lipophilicity. However, recent evidence suggests that their uptake may alternatively or additionally be regulated via the activity of specific membrane transporters. A microvillus membrane fatty acid–binding protein (MVM-FABP) provides for the uptake of long-chain fatty acids across the brush border. Likewise, Niemann-Pick C1–like 1 (NPC1L1) has recently been identified as an uptake pathway for cholesterol in enterocytes and may be a therapeutic target in patients who suffer from pathological increases in circulating cholesterol (hypercholesterolemia). However, uptake of cholesterol overall is relatively inefficient because this molecule, along with plant sterols, can also be actively effluxed from enterocytes back into the cytosol by a heterodimeric complex of two ATP-binding cassette (ABC) transporters termed ABC G5 and G8. Finally, the glycerol backbone of triglycerides may be transported into intestinal epithelial cells by a number of different aquaglyceroporins.

Arelativelynewtreatmentforhypercholesterolemiatargetsabsorptionofcholesterol,eitherderivedfromthedietorinbile,acrossthesmallintestinalepithelium.EzetimibeisadrugthatspecificallyblockscellularuptakeofcholesterolbyinhibitingtheactivityoftheNPC1L1proteinexpressedintheapicalmembraneofenterocytes.Inconjunctionwithotherdrugsdesignedtocounteratherosclerosis,thismaybeausefuladjunctinthatitcaninterrupttheenterohepaticcirculationaswellaspreventabsorptionofdietarycholesterol.Clinicalstudiessuggestthatezetimibemaysynergisticallyimprovetheefficacyofotherstrategiesdesignedtoreducecirculatinglevelsoflow-densitylipoproteincholesterolinthoseatriskforcardiovasculardisease,suchastheuseofstatindrugs.

Lipids also differ from carbohydrates and proteins in terms of their fate after absorption into the enterocyte. Unlike monosaccharides and amino acids, which leave the enterocyte in molecular form and enter the portal circulation, the products of lipolysis are reesterified in the enterocyte to form triglycerides, phospholipids, and cholesterol esters. These metabolic events take place in the smooth endoplasmic reticulum. Concurrently the enterocyte synthesizes a series of proteins known as apolipoproteins in the rough endoplasmic reticulum. These proteins are then combined with the resynthesized lipids to form a structure known as a chylomicron, which consists of a lipid core (predominantly triglyceride with much less cholesterol, phospholipid, and fat-soluble vitamin esters) coated by the apolipoproteins. The chylomicrons are then exported from the enterocyte by a process of exocytosis. However, on entering the lamina propria, they are too large (≈750–5000 Å in diameter) to permeate through the intercellular spaces of the mucosal capillaries. Instead they are taken up into lymphatics in the lamina propria and therefore bypass the portal circulation and, at least for their first pass, the liver. Eventually, chylomicrons in the lymph enter the bloodstream via the thoracic duct and then serve as the vehicle to transport lipids around the body for use by cells in other organs. The only exception to this chylomicron-mediated transport is for medium-chain fatty acids. These acids are relatively water soluble and can also permeate enterocyte tight junctions appreciably, which means that they bypass the intracellular processing steps described earlier and are not packaged into chylomicrons. They therefore enter the portal circulation and are more readily available to other tissues. A diet rich in medium-chain triglycerides may be of particular benefit in patients with inadequate bile acid pools.

The foregoing description of digestion has stressed that these processes take place in the small intestine in an aqueous •Fig. 30.16 OverallfluidbalanceinthehumanGItract.About1to2Lofwaterisingested,and8LofvarioussecretionsenterstheGItract.Ofthistotal,mostisabsorbedinthesmallintestine.About2Lispassedontothecolon,thevastmajorityofwhichisabsorbedinhealth.(FromVanderAJetal.Human Physiology. 6thed.NewYork:McGraw-Hill;1994.) milieu. The fluidity of intestinal contents, especially in the small intestine, is important in allowing the meal to be propelled along the length of the intestine and to permit digested nutrients to diffuse to their site of absorption. Part of this fluid is derived from oral intake, but in most adults this consists of only about 1 to 2 L/day derived from both food and drink (Fig. 30.16 ). Additional fluid is supplied by the stomach and the small intestine themselves, as well as the organs that drain into the GI tract. In total, these secretions add another 8 L, which means that the intestine is presented with approximately 9 to 10 L of fluid on a daily basis. However, in health only about 2 L of this load is passed to the colon for reabsorption, and eventually only 100 to 200 mL exits in stool. Thus the intestine absorbs water overall. During the postprandial period, such absorption is promoted in the small intestine predominantly via the osmotic effects of nutrient absorption. An osmotic gradient is established across the intestinal epithelium that simultaneously drives movement of water across the tight junctions. The generic mechanism for nutrient-driven Na+ and water absorption in the small intestine is diagrammed in

Fig. 30.17 . Moreover, in the period between meals, when nutrients are absent, fluid absorption can still occur via the coupled uptake of Na+ and Cl− mediated by the cooperative interaction of a Na+/H+ antiporter (NHE-3) and a Cl−/ HCO3 − antiporter (see

Fig. 30.17

Even though net water and electrolyte transport in the small intestine is predominantly absorptive, this does not imply that the tissue fails to participate in electrolyte secretion. Secretion is regulated in response to signals derived from the luminal contents and in response to deformation of the mucosa and intestinal distention. Critical secretagogues include acetylcholine, VIP, prostaglandins, and serotonin. Secretion ensures that the intestinal contents are appropriately fluid while digestion and absorption are ongoing and is important to lubricate the passage of food particles along the length of the intestine. For example, some clinical evidence suggests that constipation and intestinal obstruction, the latter observed in cystic fibrosis, can result when secretion is abnormally low. The majority of the intestinal •Fig. 30.17 MechanismsofNaClabsorptioninthesmallintestine.DRA,downregulatedinadenoma;GLUT2,glucosetransporter2;KCC1,potassium/chloridecotransporter1;NHE-3,sodium/hydrogenexchanger3;PAT1,putativeaniontransporter1;SGLT1,sodium/glucosetransporter1.SGLT1H˜2K˜KCC1NHE 3DRA?PAT1?ATPHCO3°GLUT2Glucose secretory flow of fluid into the lumen is driven by active secretion of chloride ions via the mechanism diagrammed in

Fig. 30.18 . In cystic fibrosis the loss of CFTR Cl channels can partially be compensated for by accessory chloride channels, such as the TMEM16A channel that is activated by elevations in cytoplasmic calcium. Some segments of the intestine may engage in additional secretory mechanisms, such as secretion of bicarbonate ions via the mechanisms shown in

Fig. 30.19.

Bicarbonate secretion protects the epithelium, particularly in the most proximal portions of the duodenum immediately downstream from the pylorus, from damage caused by acid and pepsin.

Absorption of Minerals and Water-Soluble Vitamins

The small intestine is also an important site for absorption of water-soluble vitamins (e.g., vitamin C, cobalamin) and minerals such as calcium, magnesium, and iron. In general these substances are taken up from the luminal contents via the activity of specific transporters. Their uptake is •Fig. 30.18 MechanismofCl−secretioninthesmallandlargeintestines.CFTR,cysticfibrosistransmembraneconductanceregula-tor;NKCC1,sodium/potassium/2chloridecotransporter1.Notethatadditionalchloridechannels(notshown),suchasthoseregulatedbyintracellularcalciumconcentration,mayalsoexistintheapicalmembraneandcontributetooveralltransport.˜Ca˜˜Cl°2K˜˜˜cAMPNKCC1ATP˜ also controlled by feedback mechanisms that sense the concentration of the relevant substrate in the circulation and adjust the expression of transporters and accessory molecules accordingly.

Motor Patterns of the Small Intestine

The smooth muscle layers in the small intestine produce motility patterns that mix chyme with the various digestive secretions and propel fluid along the length of the intestine so that nutrients (along with water and electrolytes) can be absorbed. Motor patterns of the small intestine during the postprandial period are directed predominantly toward mixing and consist largely of segmenting and retropulsive contractions that retard the meal while digestion is still ongoing. Segmentation is a stereotypical pattern of rhythmic contractions that is displayed in

Fig. 30.20 and presumably reflects programmed activity of the enteric nervous system superimposed on the basic electrical rhythm. Hormonal mediators of this fed pattern of motility are poorly defined, although CCK contributes. CCK also plays important roles in slowing gastric emptying when the meal is in the small intestine, as described at the beginning of this chapter. This makes sense as a mechanism to match nutrient delivery to the available capacity to digest and absorb the components of the meal.

•Fig. 30.19 Mechanismsofbicarbonatesecretionintheduodenum.CA,carbonicanhydrase;CFTR,cysticfibrosistransmembranecon-ductanceregulator;DRA,downregulatedinadenoma;NBC-1,sodium/bicarbonatecotransporter1;NHE-1,sodium/hydrogenexchanger-1;PAT1,putativeaniontransporter1.3Na˜Na˜Na˜H˜Cl°Cl°K˜NBC-1 NHE-1 DRA? PAT1? CFTR ATP CO2 + H2O CA HCO3 °HCO3 °

After the meal has been digested and absorbed, it is desirable to clear any undigested residues from the lumen to prepare the intestine for the next meal. Such clearance is effected by peristalsis (

Fig. 30.21 ), a coordinated sequence of contraction occurring above the intestinal contents and relaxation below that permits the contents to be conveyed over considerable distances. Peristalsis reflects the action of acetylcholine and substance P released orad to a site of intestinal distention, which serves to contract the circular muscle, as well as the inhibitory effects of VIP and nitric oxide on the caudad side. Like segmentation, peristalsis originates when action potentials generated by intrinsic innervation are superimposed on sites of cellular depolarization dictated by the basic electrical rhythm. The peristaltic motor patterns that occur during fasting, moreover, are organized into a sequence of phases known as the migrating motor complex (

Fig. 30.22 ). Phase I of the MMC is characterized by relative quiescence, whereas small disorganized contractions begin to occur during phase II. During phase III, which lasts about 10 minutes, large contractions that •Fig. 30.20 A,Radiographicviewshowingthestomachandsmallintestinefilledwithbariumcontrastmediuminanormalindividual.Notethesegmentationoftheintestine.B,Sequenceofsegmentalcontractionsinthesmallintestine.Lines1to4representsequentialtimepoints.Thedottedlinesindicatewherecontractionswilloccurnext;thearrowsdepictthedirectionofmovementoftheintestinalcontents.(A,FromGardenerEMetal.Anatomy: A Regional Study of Human Structure. 4thed.Philadelphia:Saunders;1975;B,redrawnfromCannonWB. Am J Physiol 1902;6:251.) •Fig. 30.21 Peristalticmotilityintheintestinepropelsintestinalcontentsalongthelengthofthesmallintestine.

propagate along the length of the intestine are stimulated by the hormone motilin and sweep any remaining gastric and intestinal contents out into the colon. The pylorus and ileocecal valve open fully during this phase, so even large undigested items can eventually pass from the body. Motility of the intestine then reverts to phase I of the MMC, with the entire cycle taking about 90 minutes in adults unless a meal is ingested, in which case the MMC is suspended. After the meal, motilin levels fall, and the MMC cannot be resumed until they rise again.

1.

On leaving the stomach, the meal enters the small intestine, which consists (sequentially) of the duodenum, jejunum, and ileum. The principal function of the small intestine is to digest and absorb the nutrients contained in the meal.

2.

The presence of chyme in the duodenum retards additional gastric emptying, thus helping match nutrient delivery to the ability of the small intestine to digest and absorb such substances.

3.

Digestion and absorption in the small intestine are aided by two digestive juices derived from the pancreas (pancreatic juice) and liver (bile). These secretions are triggered by hormonal and neural signals activated by the presence of the meal in the small intestine.

4.

Pancreatic secretions arise from the acini and contain various proteins capable of digesting the meal or acting as important cofactors. The secretion is diluted and alkalinized as it passes through the pancreatic ducts.

5.

Bile is produced by the liver and stored in the gallbladder until needed in the postprandial period. Bile acids, important components of bile, are biological detergents that solubilize the products of lipid digestion.

•Fig. 30.22 Migratingmotorcomplexesintheduodenumandjejunumasrecordedfromafastinghumansubjectbymanometry.D1,D2,J1,J2,andJ3indicatesequentialrecordingpointsalongthelengthoftheduodenumandjejunum.Theintensecontractions(phaseIII)propagateaborally.(RedrawnfromSofferEEetal. Am J Gastroenterol 1998;93:1318.) 6.

Carbohydrates and proteins, water-soluble macromolecules, are digested and absorbed by broadly analogous mechanisms. Lipids, the third macronutrient, require special mechanisms to transfer the products of lipolysis to the epithelial surface where they can be absorbed. The small intestine also absorbs fatand water-soluble vitamins, as well as minerals such as calcium, magnesium, and iron.

7.

The small intestine transfers large volumes of fluid into and out of the lumen on a daily basis to facilitate digestion and absorption of nutrients, driven by active transport of ions and other electrolytes.

8.

The motor patterns of the small intestine vary depending on whether a meal has been ingested. Immediately after a meal, motility is directed to retaining the meal in the small intestine, mixing it with digestive juices, and providing sufficient time for absorption of nutrients. During fasting, a “housekeeper” complex of intense contractions (the migrating motor complex) sweeps periodically along the length of the stomach and small intestine to clear them of undigested residues.

Abumrad NA, Davidson NO. Role of the gut in lipid homeostasis. Physiol Rev. 2012;92:1061-1085.

Argent BE, et al. Cell physiology of the pancreatic ducts. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012.

Barrett KE, Keely SJ. Electrolyte secretion and absorption in the small intestine and colon. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015.

Bharucha AE, Hasler WL. Motility of the small intestine and colon. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015.

Ganapathy V. Protein digestion and absorption. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012.

Liddle RA. Regulation of pancreatic secretion. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012.

Wright EM, Sala-Rabanal M, Loo DDF, Hirayama BA. Sugar absorption. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012.

Upon completion of this chapter the student should be able to answer the following questions: 1.

What are the structures of the anatomy of the colon and rectum, and what is the role of the large intestine in storing and desiccating the residues of a meal?

2.

What are the motility patterns of the colon that provide for its storage function, and what reflexes signal to the colon from more proximal portions of the GI tract?

3.

What is the role of intestinal microorganisms in metabolism and host defense?

4.

What are the mechanisms that provide for defecation, and how it can be delayed until convenient?

Overview of the Large Intestine

The most distal segment of the gastrointestinal (GI) tract, the large intestine, comprises the cecum; ascending, transverse, and descending portions of the colon; rectum; and anus (

Fig. 31.1 ). The primary functions of the large intestine are to digest and absorb components of the meal that cannot be digested or absorbed more proximally, reabsorb the remaining fluid that was used during movement of the meal along the GI tract, and store the waste products of the meal until they can conveniently be eliminated from the body. In fulfilling these functions, the large intestine uses characteristic motility patterns and expresses transport mechanisms that drive the absorption of fluid, electrolytes, and other solutes from the stool. The large intestine also contains a unique biological ecosystem known as the microbiota, consisting of many trillions of commensal bacteria and other microorganisms that engage in a lifelong symbiotic relationship with their human host. These microorganisms can metabolize components of the meal that are not digested by host enzymes and make their products available to the body via a process known as fermentation. Colonic bacteria also metabolize other endogenous substances such as bile acids and bilirubin, thereby influencing their disposition. There is emerging evidence that the colonic microbiota is critically involved in promoting development of the normal colonic epithelium and in stimulating its differentiated functions.

In addition the microbiota can detoxify xenobiotics (substances originating outside the body, such as drugs) and protect the colonic epithelium from infection by invasive pathogens. Finally, the colon is both the recipient and the source of signals that allow it to communicate with other GI segments to optimally integrate function. For example, when the stomach is filled with freshly masticated food, the presence of the meal triggers a long reflex arc that results in increased colonic motility (the gastrocolic reflex) and eventually evacuation of the colonic contents to make way for the residues of the next meal. Similarly the presence of luminal contents in the colon causes release of both endocrine and neurocrine mediators that slow propulsive motility and decrease electrolyte secretion in the small intestine. This negative feedback mechanism matches the rate of delivery of colonic contents to the segment’s capacity to process and absorb the useful components. Details of the signals that mediate this crosstalk between the colon and other components of the GI system are reviewed in the next section.

The colon is regulated primarily, though not exclusively, by neural pathways. Colonic motility is influenced by local reflexes that are generated by filling of the lumen, thereby initiating distention and the activation of stretch receptors. These regulatory pathways exclusively involve the enteric nervous system. Local reflexes triggered by distortion of the colonic epithelium and produced, for example, by passage of a bolus of fecal material stimulate short bursts of Cl− and fluid secretion mediated by 5-hydroxytryptamine (5-HT) from enteroendocrine cells and acetylcholine from enteric secretomotor nerves. On the other hand, colonic function and motility responses in particular are also regulated by long reflex arcs originating more proximally in the GI tract or in other body systems. One example is the gastrocolic reflex. Distention of the stomach activates a generalized increase in colonic motility and mass movement of fecal material, as described in more detail later. This reflex has both chemosensitive and mechanosensitive components at its site of origin and involves release of 5-HT and acetylcholine. Similarly the orthocolic reflex is activated on rising from bed and promotes a morning urge to defecate in many individuals.

The colon is relatively poorly supplied with cells that release bioactive peptides and other regulatory factors. Exceptions are enterochromaffin cells, which release 5-HT, and cells that synthesize peptide YY, so named because its sequence contains two adjacent tyrosine residues. Peptide YY is synthesized by enteroendocrine cells localized in the terminal ileum and colon and is released in response to lipid in the lumen. It decreases gastric emptying and intestinal propulsive motility. Peptide YY also reduces Cl− and thus fluid secretion by intestinal epithelial cells. Thus peptide YY has been characterized as an “ileal brake” in that it is released if nutrients, especially fat, are not absorbed by the time the meal reaches the terminal ileum and proximal part of the colon. By reducing propulsion of the intestinal contents, in part by limiting their fluidity and distention-induced motility, peptide YY provides more time for the meal to be retained in the small intestine where its constituent nutrients can be digested and absorbed.

Patterns of Colonic Motility

To appreciate colonic motility the functional anatomy of the colonic musculature will be reviewed first, followed by a discussion of the regulation of colonic motility.

Functional Anatomy of the Colonic Musculature

As in other segments of the intestine, the colon consists of functional layers, with a columnar epithelium most closely apposed to the lumen, which is then underlaid by the lamina propria, serosa, and muscle layers. Similarly the colonic mucosa is surrounded by continuous layers of circular muscle that can occlude the lumen. Indeed, at intervals •Fig. 31.1 Majoranatomicsubdivisionsofthecolon.Sigmoid colon Cecum Rectum Ileocecal valve Terminal ileum •Fig. 31.2 Radiographshowingaprominenthaustralpatterninthecolonofanormalindividual.(FromKeatsTE.An Atlas of Normal Roentgen Variants. 2nded.StLouis:Mosby–YearBook;1979.) the circular muscle contracts to divide the colon into segments called haustra. These haustra are readily appreciated if the colon is viewed at laparotomy or by x-ray imaging as shown in

Fig. 31.2 . The arrangement of the majority of the longitudinal muscle fibers, however, is distinct from •Fig. 31.3 Anatomyoftherectumandanalcanal.

that in the small intestine. Three nonoverlapping bands of longitudinal muscle known as the taeniae coli extend along the length of the colon.

Although the circular and longitudinal muscle layers of the colon are electrically coupled, this process is less efficient than in the small intestine. Thus propulsive motility in the colon is less effective than in the small intestine. Activity of the enteric nervous system also provides for the segmenting contractions that form the haustra. Contents can be moved back and forward between haustra, which is a means of retarding passage of the colonic contents and maximizing their contact time with the epithelium. In contrast, when rapid propulsion is called for, the contractions forming the haustra relax and the contour of the colon is smoothened.

The colon terminates in the rectum, which is joined to the colon at an acute angle (the rectosigmoid junction ) (Fig.

31.3 ). The rectum lacks circular muscle and is surrounded only by longitudinal muscle fibers. It is a reservoir wherein feces can be stored before defecation. Muscular contractions also form functional “valves” in the rectum that retard movement of feces and are important in delaying the loss of feces until it is convenient, at least in adults. The rectum in turn joins the anal canal, distinguished by the fact that it is surrounded not only by smooth muscle but also by striated (skeletal) muscle. The combination of these muscle layers functionally accounts for two key sphincters that control evacuation of solid waste and flatus from the body. The internal anal sphincter is composed of a thickened band of circular muscle, whereas the external anal sphincter is made up of three different striated muscle structures in the pelvic cavity that wrap around the anal canal. These latter muscles are distinctive because they maintain a significant level of basal tone and can be contracted further either voluntarily or reflexively when abdominal pressure increases abruptly (e.g., when lifting a heavy object).

Hirschsprung’s disease isaconditioninwhichasegmentofthecolonremainspermanentlycontractedandresultsinobstruction.Itistypicallydiagnosedininfancyandaffectsupto1in5000livebirthsintheUnitedStates.Thebasisofthediseaseisfailureoftheentericnervoussystemtodevelopnormallyduringfetallife.Duringorganogenesis,cellsdestinedtobecomeentericneuronsmigrateoutfromtheneuralcrestandpopulatethegutsequentiallyfrommouthtoanus.Insomeindividualsthismigrationterminatesprematurelybecauseofabnormalitiesinthemechanismsthatwouldotherwisedrivethisprocess.Mutationsinglial-derivedneurotrophicfactorandendothelinIII,aswellasintheirreceptors,havebeendescribedinindividualssufferingfromthisdisease,andtheaffectedsegmentcompletelylackstheplexusesoftheentericnervoussystemandassociatedganglia.ArelativedeficiencyofinterstitialcellsofCajalisalsoseenintheaffectedsegment,andoverallcontrolofmotilityismarkedlyimpaired.Inmostindividualsthesymptomscanbecompletelyalleviatedbysurgicalexcisionoftheaffectedsegment.

Contraction of the smooth muscle layers in the proximal part of the colon is stimulated by vagal input as well as by the enteric nervous system. On the other hand, the remainder of the colon is innervated by the pelvic nerves, which also control the caliber of the internal anal sphincter. Voluntary input from the spinal cord via branches of the pudendal nerves regulates contraction of the external anal sphincter and muscles of the pelvic floor. The ability to control these structures is learned during toilet training. This voluntary control distinguishes the anal canal from most of the GI system, with the exception of the striated muscle in the esophagus that regulates swallowing.

Consistent with its primary function, the two predominant motility patterns of the large intestine are directed not to propulsion of the colonic contents but rather to mixing of the contents and retarding their movement, thereby providing them with ample time in contact with the epithelium. Two distinctive forms of colonic motility have been identified. The first is referred to as short-duration contractions, which are designed to provide for mixing. These contractions originate in the circular muscle and are stationary pressure waves that persist for 8 seconds on average. Long-duration contractions, in contrast, are produced by the taeniae coli, last for 20 to 60 seconds, and may propagate over short distances. Notably, however, propagation may move orally as well as aborally, particularly in the more proximal segments of the colon. Both motility patterns are thought to originate largely in response to local conditions such as distention. Note that the basal electrical rhythm that governs the rate and origination sites of smooth muscle contraction in the small intestine does not traverse the ileocecal valve to continue into the colon.

On the other hand, probably as a result of both local influences and long reflex arcs, approximately 10 times per day in healthy individuals the colon engages in a motility pattern that is of high intensity and sweeps along the length of the large intestine from the cecum to the rectum. Such contractions, called high-amplitude propagating contractions, move exclusively in an aboral direction and are designed to clear the colon of its contents. However, although such a motility pattern can clearly be associated with defecation, it does not necessarily result in defecation for reasons discussed later.

It is also important to note that there is considerable variability among individuals with respect to the rate at which colonic contents are transported from the cecum to the rectum. Although small intestinal transit times are relatively constant in healthy adults, the contents may be retained in the large intestine anywhere from hours to days without signifying dysfunction. This also accounts for significant variation among individuals in their normal patterns of defecation and mandates careful elicitation of a patient’s history before diagnosing bowel dysfunction.

Transport Mechanisms in the Colon

The major role of the colonic epithelium is to either absorb or secrete electrolytes and water rather than nutrients. Secretion, which is confined to the crypts, maintains the sterility of the crypts, which might otherwise become stagnant. This is important because the stem cells that renew the epithelium are located at the base of the crypt. The stem cells give rise to daughter cells that migrate out of the crypts and acquire the differentiated properties of surface cells that are responsible for water and electrolyte absorption. The colonic epithelium also absorbs short-chain fatty acids salvaged from nonabsorbed carbohydrates by colonic bacteria. Indeed, one such short-chain fatty acid, butyrate, is a critical energy source for colonocytes. A reduction in butyrate levels in the lumen (as a result of changes in colonic microbiota caused by administration of broad-spectrum antibiotics) may induce epithelial dysfunction.

The colon receives 2 L of fluid each day and absorbs 1.8 L, thus leaving 200 mL of fluid to be lost in stool. The colon has a considerable reserve capacity for fluid absorption and can absorb up to three times its normal fluid load without loss of excessive fluid in the stool. Therefore any illness that results in stimulation of active fluid secretion in the small intestine will cause diarrhea only when the reserve capacity of 4 to 6L is exceeded.

Absorption and secretion of water by the colon are passive processes driven by absorption or secretion of electrolytes and other solutes. Quantitatively, fluid absorption by the

Irritable bowel syndrome isthenamegiventoaheterogeneouscollectionoffunctionaldisorderswhosesuffererscomplainofdiarrhea,constipation,oralternatingpatternsofboth,oftenwithaccompanyingpainanddistention.Theetiologyofthesedisordersisstillnotfullyunderstoodbutmayinvolveinpartaconditionofvisceral hypersensitivity inwhichtheindividualperceivesnormalsignalsoriginatingfromthebowel(e.g.,inresponsetodistention)aspainful.Thishypersensitivitymaybeattheleveloftheentericorcentralnervoussystem(orboth)andcanbetriggeredbyavarietyoffactorssuchaspreviousinfections,childhoodabuse,orpsychiatricdisorders.Mosttreatmentsfocusonsymptomaticrelief,butthereisthepromiseofmoreeffectivetherapiesaswelearnmoreabouttheunderlyingcausesofthecondition.Treatmentofpatientswithirritableboweldisorders,whichareoftenrefractorytotherapy,formsamajorpartofthepracticeofmanygastroenterologists.

Thecolonicepitheliumturnsoverrapidlyinhealth,thuslimitingtheaccumulationofgeneticdamage.However,thisrapidturnover,aswellasfrequent/prolongedexposuretobacteriallysynthesizedorenvironmentaltoxins,orboth,makesthelargeintestineespeciallyvulnerabletomalignancy.ColoncancerissecondinprevalenceonlytolungcancerinmenintheUnitedStatesandthirdbehindlungandbreastcancerinwomen.Withthedecreasedincidenceofcigarettesmokingandthereforelungcancer,coloncancermayassumeevengreatersignificance.Coloncancerariseswhennormalgeneticcontrolsontherateofepithelialproliferationaresubverted;initiallythisleadstogrowthofapolypandeventually,ifnotremoved,toaninvasivetumorthatmaymetastasizetootherpartsofthebody.Coloncancercanbesubdividedaccordingtothebasicnatureoftheunderlyingmoleculardefect,whichcanincludeoverexpressionofgrowthstimulatoryfactorsoramutationthatpreventsthecellsfromrespondingtofactorsthatwouldnormallybegrowthsuppressive.However,coloncancermortalitycanbereducedverysubstantiallybyearlydetectionandremovalofpolypswithmalignantpotential.Thishasdrivencurrentguidelinesforincreasedscreeningofevenasymptomaticmiddle-agedindividualsforcolonicabnormalitiesviacolonoscopy(inwhichaflexiblefiberoptictubeisinsertedintothecolontoinspectitsinterior),screeningforthepresenceofso-calledoccult(orhidden)bloodinthestoolderivedfromableedingpolyportumor,ornoninvasiveimagingtechniquessuchascomputedtomographyscans.

colon is driven by three transport processes. The first is electroneutral NaCl absorption, which is mediated by the same mechanism that drives NaCl absorption in the intestine (see

Fig. 30.17 ). NaCl absorption is stimulated by various growth factors, such as epidermal growth factor, and is inhibited by hormones and neurotransmitters that increase levels of cyclic adenosine monophosphate (cAMP) in colonic surface epithelial cells.

•Fig. 31.4 Mechanismofshort-chainfattyacid(SCFA)uptakebycolonocytes.ATP,adenosinetriphosphate;SMCT1,sodium/monocarboxylatecotransporter1.3Na˜SMCT1 SCFA used by colonocyte for intracellular metabolism ATP

The second transport process that drives fluid absorption in the colon is absorption of short-chain fatty acids, including acetate, propionate, and butyrate. These molecules are absorbed from the lumen by surface (and perhaps crypt) epithelial cells in an Na+-dependent fashion by a family of symporters related to the Na+-glucose symporter in the small intestine, and known as sodium-monocarboxylate transporters (SMCTs). Uptake of short-chain fatty acids by SMCTs located in the apical plasma membrane is driven by the low intracellular [Na+] established by the basolateral Na+,K+-ATPase (

Fig. 31.4 ). These short-chain fatty acids are used for energy by colonocytes. In addition, butyrate regulates expression of specific genes in colonic epithelial cells and may suppress development of a malignant phenotype. Expression of SMCT1 (also identified as SLC5A8) is reduced in some colon cancers, thereby leading to a reduction in butyrate uptake, which may contribute to malignant transformation.

The third absorptive process of major significance in the colon is absorption of Na+ (

Fig. 31.5 ). This transport process is predominantly localized to the distal part of the colon and is driven by the epithelial Na+ channel (ENaC), which is also involved in reabsorption of Na+ in the kidney. When the channel is opened in response to activation by neurotransmitters or hormones, or both, Na+ flows into the colonocyte cytosol and is then transported across the basolateral membrane by Na+,K+-ATPase. Cl− ions follow passively via the intercellular tight junctions to maintain electrical neutrality. Water is absorbed across the tight junctions as a result of the transepithelial osmotic gradient due to solute absorption. This mode of Na+ absorption is the •Fig. 31.5 ElectrogenicNa+ absorptioninthecolon.ENaC,epithelialsodiumchannel.

last line of defense to prevent excessive loss of water in stool, given its strategic location in the distal part of the colon. Indeed, patients suffering from bowel inflammation often show markedly diminished expression of ENaC, perhaps accounting for their diarrheal symptoms. We also know that expression of ENaC can be acutely regulated in response to whole-body Na+ balance. Thus in situations of reduced Na+ intake, the hormone aldosterone increases ENaC expression in both the colon and kidney, thereby promoting retention of Na+ .

Adequate hydration of the colonic contents is determined by the balance between water absorption and secretion. Fluid secretion in the colon is driven by Cl− ion secretion, by the same mechanism driving fluid secretion in the small intestine, and is subject to the same regulation (see

Fig. 30.18).

Indeed, some cases of constipation may reflect abnormalities in epithelial transport, and constipation that results from abnormally slow motility can be treated by agents that stimulate Cl− secretion. Conversely, excessive Cl− secretion can be one mechanism underlying diarrhea.

The remnants of the meal entering the colon interact with a vast assortment of bacteria and other microorganisms. This enteric microbial ecosystem is established shortly after birth, matures as the child grows, and fluctuates in predictable ways in healthy individuals depending on factors such as diet or circadian rhythms. More drastic perturbations in the microbiota can be provoked by antibiotics or introduction of an aggressive pathogen and may then take considerable time to resolve. The enteric bacterial ecosystem contributes to GI physiology in a surprising number of ways. Indeed, the large intestine (and to a lesser extent the distal portion of the small intestine) is an unusual organ in that it maintains a symbiotic relationship with such an extensive microbiota, whereas other body compartments are largely sterile.

Diarrhealdiseasesareamajorcauseofinfantmortalityworldwideandareusuallytheresultofinadequateaccesstocleanfoodandwater.Evenindevelopedcountries,diarrhealdiseasescausesubstantialsufferingandoccasionalwell-publicizeddeathsandcarryasubstantialeconomicburdenbecauseoftheirprevalence.Infectiousdiarrheaiscausedbyanumberoforganisms,withseveral(e.g.,choleraorpathogenicstrainsofEscherichia coli)capableofelaboratingtoxinsthattriggerexcessiveincreasesinactiveCl− secretionbysmallandlargeintestinalepithelialcells.Diarrheacanalsoresultwhennutrientsarenotappropriatelydigestedandabsorbedinthesmallintestine(e.g.,lactoseintolerance)orasaresultofcolonicinflammation.Inmostdiarrhealdiseases,colonicNaClandNa+ absorptionaredownregulatedatthesametimeCl− secretionmaybestimulated,thusfurtherworseningfluidloss.Ontheotherhand,nutrient-linkedNa+ absorptiveprocessestypicallyremainintact.Thisprovidestherationalefortheeffectivenessoforalrehydrationsolutions,whichareprepackagedmixturesofsaltandglucose.UptakeofNa+ andglucosefromthesesolutions,mediatedbySGLT1(see ),restoresfluidabsorption.Thesesolutionssavelivesinareaswherediarrheaisprevalentandtheabilitytorehydratepatientswithsterileintravenoussolutionsislimitedorabsent.

The colonic microbiota is not essential to life because animals raised in germ-free conditions apparently develop normally and are able to reproduce. However, in such animals the mucosal immune system is immature, and intestinal epithelial cells differentiate more slowly. Importantly the colonic microbiota provides benefits to the host in that the constituent microbes are capable of performing metabolic reactions that do not take place in mammalian cells. Bacterial enzymes act on both endogenous and exogenous substrates. They form secondary bile acids and deconjugate bile acids that have escaped uptake in the terminal ileum, so they can be reabsorbed. They convert bilirubin into urobilinogen (see ) and salvage nutrients that are resistant to pancreatic and brush border hydrolases, such as dietary fiber. A summary of the metabolic contributions of the colonic microbiota is provided in

Table 31.1 . Microbial metabolism can also be exploited for pharmacological purposes. A drug targeted to the colon, for example, can be conjugated in such a way that it will become bioavailable only after it is acted on by bacterial enzymes. Bacterial enzymes may also detoxify some dietary carcinogens, but equally they may generate toxic or carcinogenic compounds from dietary substrates.

Commensal microorganisms also play a critical role in limiting the growth or invasion (or both) of pathogenic microorganisms. They fulfill this antimicrobial role via a number of different mechanisms—by synthesizing and secreting compounds that inhibit the growth of competitor organisms or that are microbicidal, by functioning as a physical barrier to prevent attachment of pathogens and their subsequent entry into colonic epithelial cells, and by triggering patterns of gene expression in the epithelium that counteract the adverse effects of pathogens on epithelial function. These mechanisms provide a basis for understanding why patients who have received broad-spectrum antibiotics, which temporarily disrupt the colonic microbiota, are susceptible to overgrowth of pathogenic organisms and associated intestinal and systemic infections. They may also shed light on the efficacy of probiotics, commensal bacteria selected for their resistance to gastric acid and proteolysis that are intentionally ingested to prevent or treat a variety of digestive disorders.

Atoxinknownasheat-stable toxin of E. coli, orSTa,isamajorcausativeagentoftraveler’sdiarrhea,whichcanbecontractedbyconsumptionofinfectedfoodorwater.Thistoxinbindstoareceptorontheapicalsurfaceofintestinalepithelialcellsknownasguanylyl cyclase C (GC-C).Inturnthisenzymegenerateslargequantitiesofintracellularcyclicguanosinemonophosphate(cGMP)thattriggerincreasedCl− secretionviaactivationofthecysticfibrosistransmembraneconductanceregulator(CFTR)Cl− channel.However,onecouldofcoursequestionwhyhumansexpressareceptorforthistoxininasitethatwouldbeaccessibletoluminalbacteriaandtheirproducts.Indeed,thisledtothehypothesisthatthereisanativeligandforGC-Cthatcouldplayaphysiologicalrole.Researcherssubsequentlypurifiedandidentifiedguanylin,ahormonesynthesizedintheintestine.Togetherwitharelatedmolecule,uroguanylin,secretedbythekidney,guanylinisanimportantregulatorofsaltandwaterhomeostasisinthebody.STahasstructuralsimilaritiestoguanylin,butithasmodificationsthatpermitittopersistintheintestinallumenforprolongedperiods.Thisisanexampleofmolecularmimicry,inwhichabacterialproducthijacksareceptorandassociatedsignalingforitsownpurposes(presumablytopropagatethetoxin-producingbacteriatoadditionalhosts).

The colonic microbiota is also notable for its contribution to formation of intestinal gas. Although large volumes of air may be swallowed in conjunction with meals, the majority of this gas returns up the esophagus via belching. However, during fermentation of unabsorbed dietary components, the microbiota generates large volumes of nitrogen, hydrogen, and carbon dioxide. Approximately 1 L of these nonodorous gases is excreted on a daily basis via the anus in all individuals, even those who do not complain of flatulence. Some individuals may generate appreciable concentrations of methane. Trace amounts of odorous compounds are also present, such as hydrogen sulfide, indole, and skatole.

The final stage in the journey taken by a meal after its ingestion is expulsion of its indigestible residues from the body in the process known as defecation. The feces also contain the remnants of dead bacteria, dead and dying epithelial cells that have been desquamated from the lining of the intestine, biliary metabolites specifically targeted for excretion (e.g., conjugates of xenobiotics [see ]), and a small amount of water. In health, the stool contains few if any useful nutrients. The presence of nutrients in stool, particularly lipid (known as steatorrhea), signifies maldigestion, malabsorption, or both. Fat in the stool is a sensitive marker of small intestinal dysfunction because it is poorly used by the colonic microbiota, but loss of carbohydrate and protein in stool can also be seen if the underlying condition worsens.

The process of defecation requires coordinated action of the smooth and striated muscle layers in the rectum and anus, as well as surrounding structures such as the pelvic floor muscles. During the mass movement of feces produced by high-amplitude propagating contractions, the rectum fills with fecal material. Expulsion of this material from the body is controlled by the internal and external anal sphincters, which contribute approximately 70% to 80% and 20% to 30% of anal tone at rest, respectively. Filling of the rectum causes relaxation of the internal anal sphincter via release of vasoactive intestinal polypeptide and generation of nitric oxide. Relaxation of the inner sphincter permits the anal sampling mechanism, which can distinguish whether the rectal contents are solid, liquid, or gaseous in nature. After toilet training, sensory nerve endings in the anal mucosa then generate reflexes that initiate appropriate activity of the external sphincter to either retain the rectal contents or permit voluntary expulsion (e.g., of flatus). If defecation is not convenient, the external sphincter contracts to prevent the loss of stool. Then with time the rectum accommodates to its new volume, the internal anal sphincter contracts again, and the external anal sphincter relaxes (

Fig. 31.6

When defecation is desired, on the other hand, adoption of a sitting or squatting position alters the relative orientation of the intestine and surrounding muscular structures by straightening the path for exit of either solid or liquid feces. Relaxation of the puborectalis muscle likewise increases the rectoanal angle. After voluntary relaxation of the external anal sphincter, rectal contractions move the fecal material out of the body, sometimes followed by additional mass movements of feces from more proximal segments of the colon (

Fig. 31.7 ). Evacuation is assisted by simultaneous contraction of muscles that increase abdominal pressure, such as the diaphragm. Voluntary expulsion of flatus, on the other hand, involves a similar sequence of events, except that there is no relaxation of the puborectalis muscle. This permits flatus to be squeezed past the acute rectoanal angle while retaining fecal material.

Cooperative activity of the external anal sphincter, puborectalis muscle, and sensory nerve endings in the anal canal is required to delay defecation until it is appropriate, even if the rectum is acutely distended with stool or intraabdominal pressure rises sharply. This explains why incontinence can develop in individuals in whom the integrity of such structures has been compromised, such as after trauma, surgical or obstetrical injuries, prolapse of the rectum, or neuropathic diseases such as long-standing diabetes. Surgical intervention may be necessary to correct muscle abnormalities in patients with the distressing condition of fecal incontinence, although many can be helped to increase external anal sphincter tone with the use of biofeedback exercises.

•Fig. 31.6 Responsesoftheinternalandexternalanalsphincterstoprolongeddistentionoftherectum.Notethattheresponsesofthesphinctersaretransientbecauseofaccommodation.(RedrawnfromShusterMMetal.Bull Johns Hopkins Hosp 1965;116:79.) •Fig. 31.7 Motilityoftherectumandanalsphinctersinresponsetorectalfillingandduringdefecation.Notethatfillingoftherectumcausesaninitialdecreaseininternalsphinctertonethatiscounterbalancedbycontractionoftheexternalsphincter.Theinternalsphincterthenaccommodatestothenewrectalvolume,therebyallowingtheexternalsphinctertorelax.Finally,defecationoccurswhentheexternalanalsphincterisrelaxedvoluntarily.(DatafromChangEBetal.Gastrointestinal, Hepatobiliary and Nutritional Physiology. Philadelphia:Lippincott-Raven;1996.) 1.

The final segment of the intestine through which the meal traverses is the large intestine, which is composed of the cecum, colon, rectum, and anus. The primary role of the large intestine is to reclaim water used during the process of digestion and absorption and to store the residues of the meal until defecation is socially convenient.

2.

Colonic motility primarily serves to mix and delay passage of the luminal contents, other than during periodic large-amplitude contractions that convey fecal material to the rectum.

Bharucha AE, Brookes SJR. Neurophysiologic mechanisms of human large intestinal motility. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012.

Bharucha AE, Hasler WL. Motility of the small intestine and colon. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015.

3.

The colon is highly active in transporting water and electrolytes as well as products salvaged from undigested components of the meal by colonic bacteria.

4.

The colon maintains a lifelong mutually beneficial relationship with a vast microbial ecosystem that metabolizes endogenous substances, nutrients, and drugs and protects the host from infection with pathogens.

5.

Defecation involves both involuntary and voluntary relaxation of muscle structures surrounding the anus and reflex pathways that control these structures.

Kau AL, et al. Human nutrition, the gut microbiome and the immune system. Nature. 2011;474:327-336.

Kendig DM, Grider JR. Serotonin and colonic motility. Neurogastroenterol Motil. 2015;27:899-905.

Koren A, Ley RE. The human intestinal microbiota and microbiome. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015.

Upon completion of this chapter the student should be able to answer the following questions: 1.

How does the liver contribute metabolic, detoxification, and excretory functions that are vital to life?

2.

What cell types make up the liver, what is their unusual arrangement, and what is the liver’s unique portal blood supply?

3.

What is the function of bile as an excretory fluid, what are the mechanisms that underlie its formation, and what is the role of the enterohepatic circulation?

4.

How is bile is stored and concentrated in the gallbladder, and what are the mechanisms that coordinate gallbladder emptying with ingestion of a meal?

5.

How are ammonia and bilirubin eliminated from the body?

Overview of the Liver and Its Functions

The liver is a large multilobed organ located in the abdominal cavity whose function is intimately associated with that of the gastrointestinal system. The liver serves as the first site of processing for most absorbed nutrients and also secretes bile acids, which as we learned in , play a critical role in absorption of lipids from the diet. In addition the liver is a metabolic powerhouse, critical for disposing of a variety of metabolic waste products and xenobiotics from the body by converting them to forms that can be excreted. The liver stores or produces numerous substances needed by the body, such as glucose, amino acids, and plasma proteins. In general, key functions of the liver can be divided into three areas: (1) contributions to whole-body metabolism, (2) detoxification, and (3) excretion of protein-bound/lipid-soluble waste products. In this chapter we discuss the structural and molecular features of the liver and biliary system that subserve these functions, as well as their regulation. Although the liver contributes in a pivotal way to the maintenance of whole-body biochemical status, a complete discussion of all the underpinning reactions is beyond the scope of this chapter. We will confine our discussion primarily to hepatic functions that relate to gastrointestinal physiology.

Metabolic Functions of the Liver

Hepatocytes contribute to metabolism of the major nutrients: carbohydrates, lipids, and proteins. Thus the liver plays an important role in glucose metabolism by engaging in gluconeogenesis, conversion of other sugars to glucose. The liver also stores glucose as glycogen at times of glucose excess (e.g., postprandial period) and then releases stored glucose into the bloodstream as it is needed. This process is referred to as the glucose buffer function of the liver. When hepatic function is impaired, glucose concentrations in blood may rise excessively after ingestion of carbohydrate; conversely, between meals, hypoglycemia may be seen because of an inability of the liver to contribute to carbohydrate metabolism and interconversion of one sugar to another.

Hepatocytes also participate in lipid metabolism. They are a particularly rich source of the metabolic enzymes engaged in fatty acid oxidation to supply energy for other body functions. Hepatocytes also convert products of carbohydrate metabolism to lipids that can be stored in adipose tissue and synthesize large quantities of lipoproteins, cholesterol, and phospholipids, the latter two being important in the biogenesis of cell membranes. In addition, hepatocytes convert a considerable portion of synthesized cholesterol to bile acids, which will be discussed in more detail later in this chapter.

The liver also plays a vital role in protein metabolism. The liver synthesizes all the nonessential amino acids (see ) that do not need to be supplied in the diet, in addition to participating in interconverting and deaminating amino acids so the products can enter biosynthetic pathways for carbohydrate synthesis. With the exception of immunoglobulins, the liver synthesizes almost all the proteins present in plasma (especially albumin, which determines plasma oncotic pressure) as well as most of the important clotting factors. Patients suffering from liver disease may develop peripheral edema secondary to hypoalbuminemia and are also susceptible to bleeding disorders. Finally, the liver is the critical site for disposal of the ammonia generated from protein catabolism. This is accomplished by converting ammonia to urea, which is •Fig. 32.1 Typicalbloodflowthroughthesplanchniccirculationinafastingadulthuman.

excreted by the kidneys. The details of this process will be discussed later in this chapter.

The Liver and Detoxification

The liver serves both as a gatekeeper, by limiting entry of toxic substances into the bloodstream, and as a garbage disposal, by extracting potentially toxic metabolic products produced elsewhere in the body and converting them to chemical forms that can be excreted. The liver fulfills these functions in part because of its unusual blood supply. Unlike all other organs, the majority of blood arriving at the liver is venous in nature and is supplied via the portal vein from the intestine (

Fig. 32.1 ). As such, the liver is strategically located to receive both absorbed nutrients and potentially harmful absorbed molecules such as drugs and bacterial toxins. Depending on the efficiency with which these molecules are extracted by hepatocytes and subjected to first-pass metabolism, little or none of the absorbed substance may make it into the systemic circulation. This is a major reason why not all pharmaceutical agents can achieve therapeutic concentrations in the bloodstream if administered orally.

CHAPTER 32 Transport and Metabolic Functions of the Liver

The liver has two levels at which it removes and metabolizes/detoxifies substances originating from the portal circulation. The first of these is physical. Blood arriving in the liver percolates among cells of the macrophage lineage, known as Kupffer cells. These cells are phagocytic and particularly important in removing particulate material from portal blood, including bacteria that may enter blood from the colon even under normal conditions. The second level of defense is biochemical. Hepatocytes are endowed with a broad array of enzymes that modify both endogenous and exogenous toxins so the products are in general more water soluble and less susceptible to reuptake by the intestine. The metabolic reactions involved are broadly divided into two classes. Phase I reactions (oxidation, hydroxylation, and other reactions catalyzed by cytochrome P450 enzymes) are followed by phase II reactions that conjugate the resulting products with another molecule (e.g., glucuronic acid, sulfate, amino acids, glutathione) to promote their excretion. The products of these reactions are then excreted into bile or returned to the bloodstream to ultimately be excreted by the kidneys. We will return to the precise mechanisms involved in detoxification of some key metabolic waste products later in this chapter.

Role of the Liver in Excretion

The kidneys play an important role in excretion of water-soluble catabolites, as discussed in the renal section. Only relatively small water-soluble catabolites can be excreted by the process of glomerular filtration. However, larger water-soluble catabolites and molecules bound to plasma proteins, including lipophilic metabolites and xenobiotics, steroid hormones, and heavy metals, cannot be filtered by the glomerulus. All these substances are potentially harmful if allowed to accumulate, so a mechanism must exist for their excretion. The mechanism involves the liver, which excretes these substances in bile. Hepatocytes take up these substances with high affinity by virtue of an array of basolateral membrane transporters, and the substances are subsequently metabolized at the level of microsomes and in the cytosol (

Table 32.1 ). Ultimately, substances destined for excretion in bile are exported across the canalicular membrane of hepatocytes via a different array of transporters. The features of bile allow solubilization of even lipophilic substances, which can then be excreted into the intestine and ultimately leave the body in feces.

Structural Features of the Liver and Biliary System

Hepatocytes, the major cell type in the liver, are arranged in anastomosing cords that form plates around which large volumes of blood circulate (

Fig. 32.2 ). The liver receives a high blood flow that is disproportionate to its mass, which ensures that hepatocytes receive high quantities of both O2 and nutrients. Hepatocytes receive more than 70% of their •Fig. 32.2 Diagrammaticrepresentationofahepaticlobule.Platesofhepatocytesarearrayedradiallyaroundacentralvein.Branchesoftheportalveinandhepaticarteryarelocatedontheperipheryofthelobuleandformthe“portaltriad”togetherwiththebileduct.Bloodfromtheportalveinandhepaticarterypercolatesaroundthehepatocytesviathesinusoidsbeforedrainingintothecentralvein.(ModifiedfromBloomW,FawcettDW.A Textbook of Histology. 10thed.Philadelphia:Saunders;1975.) blood supply at rest via the portal vein (rising to more than 90% in the postprandial period).

The plates of hepatocytes that constitute the liver parenchyma are supplied by a series of sinusoids, which are low-resistance cavities supplied by branches of both the portal vein and hepatic artery. The sinusoids are unlike the capillaries that perfuse other organs. During fasting, many sinusoids are collapsed, but more can gradually be recruited as portal blood flow increases during the period after a meal when absorbed nutrients are transported to the liver. The low resistance of the sinusoidal cavities means that blood flow through the liver can increase considerably without •Fig. 32.3 Interrelationshipsofthemajorcelltypesintheliver.

a concomitant increase in pressure. Eventually the blood drains into central branches of the hepatic vein.

The sinusoids are also unusual in terms of the endothelial cells that line their walls (

Fig. 32.3 ). Hepatic endothelial cells contain specialized openings known as fenestrations that are large enough to permit passage of molecules as big as albumin. Sinusoidal endothelial cells also lack a basement membrane, which might otherwise pose a diffusion barrier. These features allow access of albumin-bound substances to the hepatocytes that will eventually take them up. The sinusoids also contain Kupffer cells. Beneath the sinusoidal endothelium and separating the endothelium from the hepatocytes is a thin layer of loose connective tissue called the space of Disse, which in health likewise poses little resistance to the movement of molecules even as large as albumin. The space of Disse is also the location of another important hepatic cell type, the stellate cell. Stellate cells serve as storage sites for retinoids and in addition are the source of key growth factors for hepatocytes.

Under abnormal conditions, stellate cells are activated to synthesize large quantities of collagen, which contributes to the hepatic dysfunction.

Ifthecirculationoftheliver(particularlyitssinusoids)iscompressedbyfibrosis,theliverlosesitsabilitytoaccommodatetheincreasesinbloodflowthatoccurafteramealwithoutaconcomitantincreaseinpressure.Becauseofthefenestrations,albuminescapesfromthecirculationandalbumin-richfluidweepsfromthesurfaceoftheliverintotheabdominalcavity,whereitoverwhelmsthelymphaticdrainage.Thisconditionisknownasascites andisreflectedinaconsiderableincreaseinthegirthofmanypatientswithliverdisease.Aspressureintheliverbuilds,newcollateralbloodvesselsforminanattempttocircumventtheobstructionandreducetheportalhypertension.Someofthesevesselsaredirectedtoabdominalstructuresand,becauseoftheirthinweakwalls,arepronetorupture.Aparticularexampleisformationofhigh-pressurecollateralstotheesophagus,whichcanthenbecomevaricesthatbleedintothelumen.Bleedingintotheesophageallumenisveryhardtocontrolandisthusamedicalemergency.Evenintheabsenceofbleeding,moreover,theformationofcollateralbloodvesselsbypassestheremainingmetaboliccapacityoftheliver,andlevelsoftoxinssuchasammoniaareincreasedandcanexertadverseeffectselsewhereinthebody.

Infectionoftheliverwithcertainvirusesoroverexposuretotoxicsubstancessuchasalcoholkillshepatocytesandactivateshepaticstellatecells,whichsynthesizeexcessiveamountsofcollagenthatresultinthehistologicappearanceoffibrosis.Iftheinsultischronic,thefibrosiseventuallybecomesirreversible,aconditionknownascirrhosis. Thefibroticscarredareascrowdoutthehepatocytemass,therebyreducingthesynthetic,metabolic,andexcretorycapacityoftheliver.Fibroticmassespressonthesinusoidsandpreventthemfromexpandingasportalbloodflowtotheliverincreasesduringthepostprandialperiod.Edemamaydevelopinpatientswithchronicliverinjuryasaresultofreducedlevelsofalbumininblood,andascitesmaythendevelop,inwhichfluidaccumulatesintheperitonealcavitysecondarytoincreasedportalpressure.Eventually,accumulationoftoxicsubstancesinthebloodstreamcanleadtojaundice,itching,andneurologicalcomplications.Ifhepaticfunctionbecomescompromisedbeyondacertainlevel,theonlyeffectivetreatmentislivertransplantation.

Hepatocytes are also the origination point for the biliary system. Although hepatocytes are considered to be epithelial cells with basolateral and apical membranes, the spatial arrangement of these two cell domains differs from that seen in simple columnar epithelium, such as that lining the gastrointestinal tract. Rather, in the liver the apical surface of the hepatocyte occupies only a small fraction of the cell membrane, and the apical membranes of adjacent cells oppose each other to form a channel between the cells known as the canaliculus (see

Fig. 32.3 ). The role of canaliculi is to drain bile from the liver, and these canaliculi drain into biliary ductules that are lined by classic columnar epithelial cells known as cholangiocytes. Ultimately the biliary ductules drain into large bile ducts that coalesce into the right and left hepatic ducts to permit exit of bile from the liver. These in turn form the common hepatic duct, from which bile can flow into either the gallbladder (via the cystic duct) or the intestine (via the common bile duct;

Fig. 32.4 ) on the basis of prevailing pressure relationships.

One other feature of the structural organization of the liver bears emphasis because of its clinical significance. Branches of the hepatic vein, hepatic artery, and bile ducts run in parallel in the so-called hepatic triad. Hepatocytes lying closest to this triad are referred to as periportal, or zone 1, and have the greatest supply of oxygen and nutrients. In contrast, hepatocytes lying closest to branches of the hepatic vein are referred to as pericentral, or zone 3. The latter cells are more sensitive to ischemia, whereas the former are more sensitive to oxidative injury. Thus the location of damaged cells on biopsy may provide clues to the cause of a given case of liver injury. Zone 1 cells are most active in detoxification functions in normal circumstances, but •Fig. 32.4 Functionalanatomyofthebiliarysystem.Right and left hepatic ducts from liver Common hepatic duct Common bile duct Cystic duct Sphincter of Oddi Duodenum Gallbladder zone 2 (intermediate between zones 1 and 3) and zone 3 cells can progressively be recruited in cases of liver disease. Conversely, zone 3 cells are thought to be most active in bile acid synthesis.

Bile is the excretory fluid of the liver that plays an important role in lipid digestion. Bile formation begins in hepatocytes, which actively transport solutes into bile canaliculi across their apical membranes. Bile is a micellar solution in which the major solutes are bile acids, phosphatidylcholine, and cholesterol in an approximate ratio of 10:3:1, respectively. Secretion of these solutes drives concomitant movement of water and electrolytes across the tight junctions that link adjacent hepatocytes to form canalicular bile. The majority of bile flow is driven by the secretion of bile acids across the apical membrane of hepatocytes via an adenosine triphosphatase (ATPase) transporter known as the bile salt export pump (BSEP; see

Table 32.1 ). The composition of the resulting fluid can be modified further as it flows through the biliary ductules (resulting in hepatic bile) and still further on storage in the gallbladder (gallbladder bile). Ultimately bile becomes a concentrated solution of biological detergents that aids in solubilization of the products of lipid digestion in the aqueous environment of the intestinal lumen, thereby enhancing the rate at which lipids are transferred to the absorptive epithelial surface. It also serves as a medium in which metabolic waste products are exported from the body.

Thoughrare,avarietyoffamilialsyndromesthataremanifestedasprogressivecholestasishavetaughtusagreatdealaboutthemolecularnatureofthetransportersthatdeliverbileconstituentsintothecanaliculus.Forexample, (PFICII)hasbeenmappedtoamutationinBSEP thatresultsinanalmosttotalabsenceofbileacidsinbile.Cholestasisdevelopsinpatientswiththisdisorder,buttheyhaverelativelylittleifanyevidenceofbileductinjury.TypeIIIPFIContheotherhandisamuchmoreaggressivediseaseinwhichcholestasisisaccompaniedbyearlyincreasesincirculatingγ-glutamyltranspeptidase.Themolecularculpritisamutationthatabolishesexpressionofmultidrug resistance protein 3 (MDR3).Intheabsenceofthistransporter,phosphatidylcholineisnolongerabletoenterbile,thusillustratingtheimportanceofthislipidinprotectingcholangiocytesfromtheinjuriouseffectsofbileacids,becausemixedmicellescannotforminitsabsence.

Bile acids are produced by hepatocytes as end products of cholesterol metabolism. Cholesterol is selectively metabolized by a series of enzymes that result in formation of bile acid (

Fig. 32.5 ). The initial and rate-limiting step is addition of a hydroxyl group to the 7 position of the steroid nucleus by the enzyme cholesterol 7α-hydroxylase. The side chain of the product of this reaction is then shortened and a carboxylic acid function added by C27 dehydroxylase to yield chenodeoxycholic acid, a dihydroxy bile acid. Alternatively the product is further hydroxylated at the 12 position and then acted on by C27 dehydroxylase to yield cholic acid, a trihydroxy bile acid. Bile acid synthesis can be up-or downregulated depending on the body’s requirements (Fig.

32.6 ). For example, if bile acid levels are reduced in the blood flowing to the liver, synthesis can be increased up to 10-fold. Conversely, feeding of bile acids profoundly suppresses new synthesis of bile acids by hepatocytes. The mechanisms underlying these changes in bile acid synthesis relate to changes in expression of the enzymes involved. Bile acids activate a variety of cell surface and nuclear receptors in hepatocytes, leading ultimately to activation of specific transcription factors that regulate enzyme abundance.

Chenodeoxycholic acid and cholic acid are defined as primary bile acids because they are synthesized by the hepatocyte (see

Fig. 32.5 ). However, each can be metabolized in the colonic lumen by bacterial enzymes to yield ursodeoxycholic and deoxycholic acid, respectively. Chenodeoxycholic acid is also converted by bacterial enzymes to form lithocholic acid, which is relatively cytotoxic. Collectively these three products of bacterial metabolism are referred to as secondary bile acids. One additional important biochemical modification occurs for both primary and secondary bile acids in the hepatocyte (see

Fig.

32.5 ). These molecules are conjugated with either glycine or taurine, which significantly depresses their pKa. The result is that conjugated bile acids are almost totally ionized at the pH prevailing in the small intestinal lumen and thus cannot passively traverse cell membranes. Consequently the conjugated bile acids are retained in the intestinal lumen until they are actively absorbed in the terminal ileum via the apical sodium-dependent bile salt transporter (asbt). Conjugated bile acids that escape this uptake step are deconjugated by bacterial enzymes in the colon, and the resulting unconjugated forms are passively reabsorbed across the colonic epithelium because they are no longer charged.

Hepatic Aspects of Enterohepatic Circulation of Bile Acids

Bile acids assist in digestion and absorption of lipids by acting as detergents rather than enzymes, and thus a significant mass of these molecules is required to solubilize all dietary lipids. Via the enterohepatic circulation, actively reabsorbed conjugated bile acids travel through the portal blood back to the hepatocyte, where they are efficiently taken up by basolateral transporters that may be Na+ dependent or independent (see

Table 32.1 ). Similarly, bile acids that are deconjugated in the colon also return to the hepatocyte, where they are reconjugated to be secreted into bile. In this way a pool of circulating primary and secondary

Cholesterol Major bile acids Cholic acid HO Primary Secondary OH OH COOH C27 dehydroxylase 7˜-Hydroxylase (12˜-hydroxylase) HO HO OH COOH Chenodeoxycholic acid Deoxycholic acid Conjugation of bile acids HO OH COOH HO OH 3 7 12 OH C O O OH C O–H CH2N H Glycine, pKa˜3.7 Taurine, pKa˜1.5 or SO2O–HN CH2CH2 H HO OH COOH Ursodeoxycholic acid HO COOH Lithocholic acid Liver Colon (bacteria) •Fig. 32.5 Structuresofthemajorprimaryandsecondarybileacidsofbile.Primarybileacidsaresynthesizedintheliver.Secondarybileacidsareproducedwhenintestinalbacteriaactonprimarybileacids.Atthebottomofthefiguretheconjugationofcholicacidwithglycineortaurineisshown.

bile acids is produced, and daily synthesis is then equal only to the minor fraction (≈10%/day, or 200–400 mg) that escapes uptake and is lost in stool (

Fig. 32.7 ). The only exception to this rule is lithocholic acid, which is preferentially sulfated in the hepatocyte rather than being conjugated with glycine or taurine. The majority of the sulfate conjugates are lost from the body after each meal because they are not substrates for asbt, thereby avoiding accumulation of a potentially toxic molecule.

Some comment should also be made with respect to the role of bile acids in whole-body cholesterol homeostasis. The pool of cholesterol in the body reflects its daily synthesis as well as the relatively minor component derived from inefficient dietary uptake, balanced against loss from the body, which can only occur in health via bile (

Fig. 32.8 ). Cholesterol can be excreted in two forms, either as the native molecule or after its conversion to bile acids. The latter account for up to a third of the cholesterol excreted per day despite enterohepatic recycling. Thus one strategy for treating hypercholesterolemia is to interrupt the enterohepatic circulation of bile acids, which drives increased conversion of cholesterol to bile acids; the bile acids are then lost from the body in feces.

As noted earlier, bile also contains cholesterol and phosphatidylcholine. Cholesterol transport across the canalicular membrane is mediated at least in part by a heterodimer of the active transporters we discussed in as participating in the efflux of cholesterol from the small intestinal epithelial cells, namely, ABC G5 and ABC G8 (see 32.1 ). Phosphatidylcholine derives from the inner leaflet of the canalicular membrane and is specifically “flipped” across the membrane by another ABC family transporter called multidrug resistance protein 3 (MDR3). Furthermore, 5.0

Limit of intestinal absorption 0.5 30 0.0 0.25 0.5 0.75 1.0 1.25 Diet Hepatic and extra-hepatic synthesis To bile As bile acids As cholesterol In health, input = output Cholesterol (g/day) 0.5 •Fig. 32.6 Relationshipbetweenratesofbileacidsynthesisandsecretion.Thebileacidsecretionratenormallyaverages30g/24h,whereasthesynthesisrateaverages0.5g/24h.Thepairsofverticalandhorizontaldottedlinesdepictthenormalrangeforbileacidsecretionandsynthesis,respectively.Increasedsecretion(simulatedbybileacidfeeding)increasestherateofreturnofbileacidstotheliverviaportalblood,whichexertsanegativefeedbackonsynthesis.Conversely,interruptionoftheenterohepaticcirculation,suchasafterilealresection,canincreasesynthesistovaluesmorethan10-foldhigherthannormal.(FromCareyMC,CahalaneMJ.In:AriasIMetal.[eds].The Liver: Biology and Pathobiology. 2nded.NewYork:RavenPress;1988.) •Fig. 32.8 Dailycholesterolbalanceinhealthyadulthumans.

•Fig. 32.9 Pathwaysforentryofsolutesintobile.(ModifiedfromBarrettKE.Gastrointestinal Physiology. NewYork:McGraw-Hill;2006.) because mixed micelles composed of bile acids, phosphatidylcholine, and cholesterol are osmotically active and the tight junctions that link adjacent hepatocytes are relatively leaky, water is drawn into the canalicular lumen, as well as other plasma solutes (e.g., Ca++, glucose, glutathione, amino acids, urea) at concentrations essentially approximating •Fig. 32.7 Relativeamountsofbileacidsindifferentbodypoolsand theenterohepaticcirculation.Therelativeamountsareindicatedbythe widthofthegreenlines.Thusthefigureillustratesthatthemajorityof thebileacidpoolcirculatesbetweentheliver,gallbladder,andsmall

Fig. 32.9 ). Finally, conjugated bilirubin, which is water soluble, and a variety of additional organic and xenobiotics are secreted into bile across the apical intestine,ratherthanbeinginthesystemiccirculation.membrane of the hepatocyte.

•Fig. 32.10 Themajortransportprocessesofcholangiocytesthatsecreteanalkaline-richfluidandreclaimusefulsubstances.AQP,aquaporin;CFTR,cysticfibrosistransmembraneconductanceregulator;GGT,γ-glutamyltranspeptidase;NKCC1,sodium/potassium/2chloridecotransporter1;NBC,sodium/bicarbonatecotransporter;NHE1,sodium/hydrogenexchanger1,SGLT-1,sodium-glucosecotransporter1;GLUT1,glucosetransporter1.

The cholangiocytes lining the biliary ductules are specifically designed to modify the composition of bile (

Fig. 32.10 ). Useful solutes (e.g., glucose, amino acids) are reclaimed by the activity of specific transporters. Chloride ions in bile are also exchanged for HCO3 − , rendering the bile slightly alkaline and reducing the risk of precipitation of Ca++ . Glutathione is broken down on the surface of cholangiocytes into its constituent amino acids by the enzyme γ-glutamyl transpeptidase (GGT), and the products are reabsorbed. Bile is also diluted at this site, in concert with ingestion of a meal, in response to hormones such as secretin that increase HCO3 − secretion and stimulate insertion of aquaporin water channels into the cholangiocyte’s apical membrane. Flow of bile is thereby increased during the postprandial period when bile acids are needed to aid in assimilation of lipid.

Role of the Gallbladder

Finally, bile enters the ducts and is conveyed toward the intestine. However, in the period between meals, outflow is blocked by constriction of the sphincter of Oddi, and thus bile is redirected to the gallbladder. The gallbladder is a muscular sac lined with high-resistance epithelial cells. During gallbladder storage, bile becomes concentrated because sodium ions are actively absorbed in exchange for protons, and bile acids, as the major anions, are too large to exit across the gallbladder epithelial tight junctions (Fig.

32.11 ). However, although the concentration of bile acids can rise more than 10-fold, bile remains isotonic because a •Fig. 32.11 Mechanismsaccountingforconcentrationofbileduringstorageinthegallbladder.NHE,sodium/hydrogenexchanger;BA−,bileacidanion;AQP1,aquaporin1.

single micelle acts as only one osmotically active particle. Any additional bile acid monomers that become available as a result of concentration are thus immediately incorporated into existing mixed micelles. This also reduces to some extent the risk that cholesterol will precipitate from bile. However, cholesterol is supersaturated in the bile of many adults, with precipitation normally being inhibited by the presence of antinucleating proteins. Prolonged storage of bile increases the chance that nucleation can occur, thus making a good case for never skipping breakfast and perhaps explaining why gallstone disease is relatively prevalent in humans.

Humansareunusuallysusceptibletogallstones, whichrepresentprecipitatedbileconstituentsthataccumulateinthegallbladderorelsewhereinthebiliarytree.GallstonesarecomposedpredominantlyofcholesterolorCa++ bilirubinate(cholesterolvs.pigmentstones,respectively).Theirsignificanceliesintheirpropensitytoobstructbiliaryflowandtherebyresultinpain,poortoleranceoflargefattymeals,retentionofbiliaryconstituents,and(ifleftuntreated)liverinjury.Insusceptibleindividuals,mechanismsthatnormallypreventnucleationofsaturatedbileareeitherdefectiveorovercome,andsmallcrystalsformandcangrowintogallstones.Humanbileisoftensupersaturatedintermsofitscholesterolcontent,thusincreasingtheriskforstoneformation,particularlyduringprolongedfasting.Cholesterolgallstonesareespeciallycommoninmiddle-agedwomenwhoareobese,particularlythosewhohavebornechildren.Thisincreasedprevalenceisapparentlydueatleastinparttotheabilityofestrogentoincreasehepaticcholesterolsecretion.Inseverecasesofgallstonediseasethegallbladdermayberemovedsurgically,whichisusuallyaccomplishedlaparoscopically.SmallgallstonesthathavelodgedinthebiliarytreecansometimesberetrievedendoscopicallybyinsertingasmallsnarethroughthesphincterofOddifromanendoscope.

Bile is secreted from the gallbladder in response to signals that simultaneously relax the sphincter of Oddi and contract the smooth muscle that encircles the gallbladder epithelium (

Fig. 32.12 ). A critical mediator of this response is cholecystokinin. In addition, intrinsic neural reflexes and vagal pathways, some of which themselves are stimulated by the ability of cholecystokinin to bind to vagal afferents, also contribute to gallbladder contractility. The net result is ejection of a concentrated bolus of bile into the duodenal lumen, where the constituent mixed micelles can aid in lipid uptake. Then, when no longer needed, the bile acids are reclaimed and reenter the enterohepatic circulation to begin the cycle again. However, the other components of bile are largely lost in stool, thus providing for their excretion from the body.

Bilirubin Formation and Excretion by the Liver

The liver is also important for excretion of bilirubin, which is a metabolite of heme that is potentially toxic to the body. Bilirubin is an antioxidant and also serves as a way to eliminate the excess heme released from the hemoglobin of senescent red blood cells. Indeed, red blood cells account for 80% of bilirubin production, with the remainder coming from additional heme-containing proteins in other tissues such as skeletal muscle and the liver. Bilirubin can cross the blood-brain barrier and, if present in excessive levels, results •Fig. 32.12 Neurohumoralcontrolofgallbladdercontractionandbiliarysecretion.ThepathwayalsoinvolvesrelaxationofthesphincterofOdditopermitoutflowofbileintotheduodenum.ACh,acetylcholine;CCK,cholecystokinin;NO,nitricoxide;VIP,vasoactiveintestinalpolypeptide.

in brain dysfunction secondary to neuronal cell death and the activation of astrocytes and microglia; it can be fatal if left untreated. Bilirubin and its metabolites are also notable for the fact that they provide color to bile, feces, and to a lesser extent urine. By the same token, when bilirubin accumulates in the circulation as a result of liver disease, it is responsible for the common symptom of jaundice, or yellowing of the skin and conjunctiva.

Bilirubin is synthesized from heme by a two-stage reaction that takes place in phagocytic cells of the reticuloendothelial system, including Kupffer cells and cells in the spleen (

Fig. 32.13 ). The enzyme heme oxygenase that is present in these cells liberates iron from the heme molecule and produces the green pigment biliverdin. This in turn can be reduced to form yellow bilirubin. Because bilirubin is essentially insoluble in aqueous solutions at neutral pH, it is transported through the bloodstream bound to albumin. When this complex reaches the liver, it enters the space of Disse, where bilirubin is selectively taken up across the basolateral membrane of hepatocytes via an OATP transporter (see

Table 32.1 ). In the microsomal compartment, bilirubin is then conjugated with one or two molecules of glucuronic acid to enhance its aqueous solubility. The reaction is catalyzed by UDP glucuronyl transferase (UGT). This enzyme is synthesized only slowly after birth, which explains why mild jaundice is relatively common in •Fig. 32.13 Conversionofhemetobilirubin.Thereactionsinsidethedashedboxoccurincellsofthereticuloendothelialsystem.NADP+,oxidizedformofnicotinamideadeninedinucleotidephosphate;NADPH,reducedformofnicotinamideadeninedinucleotidephosphate.

newborn infants. The bilirubin conjugates are then secreted into bile by a multidrug resistance–associated protein (MRP2) located in the canalicular membrane. Notably the conjugated forms of bilirubin cannot be reabsorbed from the intestine, thereby ensuring they can be excreted. However, transport of bilirubin across the hepatocyte (and indeed its initial uptake from the bloodstream) is relatively inefficient, so some conjugated and unconjugated bilirubin is present in plasma even under normal conditions. Both circulate bound to albumin, but the conjugated form is bound more loosely and thus can enter the urine.

In the colon, bilirubin conjugates are deconjugated by bacterial enzymes, whereupon the bilirubin liberated is metabolized by bacteria to yield urobilinogen, which is reabsorbed, and urobilins and stercobilins, which are excreted. Absorbed urobilinogen in turn can be taken up by hepatocytes and reconjugated, thus giving the molecule yet another chance to be excreted.

Measurement of bilirubin in plasma, as well as assessment of whether it is unconjugated or conjugated, is an important tool in the evaluation of liver disease. The presence of unconjugated bilirubin, which is essentially fully albumin bound and cannot be excreted in urine, reflects either loss of UGT (or a normal temporary delay in its maturation in infants) or a sudden oversupply of heme that overwhelms the conjugation mechanism (such as occurs in transfusion reactions or in Rhesus-incompatible newborns). Conjugated bilirubinemia on the other hand is characterized by the presence of bilirubin in urine, to which it imparts a dark coloration. This is indicative of genetic defects in the transporter that mediates bilirubin glucuronide/diglucuronide secretion into the canaliculus, or it may be due to blockage to the flow of bile, perhaps

CHAPTER 32 Transport and Metabolic Functions of the Liver caused by an obstructing gallstone. In both cases, bilirubin conjugates are formed in the liver, but with no means of exit they regurgitate back into plasma for urinary excretion.

Crigler-Najjar syndrome isaconditionassociatedwithmutationsinthehepatocyteenzymeUGT.IntypeICrigler-Najjarsyndrome,acongenitalmissensemutationresultsincompletelackofthisenzyme,whereaspatientswithtypeIICrigler-NajjarsyndromehaveamildermutationthatreducesUGTlevelstoabout10%ofthoseseeninnormalindividuals.Thuswithvaryingdegreesofseverity,Crigler-Najjarsyndromeimpairstheabilityofhepatocytestoconjugatebilirubin.Theunconjugatedbilirubinregurgitatesbackintothecirculationandbindstoalbumin,withanassociatedriskofneurologicalinjuryiflevelsriseprecipitously.TheonlyeffectivetreatmentoftypeICrigler-Najjarsyndromeatpresentislivertransplantation,althoughgenetherapymaybeapromisingoptioninthenearfuture.ThosewithtypeIIdiseasecansometimesbemanagedeffectivelywithbluelight.Thisconvertscirculatingunconjugatedbilirubintoformsthataremorewatersolubleandthuslessfirmlyboundtoalbumin,whichcanbeexcretedinurine.

Ammonia Handling by the Liver

Ammonia (NH3) is a small neutral metabolite that arises from protein catabolism and bacterial activity and is highly membrane permeant. The liver is a critical contributor to prevention of ammonia accumulation in the circulation, which is important because like bilirubin, ammonia is toxic to the central nervous system. The liver eliminates ammonia from the body by converting it to urea via a series of enzymatic reactions known as the urea, or Krebs-Henseleit, cycle (Fig. 32.14 ). The liver is the only tissue in the body that can convert ammonia to urea.

Ammonia is derived from two major sources. Approximately 50% is produced in the colon by bacterial ureases. Because the colonic lumen is normally slightly acidic, some of this ammonia is converted to the ammonium ion (NH4 +), which renders it impermeant to the colonic epithelium and therefore allows it to be excreted in stool. However, the remainder of the ammonia generated crosses the colonic epithelium passively and is transported to the liver via the portal circulation. The other major source of ammonia (≈40%) is the kidney (see ). A small amount of ammonia (≈10%) is derived from deamination of amino acids in the liver, by metabolic processes in muscle cells, and via release of glutamine from senescent red blood cells.

The “mass balance” for ammonia handling in a healthy adult is presented in

Fig. 32.15 . As just noted, ammonia is a small neutral molecule that readily crosses cell membranes without the benefit of a specific transporter, although some membrane proteins transport ammonia, including certain aquaporins. Whatever the mechanism for transport, the physicochemical properties of ammonia ensure that it is efficiently extracted from portal and systemic circulation •Fig. 32.14 Theureacycle.ADP,adenosinediphosphate;ATP,adenosinetriphosphate.

If the metabolic capacity of the liver is compromised acutely, coma and death can rapidly ensue. In chronic liver disease, patients may experience a gradual decline in mental function that reflects the action of both ammonia and other toxins that cannot be cleared by the liver, in a condition known as hepatic encephalopathy. Development of confusion, dementia, and eventually coma in a patient with liver disease is evidence of significant progression, and these symptoms can prove fatal if left untreated.

Clinical Assessment of Liver Function

Given the importance of the liver for homeostasis, tests of liver function are a mainstay of clinical diagnosis. Such tests have several goals: (1) to assess whether hepatocytes have been injured or are dysfunctional, (2) to determine whether bile excretion has been interrupted, and (3) to evaluate whether cholangiocytes have been injured or are dysfunctional. Liver function tests are also used to monitor responses to therapy or rejection reactions after liver trans •Fig. 32.15 Ammoniahomeostasisinhealth.(RedrawnfromBarrettKE.Gastrointestinal Physiology. NewYork:McGraw-Hill;2006.) by hepatocytes, where it then enters the urea cycle to be converted to urea (see

Fig. 32.14 ) and is subsequently transported back into the systemic circulation. Urea is a small neutral molecule that is readily filtered at the glomerulus, and it is reabsorbed by the kidney tubules such that approximately 50% of the filtered urea is excreted in urine (see ). Urea that enters the colon is either excreted or metabolized to ammonia via colonic bacteria, with the resulting ammonia being reabsorbed or excreted.

plantation. However, not all such tests measure function directly. Nevertheless, liver function tests are discussed briefly because of their link to hepatic physiology.

Tests for hepatocyte injury rely on markers that are specific for this cell type. When hepatocytes are killed by necrotic responses to inflammation or infection, for example, they release enzymes that include alanine aminotransferase (ALT) and aspartate aminotransferase (AST). These enzymes, which are essential to interconvert amino acids, are easily measured in serum and indicate hepatocyte injury, although AST may also be released after injury to other tissues, including the heart. Two other tests are markers of injury to the biliary system. Alkaline phosphatase is expressed in the canalicular membrane, and elevations of this enzyme in plasma suggest localized obstruction to bile flow. Similarly, increased levels of GGT are seen when there is damage to cholangiocytes.

Measurement of bilirubin in the circulation or in urine also provides insight into liver function. In addition, measurement of any of the other characteristic secreted products of the liver can be used to diagnose liver disease. Clinically the most common tests are measurements of serum albumin and a blood clotting parameter, the prothrombin time. If results of these tests are abnormal, when considered together with other aspects of the clinical picture, a diagnosis of liver

CHAPTER 32 Transport and Metabolic Functions of the Liver disease may be established. Blood glucose and ammonia levels are frequently monitored in patients with chronic liver disease. Finally, imaging tests and histological examination of biopsy specimens of liver parenchyma, usually obtained percutaneously, are also important in evaluating and monitoring patients with suspected or proven liver disease.

1.

Vital functions of the liver include carbohydrate, lipid, and protein metabolism and synthesis; detoxification of unwanted substances; and excretion of circulating substances that are lipid soluble and carried in the bloodstream bound to albumin. The liver also synthesizes the majority of plasma proteins, including albumin.

2.

Liver function depends on its unique anatomy, its constituent cell types (especially hepatocytes), and the unusual arrangement of its blood supply.

3.

Substances are excreted from the liver in bile. Bile flow is driven by the presence of bile acids, which are

Feranchak AP. Bile secretion and cholestasis. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015.

Kanel GC. Liver: anatomy, microscopic structure, and cell types. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015.

amphipathic end products of cholesterol metabolism that are produced by hepatocytes. Bile acids circulate between the liver and intestine to conserve their mass, and water-insoluble metabolites (e.g., cholesterol) are carried in bile in the form of mixed micelles.

4.

Bile is stored in the gallbladder between meals, where it is concentrated and released when hormonal and neural signals simultaneously contract the gallbladder and relax the sphincter of Oddi.

5.

The liver is critical for disposing of certain substances that would be toxic if allowed to accumulate in the bloodstream, including bilirubin and ammonia.

Parekh PJ, Balart LA. Ammonia and its role in the pathogenesis of hepatic encephalopathy. Clin Liver Dis. 2015;19:529-537.

Wolkoff AW. Organic anion uptake by hepatocytes. Compr Physiol. 2014;4:1715-1735.

Wu T, et al. Gut motility and enteroendocrine secretion. Curr Opin Pharmacol. 2013;13:928-934.

SECTION 7The Renal System BRUCE A. STANTON AND BRUCE M. KOEPPEN

Chapter 36 Potassium, Calcium, and Phosphate Homeostasis

Elements of Renal Function

Chapter 34 Solute and Water Transport Along the Nephron: Tubular Function

Chapter 35 Control of Body Fluid Osmolality and Volume

Chapter 37 Role of the Kidneys in the Regulation of Acid-Base Balance

Upon completion of this chapter the student should be able to answer the following questions: 1.

Which structures in the glomerulus are filtration barriers to plasma proteins?

2.

What is the physiological significance of the juxtaglomerular apparatus?

3.

What blood vessels supply the kidneys?

4.

What nerves innervate the kidneys?

5.

What is the location of the kidneys, and what are their gross anatomical features?

6.

What are the different parts of the nephron, and what is their locations within the cortex and medulla?

7.

What are the major components of the glomerulus, and what are the cell types located in each component?

8.

How can the concepts of mass balance be used to measure the glomerular filtration rate (GFR)?

9.

Why can inulin clearance and creatinine clearance be used to measure GFR?

10.

Why is plasma creatinine concentration used clinically to monitor GFR?

11.

What are elements of the glomerular filtration barrier, and how do they determine how much protein enters Bowman’s space?

12.

What Starling forces are involved in formation of the glomerular ultrafiltrate, and how do changes in each force affect GFR?

13.

What is autoregulation of renal blood flow and GFR, and which factors and hormones are responsible for autoregulation?

14.

Which hormones regulate renal blood flow?

15.

Why do hormones influence renal blood flow despite autoregulation?

Overview of Renal Function

The kidney presents in the highest degree the phenomenon of sensibility, the power of reacting to various stimuli in a direction, which is appropriate for the survival of the organism; a power of adaptation which almost gives one the idea that its component parts must be endowed with intelligence.

E. STARLING—1909

Certainly, mental integrity is a sine qua non of the free and independent life. But let the composition of our internal environment suffer change, let our kidneys fail for even a short time to fulfill their tasks, and our mental integrity, or personality is destroyed.

HOMER W. SMITH—1939

As both Starling and Smith recognized, the kidneys are more appropriately considered regulatory rather than excretory organs. The kidneys regulate (1) body fluid osmolality and volumes, (2) electrolyte balance, and (3) acid-base balance. In addition the kidneys excrete metabolic products and foreign substances and produce and secrete hormones.

Control of body fluid osmolality is important for maintenance of normal cell volume in all tissues of the body. Control of body fluid volume is necessary for normal function of the cardiovascular system. The kidneys are also essential in regulating the amount of several important inorganic ions in the body, including Na+, K+, Cl−, bicarbonate (HCO3 −), hydrogen (H+), Ca++ , and inorganic phosphate (Pi). Excretion of these electrolytes must be equal to daily intake to maintain appropriate total body balance. If intake of an electrolyte exceeds its excretion, the amount of this electrolyte in the body increases and the individual is in positive balance for that electrolyte. Conversely if excretion of an electrolyte exceeds its intake, its amount in the body decreases and the individual is in negative balance for that electrolyte. For many electrolytes the kidneys are the sole or principal route for excretion from the body.

Another important function of the kidneys is regulation of acid-base balance. Many metabolic functions of the body are exquisitely sensitive to pH. Thus the pH of body fluids must be maintained within narrow limits. Normal pH is maintained by buffers within body fluids and by the coordinated action of the lungs, liver, and kidneys.

The kidneys excrete a number of the end products of metabolism. These waste products include urea (from amino acids), uric acid (from nucleic acids), creatinine (from muscle creatine), end products of hemoglobin metabolism, and metabolites of hormones. The kidneys eliminate these substances from the body at a rate that matches their production. Thus the kidneys regulate hormone concentrations within body fluids. The kidneys also represent an important route for elimination of foreign substances such as drugs, toxins (e.g., pesticides), and other chemicals from the body.

Finally, the kidneys are important endocrine organs that produce and secrete renin, calcitriol, and erythropoietin. Renin is not a hormone but an enzyme that activates the renin-angiotensin-aldosterone system, which helps regulate blood pressure and Na+ and K+ balance. Calcitriol, a metabolite of vitamin D3, is necessary for normal absorption of Ca++ by the gastrointestinal tract and for its deposition in bone (see ). In patients with renal disease the kidneys’ ability to produce calcitriol is impaired and levels of this hormone are reduced. As a result, Ca++ absorption by the intestine is decreased, which over time contributes to the bone formation abnormalities seen in patients with chronic renal disease. Another consequence of many kidney diseases is a reduction in erythropoietin production and secretion. Erythropoietin stimulates red blood cell formation by bone marrow. Decreased erythrocyte production contributes to the anemia that occurs in chronic kidney disease (CKD), a progressive loss in kidney function over a period of months or years.

A large variety of diseases impair kidney function and result in renal failure. In some instances the impairment in renal function is transient, but in many cases renal function declines progressively. Patients in whom the glomerular filtration rate (GFR) is less than 10% of normal are said to have end-stage renal disease (ESRD) and must receive renal replacement therapy in the form of either dialysis or kidney transplantation to survive.

To understand the mechanisms that contribute to renal disease, it is first necessary to understand the normal physiology of renal function. Thus in the following chapters in this section of the book, various aspects of renal function are considered.

Functional Anatomy of the Kidneys

Structure and function are closely linked in the kidneys. Consequently an appreciation of the gross anatomical and histological features of the kidneys is a prerequisite for understanding their functions.

The kidneys are paired organs that lie on the posterior wall of the abdomen behind the peritoneum on either side of the vertebral column. In an adult human, each kidney weighs between 115 and 170 g and is approximately 11 cm long, 6 cm wide, and 3 cm thick.

The gross anatomical features of the human kidney are illustrated in

Fig. 33.1 . The medial side of each kidney contains an indentation through which pass the renal artery and vein, nerves, and pelvis. If a kidney were cut in half, two regions would be evident: an outer region called the cortex and an inner region called the medulla. The cortex and medulla are composed of nephrons (the functional units of the kidney), blood vessels, lymphatics, and nerves. The

Kidney disease is a major health problem worldwide. In the United States alone:

CKD affects over 23 million patients and accounts for more than 90,000 deaths annually.

Kidney diseases represent the ninth leading cause of death.

The health care cost for CKD in Medicare patients alone exceeds $44 billion per year.

Each year, kidney disease is diagnosed in over 3 million new patients.

Over 500,000 people are treated for ESRD every year.

Approximately 275,000 patients with ESRD are maintained on either hemodialysis or peritoneal dialysis.

Diabetes and hypertension are the leading causes of ESRD.

ESRD secondary to diabetes is increasing at an annual rate of more than 11% per year.

More than 17,000 kidney transplants are performed each year. Unfortunately, in excess of 100,000 patients are awaiting kidney transplants.

Urinary tract infections (8.3 million visits annually), urolithiasis (i.e., kidney and urinary tract stones; 1.3 million visits annually), interstitial cystitis (i.e., inflammation of the urinary bladder; 700,000 patients), and urinary incontinence (13 million adults affected, mostly older than 65) are also major health care problems. Individuals with ESRD must undergo renal replacement therapy, which includes peritoneal dialysis, hemodialysis, and kidney transplantation. Both peritoneal dialysis and hemodialysis, as their names suggest, rely on the ability to remove small dialyzable molecules from the blood—including metabolic waste products normally removed by intact kidneys—via diffusion across a selectively permeable membrane into a solution lacking these substances, thereby mitigating both their accumulation and associated adverse health effects. In addition, dialysis helps reestablish both fluid and electrolyte balance via removal of excess fluid, correction of acid-base changes, and normalization of plasma electrolyte concentrations). In peritoneal dialysis, the peritoneal membrane lining the abdominal cavity acts as a dialyzing membrane. Several liters of a defined dialysis solution are typically introduced into the abdominal cavity, and small molecules in blood diffuse across the peritoneal membrane into the solution, which can then be iteratively removed, discarded, and replaced. In hemodialysis, a patient’s blood is pumped through an extracorporeal artificial kidney in which blood is separated from a defined dialysis solution by an artificial semipermeable membrane that allows small molecules to diffuse from the blood down their concentration gradient into the dialysis solution, thereby removing small molecules associated with adverse health effects if allowed to accumulate in patients without functioning kidneys. Patients who are candidates for renal transplantation are often treated with dialysis until an appropriate donor kidney can be procured. Although anemia has historically been a significant problem in ESRD patients owing to severely reduced endogenous erythropoietin production, this problem can now be easily corrected in patients undergoing chronic dialysis via administration of erythropoiesis-stimulating agents (e.g., recombinant human erythropoietin).

CHAPTER 33 Elements of Renal Function 583 • Fig. 33.1 Structure of a human kidney, cut open to show the internal structures. (Modified From Boron WF, Boulpaep EL. Medical Physiology. 2nd ed. Philadelphia: Saunders Elsevier; 2009.) medulla in the human kidney is divided into conical masses called renal pyramids. The base of each pyramid originates at the corticomedullary border, and the apex terminates in a papilla, which lies within a minor calyx. Minor calyces collect urine from each papilla. The numerous minor calyces expand into two or three open-ended pouches, the major calyces. The major calyces in turn feed into the pelvis. The pelvis represents the upper expanded region of the ureter, which carries urine from the pelvis to the urinary bladder. The walls of the calyces, pelvis, and ureters contain smooth muscle that contracts to propel the urine toward the urinary bladder.

Blood flow to the two kidneys is equivalent to about 25% (1.25 L/min) of the cardiac output in resting individuals. However, the kidneys constitute less than 0.5% of total body weight. As illustrated in

Fig. 33.2 (left), the renal artery branches progressively to form the interlobar artery, the arcuate artery, the interlobular artery, and the afferent arteriole, which leads into the glomerular capillaries. The glomerular capillaries come together to form the efferent arteriole, which leads into a second capillary network, the peritubular capillaries, which supply blood to the nephron. The vessels of the venous system run parallel to the arterial vessels and progressively form the interlobular vein, arcuate vein, interlobar vein, and renal vein, which courses beside the ureter.

Ultrastructure of the Nephron

The functional unit of the kidneys is the nephron. Each human kidney contains approximately 1.2 million nephrons, which are essentially hollow tubes composed of a single epithelial cell layer. The nephron consists of a renal corpuscle, proximal tubule, loop of Henle, distal tubule, and collecting duct system (Fig. 33.3

Fig. 33.2 ). The renal corpuscle consists of glomerular capillaries enclosed within Bowman’s capsule. The proximal tubule exits this structure and initially forms several coils, followed by a straight piece that descends toward the medulla. The next segment is the loop of Henle, which is composed of aThe organization of the nephron is actually more complicated than presented here. However, for simplicity and clarity of presentation in subsequent chapters, the nephron is divided into five segments. The collecting duct system is not actually part of the nephron. However, again for simplicity, we consider the collecting duct system part of the nephron. bAlthough the renal corpuscle is composed of glomerular capillaries and Bowman’s capsule, the term glomerulus is commonly used to described the renal corpuscle.

• Fig. 33.2 Left, Organization of the vascular system of the human kidney. 1, interlobar arteries; 1a, interlobar vein; 2, arcuate arteries; 2a, arcuate veins; 3, interlobular arteries; 3a, interlobular veins; 4, stellate vein; 5, afferent arterioles; 6, efferent arterioles; 7a, 7b, glomerular capillary networks; 8, descending vasa recta; 9, ascending vasa recta. Right, Organization of the human nephron. A superficial nephron is illustrated on the left and a juxtamedullary (JM) nephron is illustrated on the right. The loop of Henle includes the straight portion of the proximal tubule (PT), descending thin limb (DTL), ascending thin limb (ATL), and thick ascending limb (TAL). B, Bowman’s capsule; CCD, cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; MD, macula densa; OMCD, outer medullary collecting duct; P, pelvis. (Modified from Kriz W, Bankir LA. Am J Physiol 1988;254:F1; and Koushanpour E, Kriz W. Renal Physiology: Principles, Structure, and Function. 2nd ed. New York: Springer-Verlag; 1986.) the straight part of the proximal tubule, the descending thin limb (which ends in a hairpin turn), the ascending thin limb (only in nephrons with long loops of Henle), and the thick ascending limb. Near the end of the thick ascending limb, the nephron passes between the afferent and efferent arterioles of the same nephron. This short segment of the thick ascending limb abutting the glomerulus is called the macula densa (see

Figs. 33.2 33.3 ). The distal tubule begins a short distance beyond the macula densa and extends to the point in the cortex where two or more nephrons join to form a cortical collecting duct. The cortical collecting duct enters the medulla and becomes the outer medullary collecting duct and then the inner medullary collecting duct.

Each nephron segment is made up of cells that are uniquely suited to perform specific transport functions (see

Fig. 33.3 ). Proximal tubule cells have an extensively amplified apical membrane (the ultrafiltrate or urine side of the cell) called the brush border, which is present only in the proximal tubule. The basolateral membrane (the interstitial or blood side of the cell) is highly invaginated. These invaginations contain many mitochondria. In contrast, the descending and ascending thin limbs of the loop of Henle have poorly developed apical and basolateral surfaces and few mitochondria. The cells of the thick ascending limb and the distal tubule have abundant mitochondria and extensive infoldings of the basolateral membrane.

The collecting duct is composed of two cell types: principal cells and intercalated cells. Principal cells have a moderately invaginated basolateral membrane and contain few mitochondria (see

Fig. 33.3 ). Principal cells play an important role in reabsorption of NaCl (see ) and secretion of K+ (see ). Intercalated cells, which play an important role in regulating acid-base balance, have a high density of mitochondria (see

Fig. 33.3 ). One population of intercalated cells secretes H+ (i.e., reabsorbs HCO3 −), and a second population secretes HCO3 − (see ). The final segment of the nephron, the inner medullary collecting duct, is composed of inner medullary collecting duct cells, which have poorly developed apical and basolateral surfaces and few mitochondria.

All cells in the nephron except intercalated cells have in their apical plasma membrane a single nonmotile primary cilium that protrudes into the tubule fluid (

Fig. 33.4 ). Primary cilia are mechanosensors (i.e., they sense changes in the rate of flow of tubule fluid) and chemosensors (i.e., they sense or respond to compounds in the surrounding fluid), and they initiate Ca++-dependent signaling pathways, including those that control kidney cell function, proliferation, differentiation, and apoptosis (i.e., programmed cell death).

Fig. 33.2 ), with approximately 10 superficial nephrons for each juxtamedullary nephron. The 585 • Fig. 33.3 Diagram of a nephron including cellular ultrastructure. Inner medulla collecting duct Ascending thin limbThick ascending limb Descending thin limb Proximal tubule Macula densa Cortical collecting duct Distal tubule Principal cell Intercalated cell OutermedullaInner medulla Cortex • Fig. 33.4 Scanning electron micrograph illustrating primary cilia (C, ≈2 to 30 µm long and 0.5 µm in diameter) in the apical plasma membrane of principal cells in the cortical collecting duct. Note that intercalated cells (IC1 and IC2) do not have cilia but have numerous microvilli. CD, collecting duct principal cells with short microvilli (arrowhead); straight ridges (open arrow) represent the cell borders between principal cells; IC1 and IC2, intercalated cells with numerous long microvilli in the apical membrane. (From Kriz W, Kaissling B. Structural organization of the mammalian kidney. In: Seldin DW, Giebisch G [eds]. The Kidney: Physiology and Pathophysiology. 3rd ed. Philadelphia: Lippincott Williams & Wilkins; 2000.) glomerulus of each superficial nephron is located in the outer region of the cortex. The corresponding loops of Henle are short, and associated efferent arterioles branch into peritubular capillaries that surround its associated nephron segments as well as adjacent nephrons. This capillary network conveys oxygen and important nutrients to the nephron segments in the cortex, delivers substances to individual nephron segments for secretion (i.e., movement of a substance from blood into tubular fluid), and serves as a pathway for return of reabsorbed water and solutes to the circulatory system. A few species, including humans,

Polycystin 1 (encoded by the PKD1 gene) and polycystin 2 (encoded by the PKD2 gene) are expressed in the membrane of primary cilia and mediate entry of Ca++ into cells. PKD1 and PKD2 are thought to play an important role in flow-dependent K+ secretion by principal cells of the collecting duct. As described in more detail in

Chapter 36, increased flow of tubule fluid in the collecting duct is a strong stimulus for secretion of K+ . Increased flow bends the primary cilium in principal cells, which activates the PKD1/PKD2 Ca++-conducting channel complex and allows Ca++ to enter the cell and increase intracellular [Ca++]. The increase in [Ca++] activates K+ channels in the apical plasma membrane, which enhances secretion of K+ from the cell into the tubule fluid.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, occurring in about 1 in 1000 people. Approximately 12.5 million people worldwide have ADPKD, which is caused primarily by mutations in PKD1 (85%–90% of cases) or PKD2 (≈15% of cases). The major phenotype of ADPKD is enlargement of the kidneys due to the presence of hundreds or thousands of space-occupying renal cysts that can be as large as 20 cm in diameter. Cysts are also seen in the liver and other organs in this condition. About 50% of patients with ADPKD progress to renal failure by the age of 60. Although it is not clear how mutations in PKD1 and PKD2 cause ADPKD, renal cyst formation may result from defects in Ca++ uptake that alter Ca++-dependent signaling pathways, including those controlling kidney cell proliferation, differentiation, and apoptosis.

also possess very short superficial nephrons whose loops of Henle never enter the medulla.

The glomerulus of each juxtamedullary nephron is located in the region of the cortex adjacent to the medulla (see

Fig. 33.2, right). When compared with superficial nephrons, juxtamedullary nephrons differ anatomically in two important ways: the loop of Henle is longer and extends deeper into the medulla, and the efferent arteriole forms not only a network of peritubular capillaries but also a series of accompanying vascular loops called the vasa recta.

Fig. 33.2 (left), the vasa recta descend into the medulla, where they form capillary networks that surround the collecting ducts and ascending limbs of the loop of Henle. The blood returns to the cortex via the ascending vasa recta. Although less than 0.7% of the renal blood flow (RBF) enters the vasa recta, these vessels serve important functions in the renal medulla that include (1) conveying oxygen and important metabolic substrates to support nephron function, (2) delivering substances to the nephron for secretion, (3) serving as a pathway for return of reabsorbed water and solutes to the circulatory system, and (4) concentrating and diluting urine (urine concentration and dilution are discussed in more detail in

Ultrastructure of the Glomerulus

The first step in urine formation begins with passive movement of a plasma ultrafiltrate from the glomerular capillaries (i.e., glomerulus) into Bowman’s space. The term ultrafiltration refers to this passive movement of fluid—similar in composition to plasma, except for the fact that the ultrafiltrate protein concentration is much lower than that in the plasma—from the glomerular capillaries into Bowman’s space. To appreciate this process, one must understand the anatomy of the glomerulus, which consists of a network of capillaries supplied by the afferent arteriole and drained by the efferent arteriole (

Figs. 33.5 33.6 ). During embryological development, the glomerular capillaries press into the closed end of the proximal tubule, forming Bowman’s capsule. As the epithelial cells thin on the outside circumference of Bowman’s capsule, they form the parietal epithelium (see

Fig. 33.5 ). The epithelial cells in contact with the capillaries thicken and develop into podocytes, which form the visceral layer of Bowman’s capsule (

Figs. 33.7–33.9 ). The visceral cells face outward at the vascular pole (i.e., where afferent and efferent arterioles enter and exit Bowman’s capsule) to form the parietal layer of Bowman’s capsule. The space between the visceral layer and the parietal layer is Bowman’s space, which at the urinary pole (i.e., where the proximal tubule joins Bowman’s capsule) of the glomerulus becomes the lumen of the proximal tubule.

The endothelial cells of glomerular capillaries are covered by a basement membrane surrounded by podocytes. The capillary endothelium, basement membrane, and foot processes of podocytes form the so-called filtration barrier (see

Figs. 33.5 and 33.7–33.9 ). The endothelium is fenestrated

Fig. 33.5 Anatomy of the glomerulus and juxtaglomerular apparatus. The juxtaglomerular apparatus is composed of the macula densa (MD) of the thick ascending limb, extraglomerular mesangial cells (EGM), and renin-and angiotensin II–producing granular cells (G) of the afferent arterioles (AA). BM, basement membrane; BS, Bowman’s space; EA, efferent arteriole; EN, endothelial cell; FP, foot processes of the podocyte; M, mesangial cells between capillaries; P, podocyte cell body (visceral cell layer); PE, parietal epithelium; PT, proximal tubule cell. (Modified from Kriz W, Kaissling B. Structural organization of the mammalian kidney. In Alpern RJ, Moe OW, Caplan M [eds]: Seldin and Giebisch’s The Kidney, 5th ed. London, Elsevier, 2013. Figure in that source based on Kriz W, Sakai T, et al. [1988]. Morphological aspects of glomerular function. In “Nephrology,” A. M. Davison, Vol. 1, Proceedings of the 10th International Congress of Nephrology, 323. Bailliere Tindall, London.)

Fig. 33.6 Scanning electron micrograph of the interlobular artery, afferent arteriole (af), efferent arteriole (ef), and glomerulus. The white bars on the afferent and efferent arterioles indicate that they are about 15 to 20 µm in diameter. (From Kimura K et al. Am J Physiol 1990;259:F936.) • Fig. 33.7 A, Electron micrograph of a podocyte surrounding a glomerular capillary. The cell body of the podocyte contains a large nucleus with three indentations. Cell processes of the podocyte form the interdigitating foot processes (FP). The arrows in the cytoplasm of the podocyte indicate the well-developed Golgi apparatus, and the asterisks indicate Bowman’s space. C, capillary lumen; GBM, glomerular basement membrane. B, Electron micrograph of the filtration barrier of a glomerular capillary. The filtration barrier is composed of three layers: the endothelium, basement membrane, and foot processes of the podocytes. Note the filtration slit diaphragm bridging the floor of the filtration slits (arrows). CL, capillary lumen. (From Kriz W, Kaissling B: Structural organization of the mammalian kidney. In: Alpern RJ, Moe OW, Caplan M [eds]: Seldin and Giebisch’s The Kidney, 5th ed. London, Elsevier, 2013.) (i.e., contains 700-Å holes, where 1 Å = 10−10 m) and freely permeable to water, small solutes (e.g., Na+ , urea, glucose), and most proteins but is not permeable to red blood cells, white blood cells, or platelets. Because endothelial cells express negatively charged glycoproteins on their surface, they minimize the filtration into Bowman’s space of albumin, the most abundant plasma protein, and most • Fig. 33.8 A, Scanning electron micrograph showing the outer surface of glomerular capillaries. This is the view that would be seen from Bowman’s space. Processes (P) of podocytes run from the cell body (CB) toward the capillaries, where they ultimately split into foot processes. Interdigitation of the foot processes creates the filtration slits. B, Scanning electron micrograph of the inner surface (blood side) of a glomerular capillary. This view would be seen from the lumen of the capillary. The fenestrations of the endothelial cells are seen as small 700-Å holes. (From Kriz W, Kaissling B: Structural organization of the mammalian kidney. In: Alpern RJ, Moe OW, Caplan M [eds]: Seldin and Giebisch’s The Kidney, 5th ed. London, Elsevier, 2013.) other plasma proteins. In addition to their role as a barrier to filtration, the endothelial cells synthesize a number of vasoactive substances (e.g., nitric oxide [NO], a vasodilator, and endothelin 1 [ET-1], a vasoconstrictor) that are important in controlling renal plasma flow (RPF). The basement • Fig. 33.9 Electron micrograph of the mesangium, the area between the glomerular capillaries containing mesangial cells. C, glomerular capillaries; cGBM, capillary glomerular basement membrane surrounded by foot processes of podocytes (PO) and endothelial cells; M, mesangial cell that gives rise to several processes, some marked by stars; mGBM, mesangial glomerular basement membrane surrounded by foot processes of podocytes and mesangial cells; US, urinary space. Note the extensive extracellular matrix surrounded by mesangial cells (triangles) (×4100). (From Kriz W, Kaissling B: Structural organization of the mammalian kidney. In: Alpern RJ, Moe OW, Caplan M [eds]: Seldin and Giebisch’s The Kidney, 5th ed. London, Elsevier, 2013.) membrane, which is a porous matrix of negatively charged proteins (type IV collagen, laminin, the proteoglycans agrin and perlecan, and fibronectin), is an important filtration barrier to plasma proteins. The basement membrane is thought to function primarily as a charge-selective filter in which the ability of proteins to cross the filter is based on charge.

The podocytes, which are endocytic, have long finger-like processes that completely encircle the outer surface of the capillaries (see

Figs. 33.7 33.8A ). The processes of the podocytes interdigitate to cover the basement membrane and are separated by apparent gaps called filtration slits (see

Figs. 33.7 33.8A ). Each filtration slit is bridged by a thin diaphragm that contains pores with a dimension of 40 × 140 Å. The filtration slit diaphragm, which appears as a continuous structure when viewed by electron microscopy (see

Fig. 33.7B ), is composed of several proteins, including nephrin (NPHS1), NEPH-1, and podocin (NPHS2), and intracellular proteins that associate with the slit diaphragm, including α-actinin-4 (ACTN4) and CD2-AP (

Figs. 33.10 33.11 ). Filtration slits, which function primarily as a size-selective filter, minimize the filtration of proteins and macromolecules that cross the basement membrane from entering Bowman’s space. Because both the basement membrane and filtration slits contain negatively charged glycoproteins, some proteins are held back (i.e., not filtered into Bowman’s space) on the basis of size and charge. For molecules with an effective molecular radius between 18 and 42 Å, cationic molecules are filtered more readily than anionic molecules.

Another important component of the renal corpuscle is the mesangium, which consists of mesangial cells and the mesangial matrix (see

Fig. 33.9 ). Mesangial cells, which possess many properties of smooth muscle cells, provide structural support for the glomerular capillaries, secrete extracellular matrix, exhibit phagocytic activity by removing macromolecules from the mesangium, and secrete prostaglandins and proinflammatory cytokines. Because they also contract and are adjacent to glomerular capillaries, mesangial cells may influence GFR by regulating blood flow through the glomerular capillaries or by altering the capillary surface area. Mesangial cells located outside the glomerulus (between the afferent and efferent arterioles) are called extraglomerular mesangial cells.

The nephrotic syndrome is produced by a variety of disorders and is characterized by increased permeability of the glomerular capillaries to proteins and by loss of normal podocyte structure, including effacement (i.e., thinning) of foot processes. The augmented permeability to proteins results in increased urinary protein excretion (proteinuria). Thus the appearance of proteins in urine can indicate kidney disease. Individuals with this syndrome often develop hypoalbuminemia as a result of the proteinuria. In addition, generalized edema is commonly seen in nephrotic individuals. Mutations in several genes encoding either slit diaphragm proteins (see

Figs. 33.10 33.11), including nephrin, NEPH-1, and podocin, or intracellular proteins that functionally interact with slit diaphragm proteins, such as CD2-AP and α-actinin 4 (ACTN4), result in proteinuria and kidney disease. For example, mutations in the nephrin gene (NPHS1) in congenital nephrotic syndrome lead to abnormal or absent slit diaphragms, causing massive proteinuria and renal failure. In addition, mutations in the podocin gene (NPHS2) cause autosomal recessive, steroid-resistant nephrotic syndrome. These naturally occurring mutations and knockout studies in mice demonstrate that nephrin, NEPH-1, podocin, CD2-AP, and α-actinin-4 play key roles in normal glomerular podocyte structure and function.

• Fig. 33.10 Anatomy of podocyte foot processes. This figure illustrates the proteins that make up the slit diaphragm between two adjacent foot processes. Nephrin and NEPH1 are membrane-spanning proteins that have large extracellular domains that interact. Podocin, also a membrane-spanning protein, organizes nephrin and NEPH1 in specific microdomains in the plasma membrane, which is important for signaling events that determine the structural integrity of podocyte foot processes. Many of the proteins that compose the slit diaphragm interact with adapter proteins inside the cell, including CD2-AP. The adapter proteins bind to the filamentous actin (F-actin) cytoskeleton, which in turn binds either directly or indirectly to proteins such as α3β1 and MAGI-1 that interact with proteins expressed by the glomerular basement membrane (GBM). α-act-4, α-actinin 4; α3β1, α3β1 integrin; α-DG, α-dystroglycan; CD2-AP, an adapter protein that links nephrin and podocin to intracellular proteins; FAT, a protocadherin that organizes actin polymerization; MAGI-1, a membrane-associated guanylate kinase protein; NHERF-2, Na+-H+ exchanger regulatory factor 2; P, paxillin; P-Cad, P-cadherin; Synpo, synaptopodin; T, talin; V, vinculin; Z, zona occludens. (Adapted from Mundel P, Shankland SJ. J Am Soc Nephrol 2002;13:3005.)

Mesangial cells are involved in development of immune complex–mediated glomerular disease. Because the glomerular basement membrane does not completely surround all glomerular capillaries (see

Fig. 33.9 ), some immune complexes can escape the blood and enter the mesangium without crossing the glomerular basement membrane. Accumulation of immune complexes induces mesangial infiltration of inflammatory cells and promotes local proinflammatory cytokine and autocoid production. These cytokines and autocoids enhance the immune complex– initiated inflammatory response, which can ultimately lead to mesangial expansion, scarring, and obliteration of the glomerulus.

Ultrastructure of the Juxtaglomerular Apparatus

The juxtaglomerular apparatus is one component of an important feedback mechanism, the tubuloglomerular feedback mechanism, described later in this chapter. Structures that make up the juxtaglomerular apparatus (see

Fig. 33.5 ) include: 1.

the macula densa of the thick ascending limb 2.

3.

reninand angiotensin II–producing granular cells of the afferent arteriole

Alport syndrome is characterized by hematuria (i.e., blood in the urine) and progressive glomerulonephritis (i.e., inflammation of the glomerular capillaries), and it accounts for 1% to 2% of all cases of ESRD. Alport syndrome is caused by mutations in type IV collagen, a major component of the glomerular basement membrane. In about 80% of patients with Alport syndrome the disease is X-linked with mutations in the COL4A5 gene. Some 15% of patients also have mutations in type IV collagen genes (COL4A3 and COL4A4); six have been identified, but the mode of inheritance is autosomal recessive. The remaining 5% of patients with Alport syndrome have autosomal dominant disease that arises from heterozygous mutations in the COL4A3 or COL4A4 genes. In Alport syndrome, the glomerular basement membrane becomes irregular in thickness and fails to serve as an effective filtration barrier to blood cells and protein.

The cells of the macula densa represent a morphologically distinct region of the thick ascending limb. This region passes through the angle formed by the afferent and efferent arterioles of the same nephron. The cells of the macula densa contact the extraglomerular mesangial cells and the granular cells of the afferent arterioles. The granular cells of the afferent arterioles contain smooth muscle myofilaments and—importantly—manufacture, store, and release renin in response to signals associated with decreased effective • Fig. 33.11 Overview of the major proteins that form the slit diaphragm. Nephrins (red) from opposite foot processes interdigitate in the center of the slit. In the slit, nephrin interacts with NEPH1 and NEPH2 (blue), FAT1 and FAT2 (green), and P-cadherin. The intracellular domains of nephrin, NEPH1, and NEPH2 interact with podocin and CD2-AP, which connect these slit diaphragm proteins with ZO-1, α-actinin 4, and actin. (Modified from Tryggvason K et al. N Engl J Med 2006;354:1387.) circulating volume and reduced renal perfusion. Renin is involved in proteolytic generation of angiotensin II and ultimately in secretion of aldosterone (see ). The juxtaglomerular apparatus is one component of the tubuloglomerular feedback mechanism involved in autoregulation of RBF and GFR.

Innervation of the Kidneys

Renal nerves regulate RBF, GFR, and salt and water reabsorption by the nephron. The nerve supply to the kidneys consists of sympathetic nerve fibers that originate in the celiac plexus. There is no corresponding parasympathetic innervation. Adrenergic fibers innervating the kidneys release norepinephrine and lie adjacent to the smooth muscle cells of the major branches of the renal artery (interlobar, arcuate, and interlobular arteries) as well as the afferent and efferent arterioles. In addition, sympathetic nerves innervate the renin-producing granular cells of the afferent arterioles. Renin secretion is stimulated by increased sympathetic activity. Nerve fibers also innervate the proximal tubule, loop of Henle, distal tubule, and collecting duct; activation of these nerves enhances Na+ reabsorption by these nephron segments.

Assessment of Renal Function

The coordinated actions of the nephron’s various segments determine the final amount of a substance that appears in urine. This represents three general processes (1) glomerular

CHAPTER 33 Elements of Renal Function

This relationship permits quantification of the amount of substance x excreted in urine versus the amount returned to the systemic circulation in renal venous blood. Thus for any substance that is neither synthesized nor metabolized, the amount that enters the kidneys is equal to the amount that leaves the kidneys in urine plus the amount that leaves the kidneys in renal venous blood.

The principle of renal clearance emphasizes the excretory function of the kidneys; it considers only the rate at which a substance is excreted into urine and not its rate of return to the systemic circulation in the renal vein. Therefore in terms of mass balance (

Eq. 33.1 ) the urinary excretion rate of substance x (Ux × V̇ ) is proportional to the plasma concentration of substance x (

Equation 33.2

To equate the urinary excretion rate of substance x to • Fig. 33.12 Mass balance relationships for the kidney. See text for definition of symbols.

its renal arterial plasma concentration, it is necessary to determine the rate at which it is removed from plasma by filtration, (2) reabsorption of the substance from tubular the kidneys. This removal rate is the clearance (Cx): fluid back into blood, and (3) (in some cases) secretion of

Equation 33.3 the substance from blood into tubule fluid. The first step in P × C = U × V˜ urine formation by the kidneys is production of an ultrafil-xa x x trate of plasma across the glomerulus. The process of glo-If

Eq. 33.3 is rearranged and the concentration of merular filtration and regulation of GFR and RBF substance x in renal artery plasma ( ) is assumed to beare discussed later in this chapter. The concept of renal clearance, which is the theoretical basis for measurement of GFR and RBF, is presented in the following section. Reabsorption and secretion are discussed in subsequent chapters.

The concept of renal clearance is based on the Fick principle (i.e., mass balance or conservation of mass).

Fig.

33.12 illustrates the various factors required to describe the mass balance relationships of a kidney. The renal artery is the single input source to the kidney for substances not synthesized by this organ, whereas the renal vein and ureter constitute the two principal output routes. In other words a nonmetabolized substance entering the renal circulation via the renal artery may only exit this circulation via the renal vein (i.e., the unfiltered fraction plus any filtered amount subsequently reabsorbed back into blood) or the ureter (the combined filtered and secreted fractions less any tubular reabsorption). The following equation defines the mass balance relationship:

Equation 33.1 where identical to its concentration in a plasma sample from any peripheral blood vessel (Px), the following relationship is obtained:

Equation 33.4

Clearance has the dimensions of volume/time, and it represents a volume of plasma from which all the substance has been removed and excreted into urine per unit time. This last point is best illustrated by considering the following example. If a substance is present in urine at a concentration of 100 mg/mL and the urine flow rate is 1 mL/min, the excretion rate for this substance is calculated as follows:

Equation 33.5

If this substance is present in plasma at a concentration of 1 mg/mL, its clearance according to

Eq. 33.4 is as follows:

Equation 33.6 are the concentrations of substance x in the renal artery and renal vein plasma, respectively

RPFa and RPFv are renal plasma flow rates in the artery and vein, respectively In other words, 100 mL of plasma will be completely Ux is the concentration of substance x in urine cleared of substance x each minute. The definition of clearV̇ is the urine flow rate ance as a volume of plasma from which all the substance . UCr x V

Amount filtered PCr x GFR = Amount excreted UCr x V .

• Fig. 33.13 Renal handling of creatinine. Creatinine is freely filtered across the glomerulus and is, to a first approximation, not reabsorbed, secreted, or metabolized by the nephron. Note that all the creatinine coming to the kidney in the renal artery does not get filtered at the glomerulus (normally, 15%–20% of plasma creatinine is filtered). The portion that is not filtered is returned to the systemic circulation in the renal vein. PCr, plasma creatinine concentration; RPF, renal plasma flow; Ucr, urinary concentration of creatinine; V., urine flow rate.

has been removed and excreted into urine per unit time is somewhat misleading because it is not a real volume of plasma; rather it is a virtual volume.

The concept of clearance is important because it can be used to measure GFR and RPF and determine whether a substance is reabsorbed or secreted along the nephron.

The GFR is equal to the sum of the filtration rates of all functioning nephrons. Thus it is an aggregate index of kidney function. A fall in GFR generally means the kidney disease is progressing, whereas recovery generally suggests recuperation. Thus serial assessment of a patient’s GFR is essential to evaluate the severity and course of kidney disease.

Creatinine is a byproduct of normal skeletal muscle creatine metabolism and is freely filtered across the glomerulus into Bowman’s space. It is normally generated by the body at a fairly constant rate, and—to a first approximation—it is not appreciably reabsorbed, secreted, or metabolized by the cells of the nephron after its filtration. Accordingly the amount of creatinine excreted in urine per minute is fairly constant at steady state (i.e., when [creatinine] is constant) and equals the amount of creatinine filtered at the glomerulus each minute (

Fig. 33.13): cFor most substances cleared from plasma by the kidneys, only a portion is actually removed and excreted in a single pass through the kidneys.

Equation 33.7 where PCr = plasma concentration of creatinine UCr = urine concentration of creatinine V̇ = urine flow

Eq. 33.7 is solved for GFR:

Equation 33.8

This equation is the same form as that for clearance (see

Eq.

33.4 ). Thus measured creatinine clearance (CrCl) can be used clinically to determine GFR at steady state. Clearance has the dimensions of volume/time, and it represents an equivalent volume of plasma from which all of the substance has been removed and excreted into urine per unit time.

Creatinine is not the only substance that can be used to measure GFR; any substance that meets the following criteria can serve as an appropriate marker. The substance must: 1.

2.

be freely filtered across the glomerulus into Bowman’s space 3.

not be reabsorbed or secreted by the nephron 4.

not be metabolized or produced by the kidney 5.

Not all creatinine (or other substances used to measure GFR) that enters the kidney in renal arterial plasma is filtered at the glomerulus. Likewise not all plasma coming into the kidneys is filtered. Although nearly all plasma that enters the kidneys in the renal artery passes through the glomerulus, approximately 10% does not. The portion of filtered plasma is termed the filtration fraction and is determined as:

Equation 33.9

Under normal conditions the filtration fraction averages 0.15 to 0.20, which means that only 15% to 20% of the plasma that enters the glomerulus is actually filtered. The remaining 80% to 85% continues on through the glomerular capillaries and into the efferent arterioles and peritubular capillaries before finally returning to the systemic circulation via the renal vein.

The first step in the formation of urine is ultrafiltration of plasma by the glomerulus. In normal adults, GFR ranges from 90 to 140 mL/min in males and from 80 to 125 mL/ min in females. Thus in 24 hours as much as 180 L of plasma is filtered by the glomeruli. The plasma ultrafiltrate is devoid of cellular elements (i.e., red and white blood cells and

Creatinine clearance (CrCl) is used to estimate GFR in clinical practice. It is synthesized at a relatively constant rate, and the amount produced is proportional to the total muscle mass. However, creatinine is not a perfect substance for measuring GFR because it is secreted to a small extent by the organic cation secretory system in the proximal tubule (see ). The error introduced by this secretory component is approximately 10%. Thus the amount of creatinine excreted in urine exceeds the amount expected from filtration alone by 10%. However, the method used to measure the plasma creatinine concentration (PCr) overestimates the true value by 10%. Consequently the two errors cancel each other, and in most clinical situations, CrCl provides a reasonably accurate measure of GFR.

A fall in GFR may be the first and only clinical sign of kidney disease. Thus measuring GFR is important when kidney disease is suspected. A 50% loss of functioning nephrons reduces GFR only by about 25%. The decline in GFR is not 50% because the remaining nephrons compensate. Because measurements of GFR are cumbersome, kidney function is usually assessed in the clinical setting by measuring the plasma concentration of creatinine (PCr),which is inversely related to GFR (

Fig. 33.14 ). However, as

Fig. 33.14 shows, GFR must decline substantially before an increase in PCr can be detected in a clinical setting. For example, a fall in GFR from 120 to 100 mL/min is accompanied by an increase in PCr from 1.0 to 1.2 mg/dL. This does not appear to be a significant change in PCr, but GFR has actually fallen by almost 20%. In the clinical setting, estimated GFR (eGFR) also includes consideration of several other factors in addition to the plasma concentration of creatinine, including age, sex, body size, and race. A free app to calculate eGFR can be downloaded at: https://www.kidney.org/apps/professionals/ platelets) and is essentially protein free. The concentration • Fig. 33.14 Relationship between GFR and plasma [creatinine] (Pcr). The amount of creatinine filtered is equal to the amount excreted; thus GFR × PCr = UCr × V. Because the production of creatinine is constant, excretion must be constant to maintain creatinine balance. Therefore if GFR falls from 120 to 60 mL/min, PCr must increase from 1 to 2 mg/dL to keep filtration of creatinine and its excretion equal to the production rate.

1.0 0.8 0.6 0.4 of salts and organic molecules (e.g., glucose, amino acids) is similar in plasma and the ultrafiltrate. Starling forces drive ultrafiltration across the glomerular capillaries, and changes in these forces alter GFR. GFR and RPF are normally held within very narrow ranges by a phenomenon called autoregulation. The next sections of this chapter review the composition of the glomerular filtrate, the dynamics of its formation, and the relationship between RPF and GFR. In addition, factors that contribute to autoregulation and regulation of GFR and RBF are discussed.

Determinants of Ultrafiltrate Composition

The glomerular filtration barrier determines the composition of the plasma ultrafiltrate. It restricts filtration of molecules on the basis of both size and electric charge (

Fig. 33.15 ). In general, neutral molecules with a radius smaller than about 0.2 • Fig. 33.15 Influence of the size and electric charge of dextran on its filterability. A value of 1 indicates that it is filtered freely, whereas a value of zero indicates that it is not filtered. The filterability of dextrans between approximately 18 and 42 Å depends on charge. Dextrans larger than 42 Å are not filtered regardless of charge, and polycationic dextrans and neutral dextrans smaller than 18 Å are freely filtered. The major proteins in plasma are albumin and immunoglobulins. Because the effective molecular radii of immunoglobulin (Ig)G (53 Å) and IgM (>100 Å) are greater than 42 Å, they are not filtered. Although the effective molecular radius of albumin is 35 Å, it is a polyanionic protein, so it does not cross the filtration barrier to a significant degree.

18 Å are freely filtered, molecules larger than about 42 Å are not filtered, and molecules between about 18 and 42 Å are filtered to varying degrees.

Fig. 33.15 shows how electric charge affects filtration of macromolecules (e.g., dextrans) by the glomerulus. Dextrans are a family of exogenous polysaccharides manufactured in various molecular weights. They can be electrically neutral or have either negative (polyanionic) or positive (polycationic) charges. As the size (i.e., effective molecular radius) of a dextran molecule increases, the rate at which it is filtered decreases. For any given molecular radius, cationic molecules are more readily filtered than anionic molecules. The reduced filtration rate for anionic molecules is explained by the presence of negatively charged glycoproteins on the surface of all components of the glomerular filtration barrier. These charged glycoproteins repel similarly charged molecules. Because most plasma proteins are negatively charged, the negative charge on the filtration barrier restricts filtration of anionic proteins more than the filtration of neutral and polyanionic proteins with a molecular radius between approximately 18 to 42 Å. For example, serum albumin, an anionic protein that has an effective molecular radius of 35.5 Å, is poorly filtered. Because the small amount of filtered albumin is normally reabsorbed avidly by the proximal tubule, almost no albumin appears in urine.

The importance of the negative charges on the filtration barrier in restricting filtration of plasma proteins is shown in

Figs. 33.15 33.16 . Removal of the negative charges from the filtration barrier causes proteins to be filtered solely on the basis of their effective molecular radius (

Fig. 33.16 ). Hence at any molecular radius between approximately 18 and 42 Å, filtration of polyanionic proteins will exceed the filtration that prevails in the normal state (in which the filtration barrier has anionic charges). In a number of glomerular diseases the negative charges on the filtration barrier are reduced because of immunological damage and inflammation. As a result, filtration of anionic proteins between approximately 18 and 42 Å in radius is increased. When the filtered proteins exceed the ability of the proximal tubule to reabsorb and catabolize them, anionic proteins begin to appear in urine (proteinuria), which is a marker of kidney disease.

Dynamics of Ultrafiltration

The forces responsible for glomerular filtration of plasma are the same as those in other capillary beds. Ultrafiltration occurs because the Starling forces (i.e., hydraulic and oncotic pressures) combine to drive fluid from the lumen of glomerular capillaries across the filtration barrier and into Bowman’s space (

Fig. 33.17 ). The hydraulic pressure inside the glomerular capillary (PGC) is oriented to promote movement of fluid from the glomerular capillary into Bowman’s space. Because the glomerular ultrafiltrate is essentially protein free under normal conditions, owing in large part to the paucity of proteins in serum smaller than 18 Å in radius 1.0 0.8 0.6 0.4 0.2 0 • Fig. 33.16 Reduction of the negative charges on the glomerular wall results in filtration of proteins on the basis of size only. In this situation the relative filterability of proteins depends only on the molecular radius. Accordingly, excretion of polyanionic proteins (18–42 Å) in urine increases because more proteins of this size are filtered.

that can be effectively filtered, the reflection coefficient (σ) for proteins across the glomerular capillary is essentially 1. Thus the oncotic pressure in Bowman’s space (πBS) is near zero. Therefore PGC is the principal force favoring filtration. In contrast, the hydraulic pressure in Bowman’s space (PBS) and the oncotic pressure in the glomerular capillary (πGC) both oppose filtration.

Fig. 33.17 , a net ultrafiltration pressure (PUF) of 17 mm Hg exists at the afferent end of the glomerulus, whereas at the efferent end it is 8 mm Hg (where PUF = PGC − PBS −πGC). Two additional points concerning Starling forces and this pressure change are important. First, PGC decreases slightly along the length of the capillary because of the resistance to flow along the length of the capillary. Second, πGC increases as plasma is filtered while protein is retained within the glomerular capillary, thereby progressively increasing the protein concentration along the length of the capillary. GFR is proportional to the sum of the Starling forces that exist across the capillaries [(PGC − PBS) −σ(πGC −πBS)] multiplied by the ultrafiltration coefficient (Kf). That is:

Equation 33.10

Kf is the product of the intrinsic permeability of the glomerular capillary and the glomerular surface area available for filtration. The rate of glomerular filtration is considerably greater in glomerular capillaries than in systemic capillaries, mainly because Kf is approximately 100 times greater in glomerular capillaries. Furthermore PGC is approximately twice as great as the hydraulic pressure in systemic capillaries.

GFR can be altered by changing Kf or by changing any of the Starling forces. In normal individuals, GFR is regulated by alterations in PGC that are mediated mainly by changes • Fig. 33.17 Idealized glomerular capillary and the Starling forces across it. The reflection coefficient (σ) for protein across the glomerular capillary is approximately 1. PBS, hydraulic pressure in Bowman’s space; PGC, hydraulic pressure in the glomerular capillary; PUF, net ultrafiltration pressure; πBS, oncotic pressure in Bowman’s space; πGC, oncotic pressure in the glomerular capillary. The negative signs for PBS and πGC indicate that these forces oppose formation of the glomerular filtrate.

in afferent or efferent arteriolar resistance. PGC is affected in three ways: 1.

Changes in afferent arteriolar resistance: A decrease in resistance increases PGC and GFR, whereas an increase in resistance decreases PGC and GFR.

2.

Changes in efferent arteriolar resistance: A decrease in resistance reduces PGC and GFR, whereas an increase in resistance elevates PGC and GFR.

3.

Changes in renal arteriolar pressure: An increase in blood pressure transiently increases PGC (which enhances GFR), whereas a decrease in blood pressure transiently decreases PGC (which reduces GFR).

Blood flow through the kidneys serves several important functions: 1.

2.

modifies the rate of solute and water reabsorption by the proximal tubule 3.

participates in concentration and dilution of urine 4.

delivers O2, nutrients, and hormones to cells along the nephron and returns CO2, reabsorbed fluid, and solutes to the general circulation 5.

Blood flow through any organ may be represented by the following equation:

A reduction in GFR in disease states is most often due to decreases in Kf because of the loss of filtration surface area. GFR also changes in pathophysiological conditions because of changes in PGC, πGC, and PBS.

1.

Changes in Kf: Increased Kf enhances GFR, whereas decreased Kf reduces GFR. Some kidney diseases reduce Kf by decreasing the number of filtering glomeruli (i.e., diminished surface area). Some drugs and hormones that dilate the glomerular arterioles also increase Kf. Similarly, drugs and hormones that constrict the glomerular arterioles also decrease Kf.

2.

Changes in PGC: With decreased renal perfusion, GFR declines because PGC falls. As previously discussed, a reduction in PGC is caused by a decline in renal arterial pressure, an increase in afferent arteriolar resistance, or a decrease in efferent arteriolar resistance.

3.

Changes in πGC: An inverse relationship exists between πGC and GFR. Alterations in πGC result from changes in protein synthesis outside the kidneys. In addition the protein loss in urine caused by some renal diseases can lead to a decrease in the plasma protein concentration and thus in πGC.

4.

Changes in PBS: Increased PBS reduces GFR, whereas decreased PBS enhances GFR. Acute obstruction of the urinary tract (e.g., a kidney stone occluding the ureter) increases PBS.

Equation 33.11 organ R = resistance to flow through that organ

Accordingly, RBF is equal to the pressure difference between the renal artery and the renal vein divided by renal vascular resistance:

Equation 33.12

The afferent arteriole, efferent arteriole, and interlobular artery are the major resistance vessels in the kidneys and thereby determine renal vascular resistance. Like most other organs, the kidneys regulate their blood flow by adjusting vascular resistance in response to changes in arterial pressure. As shown in

Fig. 33.18 these adjustments are so precise that blood flow remains relatively constant as arterial blood pressure changes between 90 and 180 mm Hg. GFR is also regulated over the same range of arterial pressures. The phenomenon whereby RBF and GFR are maintained relatively constant between blood pressures of 90 and 180 mm Hg, namely autoregulation, is achieved by changes in vascular resistance, mainly through the afferent arterioles of the kidneys. Because both RBF and GFR are regulated over the • Fig. 33.18 Relationship between arterial blood pressure and RBF and between arterial blood pressure and GFR. Autoregulation maintains GFR and RBF relatively constant as blood pressure changes from 90 to 180 mm Hg.

same range of pressures and because RBF is an important determinant of GFR, it is not surprising that the same mechanisms regulate both flows.

Two mechanisms are responsible for autoregulation of RBF and GFR: one mechanism that responds to changes in arterial pressure and another that responds to changes in [NaCl] in tubular fluid. Both regulate the tone of the afferent arteriole. The pressure-sensitive mechanism, the so-called myogenic mechanism, is related to an intrinsic property of vascular smooth muscle: the tendency to contract when stretched. Accordingly, when arterial pressure rises and the renal afferent arteriole is stretched, the smooth muscle contracts in response. Because the increase in resistance of the arteriole offsets the increase in pressure, RBF, and therefore GFR, remains constant. (That is, RBF is constant if ΔP/R is kept constant [see

Eq. 33.11

The second mechanism responsible for autoregulation of GFR and RBF is the [NaCl]-dependent mechanism known as tubuloglomerular feedback. This mechanism involves a feedback loop in which a change in GFR leads to alteration in the concentration of NaCl in tubular fluid, which is sensed by the macula densa of the juxtaglomerular apparatus and converted into signals that affect afferent arteriolar resistance and thus the GFR (Fig. 33.19 ). For example, when the GFR increases and causes [NaCl] in tubular fluid in the loop of Henle to rise, more NaCl enters the macula densa cells in this segment (Fig. 33.20 ). This leads to an increase in formation and release of adenosine triphosphate (ATP) and adenosine (a metabolite of ATP) by macula densa cells, which causes vasoconstriction of the afferent arteriole and normalization of GFR. In contrast, when GFR and [NaCl] in tubule fluid decrease, less NaCl enters the macula densa cells, and both ATP and adenosine • Fig. 33.19 Tubuloglomerular feedback. An increase in GFR (1) increases [NaCl] in tubule fluid in the loop of Henle (2). The increase in [NaCl] is sensed by the macula densa and converted to a signal (3) that increases the resistance of the afferent arteriole (RA) (4), which decreases GFR. A decrease in GFR has the opposite effects. (Modified from Cogan MG. Fluid and Electrolytes: Physiology and Pathophysiology. Norwalk, CT: Appleton & Lange; 1991.) production and release decline. The fall in [ATP] and [adenosine] results in afferent arteriolar vasodilation, which returns GFR to normal. NO, a vasodilator produced by the macula densa, attenuates tubuloglomerular feedback, whereas angiotensin II enhances tubuloglomerular feedback. Thus the macula densa may release both vasoconstrictors (e.g., ATP and adenosine) and a vasodilator (e.g., NO) that oppose each other’s action at the level of the afferent arteriole. Production plus release of either vasoconstrictors or vasodilators ensures exquisite control over tubuloglomerular feedback.

Fig. 33.20 also illustrates the role of the macula densa in controlling renin secretion by granular cells of the afferent arteriole. This aspect of function of the juxtaglomerular apparatus is considered in detail in

Because animals engage in many activities that can change arterial blood pressure, mechanisms that maintain RBF and GFR relatively constant despite changes in arterial pressure are highly desirable. If GFR and RBF were to rise or fall suddenly in proportion to changes in blood pressure, urinary excretion of fluid and solute would also change suddenly. Such changes in excretion of water and solutes without comparable changes in intake would alter fluid and electrolyte balance (the reason for which is discussed in ). Accordingly, autoregulation of GFR and RBF provides an effective means for uncoupling renal function from arterial pressure, and it ensures that fluid and solute excretion remain relatively constant.

Three points concerning autoregulation should be noted: 1. Autoregulation is absent when arterial pressure is less than 90 mm Hg.

• Fig. 33.20 Cellular mechanism whereby an increase in delivery of NaCl to the macula densa causes vasoconstriction of the afferent arteriole of the same nephron (i.e., tubuloglomerular feedback). An increase in GFR elevates [NaCl] in tubule fluid at the macula densa. This in turn enhances uptake of NaCl across the apical cell membrane of macula densa cells via the 1Na+-1K+-2Cl− (NKCC2) symporter, which leads to an increase in [ATP] and [adenosine] (ADO) release. ATP binds to P2X receptors and adenosine binds to adenosine A1 receptors in the plasma membrane of smooth muscle cells surrounding the afferent arteriole, both of which increase intracellular [Ca++]. The rise in [Ca++] induces vasoconstriction of the afferent arteriole, thereby returning GFR to normal levels. Note that ATP and adenosine also inhibit renin release by granular cells in the afferent arteriole. This too results from an increase in intracellular [Ca++] as a reflection of electrical coupling of the granular and vascular smooth muscle (VSM) cells. When GFR is reduced, [NaCl] in tubule fluid falls, as does uptake of NaCl into macula densa cells. This in turn decreases release of ATP and adenosine by the macula densa, which decreases intracellular [Ca++] in smooth muscle cells and thereby increases GFR and stimulates renin release by granular cells. In addition a decrease in entry of NaCl into macula densa cells enhances production of PGE2, which also stimulates renin secretion by granular cells. As discussed in detail in , renin increases plasma [angiotensin II], a hormone that enhances NaCl and water retention by the kidneys. (Modified from Persson AEG et al. Acta Physiol Scand 2004;181:471.) 2.

Autoregulation is not perfect; RBF and GFR do change slightly as arterial blood pressure varies.

3.

Despite autoregulation, RBF and GFR can be changed by several hormones and by alterations in sympathetic nerve activity that change in response to alterations in the extracellular fluid volume (ECFV) (

Table 33.1

Regulation of Renal Blood Flow and Glomerular Filtration Rate

Table 33.1 ). As already discussed, the myogenic mechanism and tubuloglomerular feedback play key roles in maintaining RBF and GFR constant when blood pressure is greater that 90 mm Hg and ECFV is in the normal range. However, when the ECFV changes sympathetic nerves, angiotensin II, prostaglandins, NO, endothelin, bradykinin, ATP, and adenosine exert major control over RBF and GFR.

Fig.

33.21 shows how changes in efferent and afferent arteriolar resistance, mediated by changes in the hormones listed in

Table 33.1 , modulate both RBF and GFR.

The afferent and efferent arterioles are innervated by sympathetic neurons; however, sympathetic tone is minimal when ECFV is normal (see ). When ECFV is reduced, sympathetic nerves release norepinephrine and dopamine, and circulating epinephrine (a catecholamine-like norepinephrine and dopamine) is secreted by the adrenal medulla. Norepinephrine and epinephrine cause vasoconstriction by

Sympathetic nerves ↓ ECFV ↓↓ Angiotensin II ↓ ECFV ↓↓ Endothelin ↑ Stretch, A-II, bradykinin, epinephrine; ↓ ECFV ↓↓

Prostaglandins (PGE1, PGE2, PGI2) ↓ ECFV; ↑ shear stress, A-II No change/↑↑ Nitric oxide (NO) ↑ Shear stress, acetylcholine, histamine, bradykinin, ↑↑

ATP Bradykinin ↑ Prostaglandins, ↓ ACE ↑↑ Natriuretic peptides (ANP, BNP) ↑ ECFV ↑ No change

A-II, angiotensin II; ACE, angiotensin-converting enzyme; ECFV, extracellular fluid volume.

Tubuloglomerular feedback (TGF) is absent in mice that do not express the adenosine receptor (A1). This underscores the importance of adenosine signaling in TGF. Studies have shown that when GFR increases and causes the concentration of NaCl in tubular fluid at the macula densa to rise, more NaCl enters cells via the 1Na+-1K+-2Cl− symporter (NKCC2) located in the apical plasma membrane (see

Fig.

33.20 ). Increased intracellular [NaCl] in turn stimulates release of ATP via ATP-conducting ion channels located in the basolateral membrane of macula densa cells. In addition, adenosine production is also enhanced. Adenosine binds to A1 receptors and ATP binds to P2X receptors located on the plasma membrane of smooth muscle cells in the afferent arteriole. Both hormones increase intracellular [Ca++], which causes vasoconstriction of the afferent artery and therefore a fall in GFR. Although adenosine is a vasodilator in most other vascular beds, it constricts the afferent arteriole in the kidney.

binding to α1-adrenoceptors, which are located mainly in afferent arterioles. Activation of α1-adrenoceptors decreases RBF and GFR. Dehydration or strong emotional stimuli (e.g., fear, pain) also activate sympathetic nerves and reduce RBF and GFR.

Angiotensin II is produced systemically as well as locally within the kidneys. It constricts the afferent and efferent arterioles and decreases both RBF and GFR.

Fig. 33.22 shows how norepinephrine, epinephrine, and angiotensin II act together to decrease RBF and GFR and thereby dThe efferent arteriole is more sensitive than the afferent arteriole to angiotensin II. Therefore with low concentrations of angiotensin II, constriction of the efferent arteriole predominates, and GFR increases and RBF decreases. However, with high concentrations of angiotensin II, constriction of both afferent and efferent arterioles occurs, and GFR and RBF both decrease (see

Fig. 33.21

Individuals with renal artery stenosis (narrowing of the lumen of the artery) caused by atherosclerosis, for example, often have elevated systemic arterial blood pressure mediated by the renin-angiotensin system. Pressure in the renal artery proximal to the stenosis is increased, but pressure distal to the stenosis is normal or reduced. Autoregulation is important in maintaining RBF, PGC, and GFR in the presence of this stenosis. Administration of drugs to lower systemic blood pressure also lowers the pressure distal to the stenosis; accordingly, RBF, PGC, and GFR fall.

Significant hemorrhage decreases ECFV and arterial blood pressure and therefore activates sympathetic innervation of the kidneys via the baroreceptor reflex (

Fig. 33.22 ). Norepinephrine causes intense vasoconstriction of the afferent and efferent glomerular arterioles and thereby decreases both RBF and GFR. The rise in sympathetic activity also increases release of epinephrine and angiotensin II, which cause further vasoconstriction and a fall in RBF. The rise in vascular resistance of the kidneys and other vascular beds increases total peripheral resistance. The resulting tendency for blood pressure to increase (blood pressure = cardiac output × total peripheral resistance) offsets the tendency of blood pressure to decrease in response to hemorrhage. Hence this system works to preserve arterial pressure at the expense of maintaining normal RBF and GFR.

increase blood pressure and ECFV (e.g., as would occur with hemorrhage).

Prostaglandins do not play a major role in regulating RBF in healthy resting individuals. However, during • Fig. 33.21 Relationship between selective changes in resistance of either the afferent arteriole or the efferent arteriole on RBF and GFR. Constriction of either the afferent or efferent arteriole increases resistance, and according to

Eq. 33.11 (Q =ΔP/R), an increase in resistance (R) decreases flow (Q) (i.e., RBF). Dilation of either the afferent or afferent arteriole increases flow (i.e., RBF). Constriction of the afferent arteriole (A) decreases PGC because less of the arterial pressure is transmitted to the glomerulus, thereby reducing GFR. In contrast, constriction of the efferent arteriole (B) elevates PGC and thus increases GFR. Dilation of the efferent arteriole (C) decreases PGC and thus decreases GFR. Dilation of the afferent arteriole (D) increases PGC because more of the arterial pressure is transmitted to the glomerulus, thereby increasing GFR. (Modified from Rose BD, Rennke KG. Renal Pathophysiology: The Essentials. Baltimore: Williams & Wilkins; 1994.) pathophysiological conditions such as hemorrhage and reduced ECFV, prostaglandins (PGI2, PGE1, and PGE2) are produced locally within the kidneys and serve to increase RBF without changing GFR. Prostaglandins increase RBF by dampening the vasoconstrictor effects of both sympathetic activation and angiotensin II. These effects are important because they prevent severe and potentially harmful vasoconstriction and renal ischemia. Synthesis of prostaglandins is stimulated by ECFV depletion and stress (e.g., surgery, anesthesia), angiotensin II, and sympathetic nerves. Nonsteroidal antiinflammatory drugs (NSAIDs), such as ibuprofen and naproxen, potently inhibit prostaglandin synthesis. Thus administration of these drugs during renal ischemia and hemorrhagic shock is contraindicated because, by blocking the production of prostaglandins, they decrease RBF and increase renal ischemia. Prostaglandins also play an increasingly important role in maintaining RBF and GFR as individuals age. Accordingly, NSAIDs can significantly reduce RBF and GFR in the elderly.

NO, an endothelium-derived relaxing factor, is an important vasodilator under basal conditions, and it counteracts the vasoconstriction produced by angiotensin II and catecholamines. When blood flow increases, greater shear force acts on endothelial cells in the arterioles and increases production of NO. In addition a number of vasoactive hormones, including acetylcholine, histamine, bradykinin, and ATP, facilitate release of NO from endothelial cells. Increased production of NO causes dilation of the afferent and efferent arterioles in the kidneys. Whereas increased levels of NO decrease total peripheral resistance, inhibition of NO production increases total peripheral resistance.

Abnormal production of NO is observed in individuals with diabetes mellitus and hypertension. Excess renal NO production in diabetes may be responsible for glomerular hyperfiltration (i.e., increased GFR) and damage to the glomerulus, problems characteristic of this disease. Elevated NO levels increase glomerular capillary pressure secondary to a fall in resistance of the afferent arteriole. The ensuing hyperfiltration is thought to cause glomerular damage. The normal response to an increase in dietary salt intake includes stimulation of renal NO production, which prevents an increase in blood pressure. In some individuals, however, NO production may not increase appropriately in response to an elevation in salt intake, so blood pressure rises.

Endothelin is a potent vasoconstrictor secreted by endothelial cells of the renal vessels, mesangial cells, and distal tubular cells in response to angiotensin II, bradykinin, epinephrine, and endothelial shear stress. Endothelin causes profound vasoconstriction of the afferent and efferent arterioles and decreases GFR and RBF. Although this potent vasoconstrictor may not influence GFR and RBF in resting subjects, production of endothelin is elevated in a number of glomerular disease states (e.g., renal disease associated with diabetes mellitus).

Kallikrein is a proteolytic enzyme produced in the kidneys. Kallikrein cleaves circulating kininogen to bradykinin, which is a vasodilator that acts by stimulating the release of NO and prostaglandins. Bradykinin increases RBF and GFR.

600 SECTION7Berne & Levy Physiology • Fig. 33.22 Pathway by which hemorrhage activates renal sympathetic nerve activity and stimulates production of angiotensin II. (Modified from Vander AJ. Renal Physiology. 2nd ed. New York: McGraw-Hill; 1980.) Hemorrhage ˜ Arterial blood pressure ˜ RBF and GFR (Intrarenal receptors) ° Renin secretion ° Plasma and renal renin ° Activity of renal sympathetic nerves ° Plasma and renal angiotensin II ° Constriction of renal arterioles Carotid sinus and aortic arch reflexes ° Tubular sodium and water reabsorption ˜ Renal excretion of sodium and water

Adenosine is produced within the kidneys and causes vasoconstriction of the afferent arteriole, thereby reducing RBF and GFR. As previously mentioned, adenosine plays an important role in tubuloglomerular feedback.

Secretion of atrial natriuretic peptide (ANP) by the cardiac atria and brain natriuretic peptide (BNP) by the cardiac ventricle increases when ECFV is expanded and myocardial wall tension is increased. Both ANP and BNP dilate the afferent arteriole and constrict the efferent arteriole. Therefore ANP and BNP produce a modest increase in GFR with little change in RBF.

Cells release ATP into the renal interstitial fluid. ATP can have bidirectional effects on both RBF and GFR. Under some conditions, ATP constricts the afferent arteriole, reduces RBF and GFR, and may play a role in tubuloglomerular feedback. Under other conditions, ATP may stimulate NO production and have directionally opposite effects, increasing both RBF and GFR.

Administration of therapeutic doses of glucocorticoids increases GFR and RBF.

Local release of histamine modulates RBF during the resting state and during inflammation and injury. Histamine decreases the resistance of the afferent and efferent arterioles and thereby increases RBF without elevating GFR.

The proximal tubule produces the vasodilator substance dopamine. Dopamine has several actions within the kidney, such as increasing RBF and inhibiting renin secretion.

• Fig. 33.23 Examples of the interactions of endothelial cells with smooth muscle and mesangial cells. ACE, angiotensin-converting enzyme; AI, angiotensin I; AII, angiotensin II. (Modified from Navar LG et al. Physiol Rev 1996;76:425.) Smooth muscle cell or mesangial cellVasodilation PGI2 PGE2 Nitric oxide Stretch Histamine Acetylcholine Bradykinin ATP A I A II Endothelin A C E Endothelial cell Vasoconstriction

Finally, as illustrated in

Fig. 33.23 , endothelial cells play an important role in regulating the resistance of the renal afferent and efferent arterioles by producing a number of paracrine hormones, including NO, prostacyclin (PGI2), endothelin, and angiotensin II. These hormones regulate contraction or relaxation of smooth muscle cells in afferent and efferent arterioles and mesangial cells. Shear stress, acetylcholine, histamine, bradykinin, and ATP stimulate production of NO, which increases GFR and RBF. Angiotensin-converting enzyme (ACE), located on the surface of endothelial cells lining the afferent arteriole and glomerular capillaries, converts angiotensin I to angiotensin II, which decreases GFR and RBF. Angiotensin II is also produced locally by granular cells in the afferent arteriole and by proximal tubular cells. PGI2 and PGE2 release by endothelial cells is stimulated by both sympathetic nerve activity and angiotensin II, resulting in increased GFR and RBF. Finally, endothelin release from endothelial cells decreases both RBF and GFR.

ACE proteolytically inactivates the vasodilatory hormone bradykinin and converts angiotensin I, an inactive hormone, to angiotensin II, an active vasoconstrictive hormone. Thus ACE increases angiotensin II levels and decreases bradykinin levels. ACE inhibitors (e.g., lisinopril, enalapril, and captopril) are used clinically to reduce systemic blood pressure in patients with hypertension by decreasing angiotensin II levels and elevating bradykinin levels. Both effects lower systemic vascular resistance, reduce blood pressure, and decrease renal vascular resistance, thereby increasing GFR and RBF. Angiotensin II receptor antagonists (e.g., losartan) are also used to treat hypertension. As their name suggests, they block the binding of angiotensin II to the angiotensin II receptor (AT1). These antagonists block the vasoconstrictor effects of angiotensin II on the afferent arteriole; thus they increase RBF and GFR. In contrast to ACE inhibitors, angiotensin II receptor antagonists do not inhibit kinin metabolism (e.g., bradykinin).

1.

The first step in urine formation is passive movement of a plasma ultrafiltrate from the glomerular capillaries into Bowman’s space. The term ultrafiltration refers to passive movement of a plasma-like fluid that has a very low concentration of proteins from the glomerular capillaries into Bowman’s space. The endothelial cells of glomerular capillaries are covered by a basement membrane that is surrounded by podocytes. The capillary endothelium, basement membrane, and foot processes of podocytes form the so-called filtration barrier.

2.

The juxtaglomerular apparatus is one component of an important feedback mechanism (i.e., tubuloglomerular feedback) that regulates RBF and GFR. The structures that make up the juxtaglomerular apparatus include the macula densa, extraglomerular mesangial cells, and reninand angiotensin II–producing granular cells.

3.

Clinically, GFR is frequently estimated using measures of plasma [creatinine] or CrCl.

4.

Autoregulation allows GFR and RBF to remain constant despite changes in arterial blood pressure between 90 and 180 mm Hg. When ECFV is altered, sympathetic nerves, catecholamines, angiotensin II, prostaglandins, NO, endothelin, natriuretic peptides, bradykinin, and adenosine exert substantial control over GFR and RBF.

Patel A. The primary cilium calcium channels and their role in flow sensing. Pflugers Arch. 2015;467:157-165.

Pollak MR, et al. The glomerulus: the sphere of influence. Clin J Am Soc Nephrol. 2014;9:1461-1469.

Schlondorff J. How many Achilles’ heels does a podocyte have? An update on podocyte biology. Nephrol Dial Transplant. 2015;30: 1091-1097.

Schnermann J. Concurrent activation of multiple vasoactive signaling pathways in vasoconstriction caused by tubuloglomerular feedback: a quantitative assessment. Annu Rev Physiol. 2015;77:301-322.

Taylor AE, Moore TM. Capillary fluid exchange. Adv Physiol Educ. 1999;22:s203-s210.

Arendshorst WJ, Navar LG. Renal circulation and glomerular hemodynamics. In: Schrier RW, et al., eds. Diseases of the Kidney and Urinary Tract. 9th ed. Philadelphia: Lippincott Williams & Wilkins; 2012.

Dworkin LD, et al. The renal circulations. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.

Kriz W, Kaissling B. Structural organization of the mammalian kidney. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012.

Lafayette RA, et al. Laboratory evaluation of renal function. In: Schrier RW, et al., eds. Diseases of the Kidney and Urinary Tract. 9th ed. Philadelphia: Lippincott Williams & Wilkins; 2012.

Madsen K, et al. Anatomy of the kidney. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.

Upon completion of this chapter the student should be able to answer the following questions: 1.

What three processes are involved in the production of urine?

2.

What is the composition of “normal” urine?

3.

What transport mechanisms are responsible for NaCl reabsorption by the nephron? Where are they located along the nephron?

4.

How is water reabsorption “coupled” to NaCl reabsorption in the proximal tubule?

5.

Why are solutes but not water reabsorbed by the thick ascending limb of Henle’s loop?

6.

What transport mechanisms are involved in secretion of organic anions and cations? What is the physiological relevance of these transport processes?

7.

What is glomerulotubular balance, and what is its physiological importance?

8.

What are the major hormones that regulate NaCl and water reabsorption by the kidneys? What is the nephron site of action of each hormone?

9.

What is the aldosterone paradox?

he formation of urine involves three basic processes: (1) ultrafiltration of plasma by the glomerulus, (2) reabsorption of water and solutes from the ultra-filtrate, and (3) secretion of selected solutes into tubular fluid. Although an average of 115 to 180 L/day in women and 130 to 200 L/day in men of essentially protein-free fluid is filtered by the human glomeruli each day, less than 1% of the filtered water and sodium chloride (NaCl) and variable amounts of other solutes are typically excreted in urine (

Table 34.1 ). By the processes of reabsorption and secretion, the renal tubules determine the volume and composition of urine (

Table 34.2 ), which in turn allows the 1Normal glomerular filtration rate (GFR) averages 115–180 L/day in women and 130–200 L/day in men. Thus the volume of the ultrafiltrate represents a volume that is approximately 10 times that of the extracellular fluid volume (ECFV). For simplicity, we assume throughout the remainder of this section that GFR is 180 L/day.

kidneys to precisely control the volume, osmolality, composition, and pH of the extracellular and intracellular fluid compartments. Transport proteins in cell membranes of the nephron mediate reabsorption and secretion of solutes and water in the kidneys. Approximately 5% to 10% of all human genes code for transport proteins, and genetic and acquired defects in transport proteins are the cause of many kidney diseases (

Table 34.3 ). In addition, numerous transport proteins are important drug targets. This chapter discusses NaCl and water reabsorption, transport of organic anions and cations, the transport proteins involved in solute and water transport, and some of the factors and hormones that regulate NaCl transport. Details on acid-base transport and on K+ , Ca++ , and inorganic phosphate (Pi) transport and their regulation are provided in through 37.

Solute and Water Reabsorption Along the Nephron

The general principles of solute and water transport across epithelial cells were discussed in

Quantitatively, reabsorption of NaCl and water represent the major function of nephrons. Approximately 25,000 mEq/day of Na+ and 179 L/day of water are reabsorbed by the renal tubules (see

Table 34.1).

In addition, renal transport of many other important solutes is linked either directly or indirectly to reabsorption of Na+ . In the following sections, the NaCl and water transport processes of each nephron segment and their regulation by hormones and other factors are presented.

The proximal tubule reabsorbs approximately 67% of filtered water, Na+ , Cl− , K+ , and most other solutes. In addition the proximal tubule reabsorbs virtually all the glucose and amino acids filtered by the glomerulus, as well as most of the HCO3 − . The key element in proximal tubule reabsorption is Na+,K+-ATPase in the basolateral membrane. Reabsorption of every substance, including water, is linked in some manner to the operation of Na+,K+-ATPase.

WaterL/day1801.5178.599.2Na+ mEq/day25,20015025,05099.4K+ mEq/day72010062086.1Ca++ mEq/day5401053098.2HCO3− mEq/day43202431899.9+ Cl− mEq/day18,00015017,85099.2Glucosemmol/day8000800100.0Ureag/day56282850.0 aThe filtered amount of any substance is calculated by multiplying the concentration of that substance in the ultrafiltrate by the glomerular filtration rate (GFR); for example, the filtered load of Na+ is calculated as [Na+]ultrafiltrate (140mEq/L) × GFR (180L/day) = 25,200mEq/day.

The composition and volume of urine can vary widely in the healthy state. These values represent average ranges. Normal water excretion typically ranges between 0.5 and 1.5L/day.Data from Valtin HV. Renal Physiology. 2nd ed. Boston: Little, Brown; 1983.

Na+ is reabsorbed by different mechanisms in the first and the second halves of the proximal tubule. In the first half of the proximal tubule, Na+ is reabsorbed primarily with bicarbonate (HCO3 −) and a number of other solutes (e.g., glucose, amino acids, Pi, lactate). In contrast, in the second half, Na+ is reabsorbed mainly with Cl− . This disparity is mediated by differences in the Na+ transport systems in the first and second halves of the proximal tubule and by differences in the composition of tubular fluid at these

Bilirubin50–130mEq/L20–70mEq/L30–50mEq/L5–12mEq/L2–18mEq/L50–130mEq/L20–40mEq/L200–400mmol/L6–20mmol/L5.0–7.0500–800mOsm/kgH2O0000000 sites. In absolute terms the first half of the proximal tubule reabsorbs significantly more Na+ than the second half.

In the first half of the proximal tubule, Na+ uptake into the cell is coupled with either H+ or organic solutes, includ ing glucose (Fig. 34.1 ). Specific transport proteins mediate entry of Na+ into the cell across the apical membrane. For example, the Na+/H+ antiporter, NHE3, (see

Fig. 34.1A ) couples entry of Na+ with extrusion of H+ from the cell. H+ secretion results in reabsorption of sodium bicarbonate (NaHCO3) (see ). Na+ also enters proximal tubule cells via several symporter mechanisms, including Na+/glucose (SGLT2), Na+/amino acid, Na+/Pi, and Na+/ lactate (see

Fig. 34.1B ). The glucose and other organic solutes that enter the cell with Na+ leave the cell across the basolateral membrane via passive transport mechanisms. Any Na+ that enters the cell across the apical membrane leaves the cell and enters the blood via Na+,K+-ATPase. Thus reabsorption of Na+ in the first half of the proximal tubule is coupled to that of HCO3 − and a number of organic molecules, and this generates a negative transepithelial voltage across the proximal tubule that provides the driving force for the paracellular reabsorption of Cl− . Reabsorption of many organic molecules, including glucose and lactate, is so avid they are almost completely removed from the tubular fluid in the first half of the proximal tubule (

Fig. 34.2 ). Reabsorption of NaHCO3 and Na+–organic solutes across the proximal tubule establishes a transtubular osmotic gradient (i.e., the osmolality of the interstitial fluid bathing the basolateral side of the cells is a few mOsm/L higher than the osmolality of tubule fluid) that provides the driving force for the passive reabsorption of water by osmosis. Because more water than Cl− is reabsorbed in the first half of the proximal tubule, the [Cl−] in tubular fluid rises along the length of the proximal tubule (see

Fig. 34.2

In the second half of the proximal tubule, Na+ reabsorption is largely accompanied by Cl− reabsorption via both transcellular and paracellular pathways (

Fig. 34.3 ). Na+ is primarily reabsorbed with Cl− rather than organic solutes or HCO3 − as the accompanying anion because the Na+ transport mechanisms in the second half of the proximal tubule

There are over 300 different solute transporter genes that form the so-called SLC (solute carrier) family of genes.AD, autosomal dominant; AR, autosomal recessive; TAL, thick ascending limb of Henle’s loop; XLR, X-linked recessive.Modified from Nachman RH, Glassock RJ. NephSAP 2010;9(3).

differ from those in the first half, and because the tubular fluid that enters the second half contains very little glucose or amino acids. In addition the high [Cl−] (140 mEq/L)

Fanconi syndrome, a renal disease that is either hereditary in tubule fluid, which is due to preferential reabsorption − or acquired, results from an impaired ability of the proximal of Na+ with HCO3 and organic solutes in the first half − tubule to reabsorb HCO3, Pi, amino acids, glucose, and of the proximal tubule, facilitates reabsorption of Cl− low-molecular-weight proteins. Because other downstream with Na+ . nephron segments cannot reabsorb these solutes and

The mechanism of transcellular Na+ reabsorption in the protein, Fanconi syndrome results in increased urinary excretion of HCO3− , amino acids, glucose, Pi, and low- second half of the proximal tubule is shown in

Fig. 34.3 molecular-weight proteins.

Na+ enters the cell across the luminal membrane primarily via the parallel operation of a Na+/H+ antiporter (NHE3) •Fig. 34.1 Na+ transport processes in the first half of the proximal tubule. These transport mechanisms are present in all cells in the first half of the proximal tubule but are separated into different cells to simplify the discussion. A, Operation of the Na+/H+ antiporter (NHE3) in the apical membrane and the Na+,K+-ATPase and HCO3− transporters, including the Na+/HCO3− symporter (NBC1; see also ) in the basolateral membrane mediates reabsorption of NaHCO3. Carbon dioxide and water combine inside the cells to form H+ and HCO3− in a reaction facilitated by the enzyme carbonic anhydrase (CA). B, Operation of the Na+/glucose symporter (SGLT2) in the apical membrane, in conjunction with Na+,K+-ATPase and the glucose transporter (GLUT2) in the basolateral membrane, mediates Na+-glucose reabsorption. Inactivating mutations in the GLUT2 gene lead to decreased glucose reabsorption in the proximal tubule and glucosuria (i.e., glucose in the urine). Though not shown, Na+ reabsorption is also coupled with other solutes, including amino acids, Pi, and lactate. Reabsorption of these solutes is mediated by the Na+/amino acid, Na+/Pi, and Na+/lactate symporters, respectively, located in the apical membrane and the Na+,K+-ATPase, amino acid, Pi, and lactate transporters, respectively, located in the basolateral membrane. Three classes of amino acid transporters have been identified in the proximal tubule: two that transport Na+ in conjunction with either acidic or basic amino acids and one that does not require Na+ and transports basic amino acids.

•Fig. 34.2 Concentration of solutes in tubule fluid as a function of length along the proximal tubule. [TF] is the concentration of the substance in tubular fluid; [P] is the concentration of the substance in plasma.

Values above 100 indicate that relatively less of the solute than water is reabsorbed, and values below 100 indicate that relatively more of the substance than water is reabsorbed.

•Fig. 34.3 Na+ transport processes in the second half of the proximal tubule. Na+ and Cl− enter the cell across the apical membrane through the operation of parallel Na+/H+ (NHE3) and Cl−-base (e.g., formate, oxalate, and bicarbonate) antiporters (CFEX). More than one Cl−-base antiporter is involved in this process, but only one is depicted. The secreted H+ and base combine in the tubular fluid to form an H-base complex that can recycle across the plasma membrane. Accumulation of the H-base complex in tubular fluid establishes a H-base concentration gradient that favors H-base recycling across the apical plasma membrane into the cell. Inside the cell, H+ and the base dissociate and recycle back across the apical plasma membrane. The net result is uptake of NaCl across the apical membrane. The base may be hydroxide ions (OH−), formate (HCO2−), oxalate, HCO3− , or sulfate. The positive transepithelial voltage in the lumen, indicated by the plus sign inside the circle in the tubular lumen, is generated by diffusion of Cl− (lumen to blood) across the tight junction. The high [Cl−] of tubular fluid provides the driving force for diffusion of Cl− . Some glucose is also reabsorbed in the second half of the proximal tubule by a mechanism similar to that described in the first half of the proximal tubule, except that the Na+/glucose symporter (SGLT1 gene) transports 2Na+ with one glucose and has higher affinity and lower capacity than the Na+/glucose symporter in the first part of the proximal tubule, depicted in

Fig. 34.1 . In addition, glucose exits the cell across the basolateral membrane via GLUT1 rather than via GLUT2 as in the first part of the proximal tubule (not shown). KCC, KCl symporter.

aAtrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) inhibit vasopressin (AVP)-stimulated water permeability.

and one or more Cl−-base antiporters (e.g., CFEX). Because the secreted H+ and base combine in the tubular fluid and reenter the cell, operation of the Na+/H+ and Cl−-base antiporters is equivalent to uptake of NaCl from tubular fluid into the cell. Na+ leaves the cell via Na+,K+-ATPase, and Cl− leaves the cell and enters the blood via a K+/ Cl− symporter (KCC) and a Cl− channel in the basolateral membrane.

Some NaCl is also reabsorbed across the second half of the proximal tubule via a paracellular route. Paracellular NaCl reabsorption occurs because the rise in [Cl−] in tubule fluid in the first half of the proximal tubule creates a [Cl−] gradient (140 mEq/L in the tubule lumen and 105 mEq/L in the interstitium). This concentration gradient favors diffusion of Cl− from the tubular lumen across the tight junctions into the lateral intercellular space. Movement of the negatively charged Cl− results in the tubular fluid becoming positively charged relative to blood. This positive transepithelial voltage causes diffusion of positively charged Na+ out of the tubular fluid across the tight junction into blood. Thus in the second half of the proximal tubule, some Na+ and Cl− are reabsorbed across the tight junctions via passive diffusion.

In summary, reabsorption of Na+ and Cl− in the proximal tubule occurs via both paracellular and transcellular pathways. Approximately 67% of the NaCl filtered each day is reabsorbed in the proximal tubule. Of this amount, two-thirds moves across the transcellular pathway, whereas the remaining one-third moves across the paracellular pathway (

Table 34.4

The proximal tubule reabsorbs 67% of the filtered water (

Table 34.5 ). The driving force for water reabsorption is a transtubular osmotic gradient established by reabsorption of solute (e.g., NaCl, Na+-glucose). Reabsorption of Na+ along with organic solutes, HCO3 − , and Cl− from tubular fluid into the lateral intercellular spaces reduces the osmolality of the tubular fluid and increases the osmolality of the lateral intercellular space. The osmotic gradient across the proximal tubule established by these transport processes is only a few mOsm/L (

Fig. 34.4 ). Because the proximal tubule is highly permeable to water, primarily owing to expression of aquaporin water channels (AQP1) in the apical and basolateral membranes, water is reabsorbed across cells by osmosis. In addition the tight junctions in the proximal tubule are also water permeable, so some water is also reabsorbed across the paracellular pathway between proximal tubular cells. Accumulation of fluid and solutes within the lateral intercellular space increases hydrostatic pressure in this compartment. The increased hydrostatic pressure forces fluid and solutes into the capillaries.

2In addition, protein oncotic pressure in the peritubular capillaries (πpc) is elevated because of the process of glomerular filtration (see ). The elevated πpc facilitates uptake of fluid and solute into the capillary.

•Fig. 34.4 Routes of reabsorption of water and solute across the proximal tubule. Transport of solutes, including Na+ , Cl− , and organic solutes, into the lateral intercellular space increases the osmolality of this compartment, which establishes the driving force for osmotic reabsorption of water across the proximal tubule. This occurs because some Na+,K+-ATPase and some transporters of organic solutes, HCO3− , and Cl− are located on the lateral cell membranes and deposit these solutes between cells. Furthermore, some NaCl also enters the lateral intercellular space via diffusion across the tight junction (i.e., paracellular pathway). An important consequence of osmotic water flow across the transcellular and paracellular pathways in the proximal tubule is that some solutes, especially K+ and Ca++ , are entrained in the reabsorbed fluid and thereby reabsorbed by the process of solvent drag.

reabsorption in the proximal tubule. The reabsorbed fluid is slightly hyperosmotic relative to plasma. However, this difference in osmolality is so small it is commonly said that proximal tubule reabsorption is isosmotic (i.e., ≈67% of both the filtered load of solute and water are reabsorbed). Indeed, there is little difference in the osmolality of tubular fluid at the start and end of the proximal tubule. An important consequence of osmotic water flow across the proximal tubule is that some solutes, especially K+ and Ca++ , are entrained in the reabsorbed fluid and thereby reabsorbed by the process of solvent drag (see

Fig. 34.4 ). Reabsorption of virtually all organic solutes, Cl− and other ions, and water is coupled to Na+ reabsorption. Therefore changes in Na+ reabsorption influence reabsorption of water and other solutes by the proximal tubule. This point will be discussed later, notably in , and is especially relevant during volume depletion when increased Na+ reabsorption by the proximal tubule is accompanied by a parallel increase in HCO3 − reabsorption, which can contribute to metabolic alkalosis (i.e., volume contraction alkalosis).

Proteins filtered by the glomerulus are reabsorbed in the proximal tubule. As mentioned previously, peptide hormones, small proteins, and small amounts of larger proteins such as albumin are filtered by the glomerulus. Overall, only a small percentage of proteins cross the glomerulus and enter Bowman’s space (i.e., the concentration of proteins in the glomerular ultrafiltrate is only ≈ 40 mg/L). However, the total amount of protein filtered per day is significant because the glomerular filtration rate (GFR) is so high:

Equation 34.1

Filtered protein = GFR ×[Protein] in the ultrafiltrate = 7200 mg/day, or 7 2. g/day

Filtered proteins are reabsorbed in the proximal tubule by endocytosis either as intact proteins or after being partially degraded by enzymes on the surface of proximal tubule cells. Once the proteins and peptides are inside the cell, enzymes digest them into their constituent amino acids, which then leave the cell across the basolateral membrane by transport proteins and are returned to the blood. Normally this mechanism reabsorbs virtually all the proteins filtered, and hence the urine is essentially protein free. However, because the mechanism is easily saturated, an increase in filtered proteins can result in proteinuria (appearance of protein in urine). Disruption of the glomerular filtration barrier to proteins increases the filtration of proteins and 610 SECTION7Berne & Levy Physiology • BOX 34.2 Some Organic Cations Secreted by Proximal Tubule • BOX 34.1 Some Organic Anions Secreted by Proximal Tubule cAMP, cGMPBile saltsHippuratesOxalateProstaglandins: PGE2, PGF2α UrateVitamins: ascorbate, folate

AcetazolamideAcyclovirAmoxicillinCaptoprilChlorothiazideFurosemideLosartanPenicillinProbenecidSalicylate (aspirin)HydrochlorothiazideSimvastatinBumetanideNonsteroidal antiinflammatory drugs (NSAIDs): indomethacin cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate.

results in proteinuria, which is frequently seen with kidney disease.

Secretion of Organic Anions and Organic Cations

Cells of the proximal tubule also secrete organic anions and organic cations into the tubule fluid. Secretion of organic anions and cations by the proximal tubule plays a key role in regulating the plasma levels of xenobiotics (e.g., a variety of antibiotics, diuretics, statins, antivirals, antineoplastics, immunosuppressants, neurotransmitters, and nonsteroidal antiinflammatory drugs [NSAIDs]) and toxic compounds derived from endogenous and exogenous sources. Many of the organic anions and cations (

Boxes 34.1 34.2 ) secreted by the proximal tubule are end products of metabolism that circulate in plasma. Many of these organic compounds are bound to plasma proteins and thus are not readily filtered. Therefore only a small fraction of these potentially toxic substances are eliminated from the body by excretion resulting from filtration alone. Thus secretion of organic anions and cations, including many toxins from the peritubular capillary into the tubular fluid, promote elimination of these compounds from plasma entering the kidneys. Hence these substances are removed from plasma by both filtration and secretion. It is important to note that when kidney function is reduced by disease, urinary excretion of organic anions and cations is severely reduced, which can lead to increased plasma levels of xenobiotics and potentially toxic accumulation of organic anions and cations.

Water channels called aquaporins (AQPs) mediate transcellular reabsorption of water across many nephron segments. To date, 13 aquaporins have been identified. The AQP family is divided into two groups based on their permeability characteristics. One group (aquaporins) is permeable to water (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, AQP8, AQP11, and AQP12). The other group (aquaglyceroporins) is permeable to water and small solutes, especially glycerol (AQP3, AQP7, AQP9, AQP10). Aquaporins form tetramers in the plasma membrane of cells, with each subunit forming a water channel. In the kidneys, AQP1 is expressed in the apical and basolateral membranes of the proximal tubule and in portions of the descending thin limb of Henle’s loop. The importance of AQP1 in renal water reabsorption is underscored by studies in which the AQP1 gene was “knocked out” in mice. These mice exhibit increased urine output (polyuria) and reduced ability to concentrate urine. In addition the osmotic water permeability of the proximal tubule is fivefold less in mice lacking APQ1 than in normal mice. AQP7 and AQP8 are also expressed in the proximal tubule. AQP2 is expressed in the apical plasma membrane of principal cells in the collecting duct, and its abundance in the membrane is regulated by arginine vasopressin (AVP) (see ). AQP3 and AQP4 are expressed in the basolateral membrane of principal cells in the collecting duct, and mice deficient in these AQPs (i.e., AQP3 and AQP4 knockout mice) have defects in the ability to concentrate urine (see ). AQPs are also expressed in many other organs in the body, including the lung, eye, skin, secretory glands, and brain, where they play key physiological roles. For example, AQP4 is expressed in cells that form the blood-brain barrier. Knockout of AQP4 affects the water permeability of the blood-brain barrier such that brain edema is reduced in AQP4-deficient mice after acute water loading and subsequent development of hyponatremia.

ATP ATP ADP ˜-KG NaDC3 OAT 1,2,3 OAT4 OA– ˜-KG MRP2/4 BCRP K+ •Fig. 34.5 Secretion of organic anion (OA−) across the proximal tubule. OA−s enter the cell across the basolateral membrane by one of three OA−/α-ketoglutarate (α-KG) antiporter mechanisms (organic anion transporters, OAT1, OAT2, OAT3). Uptake of α-KG into the cell against its chemical concentration gradient is driven by movement of Na+ into the cell via the Na+-dicarboxylate transporter (NaDC3). The [Na+] inside the cell is low because of the Na+,K+-ATPase in the basolateral membrane, which transports Na+ out of the cell in exchange for K+ . The α-KG recycles across the basolateral membrane on the OATs in exchange for OA− . OA−s leave the cell across the apical membrane by multidrug drug resistance proteins (MRP2 and 4), and by breast cancer resistance protein (BCRP), which require ATP. OAT4 in the apical membrane reabsorbs urate, an organic anion.

The endocytosis of proteins by the proximal tubule is mediated by apical membrane proteins that specifically bind proteins and peptides in tubule fluid. These receptors, called multiligand endocytic receptors, can bind a wide range of peptides and proteins and thereby mediate their endocytosis. Megalin and cubilin mediate protein and peptide endocytosis in the proximal tubule. Both are glycoproteins, with megalin being a member of the low-density lipoprotein receptor gene family.

Fig. 34.5 illustrates the mechanisms of organic anion (OA−) transport across the proximal tubule. These secretory pathways have maximum transport rates, low specificity (i.e., they transport many OA−s), and are responsible for secretion of the OA−s listed in

Box 34.1.

OA−s are taken up into the cell across the basolateral membrane against their chemical gradient in exchange for α-ketoglutarate (α-KG) via several OA−/α-KG antiporters, including OAT1, OAT2, and OAT3. α-KG accumulates inside the cells via metabolism of glutamate and by a Na+/α-KG symporter (i.e., the Na+/dicarboxylate transporter [NaDC3]) also present in the basolateral membrane. Thus uptake of OA− into the cell against an electrochemical gradient is coupled to the

Urinalysis is an important and routine tool for detection of kidney disease. A thorough analysis of urine includes macroscopic, microscopic, and biochemical assessments. This is performed by visual assessment of the urine, microscopic examination of urinary sediment, and biochemical evaluation of urinary composition using dipstick reagent strips. The dipstick test is both inexpensive and fast (i.e., <5 minutes) and tests urine for both pH and the presence of many substances (e.g., bilirubin, blood, glucose, ketones, protein). It is normal to find trace amounts of protein in urine, particularly concentrated urine. Urinary proteins are derived from two principal sources: (1) filtration exceeding the reabsorptive capacity of the proximal tubule and (2) synthesis and secretion of Tamm-Horsfall glycoprotein by the thick ascending limb of Henle’s loop. Because the mechanism for protein reabsorption is “upstream” of the thick ascending limb (i.e., in the proximal tubule), the secreted Tamm-Horsfall glycoprotein appears in urine. However, proteinuria in greater than trace amounts is often indicative of renal disease.

exit of α-KG out of the cell, down its chemical gradient generated by the Na+/α-KG symporter mechanism. The exit of OA−s across the luminal membrane into the tubular fluid are mediated by multidrug resistance proteins 2 and 4 (MRP2/4) and breast cancer resistance protein 1 (BCRP), which require adenosine triphosphate (ATP) for their operation. Recent studies reveal that OAT4 mediates reabsorption of the organic anion urate, the end product of purine catabolism, by the proximal tubule (see

Fig. 34.5 •Fig. 34.6 Organic cation (OC+) secretion across the proximal tubule. OC+s enter the cell across the basolateral membrane primarily by OCT2. Uptake of OC+s into the cell against their chemical concentration gradient is driven by the cell-negative potential difference. OC+s leave the cell across the apical membrane in exchange with H+by electrically neutral multidrug and toxin transporters (MATE1 and MATE2-K) and by multidrug resistance protein (MDR1), which requires ATP. ATP OCT2MATE MDR1 Na+ OC+ OC+ H+ K+ Blood Tubular fluid OC+ ATP ADP

Fig. 34.6 illustrates the mechanism of organic cation (OC+) transport across the proximal tubule. Organic cations, including xenobiotics such as the antidiabetic agent metformin, the antiviral agent lamivudine, and the anticancer drug oxaliplatin, and many important monoamine neurotransmitters including dopamine, epinephrine, histamine, and norepinephrine are secreted by the proximal tubule. Organic cations are taken up into the cell across the basolateral membrane, primarily by the organic cation transporter 2 (OCT2). Uptake of organic cations is driven by the magnitude of the cell-negative potential difference across the basolateral membrane. Organic cation transport across the luminal membrane into the tubular fluid, which is the rate-limiting step in secretion, is mediated primarily by electroneutral multidrug and toxin extrusion transporters (MATEs) and MDR1 (also known as P-glycoprotein), which requires ATP for its operation. These transport mechanisms are nonspecific, and several organic cations usually compete for secretion via a given transport pathway.

Henle’s loop reabsorbs approximately 25% of the filtered NaCl and 15% of the filtered water. Reabsorption of NaCl

Because many organic anions compete for the same secretory pathways, elevated plasma levels of one transported anion often inhibit secretion of the others. For example, infusing p-aminohippuric acid (PAH) can reduce secretion of penicillin by the proximal tubule. Because the kidneys are responsible for eliminating penicillin, infusion of PAH into individuals receiving penicillin reduces penicillin excretion and thereby extends its biological half-life. In World War II, when penicillin was in short supply, hippurates were given with penicillin to extend its therapeutic effect. Similar competition is observed for organic cation secretion by the proximal tubule, and elevated plasma levels of one transported cation species can inhibit secretion of the other competing cations. For example, the histamine H2antagonist cimetidine used to treat gastric ulcers is secreted via organic cation transport mechanisms in the proximal tubule. If cimetidine is given to patients receiving procainamide (a drug used to treat cardiac arrhythmias), cimetidine reduces urinary excretion of procainamide (also an organic cation) by direct competition for a common secretory pathway. As a consequence, coadministration of cationic drugs competing for the same pathway can increase the plasma concentration of both drugs to levels much higher than those observed when the drugs are given alone. This effect can lead to drug toxicity.

•Fig. 34.7 Transport mechanisms for NaCl reabsorption in the thick ascending limb of the loop of Henle. The positive voltage in the lumen plays a major role in driving the passive paracellular reabsorption of cations. Because the apical membrane is conductive primarily to K+ , the apical membrane voltage is more negative than the basolateral membrane voltage, which is conductive to K+ and Cl− , thereby resulting in a lumen positive transepithelial potential. Mutations in the apical membrane K+ channel (ROMK), the apical membrane 1Na+/1K+/2Cl− symporter (NKCC2), or the basolateral Cl− channel (ClCNKB) cause Bartter syndrome (see the clinical box on Bartter syndrome). CA, carbonic anhydrase.

in the loop of Henle occurs in both the thin and thick ascending limbs, whereas the descending thin limb does not reabsorb NaCl. In contrast, water reabsorption mediated by AQP1 water channels is exclusively restricted to the descending thin limb, whereas the ascending limb is impermeable to water. In addition, divalent cations (e.g., Ca++ and Mg++) and HCO3 − are also reabsorbed in the loop of Henle (see for more details).

The thin ascending limb reabsorbs NaCl by a passive mechanism. Reabsorption of water, but not NaCl, in the descending thin limb increases [NaCl] in the tubule fluid entering the ascending thin limb. As the NaCl-rich fluid moves toward the cortex, NaCl diffuses out of the tubule lumen across the ascending thin limb and into the medullary interstitial fluid, down a concentration gradient directed from the tubule fluid to the interstitium (see for details).

The key element in reabsorption of solute by the thick ascending limb is Na+,K+-ATPase in the basolateral membrane (Fig. 34.7 ). As with reabsorption in the proximal tubule, reabsorption of every solute by the thick ascending limb is linked to Na+,K+-ATPase activity. This transporter maintains a low intracellular [Na+], which provides a favorable chemical gradient for the movement of Na+ from tubular fluid into the cell. This movement of Na+ across the apical membrane into the cell is mediated by the 1Na+/1K+/2Cl− symporter (NKCC2), which couples the movement of 1Na+ with 1K+ and 2Cl− . Using the potential energy released by the downhill movement of Na+ and Cl− , this symporter drives the uphill movement of K+ into the cell. K+ channels (ROMK and Maxi-K) in the apical plasma membrane play an important role in reabsorption of NaCl by the thick ascending limb. These K+ channels allow the K+ transported into the cell via the 1Na+/1K+/2Cl− symporter to recycle back into tubule fluid. Because the [K+] in tubule fluid is relatively low, K+ recycling is required for continued operation of the 1Na+/1K+/2Cl− symporter. A Na+/H+ anti-porter (NHE3) in the apical cell membrane also mediates Na+ reabsorption as well as H+ secretion (HCO3 − reabsorption) in the thick ascending limb (see also ). The operation of the Na+/H+ antiporter in the apical membrane results in cellular uptake of Na+ in exchange for H+ . The production of H+ inside cells generates HCO3 −, which exits the cell across the basolateral membrane via a Cl−/HCO3 − antiporter (AE2). Na+ leaves the cell across the basolateral membrane via the Na+,K+-ATPase, whereas K+ and Cl− leave the cell via separate pathways in the basolateral membrane (i.e., K+ and Cl− channels and the K+/Cl− symporter).

614 SECTION7Berne & Levy Physiology •Fig. 34.8 Transport mechanism for NaCl reabsorption in the early segment of the distal tubule. This segment is impermeable to water. Mutations in the apical membrane NaCl symporter (NCC) cause Gitelman syndrome. ATP H2O NCC Na+ K+ K+ K+ Na+ Cl– Blood Tubular fluid Cl–

The voltage across the thick ascending limb is important for reabsorption of several cations. The tubular fluid is positively charged relative to blood because of the unique location of transport proteins in the apical and basolateral membranes. Two points are important: (1) increased NaCl transport by the thick ascending limb increases the magnitude of the positive voltage in the lumen, and (2) this voltage is an important driving force for reabsorption of several cations, including Na+ , K+ , Mg++ , and Ca++ , across the paracellular pathway (see

Fig. 34.7 ). The importance of the paracellular pathway to solute reabsorption is underscored by the observation that inactivating mutations of the tight junction protein claudin-16 reduce reabsorption of Mg++ and Ca++ by the ascending thick limb, even in the presence of a lumen positive transepithelial voltage.

In summary, NaCl reabsorption across the thick ascending limb occurs via transcellular and paracellular pathways. Fifty percent of NaCl reabsorption is transcellular, and 50% is paracellular. Because the thick ascending limb does not reabsorb water, owing to a lack of water channels (i.e., AQPs), reabsorption of NaCl and other solutes reduces the osmolality of tubular fluid to less than 150 mOsm/ kg H2O. Thus because the thick ascending limb of Henle’s loop produces a fluid that is dilute relative to plasma, this segment and the adjacent distal tubule (as discussed next) are often collectively referred to as the “diluting segments.”

The distal tubule and collecting duct reabsorb approximately 8% of the filtered NaCl, secrete variable amounts of K+ and H+ , and reabsorb a variable amount of water (≈8%–17%). The initial segment of the distal tubule (early distal tubule) reabsorbs Na+, Cl−, and Ca++ and is impermeable to water (

Fig. 34.8 ). Entry of NaCl into the cell across the apical membrane is mediated by a Na+/Cl− symporter,

Chapter 2, epithelial cells are joined at their apical surfaces by tight junctions (zonula occludens). A number of proteins have now been identified as components of the tight junction, including proteins that span the membrane of one cell and link to the extracellular portion of the same molecule in the adjacent cell (e.g., occludin and claudins), as well as cytoplasmic linker proteins (e.g., ZO-1, ZO-2, and ZO-3) that link the membrane-spanning proteins to the cytoskeleton of the cell. Of these junctional proteins, claudins appear to be major determinants of the permeability characteristics of tight junctions. For example, claudin-16 and claudin-19 are critical determinants of divalent cation permeability of the tight junctions in the thick ascending limb of Henle’s loop. Mutations in human claudin-16 and claudin19 cause familial hypomagnesemia (i.e., low plasma [Mg++]) with hypercalciuria (i.e., increased Ca++ in the urine) and nephrocalcinosis (i.e., calcification of the kidney). Claudin-2 is permeable to water and may be responsible for paracellular water reabsorption across the proximal tubule. Claudin-4 has been shown in cultured kidney cells to control the permeability of the tight junction to Na+ , whereas claudin-15 determines whether a tight junction is permeable to cations or anions. Thus the permeability characteristics of the tight junctions in different nephron segments are determined at least in part by the specific claudins expressed by the cells in that segment.

AVP, water is reabsorbed. By contrast, in the absence of AVP the late distal tubule and collecting duct reabsorb little

Bartter syndrome is a set of autosomal recessive genetic diseases characterized by hypokalemia, metabolic alkalosis, and hyperaldosteronism (see

Table 34.3 ). Inactivating mutations in the gene coding for the 1Na+/1K+/2Cl− symporter (NKCC2), the apical K+ channel (ROMK), or the basolateral Cl− channel (ClCNKB) decrease both NaCl reabsorption and K+ reabsorption by the ascending thick limb, which in turn causes hypokalemia (i.e., low plasma [K+]) and a decrease in ECFV. The fall in ECFV stimulates aldosterone secretion, which in turn stimulates NaCl reabsorption and H+ secretion by the distal tubule and collecting duct.

Fig. 34.8 ). Na+ leaves the cell via the action of Na+,K+-ATPase, and Cl− leaves the cell via diffusion through Cl− channels and a K+/Cl− symporter (KCC4). Thus dilution of tubular fluid begins in the thick ascending limb and continues in the early segment of the distal tubule.

The last segment of the distal tubule (late distal tubule) and the collecting duct are composed of three cell types: principal cells and two types of intercalated cells. As illustrated in

Fig. 34.9, principal cells reabsorb NaCl and water and secrete K+ . Both Na+ reabsorption and K+ secretion by these cells depend on the activity of Na+,K+-ATPase in the basolateral membrane. By maintaining a low intracellular [Na+], the Na+,K+-ATPase provides a favorable chemical gradient for movement of Na+ from tubular fluid into the cell. Because Na+ enters the cell across the apical membrane via diffusion through epithelial Na+-selective channels (ENaCs), the negative voltage inside the cell facilitates entry of Na+, which then exits the cell and enters the blood via the basolateral membrane Na+,K+-ATPase. Reabsorption of Na+ generates a negative luminal voltage across the late distal tubule and collecting duct, which provides the driving force for paracellular reabsorption of Cl− . Intercalated cells secrete either H+ or HCO3 − and play important roles in acid-base homeostasis (see ). The α-intercalated cell (see

Fig. 34.9 , center) secretes H+ and reabsorbs both HCO3 − and K+ and is thus important in regulating acid-base balance (see ). α-Intercalated cells reabsorb K+ by the operation of an H+,K+-ATPase (HKA) located in the apical plasma membrane. In contrast, β-intercalated cells (see

Fig. 34.9 , bottom) secrete HCO3 − and reabsorb both H+ and Cl− . Chloride enters the β-intercalated cell across the apical membrane via a Cl−/HCO3 − antiporter (pendrin) and leaves the cell across the basolateral membrane via a Cl− channel. A variable amount of water is reabsorbed across principal cells in the late distal tubule and collecting duct. Water reabsorption in these segments is mediated by the AVP-regulated AQP2 water channel located in the apical plasma membrane and by AQP3 and AQP4 located in the basolateral membrane of principal cells. In the presence of water (see

K+ is secreted from blood into tubular fluid by principal cells in two steps (see

Fig. 34.9, top).

First, uptake of K+ across the basolateral membrane is mediated by the action of Na+,K+-ATPase. Second, K+ leaves the cell via passive diffusion. Because [K+] inside principal cells is high (≈150 mEq/L) and [K+] in tubular fluid is low (≈10 mEq/L), K+ diffuses down its concentration gradient through apical cell membrane K+ channels (ROMK and BK) into tubular fluid. Although the negative potential inside these cells favors intracellular K+ retention, the electrochemical gradient across the apical membrane promotes secretion of K+ from the cell into tubular fluid (see ). In contrast, K+ reabsorption by α cells is mediated by an H+,K+-ATPase (HKA) located in the apical cell membrane (see

Fig. 34.9 , center). As a consequence these distal nephron segments possess the ability to both secrete and reabsorb K+ via independently regulated mechanisms, which contrasts with the general tendency to reabsorb Na+ along most nephron segments.

Regulation of NaCl and Water Reabsorption

Quantitatively, angiotensin II, aldosterone, catecholamines, natriuretic peptides, and uroguanylin are the most important hormones regulating NaCl reabsorption and thereby urinary NaCl excretion (

Table 34.6).

However, other hormones (including dopamine and adrenomedullin), Starling forces, and the phenomenon of glomerulotubular balance also influence NaCl reabsorption. AVP is the only major hormone that directly regulates the amount of water excreted by the kidneys.

Angiotensin II has a potent stimulatory effect on the isosmotic reabsorption of NaCl and water in the proximal tubule. It also stimulates reabsorption of Na+ in the thick ascending limb of Henle’s loop, as well as the late distal tubule and collecting duct. A decrease in ECFV activates the renin-angiotensin-aldosterone system (see for more details), thereby increasing the plasma concentration of angiotensin II.

Aldosterone is synthesized by the glomerulosa cells of the adrenal cortex and stimulates reabsorption of NaCl by the thick ascending limb of Henle’s loop, the late distal tubule, and the collecting duct. Most of aldosterone’s effect on NaCl reabsorption reflects its action on the late distal tubule and collecting duct. Aldosterone enhances reabsorption of NaCl across principal cells in these segments by four mechanisms: (1) increasing the amount of Na+,K+-ATPase in the basolateral membrane; (2) increasing expression of the sodium channel (ENaC) in the apical cell membrane; (3) elevating Sgk1 (serum glucocorticoid-stimulated kinase; see the molecular box) levels, which also increases the expression of ENaC in the apical cell membrane; and (4) stimulating •Fig. 34.9 Transport pathways in principal cells, α-intercalated cells, and β-intercalated cells of the late segment of the distal tubule and collecting duct. CA, carbonic anhydrase. Principal cells reabsorb Na+and secrete K+. K+is secreted by two types of K+channels (ROMK and BK) and by a K+/Cl−symporter (KCC1). α-Intercalated cells secrete H+and reabsorb HCO3−and K+, and β-intercalated cells secrete HCO3−and reabsorb H+and Cl−. CO2 + H2O HCO− 3 CA Cl− Cl− H+ ˙-Intercalated cell Pendrin V-ATPase Blood

CAP1 (channel-activating protease, also called prostatin), a serine protease that directly activates ENaCs by proteolysis. Taken together, these actions increase Na+ uptake across the apical cell membrane and facilitate Na+ exit from the cell interior into blood. The increase in reabsorption of Na+ generates a negative transepithelial luminal voltage across the late distal tubule and the collecting duct. This negative voltage in the lumen provides the electrochemical driving force for reabsorption of Cl− across the tight junctions. Aldosterone also stimulates secretion of K+ by the late distal tubule and collecting duct (collectively referred to as the aldosterone-sensitive distal nephron (ASDN)

Angiotensin II↑ReninPT, TAL, DT/CD↑NaCl and H2O reabsorptionAldosterone↑Angiotensin II, ↑[K+]pTAL, DT/CD↑NaCl and H2O reabsorption

ANP, BNP, urodilatin↑ECFVCD↓H2O and NaCl reabsorptionUroguanylin, guanylinOral ingestion of NaClPT, CD↓H2O and NaCl reabsorptionSympathetic nerves↓ECFVPT, TAL, DT/CD↑NaCl and H2O reabsorption

Dopamine↑ECFVPT↓H2O and NaCl reabsorptionAVP↑Posm, ↓ECFVDT/CD↑H2O reabsorption aAll these hormones act within minutes, except aldosterone, which exerts its action on reabsorption of NaCl with a delay of 1 hour. Aldosterone achieves its maximal effect after a few days.bThe effect on reabsorption of H2O does not include the thick ascending limb.ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide, BP, blood pressure; CD, collecting duct; DT, distal tubule; ECFV, extracellular fluid volume; [K+]p, plasma K+ concentration; Posm, plasma osmolality; PT, proximal tubule; TAL, thick ascending limb.

Chapter 36]).

Aldosterone secretion is increased by hyperkalemia and by hypovolemia (i.e., reduced ECFV via increased angiotensin II following activation of the renin-angiotensin system [RAS]; see for more details). Aldosterone secretion is decreased by hypokalemia and natriuretic peptides (discussed in more detail next). Through its stimulation of NaCl reabsorption in the collecting duct, aldosterone also indirectly increases water reabsorption by this nephron segment.

The aldosterone paradox. As noted earlier, aldosterone stimulates both NaCl reabsorption and K+ secretion by the collecting duct. Although both a reduction in the ECFV (i.e., hypovolemia, see ) increase aldosterone levels, the physiological response of the kidneys differs in these two conditions. In the setting of ECFV depletion, NaCl excretion by the kidneys is reduced to restore ECFV, without an accompanying change in K+ excretion. By contrast, during hyperkalemia, K+ excretion by the kidneys is increased to normalize plasma [K+], albeit without an accompanying change in NaCl excretion. This phenomenon—the apparent independent effects of aldosterone on urinary Na+ and K+ excretion—is called the aldosterone paradox. The paradox can be explained by the observation that ECFV depletion increases aldosterone release via activation of the RAS, whereas hyperkalemia directly stimulates adrenal release of aldosterone without a requirement for RAS activation. As such, aldosterone increases in both conditions, whereas angiotensin II levels increase only during ECFV depletion and not during hyperkalemia. It is the differential regulation of transport processes in the distal tubule and collecting duct by aldosterone and angiotensin II that accounts for this paradox. The integrated physiological response to a reduction in ECFV is depicted in

Fig. 34.10A . During hypovolemia, angiotensin II stimulates NaCl reabsorption by the proximal tubule (not shown in

Figure 34.10A ), and by the early distal tubule by activating WNK (with no lysine [K] kinase), which enhances NaCl reabsorption by activating the Na+/Cl− symporter (NCC). Aldosterone stimulates ENaC-mediated Na+ reabsorption in principal cells of the collecting duct by activating SGK1, which increases ENaC abundance in the apical plasma membrane. In parallel, angiotensin II activates WNK in principal cells, which inhibits K+ secretion via ROMK, thereby preventing increased K+ excretion despite elevated aldosterone levels, which would be expected to promote K+ secretion. Angiotensin II stimulation of proximal tubule NaCl and water reabsorption also reduces delivery of NaCl and fluid to the collecting duct, which also suppresses K+ secretion in this segment (see for more details). The corresponding integrated physiological response to hyperkalemia is depicted in

Fig. 34.10B . During hyperkalemia, aldosterone stimulates ROMK mediated K+ secretion by principal cells in the collecting duct by activating WNK. Because the early distal tubule is not directly responsive to aldosterone, this hormone does not stimulate NaCl reabsorption in this segment. In fact, because angiotensin II levels are not elevated by hyperkalemia, the basal activity of WNK in the early distal tubule is low, resulting in decreased NaCl reabsorption via NCC (see

Fig. 34.10B ). This effect of WNK on early distal tubule NaCl reabsorption offsets the stimulatory effect of aldosterone, via SGK1, on ENaC-mediated Na+ reabsorption in principal cells of the collecting duct.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) inhibit NaCl and water reabsorption. Secretion of ANP by the cardiac atria and BNP by the cardiac ventricles are both stimulated by increased ECFV and increased myocardial wall pressure. ANP and BNP reduce blood pressure by decreasing total peripheral resistance and enhancing urinary excretion of both NaCl and water, primarily by increasing RBF and GFR. These natriuretic peptides vasodilate the afferent arterioles and vasoconstrict the efferent arterioles, which increases GFR and thus filtration of NaCl, thereby increasing NaCl excretion (see later discussion of glomerulotubular balance for the mechanism). In addition the increase in RBF decreases the concentration of NaCl in the medullary interstitium, which in turn reduces passive NaCl reabsorption by the thin ascending •Fig. 34.10 Aldosterone paradox (see text for details). +, stimulate; −, inhibit; AII, angiotensin II; NCC, NaCl symporter; ENaC, epithelial sodium channel; ROMK, K+ channel; WNK, with no lysine[K] kinases. There are four WNKs in the kidney; details are not given in the figure for clarity and because some of the details of WNK regulation of NCC, ENaC, and ROMK have not been elucidated.

limb of Henle’s loop (see earlier discussion for details on NaCl reabsorption by this segment). ANP and BNP also inhibit NaCl reabsorption by the medullary portion of the collecting duct and inhibit AVP-stimulated water reabsorption across the collecting duct. Moreover, ANP and BNP also reduce secretion of AVP from the posterior pituitary. These actions of ANP and BNP are mediated by the activation of membrane-bound guanylyl cyclase receptors, which increases intracellular levels of the second messenger cyclic guanine monophosphate (cGMP). ANP is a more profound natriuretic and diuretic agent than BNP.

Urodilatin and ANP are encoded by the same gene and have similar amino acid sequences. Urodilatin is a 32–amino acid hormone that differs from ANP by the addition of four amino acids to the amino terminus. Urodilatin is secreted by the distal tubule and collecting duct and is not present in the systemic circulation; thus urodilatin influences only the function of the kidneys. Secretion of urodilatin is stimulated by a rise in blood pressure and an increase in ECFV. It inhibits NaCl and water reabsorption across the medullary portion of the collecting duct. Urodilatin is a more potent natriuretic and diuretic hormone than ANP because some of the ANP that enters the kidneys in blood is degraded by a neutral endopeptidase that has no corresponding effect on urodilatin.

Uroguanylin and guanylin are produced by neuroendocrine cells in the intestine in response to oral ingestion of NaCl. These hormones enter the circulation and inhibit NaCl and water reabsorption by the kidneys via activation of membrane-bound guanylyl cyclase receptors, which

Sgk1 (serum glucocorticoid-stimulated kinase), a serine/threonine kinase, plays an important role in maintaining NaCl and K+ homeostasis by regulating excretion of both NaCl and K+ by the kidneys. Studies in Sgk1 knockout mice reveal that this kinase is required for animals to survive severe NaCl restriction and K+ loading. NaCl restriction and K+ loading enhance plasma [aldosterone], which rapidly (in minutes) increases Sgk1 protein expression and phosphorylation. Phosphorylated Sgk1 enhances ENaC-mediated Na+ reabsorption in the collecting duct, primarily by increasing the number of ENaCs in the apical plasma membrane of principal cells and also by increasing the number of Na+,K+-ATPase pumps in the basolateral membrane. Phosphorylated Sgk1 inhibits Nedd4-2, a ubiquitin ligase that monoubiquitinylates ENaC subunits, thereby targeting them for endocytic removal from the plasma membrane and subsequent destruction in lysosomes. Inhibition of Nedd4-2 by Sgk1 reduces the monoubiquitinylation of ENaC, thereby reducing endocytosis and increasing the number of channels in the membrane. Sgk1 induces the translocation of K+ channels (ROMK) from an intracellular pool to the plasma membrane, and thereby enhances ROMK-mediated K+ secretion by principal cells. These effects of Sgk1 precede the aldosterone-stimulated increase in ENaC, ROMK, and Na+,K+-ATPase abundance, which leads to a delayed (>4 hours) secondary increase in NaCl and K+ transport by the collecting duct. Activating polymorphisms in Sgk1 cause an increase in blood pressure, presumably by enhancing NaCl reabsorption by the collecting duct, which increases the ECFV and thereby blood pressure.

increase intracellular [cGMP]. The involvement of these gut-derived hormones helps explain why the natriuretic response of the kidneys to an oral NaCl load is more pronounced than when delivered intravenously.

Catecholamines stimulate reabsorption of NaCl. Catecholamines released from the sympathetic nerves (norepinephrine) and the adrenal medulla (epinephrine) stimulate reabsorption of NaCl and water by the proximal tubule, thick ascending limb of the loop of Henle, distal tubule, and collecting duct. Although sympathetic nerves are not active when ECFV is normal, when ECFV declines (e.g., after hemorrhage), sympathetic nerve activity rises and dramatically stimulates reabsorption of NaCl and water by these four nephron segments.

Dopamine, a catecholamine, is released from dopaminergic nerves in the kidneys and is also synthesized by cells of the proximal tubule. The action of dopamine is opposite that of norepinephrine and epinephrine. Secretion of dopamine is stimulated by an increase in ECFV, and its secretion directly inhibits reabsorption of NaCl and water in the proximal tubule.

Adrenomedullin is a 52–amino acid peptide hormone that is produced by a variety of organs, including the kidneys. Adrenomedullin induces a marked diuresis and natriuresis, and its secretion is stimulated by congestive heart failure and hypertension. The major effect of adrenomedullin on the kidneys is to increase GFR and renal blood flow and

Liddle syndrome is a rare genetic disorder characterized by an increase in blood pressure (i.e., hypertension) secondary to an increase in ECFV. Liddle syndrome is caused by activating mutations in either the β or γ subunit of the epithelial Na+ channel (ENaC). These mutations increase the number of Na+ channels in the apical cell membrane of principal cells and thereby the amount of Na+ reabsorbed. In Liddle syndrome, the rate of renal Na+ reabsorption is inappropriately high, which leads to an increase in ECFV and hypertension. There are two different forms of pseudohypoaldosteronism (PHA) (i.e., the kidneys reabsorb NaCl as they do when aldosterone levels are low; however, in PHA, aldosterone levels are elevated). The autosomal recessive form is caused by inactivating mutations in the α, β, or γ subunit of ENaC. The cause of the autosomal dominant form is an inactivating mutation in the mineralocorticoid receptor. Pseudohypoaldosteronism is characterized by an increase in Na+ excretion, a reduction in ECFV, hyperkalemia, and hypotension. Some individuals with expanded ECFV and elevated blood pressure are treated with drugs that inhibit angiotensin-converting enzyme (ACE) (e.g., captopril, enalapril, lisinopril) and thereby lower fluid volume and blood pressure. Inhibition of ACE blocks degradation of angiotensin I to angiotensin II and thereby lowers plasma angiotensin II levels. The decline in plasma angiotensin II concentration has three effects. First, NaCl and water reabsorption by the nephron (especially the proximal tubule) falls. Second, aldosterone secretion decreases, thus reducing NaCl reabsorption in the thick ascending limb, distal tubule, and collecting duct. Third, because angiotensin is a potent vasoconstrictor, a reduction in its concentration permits the systemic arterioles to dilate and thereby lower arterial blood pressure. ACE also degrades the vasodilator hormone bradykinin; thus ACE inhibitors increase the concentration of bradykinin, a vasodilatory hormone. ACE inhibitors decrease ECFV and the arterial blood pressure by promoting renal NaCl and water excretion and by reducing total peripheral resistance.

thereby indirectly stimulate excretion of NaCl and water (see earlier discussion about ANP and BNP).

Arginine vasopressin (AVP) regulates water reabsorption. It is the most important hormone that regulates reabsorption of water in the kidneys (see ). This hormone is secreted by the posterior pituitary gland in response to an increase in plasma osmolality (1% or more) or a decrease in ECFV (>5%–10% of normal). AVP increases the permeability of the collecting duct to water. It increases reabsorption of water by the collecting duct because of the osmotic gradient that exists across the wall of the collecting duct (see

Chapter 35). AVP has little effect on urinary NaCl excretion.

Starling forces regulate reabsorption of NaCl and water across the proximal tubule. As previously described, Na+ , Cl−, HCO3 −, amino acids, glucose, and water are transported into the intercellular space of the proximal tubule. Starling forces between this space and the peritubular capillaries facilitate movement of the reabsorbed fluid into the capillaries. Starling forces across the wall of peritubular capillaries •Fig. 34.11 Starling forces modify proximal tubule solute and water reabsorption. (1) Solute and water are reabsorbed across the apical membrane. This solute and water then cross the lateral cell membrane. Some solute and water reenters the tubule fluid (3), and the remainder enters the interstitial space and then flows into the capillary (2). The width of the arrows is directly proportional to the amount of solute and water moving by pathways 1 to 3. Starling forces across the capillary wall determine the amount of fluid flowing through pathway 2 versus pathway 3. Transport mechanisms in the apical cell membranes determine the amount of solute and water entering the cell (pathway 1). Pi, interstitial hydrostatic pressure; Ppc, peritubular capillary hydrostatic pressure; πi, interstitial fluid oncotic pressure; πpc, peritubular capillary oncotic pressure. Thin arrows across the capillary wall indicate the direction of water movement in response to each force.

consist of hydrostatic pressure in the peritubular capillary (Ppc) and lateral intercellular space (Pi) and oncotic pressure in the peritubular capillary (πpc) and lateral intercellular space (πi). Thus reabsorption of water as a result of transport of Na+ from tubular fluid into the lateral intercellular space is modified by the Starling forces. Accordingly:

Equation 34.2 where J is flow (positive numbers indicate flow from the intercellular space into blood). Starling forces that favor movement from the interstitium into the peritubular capillaries are πpc and Pi (Fig. 34.11 ). The opposing Starling forces are πi and Ppc. Normally the sum of the Starling forces favors movement of solute and water from the interstitial space into the capillary. However, some of the solutes and fluid that enter the lateral intercellular space leak back into the proximal tubular fluid. Starling forces do not affect transport by the loop of Henle, distal tubule, and collecting duct because these segments are less permeable to water than the proximal tubule is.

A number of factors can alter the Starling forces across the peritubular capillaries surrounding the proximal tubule. For example, dilation of the efferent arteriole increases Ppc, whereas constriction of the efferent arteriole decreases it.

An increase in Ppc inhibits solute and water reabsorption by increasing back-leak of NaCl and water across the tight junction, whereas a decrease stimulates reabsorption by decreasing back-leak across the tight junction.

Peritubular capillary oncotic pressure (πpc) is partially determined by the rate of formation of the glomerular ultrafiltrate. For example, if one assumes a constant plasma flow in the afferent arteriole, the plasma proteins become less concentrated in the plasma that enters the efferent arteriole and peritubular capillary as less ultrafiltrate is formed (i.e., as GFR decreases). Hence, πpc decreases. Thus πpc is directly related to the filtration fraction (FF = GFR/renal plasma flow [RPF]). A fall in the FF resulting from a decrease in GFR, at constant RPF, decreases πpc. This in turn increases the backflow of NaCl and water from the lateral intercellular space into tubular fluid and thereby decreases net reabsorption of solute and water across the proximal tubule. An increase in FF has the opposite effect.

The importance of Starling forces in regulating solute and water reabsorption by the proximal tubule is underscored by the phenomenon of glomerulotubular (G-T) balance. Spontaneous changes in GFR markedly alter the filtered amount of Na+ (filtered Na+= GFR × [Na+] in the filtered fluid). Without rapid adjustments in Na+ reabsorption to counter the changes in filtration of Na+ , urinary excretion of Na+ would fluctuate widely and disturb the Na+ balance of the body and thus alter ECFV and blood pressure (see for more details). However, spontaneous changes in GFR do not alter Na+ excretion in urine or Na+ balance when ECFV is normal because of the phenomenon of G-T balance. When body Na+ balance is normal (i.e., ECFV is normal), G-T balance refers to the fact that reabsorption of Na+ and water increases in proportion to the increase in GFR and filtered amount of Na+ . Thus a constant fraction of the filtered Na+ and water is reabsorbed from the proximal tubule despite variations in GFR. The net result of G-T balance is to reduce the impact of changes in GFR on the amount of Na+ and water excreted in urine when ECFV is normal.

Two mechanisms are responsible for G-T balance. One is related to the oncotic and hydrostatic pressure differences between the peritubular capillaries and the lateral intercellular space (i.e., Starling forces). For example, an increase in the GFR (at constant RPF) raises the protein concentration in glomerular capillary plasma above normal. This protein-rich plasma leaves the glomerular capillaries, flows through the efferent arterioles, and enters the peritubular capillaries. The increased πpc augments the movement of 1.

The four major segments of the nephron (proximal tubule, Henle’s loop, distal tubule, and collecting duct) determine the composition and volume of urine by the processes of selective reabsorption of solutes and water and secretion of some solutes.

2.

Tubular reabsorption of substances filtered by the glomerulus allows the kidneys to retain substances that are essential and regulate their levels in plasma by altering the degree to which they are reabsorbed. Reabsorption of Na+ , Cl− , other anions, and organic anions and cations together with water constitutes the major function of the nephron. Approximately 25,200 mEq of Na+ and 179 L of water are reabsorbed each day. Proximal tubule cells reabsorb 67% of the glomerular ultrafiltrate, and cells of Henle’s loop reabsorb about 25% of the NaCl that was filtered and about 15% of the water that was filtered. The distal segments of the nephron (distal tubule and collecting duct system) have a more limited reabsorptive capacity. However, although the proximal tubule reabsorbs

Brown D, et al. New insights into the dynamic regulation of water and acid-base balance by renal epithelial cells. Am J Physiol Cell Physiol. 2012;302:C1421-C1433.

Dantzler WH, et al. Urine-concentrating mechanism in the inner medulla: function of the thin limbs of the loops of Henle. Clin J Am Soc Nephrol. 2014;9:1781-1789.

solute and fluid from the lateral intercellular space into the peritubular capillaries. This action increases net solute and water reabsorption by the proximal tubule.

The second mechanism responsible for G-T balance is initiated by an increase in the filtered amount of glucose and amino acids. As discussed earlier, reabsorption of Na+ in the first half of the proximal tubule is coupled to that of glucose and amino acids. The rate of Na+ reabsorption therefore partially depends on the filtered amount of glucose and amino acids. As the GFR and filtered amount of glucose and amino acids increase, reabsorption of Na+ and water also rises.

In addition to G-T balance, another mechanism minimizes changes in the filtered amount of Na+ . As discussed in , an increase in GFR (and thus in the amount of Na+ filtered by the glomerulus) activates the tubuloglomerular feedback mechanism. This action returns the GFR and filtration of Na+ to normal values. Thus spontaneous changes in GFR (e.g., caused by changes in posture and blood pressure) increase the amount of Na+ filtered for only a few minutes. The mechanisms that underlie G-T balance maintain urinary Na+ excretion constant and thereby maintain Na+ homeostasis (and ECFV and blood pressure) until the GFR returns to normal.

the largest fraction of the filtered solutes and water (i.e., 67%), final adjustments in the composition and volume of urine and most of the regulation by hormones and other factors occur primarily in the distal tubule and collecting duct.

3.

Secretion of substances from the blood into tubular fluid is a means for excreting various byproducts of metabolism, and it also serves to eliminate exogenous organic anions and cations (e.g., drugs) and toxins from the body. Many organic anions and cations are bound to plasma proteins and are therefore unavailable for ultrafiltration. Thus secretion is their major route of excretion in urine.

4.

Various hormones (including angiotensin II, aldosterone, AVP, natriuretic peptides [ANP, BNP, and urodilatin], uroguanylin, and guanylin), sympathetic nerves, dopamine, and Starling forces regulate reabsorption of NaCl by the kidneys. AVP is the major hormone that regulates water reabsorption.

Divers J, Freedman BI. Genetics in kidney disease in 2013: susceptibility genes for renal and urological disorders. Nat Rev Nephrol. 2014;10:69-70.

Hoenig MP, Zeidel ML. Homeostasis, the milieu interieur, and the wisdom of the nephron. Clin J Am Soc Nephrol. 2014;9: 1272-1281.

Knepper MA, et al. Molecular physiology of water balance. N Engl J Med. 2015;372:1349-1358.

Kortenoeven ML, et al. Vasopressin regulation of sodium transport in the distal nephron and collecting duct. Am J Physiol Renal Physiol. 2015;309:F280-F299.

McCormick JA, Ellison DH. Distal convoluted tubule. Compr Physiol. 2015;5:45-98.

Mount DB. Thick ascending limb of the loop of Henle. Clin J Am Soc Nephrol. 2014;9:1974-1986.

Palmer BF. Regulation of potassium homeostasis. Clin J Am Soc Nephrol. 2015;10:1050-1060.

Palmer LG, Schnermann J. Integrated control of Na transport along the nephron. Clin J Am Soc Nephrol. 2015;10:676-687.

Pearce D, et al. Collecting duct principal cell transport processes and their regulation. Clin J Am Soc Nephrol. 2015;10:135-146.

Pelis RM, Wright SH. Renal transport of organic anions and cations. Compr Physiol. 2011;1:1795-1835.

Pluznick JL, Caplan MJ. Chemical and physical sensors in the regulation of renal function. Clin J Am Soc Nephrol. 2015;10:1626-1635.

Roy A, et al. Collecting duct intercalated cell function and regulation. Clin J Am Soc Nephrol. 2015;10:305-324.

Theilig F, Wu Q. ANP-induced signaling cascade and its implications in renal pathophysiology. Am J Physiol Renal Physiol. 2015;308:F1047-F1055.

Weiner ID, et al. Urea and ammonia metabolism and the control of renal nitrogen excretion. Clin J Am Soc Nephrol. 2015;10: 1444-1458.

Brown D, Nielsen S. The cell biology of vasopressin action. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.

Burckhardt G, Koepsell H. Organic anion and cation transporters in renal elimination of drugs. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012.

Christensen EI, et al. Renal filtration, transport, and metabolism of albumin and albuminuria. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012.

Gamba G, Schild L. Sodium chloride transport in the loop of Henle, distal convoluted tubule, and collecting duct. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012.

Giebisch G, Satlin L. Regulation of potassium excretion. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012.

Moe OW, et al. Renal transport of glucose, amino acids, sodium, chloride, and water. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.

Preisig P, et al. Cellular mechanisms of renal tubular acidification. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012.

Sands JM, Layton H. The urine concentrating mechanism and urea transporters. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012.

Upon completion of this chapter the student should be able to answer the following questions: 1.

Why do changes in water balance result in alterations in the [Na+] of the extracellular fluid (ECF)?

2.

How is the secretion of arginine vasopressin (AVP) controlled by changes in the osmolality of body fluids and in blood volume and pressure?

3.

What are the cellular events associated with the action of AVP on the collecting duct, and how do they lead to an increase in the water permeability of this segment of the nephron?

4.

What is the role of Henle’s loop in the production of both dilute and concentrated urine?

5.

What is the composition of the medullary interstitial fluid, and how does it participate in the process of producing concentrated urine?

6.

What are the roles of the vasa recta in the process of diluting and concentrating urine?

7.

How is the diluting and concentrating ability of the kidneys quantitated?

8.

Why do changes in Na+ balance alter the volume of extracellular fluid?

9.

What is the effective circulating volume, how is it influenced by changes in Na+ balance, and how does it influence renal Na+ excretion?

10.

What are the mechanisms by which the body monitors the effective circulating volume?

11.

What are the major signals acting on the kidneys to alter their excretion of Na+?

12.

How do changes in extracellular fluid volume alter Na+ transport in the different segments of the nephron, and how do these changes in transport regulate renal Na+ excretion?

13.

What are the mechanisms involved in formation of edema, and what role do the kidneys play in this process?

he kidneys maintain the osmolality and volume of the body fluids within a narrow range by regulating excretion of water and NaCl, respectively. This chapter discusses the regulation of renal water excretion (urine concentration and dilution) and NaCl excretion. The composition and volumes of the various body fluid compartments are reviewed in

Control of Body Fluid Osmolality: Urine Concentration and Dilution , water constitutes approximately 60% of the healthy adult human body. Body water is divided into two major compartments—intracellular fluid (ICF) and extracellular fluid (ECF)—that are in osmotic equilibrium because of the high permeability of most cell membranes to water via aquaporins (e.g., AQP1). Water intake into the body generally occurs orally. This may be water contained in beverages as well as water generated during metabolism of ingested foods (e.g., carbohydrates). In many clinical situations, intravenous infusion is an important route of water entry.

The kidneys are responsible for regulating water balance and under most conditions are the major route for elimination of water from the body (

Table 35.1 ). Other routes of water loss from the body include evaporation from cells of the skin and respiratory passages. Collectively, water loss by these routes is termed insensible water loss because the individual is unaware of its occurrence. Production of sweat accounts for the loss of additional water. Water loss by this mechanism can increase dramatically in a hot environment, with exercise, or in the presence of fever ( 35.2 ). Finally, water can be lost from the gastrointestinal tract. Fecal water loss is normally small (≈100 mL/day) but can increase dramatically with diarrhea (e.g., 20 L/ day with cholera). Vomiting can also cause gastrointestinal water losses.

Although water loss from sweating, defecation, and evaporation from the lungs and skin can vary depending on the environmental conditions or during pathological conditions, loss of water by these routes cannot be regulated. In contrast, renal excretion of water is tightly regulated to maintain whole-body water balance. Maintenance of water balance requires that water intake and loss from the body be precisely matched. If intake exceeds losses, positive water balance exists. Conversely, when intake is less than losses, for review of (with its anions Cl− and HCO3 −), these disorders also result steady-state balance). in alterations in plasma or serum [Na+] (

Fig. 35.2 ). One

When water intake is low or water losses increase, the kidneys conserve water by producing a small volume of urine that is hyperosmotic with respect to plasma. When water intake is high, a large volume of hypoosmotic urine is produced. In a normal individual, urine osmolality (Uosm) can vary from approximately 50 to 1200 mOsm/kg H2O, and the corresponding urine volume can vary from approximately 18 L/day to 0.5 L/day. Importantly the kidneys can regulate excretion of water separately from excretion of total solute (

Fig. 35.1 ). The ability to regulate water excretion separate from excretion of solutes (e.g., Na+ , K+ , urea, etc.) is necessary for survival because it allows water balance to be achieved without upsetting the other homeostatic functions of the kidneys.

It is important to recognize that disorders of water balance are manifested by alterations in body fluid osmolality, which are usually measured by changes in plasma osmolality (POSM). Because the major determinant of plasma osmolality is Na+ aFluid intake varies widely for both social and cultural reasons.

of the most common fluid and electrolyte disorders seen in clinical practice is an alteration in serum [Na+]. When an abnormal serum [Na+] is found in an individual, it is tempting to suspect a problem in Na+ balance. However, the problem most often relates to water balance, not Na+ balance. As described later, changes in Na+ balance result in alterations in the volume of ECF, not its osmolality.

The following sections discuss the mechanisms by which the kidneys excrete either hypoosmotic (dilute) or hyperosmotic (concentrated) urine. Control of arginine vasopressin secretion and its important role in regulating excretion of water by the kidneys are also explained (see also • Fig. 35.1 Relationships between plasma AVP levels, and urine osmolality, urine flow rate, and total solute excretion. Max, maximum; Min, minimum. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) aIn hot weather and during prolonged heavy exercise, water balance is maintained by increased water ingestion. Decreased excretion of water by the kidneys alone is insufficient to maintain water balance.

Kidneys excrete 1 L of water Kidneys excrete hyperosmotic in hypoosmotic urine, urine as the individual drinks returning volume to 14 L and water, returning volume to 14 L restoring [Na˜] and osmolality and restoring [Na˜] and osmolality to normal. to normal.

• Fig. 35.2 Response to changes in water balance. Illustrated are the effects of adding or removing 1 L of water from the ECF of a 70-kg individual. Positive Water Balance: (1) Addition of 1 L of water increases the ECFV and reduces its osmolality. The [Na+] is also decreased (hyponatremia). (2) The normal renal response is to excrete 1 L of water as hypoosmotic urine. (3) As a result of the renal excretion of water, the ECFV, osmolality, and [Na+] are returned to normal. Negative Water Balance: (4) The loss of 1 L of water from the ECF decreases its volume and increases its osmolality. The [Na+] is also increased (hypernatremia). (5) The renal response is to conserve water by excreting a small volume of hyperosmotic urine. (6) With ingestion of water, stimulated by thirst, and conservation of water by the kidneys, the ECFV, osmolality, and [Na+] are returned to normal. Size of the boxes indicates relative ECFV. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

In the clinical setting, hypoosmolality (a reduction in plasma osmolality) shifts water into cells, and this process results in cell swelling (see ). Symptoms associated with hypoosmolality are related primarily to swelling of brain cells. For example, a rapid fall in Posm can alter neurological function and thereby cause nausea, malaise, headache, confusion, lethargy, seizures, and coma. When Posm is increased (i.e., hyperosmolality), water is lost from cells. Symptoms of an increase in Posm are also primarily neurological and include lethargy, weakness, seizures, coma, and even death.

Symptoms associated with changes in body fluid osmolality vary depending on how quickly osmolality is changed. Rapid changes in osmolality (i.e., over hours) are less well tolerated than changes that occur more gradually (i.e., over days to weeks). Indeed, individuals who have developed alterations in their body fluid osmolality over an extended period of time may be entirely asymptomatic. This reflects the ability of cells over time to either eliminate intracellular osmoles, as occurs with hypoosmolality, or to generate new intracellular osmoles in response to hyperosmolality and thus minimize changes in cell volume of the neurons (see several nephron segments express a different AVP receptor (V2) that mediates the kidneys’ ability to regulate the volume and osmolality of urine. When plasma AVP levels are low, a large volume of urine is excreted (diuresis), and the urine osmolality is less than that of plasma (i.e., dilute).

When plasma levels of AVP are high, a small volume of urine is excreted (antidiuresis), and the urine osmolality is greater than that of plasma (i.e., concentrated).

AVP is a small peptide that is 9 amino acids in length (arginine is found at position 8). It is synthesized in neuroendocrine cells located within the supraoptic and paraventricular nuclei of the hypothalamus.

The synthesized hormone is packaged in granules that are transported down the axon of the cell and stored in nerve terminals located in the neurohypophysis (posterior pituitary). The anatomy of the hypothalamus and pituitary gland are shown in

Fig.

35.3

Secretion of AVP by the posterior pituitary can be influenced by several factors. The primary physiological regulators of AVP secretion are (1) the osmolality of body fluids (osmotic) and (2) the volume and pressure of the vascular system (hemodynamic, or nonosmotic). Other factors that can alter AVP secretion include nausea (stimulates), atrial

The human form of vasopressin is arginine vasopressin (AVP), which is also known as antidiuretic hormone (ADH). AVP, acting though V1 receptors, causes vascular smooth muscle contraction. As described subsequently, aDiuresis is the term used for excretion of a large volume of urine. This may reflect either excretion of a large volume of water (water diuresis), or excretion of a large amount of solute (solute diuresis). bNeurons within the supraoptic and paraventricular nuclei synthesize either AVP or the related peptide oxytocin. AVP-secreting cells predominate in the supraoptic nucleus, whereas oxytocin-secreting neurons are primarily found in the paraventricular nucleus.

626 SECTION7Berne & Levy Physiology • Fig. 35.3 Anatomy of the hypothalamus and pituitary gland (midsagittal section) depicting the pathways for AVP section. Also shown are pathways involved in regulating AVP secretion. Afferent fibers from the baroreceptors are carried in the vagus and glossopharyngeal nerves. The inset box illustrates an expanded view of the hypothalamus and pituitary gland. Vagus and glossopharyngeal nerves Vasomotor center (medulla oblongata) Cerebellum Baroreceptor input Pituitary Posterior lobe AVP Paraventricular neuronsOsmoreceptors Supraoptic neurons Optic chiasm Anterior lobe natriuretic peptide (inhibits), and angiotensin II (stimulates). A number of drugs, prescription and nonprescription, also affect AVP secretion. For example, nicotine stimulates secretion, whereas ethanol inhibits secretion.

Osmotic Control of AVP Secretion

Changes in the osmolality of body fluids (changes as minor as 1% are sufficient) play the most important role in regulating AVP secretion. The receptors that monitor changes in osmolality of body fluids (termed osmoreceptors) are distinct from the cells that synthesize and secrete AVP, and are located in the organum vasculosum of the lamina terminalis (OVLT) of the hypothalamus.

The osmoreceptors sense changes in body fluid osmolality by either shrinking or swelling. Recent studies have provided evidence that transient receptor potential vanilloid (TRVP) cation channels are involved in the response of the cells to changes in body fluid osmolality. The osmoreceptors respond only to solutes in plasma that are effective osmoles (see ). For example, urea is an ineffective osmole when the function of osmoreceptors is considered. Thus elevation of the plasma urea concentration alone has little effect on AVP secretion.

When the effective osmolality of the plasma increases, the osmoreceptors send signals to the AVP synthesizing/ secreting cells located in the supraoptic and paraventricular nuclei of the hypothalamus, and AVP synthesis and secretion are stimulated. Conversely, when the effective cAngiotensin II also stimulates AVP secretion; the cells that mediate this response are located in the subfornical organ (SFO).

The gene for AVP is found on chromosome 20. It contains approximately 2000 base pairs with three exons and two introns. The gene codes for a preprohormone that consists of a signal polypeptide, the AVP molecule, neurophysin, and a glycopeptide (copeptin). As the cell processes the preprohormone the signal peptide is cleaved off in the rough endoplasmic reticulum. Once packaged in neurosecretory granules, the preprohormone is further cleaved into AVP, neurophysin, and copeptin molecules. The neurosecretory granules are then transported down the axon to the posterior pituitary and stored in the nerve endings until released. When the neurons are stimulated to secrete AVP, the action potential opens Ca++ channels in the nerve terminal, which raises the intracellular [Ca++] and causes exocytosis of the neurosecretory granules. All three peptides are secreted in this process. Neurophysin and copeptin do not have an identified physiological function.

osmolality of the plasma is reduced, secretion is inhibited. Because AVP is rapidly degraded in the plasma, circulating levels can be reduced to zero within minutes after secretion is inhibited. As a result the AVP system can respond rapidly to fluctuations in body fluid osmolality.

Fig. 35.4A illustrates the effect of changes in plasma osmolality on circulating AVP levels. The slope of the relationship is quite steep and accounts for the sensitivity of this system. The set point of the system is the plasma osmolality value at which AVP secretion begins to increase. Below this set point, virtually no AVP is released. The set point varies

CHAPTER 35 Control of Body Fluid Osmolality and Volume among individuals and is genetically determined. In healthy adults it varies from 275 to 290 mOsm/kg H2O (average ≈ 280–285 mOsm/kg H2O). Several physiological factors can also change the set point in a given individual. As discussed later, alterations in blood volume and pressure can shift it. In addition, pregnancy is associated with a decrease in the set point. The mechanism responsible for the set-point shift with pregnancy is not completely understood but is likely due to hormone levels (e.g., relaxin and chorionic gonadotropin) that are elevated during pregnancy.

Hemodynamic (Nonosmotic) Control of AVP Secretion

A decrease in blood volume or pressure also stimulates AVP secretion. The receptors responsible for this response are located in both the low-pressure (left atrium and large pulmonary vessels) and the high-pressure (aortic arch and carotid sinus) sides of the circulatory system. Because the low-pressure receptors are located in the high-compliance side of the circulatory system (i.e., venous), and because the majority of blood is in the venous side of the circulatory system, these low-pressure receptors can be viewed as responding to overall vascular volume. The high-pressure receptors respond to arterial pressure. Both groups of receptors are sensitive to stretch of the wall of the structure in which they are located (e.g., cardiac atrial and aortic arch) and are termed baroreceptors. Signals from these receptors are carried in afferent fibers of the vagus and glossopharyngeal nerves to the brainstem (solitary tract nucleus of the medulla oblongata), which is part of the center that ). Signals are then relayed from the brainstem to the

AVP secretory cells of the supraoptic and paraventricular hypothalamic nuclei. The sensitivity of the baroreceptor system is less than that of the osmoreceptors, and a 5% to 10% decrease in blood volume or pressure is required before AVP secretion is stimulated. This is illustrated in

Fig.

35.4B . A number of substances have been shown to alter the secretion of AVP through their effects on blood pressure. These include bradykinin and histamine, which lower pressure and thus stimulate AVP secretion, and norepinephrine, which increases blood pressure and inhibits AVP secretion.

Alterations in blood volume and pressure also affect the response to changes in body fluid osmolality (see

Fig.

35.4C ). With a decrease in blood volume or pressure, the set point is shifted to lower osmolality values and the slope of the relationship is steeper. In terms of survival of the individual this means that faced with circulatory collapse, the kidneys will continue to conserve water, even though by doing so they reduce the osmolality of the body fluids. With an increase in blood volume or pressure, the opposite occurs. The set point is shifted to higher osmolality values and the slope is decreased.

AVP Actions on the Kidneys

The primary action of AVP on the kidneys is to enhance absorption of water from the tubular fluid by increasing • Fig. 35.4 Osmotic and hemodynamic (nonosmotic) control of AVP secretion. A, Effect of changes in plasma osmolality (constant blood volume and pressure) on plasma AVP levels. B, Effect of changes in blood volume or pressure (constant plasma osmolality) on plasma AVP levels. C, Interactions between osmolar and blood volume and pressure stimuli on plasma AVP levels.

Inadequate release of AVP from the posterior pituitary results in excretion of a large volume of dilute urine (polyuria). To compensate for this loss of water the individual must ingest a large volume of water (polydipsia) to maintain constant body fluid osmolality. If the individual is deprived of water, body fluids will become hyperosmotic. This condition is called central diabetes insipidus or pituitary diabetes insipidus. Central diabetes insipidus can be inherited, although this is rare. It occurs more commonly after head trauma and with brain neoplasms or infections. Individuals with central diabetes insipidus have a urine-concentrating defect that can be corrected by administration of exogenous AVP. The inherited (autosomal dominant) form of central diabetes insipidus is due to numerous mutations in all regions of the AVP gene (i.e., AVP, copeptin and neurophysin). The human placenta produces a cysteine aminopeptidase that degrades AVP. In some women the levels of this vasopressinase result in diabetes insipidus. The associated polyuria can be treated by administration of the synthetic AVP analogue desmopressin (DDAVP).

The syndrome of inappropriate AVP (ADH) secretion (SIADH) is a common clinical problem characterized by plasma AVP levels that are elevated above what would be expected on the basis of body fluid osmolality and blood volume and pressure—hence the term inappropriate AVP (ADH) secretion (this is alternatively named syndrome of inappropriate antidiuresis [SIAD]). In addition the collecting duct overexpresses water channels (see below), thus augmenting the effect of AVP to stimulate water retention by the kidney. Individuals with SIADH retain water, and their body fluids become progressively hypoosmotic. In addition, their urine is more hyperosmotic than expected based on the low body-fluid osmolality. SIADH can be caused by infections and neoplasms of the brain, drugs (e.g., antitumor drugs), pulmonary diseases, and carcinoma of the lung. Many of these conditions stimulate AVP secretion by altering neural input to the AVP secretory cells. By contrast, small cell carcinoma of the lung produces and secretes a number of peptides including AVP. Recently, AVP receptor antagonists (e.g., the nonpeptide antagonists conivaptan [Vaprisol] and tolvaptan [Samsca and Jinarc]) have been developed that can be used to treat SIADH and other conditions in which AVP-dependent water retention by the kidneys occurs (e.g., congestive heart failure and hepatic cirrhosis).

the water permeability of the latter portion of the distal tubule and collecting duct. In addition, and importantly, AVP increases the permeability of the medullary portion of the collecting duct to urea. Finally, AVP stimulates NaCl reabsorption by the thick ascending limb of Henle’s loop, distal tubule, and collecting duct.

In the absence of AVP, the apical membrane of principal cells (see ), located in the latter portion of the distal tubule and along the collecting duct, is relatively impermeable to water. This reflects the fact that in the absence of AVP the apical membrane of these cells contains few water channels (aquaporins). Thus in the absence of AVP, little water is reabsorbed by these nephron segments. Binding of AVP to the V2 receptor located in the basolateral membrane of principal cells results in the insertion of aquaporin (AQP2) water channels into the apical membrane, allowing water to enter the cell from the tubule lumen. This water then exits the cell across the basolateral membrane, which is always freely permeable to water owing to the presence of AQP3 and AQP4 water channels. Thus in the presence of AVP, water is reabsorbed from the tubule lumen.

The gene for the V2 receptor is located on the X chromosome. It codes for a 371–amino acid protein that is in the family of receptors that have seven membrane spanning domains and are coupled to heterotrimeric G proteins. As shown in

Fig. 35.5 , binding of AVP to its receptor on the basolateral membrane activates adenylcyclase. The increase in intracellular cyclic adenosine monophosphate (cAMP) then activates protein kinase A (PKA), which results in phosphorylation of AQP2 water channels and also results in increased transcription of the AQP2 gene via activation of a cAMP-response element (CRE). Vesicles containing phosphorylated AQP2 move toward the apical membrane along microtubules driven by the molecular motor dynein. Once near the apical membrane, proteins called SNAREs interact with vesicles containing AQP2 and facilitate fusion of these vesicles with the membrane. Addition of AQP2 to the membrane allows water to enter the cell driven by the osmotic gradient (lumen osmolality < cell osmolality). The water then exits the cell across the basolateral membrane through AQP3 and AQP4 water channels, which are constitutively present in the basolateral membrane. When the V2 receptor is not occupied by AVP, the AQP2 water channels are removed from the apical membrane by clathrinmediated endocytosis, thus rendering the apical membrane impermeable to water. The endocytosed AQP2 molecules may be either stored in cytoplasmic vesicles, ready for reinsertion into the apical membrane when AVP levels in the plasma increase, or degraded.

AVP also regulates long-term expression of AQP2 (and AQP3). When large volumes of water are ingested over an extended period of time (e.g., psychogenic polydipsia), the abundance of AQP2 and AQP3 in principal cells is reduced. As a consequence, when water ingestion is restricted, these individuals cannot maximally concentrate their urine. Conversely, in states of restricted water ingestion, AQP2 and AQP3 protein expression in principal cells increases, thereby facilitating excretion of maximally concentrated urine.

It is also clear that expression of AQP2 (and in some instances also AQP3) varies in pathological conditions associated with disturbances in urine concentration and dilution. AQP2 expression is reduced in a number of conditions associated with impaired urine concentrating ability (e.g., hypercalcemia, hypokalemia). By contrast, in conditions associated with water retention (e.g., congestive heart failure, hepatic cirrhosis, pregnancy) AQP2 expression is increased.

CHAPTER 35 Control of Body Fluid Osmolality and Volume 629 • Fig. 35.5 Action of AVP via the V2 receptor on the principal cell of the late distal tubule and collecting duct. See text for details. AC, adenylcyclase; cAMP, cyclic adenosine monophosphate; CREB-P, phosphorylated cAMP response element binding protein; CRE, cAMP response element; P, phosphorylated proteins; AQP2, aquaporin 2; AP2, aquaporin 2 gene. (Adapted from Brown D, Nielsen S. The cell biology of vasopressin action. In: Brenner BM [ed]. The Kidney. 7th ed. Philadelphia: Saunders; 2004.)

AVP also increases the permeability of the terminal portion of the inner medullary collecting duct to urea. This results in an increase in urea reabsorption and an increase in the osmolality of the medullary interstitial fluid, which as described below is needed for maximal urine concentration. The cells of the collecting duct express two types of urea transporters, UT-A1 and UT-A3. UT-A1 is localized to the apical membrane, and UT-A3 is localized primarily to the basolateral membrane. AVP, acting through the cAMP/PKA cascade, increases expression of both UT-A1 and UT-A3. Increasing the osmolality of the interstitial fluid of the renal medulla also increases the permeability of the inner medullary collecting duct to urea. This effect is mediated by the phospholipase C/protein kinase C (PKC) pathway, which increases UT-A1 and UT-A3 expression.

AVP also stimulates reabsorption of NaCl by the thick ascending limb of Henle’s loop and by the distal tubule and cortical segment of the collecting duct. This increase in Na+ reabsorption is associated with increased abundance of three Na+ transporters: the 1Na+/1K+/2Cl− symporter (thick ascending limb of Henle’s loop), the Na+/Cl− symporter (distal tubule), and the Na+ channel (ENaC, in the latter portion of the distal tubule and collecting duct). Stimulation of thick ascending limb NaCl transport may help maintain the hyperosmotic medullary interstitium that is necessary for absorption of water from the medullary portion of the collecting duct (see below).

In addition to affecting the secretion of AVP, changes in plasma osmolality and blood volume or pressure lead to alterations in the perception of thirst. When body fluid osmolality is increased or the blood volume or pressure is reduced, the individual perceives thirst. Of these stimuli, hypertonicity is the more potent. An increase in plasma osmolality of only 2% to 3% produces a strong desire to drink, whereas decreases in blood volume and pressure in the range of 10% to 15% are required to produce the same response.

As already discussed, there is a genetically determined threshold for AVP secretion (i.e., a body fluid osmolality above which AVP secretion increases). Similarly there is a genetically determined threshold for triggering the sensation of thirst. However, the thirst threshold is higher than the threshold for AVP secretion. On average the threshold for AVP secretion is approximately 285 mOsm/kg H2O, whereas the thirst threshold is approximately 295 mOsm/ kg H2O. Because of this difference, thirst is stimulated at a body fluid osmolality where AVP secretion is already maximal.

The neural centers involved in regulating water intake (the thirst center) are located in the same region of the hypothalamus involved with regulating AVP secretion. However, it is not certain whether the same cells serve both functions. Indeed the thirst response, like the regulation of

The collecting ducts of some individuals do not respond normally to AVP. These individuals cannot maximally concentrate their urine and consequently have polyuria and polydipsia. This clinical entity is termed nephrogenic diabetes insipidus to distinguish it from central diabetes insipidus. Nephrogenic diabetes insipidus can result from a number of systemic disorders and more rarely occurs as a result of inherited disorders. Many of the acquired forms of nephrogenic diabetes insipidus are the result of decreased expression of AQP2 in the collecting duct. Decreased expression of AQP2 has been documented in the urine concentrating defects associated with hypokalemia, lithium ingestion (35% of individuals who take lithium for bipolar disorder develop some degree of nephrogenic diabetes insipidus), ureteral obstruction, low-protein diet, and hypercalcemia. The inherited forms of nephrogenic diabetes insipidus reflect mutations in the AVP receptor (V2 receptor) gene or the AQP2 gene. Approximately 90% of hereditary forms of nephrogenic diabetes insipidus are the result of mutations in the V2 receptor gene, with the other 10% the result of mutations in the AQP2 gene. Since the gene for the V2 receptor is located on the X chromosome, these inherited forms of nephrogenic diabetes insipidus are X-linked. The gene coding for AQP2 is located on chromosome 12 and is inherited as both an autosomal recessive as well as an autosomal dominant defect. As noted in , aquaporins exist as homotetramers. This homotetramer formation explains the difference between the two forms of AQP2-related nephrogenic diabetes insipidus. In the recessive form, heterozygotes produce both normal AQP2 and defective AQP2 molecules. The defective AQP2 monomer is retained in the endoplasmic reticulum of the cell, and thus the homotetramers that do form only contain normal molecules. Thus mutations in both alleles are required to produce nephrogenic diabetes insipidus. In the autosomal dominant form, the defective monomers can form tetramers with normal monomers as well as defective monomers. However, tetramers containing defective monomers are unable to traffic to the apical membrane.

Recently, individuals have been found that have activating (gain-of-function) mutations in the V2 receptor gene. Thus the receptor is constitutively activated even in the absence of AVP. These individuals have laboratory findings similar to those seen in SIADH, including reduced plasma osmolality, hyponatremia (reduced plasma [Na+]), and urine more concentrated than would be expected from the reduced body fluid osmolality. However, unlike SIADH where circulating levels of AVP are elevated and thus responsible for water retention by the kidneys, these individuals have undetectable levels of AVP in their plasma. This new clinical entity has been termed nephrogenic syndrome of inappropriate antidiuresis.

AVP secretion, only occurs in response to effective osmoles (e.g., NaCl). Even less is known about the pathways involved in the thirst response to decreased blood volume or pressure, but it is believed that the pathways are the same as those involved in the volume and pressure-related regulation of AVP secretion. Angiotensin II acting on cells of the thirst center also evokes the sensation of thirst. Because angiotensin II levels are increased when blood volume and pressure are reduced, this effect of angiotensin II contributes to the homeostatic response that restores and maintains body fluids at their normal volume.

The sensation of thirst is satisfied by the act of drinking, even before sufficient water is absorbed from the gastrointestinal tract to correct the plasma osmolality. It is interesting to note that cold water is more effective in reducing the thirst sensation. Oropharyngeal and upper gastrointestinal receptors appear to be involved in this response. However, relief of the thirst sensation via these receptors is short lived, and thirst is only completely satisfied when the plasma osmolality or blood volume or pressure is corrected.

It should be apparent that the AVP and thirst systems work in concert to maintain water balance. An increase in plasma osmolality evokes drinking and, via AVP action on the kidneys, conservation of water. Conversely, when plasma osmolality is decreased, thirst is suppressed and, in the absence of AVP, renal water excretion is enhanced. However, most of the time fluid intake is dictated by cultural factors and social situations. This is especially the case when thirst is not stimulated. In this situation, maintaining normal body fluid osmolality relies solely on the ability of the kidneys to excrete water. How the kidney accomplishes this is discussed in detail in the following sections of this chapter.

Renal Mechanisms for Dilution and Concentration of Urine

As already noted, water excretion is regulated separately from solute excretion. For this to occur the kidneys must be able to excrete urine that is either hypoosmotic or hyper-osmotic with respect to body fluids. This ability to excrete urine of varying osmolality in turn requires that solute be separated from water at some point along the nephron. As discussed in , reabsorption of solute in the proximal tubule results in reabsorption of a proportional amount of water. Hence solute and water are not separated in this portion of the nephron. Moreover, this proportionality between proximal tubule water and solute reabsorption occurs regardless of whether the kidneys excrete dilute or concentrated urine. Thus the proximal tubule reabsorbs a large portion of the filtered amount of solute and water, but it does not produce dilute or concentrated tubular fluid. The loop of Henle, in particular the thick ascending limb, is the major site where solute and water are separated. Thus excretion of both dilute and concentrated urine requires normal function of the loop of Henle.

Excretion of hypoosmotic urine is relatively easy to understand. The nephron must simply reabsorb solute from the tubular fluid and not allow water reabsorption to also occur. As just noted, and as described in greater detail later, reabsorption of solute without concomitant water reabsorption occurs in the ascending limb of Henle’s loop. Under appropriate conditions (i.e., in the absence of AVP) the distal tubule and collecting duct also dilute the tubular fluid by reabsorbing solute but not water.

Excretion of hyperosmotic urine is more complex and thus more difficult to understand. This process in essence

With adequate access to water, the thirst mechanism can prevent development of hyperosmolality. This mechanism is responsible for the polydipsia seen in response to the polyuria of both central and nephrogenic diabetes insipidus. Most individuals ingest water/beverages even in the absence of the thirst sensation. Normally the kidneys are able to excrete this excess water because they can excrete up to 18 L/day of urine. However, in some instances, the volume of water ingested exceeds the kidneys’ capacity to excrete water, especially over short periods of time. When this occurs, body fluids become hypoosmotic.

An example of how water intake can exceed the capacity of the kidneys to excrete water is long-distance running. A study of participants in the Boston Marathon found that 13% of the runners developed hyponatremia during the course of the race.

This reflected the practice of some runners of ingesting water or other hypotonic drinks during the race to remain “well hydrated.” In addition, water is produced from the metabolism of glycogen and triglycerides used as fuels by the exercising muscle. Because over the course of the race they ingested (and generated through metabolism) more water than their kidneys were able to excrete or was lost by sweating, hyponatremia developed. In some racers the hyponatremia was severe enough to elicit the neurological symptoms described previously.

The maximum amount of water that can be excreted by the kidneys (e.g., 18 L/day) depends on the amount of solute excreted, which in turn depends on food intake. For example, with maximally dilute urine (Uosm = 50 mOsm/kg H2O), the maximum urine output of 18 L/day will be achieved only if the solute excretion rate is 900 mmol/day:

If solute excretion is reduced, as commonly occurs in the elderly with reduced food intake, the maximum urine output will decrease. For example, if solute excretion is only 400 mmol/day, a maximum urine output (at Uosm = 50 mOsm/kg H2O) of only 8 L/day can be achieved. Thus individuals with reduced food intake have a reduced capacity to excrete water.

dAlmond CS et al. Hyponatremia among runners in the Boston Marathon. N Engl J Med 2005;352:1150-1556.

involves removing water from the tubular fluid without solute. Because water movement is passive, driven by an osmotic gradient, the kidney must generate a hyperosmotic compartment into which water is reabsorbed, without solute, osmotically from the tubular fluid. The hyperosmotic compartment in the kidney that serves this function is the interstitium of the renal medulla. Henle’s loop is critical for generating the hyperosmotic medullary interstitium. Once established, this hyperosmotic compartment drives water reabsorption from the collecting duct and thereby concentrates urine.

Fig. 35.6 summarizes tubular fluid osmolality at several points along the nephron, both in the absence and presence

CHAPTER 35 Control of Body Fluid Osmolality and Volume of AVP. Note that tubular fluid entering the loop of Henle from the proximal tubule is isoosmotic with respect to plasma and is so regardless of the absence or presence of AVP. Also, tubular fluid leaving the thick ascending limb is hypoosmotic with respect to plasma, both in the absence and presence of AVP. The osmolality of tubular fluid along the collecting duct is hypoosmotic with respect to plasma in the absence of AVP and becomes progressively hyperosmotic (i.e., from cortex to inner medulla) in the presence of AVP.

Establishment and maintenance of the hyperosmotic medullary interstitium has been a subject of study for more than 50 years. Despite this intense study, the most accepted model for how the medullary osmotic gradient is established, especially within the inner medulla, is incomplete and not consistent with more recent experimental findings regarding the transport properties of the nephron segments in this region of the kidney. With the caveat that the current model needs refinement, it is presented here because it embodies some fundamental concepts that underlie the process.

In the current model the medullary interstitial osmotic gradient is established by a process termed countercurrent multiplication. By this process, solute (principally NaCl) is reabsorbed without water from the ascending limb of Henle’s loop into the surrounding medullary interstitium. This decreases the osmolality in the tubular fluid and raises the osmolality of the interstitium at this point. The increased osmolality of the interstitium then causes water to be reabsorbed from the descending limb of Henle’s loop, thus increasing the tubular fluid osmolality in this segment. Thus at any point along the loop of Henle the fluid in the ascending limb has an osmolality less than fluid in the adjacent descending limb. This osmotic difference was termed the single effect. Because of the countercurrent flow of tubular fluid in the descending (fluid flowing into the medulla) and ascending (fluid flow out of the medulla) limbs, this single effect could be multiplied, resulting in an osmotic gradient within the medullary interstitium, where the tip of the papilla has an osmolality of 1200 mOsm/kg H2O compared to 300 mOsm/kg H2O at the corticomedullary junction.

Fig. 35.7 schematically depicts the processes for excreting a dilute urine (water diuresis) as well as a concentrated urine (antidiuresis). Three key concepts underlie these processes: 1.

Urine is concentrated by AVP-dependent reabsorption of water from the collecting duct.

2.

Reabsorption of NaCl from the ascending limb of Henle’s loop dilutes the tubular fluid and at the same time generates a high [NaCl] in the medullary interstitium (up to 600 mmol/L at the tip of the papilla), which then drives water reabsorption from the collecting duct.

3.

Urea accumulates in the medullary interstitium (up to 600 mmol/L), which allows the kidneys to excrete urine with the same high urea concentration. This allows large amounts of urea to be excreted with relatively little water. First, how the kidneys excrete dilute urine (water diure sis) when AVP levels are low or zero is considered. The following numbers refer to those encircled in

Fig. 35.7A + AVP: 140 − AVP: 140 + AVP: 290 Cortex + AVP: 1200 + AVP: 1200 − AVP: 600 − AVP: 50 • Fig. 35.6 Tubular fluid osmolality along the nephron in the presence (+AVP) and in the absence (−AVP) of arginine vasopressin. See text for details. (Adapted from Sands JM et al. Urine concentration and dilution. In: Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.) 1.

Fluid entering the descending thin limb of the loop of Henle from the proximal tubule is isosmotic with respect to plasma. This reflects the essentially isosmotic nature of solute and water reabsorption in the proximal tubule (see ). (NOTE: Water is reabsorbed from the segments of the proximal tubule via AQP1.) 2.

Water is reabsorbed from the thin descending limb of Henle’s loop. Most of this water is reabsorbed in the outer medulla, thereby limiting the amount of water added to the deepest part of the inner medullary interstitial space and thus preserving the hyperosmolality of this region of the medulla. (NOTE: Water is reabsorbed via AQP1.) 3.

In the inner medulla the terminal portion of the descending thin limb and all of the thin ascending limb is impermeable to water. (NOTE: AQP1 is not expressed.) These same nephron segments express the Cl− channel CLC-K1, which mediates Cl− reabsorption, with Na+ following via the paracellular pathway. This passive reabsorption of NaCl without concomitant water reabsorption begins the process of diluting the tubular fluid.

4.

The thick ascending limb of the loop of Henle is also impermeable to water and actively reabsorbs NaCl from the tubular fluid and thereby dilutes it further (see ). Dilution occurs to such a degree that this segment is often referred to as the diluting segment of the kidney. Fluid leaving the thick ascending limb is hypoosmotic with respect to plasma (see

Fig. 35.6 5.

The distal tubule and cortical portion of the collecting duct actively reabsorb NaCl. In the absence of AVP these segments are not permeable to water (i.e., AQP2 is not present in the apical membrane of the cells). Thus when AVP is absent or present at low levels (i.e., decreased plasma osmolality), the osmolality of tubule fluid in these segments is reduced further because NaCl is reabsorbed without water. Under this condition, fluid leaving the cortical portion of the collecting duct is hypoosmotic with respect to plasma (see

Fig. 35.6 6.

The medullary collecting duct actively reabsorbs NaCl. Even in the absence of AVP, this segment is slightly permeable to water and some water is reabsorbed.

7.

The urine has an osmolality as low as approximately 50 mOsm/kg H2O and contains low concentrations of NaCl. The volume of urine excreted can be as much as 18 L/day, or approximately 10% of the glomerular filtration rate (GFR). Next, how the kidneys excrete concentrated urine (antidiuresis) when plasma osmolality and plasma AVP levels are high is considered. The following numbers refer to those encircled in

Fig. 35.7B : 1–4. These steps are similar to those for production of dilute urine. An important point in understanding how a concentrated urine is produced is to recognize that while reabsorption of NaCl by the ascending thin and thick limbs of the loop of Henle dilutes the tubular fluid, the reabsorbed NaCl accumulates in the medullary interstitium and raises the osmolality of this compartment.

• Fig. 35.7 Schematic of nephron segments involved in urine dilution and concentration. Henle’s loops of juxtamedullary nephrons are shown. A, Mechanism for excretion of dilute urine (water diuresis). AVP) is absent and the collecting duct is essentially impermeable to water. Note also that during a water diuresis the osmolality of the medullary interstitium is reduced as a result of increased vasa recta blood flow and entry of some urea into the medullary collecting duct. B, Mechanism for excretion of a concentrated urine (antidiuresis). Plasma AVP levels are maximal and the collecting duct is highly permeable to water. Under this condition the medullary interstitial gradient is maximal. See text for details.

Accumulation of NaCl in the medullary interstitium is crucial for production of urine hyperosmotic to plasma, because it provides the osmotic driving force for water reabsorption by the medullary collecting duct. As already noted, AVP stimulates NaCl reabsorption by the thick ascending limb of Henle’s loop. This is thought to maintain the medullary interstitial gradient at a time when water is being added to this compartment from the medullary collecting duct, which would tend to dissipate the gradient.

5.

Because of NaCl reabsorption by the ascending limb of the loop of Henle, the fluid reaching the collecting duct is hypoosmotic with respect to the surrounding interstitial fluid. Thus an osmotic gradient is established across the collecting duct. In the presence of AVP, which increases the water permeability of the latter portion of the distal tubule and the collecting duct by causing insertion of AQP2 into the luminal membrane of the cells, water diffuses out of the tubule lumen and the tubule fluid osmolality increases. This diffusion of water out of the lumen of the collecting duct begins the process of urine concentration. The maximum osmolality the fluid in the distal tubule and cortical portion of the collecting duct can attain is approximately 290 mOsm/kg H2O (i.e., the same as plasma), which is the osmolality of the interstitial fluid and plasma within the cortex of the kidney.

6.

As the tubular fluid descends deeper into the medulla, water continues to be reabsorbed from the collecting duct, increasing the tubular fluid osmolality to 1200 mOsm/ kg H2O at the tip of the papilla.

7.

The urine produced when AVP levels are elevated has an osmolality of 1200 mOsm/kg H2O and contains high concentrations of urea and other nonreabsorbed solutes. Urine volume under this condition can be as low as 0.5 L/day.

In comparing the two conditions just described, it should be apparent that a relatively constant volume of dilute tubular fluid is delivered to the AVP-sensitive portions of the nephron (latter portion of the distal tubule and collecting duct). Plasma AVP levels then determine the amount of water reabsorbed by these segments. When AVP levels are low, a relatively small volume of water is reabsorbed by these segments and a large volume of hypoosmotic urine is excreted (up to 10% of the filtered water). When AVP levels are high, a large volume of water is reabsorbed by these same segments and a small volume of hyperosmotic urine is excreted (<1% of filtered water). During antidiuresis, most of the water is reabsorbed in the distal tubule and cortical and outer medullary portions of the collecting duct. Thus a relatively small volume of fluid reaches the inner medullary collecting duct where it is then reabsorbed. This distribution of water reabsorption along the length of the collecting duct (i.e., cortex > outer medulla > inner medulla) allows for maintenance of a hyperosmotic interstitial environment in the inner medulla by minimizing the amount of water entering this compartment.

• Fig. 35.8 The medullary interstitial gradient comprises primarily NaCl and urea. The concentrations for NaCl and urea depicted reflect those found in the antidiuretic state (i.e., excretion of hyperosmotic urine). See text for details. (Adapted from Sands JM et al. Urine concentration and dilution. In: Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Elsevier; 2012.) 100200300400500600Concentration(mmol/L)CortexO termed llaInnerMed llaUreaNacl

As noted earlier, the interstitial fluid of the renal medulla is critically important in concentrating urine. The osmotic pressure of the interstitial fluid provides the driving force for reabsorbing water from both the descending thin limb of the loop of Henle and the collecting duct. The principal solutes of the medullary interstitial fluid are NaCl and urea, but the concentration of these solutes is not uniform throughout the medulla (i.e., a gradient exists from cortex to papilla). Other solutes also accumulate in the medullary interstitium (e.g., NH4 + and K+), but the most abundant solutes are NaCl and urea. For simplicity, this discussion assumes that NaCl and urea are the only solutes.

Fig. 35.8 , NaCl and urea accumulate in the renal medulla, and the interstitial fluid at the tip of the papilla of the inner medulla reaches a maximum osmolality of 1200 mOsm/kg H2O, with approximately 600 mOsm/kg H2O attributable to NaCl (300 mmol/L) and 600 mOsm/ kg H2O attributable to urea (600 mmol/L). Establishment of the NaCl gradient is essentially complete at the transition between the outer and inner medulla.

The medullary gradient for NaCl results from accumulation of NaCl reabsorbed by the nephron segments in the medulla during countercurrent multiplication. The most important segment in this regard is the ascending limb of the loop of Henle. Urea accumulation within the medullary interstitium is more complex and occurs most effectively when hyperosmotic urine is excreted (i.e., antidiuresis). When dilute urine is produced, especially over extended periods, the osmolality of the medullary interstitium declines (see

Fig. 37.7A ). This reduced osmolality is almost entirely caused by a decrease in the concentration of urea. This decrease reflects washout by the vasa recta (discussed later) and diffusion of urea from the interstitium into the tubular fluid within the medullary portion of the collecting duct, which is permeable to urea even in the absence of AVP. (NOTE: The cortical and outer medullary portions of the collecting duct have a low permeability to urea, whereas the inner medullary portion has a relatively high permeability because of the presence of the urea transporters UT-A1 and UT-A3, the expression of which is increased by AVP.) Some of this reabsorbed urea is secreted into the thin descending limbs of Henle’s loops via the urea transporter UT-A2, and some enters the vasa recta via the UT-B transporter. The urea that is secreted into the descending thin limbs of Henle’s loops is then trapped in the nephron until it again reaches the medullary collecting duct, where it can reenter the medullary interstitium. Thus urea recycles from the interstitium to the nephron and back into the interstitium. This process of urea recycling facilitates accumulation of urea in the medullary interstitium, where it can attain a concentration at the tip of the papilla of 600 mmol/L.

As described, the hyperosmotic medulla is essential for concentrating the tubular fluid within the collecting duct. Because water reabsorption from the collecting duct is driven by the osmotic gradient established in the medullary interstitium, urine can never be more concentrated than that of the interstitial fluid in the papilla. Thus any condition that reduces the medullary interstitial osmolality impairs the ability of the kidneys to maximally concentrate urine. Urea within the medullary interstitium contributes to the total osmolality of the urine. However, because the inner medullary collecting duct is highly permeable to urea, especially in the presence of AVP, urea cannot drive water reabsorption across this nephron segment. Instead, urea in the tubular fluid and medullary interstitium equilibrate, and a small volume of urine with a high concentration of urea is excreted.

It is the medullary interstitial NaCl concentration that is responsible for reabsorbing water from the medullary collecting duct and thereby concentrating the nonurea solutes (e.g., NH4 + salts, K+ salts, creatinine) in the urine.

The vasa recta, the capillary networks that supply blood to the medulla, are highly permeable to solute and water. As with the loop of Henle, the vasa recta form a parallel set of hairpin loops within the medulla (see ). Not only do the vasa recta bring nutrients and oxygen to the medullary nephron segments, but more importantly they also remove the excess water and solute that is continuously added to the medullary interstitium by these nephron seg eOn a typical diet the kidneys must excrete 450 mmol/day of urea. At a maximal urine [urea] of 600 mmol/L this amount of urea can be excreted in less than 1 L of urine. However, if the maximal urine [urea] is reduced because of a decrease in the medullary interstitial fluid [urea], a larger urine volume would be needed to excrete the 450 mmol/day of urea (e.g., 2.25 L of urine would be required if the maximal urine [urea] was only 200 mM).

CHAPTER 35 Control of Body Fluid Osmolality and Volume ments. The ability of the vasa recta to maintain the medullary interstitial gradient is flow dependent. A substantial increase in vasa recta blood flow dissipates the medullary gradient (i.e., washout of osmoles from the medullary interstitium). Alternatively, reduced blood flow reduces oxygen delivery to the nephron segments within the medulla. Because transport of salt and other solutes requires oxygen and ATP, reduced medullary blood flow decreases salt and solute transport by nephron segments in the medulla. As a result the medullary interstitial osmotic gradient cannot be maintained.

Assessment of Renal Diluting and Concentrating Ability

Assessment of renal water handling includes measurements of urine osmolality and the volume of urine excreted. The range of urine osmolality is from 50 to 1200 mOsm/kg H2O. The corresponding range in urine volume is 18 L to as little as 0.5 L/day. These ranges are not fixed and vary from individual to individual. They can also be affected by disease processes, and as noted previously they are dependent on the amount of solute the kidneys must also excrete.

The ability of the kidneys to dilute or concentrate urine requires the separation of solute and water (i.e., the single effect of the countercurrent multiplication process). This separation of solute and water in essence generates a volume of water that is “free of solute.” When urine is dilute, solute-free water is excreted from the body. When urine is concentrated, solute-free water is returned to the body (i.e., conserved).

For the kidneys to maximally excrete solute-free water (i.e., 18 L/day) the following conditions must be met: 1.

AVP must be absent; without it the collecting duct does not reabsorb a significant amount of water.

2.

The tubular structures that separate solute from water (i.e., dilute the tubule fluid) must function normally. In the absence of AVP the following nephron segments can dilute the luminal fluid: ascending thin limb of Henle’s loop thick ascending limb of Henle’s loop

Because of its high transport rate, the thick ascending limb is quantitatively the most important nephron segment involved in the separation of solute and water.

3. An adequate amount of tubular fluid must be delivered to the aforementioned nephron sites for maximal separation of solute and water. Factors that reduce delivery (e.g., decreased GFR or enhanced proximal tubule reabsorption) impair the kidneys’ ability to maximally excrete solute-free water. Similar requirements also apply to conservation of water by the kidneys. For the kidneys to conserve water maximally (6–8 L/day) the following conditions must be met: 1. An adequate amount of tubular fluid must be delivered to those nephron segments that separate solute from water; the most important segment in this regard is the

In response to elevated AVP levels In response to decreased AVP levelsthe kidneys retain 1 L of water the kidneys excrete 1 L of waterreturning osmolality and [Na˜] returning osmolality and [Na˜]to normal, but increasing ECF volume.

to normal, but reducing ECF volume.

• Fig. 35.9 Impact of changes in Na+ balance on ECFV. (1) Addition of NaCl (without water) to the ECF increases the [Na+] and osmolality. (2) The increase in ECF osmolality stimulates secretion of AVP from the posterior pituitary, which than acts on the kidneys to conserve water. (3) Decreased renal excretion of water together with water ingestion restore plasma osmolality and plasma [Na+] to normal. However, the ECFV is now increased by 1 L. (4) Removal of NaCl (without water) from the ECF decreases plasma [Na+] and plasma osmolality. (5) The decrease in ECF osmolality inhibits AVP secretion. In response to the decrease in plasma AVP the kidneys excrete water. (6) Increased renal excretion of water returns the plasma [Na+] and plasma osmolality to normal. However, ECFV is now decreased by 1 L. As illustrated, changes in Na+ balance alter ECFV because of the efficiency of the AVP system to maintain normal body fluid osmolality. (Adapted from Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) thick ascending limb of Henle’s loop. Delivery of tubular fluid to Henle’s loop depends on GFR and proximal tubule reabsorption.

2.

Reabsorption of NaCl by the nephron segments must be normal; again, the most important segment is the thick ascending limb of Henle’s loop.

3.

A hyperosmotic medullary interstitium must be present. The interstitial fluid osmolality is maintained by NaCl reabsorption by Henle’s loop (conditions 1 and 2) and by effective accumulation of urea. Urea accumulation in turn depends on adequate dietary protein intake.

4.

Maximum levels of AVP must be present and the collecting duct must respond normally to AVP.

Control of Extracellular Fluid Volume and Regulation of Renal NaCl Excretion

The major solutes of ECF are the salts of Na+ (see also the major determinant of ECF osmolality, alterations in Na+ balance are commonly assumed to disturb ECF osmolality. However, under normal circumstances this is not the case because the AVP and thirst systems maintain body fluid osmolality within a very narrow range (discussed earlier). As illustrated in

Fig. 35.9 , adding or removing NaCl from ECF changes this body fluid compartment’s volume and not the [Na+] (compare initial condition and final conditions). For example, addition of NaCl to the ECF (without water) increases the [Na+] and osmolality of this compartment. (ICF osmolality also increases because of osmotic equilibration with the ECF.) In response, AVP secretion and thirst are stimulated, and as a result water is ingested and renal water loss is reduced. This restores plasma osmolality (and serum [Na+]) to their initial values, but the volume of the ECF is now increased. The opposite occurs when NaCl is lost from the ECF. Changes in ECF volume (ECFV) can be monitored by measuring body weight, because 1 L of ECF equals 1 kg of ). Of these, NaCl is the most abundant. Because NaCl is body weight.

The kidneys are the major route for excretion of NaCl from the body. Only about 10% of the Na+ lost from the body each day does so by nonrenal routes (e.g., in perspiration and feces). As such, the kidneys are critically important in regulating ECFV. Under normal conditions the kidneys keep ECFV constant (a state termed euvolemia) by adjusting the excretion of NaCl to match the amount ingested in the diet. If ingestion exceeds excretion, ECFV increases above normal (volume expansion), whereas the opposite occurs if excretion exceeds ingestion (volume contraction).

The typical diet contains approximately 140 mEq/day of Na+ (8 g of NaCl), and thus daily Na+ excretion in urine is also about 140 mEq/day. However, the kidneys can vary excretion of Na+ over a wide range. Excretion rates as low as 10 mEq/day can be attained when individuals are placed on a low-salt diet. Conversely, the kidneys can increase their excretion rate to more than 1000 mEq/day when challenged by ingestion of a high-salt diet. These changes in Na+ excretion can occur with only modest changes in the ECFV and Na+ content of the body.

The response of the kidneys to abrupt changes in NaCl intake typically takes several hours to several days, depending on the magnitude of the change. During this transition period the intake and excretion of Na+ are not matched as they are in the steady state. Thus the individual experiences either positive Na+ balance (intake > excretion) or negative Na+ balance (intake < excretion). However, by the end of the transition period a new steady state is established and intake once again equals excretion.

This section reviews the physiology of the receptors that monitor ECFV and explains the various signals that act on the kidneys to regulate NaCl excretion and thereby ECFV. In addition, responses of the various portions of the nephron to these signals is considered.

Concept of Effective Circulating Volume , the ECF is subdivided into two compartments: blood plasma and interstitial fluid. Plasma volume is a determinant of vascular volume and thus blood pressure and cardiac output. Maintenance of Na+ balance, and thus ECFV, involves a complex system of sensors and effector signals that act primarily on the kidneys to regulate NaCl excretion. As can be appreciated from the dependency of vascular volume, blood pressure, and cardiac output on ECFV, this complex system is designed to ensure adequate tissue perfusion. Because the primary sensors of this system are located in the large vessels of the vascular system, changes in vascular volume, blood pressure, and cardiac output are the principal factors regulating renal NaCl excretion (discussed later). In a healthy individual, changes in ECFV result in parallel changes in vascular volume, blood pressure, and cardiac output. Thus a decrease in ECFV results in reduced vascular volume, blood pressure, and cardiac output. Conversely, an increase in ECFV results in increased vascular volume, blood pressure, and cardiac output. The degree to which these cardiovascular parameters

CHAPTER 35 Control of Body Fluid Osmolality and Volume change is dependent upon the degree of ECFV contraction or expansion and the effectiveness of cardiovascular reflex mechanisms (see ). When a person is in negative Na+ balance, ECFV is decreased and renal NaCl excretion is reduced. Conversely, with positive Na+ balance there is an increase in ECFV, which results in enhanced renal NaCl excretion (i.e., natriuresis).

However, in some pathological conditions (e.g., congestive heart failure, hepatic cirrhosis), renal excretion of NaCl does not reflect the ECFV. In both of these situations the volume of the ECF is increased. However, instead of increased renal NaCl excretion, as would be expected, renal excretion of NaCl is reduced. To explain renal Na+ handling in these situations, it is necessary to understand the concept of effective circulating volume (ECV). Unlike ECF, ECV is not a measurable and distinct body fluid compartment. Effective circulating volume refers to that portion of the ECF that is contained within the vascular system and is “effectively” perfusing the tissues (effective blood volume and effective arterial blood volume are other commonly used terms). More specifically the ECV reflects the activity of volume sensors located in the vascular system (discussed later).

Patients with congestive heart failure frequently have an increase in ECFV that manifests as increased plasma volume and accumulation of interstitial fluid in the lungs (pulmonary edema) and peripheral tissues (generalized edema). This excess fluid is the result of NaCl and water retention by the kidneys. The kidneys’ response (i.e., retention of NaCl and water) is paradoxical because the ECFV is increased. However, this fluid is not in the vascular system but rather in the interstitial fluid compartment. In addition, blood pressure and cardiac output may be reduced because of poor cardiac performance. Therefore the sensors located in the vascular system respond as they do in ECFV contraction and cause NaCl and water retention by the kidneys. In this situation the ECV, as monitored by the volume sensors, is decreased.

In healthy individuals, ECV varies directly with ECFV and in particular the volume of the vascular system (arterial and venous), the arterial blood pressure, and cardiac output. However, as noted this is not the case in certain pathological conditions. In the remaining sections of this chapter the relationship between ECFV and renal NaCl excretion in healthy adults—where changes in ECV and ECFV occur in parallel—are examined.

ECFV (or ECV) is monitored by multiple sensors. A number of these sensors are located in the vascular system, and they monitor its fullness and pressure. These receptors are typically called vascular volume receptors, or because they respond to pressure-induced stretch of the walls of the structure in which they are located (e.g., blood vessels or the cardiac atria and ventricles), they are also referred to as baroreceptors.

Volume Sensors in the Low-Pressure Cardiopulmonary Circuit

Volume sensors (i.e., baroreceptors) are located within the walls of the left and right atria, right ventricle, and large pulmonary vessels, and they respond to distention of these structures (see 19).

Because the low-pressure side of the circulatory system has a high compliance, these sensors respond mainly to the “fullness” of the vascular system. These baroreceptors send signals to the brainstem via afferent fibers in the glossopharyngeal and vagus nerves (cranial nerves IX and X). The activity of these sensors modulates both sympathetic nerve outflow and AVP secretion. For example, a decrease in filling of the pulmonary vessels and cardiac atria increases sympathetic nerve activity and stimulates AVP secretion. Conversely, distention of these structures decreases sympathetic nerve activity. In general, 5% to 10% changes in blood volume and pressure are necessary to evoke a response.

The cardiac atria possess an additional mechanism related to control of renal NaCl excretion. The myocytes of the atria synthesize and store a peptide hormone, atrial natriuretic peptide (ANP). It is released when the atria are distended and, via mechanisms outlined later in this chapter, reduces blood pressure and increases excretion of NaCl and water by the kidneys. The ventricles of the heart also produce a natriuretic peptide, brain natriuretic peptide (BNP), so named because it was first isolated from the brain. Like ANP, BNP is released from myocytes by distention of the ventricles. Its actions are similar to those of ANP.

Volume Sensors in the High-Pressure Arterial Circuit

Baroreceptors are also present in the arterial side of the circulatory system, located in the wall of the aortic arch, carotid sinus, and afferent arterioles of the kidneys. The aortic arch and carotid baroreceptors send input to the brainstem via afferent fibers in the glossopharyngeal and vagus nerves (cranial nerves IX and X). The response to this input alters sympathetic outflow and AVP secretion. Thus a decrease in blood pressure increases sympathetic nerve activity and AVP secretion. An increase in pressure tends to reduce sympathetic nerve activity (and activate parasympathetic nerve activity). The sensitivity of the high-pressure baroreceptors is similar to that in the low-pressure side of the vascular system; 5% to 10% changes in pressure are needed to evoke a response.

The juxtaglomerular apparatus (JGA) of the kidneys (see ), particularly the afferent arteriole, responds fThe liver and central nervous system also have sensors that respond to changes in blood pressure and [Na+] and then signal the kidneys to alter NaCl excretion. These systems do not appear to be as important as vascular receptors in monitoring changes in ECFV and effecting changes in renal NaCl excretion and are not considered here.

directly to changes in pressure. If perfusion pressure in the afferent arteriole is reduced, renin is released from the granular cells. By contrast, renin secretion is suppressed when perfusion pressure is increased. As described later in this chapter, renin determines blood levels of angiotensin II and aldosterone, both of which reduce renal NaCl excretion.

Constriction of a renal artery (e.g., by an atherosclerotic plaque) reduces perfusion pressure to that kidney. This reduced perfusion pressure is sensed by the afferent arteriole of the JGA and results in renin secretion. The elevated renin levels increase the production of angiotensin II, which in turn increases systemic blood pressure via its vasoconstrictor affect on arterioles throughout the vascular system. The increased systemic blood pressure is sensed by the JGA of the contralateral kidney (i.e., the kidney without stenosis of its renal artery), and renin secretion from that kidney is suppressed. In addition the high levels of angiotensin II also inhibit renin secretion by the contralateral kidney (negative feedback). Treatment of patients with constricted renal arteries includes surgical repair of the stenotic artery, administration of angiotensin II receptor blockers, or administration of an inhibitor of angiotensin-converting enzyme (ACE). The ACE inhibitor blocks conversion of angiotensin I to angiotensin II.

When the vascular volume sensors have detected a change in ECFV, they send signals to the kidneys, which results in an appropriate adjustment in NaCl and water excretion. Accordingly, when the ECFV is expanded, renal NaCl and water excretion are increased. Conversely, when the ECFV is contracted, renal NaCl and water excretion are reduced. The signals involved in coupling the volume sensors to the kidneys are both neural and hormonal. These are summarized in

Box 35.1 , as are their effects on renal NaCl and water excretion.

, sympathetic nerve fibers innervate the afferent and efferent arterioles of the glomerulus as well as the cells of the nephron. With ECFV contraction, activation of the lowand high-pressure vascular baroreceptors results in stimulation of sympathetic nerve activity, including those fibers innervating the kidneys. This stimulation has the following effects: 1.

The afferent and efferent arterioles are constricted (mediated by α-adrenergic receptors). This vasoconstriction (the effect is greater on the afferent arteriole) decreases the hydrostatic pressure within the glomerular capillary lumen, which results in a decrease in GFR. With this decrease in GFR, the filtered amount of Na+ is reduced.

2.

Renin secretion is stimulated by the cells of the afferent arterioles (mediated by β-adrenergic receptors). As • BOX 35.1 Signals Involved in Control of Renal NaCl and Water Excretion

Renal Sympathetic Nerves (↑Activity: reabsorption along the nephron

Renin-Angiotensin-Aldosterone (↑Secretion:

II stimulates Na+ reabsorption along the nephron stimulates Na+ reabsorption in the distal tubule and collecting duct and to a lesser degree in the thick ascending limb of Henle’s loop

Natriuretic Peptides: ANP, BNP & Urodilatin (↑Secretion: ↑NaCl Excretion) and water reabsorption by the collecting duct secretion and inhibition of AVP action on the distal tubule and collecting duct

AVP (↑Secretion: ↓H2O Excretion) ↑H2O reabsorption by the distal tubule and collecting duct described later, renin ultimately increases the circulating levels of angiotensin II and aldosterone, both of which stimulate Na+ reabsorption by the nephron.

3. NaCl reabsorption along the nephron is directly stimulated (mediated by α-adrenergic receptors on the cells of the nephron). Because of the large amount of Na+ reabsorbed by the proximal tubule, the effect of increased sympathetic nerve activity is quantitatively most important for this segment. As a result of these actions, increased renal sympathetic nerve activity decreases NaCl excretion, an adaptive response that works to restore ECFV to normal. With ECFV expansion, renal sympathetic nerve activity is reduced. This generally reverses the effects just described.

Cells in the afferent arterioles (juxtaglomerular cells, also known as granular cells) are the site of synthesis, storage, and release of the proteolytic enzyme renin. Three factors are important in stimulating renin secretion: 1.

Perfusion pressure. The afferent arteriole behaves as a high-pressure baroreceptor. When perfusion pressure to the kidneys is reduced, renin secretion is stimulated. Conversely, an increase in perfusion pressure inhibits renin release.

2.

Sympathetic nerve activity. Activation of the sympathetic nerve fibers that innervate the afferent arterioles increases renin secretion (mediated by β-adrenergic receptors).

CHAPTER 35 Control of Body Fluid Osmolality and Volume

Renin secretion is decreased as renal sympathetic nerve activity is decreased.

3. Delivery of NaCl to the macula densa. Delivery of NaCl to the macula densa regulates GFR by a process termed tubuloglomerular feedback (see ). In addition the macula densa plays a role in renin secretion. When NaCl delivery to the macula densa is decreased, renin secretion is enhanced. Conversely, an increase in NaCl delivery inhibits renin secretion. It is likely that macula densa–mediated renin secretion helps maintain systemic arterial pressure under conditions of a reduced vascular volume. For example, when vascular volume is reduced, perfusion of body tissues (including the kidneys) decreases. This in turn decreases GFR and the filtered amount of NaCl. The reduced delivery of NaCl to the macula densa stimulates renin secretion, which acts through angiotensin II (a potent vasoconstrictor) to increase blood pressure and thereby maintain tissue perfusion.

Although many tissues express renin (e.g., brain, heart, adrenal gland), juxtaglomerular (JG) cells are the primary source of circulating renin; they are located in the afferent arterioles of the kidneys. Renin secretion is stimulated by a decrease in intracellular [Ca++], a response opposite that of most secretory cells, where secretion is stimulated by an increase in intracellular [Ca++]. Renin secretion is also stimulated by an increase in intracellular cAMP levels. Conversely, anything that increases intracellular [Ca++] will inhibit renin secretion. Renin secretion is also inhibited by increases in intracellular cyclic guanosine monophosphate (cGMP).

Stretch of the afferent arteriole, angiotensin-II, and endothelin increase intracellular [Ca++] and thus inhibit renin secretion by JG cells. The stimulatory effect of sympathetic nerve activity on renin secretion is mediated by norepinephrine, which increases intracellular cAMP (via β-adrenergic receptors). Prostaglandin E2 also increases JG cell cAMP levels and therefore stimulates renin secretion. Natriuretic peptides and nitric oxide (NO) inhibit renin secretion by increasing intracellular cGMP.

Control of renin secretion by the macula densa is complex and appears to involve several paracrine factors, including ATP, adenosine, and prostaglandin E2 (see

Fig. 35.10 summarizes the essential components of the renin-angiotensin-aldosterone system (RAAS). Renin alone does not have a physiological function; it functions solely as a proteolytic enzyme. Its substrate is a circulating protein, angiotensinogen, which is produced by the liver. Angiotensinogen is cleaved by renin to yield a 10–amino acid peptide, angiotensin I. Angiotensin I also has no known physiological function, and it is cleaved to an 8–amino acid peptide, angiotensin II, by angiotensin-converting enzyme (ACE) found on the surface of vascular endothelial cells. Lung and renal endothelial cells are important sites for the conversion of angiotensin I to angiotensin II. ACE also • Fig. 35.10 Schematic representation of the essential components of the renin-angiotensin-aldosterone system (RAAS). Activation of this system results in a decrease in excretion of Na+ and water by the kidneys. NOTE: Angiotensin I is converted to angiotensin II by an angiotensin-converting enzyme that is present on all vascular endothelial cells. As shown, the endothelial cells within the lungs play a significant role in this conversion process. See text for details.

degrades bradykinin, a potent vasodilator. Angiotensin II has several important physiological functions: 1.

stimulation of aldosterone secretion by the adrenal cortex 2.

arteriolar vasoconstriction, which increases blood pressure 3.

stimulation of AVP secretion and thirst 4.

enhancement of NaCl reabsorption by the proximal tubule, thick ascending limb of Henle’s loop, the distal tubule, and the collecting duct (Of these segments the effect on the proximal tubule is quantitatively the largest.) Angiotensin II is an important secretagogue for aldoste rone. An increase in plasma K+ concentration is the other important stimulus for aldosterone secretion (see ). Aldosterone is a steroid hormone produced by the glomerulosa cells of the adrenal cortex and acts in a number of ways on the kidneys (see also

Chapters 34, 36, and ). With regard to regulation of the ECFV, aldosterone reduces NaCl excretion by stimulating its reabsorption by several nephron segments. Most importantly it stimulates NaCl reabsorption in the aldosterone-sensitive distal nephron (ASDN), which consists of the latter portion of the distal tubule and the collecting duct. To a lesser extent it also stimulates NaCl reabsorption by the thick ascending limb of Henle’s loop and the early portion of the distal tubule.

Aldosterone sensitivity is conferred by the presence of mineralocorticoid receptors as well as the enzyme 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2). Because the mineralocorticoid receptor also binds glucocorticoids, 11β-HSD2 is required for aldosterone specificity because it metabolizes glucocorticoids and thus prevents them from binding to the mineralocorticoid receptor. The aldosterone-sensitive distal nephron expresses both the mineralocorticoid receptor and 11β-HSD2.

). In the ASDN it increases the abundance and activity of ENaC in the apical membrane of principal cells. This increases Na+ entry into cells across the apical membrane. Extrusion of Na+ from cells across the basolateral membrane occurs by Na+,K+-ATPase, the abundance of which is also increased by aldosterone. Thus aldosterone increases reabsorption of NaCl from the tubular fluid by distal nephron segments, whereas reduced levels of aldosterone decreases the amount of NaCl reabsorbed by these segments. Although the principal site of action of aldosterone is the ASDN, aldosterone also increases the abundance of the Na+/Cl− symporter in the distal tubule and the Na+/K+/2Cl− symporter in the thick ascending limb of Henle’s loop.

Box 35.1 , activation of the RAAS, as occurs with ECFV depletion, decreases excretion of NaCl

Diseases of the adrenal cortex can alter aldosterone levels and thereby impair the ability of the kidneys to maintain Na+ balance and euvolemia. With decreased secretion of aldosterone (hypoaldosteronism), the reabsorption of NaCl, mainly by the aldosterone-sensitive distal nephron, is reduced and NaCl is lost in the urine. Because urinary NaCl loss can exceed the amount of NaCl ingested in the diet, negative Na+ balance ensues and ECFV decreases. In response to ECFV contraction, sympathetic tone is increased and levels of renin, angiotensin II, and AVP are elevated. With increased aldosterone secretion (hyperaldosteronism) the effects are the opposite; NaCl reabsorption by the aldosterone-sensitive distal nephron is enhanced and excretion of NaCl is reduced. Consequently, ECFV is increased, sympathetic tone is decreased, and levels of renin, angiotensin II, and AVP are decreased. As described later, ANP and BNP levels are also elevated in this setting.

by the kidneys. The RAAS is suppressed with ECFV expansion, and renal NaCl excretion is therefore enhanced.

The body produces a number of substances that act on the kidneys to increase NaCl excretion (see ). Of these, natriuretic peptides produced by the heart and kidneys are best understood and will be the focus of the following discussion.

The heart produces two natriuretic peptides. Atrial myocytes produce and store the peptide hormone ANP, and ventricular myocytes produce and store BNP. Both peptides are secreted when the heart dilates (i.e., during volume expansion, with heart failure), and they relax vascular smooth muscle and promote NaCl and water excretion by the kidneys. The kidneys also produce a related natriuretic peptide termed urodilatin. Its actions are limited to promoting NaCl excretion by the kidneys. In general the actions of these natriuretic peptides as they relate to renal NaCl and water excretion antagonize those of the RAAS. These actions include: 1.

Vasodilation of the afferent and vasoconstriction of the efferent arterioles of the glomerulus. This increases the GFR and the filtered amount of NaCl.

2.

Inhibition of renin secretion by the afferent arterioles.

3.

Inhibition of aldosterone secretion by the glomerulosa cells of the adrenal cortex. This occurs via two mechanisms: (a) inhibition of renin secretion by the juxtaglomerular cells, thereby reducing angiotensin II– induced aldosterone secretion, and (b) direct inhibition of aldosterone secretion by the glomerulosa cells of the adrenal cortex.

4.

Inhibition of NaCl reabsorption by the collecting duct, which is also caused in part by reduced levels of aldosterone. However, natriuretic peptides increase cGMP, which inhibits cation channels in the apical membrane of medullary collecting duct cells and thereby decreases NaCl reabsorption.

CHAPTER 35 Control of Body Fluid Osmolality and Volume 5. Inhibition of AVP secretion by the posterior pituitary and AVP action on the collecting duct. These effects decrease water reabsorption by the collecting duct and thus increase excretion of water in the urine. The net effect of natriuretic peptides is to increase excre tion of NaCl and water by the kidneys. Hypothetically a reduction in circulating levels of these peptides would be expected to decrease NaCl and water excretion, but convincing evidence for this has not been reported.

As discussed previously, a decreased ECFV stimulates AVP secretion by the posterior pituitary. Elevated levels of AVP decrease water excretion by the kidneys, which serves to reestablish euvolemia.

Control of NaCl Excretion During Euvolemia

Maintenance of Na+ balance and therefore euvolemia requires precise matching of the amount of NaCl ingested with the amount excreted from the body. As already noted the kidneys are the major route for NaCl excretion. Accordingly, in a euvolemic individual we can equate daily urine NaCl excretion with daily NaCl intake.

As noted, the amount of NaCl excreted by the kidneys can vary widely. Under conditions of salt restriction (i.e., low-NaCl diet), virtually no NaCl appears in the urine. Conversely, in individuals who ingest large quantities of NaCl, renal NaCl excretion can exceed 1000 mEq/day. The time course for adjustment of renal NaCl excretion varies (hours to days) and depends on the magnitude of the change in NaCl intake. Acclimation to large changes in NaCl intake requires a longer time than acclimation to small changes in intake.

The general features of NaCl transport along the nephron are illustrated in

Fig. 35.11 . Most (67%) of the Na+ filtered by the glomerulus is reabsorbed by the proximal tubule. An additional 25% is reabsorbed by the thick ascending limb of the loop of Henle, and the remainder by the distal tubule and collecting duct.

In a normal adult the filtered amount (load) of Na+ is approximately 25,000 mEq/day:

Equation 35.1

Filtered load of Na+= (GFR) × (Plasma [Na+ ]) = (180 L/day) × (140 mEq/L) , mEq/day

With a typical diet, less than 1% of this filtered load is excreted in urine (≈140 mEq/day).

Because of the large filtered load of Na+ , small changes in its reabsorption by the nephron can profoundly affect Na+ balance and thus ECFV. For example, an increase in Na+ excretion from 1% gThe percentage of the filtered load excreted in urine is termed fractional excretion. In this example the fractional excretion of Na+ is 140 mEq/day ÷ 25,200 mEq/day = 0.005, or 0.5%.

• Fig. 35.11 Segmental Na+ reabsorption. The percentage of the filtered load of Na+ reabsorbed by each nephron segment is indicated. PT, proximal tubule; TAL, thick ascending limb; DT, distal tubule; CD, cortical collecting duct.

to 3% of the filtered load represents an additional loss of approximately 500 mEq/day of Na+ . Because the ECF Na+ concentration is 140 mEq/L, such a Na+ loss would decrease the ECFV by more than 3 L (i.e., water excretion would parallel the loss of Na+ to maintain body fluid osmolality constant: 500 mEq/day ÷ 140 mEq/L = 3.6 L/day of fluid loss). Such fluid loss in a 70-kg individual would represent a 26% decrease in ECFV.

In euvolemic individuals the nephron segments distal to the loop of Henle (distal tubule and collecting duct) are the main nephron segments where Na+ reabsorption is adjusted to maintain excretion at a level appropriate for dietary intake. However, this does not mean the other portions of the nephron are not involved in this process. Because the reabsorptive capacity of the distal tubule and collecting duct is limited, these other portions of the nephron (i.e., proximal tubule and loop of Henle) must reabsorb the bulk of the filtered load of Na+ . Thus during euvolemia, Na+ handling by the nephron can be explained by two general processes: 1.

Na+ reabsorption by the proximal tubule and loop of Henle is regulated so that a relatively constant portion of the filtered load of Na+ is delivered to the distal tubule. The combined action of the proximal tubule and loop of Henle reabsorbs approximately 92% of the filtered load of Na+ , and thus 8% of the filtered load is delivered to the distal tubule.

2.

Reabsorption of this remaining portion of the filtered load of Na+ by the distal tubule and collecting duct is regulated so that the amount of Na+ excreted in the urine matches the amount ingested in the diet. Thus these later nephron segments make final adjustments in Na+ excretion to maintain the euvolemic state.

Mechanisms for Maintaining Constant Delivery of NaCl to the Distal Tubule

A number of mechanisms maintain a constant delivery of Na+ to the beginning of the distal tubule. These processes are autoregulation of the GFR (and thus the filtered load of Na+), glomerulotubular balance, and load dependency of Na+ reabsorption by the loop of Henle.

Autoregulation of the GFR (see ) allows maintenance of a relatively constant filtration rate over a wide range of perfusion pressures. Because the filtration rate is constant, the filtered load of Na+ is also constant.

Despite the autoregulatory control of GFR, small variations occur. If these changes are not compensated for by an appropriate adjustment in Na+ reabsorption by the nephron, Na+ excretion would change markedly. Fortunately, Na+ reabsorption in the euvolemic state, especially by the proximal tubule, changes in parallel with changes in GFR. This phenomenon is termed glomerulotubular balance. Thus if GFR increases, the amount of Na+ reabsorbed by the proximal tubule also increases. The opposite occurs if GFR decreases (see for a more detailed description of glomerulotubular balance).

The final mechanism that helps maintain constant delivery of Na+ to the distal tubule and collecting duct involves the ability of the loop of Henle to increase its reabsorptive rate in response to increased delivery of Na+ .

Regulation of Distal Tubule and Collecting Duct NaCl Reabsorption

When delivery of Na+ is constant, small adjustments in Na+ reabsorption, primarily by the ASDN, are sufficient to balance excretion with intake. Aldosterone is the primary regulator of Na+ reabsorption by the ASDN and thus of NaCl excretion. When aldosterone levels are elevated, Na+ reabsorption by these segments is increased (Na+ excretion is decreased). When aldosterone levels are decreased, Na+ reabsorption is decreased (NaCl excretion is increased). Other factors have been shown to alter Na+ reabsorption by the ASDN (e.g., angiotensin II and natriuretic peptides), but their role during euvolemia is unclear.

As long as variations in dietary intake of NaCl are minor, the mechanisms previously described can regulate renal Na+ excretion appropriately and thereby maintain euvolemia. However, these mechanisms cannot effectively handle significant changes in NaCl intake. When NaCl intake changes significantly, ECFV expansion or contraction occurs. In such cases, additional factors act on the kidneys to adjust Na+ excretion and thereby reestablish the euvolemic state.

Control of NaCl Excretion With Volume Expansion

During ECFV expansion, the high-pressure and low-pressure vascular volume sensors send signals to the kidneys that result in increased excretion of NaCl and water. The signals acting on the kidneys include: ˜ AVP ° ANP and BNP Heart Brain ˜ Aldosterone Lung Adrenal gland ° Na+, H2O excretion ˜ Renin ° Urodilatin ˜ Sympathetic activity 2 1 3 ˜ Angiotensin I ˜ Angiotensin II Volume expansion UNa+V = °GFR ˜PNa+ – ˜R • Fig. 35.12 Integrated response to ECFV expansion. Numbers refer to the description of the response in the text. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; GFR, glomerular filtration rate; PNa+, plasma [Na+]; R, tubular reabsorption of Na+; UNa+V, Na+ excretion rate.

1.

decreased activity of renal sympathetic nerves 2.

release of ANP and BNP from the heart and urodilatin in the kidneys 3.

inhibition of AVP secretion from the posterior pituitary and decreased AVP action on the collecting duct 4.

decreased renin secretion and thus decreased production of angiotensin II 5.

decreased aldosterone secretion, which is caused by reduced angiotensin II levels, and elevated natriuretic peptide levels The integrated response of the nephron to these signals is

Fig. 35.12 . Three general responses to ECFV expansion occur (the numbers match those encircled in

Fig. 35.12 1. GFR increases. GFR increases mainly as a result of the decrease in sympathetic nerve activity. Sympathetic fibers innervate the afferent and efferent arterioles of the glomerulus and control their diameter. Decreased sympathetic nerve activity leads to arteriolar dilation and an increase in renal plasma flow (RPF). Because the effect appears to be greater on the afferent arterioles, the hydrostatic pressure within the glomerular capillary is increased, thereby increasing the GFR. Because RPF increases to a greater degree than GFR, the filtration fraction (GFR/RPF) decreases. Natriuretic peptides also increase GFR by dilating the afferent arterioles and constricting the efferent arterioles. Thus the increased natriuretic peptide levels that occur during ECFV expansion contribute to this response. With the increase in GFR, the filtered load of Na+ increases.

2. The reabsorption of Na+ decreases in the proximal tubule and loop of Henle. Several mechanisms reduce Na+ reabsorption by the proximal tubule. Because activation of the sympathetic nerve fibers that innervate this nephron segment stimulates proximal tubule Na+ reabsorption, the decreased sympathetic nerve activity that results from ECFV expansion decreases Na+ reabsorption. In addition, angiotensin II directly stimulates Na+ reabsorption by the proximal tubule. Because angiotensin II levels are also reduced under this condition, proximal tubule Na+ reabsorption decreases as a result. Starling forces across the proximal tubule also change. The elevated hydrostatic pressure within the glomerular capillaries in ECFV expansion also tends to increase the hydrostatic pressure within the peritubular capillaries. In addition the decrease in filtration fraction reduces the peritubular oncotic pressure. These alterations in the capillary Starling forces (i.e., hydrostatic and oncotic) reduce the absorption of solute (e.g., NaCl) and water from the lateral intercellular space and thus reduce tubular reabsorption of NaCl (see for a complete description of this mechanism). Both the increase in the filtered load and the decrease in NaCl reabsorption by the proximal tubule result in delivery of more NaCl to the loop of Henle. Because activation of sympathetic nerves and angiotensin II and aldosterone stimulate NaCl reabsorption by the thick ascending limb of the loop of Henle, the reduced nerve activity and low angiotensin II and aldosterone levels that occur with ECFV expansion reduce NaCl reabsorption by the thick ascending limb. Thus the fraction of the filtered load delivered to the distal tubule is increased.

3. Na+ reabsorption decreases in the distal tubule and collecting duct. As noted, the amount of Na+ delivered to the distal tubule exceeds that observed in the euvolemic state (i.e., the amount of Na+ delivered to the distal tubule varies in proportion to the degree of ECFV expansion). This increased load of Na+ overwhelms the reabsorptive capacity of the distal tubule and the collecting duct, and this capacity is also impaired by reduced levels of angiotensin II and aldosterone, as well as increased levels of natriuretic peptides. The final component in the response to ECFV expansion is increased excretion of water. As Na+ excretion increases, plasma osmolality begins to fall. This decreases secretion of AVP. AVP secretion is also decreased in response to the elevated levels of natriuretic peptides. In addition, natriuretic peptides inhibit the action of AVP on the collecting duct. Together, these effects decrease water reabsorption by the collecting duct and thereby increase water excretion by the kidneys. Thus excretion of NaCl and water occurs in concert; euvolemia is restored and body fluid osmolality remains constant. The time course of this response (hours to days) depends on the magnitude of the ECFV expansion. Thus if the degree of ECFV expansion is small, the mechanisms just described generally restore euvolemia within 24 hours. However, with large degrees of ECFV expansion, the response can take several days.

In brief, the renal response to ECFV expansion involves the integrated action of all parts of the nephron; (1) the amount of Na+ filtered is increased, (2) Na+ reabsorption by the proximal tubule and loop of Henle is reduced (glomerulotubular balance does not occur under this condition), and (3) reabsorption of Na+ by the distal tubule and collecting duct is decreased (while the delivery of Na+ is increased), primarily secondary to a reduction in aldosterone. In summary, ECFV expansion leads to a reduction in Na+ reabsorption by both the proximal tubule and the ASDN, which results in excretion of a larger fraction of the filtered Na+ , thereby restoring euvolemia.

Control of NaCl Excretion With Volume Contraction

During ECFV contraction, the high-pressure and low-pressure vascular volume sensors send signals to the kidneys to reduce NaCl and water excretion and thereby restore ECFV. The signals that act on the kidneys include: 1.

2.

increased secretion of renin, which results in elevated angiotensin II levels and thus increased secretion of aldosterone by the adrenal cortex 3.

inhibition of ANP and BNP secretion by the heart and urodilatin production by the kidneys 4.

stimulation of AVP secretion by the posterior pituitary

The integrated response of the nephron to these signals is illustrated in

Fig. 35.13 . The general response is as follows (the numbers correlate with those encircled in

Fig. 35.13): 1.

GFR decreases. Afferent and efferent arteriolar constriction occurs as a result of increased renal sympathetic nerve activity. The effect appears to be greater on the afferent than on the efferent arteriole, because the hydrostatic pressure in the glomerular capillaries fall, which thereby decreases GFR. Because RPF decreases more than GFR, filtration fraction increases. The decrease in GFR reduces the filtered amount of Na+ .

2.

Na+ reabsorption by the proximal tubule and loop of Henle is increased. Several mechanisms augment Na+ reabsorption in the proximal tubule. For example, increased sympathetic nerve activity and angiotensin II levels directly stimulate Na+ reabsorption. The decreased hydrostatic pressure within the glomerular capillaries also leads to a decrease in the hydrostatic pressure within the peritubular capillaries. In addition, as just noted, the increased filtration fraction results in an increase in the peritubular oncotic pressure. These alterations in the capillary Starling forces facilitate movement of fluid from the lateral intercellular space into the capillary and thereby stimulate reabsorption of NaCl and water by the proximal tubule (see for a complete description of this mechanism). Increased sympathetic nerve activity as well as elevated levels of angiotensin II and aldosterone stimulate Na+ reabsorption by the thick ascending limb.

3.

Na+ reabsorption by the distal tubule and collecting duct is enhanced. The small amount of Na+ that is delivered to the ASDN, owing to decreased filtration and increased reabsorption by the proximal tubule and loop of Henle, is almost completely reabsorbed because transport in the segments of the ASDN is enhanced. Stimulation of Na+ reabsorption by the ASDN is primarily the result of increased aldosterone levels, although increased sympathetic nerve activity and increased angiotensin II levels may also contribute to this response. Lastly, levels of natriuretic peptides, which inhibit collecting duct reabsorption, are reduced. Finally, water reabsorption by the latter portion of the distal tubule and the collecting duct is enhanced by AVP, the levels of which are elevated through activation of the lowand high-pressure vascular volume sensors as well as by the elevated levels of angiotensin II. As a result, water excretion is reduced. Because both water and Na+ are retained by the kidneys in equal proportions, euvolemia is reestablished and ˜ AVP ° ANP and BNP Heart Brain ˜ Aldosterone Lung Adrenal gland ° Na+, H2O excretion ˜ Renin ˜ Sympathetic activity 2 1 3 ˜ Angiotensin I ˜ Angiotensin II Volume contraction • Fig. 35.13 Integrated response to ECFV contraction. Numbers refer to the description of the response in the text. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; GFR, glomerular filtration rate; PNa+, plasma [Na+]; R, tubular reabsorption of Na+; UNa+V, Na+ excretion rate.

body fluid osmolality remains constant. The time course of this restoration of ECFV (hours to days) and the degree to which euvolemia is attained depend on the magnitude of ECFV contraction as well as the dietary intake of Na+ . Euvolemia can be restored more rapidly if additional NaCl is ingested in the diet.

In brief, the nephron’s response to ECFV contraction involves the integrated action of all its segments: (1) the 1.

Regulation of body fluid osmolality (i.e., steady-state balance) requires that the amount of water added to the body exactly matches the amount lost from the body. Water is lost from the body by several routes (e.g., during respiration, with sweating, and in feces). The kidneys are the only regulated route of water excretion. Excretion of water by the kidneys is regulated by AVP secreted from the posterior pituitary. When AVP levels are high, the kidneys excrete a small volume of hyperosmotic urine. When AVP levels are low, a large volume of hypoosmotic urine is excreted.

2.

Disorders of water balance alter body fluid osmolality. Because Na+ with its anions are the major determinant filtered amount of Na+ is decreased, (2) reabsorption in the proximal tubule and loop of Henle is enhanced (GFR is decreased, whereas proximal reabsorption is increased, and thus glomerulotubular balance does not occur under this condition), and (3) delivery of Na+ to the ASDN is reduced. This decreased delivery, together with enhanced Na+ and water reabsorption by the ASDN, virtually eliminates Na+ from the urine and reduces urine volume.

of ECF osmolality, disorders of water balance manifest as changes in ECF [Na+]. Positive water balance (intake > excretion) results in a decrease in body fluid osmolality and hyponatremia. Negative water balance (intake < excretion) results in an increase in body fluid osmolality and hypernatremia.

3. The volume of the ECF is determined by the amount of Na+ in this compartment. To maintain a constant ECFV (i.e., euvolemia) NaCl excretion must match NaCl intake. The kidneys are the major route for regulating excretion of NaCl from the body. Volume sensors located primarily in the vascular system monitor volume and pressure. When ECFV expansion occurs, neural and hormonal signals are sent to the kidneys to increase excretion of NaCl and water and thereby restore euvolemia. When ECFV contraction occurs, neural and hormonal signals are sent to the kidneys to decrease NaCl and water excretion and

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Nielsen S, et al. Aquaporin water channels in mammalian kidney. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013.

Robertson GL. Thirst and vasopressin. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013.

Sands JM, et al. Urine concentration and dilution. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.

Sands JM, Layton HE. The urine concentrating mechanism and urea transporters. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013.

Sands JM, Layton HE. Advances in understanding the urine-concentrating mechanism. Annu Rev Physiol. 2014;76:387-409.

Schrier RW. Systemic arterial vasodilation, vasopressin, and vasopres sinase in pregnancy. J Am Soc Nephrol. 2010;21:570-572. Sterns RH. Disorders of plasma sodium–causes, consequences, and correction. N Engl J Med. 2015;372:55-65.

thereby restore euvolemia. The sympathetic nervous system, the RAAS, and natriuretic peptides are important components of the system that maintain steady-state Na+ balance.

Verbalis JG. Disorders of water balance. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.

Eladari D, et al. A new look at electrolyte transport in the distal tubule. Annu Rev Physiol. 2012;74:325-349.

Gamba G, et al. Sodium chloride transport in the loop of Henle, distal convoluted tubule, and collecting duct. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013.

Mount DB. Transport of sodium, chloride, and potassium. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.

Mount DB. Thick ascending limb of the loop of Henle. Clin J Am Soc Nephrol. 2014;9:1974-1986.

Palmer LG, Schnermann J. Integrated control of Na transport along the nephron. Clin J Am Soc Nephrol. 2015;10:676-687.

Pearce D, et al. Aldosterone regulation of transport. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.

Pearce D, et al. Collecting duct principal cell transport processes and their regulation. Clin J Am Soc Nephrol. 2015;10:135-146.

Slotki IN, Skorecki KL. Disorders of sodium balance. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.

Staub O, Loffing J. Mineralocorticoid action in aldosterone sensitive distal nephron. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013.

Subramanya AR, Ellison DH. Distal convoluted tubule. Clin J Am Soc Nephrol. 2014;9:2147-2163.

Vesley DL. Natriuretic hormones. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013.

Weinstein AM. Sodium and chloride transport: proximal nephron. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013.

Potassium, Calcium, and Phosphate Homeostasis

Upon completion of this chapter the student should be able to answer the following questions: 1.

How does the body maintain K+ homeostasis?

2.

What is the distribution of K+ within the body compartments? Why is this distribution important?

3.

What are the hormones and factors that regulate plasma K+ levels? Why is this regulation important?

4.

How do the various segments of the nephron transport K+ , and how does the mechanism of K+ transport by these segments determine how much K+ is excreted in the urine?

5.

Why are the distal tubule and collecting duct so important in regulating K+ excretion?

6.

How do plasma K+ levels, aldosterone, vasopressin, tubular fluid flow rate, and acid-base balance influence K+ excretion?

7.

What is the physiological importance of calcium (Ca++) and inorganic phosphate (Pi)?

8.

How does the body maintain Ca++ and Pi homeostasis?

9.

What roles do the kidneys, intestinal tract, and bone play in maintaining plasma Ca++ and Pi levels?

10.

What hormones and factors regulate plasma Ca++ and Pi levels?

11.

What are the cellular mechanisms responsible for Ca++ and Pi reabsorption along the nephron?

12.

What hormones regulate renal Ca++ and Pi excretion by the kidneys?

13.

What is the role of the calcium-sensing receptor (CaSR)?

14.

What are some of the more common clinical disorders of Ca++ and Pi homeostasis?

15.

What is the role of the kidneys in the production of calcitriol (active form of vitamin D)?

16.

What effects do loop and thiazide diuretics have on Ca++ excretion?

Potassium (K+) is one of the most abundant cations in the body, and it is critical for many cell functions, including regulation of cell volume, regulation of intracellular pH, synthesis of DNA and protein, growth, enzyme function, resting membrane potential, and cardiac and neuromuscular activity. Despite wide fluctuations in dietary K+ intake, [K+] in cells and extracellular fluid (ECF) remains remarkably constant. Two sets of regulatory mechanisms safeguard K+ homeostasis. First, several mechanisms regulate [K+] in the ECF. Second, other mechanisms maintain the amount of K+ in the body constant by adjusting renal K+ excretion to match dietary K+ intake. It is the kidneys that regulate excretion of K+ .

Total body [K+] is 50 mEq/kg of body weight, or 3600 mEq for a 70-kg individual. Ninety-eight percent of the K+ in the body is located within cells, where the average [K+] is 150 mEq/L. High intracellular [K+] is required for many cell functions, including cell growth and division and volume regulation. Only 2% of total body [K+] is located in the ECF, where its normal concentration is approximately 4 mEq/L. A [K+] in ECF that exceeds 5.0 mEq/L constitutes hyperkalemia. Conversely, a [K+] in ECF of less than 3.5 mEq/L constitutes hypokalemia.

Hypokalemia is one of the most common electrolyte disorders in clinical practice and can be observed in as many as 20% of hospitalized patients. The most frequent causes of hypokalemia include administration of diuretic drugs, surreptitious vomiting (e.g., bulimia), and severe diarrhea. Gitelman syndrome (a genetic defect in the Na+/ Cl− symporter in the apical membrane of distal tubule cells) also causes hypokalemia (see Chapter 36). Hyperkalemia is also a common electrolyte disorder and is seen in 1% to 10% of hospitalized patients. Hyperkalemia often occurs in patients with renal failure, in patients taking drugs such as angiotensin-converting enzyme (ACE) inhibitors and K+-sparing diuretics, in patients with hyperglycemia (i.e., high blood sugar), and in the elderly. Pseudohyperkalemia, a falsely high plasma [K+], is caused by traumatic lysis of red blood cells during blood drawing. Red blood cells, like all cells, contain K+ , and lysis of red blood cells releases K+ into plasma, thereby artificially elevating plasma [K+].

The large concentration difference of K+ across cell membranes (≈146 mEq/L) is maintained by the operation of Na+,K+-ATPase. This [K+] gradient is important in • Fig. 36.2 Electrocardiographs from individuals with varying plasma • Fig. 36.1 Effects of variations in plasma [K+] on resting membrane potential of skeletal muscle. Hyperkalemia causes membrane potential to become less negative, which decreases excitability by inactivating the fast Na+ channels responsible for the depolarizing phase of the action potential. Hypokalemia hyperpolarizes the membrane potential and thereby reduces excitability because a larger stimulus is required to depolarize the membrane potential to the threshold potential. Resting indicates “normal” resting membrane potential. Normal threshold indicates the membrane threshold potential.

maintaining the potential difference across cell membranes. Thus K+ is critical for the excitability of nerve and muscle cells, as well as for the contractility of cardiac, skeletal, and smooth muscle cells (

Fig. 36.1 [K+]. See text for details. (Modified from Barker L et al. Principles of Ambulatory Medicine. 5th ed. Baltimore: Williams & Wilkins; 1999.)

This rise in plasma [K+], which could have deleterious effects on the electrical activity of the heart and other excitable tissues, is prevented by the rapid (minutes) uptake of K+ into cells. Because excretion of K+ by the kidneys

Cardiac arrhythmias are produced by both hypokalemia and hyperkalemia. The electrocardiogram (ECG;

Fig. 36.2 ) monitors the electrical activity of the heart and is a fast and easy way to determine whether changes in plasma [K+] influence the heart and other excitable cells. In contrast, measurement of plasma [K+] by the clinical laboratory requires a blood sample, and values are often not immediately available. The first sign of hyperkalemia is the appearance of tall, thin T waves on the ECG. Further increases in plasma [K+] prolong the PR interval, depress the ST segment, and lengthen the QRS interval of the ECG. Finally, as plasma [K+] approaches 10 mEq/L, the P wave disappears, the QRS interval broadens, the ECG appears as a sine wave, and the ventricles fibrillate (i.e., manifest rapid, uncoordinated contractions of muscle fibers). Hypokalemia prolongs the QT interval, inverts the T wave, and lowers the ST segment of the ECG.

After a meal the K+ absorbed by the gastrointestinal (GI) tract enters the ECF within minutes (Fig. 36.3 ). If the K+ ingested during a normal meal (≈33 mEq) were to remain in the ECF compartment (14 L), plasma [K+] would increase by 2.4 mEq/L (33 mEq added to 14 L of ECF):

Equation 36.1 14L = 2 4. mEq after a meal is relatively slow (hours), uptake of K+ by cells is essential to prevent life-threatening hyperkalemia. Maintaining total body [K+] constant requires that all the K+ absorbed by the GI tract eventually be excreted by the kidneys. This process requires about 6 hours.

Regulation of Plasma [K+]

Several hormones, including epinephrine, insulin, and aldosterone, increase uptake of K+ into skeletal muscle, liver, bone, and red blood cells (Box 36.1

Fig. 36.3 ) by stimulating Na+,K+-ATPase, the 1Na+/1K+/2Cl− symporter, and the Na+/Cl− symporter in these cells. Acute stimulation of K+ uptake (i.e., within minutes) is mediated by increased activity of existing Na+,K+-ATPase, 1Na+/1K+/2Cl− , and Na+/Cl− transporters, whereas a chronic increase in K+ uptake (i.e., within hours to days) is mediated by an increase in the quantity of Na+,K+-ATPase. The rise in plasma [K+] that follows K+ absorption by the GI tract stimulates secretion of insulin from the pancreas, release of aldosterone from the adrenal cortex, and secretion of epinephrine from the adrenal medulla (see

Fig. 36.3 ). In contrast, a decrease in plasma [K+] inhibits release of these hormones. Whereas insulin and epinephrine act within a few minutes, aldosterone requires about an hour to stimulate uptake of K+ into cells.

• BOX 36.1 Major Factors, Hormones, and Drugs Influencing Distribution of K+Between Intracellular and Extracellular Fluid Compartments

Physiological: Keep Plasma [K+] Constant

Pathophysiological: Displace Plasma [K+] From Normal

CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis

Catecholamines affect the distribution of K+ across cell membranes by activating α-and β2-adrenergic receptors. Stimulation of α-adrenoceptors releases K+ from cells, especially in the liver, whereas stimulation of β2-adrenoceptors promotes K+ uptake by cells.

For example, activation of β2-adrenoceptors after exercise is important in preventing hyperkalemia. The rise in plasma [K+] after a K+-rich meal is greater if the patient has been pretreated with propranolol, a β-adrenoceptor antagonist. Furthermore, release of epinephrine during stress (e.g., myocardial ischemia) can rapidly lower plasma [K+].

Insulin also stimulates uptake of K+ into cells. The importance of insulin is illustrated by two observations. First, the rise in plasma [K+] after a K+-rich meal is greater in patients with diabetes mellitus (i.e., insulin deficiency) than in healthy people. Second, insulin (and glucose to prevent insulin-induced hypoglycemia) can be infused to correct hyperkalemia. Insulin is the most important hormone that shifts K+ into cells after ingestion of K+ in a meal. In patients with chronic kidney disease, although insulin-stimulated glucose uptake into cells is impaired, insulin stimulation of K+ uptake into cells is normal.

• Fig. 36.3 Overview of K+homeostasis. An increase in plasma insulin, epinephrine, or aldosterone stimulates movement of K+into cells and decreases plasma [K+], whereas a fall in plasma concentration of these hormones has the opposite effect and increases plasma [K+]. The amount of K+in the body is determined by the kidneys. An individual is in K+balance when dietary intake and urinary output (plus output by the GI tract) are equal. Excretion of K+by the kidneys is regulated by plasma [K+], aldosterone, and AVP. Diet 100 mEq of K+/day Feces 5–10 mEq of K+/day Urine 90–95 mEq of K+/day Intestinal absorption 90 mEq of K+/day Extracellular fluid 65 mEq of K+ Tissue stores 3435 mEq of K+ Insulin Epinephrine Aldosterone Plasma [K+] AVP Aldosterone

Aldosterone, like catecholamines and insulin, also promotes uptake of K+ into cells. A rise in aldosterone levels (e.g., primary aldosteronism) causes hypokalemia, whereas a fall in aldosterone levels (e.g., Addison’s disease) causes hyperkalemia. As discussed later and as illustrated in

Fig.

36.3 , aldosterone also stimulates urinary K+ excretion. Thus aldosterone alters plasma [K+] by acting on uptake of K+ into cells and altering urinary K+ excretion.

Box 36.1 ). These factors are not involved in regulation of plasma [K+] but rather alter the movement of K+ between the intracellular fluid (ICF) and ECF and thus cause development of hypokalemia or hyperkalemia.

Metabolic acidosis increases plasma [K+], whereas metabolic alkalosis decreases it. Respiratory alkalosis causes hypokalemia. In contrast, respiratory acidosis has little or no effect on plasma [K+]. Metabolic acidosis produced by addition of inorganic acids (e.g., HCl, H2SO4) increases plasma [K+] much more than an equivalent acidosis produced by accumulation of organic acids (e.g., lactic acid, acetic acid, ketoacids). The reduced pH (i.e., increased [H+]) promotes movement of H+ into cells and the reciprocal movement of K+ out of cells to maintain electroneutrality. This effect of acidosis occurs in part because acidosis inhibits the transporters that accumulate K+ inside cells, including Na+,K+-ATPase and the 1Na+/1K+/2Cl− symporter. In addition, movement of H+ into cells occurs as the cells buffer changes in [H+] of the ECF (see

Chapter 37).

As H+ moves across cell membranes, K+ moves in the opposite direction, and thus cations are neither gained nor lost across cell membranes. Metabolic alkalosis has the opposite effect; plasma [K+] decreases as K+ moves into cells and H+ exits.

Although organic acids produce a metabolic acidosis, they do not cause significant hyperkalemia. Two explanations have been suggested for the reduced ability of organic acids to cause hyperkalemia. First, the organic anion may enter the cell with H+ and thereby eliminate the need for K+-H+ exchange across the membrane. Second, organic anions may stimulate insulin secretion, which moves K+ into cells. This movement may counteract the direct effect of the acidosis, which moves K+ out of cells.

The osmolality of plasma also influences the distribution of K+ across cell membranes. An increase in the osmolality of ECF enhances the release of K+ by cells and thus increases extracellular [K+]. Plasma [K+] may increase by 0.4 to 0.8 mEq/L with a 10 mOsm/kg H2O elevation in plasma osmolality. In patients with diabetes mellitus who do not take insulin, plasma [K+] is often elevated, in part because of the lack of insulin and in part because of the increase in plasma [glucose] (i.e., from a normal value of ≈ 100 mg/ dL to as high as ≈ 1200 mg/dL in some cases), which increases plasma osmolality. Hypoosmolality has the opposite action. The alterations in plasma [K+] associated with changes in osmolality are related to changes in cell volume. For example, as plasma osmolality increases, water leaves cells because of the osmotic gradient across the plasma membrane (see

Chapter 1).

Water leaves cells until the intracellular osmolality equals that of ECF. This loss of water shrinks cells and causes [K+] in cells to rise. The rise in intracellular [K+] provides a driving force for the exit of K+ from cells. This sequence increases plasma [K+]. A fall in plasma osmolality has the opposite effect.

Cell lysis causes hyperkalemia as a result of addition of intracellular K+ to ECF. Severe trauma (e.g., burns) and some conditions such as tumor lysis syndrome (i.e., chemotherapy-induced destruction of tumor cells) and rhabdomyolysis (i.e., destruction of skeletal muscle) destroy cells and release K+ and other cell solutes into ECF. In addition, gastric ulcers may cause seepage of red blood cells into the GI tract. The blood cells are digested, and the K+ released from the cells is absorbed and can cause hyperkalemia.

More K+ is released from skeletal muscle cells during exercise than during rest. The ensuing hyperkalemia depends on the degree of exercise. In people walking slowly, plasma [K+] increases by 0.3 mEq/L. With vigorous exercise, plasma [K+] may increase by 2.0 or more mEq/L.

K+ Excretion by the Kidneys

The kidneys play a major role in maintaining K+ balance. As illustrated in

Fig. 36.3 the kidneys excrete 90% to 95% of the K+ ingested in the diet. Excretion equals intake even when intake increases by as much as 10-fold. This balance in urinary excretion and dietary intake underscores the importance of the kidneys in maintaining K+ homeostasis. Although small amounts of K+ are lost each day in feces and sweat (≈5%–10% of the K+ ingested in the diet), except during severe diarrhea this amount is essentially constant, is not regulated, and therefore is relatively less important than the K+ excreted by the kidneys. K+ secretion from blood into tubular fluid by cells of the distal tubule and collecting duct system is the key factor in determining urinary K+ excretion (

Fig. 36.4

CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis 3% 10% to 50% 9% 5% to 30% • Fig. 36.4 K+ transport along the nephron. Excretion of K+ depends on the rate and direction of K+ transport by the late segment of the distal tubule and collecting duct. Percentages refer to the amount of filtered K+ reabsorbed or secreted by each nephron segment. Arrows indicate direction of transport. Left, Dietary K+ depletion. An amount of K+ equal to 1% of the filtered load of K+ is excreted. Right, Normal and increased dietary K+ intake. An amount of K+ equal to 15% to 80% of the filtered load is excreted. CCD, cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; PT, proximal tubule;

TAL, thick ascending limb.

Exercise-induced changes in plasma [K+] do not usually produce symptoms and are reversed after several minutes of rest. However, vigorous exercise can lead to life-threatening hyperkalemia in individuals (1) who have endocrine disorders that affect release of insulin, epinephrine (a β-adrenergic agonist), or aldosterone; (2) whose ability to excrete K+ is impaired (e.g., renal failure); or (3) who take certain medications, such as β1-adrenergic blockers. For example, during vigorous exercise, plasma [K+] may increase by at least 2 to 4 mEq/L in individuals who take β1-adrenergic receptor antagonists for hypertension. Because acid-base balance, plasma osmolality, cell lysis, and exercise do not maintain plasma [K+] at a normal value, they do not contribute to K+ homeostasis (see

Box 36.1 ). The extent to which these pathophysiological states alter plasma [K+] depends on the integrity of the homeostatic mechanisms that regulate plasma [K+] (e.g., secretion of epinephrine, insulin, and aldosterone).

Because K+ is not bound to plasma proteins, it is freely filtered by the glomerulus. When individuals ingest 100 mEq of K+ per day, urinary K+ excretion is about 15% of the amount filtered. Accordingly K+ must be reabsorbed along the nephron. When dietary K+ intake increases, however, K+ excretion can exceed the amount filtered. Thus K+ can also be secreted.

The proximal tubule reabsorbs about 67% of the filtered K+ under most conditions. Approximately 20% of the filtered K+ is reabsorbed by the loop of Henle, and as with the proximal tubule, the amount reabsorbed is a constant fraction of the amount filtered. In contrast to these segments, which can only reabsorb K+ , the distal tubule and collecting duct are able to reabsorb or secrete K+ . The rate of K+ reabsorption or secretion by the distal tubule and collecting duct depends on a variety of hormones and factors. When 100 mEq/day of K+ is ingested, it is secreted by these nephron segments. A rise in dietary K+ intake increases K+ secretion. K+ secretion can increase the amount of K+ that appears in urine so that it approaches 80% of the amount filtered (see

Fig. 36.4 , right panel). In contrast, a low-K+ diet activates K+ reabsorption along the distal tubule and collecting duct so that urinary excretion falls to about 1% of the K+ filtered by the glomerulus (see

Fig. 36.4 , left panel). The kidneys cannot reduce K+ excretion to the same low levels as they can for Na+ (i.e., 0.2%). Therefore hypokalemia can develop in individuals placed on a K+-deficient diet. Because the magnitude and direction of K+ transport by the distal tubule and collecting duct are variable, the overall rate of urinary K+ excretion is determined by these tubular segments.

Cellular Mechanism of K+ Secretion by Principal Cells and Intercalated Cells

Fig. 36.5A illustrates the cellular mechanisms of K+ secretion by principal cells in the late segment of the distal • Fig. 36.5 Cellular mechanism of K+ secretion by principal cells (A) and α-intercalated cells (B) in the late segment of the distal tubule and collecting duct. α-Intercalated cells contain very low levels of Na+,K+-ATPase in the basolateral membrane (not shown). K+ depletion increases K+ reabsorption by α-intercalated cells by stimulating the H+,K+-ATPase (HKA).

In individuals with advanced renal disease, the kidneys are unable to eliminate ingested K+ from the body. Therefore plasma [K+] rises. The resulting hyperkalemia reduces the resting membrane potential (i.e., the voltage becomes less negative), and this reduced potential decreases the excitability of neurons, cardiac cells, and muscle cells by inactivating fast Na+ channels, which are critical for the depolarization phase of the action potential (see

Fig. 36.1 ). Severe rapid increases in plasma [K+] can lead to cardiac arrest and death. In contrast, in patients taking diuretic drugs for hypertension, urinary K+ excretion often exceeds dietary K+ intake. Accordingly K+ balance is negative and hypokalemia develops. This decline in extracellular [K+] hyperpolarizes the resting cell membrane (i.e., the voltage becomes more negative) and reduces the excitability of neurons, cardiac cells, and muscle cells. Severe hypokalemia can lead to paralysis, cardiac arrhythmias, and death. Hypokalemia can also impair the ability of the kidneys to concentrate urine and can stimulate renal production of NH4 + , which affects acid-base balance (see ). Therefore maintenance of high intracellular [K+], low extracellular [K+], and a high [K+] gradient across cell membranes is essential for a number of cellular functions.

CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis tubule and the collecting duct. Secretion from blood into the tubule lumen is a two-step process: (1) uptake of K+ from blood across the basolateral membrane by Na+,K+-ATPase and (2) diffusion of K+ from the cell into tubular fluid via K+ channels (ROMK and BK). A K+/ Cl− symporter (KCC1) in the apical plasma membrane also secretes K+ . Na+,K+-ATPase creates a high intracellular [K+] that provides the chemical driving force for exit of K+ across the apical membrane through K+ channels. Although K+ channels are also present in the basolateral membrane, K+ preferentially leaves the cell across the apical membrane and enters the tubular fluid. K+ transport follows this route for two reasons. First, the electrochemical gradient of K+ across the apical membrane favors its downhill movement into tubular fluid. Second, the permeability of the apical membrane to K+ is greater than that of the basolateral membrane. Therefore K+ preferentially diffuses across the apical membrane into tubular fluid. K+ secretion across the apical membrane via the K+/Cl− symporter is driven by the favorable concentration gradient of K+ between the cell and tubular fluid. The three major factors that control the rate of K+ secretion by the late segment of the distal tubule and the collecting duct are: 1.

the activity of Na+,K+-ATPase 2.

the driving force (electrochemical gradient for K+ channels and the chemical concentration gradient for the K+/Cl− symporter) for movement of K+ across the apical membrane 3.

the permeability of apical membrane K+ channels to K+ Every change in K+ secretion by principal cells results from an alteration in one or more of these factors (Fig. 36.6 α-Intercalated cells reabsorb K+ by a H+ , K+-ATPase transport mechanism (HKA) located in the apical membrane (see

Fig. 36.5B ). This transporter mediates K+ uptake across the apical plasma membrane in exchange for H+ . K+ exit from intercalated cells into the blood is mediated by a K+ channel. Reabsorption of K+ is activated by a low-K+ diet.

Regulation of K+ Secretion by the Distal Tubule and Collecting Duct

Regulation of K+ excretion is achieved mainly by alterations in K+ secretion by principal cells of the late segment of the distal tubule and collecting duct. Plasma [K+] and aldosterone are the major physiological regulators of K+ secretion. Ingestion of a K+-rich meal also stimulates renal K+ excretion by a mechanism involving an unknown gut-dependent mechanism. Arginine vasopressin (AVP), also stimulates K+ secretion; however, it is less important than plasma [K+] and aldosterone. Other factors, including the flow rate of tubular fluid and acid-base balance, influence secretion of K+ by the distal tubule and collecting duct. However, they are not homeostatic mechanisms, because they disturb K+ balance (

Box 36.2

Plasma [K+] is an important determinant of K+ secretion by the distal tubule and collecting duct. Hyperkalemia (e.g., resulting from a high-K+ diet or from rhabdomyolysis)

Fig. 36.6 Cellular mechanism of K+ secretion by principal cells. The numbers indicate where K+ secretion is regulated. 1, Na+,K+-ATPase; 2, electrochemical gradient of K+ across the apical membrane; 3, permeability of the apical membrane to K+ .

Fig. 36.7 Effects of aldosterone on secretion of K+ by principal cells in the late segment of the distal tubule and collecting duct. Numbers refer to the five effects of aldosterone discussed in text.

K+ K+ ˜ K+ permeability + ˜ CAP ˜ SGK˜ ENaC ˜Na+,K+-ATPase + + Na+ ATP 2 1 3 4 5

IN THE CLINIC • BOX 36.2 Major Factors and Hormones Influencing K+Excretion

Physiological: Keep K+ Balance Constant

Pathophysiological: Displace K+ Balance

Flow rate of tubule fluid Acid-base disorders Glucocorticoids stimulates secretion of K+ within minutes. Several mechanisms are involved. First, hyperkalemia stimulates Na+ , K+-ATPase and thereby increases K+ uptake across the basolateral membrane. This uptake raises intracellular [K+] and increases the electrochemical driving force for exit of K+ across the apical membrane. Second, hyperkalemia also increases the permeability of the apical membrane to K+ . Third, hyperkalemia stimulates secretion of aldosterone by the adrenal cortex, which as discussed later, acts synergistically with plasma [K+] to stimulate secretion of K+ . Fourth, hyperkalemia also increases the flow rate of tubular fluid, which as discussed later, stimulates secretion of K+ by the distal tubule and collecting duct.

Hypokalemia (e.g., caused by a low-K+ diet or loss of K+ in diarrhea fluid) decreases K+ secretion via actions opposite those described for hyperkalemia. Hence hypokalemia inhibits Na+,K+-ATPase, decreases the electrochemical driving force for efflux of K+ across the apical membrane, reduces permeability of the apical membrane to K+ , and decreases plasma aldosterone levels.

Chronic hypokalemia (plasma [K+] < 3.5 mEq/L) occurs most often in patients who receive diuretics for hypertension. Hypokalemia also occurs in patients who vomit, undergo nasogastric suction, have diarrhea, abuse laxatives, or have hyperaldosteronism. Hypokalemia occurs because excretion of K+ by the kidneys exceeds dietary intake of K+ . Vomiting, nasogastric suction, diuretics, and diarrhea can all decrease ECF volume (ECFV), which in turn stimulates secretion of aldosterone (see ). Because aldosterone stimulates excretion of K+ by the kidneys, its action contributes to development of hypokalemia.

Chronic hyperkalemia (plasma [K+] > 5.0 mEq/L) occurs most frequently in individuals with reduced urine flow, low plasma aldosterone levels, and renal disease in which the glomerular filtration rate (GFR) falls below 20% of normal. In these individuals, hyperkalemia occurs because excretion of K+ by the kidneys is less than dietary intake of K+ . Less common causes of hyperkalemia occur in people with deficiencies in insulin, epinephrine, and aldosterone secretion or in people with metabolic acidosis caused by inorganic acids.

Chronically elevated (i.e., ≥24 hours) plasma aldosterone levels enhance secretion of K+ across principal cells in the late segment of the distal tubule and collecting duct (i.e., aldosterone-sensitive distal nephron [ASDN]) via five mechanisms (Fig. 36.7 ): (1) by increasing the amount of Na+,K+-ATPase in the basolateral membrane; (2) by increasing expression of the epithelial sodium channel (ENaC) in the apical cell membrane; (3) by elevating SGK1 (serum glucocorticoid-stimulated kinase) levels, which also increases expression of ENaC in the apical membrane and activates K+ channels; (4) by stimulating CAP1 (channelactivating protease, also called prostatin), which directly activates ENaC; and (5) by stimulating the permeability of the apical membrane to K+ . Aldosterone increases the permeability of the apical membrane to K+ by increasing the number of K+ channels in the membrane. However, the cellular mechanisms involved in this response are not completely known. Increased expression of Na+,K+-ATPase facilitates uptake of K+ across the basolateral membrane into cells and thereby elevates intracellular [K+]. The increase in the number and activity of Na+ channels enhances entry of Na+ into the cell from tubular fluid, an effect that depolarizes the apical membrane voltage. Depolarization of the apical membrane and increased intracellular [K+] enhance the electrochemical driving force for secretion of K+ from the cell into the tubule fluid. Taken together, these actions increase uptake of K+ into the cell across the basolateral membrane and enhance exit of K+ from the cell across the apical membrane. Secretion of aldosterone is increased by hyperkalemia and by angiotensin II (after activation of the renin-angiotensin system). Secretion of aldosterone is decreased by hypokalemia and natriuretic peptides released from the heart.

CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis

Although an acute (e.g., within hours) increase in aldosterone levels enhances the activity of Na+,K+-ATPase, K+ excretion does not increase immediately. The reason for this relates to the effect of aldosterone on Na+ reabsorption and tubular flow. Aldosterone stimulates reabsorption of Na+ and water and thus decreases tubular flow. Reduction in flow in turn decreases K+ secretion (as discussed in more detail later). However, chronic stimulation of Na+ reabsorption increases the ECFV and thereby returns tubular flow to normal. These actions allow a direct stimulatory effect of aldosterone on the ASDN to enhance K+ excretion.

Although AVP does not affect urinary K+ excretion, this hormone does promote secretion of K+ by the ASDN (Fig.

36.8 ). AVP increases the electrochemical driving force for exit of K+ across the apical membrane of principal cells by stimulating uptake of Na+ across the apical membrane of these cells. The increased Na+ uptake reduces the electrical potential difference across the apical membrane (i.e., the interior of the cell becomes less negatively charged). Despite this effect, AVP does not change K+ secretion by these nephron segments. The reason for this relates to the effect of AVP on tubular fluid flow. AVP decreases flow of tubular fluid by stimulating water reabsorption. The decrease in tubular flow in turn reduces secretion of K+ (explained later). The inhibitory effect of decreased flow of tubular fluid offsets the stimulatory effect of AVP on the electrochemical driving force for exit of K+ across the apical membrane (see

Fig. 36.8 ). If AVP did not increase the electrochemical gradient favoring K+ secretion, urinary K+ excretion would fall as AVP levels increased and urinary flow rates decreased. Hence K+ balance would change in • Fig. 36.8 Opposing effects of AVP and urine flow rate on secretion of K+by the ASDN. K+secretion is stimulated by an increase in urinary flow rate and reduced by a fall in AVP levels. In contrast, K+secretion is reduced by a decrease in urinary flow rate and increased by a rise in AVP levels. Because the effects of flow and AVP oppose each other, net K+secretion is not affected by water diuresis or antidiuretics. ˜ Urinary flow rate Water diuresis DT/CCD K+ secretion ˛ AVP levels Constant K+ balance ˛ Urinary flow rate DT/CCD K+ secretion ˜ AVP levels Antidiuresis + _ _ + response to alterations in water balance. Thus the effects of AVP on the electrochemical driving force for exit of K+ across the apical membrane and on tubule flow enable urinary K+ excretion to be maintained constant despite wide fluctuations in water excretion.

Plasma [K+], aldosterone, and AVP play important roles in regulating K+ balance; however, the factors and hormones discussed next perturb K+ balance (see

Box 36.2

Flow of Tubular Fluid

A rise in the flow of tubular fluid (e.g., with diuretic treatment, ECFV expansion) stimulates secretion of K+ within minutes, whereas a fall (e.g., ECFV contraction caused by hemorrhage, severe vomiting, or diarrhea) reduces secretion of K+ by the late segment of the ASDN. Increments in tubular fluid flow are more effective in stimulating secretion of K+ as dietary K+ intake is increased. Recent studies on the primary cilium in principal cells have elucidated some of the mechanisms whereby increased flow stimulates secretion of K+ (

Fig. 36.9 ). Increased flow bends the primary cilium in principal cells, which activates the PKD1/PKD2 Ca++-conducting channel complex. This allows more Ca++ to enter principal cells and increases intracellular [Ca++]. The increase in [Ca++] activates BK K+ channels in the apical plasma membrane, which enhances K+ secretion from the cell into the tubule fluid. Increased flow may also stimulate secretion of K+ by other mechanisms. As flow increases, such as after administration of diuretics or as the result of an increase in ECFV, so does the [Na+] of tubule fluid. This increase in [Na+] facilitates entry of Na+ across the apical membrane of ASDN cells, thereby decreasing the cells’

K+ K+ ° Flow stimulates Na+ entry, which reduces Vm ° Flow Cilia bend activates PKD1/PKD2 and Ca++ entry ° Flow bends cilia ° Ca++ activates Na+ Ca++ ATP 1 25 4 3 • Fig. 36.9 Cellular mechanism whereby an increased flow rate of tubule fluid stimulates secretion of K+ by principal cells. See text for details.

interior negative membrane potential. This depolarization of the cell membrane potential increases the electrochemical driving force that promotes secretion of K+ across the apical cell membrane into tubule fluid. In addition, increased uptake of Na+ into cells activates the Na+,K+-ATPase in the basolateral membrane, thereby increasing uptake of K+ across the basolateral membrane and consequently elevating [K+]. However, it is important to note that an increase in flow rate during a water diuresis does not have a significant effect on excretion of K+ , most likely because during a water diuresis the [Na+] of tubule fluid does not increase as flow rises.

Another factor that modulates secretion of K+ is the [H+] of ECF. Acute alterations (within minutes to hours) in the pH of plasma influences secretion of K+ by the ASDN. Alkalosis (i.e., plasma pH above normal) increases secretion of K+ , whereas acidosis (i.e., plasma pH below normal) decreases it. An acute acidosis reduces K+ secretion via two mechanisms: (1) it inhibits Na+,K+-ATPase and thereby reduces cell [K+] and the electrochemical driving force for exit of K+ across the apical membrane, and (2) it reduces the permeability of the apical membrane to K+ . Alkalosis has the opposite effects.

The effect of metabolic acidosis on excretion of K+ is time dependent. As already noted, and illustrated in

Fig. 36.10 , acute metabolic acidosis reduces K+ excretion. However, when metabolic acidosis lasts for several days, urinary K+ excretion is stimulated (see

Fig. 36.10 ). This occurs because chronic metabolic acidosis decreases reabsorption of water and solutes (e.g., NaCl) by the proximal tubule by

ROMK is the primary channel in the apical membrane of principal cells that mediates K+ secretion. Four ROMK subunits make up a single channel. In addition the BK K+ channel, which is activated by elevations in intracellular [Ca++], is also expressed in the apical membrane. The BK channel mediates the flow-dependent increase in K+ secretion discussed earlier. Interestingly, knockout of the KCNJ1 gene (ROMK) causes increased excretion of NaCl and K+ by the kidneys, thereby leading to reduced ECFV and hypokalemia. Although this effect is somewhat perplexing, it should be noted that ROMK is also expressed in the apical membrane of the thick ascending limb (TAL) of Henle’s loop, where it plays a very important role in recycling of K+ across the apical membrane, an effect that is critical for operation of the Na+/ K+/2Cl− symporter (see

Fig. 36.7 ). In the absence of ROMK, reabsorption of NaCl by the TAL is reduced, which leads to loss of NaCl in urine. Reduction of NaCl reabsorption by the TAL also reduces the positive transepithelial luminal voltage, which is the driving force for reabsorption of K+ by this nephron segment. Thus the reduction in paracellular K+ reabsorption by the TAL increases urinary K+ excretion even when the cortical collecting duct is unable to secrete the normal amount of K+ because of a lack of ROMK channels. The cortical collecting duct, however, does secrete K+ even in ROMK knockout mice via the flow and Ca++-dependent BK K+ channel expressed in the apical membrane of principal cells.

inhibiting Na+,K+-ATPase. Hence the flow of tubular fluid is augmented along the ASDN. Inhibition of water and NaCl reabsorption by the proximal tubule also decreases ECFV and thereby stimulates secretion of aldosterone. In addition, chronic acidosis caused by inorganic acids increases plasma [K+], which stimulates secretion of aldosterone. The rise in tubular fluid flow, plasma [K+], and aldosterone levels offsets the effects of acidosis on cell [K+] and apical membrane permeability, and K+ secretion rises. Thus metabolic acidosis may either inhibit or stimulate excretion of K+ , depending on the duration of the disturbance. As noted, acute metabolic alkalosis stimulates excretion of K+ . Chronic metabolic alkalosis, especially in association with ECFV contraction, significantly increases renal K+ excretion because of the associated increased levels of aldosterone.

CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis • Fig. 36.10 Acute versus chronic effect of metabolic acidosis on excretion of K+. See text for details. ECV, effective circulating volume. ˛ Na+,K+-ATPase activity ˛ K+ permeability of apical membrane ˛ Apical membrane K+ gradient Metabolic acidosis Acute Chronic Distal tubule and collecting duct principal cells ˜ Na+,K+-ATPase activity ˜ K+ permeability of apical membrane ˜ K+ secretion ˜ K+ excretion Skeletal muscle cell Proximal tubule cell ˛ H+/K+ exchange ˜ NaCl and H2O reabsorption ˜ ECV ˛ Plasma [K+] ˛ Tubule fluid flow rate Distal tubule and collecting duct principal cells ˛ Aldosterone ˛ Aldosterone ˛ K+ secretion ˛ K+ excretion

Glucocorticoids increase urinary K+ excretion. This effect is mediated in part by an increase in GFR, which enhances the urinary flow rate, a potent stimulus of K+ excretion, and by stimulation of SGK1 activity (see earlier).

As discussed, the rate of urinary K+ excretion is frequently determined by simultaneous changes in hormone levels, acid-base balance, or the flow rate of tubule fluid ( 36.1 ). The powerful effect of flow often enhances or opposes the response of the ASDN to hormones and changes in acid-base balance. This interaction can be beneficial in the case of hyperkalemia, in which the increase in flow enhances excretion of K+ and thereby restores K+ homeostasis. However, this interaction can also be detrimental, as in the case of alkalosis, in which changes in flow and acid-base status alter K+ homeostasis.

Overview of Calcium and Inorganic Phosphate Homeostasis are multivalent ions that subserve many complex and vital functions. Ca++ is an important cofactor in many enzymatic reactions; it is a key second messenger in numerous signaling pathways; it plays an important role in neural transduction, blood clotting, and muscle contraction; and it is a critical component of the extracellular matrix, cartilage, teeth, and bone. Pi, like Ca++ , is a key component of bone. Pi is essential for metabolic aAt physiological pH, inorganic phosphate exists as HPO4 − and H2PO4 − (pK = 6.8). For simplicity, we collectively refer to these ion species as Pi.

Modified from Field MJ et al. In Narins R (ed). Textbook of Nephrology: Clinical Disorders of Fluid and Electrolyte Metabolism. 5th ed. New York: McGraw-Hill; 1994.

The cellular mechanisms whereby changes in the K+ content of the diet and acid-base balance regulate secretion of K+ by the early segment of the ASDN have recently been elucidated. Elevated K+ intake increases secretion of K+ by several mechanisms, all related to increased serum [K+]. Hyperkalemia increases the activity of the ROMK channel in the apical plasma membrane of principal cells. Moreover, hyperkalemia inhibits reabsorption of NaCl and water by the proximal tubule, thereby increasing the ASDN flow rate, a potent stimulus to secretion of K+ . Hyperkalemia also enhances [aldosterone], which increases K+ secretion by three mechanisms. First, aldosterone increases the number of K+ channels in the apical plasma membrane. Second, aldosterone stimulates uptake of K+ across the basolateral membrane by increasing the number of Na+,K+-ATPase pumps, thereby enhancing the electrochemical gradient driving secretion of K+ across the apical membrane. Third, aldosterone increases movement of Na+ across the apical membrane, which depolarizes the apical plasma membrane voltage and thus increases the electrochemical gradient, promoting secretion of K+ . A low-K+ diet dramatically reduces secretion of K+ by the ASDN by increasing the activity of protein tyrosine kinase, which causes ROMK channels to be endocytosed from the apical plasma membrane, thereby reducing K+ secretion. Acidosis decreases secretion of K+ by inhibiting the activity of ROMK channels, whereas alkalosis stimulates secretion of K+ by enhancing ROMK channel activity.

processes, including formation of adenosine triphosphate (ATP), and it is an important component of nucleotides, nucleosides, and phospholipids. Phosphorylation of proteins is an important mechanism of cellular signaling, and Pi is an important buffer in cells, plasma, and urine.

In adults the kidneys play important roles in regulating total body Ca++ and Pi by excreting the amount of Ca++ and Pi that is absorbed by the intestinal tract (normal bone remodeling results in no net addition of Ca++ and Pi to, or Ca++ and Pi release from, bone). If plasma concentrations of Ca++ and Pi decline substantially, intestinal absorption, bone resorption (i.e., loss of Ca++ and Pi from bone), and renal tubular reabsorption increase and return plasma concentrations of Ca++ and Pi to normal levels. During growth and pregnancy, intestinal absorption exceeds urinary excretion, and these ions accumulate in newly formed fetal tissue and bone. In contrast, bone disease (e.g., osteoporosis) or a decline in lean body mass increases urinary Ca++ and Pi loss without a change in intestinal absorption. These conditions produce a net loss of Ca++ and Pi from the body. Finally, during chronic renal failure, Pi accumulates in the body because absorption by the intestinal tract exceeds excretion in the urine. This can lead to accumulation of Pi in the body and changes in bone (see the In The Clinic box discussion of chronic renal failure).

This brief introduction reveals that the kidneys, in conjunction with the GI tract and bone, play a major role in maintaining plasma Ca++ and Pi levels as well as Ca++ and Pi balance (see ). Accordingly this section of the chapter discusses Ca++ and Pi handling by the kidneys, with an emphasis on the hormones and factors that regulate urinary excretion.

Cellular processes in which Ca++ plays an important role include bone formation, cell division and growth, blood coagulation, hormone-response coupling, and electrical stimulus-response coupling (e.g., muscle contraction, neurotransmitter release). Nearly 99% of Ca++ is stored in bone and teeth, approximately 1% is found in ICF, and 0.1% in ECF. The total Ca++ concentration ([Ca++]) in plasma is 10 mg/dL (2.5 mM or 5 mEq/L), and its concentration is normally maintained within very narrow limits. Approximately 50% of the Ca++ in plasma is ionized, 40% is bound to plasma proteins (mainly albumin), and 10% is complexed to several anions, including Pi, HCO3 − , citrate, and SO42− (Fig. 36.11 ). The pH of plasma influences this distribution (

Fig. 36.12). Acidemia increases the

CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis 1.25 mmol/L E A S 0.25 mmol/L R 1.0 mmol/L D • Fig. 36.12 Effect of pH on plasma [Ca++]. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) • Fig. 36.11 Distribution of Ca++ in plasma. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) • Fig. 36.13 Effect of Ca++ on nerve and muscle excitability. (From Koeppen BM, Stanton BA. Renal

Physiology. 5th ed. Philadelphia: Elsevier; 2013.) percentage of ionized Ca++ at the expense of Ca++ bound to proteins, whereas alkalemia decreases the percentage of ionized Ca++, again by altering the Ca++ bound to proteins. Individuals with alkalemia are susceptible to tetany (tonic muscular spasms), whereas individuals with acidemia are less susceptible to tetany, even when total plasma Ca++ levels are reduced. The increase in [H+] in patients with metabolic acidosis causes more H+ to bind to plasma proteins, Pi, HCO3 − , citrate, and SO42− , thereby displacing Ca++ . This displacement increases the plasma concentration of ionized Ca++ . In alkalemia the [H+] of plasma decreases. Some H+ ions dissociate from plasma proteins, Pi, HCO3 − , citrate, and SO42− in exchange for Ca++ , thereby decreasing the plasma concentration of ionized Ca++ . In addition, the plasma albumin concentration also affects ionized plasma [Ca++].

Hypoalbuminemia increases the ionized [Ca++], whereas hyperalbuminemia decreases ionized plasma [Ca++]. The total measured plasma [Ca++] does not reflect the total ionized [Ca++], which is the physiologically relevant measure of plasma [Ca++]. A low ionized plasma [Ca++] (hypocalcemia) increases the excitability of nerve and muscle cells and can lead to hypocalcemic tetany. Tetany associated with hypocalcemia occurs because hypocalcemia causes the threshold potential to shift to more negative values (i.e., closer to the resting membrane voltage) (

Fig.

36.13). An elevated ionized plasma [Ca • Fig. 36.14 Overview of Ca++ homeostasis. PTH, parathyroid hormone. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) arrhythmias, lethargy, disorientation, and even death.

This effect of hypercalcemia occurs because an elevated plasma [Ca++] causes the threshold potential to shift to less negative values (i.e., farther from the resting membrane voltage). Plasma [Ca++] is regulated within a very narrow range, primarily by parathyroid hormone (PTH), calcitriol (1,25-dihydroxyvitamin D), the active metabolite of vitamin D3, and plasma Ca++ , and this regulation will be discussed next.

Within cells, Ca++ is sequestered in the endoplasmic reticulum and mitochondria, or it is bound to proteins. Thus the free intracellular [Ca++] is very low (≈100 nM). The large concentration gradient for [Ca++] across cell membranes is maintained by a Ca++-ATPase pump (PMCa1b) in all cells and by a 3Na+/Ca++ exchanger (NCX1) in some cells.

Overview of Calcium Homeostasis

Ca++ homeostasis depends on two factors: (1) the total amount of Ca++ in the body and (2) the distribution of Ca++ between bone and ECF. The total body Ca++ level is determined by the relative amounts of Ca++ absorbed by the intestinal tract and excreted by the kidneys (Fig.

36.14 ). The intestinal tract absorbs Ca++ through an active carrier-mediated transport mechanism that is stimulated by calcitriol, the active metabolite of vitamin D3 that is produced in the proximal tubule of the kidneys. Net Ca++ absorption by the intestine is normally 200 mg/day, but it can increase to 600 mg/day when calcitriol levels rise. In adults, Ca++ excretion by the kidneys equals the amount absorbed by the GI tract (200 mg/day), and it changes in bIn clinical practice the terms hypercalcemia and hypocalcemia are often used to describe a high or low total plasma [Ca++], respectively, even though this usage is not the physiologically correct usage of hypercalcemia and hypocalcemia.

parallel with intestinal absorption. Thus in adults, Ca++ balance is maintained because the amount of Ca++ ingested in an average diet (1000 mg/day) equals the amount lost in feces (800 mg/day, the amount that escapes absorption by the intestinal tract) plus the amount excreted in urine (200 mg/day).

The second factor that controls Ca++ homeostasis is the distribution of Ca++ between bone and ECF (see

Fig. 36.14 ). Two hormones (PTH and calcitriol ) regulate the distribution of Ca++ between bone and ECF and thereby, in concert with the kidneys, regulate plasma [Ca++]. PTH is secreted by the parathyroid glands, and its secretion is stimulated by a decline in plasma [Ca++] (i.e., hypocalcemia). Plasma Ca++ is an agonist of the calcium sensing receptor (CaSR), which is located in the plasma membrane of chief cells in parathyroid glands (discussed later). Hypercalcemia activates the CaSR, which decreases PTH release, whereas hypocalcemia reduces CaSR activity that in turn increases PTH release. PTH increases plasma [Ca++] by: (1) stimulating bone resorption, (2) increasing Ca++ reabsorption by the distal tubule of the kidney, and (3) stimulating the production of calcitriol, which in turn increases Ca++ absorption by the intestinal tract and facilitates PTH-mediated bone resorption. Production of calcitriol in the kidney is stimulated by hypocalcemia and hypophosphatemia. Calcitriol increases plasma [Ca++], primarily by stimulating Ca++ absorption from the intestinal tract. It also facilitates the action of PTH on bone and enhances Ca++ reabsorption in the kidneys by increasing expression of key Ca++ transport and binding proteins in the kidneys (details discussed later). In addition, cCalcitonin is secreted by thyroid C cells (parafollicular cells), and its secretion is stimulated by hypercalcemia. Calcitonin decreases plasma [Ca++], mainly by stimulating bone formation (i.e., deposition of Ca++ in bone). Although it plays an important role in Ca++ homeostasis in lower vertebrates, calcitonin plays only a minor role in Ca++ homeostasis in humans, so it will not be discussed further.

hypercalcemia activates the CaSR in the TAL of Henle’s loop, inhibiting Ca++ reabsorption in this segment, resulting in an increase in urinary Ca++ excretion and thereby a reduction in plasma [Ca++]. Hypocalcemia has the opposite effect. Importantly, regulation of Ca++ excretion by the kidneys is one of the major ways the body regulates plasma [Ca++].

Conditions that lower PTH levels (i.e., hypoparathyroidism after parathyroidectomy for an adenoma) reduce plasma [Ca++], which can cause hypocalcemic tetany (intermittent muscular contractions). In severe cases, hypocalcemic tetany can cause death by asphyxiation. Hypercalcemia can also cause lethal cardiac arrhythmias and decreased neuromuscular excitability. Clinically the most common causes of hypercalcemia are primary hyperparathyroidism and malignancy-associated hypercalcemia. Primary hyperparathyroidism results most often from overproduction of PTH caused by a benign tumor of the parathyroid glands. In contrast, malignancy-associated hypercalcemia, which occurs in 10% to 20% of all patients with cancer, is caused by secretion of parathyroid hormone–related peptide (PTHrP), a PTH-like hormone secreted by carcinomas in various organs. Increased levels of PTH and PTHrP cause hypercalcemia and hypercalciuria.

Calcium Transport Along the Nephron

The Ca++ available for glomerular filtration consists of the ionized fraction and the amount complexed with anions. Thus about 60% of the Ca++ in plasma is available for glomerular filtration. Normally 99% of filtered Ca++ is reabsorbed by the nephron (Fig. 36.15 ). The proximal tubule reabsorbs about 50% to 60% of the filtered Ca++ . Another 15% is reabsorbed in the loop of Henle (mainly the cortical • Fig. 36.15 Overview of Ca++ transport along the nephron. Percentages refer to amount of filtered Ca++ reabsorbed by each segment. CCD, cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; PT, proximal tubule; TAL, thick ascending limb.

CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis portion of the TAL), about 10% to 15% is reabsorbed by the distal tubule, and less than 1% is reabsorbed by the collecting duct. About 1% (200 mg/day) is excreted in urine. This fraction is equal to the net amount absorbed daily by the intestinal tract.

Ca++ reabsorption by the proximal tubule occurs primarily via the paracellular pathway. This passive paracellular reabsorption of Ca++ is driven by the lumen-positive trans-epithelial voltage (Vte) across the second half of the proximal tubule and by a favorable concentration gradient of Ca++ , both of which are established by transcellular sodium and water reabsorption in the first half of the proximal tubule (see

Ca++ reabsorption by the loop of Henle also occurs primarily via the paracellular pathway. Like the proximal tubule, Ca++ and Na+ reabsorption in the TAL parallel each other. These processes are parallel because of the significant component of Ca++ reabsorption that occurs via passive paracellular reabsorption secondary to Na+ reabsorption that generates a lumen-positive Vte. Loop diuretics inhibit Na+ reabsorption by the TAL of the loop of Henle, and in so doing reduce the magnitude of the lumen-positive Vte (see ). This action in turn inhibits reabsorption of Ca++ via the paracellular pathway. Thus loop diuretics are used to increase renal Ca++ excretion in patients with hypercalcemia.

Mutations in the tight junction protein claudin-16 (CLDN16) reduce the permeability of the paracellular pathway to Ca++ and Mg++ and thereby reduce the diffusive reabsorptive movement of Ca++ and Mg++ across tight junctions in the TAL of Henle’s loop. Familial hypomagnesemic hypercalciuria is caused by mutations in claudin-16, which is a component of the tight junctions in TAL cells. This disorder is characterized by enhanced excretion of Ca++ and Mg++ due to a fall in passive reabsorption of these ions across the paracellular pathway in the TAL. Affected individuals have high levels of Ca++ in their urine, which leads to stone formation (nephrolithiasis).

In the distal tubule where the voltage in the tubule lumen is electrically negative with respect to blood, reabsorption of Ca++ is entirely active because Ca++ is reabsorbed against its electrochemical gradient (Fig. 36.16 ). Thus Ca++ reabsorption by the distal tubule is exclusively transcellular. Calcium enters the cell across the apical membrane through Ca++-permeable epithelial ion channels (TRPV5). Inside the cell, Ca++ binds to calbindin-D28K. The calbindin-Ca++ complex carries Ca++ across the cell and delivers Ca++ to the basolateral membrane, where it is extruded from the cell primarily by the 3Na+/1Ca++ antiporter (NCX1); however, plasma membrane Ca++-ATPase isoform 1b (PMCA1b) may also contribute. Urinary Na+ and Ca++ excretion usually change in parallel. However, excretion of these ions does not always change in parallel, because reabsorption of Ca++ and Na+ by the distal tubule is independent and differentially • Fig. 36.16 Cellular mechanism of Ca++ reabsorption by the distal tubule. Ca++ is reabsorbed exclusively by a cellular pathway. Ca++ enters the cell across the apical membrane via a Ca++-permeable ion channel (TRPV5). Inside cells, Ca++ binds to calbindin (calbindin-D28K), and the Ca++-calbindin complex diffuses across the cell to deliver Ca++ to the basolateral membrane. Ca++ is transported across the basolateral membrane primarily by a 3 (or 4) Na+/Ca++ antiporter (NCX1) and also by a Ca++-H+-ATPase (PMCa1b). Claudin 8 (CLDN8) is a tight junction protein that is impermeable to Ca++ and thereby prevents the back diffusion of Ca++ across the tight junction into the tubule lumen, which is electrically negative compared to the blood side of the cell.

regulated. For example, thiazide diuretics inhibit Na+ reabsorption by the distal tubule and stimulate Ca++ reabsorption by this segment. Accordingly the net effects of thiazide diuretics are to increase urinary Na+ excretion and reduce urinary Ca++ excretion. Because thiazide diuretics reduce urinary Ca++ excretion, they are often given to reduce urinary [Ca++] in individuals who produce Ca++-containing kidney stones.

Regulation of Urinary Calcium Excretion

Table 36.2 ). Of these, PTH exerts the most powerful control on renal Ca++ excretion; it is the primary hormone/ factor responsible for maintaining Ca++ homeostasis. Overall this hormone stimulates Ca++ reabsorption by the kidneys (i.e., reduces Ca++ excretion). Although PTH inhibits reabsorption of NaCl and fluid, and therefore Ca++ reabsorption by the proximal tubule, PTH stimulates Ca++ reabsorption by the TAL of the loop of Henle and the distal tubule. Thus the net effect of PTH is to enhance renal Ca++ reabsorption. Changes in plasma [Ca++] also regulate urinary Ca++ excretion, with hypercalcemia increasing excretion and hypocalcemia decreasing excretion. Hypercalcemia increases urinary Ca++ excretion by: (1) reducing proximal tubule Ca++ reabsorption (reduced paracellular reabsorption due to increased interstitial fluid [Ca++]); (2) inhibiting Ca++ reabsorption by the TAL of the loop of Henle via activation of the CaSR located in the basolateral membrane of these cells (NaCl reabsorption is decreased, thereby reducing the magnitude of the lumen-positive Vte); and (3) suppressing Ca++ reabsorption by the distal tubule by reducing PTH levels. As a result, urinary Ca++ excretion increases. Hypocalcemia has the opposite effect on urinary Ca++ excretion, primarily by increasing Ca++ reabsorption by the proximal tubule and TAL. Calcitriol enhances Ca++ reabsorption by the distal tubule, but it is less effective than PTH.

Several factors disturb Ca++ excretion. An increase in plasma [Pi] concentration (e.g., caused by a dramatic increase in dietary intake of Pi or by reduced kidney function) elevates PTH levels both directly and by decreasing the ionized plasma [Ca++] and thereby decreases Ca++ excretion. A decline in plasma [Pi] (e.g., caused by dietary Pi depletion) has the opposite effect (NOTE: with normal kidney function, changes in dietary Pi intake over a sevenfold range has no effect on plasma [Pi]). Changes in ECFV alter Ca++ excretion mainly by affecting NaCl and fluid reabsorption in the proximal tubule. Volume contraction increases NaCl and water reabsorption by the proximal tubule and thereby enhances Ca++ reabsorption. Accordingly, urinary Ca++ excretion declines. Volume expansion has the opposite effect. Acidemia increases Ca++ excretion, whereas alkalemia decreases excretion. Regulation of Ca++ reabsorption by pH

CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis aPTH inhibits Ca++ reabsorption by the proximal tubule but stimulates reabsorption by the TAL and distal tubule. Overall the net effect is to increase Ca++ reabsorption and thereby reduce urinary Ca++ excretion. CaSR, calcium-sensing receptor. Modified from Mount DB, Yu A. Transport of inorganic solutes: sodium, chloride, potassium, magnesium, calcium and phosphate. In: Brenner BM (ed). Brenner and Rector’s The Kidney. 8th ed. Philadelphia: Saunders; 2008.

occurs primarily in the distal tubule. Alkalosis stimulates the apical membrane Ca++ channel (TRPV5), thereby increasing Ca++ reabsorption. By contrast, acidosis inhibits the same channel, thereby reducing Ca++ reabsorption. Finally, as noted earlier, loop diuretics inhibit Ca++ reabsorption by the TAL, and thiazide diuretics stimulate Ca++ reabsorption by the distal tubule.

The calcium-sensing receptor (CaSR) is a receptor expressed in the plasma membrane of cells involved in regulating Ca++ homeostasis; it senses small changes in extracellular [Ca++]. Ca++ binds to CaSR receptors in PTH-secreting cells of the parathyroid gland and calcitriol-producing cells of the proximal tubule. Activation of the receptor by an increase in plasma [Ca++] results in inhibition of PTH secretion and production of calcitriol by the proximal tubule. Moreover, reduction in PTH secretion also contributes to decreased production of calcitriol because PTH is a potent stimulus of calcitriol synthesis. By contrast, a fall in plasma [Ca++] has the opposite effect on PTH and calcitriol secretion.

The CaSR also maintains Ca++ homeostasis by directly regulating Ca++ excretion by the kidneys. CaSRs in the TAL respond directly to changes in plasma [Ca++] and regulate Ca++ absorption. An increase in plasma [Ca++] activates CaSR in the TAL and inhibits Ca++ absorption, thereby stimulating urinary Ca++ excretion. By contrast, a fall in plasma [Ca++] leads to an increase in Ca++ absorption by the TAL and a corresponding decrease in urinary Ca++ excretion. Thus the direct effect of plasma [Ca++] on CaSRs in the TAL acts in concert with changes in PTH, which regulates Ca++ reabsorption by the distal tubule to regulate urinary Ca++ excretion and thereby maintain Ca++ homeostasis.

Mutations in the gene coding for CaSR cause disorders in Ca++ homeostasis. Familial hypocalciuric hypercalcemia (FHH) is a haploinsufficient state caused by an inactivating mutation of CaSR. The hypercalcemia is caused by deranged Ca++-regulated PTH secretion (i.e., the set point for Ca++-regulated PTH secretion is shifted such that PTH levels are elevated at any level of plasma [Ca++], and are not suppressed in the setting of hypercalcemia). The hypocalciuria is caused by enhanced Ca++ reabsorption in the TAL and distal tubule owing to elevated PTH levels and defective CaSR regulation of Ca++ transport in the kidneys. Autosomal-dominant hypoparathyroidism is caused by an activating mutation in CaSR. Activation of CaSRs causes deranged Ca++-regulated PTH secretion (i.e., the set point for Ca++-regulated PTH secretion is shifted such that PTH levels are decreased at any level of plasma [Ca++]). Hypercalciuria results and is caused by decreased PTH levels and defective CaSR-regulated Ca++ transport in the kidneys.

Pi is an important component of many organic molecules, including DNA, RNA, ATP, nucleotides, nucleosides, and phospholipids and intermediates of metabolic pathways. Like Ca++ it is a major constituent of bone. Its concentration in plasma is an important determinant of bone formation and resorption. In addition, urinary Pi is an important buffer (i.e., it is one of many titratable acids) involved in the maintenance of acid-base balance (see ). Some 85% of Pi is located in bone and teeth, 14% is located in the ICF, and 1% is located in the ECF. Normal plasma • Fig. 36.17 Overview of Pi homeostasis. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) [Pi] is 3 to 4 mg/dL (1–1.5 mM). Pi in plasma is ionized (45%), complexed (30%), and bound to protein (25%). Phosphate deficiency causes muscle weakness, rhabdomyolysis, and reduced bone mineralization resulting in rickets (in children) and osteomalacia (in adults).

Overview of Phosphate Homeostasis

A general scheme of Pi homeostasis is shown in

Fig. 36.17 . Maintenance of Pi homeostasis depends on two factors: (1) the amount of Pi in the body and (2) the distribution of Pi between the ICF and ECF compartments. Total body Pi levels are determined by the relative amount of Pi absorbed by the intestinal tract versus the amount excreted by the kidneys. Pi absorption by the intestinal tract occurs via active and passive mechanisms; Pi absorption increases as dietary Pi rises, and it is stimulated by calcitriol. Despite variations in Pi intake between 800 and 1500 mg/day, in adults (i.e., the steady state) the kidneys maintain total body Pi balance constant by excreting an amount of Pi in the urine equal to the amount absorbed by the intestinal tract (normal bone remodeling results in no net addition of Pi to, or Pi release from, bone). By contrast, during growth, Pi is accumulated in the body. Renal Pi excretion is the primary mechanism by which the body regulates Pi balance and thereby Pi homeostasis.

The second factor that maintains Pi homeostasis is distribution of Pi among bone and the ICF and ECF compartments. Regulation of plasma [Pi] is controlled by two hormones, PTH and calcitriol, in concert with the kidneys (see

Fig. 36.17 ). Release of Pi from bone is stimulated by the same hormones (i.e., PTH and calcitriol) that release Ca++ from this pool. Thus release of Pi from bone is always accompanied by release of Ca++ .

The kidneys also make an important contribution to maintaining plasma [Pi] within a narrow range (1–1.5 mM).

Pi excretion by the kidneys is regulated by PTH and calcitriol (see

Fig. 36.17 ). PTH increases Pi excretion, whereas calcitriol inhibits Pi excretion. Plasma [Pi] is determined by: (1) intestinal absorption, (2) storage in bone, and (3) Pi excretion by the kidneys. Maintenance of plasma [Pi] is essential for optimal Ca++-Pi complex formation required for bone mineralization without deposition of Ca++-Pi in vascular and other soft tissues.

A rise in plasma [Pi] directly stimulates PTH synthesis and release and also decreases the ionized [Ca++], which stimulates PTH release by its interaction with the CaSR. PTH enhances urinary Pi excretion by inhibiting proximal tubule Pi reabsorption. Hyperphosphatemia also decreases calcitriol production by the proximal tubule, which leads to a reduction in Pi absorption by the intestine. Both the increase in PTH and the decrease in calcitriol reduce  plasma [Pi].

Phosphate Transport Along the Nephron

Fig. 36.18 summarizes Pi transport by the various portions of the nephron. The proximal tubule reabsorbs 80% of the Pi filtered by the glomerulus; the loop of Henle, distal tubule, and collecting duct reabsorb negligible amounts of Pi. Therefore approximately 20% of the Pi filtered across the glomerular capillaries is excreted in urine.

Pi reabsorption by the proximal tubule occurs by a transcellular route (

Fig. 36.19 ). Pi uptake across the apical membrane of the proximal tubule occurs via two Na+/Pi symporters (IIa and IIc). Type IIa transports 3Na+ with one divalent Pi (HPO42−), and carries positive charge into the cell. Type IIc transports 2Na+ with one monovalent Pi (H2PO4 −) and is electrically neutral. Pi exits across the basolateral membrane by a Pi-inorganic anion antiporter that has not been characterized.

CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis • Fig. 36.18 Pi transport along the nephron. Pi is reabsorbed primarily by the proximal tubule. Percentages refer to the amount of the filtered Pi reabsorbed by each nephron segment. Approximately 20% of the filtered Pi is excreted. CCD, cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; PT, proximal tubule; TAL, thick ascending limb.

In patients with chronic renal failure, the kidneys cannot excrete Pi. Because of continued Pi absorption by the intestinal tract, Pi accumulates in the body and plasma [Pi] rises. The excess Pi complexes with Ca++ and reduces the ionized plasma [Ca++]. Pi accumulation also decreases production of calcitriol. This response reduces Ca++ absorption by the intestine, an effect that further reduces plasma [Ca++]. This reduction in plasma [Ca++] increases PTH secretion and Ca++ release from bone. These actions result in renal osteodystrophy (i.e., increased bone resorption with replacement by fibrous tissue, which renders bone more susceptible to fracture). Chronic hyperparathyroidism (i.e., elevated PTH levels due the fall in plasma [Ca++]) during chronic renal failure can lead to metastatic calcifications in which Ca++ and Pi precipitate in arteries, soft tissues, and viscera. Deposition of Ca++ and Pi in the heart may cause myocardial failure. Prevention and treatment of hyperparathyroidism and Pi retention include a low-Pi diet or administration of a “phosphate binder” (i.e., an agent that forms insoluble Pi salts and thereby renders Pi unavailable for absorption by the intestinal tract) in the diet. Supplemental Ca++ and calcitriol are also prescribed to increase plasma [Ca++].

• Fig. 36.19 Cellular mechanisms of Pi reabsorption by the proximal tubule. The apical transport pathway contains two Na+/Pi symporters, one that transports three Na+for each Pi (IIa) and one that transports two Na+for each Pi (IIc). Pi leaves the cell across the basolateral membrane by an unknown mechanism. ATP, adenosine triphosphate. TubularfluidBloodPiPiK˜Na˜2Na˜Pi3Na˜ATPIIaIIc?

Fibroblast growth factor 23 (FGF-23) increases renal Pi excretion and thereby contributes to regulation of plasma [Pi] (see

Fig. 36.17 ). FGF-23 is secreted by osteocytes and osteoblasts and inhibits Pi reabsorption and calcitriol production by the proximal tubule. Secretion of FGF-23 is stimulated by sustained hyperphosphatemia, PTH, and calcitriol. Activating mutations in the FGF23 gene cause hypophosphatemia, low plasma calcitriol, and rickets/osteomalacia, whereas inactivating mutations cause hyperphosphatemia, high serum calcitriol, and calcification of soft tissue.

Regulation of Urinary Phosphate Excretion

Table 36.3

Fig. 36.20 ). Increased plasma Pi reduces

TABLE Summary of Hormones and Factors 36.3 Affecting Pi Reabsorption by Proximal Tubule

PTH Decrease FGF-23 Decrease Phosphate loading Decrease Phosphate depletion Increase Metabolic acidosis: chronic Decrease Metabolic alkalosis: chronic Increase ECFV expansion Decrease Growth hormone Increase Glucocorticoids Decrease • Fig. 36.20 Overview of the major hormones regulating plasma [Pi]. FGF-23, fibroblast growth factor 23; PTH, parathyroid hormone. Dashed lines indicate negative feedback. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) plasma [Ca++] and therefore increases plasma PTH, which increases Pi excretion by the kidneys. PTH, the most important hormone that controls Pi excretion, inhibits Pi reabsorption by the proximal tubule and thereby increases Pi excretion. PTH reduces Pi reabsorption by stimulating endocytic removal of Na+/Pi transporters from the brush border membrane of the proximal tubule. Increased plasma Pi also increases FGF-23, which inhibits Pi reabsorption and calcitriol production by the proximal tubule. Elevated plasma Pi also suppresses calcitriol production, which results in a decrease in intestinal Pi reabsorption. Dietary Pi intake also regulates Pi excretion by mechanisms unrelated to changes in PTH levels. Pi loading increases excretion, whereas Pi depletion decreases it. Changes in dietary Pi intake modulate Pi transport by altering the transport rate of each Na+/Pi symporter and the number of symporters in the apical membrane of the proximal tubule.

ECFV also affects Pi excretion. Expansion of the ECF enhances Pi excretion by: (1) increasing GFR and thus the filtered load of Pi; (2) decreasing Na/Pi coupled reabsorption, which reduces ECFV, and (3) reducing plasma [Ca++], thereby increasing PTH, which inhibits Pi reabsorption in the proximal tubule. Acid-base balance also influences Pi excretion; chronic acidosis increases Pi excretion, and chronic alkalosis decreases it. These effects of acid-base balance, like the effect of PTH, are mediated by changes of expression of the Na /Pi symporters in the apical membrane. Systemic acidosis increases glucocorticoid secretion, and glucocorticoids increase excretion of Pi by inhibiting Pi reabsorption by the proximal tubule. This inhibition, together with the direct effect of acidosis on Pi reabsorption by the proximal tubule, enables the distal tubule and collecting duct to secrete more H+ as titratable acid and to generate more HCO3 − because Pi is an important urinary buffer. Growth hormone decreases Pi excretion.

Klotho, which was identified in 1997, is highly expressed in the early distal tubule of the kidney. Klotho knockout mice have a phenotype that resembles chronic kidney disease (CKD), including soft tissue calcification, hyperphosphatemia, and elevated plasma FGF-32. Klotho exists as both a membrane-bound and a soluble protein. The membrane-bound form is a coreceptor for FGF-23, thus Klotho promotes Pi excretion by the kidneys and reduces serum levels of 1,25-dihydroxyvitamin D3. Soluble circulating Klotho has a number of additional functions, including modulating ion transport and Wnt signal transduction, inhibiting reninangiotensin signaling, and modulating FGF-23 regulation of PTH production. A considerable body of experimental data suggests that Klotho may be a biomarker for CKD, and that Klotho deficiency may contribute to development of CKD. Moreover, experimental data also suggest that Klotho therapy may slow the progression of CKD.

CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis 667 • Fig. 36.21 Overview of the major hormones regulating plasma [Ca++]. Dashed lines indicate negative feedback. FGF-23, fibroblast growth factor 23; PTH, parathyroid hormone. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

Integrative Review of Parathyroid Hormone and Calcitriol on Ca++ and Pi Homeostasis

Fig. 36.21 , PTH has numerous effects on Ca++ and Pi homeostasis. Hypocalcemia is the major stimulus of PTH secretion. PTH stimulates bone resorption, increases urinary Pi excretion, decreases urinary Ca++ excretion, and stimulates production of calcitriol, which stimulates Ca++ and Pi absorption by the intestine. Because changes in Pi handling in bone, intestines, and kidneys tend to balance out, PTH increases plasma [Ca++] while having little effect on plasma [Pi]. Overall a rise in plasma PTH levels in response to hypocalcemia returns plasma [Ca++] to the normal range. A decline in plasma [Ca++] has the opposite effect.

Calcitriol (the active form of vitamin D) also plays an important role in Ca++ and Pi homeostasis (

Fig. 36.22

The primary action of calcitriol is to stimulate Ca++ and Pi absorption by the intestine. To a lesser degree it acts with PTH to release Ca++ and Pi from bone and to decrease Ca++ excretion by the kidneys. The net effect of calcitriol is to increase plasma [Ca++] and [Pi]. Thus the major stimuli of calcitriol production are hypocalcemia via PTH and hypophosphatemia (i.e., a low plasma [Pi]).

In the absence of glucocorticoids (e.g., in Addison’s disease), excretion of Pi is depressed, as is the ability of the kidneys to excrete titratable acid and to generate new HCO3 − . Growth hormone increases reabsorption of Pi by the proximal tubule. As a result, growing children are in positive Pi balance and have a higher plasma [Pi] than adults, and this elevated [Pi] is important for bone formation.

• Fig. 36.22 Activation of calcitriol (vitamin D3) and its effect on Ca++ and Pi homeostasis. Hypocalcemia (via PTH) and hypophosphatemia are the major stimuli of the metabolism of calcifediol to calcitriol in the kidneys. The net effect of calcitriol is to increase plasma [Ca++] and [Pi]. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) 1.

K+ homeostasis is maintained by the kidneys, which adjust K+ excretion to match dietary K+ intake, and by the hormones insulin, epinephrine, and aldosterone, which regulate the distribution of K+ between the ICF and ECF compartments. Other events, such as cell lysis, exercise, and changes in acid-base balance and plasma osmolality, disturb K+ homeostasis and plasma [K+].

2.

Excretion of K+ by the kidneys is determined by the rate and direction of K+ transport by the distal tubule and collecting duct. Secretion of K+ by these tubular segments is regulated by plasma [K+], aldosterone, and AVP. In contrast, changes in tubular fluid flow and acid-base disturbances perturb K+ excretion by the kidneys. In K+-depleted states, K+ secretion is inhibited and the distal tubule and collecting duct reabsorb K+ .

3.

The kidneys, in conjunction with the intestinal tract and bone, play a vital role in regulating plasma [Ca++] and [Pi].

4.

Plasma [Ca++] is regulated by PTH and calcitriol. Calcitonin is not a major regulatory hormone in humans. Ca++ excretion by the kidneys is regulated by PTH, plasma [Ca++], and calcitriol and is altered by changes in acid-base status, ECFV, and plasma Pi.

5.

Ca++ reabsorption by the TAL and distal tubule are regulated by PTH and calcitriol, both of which stimulate Ca++ reabsorption, and by plasma [Ca++].

6.

Plasma [Pi] is regulated by PTH, FGF-23, and calcitriol. Pi excretion is regulated by PTH, FGF-23, dietary phosphate, and growth hormone and is  altered by acid base balance, ECFV expansion,  and glucocorticoids. Bone tumors secrete FGF-23, which enhances renal Pi excretion and thereby causes hypophosphatemia, hyperphosphatemia,  and a defect in bone mineralization (i.e.,  osteomalacia).

Biber J, et al. Phosphate transporters and their function. Annu Rev Physiol. 2013;75:535-550. Christov M, Juppner H. Insights from genetic disorders of phosphate homeostasis. Semin Nephrol. 2013;33:143-157.

Miller T. Control of renal calcium, phosphate, electrolyte, and water excretion by the calcium-sensing receptor. Best Pract Res Clin Endocrinol Metab. 2013;27:345-358.

Palmer BF. Regulation of potassium homeostasis. Clin J Am Soc Nephrol. 2015;10:1050-1060.

Patel A. The primary cilium calcium channels and their role in flow sensing. Pflugers Arch. 2015;467:157-165.

Pearce D, et al. Collecting duct principal cell transport processes and their regulation. Clin J Am Soc Nephrol. 2015;10:135-146.

Bernardo JF, Friedman PA. Renal calcium metabolism. In. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney:

CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis

Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013:2225.

Berndt TJ, Kumar R. Clinical disturbances of phosphate homeostasis. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013:2369.

Malnic G, et al. Regulation of K+ excretion. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013:1659.

Murer H, et al. Proximal tubular handling of phosphate. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013:2351.

Smogorzewski MJ, et al. Disorders of calcium, magnesium, and phosphate balance. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012:689.

Wang W, Huang CL. The molecular biology of renal K+ channels. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013: 1601.

Upon completion of this chapter the student should be able to answer the following questions: 1.

How does HCO3 − operate as a buffer, and why is it an important buffer of the extracellular fluid?

2.

How does metabolism of food produce acid and alkali, and what effect does the composition of the diet have on systemic acid-base balance?

3.

What is the difference between volatile and nonvolatile acids, and what is net endogenous acid production (NEAP)?

4.

How do the kidneys and lungs contribute to systemic acid-base balance, and what is renal net acid excretion (RNAE)?

5.

Why are urinary buffers necessary for excretion of acid by the kidneys?

6.

What are the mechanisms for H+ transport in the various segments of the nephron, and how are these mechanisms regulated?

7.

How do the various segments of the nephron contribute to the process of reabsorbing the filtered HCO3 −?

8.

How do the kidneys produce new HCO3 −?

9.

How is ammonium produced by the kidneys, and how does its excretion contribute to renal acid excretion?

10.

What are the major mechanisms by which the body defends itself against changes in acid-base balance?

11.

What are the differences between simple metabolic and respiratory acid-base disorders, and how are they differentiated by arterial blood gas measurements?

he concentration of H+ in body fluids is low compared with that of other ions. For example, Na+ is present at a concentration some three million times greater than that of H+ ([Na+] = 140 mEq/L; [H+] = 40 nEq/L). Because of the low [H+] of the body fluids, it is commonly expressed as the negative logarithm, or pH.

Virtually all cellular, tissue, and organ processes are sensitive to pH. Indeed, life cannot exist outside of a range of extracellular fluid (ECF) pH from 6.8 to 7.8 (160–16 nEq/L of H+). Normally the pH of ECF is maintained between 7.35 and 7.45. As described in the pH of intracellular fluid (ICF) is slightly lower (7.1–7.2) but also tightly regulated.

Each day, acid and alkali are ingested in the diet. Also, cellular metabolism produces a number of substances that have an impact on the pH of body fluids. Without appropriate mechanisms to deal with this daily acid and alkali load and thereby maintain acid-base balance, many processes necessary for life could not occur. This chapter reviews the maintenance of whole-body acid-base balance. Although the emphasis is on the role of the kidneys in this process, the roles of the lungs and liver are also considered. In addition the impact of diet and cellular metabolism on acid-base balance is presented. Finally, disorders of acid-base balance are considered, primarily to illustrate the physiological processes involved. Throughout this chapter, acid is defined as any substance that adds H+ to body fluids, whereas alkali is defined as a substance that removes H+ from body fluids.

The HCO3 − Buffer System

Bicarbonate (HCO3 −) is an important buffer of the ECF. With a normal plasma [HCO3 −] of 23 to 25 mEq/L and a volume of 14 L (for a 70-kg individual) the ECF can potentially buffer 350 mEq of H+ . The HCO3 − buffer system differs from the other buffer systems of the body (e.g., phosphate) because it is regulated by both the lungs and kidneys. This is best appreciated by considering the following reaction:

Equation 37.1

As indicated the first reaction (hydration/dehydration of CO2) is the rate-limiting step. This normally slow reaction is greatly accelerated in the presence of carbonic anhydrase.

The second reaction, ionization of H2CO3 to H+ and HCO3 − , is virtually instantaneous.

aCarbonic anhydrase (CA) actually catalyzes the following reaction: (e.g., citrate) •Fig. 37.1 Overviewofacid-basebalance.Thelungsandkidneysworktogethertomaintainacid-basebalance.ThelungsexcreteCO2(volatileacid),andthekidneysexcreteacid(renalnetacidexcretion[RNAE])equaltonetendogenousacidproduction(NEAP),whichreflectsdietaryintake,cellularmetabolism,andlossofacidandalkali(e.g.,HCO3− lossinfeces)fromthebody.Seetextfordetails.(FromKoeppenBM,StantonBA.Renal Physiology. 5thed.Philadelphia:Elsevier;2013.)

The Henderson-Hasselbalch equation is used to quantitate how changes in CO2 and HCO3 − effect pH:

Equation 37.2

Equation 37.3 [HCO3 − ]pH = 6 1. + log 0 03. PCO2

In these equations the amount of CO2 is determined from the partial pressure of CO2 (PCO2) and its solubility (α) in solution. For plasma at 37°C, α has a value of 0.03. Also, pK′ is the negative logarithm of the overall dissociation constant for the reaction in

Eq. 37.1 and has a value for plasma at 37°C of 6.1. Alternatively the relationship between HCO3 − and CO2 on the [H+] can be determined as follows:

Equation 37.4

Inspection of Eqs. 36.3 and 36.4 show that pH and [H+] vary when either [HCO3 −] or PCO2 are altered. Disturbances of acid-base balance that result from a change in [HCO3 −] are termed metabolic acid-base disorders, whereas those resulting from a change in PCO2 are termed respiratory acid-base disorders. These disorders are considered in more detail in a subsequent section. The kidneys are primarily responsible for regulating the [HCO3 −] of ECF, whereas the lungs control the PCO2.

Overview of Acid-Base Balance

The diet of humans contains many constituents that are either acid or alkali. In addition, cellular metabolism produces acid and alkali. Finally, alkali is normally lost each day in feces. As described later, although diet dependent, the net effect of these processes is the addition of acid to body fluids. For acid-base balance to be maintained, acid must be excreted from the body at a rate equivalent to its addition. If acid addition exceeds excretion, acidosis results. Conversely, if acid excretion exceeds addition, alkalosis results.

Fig. 37.1 , the major constituents of the diet are carbohydrates and fats. When tissue perfusion is adequate, O2 is available to tissues, and insulin is present at normal levels, carbohydrates and fats are metabolized to CO2 and H2O. On a daily basis, 15 to 20 moles of CO2 are generated through this process. Normally this large quantity of CO2 is effectively eliminated from the body by the lungs. Therefore this metabolically derived CO2 has no impact on acid-base balance. CO2 is usually termed volatile acid because it has the potential to generate H+ after hydration with H2O (see

Eq. 36.1 ). Acid not derived directly from hydration of CO2 is termed nonvolatile acid (e.g., lactic acid).

The cellular metabolism of other dietary constituents also has an impact on acid-base balance. For example, cysteine and methionine, sulfur-containing amino acids, yield sulfuric acid when metabolized, whereas hydrochloric acid results from metabolism of lysine, arginine, and histidine. A portion of this nonvolatile acid load is offset by production of HCO3 − through metabolism of the amino acids aspartate and glutamate. On average the metabolism of dietary amino acids yields net nonvolatile acid production. Metabolism of certain organic anions (e.g., citrate) results in production of HCO3 − , which offsets nonvolatile acid production to some degree. Overall in individuals ingesting a meat-containing diet, acid production exceeds HCO3 − production. In contrast, a vegetarian diet produces less nonvolatile acid. In addition to the metabolically derived acids and alkalis, the foods ingested contain acid and alkali. For example, the presence of phosphate (H2PO4 −) in ingested food increases the dietary acid load. Finally, during digestion, some HCO3 − is normally lost in feces. This loss is equivalent to the addition of nonvolatile acid to the body. In an individual ingesting a meat-containing diet, dietary intake, cellular metabolism, and fecal HCO3 − loss result in addition of approximately 0.7 to 1.0 mEq/kg body weight of nonvolatile acid to the body each day (50–100 mEq/day for most adults). This acid, referred to as net endogenous acid production (NEAP), results in an equivalent loss of HCO3 − from the body that must be replaced.

Wheninsulinlevelsarenormal,carbohydratesandfatsarecompletelymetabolizedtoCO2+ H2O.However,ifinsulinlevelsareabnormallylow(e.g.,diabetesmellitus),cellularmetabolismleadstoproductionofseveralorganicketoacids(e.g.,β-hydroxybutyricacidandacetoaceticacidfromfattyacids).

IntheabsenceofadequatelevelsofO2(hypoxia),anaerobicmetabolismbycellscanalsoleadtoproductionoforganicacids(e.g.,lacticacid)ratherthanCO2+ H2O.Thisfrequentlyoccursinnormalindividualsduringvigorousexercise.Poortissueperfusion,suchasoccurswithreducedcardiacoutput,canalsoleadtoanaerobicmetabolismbycellsandthustoacidosis.IntheseconditionstheorganicacidsaccumulateandthepHofbodyfluidsdecreases(acidosis).Treatment(e.g.,administrationofinsulininthecaseofdiabetes)orimproveddeliveryofadequatelevelsofO2totissues(e.g.,inthecaseofpoortissueperfusion)resultsinthemetabolismoftheseorganicacidstoCO2+ H2O,whichconsumesH+ andtherebyhelpscorrecttheacid-basedisorder.

Nonvolatile acids do not circulate throughout the body but are immediately neutralized by the HCO3 − in ECF.

Equation 37.5

Equation 37.6

This neutralization process yields the Na+ salts of the strong acids and removes HCO3 − from the ECF. Thus HCO3 − minimizes the effect of these strong acids on the pH of ECF. As noted previously, ECF contains approximately 350 mEq of HCO3 − . If this HCO3 − were not replenished, the daily production of nonvolatile acids (≈70 mEq/day) would deplete the ECF of HCO3 − within 5 days. To maintain acid-base balance the kidneys must replenish the HCO3 − lost by neutralization of the nonvolatile acids, a process termed renal net acid excretion (RNAE).

Net Acid Excretion by the Kidneys

Under steady-state conditions, NEAP must equal RNAE to maintain acid-base balance. Although NEAP varies from individual to individual and from day to day in anyone individual, it is not regulated. Instead the kidneys regulate RNAE to match NEAP and in so doing replenish the HCO3 − (new HCO3 −) lost by neutralization of nonvolatile acids. In addition the kidneys must prevent the loss of HCO3 − in urine. This latter task is quantitatively more important because the filtered load of HCO3 − is approximately 4320 mEq/day (24 mEq/L × 180 L/day = 4320 mEq/day), compared with only 50 to 100 mEq/day needed to balance NEAP.

Both reabsorption of filtered HCO3 − and excretion of acid are accomplished via H+ secretion by the nephrons. Thus in a single day the nephrons must secrete approximately 4390 mEq of H+ into the tubular fluid. Most of the secreted serves to reabsorb the filtered load of HCO3 . Only 50 to 100mEq of H+ , an amount equivalent to NEAP, is excreted in urine. As a result of this acid excretion, urine is normally acidic.

The kidneys cannot excrete urine more acidic than pH 4.0 to 4.5. Even at a pH of 4.0, only 0.1 mEq/L of H+ can be excreted. Therefore to excrete sufficient acid, the kidneys excrete H+ with urinary buffers such as phosphate (Pi).

Other constituents of urine can also serve as buffers (e.g., creatinine), although their role is less important than Pi. Collectively the various urinary buffers are termed titratable acid. This term is derived from the method by which these buffers are quantitated in the laboratory. Typically, alkali (OH−) is added to a urine sample to titrate its pH to that of plasma (i.e., 7.4). The amount of alkali added is equal to the H+ titrated by these urine buffers and is termed titratable acid.

Excretion of H+ as a titratable acid is insufficient to balance NEAP. An additional and important mechanism by which the kidneys contribute to maintenance of acid-base balance is through synthesis and excretion of ammonium (NH4 +). The mechanisms involved in this process are discussed in more detail later in this chapter. With regard to renal regulation of acid-base balance, each NH4 + excreted in urine results in the return of one HCO3 − to the systemic circulation, which replenishes the HCO3 − lost during neutralization of the nonvolatile acids. Thus production and excretion of NH4 + , like excretion of titratable acid, is equivalent to excretion of acid by the kidneys.

In brief the kidneys contribute to acid-base homeostasis by reabsorbing the filtered load of HCO3 − and excreting an bThe titration reaction is: HPO42−+ H+↔ H2PO4 − . This reaction has a pK of approximately 6.8.

amount of acid equivalent to NEAP. This process can be quantitated as follows:

Equation 37.7 where (UNH4+× V˜ ) and (UTA × V˜ ) are the rates of excretion (mEq/day) of NH4 + and titratable acid (TA), and (UHCO3−× V˜ ) is the amount of HCO3 − lost in urine (equivalent to adding H+ to the body).

Again, maintenance of acid-base balance means that net acid excretion must equal nonvolatile acid production. Under most conditions, very little HCO3 − is excreted in urine. Thus net acid excretion essentially reflects titratable acid and NH4 + excretion. Quantitatively, titratable acid accounts for approximately one-third and NH4 + for two-thirds of RNAE.

HCO3 − Reabsorption Along the Nephron

Eq. 37.7 , net acid excretion is maximized when little or no HCO3 − is excreted in urine. Indeed, under most circumstances, very little HCO3 − appears in urine. Because HCO3 − is freely filtered at the glomerulus, approximately 4320 mEq/day is delivered to the nephrons and is then reabsorbed.

Fig. 37.2 summarizes the contribution of each nephron segment to reabsorption of filtered HCO3 − .

The proximal tubule reabsorbs the largest portion of the filtered load of HCO3 − .

Fig. 37.3 summarizes the primary cThis equation ignores the small amount of free H+ excreted in urine. As already noted, urine with a pH = 4.0 contains only 0.1 mEq/L of H+ .

transport processes involved. H+ secretion across the apical membrane of the cell occurs by both a Na+/H+ antiporter and H+-ATPase (V-type). The Na+/H+ antiporter (NHE3) is the predominant pathway for H+ secretion (accounts for ≈ two-thirds of HCO3 − reabsorption) and uses the lumen-to-cell [Na+] gradient to drive this process (i.e., •Fig. 37.2 SegmentalreabsorptionofHCO3− .ThefractionofthefilteredloadofHCO3− reabsorbedbythevarioussegmentsofthenephronisshown.NormallytheentirefilteredloadofHCO3− isreabsorbedandlittleornoHCO3− appearsintheurine.CCD,corticalcollectingduct;DT,distaltubule;IMCD,innermedullarycollectingduct;PT,proximaltubule;TAL,thickascendinglimb.

•Fig. 37.3 CellularmechanismforreabsorptionoffilteredHCO3− bycellsoftheproximaltubule.OnlytheprimaryH+ andHCO3− transportersareshown.ATP,adenosinetriphosphate;CA,carbonicanhydrase.

secondary active secretion of H+). Within the cell, H+ and HCO3 − are produced in a reaction catalyzed by carbonic anhydrase (CA-II). The H+ is secreted into the tubular fluid, whereas the HCO3 − exits the cell across the basolateral membrane and returns to the peritubular blood. HCO3 − movement out of the cell across the basolateral membrane is coupled to other ions. The majority of HCO3 − exits via a symporter that couples the efflux of Na+ with HCO3 − (sodium bicarbonate symporter, NBC1). Some HCO3 − exits the cell by other transporters, but they are not as important as the Na+/HCO3 − symporter. As noted in

Fig. 37.3 , carbonic anhydrase (CA-IV) is also present in the brush border and basolateral membrane of the cell. The brush border enzyme catalyzes dehydration of H2CO3 in the luminal fluid, whereas the enzyme localized to basolateral membrane facilitates HCO3 − exit from the cell. The movement of CO2 into and out of the cell occurs via AQP1, which is localized to both the luminal and basolateral membranes.

The cellular mechanism for HCO3 − reabsorption by the thick ascending limb (TAL) of the loop of Henle is very similar to that in the proximal tubule. H+ is secreted by a Na+/H+ antiporter and H+-ATPase. Like in the proximal tubule, the Na+/H+ antiporter (NHE3) is the predominant pathway for H+ secretion. HCO3 − exit from the cell involves both a Na+/HCO3 − symporter (NBC1) and a Cl−/HCO3 − antiporter (anion exchanger, AE-2). Some HCO3 − may also exit the cell through Cl− channels present in the basolateral membrane.

The distal tubule and collecting duct reabsorb the small amount of HCO3 − that escapes reabsorption by the proximal tubule and loop of Henle.

Fig. 37.4 shows the cellular mechanism of H+/HCO3 − transport by the intercalated cells located within these segments (see also

One type of intercalated cell secretes H+ (reabsorbs HCO3 −) and is called the A-or α-intercalated cell. Within this cell, H+ and HCO3 − are produced by hydration of CO2; this reaction is catalyzed by carbonic anhydrase (CA-II). H+ is secreted into the tubular fluid via two mechanisms. The first involves an apical membrane H+-ATPase (V-type). The second couples secretion of H+ with reabsorption of via an H+-K+-ATPase similar to those found in the stomach and colon (HKα1 and HKα2). HCO3 − exits the cell across the basolateral membrane in exchange for Cl− (via a Cl−/HCO3 − antiporter, AE-1) and enters the peritubular capillary blood.

A second population of intercalated cells secretes HCO3 − rather than H+ into the tubular fluid (also called B-or dHere and in the remainder of the chapter we focus on the function of intercalated cells. The early portion of the distal tubule, which does not contain intercalated cells, also reabsorbs HCO3 − . The cellular mechanism appears to involve an apical membrane Na+/H+ antiporter (NHE2) and a basolateral Cl−/HCO3 − antiporter (AE2).

β-intercalated cells).

In these cells the H+-ATPase (V-type) is located in the basolateral membrane and the Cl−/HCO3 − antiporter is located in the apical membrane (see

Fig. 37.4).

However, the apical membrane Cl−/HCO3 − antiporter is different from the one found in the basolateral membrane of the H+-secreting intercalated cells and has been identified as pendrin. The activity of the HCO3 −-secreting intercalated cell is increased during metabolic alkalosis, when the kidneys must excrete excess HCO3 − . However, under most conditions (e.g., ingestion of a meat-containing diet) H+ secretion predominates in these segments.

The apical membrane of collecting duct cells is not very permeable to H+ , and thus the pH of tubular fluid can become quite acidic. Indeed, the most acidic tubular fluid along the nephron (pH = 4.0–4.5) is produced there. In comparison the permeability of the proximal tubule to H+ and HCO3 − is much higher, and the tubular fluid pH falls to only 6.5 in this segment. As explained later the ability of the collecting duct to lower the pH of the tubular fluid is critically important for excretion of urinary titratable acids and NH4 + .

Regulation of H+ Secretion

A number of factors influence secretion of H+ and thus reabsorption of filtered HCO3 − by the cells of the nephron. From a physiological perspective the primary factor that regulates H+ secretion by the nephron is a change in systemic acid-base balance. Thus acidosis stimulates RNAE, whereas RNAE is reduced during alkalosis.

The response of the kidneys to changes in acid-base balance includes both immediate changes in the activity and/or number of transporters in the membrane and longer-term changes in the synthesis of transporters. For example, with metabolic acidosis, H+ secretion is stimulated by multiple mechanisms, depending on the particular nephron segment. First, the decrease in intracellular pH that occurs with acidosis will create a more favorable cell-to–tubular fluid H+ gradient and thereby make secretion of H+ across the apical membrane more energetically favorable. Second, the decrease in pH may lead to allosteric changes in transport proteins, thereby altering their kinetics. Lastly, transporters may be shuttled to the membrane from intracellular vesicles. With long-term acidosis the abundance of transporters increases, either by increased transcription of appropriate transporter genes or by increased translation of transporter mRNA.

eA third group of intercalated cells shares features of both H+-secreting and HCO3 −-secreting intercalated cells. The precise function of this cell type in acid-base transport is not fully understood. fTraditionally it was believed that intercalated cells were only involved in acid-base transport. There is now good evidence that NaCl reabsorption is also carried out by intercalated cells (B type). Reabsorption of NaCl occurs by the tandem operation of an apical membrane Cl−/HCO3 − anti-porter (pendrin) and an apical membrane Na+/HCO3 −/2Cl− antiporter (NDCBE). This mechanism of NaCl reabsorption is inhibited by thiazide diuretics.

•Fig. 37.4 CellularmechanismsforreabsorptionandsecretionofHCO3− byintercalatedcellsofthedistaltubuleandcollectingduct.OnlytheprimaryH+ andHCO3− transportersareshown.ATP,adenosinetriphosphate;CA,carbonicanhydrase.

•Fig. 37.5 ExamplesofcellularH+ andHCO3− sensors.ATP,adenosinetriphosphate;cAMP,cyclicadenosinemonophosphate;GPCR,Gprotein–coupledreceptor;IRR,insulinreceptor–relatedreceptor;Pyk2,nonreceptortyrosinekinase;sAC,solubleadenylylcyclase.(Adaptedfrom:LevinLR,BuckJ.Annu Rev Physiol 2015;77:347.)

Fig.37.5 ).Forexample,aGprotein–coupledH+ receptor(GPCR–GPR4)hasbeenlocalizedtothecollectingduct.ActivationofthisreceptorbyanincreaseinECF[H+]stimulatesH+ secretion.Alsointhecollectingduct,HCO3−-secretingintercalatedcells(B-orβ-ICs)expressabasolateralinsulin-relatedreceptor(IRR)thatisatyrosinekinase.ItisactivatedbyanincreaseinECF[HCO3−]andstimulatesHCO3− secretionbythecell.Asolubleadenylylcyclase(sAC)regulatedbyintracellularHCO3− appearstoalsoplayarollinregulatingcollectingductH+ secretion.Intheproximaltubule,basolateralmembranereceptortyrosinekinases(ErbB1andErbB2)sensechangesinECFPCO2.ActivationofthesereceptorsbyanincreaseinPCO2resultsingenerationofangiotensinII,whichactingfromthelumenviaAT-1Areceptors,stimulatesH+ secretion/HCO3− reabsorption.Alsointheproximaltubule,thenonreceptortyrosinekinase(Pyk2)sensesintracellular[H+].Whenitisactivatedbyanincreaseinintracellular[H+],H+ secretion/HCO3− reabsorptionisstimulated.Finally,thegatingofseveralionchannels(e.g.,therenaloutermedullaryK+ channel[ROMK])iseffectedbychangesineitherECForICFpH.Thesetoohavethepotentialtoserveascellularacid-basesensors.

Although some of the effects just described may be attributable directly to acidosis, many of these changes in cellular H+ transport are mediated by hormones or other factors. Three known mediators of the renal response to acidosis are endothelin, cortisol, and angiotensin II. Endothelin (ET-1)

Intheproximaltubule,metabolicacidosisincreasesthetransportkineticsoftheNa+/H+ antiporter(NHE3)andincreasesapicalmembraneexpressionoftheNa+/H+ antiporter,H+-ATPase,andthebasolateralNa+/3HCO3− symporter(NBCe1).Inthecollectingduct,acidosisleadstoexocyticinsertionofH+-ATPaseintotheapicalmembraneofintercalatedcells.Withlong-termacidosistheabundanceofkeyacid-basetransportersisincreasedintheproximaltubule(NHE3andNBCe1)andincollectingductintercalatedcells(H+-ATPaseandAE1).Lastly,acidosisdecreasesexpressionoftheCl−/HCO3− antiporterpendrininHCO3−-secretingintercalatedcells.

is produced by endothelial and proximal tubule cells. With acidosis, ET-1 secretion is enhanced. In the proximal tubule, ET-1 stimulates phosphorylation and subsequent insertion of the Na+/H+ antiporter into the apical membrane, and insertion of the Na+/3HCO3 − symporter into the basolateral membrane. ET-1 may mediate the response to acidosis in other nephron segments as well. Acidosis also stimulates secretion of the glucocorticoid hormone cortisol by the adrenal cortex. Cortisol increases the abundance of the Na+/ H+ antiporter and Na+/3HCO3 − symporter in the proximal tubule. Angiotensin II is produced in proximal tubule cells in response to acidosis. It is secreted into the tubular fluid, where it binds to the angiotensin I receptor and thereby stimulates H+ secretion/HCO3 − reabsorption by the proximal tubule. Both cortisol and angiotensin II also stimulate production and secretion of NH4 + by the proximal tubule, which as described later is an important component of the kidney’s response to acidosis.

Acidosis also stimulates secretion of parathyroid hormone (PTH). PTH inhibits phosphate (Pi) reabsorption by the proximal tubule (see ). In so doing, more Pi is delivered to the distal nephron, where it serves as a urinary buffer and thus increases the capacity of the kidneys to excrete titratable acid.

The response of the kidneys to alkalosis is less well characterized. RNAE is decreased because of increased urinary HCO3 − excretion and because excretion of titratable acid and NH4 + are reduced. The factors that regulate this response are not well characterized.

Other factors not necessarily related to maintaining acid-base balance can influence secretion of H+ by the cells of the nephron. Because a significant H+ transporter in the nephron is the Na+/H+ antiporter, factors that alter Na+ reabsorption can secondarily affect H+ secretion. For example, with volume contraction (negative Na+ balance), Na+ reabsorption by the nephron is increased (see ), including reabsorption of Na+ via the Na+/H+ antiporter. As a result, H+ secretion is enhanced. This occurs by several mechanisms. One mechanism involves the renin-angiotensinaldosterone system, which is activated by volume contraction. As noted earlier, angiotensin II acts on the proximal tubule to stimulate the apical membrane Na+/H+ antiporter as well as the basolateral Na+/3HCO3 − symporter. To a lesser degree, angiotensin II stimulates H+ secretion in the TAL of Henle’s loop and the early portion of the distal tubule, a process also mediated by the Na+/H+ antiporter. Aldosterone’s primary action on the distal tubule and collecting duct is to stimulate Na+ reabsorption by principal cells (see ). However, it also stimulates intercalated cells in these segments to secrete H+ . This effect is both indirect and direct. By stimulating Na+ reabsorption by principal cells, aldosterone hyperpolarizes the transepithelial voltage (i.e., the lumen becomes more electrically negative). This change in transepithelial voltage then facilitates secretion of H+ by intercalated cells. In addition to this indirect effect, aldosterone (and angiotensin II) act directly on intercalated cells to stimulate H+ secretion via H+-ATPase and H+,K+-ATPase.

Another mechanism by which ECF volume (ECFV) contraction enhances H+ secretion (HCO3 − reabsorption) is through changes in peritubular capillary Starling forces. As described in , ECFV contraction alters the peritubular capillary Starling forces such that overall proximal tubule reabsorption is enhanced. With this enhanced reabsorption, more of the filtered load of HCO3 − is reabsorbed.

Potassium balance influences secretion of H+ by the proximal tubule. Hypokalemia stimulates and hyperkalemia inhibits H+ secretion. It is thought that K+-induced changes in intracellular pH are responsible at least in part for this effect, with hypokalemia acidifying and hyperkalemia alkalinizing the cells. Hypokalemia also stimulates H+ secretion by the collecting duct. This occurs as a result of increased expression of the H+,K+-ATPase in intercalated cells.

Formation of New HCO3 −

As discussed previously, reabsorption of the filtered load of HCO3 − is important for maximizing RNAE. However, HCO3 − reabsorption alone does not replenish the HCO3 − lost during neutralization of the nonvolatile acids produced during metabolism. To maintain acid-base balance, the kidneys must replace this lost HCO3 − with new HCO3 − . Generation of new HCO3 − occurs by excretion of titratable acid and by synthesis and excretion of NH4 + .

Production of new HCO3 − as a result of titratable acid excretion is depicted in

Fig. 37.6 . Because of HCO3 − reabsorption by the proximal tubule and loop of Henle, fluid reaching the distal tubule and collecting duct normally contains little HCO3 − . Thus when H+ is secreted it will combine with non-HCO3 − buffers (primarily Pi) and be excreted as titratable acid. Because the H+ was produced inside the cell from hydration of CO2, a HCO3 − is also produced. This HCO3 − is returned to the ECF as new HCO3 − . As noted, Pi excretion increases with acidosis. However, even with increased Pi available for titratable acid formation, this response is insufficient to generate the required amount of new HCO3 − . The remainder of new HCO3 − generation occurs as a result of NH4 + generation and excretion.

NH4 + is produced by the kidneys, and its synthesis and subsequent excretion adds HCO3 − to ECF. Importantly, this process is regulated in response to the acid-base requirements of the body.

NH4 + is produced in the kidneys via metabolism of glutamine. Essentially the kidneys metabolize glutamine, excrete NH4 + , and add HCO3 − to the body. However, formation of new HCO3 − via this process depends on the kidneys’ ability to excrete NH4 + in urine. If NH4 + is not excreted in urine but instead enters the systemic circulation, it is converted into urea by the liver. This conversion process generates H+ , which is then buffered by HCO3 − . Thus production of urea from renal-generated NH4 + consumes HCO3 − and negates formation of HCO3 − through synthesis and excretion of NH4 + by the kidneys. However, normally the kidneys excrete NH4 + in urine and thereby produce new HCO3 − .

The process by which the kidneys excrete NH4 + is complex.

Fig. 37.7 illustrates the essential features of this process. NH4 + is produced from glutamine in the cells of the proximal tubule, a process termed ammoniagenesis. Each glutamine molecule produces two molecules of NH4 + and the divalent anion 2-oxoglutarate2− . Metabolism of this anion ultimately provides two molecules of HCO3 − . HCO3 − exits the cell across the basolateral membrane and enters the peritubular blood as new HCO3 − . NH4 + exits the cell across the apical membrane and enters the tubular fluid. The primary mechanism for NH4 + secretion into the tubular fluid involves the Na+/H+ antiporter, with NH4 + substituting for H+ . In addition, some NH3 can diffuse out of the cell into the tubular fluid, where it is protonated to NH4 + .

A significant portion of the NH4 + secreted by the proximal tubule is reabsorbed by the loop of Henle. The TAL is the primary site of this NH4 + reabsorption, with NH4 + substituting for K+ on the 1Na+/1K+/2Cl− symporter. In addition the lumen-positive transepithelial voltage in this segment drives paracellular reabsorption of NH4 + .

678 SECTION7Berne & Levy Physiology •Fig. 37.6 GeneralschemeforexcretionofH+withnon-HCO3−urinarybuffers(titratableacid).Theprimaryurinarybufferisphosphate(HPO42−).AH+-secretingintercalatedcellisshown.Forsimplicity,onlytheH+-ATPaseisdepicted.H+secretionbytheH+,K+-ATPasealsotitratesluminalbuffers.ATP,adenosinetriphosphate;CA,carbonicanhydrase.Tubular fluid Blood Cl– CO2 + H2O Buffer + H+ H-Buffer H˜ATP CA HCO3 – HCO3 –

The NH4 + reabsorbed by the TAL of the loop of Henle accumulates in the medullary interstitium. From there it is then secreted into the tubular fluid by the collecting duct.

The cells of the collecting duct express two NH3 membrane transporters known as Rhesus (Rh) glycoproteins (RhBG and RhCG).

RhBG is present in the basolateral membrane of H+-secreting intercalated cells and principal cells, and RhCG is present in both the apical and basolateral membranes of these cells. As depicted in

Fig. 37.7 , NH3 is transported across the collecting duct, a process traditionally termed nonionic diffusion. The secreted NH3 is protonated in the tubule lumen as a result of intercalated H+ secretion. Because the apical membrane has a low permeability to NH4 + it is effectively trapped in the tubular lumen, a process traditionally termed diffusion trapping.

H+ secretion by the collecting duct is critical for excretion of NH4 + . If collecting duct H+ secretion is inhibited, the NH4 + reabsorbed by the TAL of Henle’s loop will not be excreted in the urine. Instead it will be returned to the systemic circulation, where as described previously, it will be converted to urea by the liver and consume HCO3 − in the process. Thus new HCO3 − is produced during the metabolism of glutamine by cells of the proximal tubule. However, the overall process is not complete until the NH4 + is excreted (i.e., production of urea from NH4 + by the liver is prevented). Thus NH4 + excretion in urine can be used as a marker of glutamine metabolism in the proximal tubule.

AssessingNH4+ excretionbythekidneysisdoneindirectlybecauseassaysofurineNH4+ arenotroutinelyavailable.Considerforexamplethesituationofmetabolicacidosis,whereintheappropriaterenalresponseistoincreasenetacidexcretion.Accordingly,littleornoHCO3− willappearinurine,urinewillbeacidic,andNH4+ excretionwillbeincreased.Toassessthis,andespeciallytheamountofNH4+ excreted,the“urinarynetcharge”or“urineaniongap”canbecalculatedbymeasuringurinaryconcentrationsofNa+ ,K+ ,andCl−:

TheconceptofurineaniongapduringametabolicacidosisassumesthatthemajorcationsinurineareNa+ ,K+ ,andNH4+ andthatthemajoranionisCl− (withurinepH< 6.5,virtuallynoHCO3− ispresent).AsaresulttheurineaniongapwillyieldanegativevaluewhenNH4+ isbeingexcreted.Indeed,theabsenceofaurineaniongaportheexistenceofapositivevalueindicatesarenaldefectinNH4+ productionandexcretion.

gBoth RhBG and RhCG transport NH3. There is some evidence that RhBG may also transport some NH4 + .

•Fig. 37.7 Production,transport,andexcretionofNH4+bythenephron.GlutamineismetabolizedtoNH4+andHCO3−intheproximaltubule.TheNH4+issecretedintothelumen,andtheHCO3−enterstheblood.ThesecretedNH4+isreabsorbedinHenle’sloop,primarilybythethickascendinglimb,andaccumulatesinthemedullaryinterstitium.NH3issecretedbythecollectingductviarhesusglycoproteins,andH+secretiontrapsNH4+inthelumen.ForeachmoleculeofNH4+excretedintheurine,amoleculeof“new”HCO3−isaddedbacktotheECF.CA,carbonicanhydrase.Tubular fluid Tubular fluid CO2 + H2O NH4 + NH4 + 2NH4 + NH4 + NH3 NH3 NH4 + NH4 + NH3 + H+ NH4 + NH4 + NH4 + NH4 + H˜NH3HCO3 – Blood Blood Na+ Na+ H+H+ Glutamine A= 2HCO3 – CA

In the net, one new HCO3 − is returned to the systemic circulation for each NH4 + excreted in the urine.

An important feature of the renal NH4 + system is that it is regulated by systemic acid-base balance. As already described, cortisol levels increase with acidosis, as does angiotensin II secretion into the lumen of the proximal tubule. Both cortisol and angiotensin II stimulate ammoniagenesis (i.e., NH4 + production from glutamine). During systemic acidosis, the enzymes in the proximal tubule cell responsible for metabolism of glutamine are stimulated. This involves synthesis of new enzyme and requires several days for complete adaptation. With increased levels of these enzymes, NH4 + production is increased, allowing enhanced production of new HCO3 − . Conversely, glutamine metabolism is reduced with alkalosis. Acidosis also increases the abundance of RhBG and RhCG in the collecting duct. Thus the ability to secrete NH4 + is also enhanced.

Response to Acid-Base Disorders

The pH of the ECF is maintained within a very narrow range (7.35–7.45).

Inspection of

Eq. 37.3 shows that the pH of hFor simplicity of presentation in this chapter, the value of 7.40 for body fluid pH is used as normal, even though the normal range is from 7.35 to 7.45. Similarly the normal range for PCO2 is 35 to 45 mm Hg. However, a PCO2 of 40 mm Hg is used as the normal value. Finally, a value of 24 mEq/L is considered a normal ECF [HCO3 −], even though the normal range is 22 to 28 mEq/L.

ECF varies when either [HCO3 −] or PCO2 are altered. As already noted, disturbances of acid-base balance that result from a change in [HCO3 −] of ECF are termed metabolic acid-base disorders, whereas those resulting from a change in PCO2 are termed respiratory acid-base disorders. The kidneys are primarily responsible for regulating [HCO3 −], whereas the lungs regulate PCO2.

When an acid-base disturbance develops, the body uses several mechanisms to defend against the change in ECF pH. These defense mechanisms do not correct the acid-base disturbance but merely minimize the change in pH imposed by the disturbance. Restoration of the blood pH to its normal value requires correction of the underlying process or processes that produced the acid-base disorder. The body has three general mechanisms to defend against changes in body fluid pH produced by acid-base disturbances: (1) extracellular and intracellular buffering, (2) adjustments in blood PCO2 via alterations in the ventilatory rate of the lungs, and (3) adjustments in RNAE.

The first line of defense against acid-base disorders is extracellular and intracellular buffering. The response of the extracellular buffers is virtually instantaneous, whereas the response to intracellular buffering is slower and can take several minutes.

Metabolic disorders that result from addition of nonvolatile acid or alkali to body fluids are buffered in both the extracellular and intracellular compartments. The HCO3 − buffer system is the principal ECF buffer. When nonvolatile acid is added to body fluids (or alkali is lost from the body), HCO3 − is consumed during the process of neutralizing the acid load, and the [HCO3 −] of ECF is reduced. Conversely, when nonvolatile alkali is added to body fluids (or acid is lost from the body), H+ is consumed, causing more HCO3 − to be produced from the dissociation of H2CO3. Consequently, [HCO3 −] increases.

Although the HCO3 − buffer system is the principal ECF buffer, Pi and plasma proteins provide additional extracellular buffering. The combined action of the buffering processes for HCO3 − , Pi, and plasma protein accounts for approximately 50% of the buffering of a nonvolatile acid load and 70% of a nonvolatile alkali load. The remainder of the buffering under these two conditions occurs intracellularly. Intracellular buffering involves movement of H+ into cells (during buffering of nonvolatile acid) or movement of H+ out of cells (during buffering of nonvolatile alkali). H+ is titrated inside the cell by HCO3 − , Pi, and the histidine groups on proteins.

Bone represents an additional source of extracellular buffering. However, with acidosis, buffering by bone results in its demineralization.

When respiratory acid-base disorders occur, the pH of body fluid changes as a result of alterations in PCO2. Virtually all buffering in respiratory acid-base disorders occurs intracellularly. When PCO2 rises (respiratory acidosis), CO2 moves into the cell, where it combines with H2O to form H2CO3. H2CO3 then dissociates to H+ and HCO3 − . Some of the H+ is buffered by cellular proteins, and HCO3 − exits the cell and raises the ECF [HCO3 −]. This process is reversed when PCO2 is reduced (respiratory alkalosis). Under this condition the hydration reaction (H2O + CO2 ↔ H2CO3) is shifted to the left by the decrease in PCO2. As a result the dissociation reaction (H2CO3 ↔ H++ HCO3 −) also shifts to the left, thereby reducing the ECF [HCO3 −].

The lungs are the second line of defense against acid-base disorders. As indicated by the Henderson-Hasselbalch equation (see

Eq. 37.2 ), changes in PCO2 alter the blood pH: a rise decreases the pH, and a reduction increases the pH.

The ventilatory rate determines the PCO2. Increased ventilation decreases PCO2, whereas decreased ventilation increases it (Fig. 37.8 ). The blood PCO2 and pH are important regulators of the ventilatory rate. Chemoreceptors located in the brainstem (ventral surface of the medulla) and periphery (carotid and aortic bodies) sense changes in PCO2 and [H+] and alter the ventilatory rate appropriately. Thus when metabolic acidosis occurs, a rise in the [H+] (decrease in pH) stimulates the ventilatory rate. Conversely, during metabolic alkalosis, a decreased [H+] (increase in pH) reduces the ventilatory rate. With maximal hyperventilation, the PCO2 can be reduced to approximately •Fig. 37.8 EffectofventilatoryrateonalveolarPCO2andthusarterialbloodPCO2.

10 mm Hg. Because hypoxia, a potent stimulator of ventilation, also develops with hypoventilation, the degree to which the PCO2 can be increased is limited. In an otherwise normal individual, hypoventilation cannot raise the PCO2 above 60 mm Hg. The respiratory response to metabolic acid-base disturbances may be initiated within minutes but may require several hours to complete.

The third and final line of defense against acid-base disorders involves the kidneys. In response to an alteration in plasma pH and PCO2, the kidneys make appropriate adjustments in the excretion of RNAE. The renal response may require several days to reach completion because it takes hours to days to increase the synthesis and activity of the proximal tubule enzymes involved in NH4 + production. In the case of acidosis (increased [H+] or PCO2), the secretion of H+ by the nephron is stimulated and the entire filtered load of HCO3 − is reabsorbed. Titratable acid excretion is increased and production and excretion of NH4 + is also stimulated, and thus RNAE is increased (

Fig. 37.9). The new HCO3 − generated during the process of net acid excretion is added to the body, and the plasma [HCO3 −] increases.

When metabolic alkalosis exists (decreased [H+]), the filtered load of HCO3 − is increased (plasma [HCO3 −] is elevated). With respiratory alkalosis (decreased PCO2), plasma [HCO3 −] is decreased and thus the filtered load is decreased. In both conditions, secretion of H+ by the nephron is inhibited. As a result, HCO3 − excretion is increased. At the same time, excretion of both titratable acid and NH4 + is decreased. Thus RNAE is decreased and HCO3 − appears in urine. Also, some HCO3 − is secreted into urine by the HCO3 −-secreting intercalated cells of the distal tubule and collecting duct. With enhanced excretion of HCO3 − , plasma [HCO3 −] decreases.

•Fig. 37.9 Responseofthenephrontoacidosis.Pi,phosphate;PTH,parathyroidhormone;Rhbg&Rhcg,rhesusglycoproteins;RNAE,renalnetacidexcretion;TA,titratableacid;V,urineflowrate.

Lossofgastriccontentsfromthebody(e.g.,vomiting,nasogastricsuctioning)producesmetabolicalkalosissecondarytothelossofHCl.Ifthelossofgastricfluidissignificant,ECFVcontractionoccurs.UnderthisconditionthekidneyscannotexcretesufficientquantitiesofHCO3− tocompensateforthemetabolicalkalosis.TheinabilityofthekidneystoexcreteHCO3− isaresultoftheneedtoreduceNa+ excretiontocorrecttheECFVcontraction.Asdescribedpreviously(see fordetails)theresponseofthekidneystovolumecontractionistoreducetheglomerularfiltrationrate,whichreducesthefilteredloadofHCO3− ,andtoincreaseNa+ reabsorptionalongthenephron.BecausealargeamountofNa+ reabsorptionoccursviatheNa+/H+ antiporter,thisresultsinanincreaseinH+ secretion(HCO3− reabsorption)bytheproximaltubule.InthissettingtheentirefilteredloadofHCO3− isreabsorbedandnewHCO3− generationmayevenbeenhanced.Thelatterresponseoccursbecausealdosterone,thelevelsofwhichareelevatedinvolumecontraction,notonlystimulatesdistalNa+ reabsorptionbutalsoH+ secretionbyintercalatedcells.ThisstimulationofH+ secretiongeneratesnewHCO3− byenhancingtitratableacidandNH4+ excretion.Thusinindividualswholosegastriccontents,metabolicalkalosisandparadoxicallyacidicurinecharacteristicallyoccur.Correctionofthealkalosisoccursonlywheneuvolemiaisreestablished.

Table 37.1 summarizes the primary alterations and the subsequent compensatory or defense mechanisms of the various simple acid-base disorders. In all acid-base disorders the compensatory response does not correct the underlying disorder but simply reduces the magnitude of the change in pH. Correction of the acid-base disorder requires treatment of its cause.

Types of Acid-Base Disorders

Metabolic acidosis is characterized by a decreased ECF [HCO3 −] and pH. It can develop via the addition of nonvolatile acid to the body (e.g., diabetic ketoacidosis), the loss of nonvolatile base (e.g., HCO3 − loss caused by diarrhea), or failure of the kidneys to excrete titratable acid and NH4 + (e.g., renal failure). As previously described, the buffering of H+ occurs in both the ECF and ICF compartments. When the pH falls, the respiratory centers are stimulated and the ventilatory rate is increased (respiratory compensation).

Finally, in metabolic acidosis, RNAE is increased. This occurs via elimination of all HCO3 − from urine (enhanced reabsorption of filtered HCO3 −) and via increased titratable acid and NH4 + excretion (enhanced production of new HCO3 −). If the process that initiated the acid-base disturbance is corrected, the enhanced RNAE will ultimately return the pH and [HCO3 −] to normal. After correction of the pH, the ventilatory rate also returns to normal.

Metabolic alkalosis is characterized by an increased ECF [HCO3 −] and pH. It can occur via the addition of nonvolatile base to the body (e.g., ingestion of antacids), as a result of volume contraction (e.g., hemorrhage), or more commonly from loss of nonvolatile acid (e.g., loss of gastric HCl because of prolonged vomiting). Buffering occurs predominantly in the ECF and to a lesser degree in the

Respiratoryacidosis↓ Respiratoryalkalosis↑ ECF,extracellularfluid;ICF,intracellularfluid;RNAE,renalnetacidexcretion

Whennonvolatileacidisaddedtobodyfluids,asindiabetic ketoacidosis,the[H+]increases(pHdecreases),andthe[HCO3−]decreases.Inadditiontheconcentrationoftheanionassociatedwiththenonvolatileacidincreases.Thischangeinanionconcentrationprovidesaconvenientwayofanalyzingthecauseofametabolicacidosisbycalculatingwhatistermedtheanion gap. TheaniongaprepresentsthedifferencebetweentheconcentrationofthemajorECFcation(Na+)andthemajorECFanions(Cl− andHCO3−):

Undernormalconditionstheaniongaprangesfrom8to16mEq/L.Itisimportanttorecognizethatananiongapdoesnotactuallyexist.Allcationsarebalancedbyanions.Thegapsimplyreflectstheparametersthataremeasured.Inreality:

IftheanionofthenonvolatileacidisCl− ,theaniongapwillbenormal(i.e.,thedecreasein[HCO3−]ismatchedbyanincreasein[Cl−]).Metabolicacidosisassociatedwithdiarrheaorrenaltubularacidosis(i.e.,defectinrenalH+ secretion)hasanormalaniongap.Incontrast,iftheanionofthenonvolatileacidisnotCl− (e.g.,lactate,β-hydroxybutyrate),theaniongapwillincrease(i.e.,thedecreasein[HCO3−]isnotmatchedbyanincreasein[Cl−]butratherbyanincreaseintheconcentrationoftheunmeasuredanion).Theaniongapisincreasedinmetabolicacidosis–associatedketoacidosis(e.g.,diabetesmellitus)withrenalfailure,lacticacidosis,oringestionoftoxinsorcertaindrugs(e.g.,largequantitiesofaspirin).Thuscalculationoftheaniongapisausefulwayofidentifyingtheetiologyofmetabolicacidosisintheclinicalsetting.

Albuminisanegativelychargedmacromolecule,anditmakesaconsiderablecontributionto“unmeasuredanions.”Asaresulttheaniongapmustbeadjustedinpatientswhohaveanabnormalserum[albumin].Foreach1g/dLchangeinserum[albumin],theaniongapneedstobeadjustedinthesamedirectionby2.5mEq/L.

ICF. The increase in the pH inhibits the respiratory centers, which reduces the ventilatory rate, thus the PCO2 is elevated (respiratory compensation).

The renal compensatory response to metabolic alkalosis is to increase excretion of HCO3 − by reducing its reabsorption along the nephron. Normally this occurs quite rapidly (minutes to hours) and effectively. Enhanced renal excretion of HCO3 − eventually returns the pH and [HCO3 −] to normal, provided the underlying cause of the initial acid-base disturbance is corrected. When the pH is corrected, the ventilatory rate also returns to normal.

Respiratory acidosis is characterized by an elevated PCO2 and reduced ECF pH. It results from decreased gas exchange across the alveoli as a result of either inadequate ventilation (e.g., drug-induced depression of the respiratory centers) or impaired gas diffusion (e.g., pulmonary edema, such as occurs in cardiovascular and lung disease). In contrast to the metabolic disorders, buffering during respiratory acidosis occurs almost entirely in the ICF compartment. The increase in PCO2 and the decrease in pH stimulate both HCO3 − reabsorption by the nephron and titratable acid and NH4 + excretion (renal compensation). Together these responses increase RNAE and generate new HCO3 − . The renal compensatory response takes several days to occur. Consequently, respiratory acid-base disorders are commonly divided into acute and chronic phases. In the acute phase the time for the renal compensatory response is not sufficient, and the body relies on ICF buffering to minimize the change in pH. In the chronic phase, renal compensation occurs. Correction of the underlying disorder returns the PCO2 to normal and RNAE decreases to its initial level.

Respiratory alkalosis is characterized by a reduced PCO2 and an increased ECF pH. It results from increased gas exchange in the lungs, usually caused by increased ventilation from stimulation of the respiratory centers (e.g., via drugs or disorders of the central nervous system). Hyperventilation also occurs at high altitude and as a result of anxiety, pain, or fear. Buffering is primarily in the ICF compartment. As with respiratory acidosis, respiratory alkalosis has both acute and chronic phases reflecting the time required for renal compensation to occur. The acute phase of respiratory alkalosis reflects intracellular buffering, whereas the chronic phase reflects renal compensation. With renal compensation, the elevated pH and reduced PCO2 inhibit HCO3 − reabsorption by the nephron and reduce titratable acid and NH4 + excretion. As a result of these effects, RNAE is reduced. Correction of the underlying disorder returns the PCO2 to normal, and renal excretion of acid then increases to its initial level.

Analysis of Acid-Base Disorders

Analysis of an acid-base disorder is directed at identifying the underlying cause so appropriate therapy can be initiated. The patient’s medical history and associated physical findings often provide valuable clues about the nature and origin of an acid-base disorder. In addition, analysis of an arterial blood sample is frequently required. Such an analysis is straightforward if approached systematically. For example, consider the following data: pH = .

The acid-base disorder represented by these values or any other set of values can be determined using the following three-step approach: 1.

Examination of the pH. When pH is considered first, the underlying disorder can be classified as either an acidosis or an alkalosis. The defense mechanisms of the body cannot correct an acid-base disorder by themselves. Thus even if the defense mechanisms are completely operative, the change in pH indicates the acid-base disorder. In the example provided, the pH of 7.35 indicates acidosis.

2.

Determination of metabolic versus respiratory disorder. Simple acid-base disorders are either metabolic or

Role of the Kidneys in the Regulation of Acid-Base Balance respiratory. To determine which disorder is present the clinician must next examine the ECF [HCO3 −] and PCO2. As previously discussed, acidosis could be the result of a decrease in [HCO3 −] (metabolic) or an increase in PCO2 (respiratory). Alternatively, alkalosis could be the result of an increase in ECF [HCO3 −] (metabolic) or a decrease in PCO2 (respiratory). For the example provided, the ECF [HCO3 −] is reduced from normal (normal = 24 mEq/L), as is the PCO2 (normal = 40 mm Hg). The disorder must therefore be metabolic acidosis; it cannot be a respiratory acidosis because the PCO2 is reduced.

3. Analysis of a compensatory response. Metabolic disorders result in compensatory changes in ventilation and thus in PCO2, whereas respiratory disorders result in compensatory changes in RNAE and thus in ECF [HCO3 −] (

Table 37.2 ). In an appropriately compensated metabolic acidosis, the PCO2 is decreased, whereas it is elevated in compensated metabolic alkalosis. With respiratory acidosis, compensation results in an elevation of the [HCO3 −]. Conversely, ECF [HCO3 −] is reduced in response to respiratory alkalosis. In this example, the PCO2 is reduced from normal, and the magnitude of this reduction (10 mm Hg decrease in PCO2 for an 8 mEq/L decrease in ECF [HCO3 −]) is as expected (see

Table 37.2 ). Therefore the acid-base disorder is a simple metabolic acidosis with appropriate respiratory compensation. A mixed acid-base disorder reflects the presence of two or more underlying causes for the acid-base disturbance. For example, consider the following data: pH = .6 96

When the three-step approach is followed, it is evident that the disturbance is an acidosis that has both a metabolic component (ECF [HCO3 −] < 24 mEq/L) and a respiratory component (PCO2 > 40 mm Hg). Thus this disorder is mixed. Mixed acid-base disorders can occur, for example, in an individual who has a history of a chronic pulmonary disease such as emphysema (i.e., chronic respiratory acidosis) and who develops an acute gastrointestinal illness with diarrhea. Because diarrhea fluid contains HCO3 − , its loss from the body results in the development of metabolic acidosis.

A mixed acid-base disorder is also indicated when a patient has abnormal PCO2 and ECF [HCO3 −] values but the pH is normal. Such a condition can develop in a patient who has ingested a large quantity of aspirin. Salicylic acid (active ingredient in aspirin) produces metabolic acidosis and at the same time stimulates the respiratory centers, causing hyperventilation and respiratory alkalosis. Thus the patient has a reduced ECF [HCO3 −] and a reduced PCO2. (NOTE: The PCO2 is lower than would occur with normal respiratory compensation of a metabolic acidosis.) 1.

The kidneys maintain acid-base balance through excretion of an amount of acid equal to the amount of nonvolatile acid produced by metabolism and the quantity ingested in the diet (termed renal net acid excretion [RNAE]). The kidneys also prevent loss of HCO3 − in urine by reabsorbing virtually all the HCO3 − filtered at the glomeruli. Both reabsorption of filtered HCO3 − and excretion of acid are accomplished by secretion of H+ by the nephrons. Acid is excreted by the kidneys in the form of titratable acid (primarily as Pi) and NH4 + . Both titratable acid and NH4 + excretion result in generation of new HCO3 − , which replenishes the ECF HCO3 − lost during neutralization of nonvolatile acids.

2.

The body uses three lines of defense to minimize the impact of acid-base disorders on body fluid pH: (1) ECF and ICF buffering, (2) respiratory compensation, and (3) renal compensation.

Curthoys NP. Renal ammonium ion production and excretion. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013.

Dubose TD Jr. Disorders of acid-base balance. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.

Hamm LL, et al. Cellular mechanisms of renal tubule acidification. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013.

Weiner ID, Verlander JW. Renal acidification mechanisms. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.

Berend K, et al. Physiological approach to assessment of acid-base disturbances. N Engl J Med. 2014;371:1434-1445.

3.

Metabolic acid-base disorders result from primary alterations in ECF [HCO3 −], which in turn results from addition of acid to or loss of alkali from the body. In response to metabolic acidosis, pulmonary ventilation is increased, which decreases PCO2. The pulmonary response to metabolic acid-base disorders occurs in a matter of minutes. RNAE is also increased, but this takes several days. An increase in ECF [HCO3 −] causes alkalosis. This rapidly (minutes to hours) decreases pulmonary ventilation, which elevates PCO2 as a compensatory response. RNAE is also decreased, but this takes several days.

4.

Respiratory acid-base disorders result from primary alterations in PCO2. Elevation of PCO2 produces acidosis, and the kidneys respond with an increase in RNAE. Conversely, a reduction of PCO2 produces alkalosis, and RNAE is reduced. The kidneys respond to respiratory acid-base disorders over several hours to days.

Breton S, Brown D. Regulation of liminal acidification by the V-ATPase. Physiology (Bethesda). 2013;28:318-329.

Brown D, Wagner CA. Molecular mechanisms of acid-base sensing in the kidney. J Am Soc Nephrol. 2012;23:774-780.

Curthoys NP, Moe OW. Proximal tubule function and response to acidosis. Clin J Am Soc Nephrol. 2014;9:1627-1638.

Hamm LL, et al. Acid-base homeostasis. Clin J Am Soc Nephrol. 2015;10:2232-2242.

Levin LR, Buck J. Physiological roles of acid-base sensors. Annu Rev Physiol. 2015;77:347-362.

Roy A, et al. Collecting duct intercalated cell function and regulation. Clin J Am Soc Nephrol. 2015;10:305-324.

Seifter JL. Integration of acid-base and electrolyte disorders. N Engl J Med. 2014;371:1821-1831.

Weiner ID, Verlander JW. Ammonia transport in the kidney by rhesus glycoproteins. Am J Physiol Renal Physiol. 2014;306:F1107-F1120.

SECTION 8The Endocrine and Reproductive Systems BRUCE A. WHITE AND JOHN R. HARRISON

Chapter 39 Hormonal Regulation of Energy Metabolism

Introduction to the Endocrine System

Chapter 40 Hormonal Regulation of Calcium and Phosphate Metabolism

Chapter 41 The Hypothalamus and Pituitary Gland

The Thyroid Gland

The Adrenal Gland

Chapter 44 The Male and Female Reproductive Systems

Upon completion of this chapter, the student should be able to answer the following questions: 1.

Name the major endocrine glands and their hormonal product or products.

2.

Map out and differentiate a simple endocrine negative feedback loop and one involving the hypothalamus, anterior pituitary and peripheral endocrine gland, and list the major endocrine glands under each type of feedback loop.

3.

Define a releasing hormone and a tropic hormone.

4.

Explain the chemical nature and the characteristics of protein/peptide hormones, catecholamine hormones, steroid hormones, and iodothyronines (thyroid hormones). Include such characteristics as site of regulation (synthesis or secretion), circulating form of hormone, subcellular localization of hormone receptor, and metabolic clearance.

5.

Integrate the concept of peripheral conversion with the function/action of a secreted hormone.

6.

Integrate the intracellular steps associated with a hormone response in a target cell.

he ability of cells to communicate with each other is an underpinning of human biology. As discussed in , cell-to-cell communication exists at various levels of complexity and distance. Endocrine signaling involves (1) the regulated secretion of an extracellular signaling molecule, called a hormone, into the extracellular fluid; (2) diffusion of the hormone into the vasculature and its circulation throughout the body; and (3) diffusion of the hormone out of the vascular compartment into the extracellular space and binding to a specific receptor within cells of a target organ. Because of the spread of hormones throughout the body, one hormone often regulates the activity of several target organs. Conversely, cells frequently express receptors for multiple hormones.

The endocrine system is a collection of glands whose function is to regulate multiple organs within the body to (1) meet the growth and reproductive needs of the organism and (2) respond to fluctuations within the internal environment, including various types of stress. The endocrine system comprises the following major glands (

Fig. 38.1

Endocrine tissues of the pancreas Parathyroid glands

Pituitary gland (in association with hypothalamic nuclei)

Gonads (testes or ovaries) These endocrine glands synthesize and secrete bioactive hormones and, with the exception of gonads, which perform both endocrine and gametogenic functions, are dedicated to hormone production (

Table 38.1 ). A transitory organ, the placenta, also performs a major endocrine function.

In addition to dedicated endocrine glands, there are endocrine cells within organs whose primary function is not endocrine (see

Table 38.1 ). These include cells within the heart that produce atrial natriuretic peptide, liver cells that produce insulin-like growth factor type 1 (IGF-1), cells within the kidney that produce erythropoietin, and numerous cell types within the gastrointestinal tract that produce gastrointestinal hormones. There also exist collections of cell bodies (called nuclei) within the hypothalamus that secrete peptides, called neurohormones, into capillaries associated with the pituitary gland.

A third subset of the endocrine system is represented by numerous cell types that express intracellular enzymes, ectoenzymes, or secreted enzymes that modify inactive precursors or less active hormones into highly active hormones (see

Table 38.1). An example is the generation of angiotensin II from the inactive polypeptide angiotensinogen by two subsequent proteolytic cleavages (see ). Another example is activation of vitamin D by two subsequent hydroxylation reactions in the liver and kidneys to produce the highly bioactive hormone 1,25-dihydroxyvitamin D (vitamin D).

Configuration of Feedback Loops Within the Endocrine System

The predominant mode of a closed feedback loop among endocrine glands is negative feedback. In a negative feedback loop, a hormone acts on one or more target organs to induce a change (either a decrease or increase) in circulating levels of a specific component, and the change in this component then inhibits secretion of the hormone. Negative feedback loops confer stability by keeping a physiological parameter (e.g., blood glucose level) within a normal range. There are also a few examples of positive feedback

CHAPTER 38 Introduction to the Endocrine System 687 • Fig. 38.1 Glands of the endocrine system.

in endocrine regulation. A closed positive feedback loop, in which a hormone increases levels of a specific component and this component stimulates secretion of the hormone, confers instability. Under the control of positive feedback loops, something has got to give; for example, positive feedback loops control processes that lead to rupture of a follicle through the ovarian wall or expulsion of a fetus from the uterus.

There are two basic configurations of negative feedback loops within the endocrine system: a physiological response–driven feedback loop (referred to simply as a response-driven feedback loop) and an endocrine axis–driven feedback loop (

Fig. 38.2).

The response-driven feedback loop is observed in endocrine glands that control blood glucose levels (pancreatic islet cells), blood Ca++ and Pi levels (parathyroid glands, kidneys), blood osmolarity and volume (hypothalamus/posterior pituitary gland), and blood Na+ , K+ , and H+ levels (zona glomerulosa of the adrenal cortex and atrial cells). In the response-driven configuration, secretion of a hormone is stimulated or inhibited by a change in the level of a specific extracellular parameter (e.g., an increase in blood glucose level stimulates insulin secretion). Alterations in hormone levels lead to changes in the physiological characteristics of target organs (e.g., decreased hepatic gluconeogenesis, increased uptake of glucose by muscle) that directly regulate the parameter (in this case, blood glucose level) in question. The change in the parameter (decreased blood glucose level) then inhibits further secretion of the hormone (i.e., insulin secretion drops as blood glucose level falls).

Much of the endocrine system is organized into endocrine axes; each axis consists of the hypothalamus, the pituitary gland, and the peripheral endocrine glands (see

Fig. 38.2 ). Thus the endocrine axis–driven feedback loop involves a three-tiered configuration. The first tier is represented by hypothalamic neuroendocrine neurons that secrete releasing hormones. Releasing hormones stimulate (or, in a few cases, inhibit) the production and secretion of tropic hormones from the pituitary gland (second tier). Tropic hormones stimulate the production and secretion of hormones from peripheral endocrine glands (third tier). The peripherally produced hormones—namely, thyroid hormone, cortisol, sex steroids, and IGF-1—typically have pleiotropic actions (e.g., multiple phenotypic effects) on numerous cell types. However, in endocrine axis–driven feedback, the primary feedback loop involves feedback inhibition of pituitary tropic hormones and hypothalamic releasing hormones by the peripherally produced hormone. In contrast to response-driven feedback, the physiological responses to the peripherally produced hormone play only a minor role in regulation of feedback within endocrine axis–driven feedback loops. From a clinical perspective, endocrine diseases are described as primary, secondary, or tertiary diseases (e.g., secondary hyperthyroidism, tertiary hypogonadism). Primary disease is a lesion in the peripheral endocrine gland; secondary disease is a lesion in the anterior pituitary gland; and tertiary disease is a lesion in the hypothalamus.

An important aspect of the endocrine axes is the ability of descending and ascending neuronal signals to modulate release of the hypothalamic releasing hormones and thereby control the activity of the axis. A major neuronal input to releasing hormone–secreting neurons comes from another region of the hypothalamus called the suprachiasmatic nucleus (SCN). SCN neurons impose a daily rhythm, called a circadian rhythm, on the secretion of hypothalamic releasing hormones and the endocrine axes that they control (

Fig. 38.3).

SCN neurons represent an intrinsic circadian clock, as evidenced by the fact that they demonstrate a spontaneous peak of electrical activity at the same time every 24 to 25 hours. The 24-to 25-hour cycle can be “entrained” by the normal environmental light-dark cycle created by the earth’s rotation, so that the periodicity of the clock appears to be environmentally controlled (

Fig. 38.4

Hormones Produced to a Significant Degree by Peripheral Conversion

LungsAngiotensinIIKidney1,25-DihydroxyvitaminD(vitaminD)Adipose,mammaryglands,otherorgansEstradiol-17β Liver,sebaceousgland,otherorgansTestosteroneGenitalskin,prostate,otherorgans5-Dihydrotestosterone(DHT)ManyorgansTriiodothyronine •Fig. 38.2 Physiologicalresponse–drivenandendocrineaxis–drivennegativefeedbackloops.Endocrine gland Pituitary gland Releasing hormone Peripheral endocrine gland Hormone Hormone Negative feedback Negative feedback Circulating component (e.g., blood glucose) Tropic hormone Physiological effects Physiological effects Hypothalamic neuroendocrine neurons Target organ(s) Target organs ENDOCRINE AXIS–DRIVEN NEGATIVE FEEDBACK PHYSIOLOGICAL RESPONSE–DRIVEN NEGATIVE FEEDBACK

Neural input is generated from specialized light-sensitive retinal cells that are distinct from rods and cones and from signals to the SCN via the retinohypothalamic tract. Under constant conditions of light or dark, however, the SCN clock becomes “free running” and slightly drifts away from a 24-hour cycle each day.

The pineal gland forms a neuroendocrine link between the SCN and various physiological processes that require circadian control. This tiny gland, close to the hypothalamus, synthesizes the hormone melatonin from the neurotransmitter serotonin, of which tryptophan is the precursor. The rate-limiting enzyme for melatonin synthesis is N-acetyltransferase. The amount and activity of this enzyme in the pineal gland vary markedly in a cyclic manner, which accounts for the cycling of melatonin secretion and its plasma levels. Synthesis of melatonin is inhibited by light and markedly stimulated by darkness (see

Fig. 38.4 ). Thus melatonin may transmit the information that nighttime has arrived, and body functions are regulated accordingly. Melatonin feedback to the SCN at dawn or dusk may also help evoke day-night entrainment of the SCN 24to 25-hour clock. Melatonin has numerous other actions, including induction of sleep.

Another important input to hypothalamic neurons and the pituitary gland is stress, either as systemic stress (e.g., hemorrhage, inflammation) or as processive stress (e.g., fear, anxiety). Major medical or surgical stress overrides the circadian clock and causes a pattern of persistent and exaggerated hormone release and metabolism that mobilizes endogenous fuels, such as glucose and free fatty acids, and augments their delivery to critical organs. Growth and reproductive processes, in contrast, are suppressed. In addition, cytokines released during inflammatory or immune responses, or both, directly regulate the release of hypothalamic releasing hormones and pituitary hormones.

Chemical Nature of Hormones

Hormones are classified biochemically as proteins/peptides, catecholamines, steroid hormones, or iodothyronines. The chemical nature of a hormone determines (1) how it is synthesized, stored, and released; (2) how it is transported in blood; (3) its biological half-life and mode of clearance; and (4) its cellular mechanism of action.

Protein and peptide hormones can be grouped into structurally related molecules that are encoded by gene families. Protein/peptide hormones obtain their specificity from their primary amino acid sequence and from posttranslational modifications, especially glycosylation.

Because protein/peptide hormones are destined for secretion outside the cell, their synthesis and processing are differently from those of proteins destined to remain within the cell or to be continuously added to the membrane (

Fig. 38.5 ). These hormones are synthesized on the polyribosome as larger preprohormones or prehormones. The nascent peptides have at their N-terminus a group of 15 to 30 amino acids called the signal peptide. The signal peptide interacts with a ribonucleoprotein particle, which ultimately directs the growing peptide chain through a pore •Fig. 38.4 Originofcircadianrhythmsinendocrineglandsecretion,metabolicprocesses,andbehavioralactivity.(ModifiedfromTurekFW.Recent Prog Horm Res. 1994;49:43.) a membrane-bound secretory vesicle that is subsequently released into the cytoplasm. The carbohydrate moiety of glycoproteins is added in the Golgi apparatus.

Most hormones are produced as prohormones. Prohormones harbor the peptide sequence of the active hormone within their primary sequence. However, prohormones are inactive or less active and require the action of endopeptidases to trim away the neighboring inactive sequences.

D Plasma ACTH Protein/peptide hormones are stored in the gland as membrane-bound secretory vesicles and are released by exocytosis through the regulated secretory pathway. Thus these hormones are not continually secreted. Rather, they are secreted in response to a stimulus through a mechanism of stimulus-secretion coupling. Regulated exocytosis requires energy, Ca++ , an intact cytoskeleton (microtubules, microfilaments), and the presence of coat proteins that specifically deliver secretory vesicles to the cell membrane.

The ultrastructure of protein hormone–producing cells is characterized by abundant rough endoplasmic reticulum and Golgi membranes and the presence of secretory vesicles •Fig. 38.3 Acircadianpacemakerdirectsnumerousendocrineand bodyfunctions,eachwithitsowndailyschedule.Thenighttimerise inplasmamelatoninmaymediatecertainothercircadianpatterns.ACTH,adrenocorticotropichormone.(DatafromSchwartzWJ.Adv Intern Med. 1994;38:81.) in the membrane of the endoplasmic reticulum located on the cisternal (i.e., inner) surface of the endoplasmic reticular membrane. Removal of the signal peptide by a signal peptidase generates a hormone or prohormone, which is then transported from the cisternae of the endoplasmic reticulum to the Golgi apparatus, where it is packaged into (

Fig. 38.6

Protein/peptide hormones are soluble in body fluids and, with the notable exceptions of IGFs and growth hormone, circulate in blood predominantly in an unbound form and therefore have short biological half-lives. Protein hormones are removed from blood primarily by endocytosis and lysosomal degradation of hormone-receptor complexes (see the section “

Cellular Responses to Hormones”). Many protein hormones are small enough to appear in urine in a physiologically active form. For example, follicle-stimulating hormone and luteinizing hormone are present in urine.

Fig. 38.5 Schematic Representation of Peptide Hormone Synthesis. Inthenucleus,theprimarygenetranscript,amessengerRNAprecursormolecule,undergoesexcisionofintrons,splicingofexons,cappingofthe5′ end,andadditionofpolyadenylation(poly-A)atthe3′ end.TheresultantmaturemessengerRNAentersthecytoplasm,whereitdirectsthesynthesisofapreprohormonepeptidesequenceonribosomes.Inthisprocess,theN-terminussignalisremoved,andtheresultantprohormoneistransferredvectoriallyintotheendoplasmicreticulum.TheprohormoneundergoesfurtherprocessingandpackagingintheGolgiapparatus.Afterfinalcleavageoftheprohormonewithinthegranules,theycontainthehormoneandcopeptidesreadyforsecretionbyexocytosis.NH2,amidogen.

Fig. 38.6 Ultrastructure of a Protein Hormone–Producing Cell. Notethepresenceofsecretoryvesiclesandroughendoplasmicreticulumintheproteinhormone–secretingcell.(FromKierszenbaumAL.Histology and Cell Biology: An Introduction to Pathology. 2nded.Philadelphia:Mosby;2007.) •Fig. 38.7 Chemicalstructuresofcatecholamines.

Proteins/peptides are readily digested in the gastrointestinal tract if administered orally. Hence they must be administered by injection or, in the case of small peptides, through a mucous membrane (sublingually or intranasally). Because proteins/peptides do not cross cell membranes readily, they signal through membrane receptors (see

Catecholamines are synthesized by the adrenal medulla and neurons and include norepinephrine, epinephrine, and dopamine (

Fig. 38.7 ). The primary hormonal products of the adrenal medulla are epinephrine and, to a lesser extent, norepinephrine. Catecholamines obtain their specificity through enzymatic modifications of the amino acid tyrosine. Catecholamines are stored in secretory vesicles that are part of the regulated secretory pathway. They are copackaged with adenosine triphosphate, Ca++, and proteins called chromogranins. Chromogranins play a role in the biogenesis of secretory vesicles and in the organization of components within the vesicles. Catecholamines are soluble in blood and circulate either unbound or loosely bound to albumin. They are similar to protein/peptide hormones in that they do not cross cell membranes readily and hence produce their actions through cell membrane receptors. Catecholamines have short biological half-lives (1 to 2 minutes) and are removed from blood primarily by cell uptake and enzymatic modification.

Steroid hormones are made by the adrenal cortex, ovaries, testes, and placenta. Steroid hormones from these glands belong to five categories: progestins, mineralocorticoids, glucocorticoids, androgens, and estrogens. Progestins,

Bioactivehormonesaregeneratedfromprohormonesthroughproteolyticcleavageoftheprohormonebyprohormone (alsocalledproprotein)convertases. Theseenzymesareexpressedinacell-specificmanner.Forexample,insulin-producingcells(betacells)ofthepancreaticisletsexpressbothPC1andPC2.Insulinisproducedaspreproinsulin,cleavedtoproinsulinintheendoplasmicreticulum,andpackagedinsecretoryvesiclesasproinsulin.Whileinthesecretoryvesicle,aportionofthecenterofthesinglechain(connectingpeptide)iscleavedsequentiallybyPC1andPC2.Thematuresecretoryvesiclecontainsandsecretesequimolaramountsofinsulinandconnectingpeptide.Sometimesprohormonescontainthesequenceofmultiplehormones.Forexample,theproteinproopiomelanocortin(POMC)containstheaminoacidsequencesofadrenocorticotropichormone(ACTH)andmelanocyte-stimulatinghormones(MSHs).PituitarycellsexpressonlyPC1andreleaseonlyACTHasabioactivepeptide.Incontrast,certainneuronalcelltypesandkeratinocytesexpressbothPC1andPC2andcanproduceMSHs.Therearealsoprohormones,calledpolyproteins, thatcontainmultiplecopiesofthesamebioactivepeptide.Forexample,thesequenceforthyrotropin-releasinghormoneisreiteratedsixtimeswithintheprepro–thyrotropin-releasinghormonesequence.RaremutationsinPC1havebeenidentifiedinhumansandareassociatedwithextremeobesityinchildhood,defectsinglucosehomeostasis,lowglucocorticoidlevels,lossofmenstrualcyclesandhypogonadism,andproblemsingastrointestinalfunction.

mineralocoricoids, and glucocorticoids are 21-carbon steroids, whereas androgens are 19-carbon steroids and estrogens are 18-carbon steroids (

Table 38.2 ). Steroid hormones also include the active metabolite of vitamin D (see ), which is a secosteroid (i.e., one of the rings has an open conformation).

Gonadotropinsarepituitaryhormones,whichareluteinizinghormoneandfollicle-stimulatinghormone.Thesehormonesareheterodimersthatconsistofacommonα subunitandauniqueβ subunit(see ).Theurineofpostmenopausalwomenisanexcellentsourceofgonadotropinsbecausepostmenopausalserumgonadotropinlevelsarehighasaresultofthelossofnegativefeedbackbyovariansteroids(see ),andthehormonesarefilteredandexcretedasintactmoleculesinurine.Athirdgonadotropinistheplacentalhormonehumanchorionicgonadotropin(hCG;see ),whichhasthesamecommonα subunitandanhCG-specificβ subunit.Itisanextremelystablehormone,andbloodhCGlevelsdoubleevery2daysduringthefirsttrimester.Accordingly,urinarylevelsofhCGalsoincreaserapidly.Pregnancy tests arebasedonimmunologicaldetectioninurineofthehCGspecificβ subunitaspartoftheintacthCGheterodimer.

Steroid hormones are synthesized by a series of enzymatic modifications of cholesterol, and a cyclopentanoperhydrophenanthrene ring (or a derivative thereof) serves as their core (

Fig. 38.8 ). The enzymatic modifications of cholesterol are of three general types: hydroxylation, dehydrogenation/ reduction, and lyase reactions. The purpose of these modifications is to produce a cholesterol derivative that is sufficiently unique to be recognized by a specific receptor. Thus progestins bind to the progesterone receptor, mineralocorticoids bind to the mineralocorticoid receptor, glucocorticoids bind to the glucocorticoid receptor, androgens bind to the androgen receptor, estrogens bind to the estrogen receptor, and the active vitamin D metabolite binds to the vitamin D receptor. The complexity of steroid hormone action is increased by the expression of multiple forms of each receptor. In addition, there is some degree of nonspecificity between steroid hormones and the receptors to which they bind. For example, glucocorticoids bind to the mineralocorticoid receptor with high affinity, and progestins, glucocorticoids, and androgens can all interact with the progesterone, glucocorticoid, and androgen receptors to some degree. As discussed later, steroid hormones are hydrophobic and pass through cell membranes easily. Accordingly, classic steroid hormone receptors are localized intracellularly and act by regulating gene expression. There is mounting evidence of the presence of plasma membrane and juxtamembrane steroid hormone receptors that mediate rapid, nongenomic actions of steroid hormones.

Steroidogenic cell types are defined as cells that can convert cholesterol to pregnenolone, which is the first reaction common to all steroidogenic pathways. Steroidogenic cells have some capacity for cholesterol synthesis but often obtain cholesterol from cholesterol-rich lipoproteins (low-density lipoproteins and high-density lipoproteins). Pregnenolone is then further modified by several enzymatic reactions. Because of their hydrophobic nature, steroid hormones and precursors can leave the steroidogenic cell easily and thus are not stored. Therefore, steroidogenesis is regulated at the level of uptake, storage, and mobilization of cholesterol and at the level of steroidogenic enzyme gene expression and activity. Steroids are not regulated at the •Fig. 38.8 A, Structureofcholesterol,theprecursorofsteroidhormones.B, Structureofsteroidhormones.

Mitochondria with tubular cristae Nucleus •Fig. 38.9 Ultrastructure of a Steroidogenic Cell. Notetheabundanceoflipiddroplets,smoothendoplasmicreticulum,andmitochondriawithtubularcristae.(FromKierszenbaumAL.Histology and Cell Biology: An Introduction to Pathology. 2nded.Philadelphia:Mosby;2007.) level of secretion of the preformed hormone. A clinical implication of this mode of secretion is that high levels of steroid hormone precursors are easily released into blood when a steroidogenic enzyme within a given pathway is inactive or absent. The ultrastructure of steroidogenic cells is distinct from proteinand catecholamine-secreting cells. Steroidogenic enzymes reside within the inner mitochondrial membrane or the membrane of the smooth endoplasmic reticulum. Thus steroidogenic cells typically contain extensive mitochondria and smooth endoplasmic reticulum (

Fig. 38.9 ). These cells also contain lipid droplets, which represent a store of cholesterol esters.

An important feature of steroidogenesis is that steroid hormones often undergo further modifications (apart from those involved in deactivation and excretion) after their release from the original steroidogenic cell. For example, estrogen synthesis by the ovary and placenta requires at least two cell types to complete the conversion of cholesterol to estrogen. This means that one cell secretes a precursor and a second cell converts the precursor to estrogen. There is also considerable peripheral conversion of active steroid hormones. For example, the testes secrete little estrogen. However, adipose, muscle, and other tissues express the enzyme for converting testosterone (a potent androgen) to estradiol-17β (a potent estrogen). Thus the overall production of a particular steroid hormone is equivalent to the sum of the secretion of this steroid hormone from a steroidogenic cell type and peripheral conversion of other steroids to the particular steroid hormone (

Fig. 38.10 ). Peripheral conversion can produce (1) a more active but similar class of hormone (e.g., conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D); (2) a less active hormone that can be reversibly activated by another tissue (e.g., conversion of cortisol to cortisone in the kidneys, followed by conversion of cortisone to cortisol in abdominal adipose tissue); or (3) a different class of hormone (e.g., conversion of testosterone to estrogen). Peripheral conversion of steroids plays an important role in several endocrine disorders (see

Because of their nonpolar nature, steroid hormones are not readily soluble in blood. Therefore, steroid hormones circulate bound to transport proteins, including albumin, but also the specific transport proteins sex hormone– binding globulin and corticosteroid-binding globulin (see the section “

Transport of Hormones in the Circulation ”). Excretion of hormones from the body typically involves inactivating modifications, followed by glucuronide or sulfate conjugation in the liver, which is often coupled to biliary excretion. These modifications also increase the water solubility of the steroid and decrease its affinity for transport proteins, thereby allowing the inactivated steroid hormone to be excreted by the kidneys. Steroid compounds are absorbed fairly readily in the gastrointestinal tract and may therefore be administered orally.

Thyroid hormones are iodothyronines (Fig. 38.11 ) that are made by the coupling of iodinated tyrosine residues through an ether linkage. Their specificity is determined by the thyronine structure, as well as by where the thyronine is iodinated. Thyroid hormones cross cell membranes by •Fig. 38.10 Peripheralconversionofsteroidhormones.

3,5,3˜5˜-Tetraiodothyronine (thyroxine, or T4) 3,5,3˜-Triiodothyronine (T3) •Fig. 38.11 Structureofthyroidhormones,whichareiodothyronines.

transport systems. They are stored extracellularly in the thyroid as an integral part of the glycoprotein molecule thyroglobulin. Thyroid hormones are sparingly soluble in blood and aqueous fluids and are transported in blood that is bound (>99%) to serum-binding proteins. A major transport protein is thyroid hormone–binding globulin. Thyroid hormones have long half-lives (7 days for thyroxine; 18 hours for triiodothyronine). Thyroid hormones are similar to steroid hormones in that the thyroid hormone receptor is intracellular and acts as a transcription factor. In fact, the thyroid hormone receptor belongs to the same gene family that includes steroid hormone receptors and vitamin D receptor. Thyroid hormones can be administered orally; the amount absorbed intact is sufficient for this to be an effective mode of therapy.

Transport of Hormones in the Circulation

A significant fraction of steroid and thyroid hormones is transported in blood that is bound to plasma proteins that are produced in a regulated manner by the liver. Protein and polypeptide hormones are generally transported free in blood. The concentrations of bound hormone, free hormone, and plasma transport protein are in equilibrium. If free hormone levels drop, hormone will be released from the transport proteins. This relationship may be expressed as follows:

Equation 38.1 where [H] = concentration of free hormone, [P] = concentration of plasma transport protein, [HP] = concentration of bound hormone, and K = the dissociation constant.

Free hormone is the biologically active form for action on the target organ, feedback control, and clearance by cellular uptake and metabolism. As a consequence, when hormonal status is evaluated, sometimes free hormone levels must be determined in addition to total hormone levels. This is particularly important because hormone transport proteins themselves are regulated by altered endocrine and disease states.

Protein binding serves several purposes. It prolongs the circulating half-life of the hormone. Many hormones cross cell membranes readily and would either enter cells or be excreted by the kidneys if they were not protein bound. The bound hormone represents a reservoir of hormone and, as such, can serve to buffer acute changes in hormone secretion. Some hormones, such as steroids, are sparingly soluble in blood, and protein binding facilitates their transport.

Cellular Responses to Hormones

Hormones are also referred to as ligands, in the context of ligand-receptor binding, and as agonists, in that their binding to the receptor is transduced into a cellular response. Receptor antagonists typically bind to a receptor and lock it in an inactive state, in which the receptor is unable to induce a cellular response. Loss or inactivation of a receptor results in hormonal resistance. Constitutive activation of a receptor leads to unregulated, hormone-independent activation of cellular processes.

Hormones regulate essentially every major aspect of cellular function in every organ system. Hormones control the growth of cells, ultimately determining their size and competency for cell division. Hormones regulate the differentiation of cells and their ability to survive or to undergo programmed cell death. They influence cellular metabolism, the ionic composition of body fluids, and cell membrane potential. Hormones orchestrate several complex cytoskeleton-associated events, including cell shape, migration, division, exocytosis, recycling/endocytosis, and cell-cell and cell-matrix adhesion. Hormones regulate the expression and function of cytosolic and membrane proteins, and a specific hormone may determine the level of its own receptor or the receptors for other hormones.

Although hormones can exert coordinated, pleiotropic control on multiple aspects of cell function, any given hormone does not regulate every function in every cell type. Rather, a single hormone controls a subset of cellular functions in only the cell types that express receptors for that hormone. Thus selective receptor expression determines which cells respond to a given hormone. Moreover, the differentiated state of a cell determines how it responds to a hormone. Thus the specificity of hormonal responses resides in the structure of the hormone itself, the receptor for the hormone, and the cell type in which the receptor is expressed. Serum hormone concentrations are typically extremely low (10−11 to 10−9 mol/L). Therefore, a receptor must have high affinity, as well as specificity, for its cognate hormone.

How does hormone-receptor binding get transduced into a cellular response? Hormone binding to a receptor induces conformational changes in the receptor. These changes are collectively referred to as a signal. The signal is transduced into the activation of one or more intracellular messengers. Messenger molecules then bind to effector proteins, which in turn modify specific cellular functions. The combination of hormone-receptor binding (signal), activation of messengers (transduction), and regulation of one or more effector proteins is referred to as a signal transduction pathway (also called simply a signaling pathway), and the final outcome is referred to as the cellular response.

Signaling pathways are usually characterized by the following properties: 1.

Multiple, hierarchical steps in which “downstream” effector proteins are dependent on and driven by “upstream” receptors, transducers, and effector proteins. This means that loss or inactivation of one or more components within the pathway leads to general resistance to the hormone, whereas constitutive activation or overexpression of components can drive a pathway in an unregulated manner.

2.

Amplification of the initial hormone-receptor binding. Amplification can be so great that maximal response to a hormone is achieved when the hormone binds to a small percentage of receptors.

3.

Activation of multiple pathways, or at least regulation of multiple cell functions, from one hormone-receptor binding event. For example, binding of insulin to its receptor activates three separate signaling pathways. Even in fairly simple pathways (e.g., glucagon activation of adenylate cyclase), divergent downstream events allow the regulation of multiple functions (e.g., post-translational activation of glycogen phosphorylase and increased phosphoenolpyruvate carboxykinase gene transcription).

4.

Antagonism by constitutive and regulated negative feedback reactions. This means that a signal is dampened or terminated (or both) by opposing reactions and that loss or gain of function of opposing components can cause hormone-independent activation of a specific pathway or hormone resistance. As discussed in , hormones signal to cells through membrane or intracellular receptors. Membrane receptors have rapid effects on cellular processes (e.g., enzyme activity, cytoskeletal arrangement) that are independent of the synthesis of new protein. Membrane receptors can also rapidly regulate gene expression through either mobile kinases (e.g., cyclic adenosine monophosphate–dependent protein kinase [PKA], mitogen-activated protein kinases [MAPKs]) or mobile transcription factors (e.g., signal transducer and activator of transcription proteins [STATs], Mothers against decapentaplegic homologs [Smads]). Steroid hormones have slower, longer term effects that involve chromatin remodeling and changes in gene expression. Increasing evidence indicates that steroid hormones have rapid, nongenomic effects as well, but these pathways are still being elucidated.

The presence of a functional receptor is an absolute requirement for hormone action, and loss of a receptor produces essentially the same symptoms as loss of hormone. In addition to the receptor, there are fairly complex pathways involving numerous intracellular messengers and effector proteins. Accordingly, endocrine diseases can arise from abnormal expression or abnormal activity, or both, of any of these signal transduction pathway components. Finally, hormonal signals can be terminated in several ways, including hormone/receptor internalization, phosphorylation/ dephosphorylation, proteosomal destruction of receptor, and generation of feedback inhibitors.

Endocrinediseasescanbebroadlycategorizedashyperfunctionorhypofunctionofaspecifichormonalpathway.Hypofunctioncanbecausedbylackofactivehormoneorbyhormone resistance asaresultofinactivationofhormonereceptorsorpostreceptordefects.Testicular feminization syndrome isadramaticformofhormoneresistanceinwhichtheandrogenreceptorismutatedandcannotbeactivatedbyandrogens.Inpatientsinwhomthediagnosisisnotmadebeforepuberty,thetestisbecomeshyperstimulatedbecauseofabrogationofthenegativefeedbackbetweenthetestisandthepituitarygland.Theincreasedandrogenlevels 1.

Endocrine signaling involves (1) regulated secretion of an extracellular signaling molecule, called a hormone, into the extracellular fluid; (2) diffusion of the hormone into the vasculature and circulation throughout the body; and (3) diffusion of the hormone out of the vascular compartment into the extracellular space and binding to a specific receptor within cells of a target organ.

2.

The endocrine system is composed of the endocrine tissue of the pancreas, the parathyroid glands, the pituitary gland, the thyroid gland, the adrenal glands, and the gonads (testes or ovaries).

3.

Negative feedback represents an important control mechanism that confers stability on endocrine systems. Hormonal rhythms are imposed on negative feedback loops.

4.

Protein/peptide hormones are produced on ribosomes and stored in endocrine cells in membrane-bound secretory granules. They typically do not cross cell

Bae YJ, Kratzsch J. Corticosteroid-binding globulin: modulating mechanisms of bioavailability of cortisol and its clinical implications. Best Pract Res Clin Endocrinol Metab. 2015;29:761-772.

havenodirectbiologicaleffectasaresultofthereceptordefect.However,theandrogensareperipherallyconvertedtoestrogens.Thusaffectedindividualsaregeneticallymale(i.e.,46,XY)buthaveastronglyfeminizedexternalphenotype,afemalesexualidentity,andusuallyasexualpreferenceformen(i.e.,heterosexualinrelationtosexualidentity).Treatmentinvolvesremovalofthehyperstimulatedtestes(whichresideintheabdomenandposeariskforcancer),estrogenreplacementtherapy,andcounselingforthepatientand,ifoneexists,thepartner/spousetoaddressinfertilityandsocial/psychologicaldistress.

membranes readily and act through cell membrane– associated receptors.

5.

Catecholamines are synthesized in the cytosol and secretory granules and do not readily cross cell membranes. They act through cell membrane– associated receptors.

6.

Steroid hormones are not stored in tissues and generally cross cell membranes relatively readily. They act through intracellular receptors.

7.

Thyroid hormones are synthesized in follicular cells and stored in follicular colloid as thyroglobulin. They cross cell membranes and associate with nuclear receptors.

8.

Some hormones act through membrane receptors, and their responses are mediated by rapid intracellular signaling pathways.

9.

Other hormones bind to nuclear receptors and act by directly regulating gene transcription.

Vija L, Ferlicot S, Paun D. Testicular histological and immunohistochemical aspects in a post-pubertal patient with 5 alpha-reductase type 2 deficiency: case report and review of the literature in a perspective of evaluation of potential fertility of these patients. BMC Endocr Disord. 2014;14:43.

Upon completion of this chapter the student should be able to answer the following questions: 1.

Explain the different requirements for and utilization by different cells of fuels during the digestive phase as opposed to the interdigestive and fasting phases.

2.

Integrate the structure, synthesis, and secretion of insulin with circulating fuel levels, especially glucose.

3.

Utilize the different signaling pathways regulated by insulin to link insulin to itscellular effects at the molecular level.

4.

Integrate the structure, synthesis, and secretion of glucagon with the levels of circulating fuels, insulin, and catecholamines.

5.

Map out and integrate the actions of insulin on the utilization and storage of glucose, free fatty acids (FFAs), and amino acids (AAs) by hepatocytes, skeletal muscle, and adipocytes during the digestive phase.

6.

Map out and integrate the actions of counterregulatory hormones (glucagon, catecholamines) on the utilization of glucose, the sparing of glucose, and the utilization of FFAs and AAs by hepatocytes, skeletal muscle, and adipocytes during the interdigestive and fasting phases.

7.

Integrate the changes in fuel utilization and hormonal signaling in hepatocytes during the interdigestive and fasting phases that allow for and promote hepatic glucose production and ketogenesis.

8.

Compare signaling pathways that have orexigenic and anorexigenic actions via the hypothalamus.

9.

Link several pathologies related to metabolism, especially those caused by the absolute or relative absence of insulin and by obesity.

Continual Energy Supply and Demand: The Challenge

There are an estimated 40 trillion cells in the human body, not including the approximately 40 trillion nonhuman cells that comprise the human microbiome. All these cells must continually perform work to stay alive. This work includes maintenance of cellular composition and structural integrity, along with the integrated synthesis and breakdown (i.e., turnover) of macromolecules and organelles. This work also involves the functions of cells that contribute to the human body as a whole (e.g., contraction of the muscle fibers of the diaphragm). Additional work is required of cells when the human body is engaged in a variety of activities, including (but not limited to) manual labor, exercise, and outdoor play; body growth spurt and maturation of the reproductive systems at puberty; pregnancy and breastfeeding; combating a serious infection or cancer; and the healing of damaged tissues/organs (e.g., a broken bone or healing from surgery). On average the resting metabolic rate of a relaxed, awake, stationary, healthy adult human accounts for about 70% of their total energy expenditure each day (

Fig. 39.1

To perform this work, cells need fuels, along with the capability to convert fuels into potential chemical energy in the form of adenosine triphosphate (ATP). Cells then convert the energy within ATP into chemical and mechanical work (see

Fig. 39.1 ). This means that the need for ATP is immediate and unending, and consequently all living cells must continually synthesize ATP. In fact, humans produce about the equivalent of their body weight in ATP daily. This places a demand on the body to continually supply fuel in some form to all cells. All fuel originates from the diet, but humans do not eat in a nonstop manner all day long. Thus the constant cellular demand for fuels to make ATP and perform work is paired with an intermittent intake of fuels. Diet-derived fuels are oxidized for ATP, but some are also stored for future use, and some are converted to other fuels that can be used by other cell types, depending on the metabolic phase, type of fuel, and cell type in question.

In trying to make sense of energy metabolism, it is important to organize one’s thinking around the following: 1. Metabolic phases. Metabolic phases refer to the hourly and daily differences in fuel usage and energy metabolism, which are dictated largely by the abundance or scarcity of certain fuels and orchestrated by phase-specific hormones. In general there are three metabolic phases (

Fig. 39.2 ): (1) the digestive or absorptive phase, which occurs during the 2 to 3 hours it takes to digest a meal; (2) the interdigestive or postabsorptive phase, which normally occurs between meals; and (3) the

Sources of fuels 1.

2.

Diet—Release of stored fuel during postabsorptive stage (especially during sleep on a daily basis, or during a fast or prolonged exercise)

ATP constant amount universal not stored Structural integrity Differentiated function Growth and division Response to stimuli and stress Output 60%–70% = Resting metabolic rate 25%–30% = Movement Glucose, FFAs, AAs, ketone bodies EnergyOxidation of fuels 60% Heat 40% •Fig. 39.1 Overviewofenergymetabolism.

•Fig. 39.2 Bloodglucoselevelsduringthethreemetabolicphases:D,digestivephase;F,fastingphase;I,interdigestivephase.B,break-fast;D,dinner;L,lunch.Red arrow indicatestheupperlimitfornormalfastingglucose;green arrow indicatesthelowerlimitfornormalfastingglucose.FFB150100BloodGlucose(mg/dL)50LDDDDII fasting phase, which most commonly occurs between the last snack before bedtime and breakfast. (In fact, physicians refer to a blood value as “fasting,” e.g., “fasting blood glucose,” if the patient abstains from eating after midnight and has blood drawn about 8 AM; prolonged fasting and starvation are more extreme forms of fasting.) Physical exertion, which imposes a heightened energy demand, is another type of metabolic phase that occurs with some frequency and regularity for some individuals. This chapter primarily compares how metabolism differs between the digestive phase and the fasting phase, and how different hormones orchestrate these metabolic differences.

2, Metabolic actions of hepatocytes, adipocytes, and skeletal myocytes. All cells are involved in energy metabolism, but these three cell types have a profound impact on whole-body metabolism. Key features with respect to metabolism of these three cell types are listed in

Table 39.1 3, Insulin and the counterregulatory hormones. Metabolism during the digestive phase is orchestrated almost entirely by insulin. During the fasting phase, insulin drops to low levels, and this alone allows for some of the metabolic adaptions associated with fasting. In addition, glucagon and catecholamines (epinephrine, norepinephrine) stimulate metabolic pathways that integrate the body’s response to an absence of ingested and absorbed fuels. These hormones are referred to as counterregulatory hormones based on their opposition to insulin. Growth hormone (see ) also contribute somewhat to fasting-phase metabolism.

Integrated Overview of Energy Metabolism

Fuels enter the body from the diet during the digestive phase. Our diet includes both monomeric and polymeric forms (the latter are converted into monomeric forms during digestion and absorption) of the following fuels: (1) monosaccharides, including glucose, fructose, and galactose; (2) long-chain free fatty acids (referred to in this chapter as simply FFAs); and (3) amino acids (AAs). The diet may also include other fuels such as ethanol.

Glucose,fed•UtilizationforATPphase•Storageasglycogen

Glucose,fasting•Breakdownofglycogenandphasereleaseofglucoseintoblood

FFA/TG,fedphase•MakeFFAsfromglucosebyDNL

FFA/TG,fasting•UtilizationforATPphase•UtilizationtoproduceKBs

SecretionofVLDLAAs,fedphase•Utilizationformultipleanabolic pathwaysAAs,fastingphase•Utilizationforgluconeogenesis

UtilizationforketogenesisKBs,fedphase•ShouldbeabsentKBs,fastingphase•SynthesisfromFFAsandsome

AA,aminoacid;ATP,adenosinetriphosphate;DNL,denovolipogenesis;FFA,freefattyacid;G3P,glycerol-3-phosphate;KB,ketonebody;TG,triglyceride;VLDL,verylow-densitylipoprotein.

During the digestive phase, absorbed fuels are partitioned and used for different purposes. Insulin (discussed in detail later) regulates essentially every aspect of metabolism during the digestive phase. Glucose is the primary fuel used for energy (i.e., ATP production) during the digestive phase (

Fig. 39.3A ). Glucose is considered a universal fuel in that most cells can perform the following: 1.

2.

“trap” and “activate” imported glucose by converting glucose into glucose-6-phosphate (G6P) through the activity of one or more hexokinases. G6P cannot pass through GLUT transporters (“trapping”) and is now a substrate for several enzymatic pathways (“activation”).

3.

metabolize G6P to pyruvate via the glycolytic pathway, which yields a small amount of ATP without requiring mitochondria or O2. Cells without mitochondria ferment pyruvate to lactate and export lactate as waste. Most cells import pyruvate into mitochondria, convert it to acetyl CoA by pyruvate dehydrogenase, and then condense acetyl CoA with oxaloacetate to form citrate. Citrate is cycled through the tricarboxylic acid (TCA) cycle back to oxaloacetate. This metabolism of pyruvate through the TCA cycle releases CO2 as waste and generates guanosine triphosphate (GTP) along with flavin adenine dinucleotide hydride (FADH2) and nicotine adenine dinucleotide hydride (NADH). FADH2 and NADH are used by the electron transport system and oxidative phosphorylation to ultimately generate relatively large amounts of ATP through a process that is absolutely dependent on O2. Cells with no or very few mitochondria (e.g., erythrocytes, lens cells of the eye) are absolutely dependent on glucose for energy. Additionally the brain can only use glucose under normal conditions, in that it has essentially no capacity for oxidation of FFAs and only marginal capacity for the use of lactate, ketone bodies (KBs), and AAs as fuels. Thus in all metabolic phases, maintenance of blood glucose above a certain minimal threshold is absolutely necessary to avoid central nervous system (CNS)-related symptoms, beginning with those caused by a hypoglycemia-activated autonomic response (e.g., nausea, sweating, cardiac arrhythmias). If blood glucose continues to fall, progression to symptoms caused by neuroglycopenia (e.g., cognitive dysfunction, loss of coordinated motor function, and ultimately even coma

RBCCNSDietG6PG6P GLY HEPATOADIPOSKM2,4,5 11 2,3 2,4,5 8 7,82,4,52,3,4,5 6 G6P G6P G3G1 1 1 1 G2 G4-mG4-mG4-m ATP A. Digestive Phase ATP ATP TG G’OL3P G6P TG GlucoseATP 6 ATP GLY ATP G6P AAs G’OL ATP ATP13 TG 11 11 12 12 FFA A,4,5 GLY ATP RBCSKMG6P KBs CNS2,4,5112 3 HEPATOFFA 9 11 10 KBs A,4,5 A,4,5 B,4,5 G4-iG4-i B,4,5 ADIPOB,4,5 GLY G6P LACT ATP G6P Glucose PROT G4-iG4-i G1G1 G3G3 G2G2 B. Fasting Phase •Fig. 39.3 A, Overview of glucose utilization during the digestive phase. GLUTtransporters:G1,GLUT1;G2,GLUT2;G3,GLUT3;G4-m,functionalGLUT4localizedtocellmembrane.Celltypes:ADIPO,adipocyte;CNS,centralnervoussystemneuronsandglia;HEPATO,hepatocyte;RBC,redbloodcell;SKM,skeletalmyocyte.Metabolites:G6P,glucose-6-phosphate;GLY,glycogen;G’OL3P,glycerol-3-phosphate;TG,triglyceride.Metabolicreactions/pathways:1,hexokinase/glucokinase;2,glycolysis;3,lactatedehydrogenase;4,TCAcycle± pyruvatedehydrogenase;5,oxidativephosphorylation;6,glycogensynthesis;7,denovolipogenesis;8,esterificationofFFAstoG’OL3PtoformTG.B, Overview of energy metabolism during the fasting phase. GLUTtransporters:seelegendforA,plus:G4-I,inactiveGLUT4withintracellularlocalization.Celltypes:seelegendforAMetabolites:seelegendforA,plus:AAs,aminoacids;FFA,freefattyacid;G’OL,glycerol;KBs,ketonebodies;LACT,lactate;PROT,protein.Metabolicreactions/pathways:seelegendforA,plus:9,glycogenolysis;10,gluconeogenesis;11,G6Pase;12,ketogenesis;13,proteolysis;A,β-oxidation;B,ketolysis.

and death) can occur. This means that whole-body metabolism during the interdigestive and fasting phases must meet the challenge of maintaining blood glucose above 60 mg/ dL (see

Fig. 39.2 , green arrow).

On the other hand, glucose levels must be maintained below a certain maximal threshold (i.e., fasting blood glucose < 100 mg/dL) to avoid glucose-induced side reactions (both enzymatic and nonenzymatic) as well as osmotic imbalances that lead to multiple forms of damage to cell function (see

Fig. 39.2 , red arrow).

Theendotheliumofthemicrovasculatureofthekidneyandretina,aswellastheendotheliumofthevasanervosumoftheautonomicnervoussystem,areparticularlysensitivetohyperglycemia.Chronicallyhighbloodglucoseresultsinpathologicallyhighintracellularlevelsofglucoseintheseendothelialcells,resultinginalteredproteinandlipidstructure,oxidativestress,andalteredsignalingpathways.Theseinsults,collectivelyreferredtoasglucotoxicity, causepathologicalchangesinintracellularandmembranecomponentsaswellasinsecretedmoleculesthateithersignaland/ormakeuptheextracellularmatrix.Indeed,glucotoxicityistherootcauseofthenephropathy, retinopathy, and peripheral neuropathy thatoccurinpoorlycontrolleddiabetesmellitus.Thereforewhole-bodymetabolismduringallmetabolicphasesmustmeetthechallengeofminimizingthemagnitudeanddurationoftheriseinbloodglucoseassociatedwithingestionofamealandmustmaintainbloodglucosebelowasafemaximalthresholdof100mg/dLduringallothertimes.Fastingbloodglucosebetween100and124mg/dLisindicativeofimpaired glucose tolerance, andvaluesat125mg/dLandaboveareevidenceofdiabetes mellitus.

Glucose is consumed by erythrocytes and the brain continually throughout all metabolic phases. In contrast, hepatocytes, skeletal myocytes, and adipocytes primarily use glucose during the digestive phase only. Insulin stimulates glycolysis and entry of pyruvate (end product of glycolysis) into the TCA cycle and oxidative phosphorylation for ATP production in hepatocytes, skeletal myocytes, and adipocytes (see

Table 39.1).

Hepatocytes express the GLUT2 isoform of the glucose transporter, which is not regulated by insulin for its insertion into the cell membrane. In contrast, skeletal myocytes and adipocytes express the GLUT4 isoform. Newly synthesized GLUT4 exists in an intracellular inactive state with GLUT4 storage vesicles (G4-i in

Fig. 39.3B ). Insulin induces translocation of GLUT4 to the cell membrane, where it can function as an active glucose transporter (G4-m in

Fig. 39.3A).

After its phosphorylation to G6P by glucokinase, hepatocytes convert some of the imported glucose into the storage form, glycogen, during the digestive phase (see

Fig.

39.3A ). Similarly, skeletal muscle converts some of the G6P from imported glucose into glycogen. Hepatocytes can only store a finite amount of glucose as glycogen. Hepatocytes also convert excess glucose into FFAs through the process of de novo lipogenesis (DNL). These FFAs are typically esterified to glycerol-3-phosphate (G3P) to form triglyceride (TG), which accumulates as intrahepatic TG during the digestive phase (see

Fig. 39.3A ). As discussed later for insulin signaling, an excessive accumulation of intrahepatic TG (i.e., fatty liver, hepatic steatosis) can result in insulin resistance.

During the digestive phase, AAs are used in multiple anabolic pathways to regenerate degraded molecules, including other AAs, proteins, nucleotides and nucleic acids, glutathione, and complex lipids.

FFAs represent the most efficient fuel type in terms of ATP molecules made per carbon of fuel. However, utilization of FFAs competes effectively with glucose utilization in the mitochondria. High FFA levels during the digestive phase would promote a greater magnitude and duration of the glucose surge, thereby contributing to hyperglycemia. Thus most of the FFAs in an average meal are prevented from entering the circulation by their reesterification into TG and packaging into chylomicrons within the intestinal enterocyte. Chylomicrons are secreted, enter lymphatic vessels, and ultimately enter the blood. Once in the general circulation, chylomicrons convey the FFAs (as TG) to the adipose tissue where they are released from chylomicronassociated TG through lipolysis, imported by adipocytes, and reesterified as intracellular TG for storage (discussed in detail later).

During the digestive phase, hepatocytes, skeletal myocytes, and adipocytes function largely independently of each other. In contrast the actions of these three cell types become highly integrated during the fasting phase to maintain adequate blood glucose levels while providing alternative energy substrates for each cell type (see

Fig.

39.3B

Table 39.1

As stated earlier, cells without mitochondria (e.g., erythrocytes) and cells of the CNS require glucose for ATP production during all metabolic phases, and thus the body must maintain blood glucose above 60 mg/dL even days or weeks after the last ingestion of food (an exception to this rule is that the brain can use KBs [discussed later] during a prolonged fasting phase). Two general processes contribute to maintenance of blood glucose during the fasting phase: hepatic glucose production and glucose sparing. Hepatic glucose production is in turn based on two metabolic pathways. The first is the rapid catabolic process of glycogenolysis (pathway 9 in

Fig. 39.3B ). Hepatocytes express the enzyme glucose-6-phosphatase (G6Pase) (pathway 11 in

Fig. 39.3B ), allowing them to convert G6P back to glucose, which can then exit the cell through a bidirectional GLUT transporter. Release of glucose derived from glycogenolysis is relatively short

Source of blood

DietaryGluconeogenesis0 Breakfast 12.00 24.00Glycogenolysis •Fig. 39.4 Relativecontributionsofthethreesourcesofbloodglucoserelativetomealsandtimeofday.Theinsetboxstressesreplacementofglycogenolysiswithgluconeogenesisduringthefastingphase(i.e.,sleep).(AdaptedfromBaynesJW,DominiczakJH[eds].Medical Biochemistry. 3rded.Philadelphia:Mosby/Elsevier;2009.) lived because the liver glycogen supply becomes exhausted by about 8 hours. The second metabolic contribution to hepatic glucose production during the fasting phase is the gradual pathway of gluconeogenesis (pathway 10 in

Fig.

39.3B ). The onset of gluconeogenesis during fasting is slower than glycogenolysis, but gluconeogenesis continues essentially nonstop throughout a fasting phase (

Fig. 39.4 ). Gluconeogenesis requires precursors, especially lactate, “gluconeogenic” AAs, and glycerol. How are these precursors supplied during the fasting phase? Lactate is continually produced by erythrocytes. Lactate is also produced by glycolytic skeletal muscle fibers during exercise (exercise tends to occur more frequently during the interdigestive and fasting phases as opposed to “on a full stomach”), although much of this lactate is utilized by aerobic skeletal muscle and cardiac muscle during exercise. But additionally the overall anabolism of the digestive phase switches over to a general catabolism during the fasting phase (see

Fig.

39.3B ). TGs within adipocytes undergo lipolysis to FFAs and glycerol, and there is a general net proteolysis with the release of AAs during the fasting state. The glycerol and gluconeogenic AAs are released from cells and circulate to the liver, where they are subsequently used for gluconeogenesis. Thus gluconeogenesis requires an integration of catabolic pathways in adipocytes and skeletal myocytes with anabolic gluconeogenesis in hepatocytes. Gluconeogenesis eventually supplants glycogenolysis and can continue as long as precursors flow into the liver.

Glucose sparing represents the other general process that contributes to maintenance of adequate circulating glucose levels during the fasting phase. Glucose sparing means the switching of fuel utilization from glucose to a nongluconeogenic fuel in most cell types, but especially in skeletal muscle, which represents the potentially largest single consumer of glucose. First, the uptake of glucose by skeletal muscle and adipocytes is greatly reduced because the GLUT4 transporter isoform exists in an intracellular

CHAPTER 39 Hormonal Regulation of Energy Metabolism

Fig. 39.3B) during the fasting phase.

Thus alternative fuels need to be delivered to skeletal muscle and adipocytes.

The nongluconeogenic fuels (i.e., cannot be used for gluconeogenesis by the liver) are FFAs and KBs. FFAs are

Fig. 39.3B are also released after packaging of intrahepatic TGs into cussed later). KBs are produced by hepatocytes from acetyl

CoA, which in turn originates from FFAs and ketogenic

AAs, both of which become abundant during the fasting phase. Thus glucose sparing depends on catabolic adipocyte metabolism, which results in lipolysis of stored TGs and release of FFAs. FFAs are imported by hepatocytes, which use FFAs to produce acetyl CoA. Protein degradation in available for ketogenesis. High levels of intramitochondrial acetyl CoA in the hepatocyte not only provides ample carbons for ATP synthesis but serves to: (1) inhibit conver sion of pyruvate to acetyl CoA, (2) promote conversion of pyruvate to oxaloacetate for gluconeogenesis, and (3) promote synthesis of KBs (see

Fig. 39.3B ). After several days of fasting, the CNS can start using KBs for energy, thereby further sparing glucose for erythrocytes. Many other cell types with mitochondria use KBs along with FFAs for ATP production, especially skeletal muscle.

The hormones that drive glycogenolysis, gluconeogen esis, lipogenesis, and hepatic ketogenesis as well as VLDL production by the liver during the fasting phase are glucagon and catecholamines. In the presence of low glucose, insulin levels fall, and that removes the inhibition by insulin of the secretion of another pancreatic hormone, glucagon. Thus diminished blood glucose causes a rise in the circulating glucagon-to-insulin ratio. Hepatocytes are the primary target organ of glucagon, which directly drives glyco genolysis, gluconeogenesis, FFA oxidation, and ketogenesis by the hepatocyte (

Fig. 39.5B).

from sympathetic innervation and epinephrine from the ) can reinforce the actions of glucagon. Adipocytes also express the glucagon receptor, as well as the β2-and β3-adrenergic receptors that respond to catecholamines in response to hypoglycemia, exertion, or certain stresses. Skeletal muscle is not a target of glucagon but does respond to catecholamines stimulation through β2-adrenergic receptors. Skeletal muscle is very responsive to intracellular Ca++ , which increases during physical exertion/movement, and to an increase in the intracellular adenosine monophosphate (AMP):ATP ratio, which activates AMP kinase.

Finally, it is important to understand that the pathways upregulated during the fasting phase are opposed by insulin-dependent pathways that are most active during the digestive phase (discussed later). Thus attenuation of insulin signaling also contributes to the ability of hepatocytes, skeletal myocytes, and adipocytes to display an integrated response to the metabolic challenges of the fasting phase.

•Fig. 39.5 TheisletsofLangerhans(endocrinepancreas)fromrat.A, Pancreashistologyshowingexocrineaciniwheredigestiveenzymesareproducedtobedeliveredtotheduodenumviathepancreaticduct,andanendocrineisletwhereinsulinandglucagonareproducedanddeliveredtothecirculationuponuptakebyarichcapillarybed.B, Stainingofendocrineisletforinsulinwithinbetacells;thesearethemostnumerouscelltypeandareprimarilylocatedcentrallywithintheislet.C, Stainingofendocrineisletforglucagonwithalphacells;thesearemuchlessnumerousthanbetacellsandareprimarilylocatedalongtheperipheryoftheislet.

•Fig. 39.6 ProinsulinisprocessedbyprohormoneconvertasesintoamatureinsulinmoleculewithtwopeptidestrandslinkedbyH-bondsandaCpeptide.Botharesecretedinequimolarratios.(FromWhiteBA,PorterfieldSP[eds].Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby/Elsevier;2013.)C peptide C peptide Insulin B B Converting enzymes Proinsulin “folded” S-S bonds formed Endoplasmic reticulum Golgi Secretory granule A A S S S S S S S S S S S S

The islets of Langerhans constitute the endocrine pancreas (

Fig. 39.6 ). Approximately 1 million islets making up about 1% to 2% of the pancreatic mass are spread throughout the exocrine pancreas (see ). The islets are composed of several cell types, each producing a different hormone. Beta cells make up about three-fourths of the cells of the islets and produce the hormone insulin (see

Fig. 39.5B ). Alpha cells account for about 10% of islet cells and secrete glucagon (see

Fig. 39.5C ). Other endocrine cell types reside within islets, but their respective hormone products are of marginal or unclear importance and thus will not be discussed further.

Blood flow to the islets is somewhat autonomous from blood flow to the surrounding exocrine pancreatic tissue. Blood flow through the islets passes from beta cells, which predominate in the center of the islet, to alpha and delta cells, which predominate in the periphery (see

Fig.

38.5B-C ). Consequently the first cells affected by circulating insulin are the alpha cells, in which insulin inhibits glucagon secretion.

Insulin is the primary anabolic hormone that dominates regulation of metabolism during the digestive phase. Insulin is a protein hormone that belongs to the gene family that includes insulinlike growth factors I and II (IGF-I, IGF-II) and relaxin. The insulin gene encodes preproinsulin. Insulin is synthesized as preproinsulin, which is converted to proinsulin as the hormone enters the endoplasmic reticulum. Proinsulin is packaged in the

CHAPTER 39 Hormonal Regulation of Energy Metabolism 705 4 (-) ATP of cell membrane • Fig. 39.7 Glucose is the primary stimulus of insulin secretion and is enhanced by sulfonylurea drugs as well as GLP-1 analogues/DPP-4 inhibitors. See text for explanation of numbered steps in glucose-stimulated insulin secretion (GSIS).

Golgi apparatus into membrane-bound secretory granules. Proinsulin contains the AA sequence of insulin plus the C (connecting) peptide. The proteases that cleave proinsulin (proprotein convertases) are packaged with proinsulin within secretory vesicles. Proteolytic processing clips out the C peptide and generates the mature hormone, which consists of two chains, an α chain and a β chain, connected by two disulfide bridges (

Fig. 39.7). A third disulfide bridge is contained within the α chain. Insulin is stored in secretory granules in zinc-bound crystals. On stimulation, the granule’s contents are released to the outside of the cell by exocytosis. Equimolar amounts of mature insulin and C peptide are released, along with small amounts of proinsulin. C peptide has no known biological activity but is useful in assessing endogenous insulin production. C peptide is more stable in blood than insulin (making it easier to assay) and helps distinguish endogenous insulin production from injected insulin, insofar as the latter has been purified from C peptide.

Insulin has a short half-life of about 5 minutes and is cleared rapidly from the circulation. It is degraded by insulin-degrading enzyme (IDE; also called insulinase) in the liver, kidney, and other tissues. Because insulin is secreted into the hepatic portal vein, it is exposed to liver IDE before it enters the peripheral circulation. About half the insulin is degraded before leaving the liver. Thus peripheral tissues are exposed to significantly less serum insulin concentrations than the liver. Recombinant human insulin and insulin analogues with different characteristics of speed of onset and duration of action and peak activity are now available. Serum insulin levels normally begin to rise within 10 minutes after ingestion of food and reach a peak in 30 to 45 minutes. The higher serum insulin level rapidly lowers blood glucose to baseline values.

Glucose is the primary stimulus of insulin secretion (“steps” in glucose-stimulated insulin secretion described in the discussion that follows refer to

Fig. 39.8 ). Entry of glucose into beta cells is facilitated by the GLUT2 transporter (step 1). Once glucose enters the beta cell, it is phosphorylated to G6P by the low-affinity hexokinase glucokinase (step 2). Glucokinase is referred to as the “glucose sensor” of the beta cell because the rate of glucose entry is correlated to the rate of glucose phosphorylation, which in turn is directly related to insulin secretion. Metabolism of G6P through glycolysis, the TCA cycle, and oxidative phosphorylation by beta cells increases the intracellular ATP : ADP ratio (step 3) and closes an ATP-sensitive K+ channel (step 4). This results in depolarization of the beta cell membrane (step 5), which opens voltage-gated Ca++ channels (step 6). Increased intracellular [Ca++] activates microtubule-mediated exocytosis of insulin/proinsulincontaining secretory granules (step 7).

Ingested glucose has a greater effect on insulin secretion than injected glucose. This phenomenon, called the incretin effect, is due to stimulation by glucose of incretin hormones from the gastrointestinal tract. One clinically relevant incretin hormone is glucagon-like peptide 1 (GLP-1), which is released by L cells of the ileum in response to glucose in the ileal lumen (

Fig. 39.9 ). As a hormone, GLP-1 enters the circulation and ultimately binds to the Gs-coupled GLP1 receptor (GLP1R) on beta cells. This GLP1R/Gs/adenylyl cyclase/protein kinase A (PKA) signaling pathway amplifies the intracellular effects of Ca++ on insulin secretion. GLP-1 is rapidly degraded in the circulation by dipeptidyl peptidase 4 (DPP-4).

706 SECTION8Berne & Levy Physiology •Fig. 39.8 Secondaryregulatorsofinsulinsecretion.Seetextforexplanationofabbreviations.Specific amino acids (Eating a meal) AA transporter ATP ˜CA2+ °cAMP ˛ CELL ˜cAMP M3R (Gq)ACh Parasympathetic innervation (Anticipating & ingesting a meal) Norepinephrine, epinephrine Sympathetic innervation adrenal medulla (Exercise, Hypoglycemia) ? ˛2AR (Gs) ˛2AR Agonists ˙2AR (Gi) ˙2AR Agonists Insulin

Fig. 39.10 ). Insulin secretion is primarily dampened by sympathetic autonomic regulation through α2-adrenergic receptors. Binding of norepinephrine or epinephrine to α2-adrenergic receptors decreases cyclic adenosine mono-phosphate (cAMP), possibly by closing Ca++ channels (see

Fig. 39.6 ). Adrenergic inhibition of insulin serves to protect against hypoglycemia, especially during exercise. Beta cells also express Gs-coupled β2-adrenergic receptors that normally play a minor role in promoting insulin secretion. Note, however, that β-adrenergic receptor agonists oppose the actions of insulin on overall metabolism and may antagonize the actions of administered insulin in diabetics. Conversely, β-blockers (i.e., β-adrenergic receptor antagonists) may increase the severity of hypoglycemic episodes in patients receiving exogenous insulin.

TheATP-sensitiveK+ channelisanoctamericproteincomplexthatcontainsfourATP-bindingsubunitscalledSUR subunits. Thesesubunitsareboundbysulfonylurea drugs, whichalsoclosetheK+ channelandarewidelyusedasoralhypoglycemicstotreathyperglycemiainpatientswithpartiallyimpairedbetacellfunction.Hypoglycemiaisasignificantsideeffectofsulfonylureadrugsifusedinexcessorincorrectlyincombinationwithotherdrugs,owingtoinappropriatelyhighreleaseofinsulin.

BothDPP-4–resistant analogues of GLP-1 andinhibitors of DPP-4 arecurrentlyapprovedfortreatmentofpatientswithtype2DMwithsomebetacellfunction.Importantlythesedrugsarepermissive totheactionsofglucoseonthebetacellandthusonlyweaklyincreaseinsulinsecretionintheabsenceofglucose.ThusGLP-1analoguesinducehypoglycemiamuchlessfrequentlythansulfonylureadrugs.

The insulin receptor (InsR) is a member of the receptor tyrosine kinase (RTK) gene family (see ). Most of the actions of insulin on metabolism involve activation of the protein kinase Akt, which in turn has pleiotropic actions on cell metabolism.

The InsR is expressed on the cell membrane as a homodimer, with each monomer containing a tyrosine kinase

B InsRec IRS protein Metabolic actions Anabolic actions of insulin • Utilization of glucose for ATP & anabolic pathways • Storage of FFAs as TG • Protein synthesis from AAs • Macromolecular synthesis (e.g., RNA, DNA, lipids) • Organelle formation • Cell proliferation/renewal Mitogenic actions Merabolic actions PIP2PIP3 RAS/ RAF MAPK/ ERK PI3K Akt

GLUT 4 Translocation Glucose import Glycolysis, Glycogenesis, PDH, DNL Glucogenolysis Gluconeogensis FFA ˜ oxidation Ketogenesis Lipolysis Proteolysis autophagy Protein synthesis Gycolysis, PPP, DNL Gluconeo-genesis, VLDL export Protein phoshatases mTORC1 SREBP-1C FOX01 Akt •Fig. 39.9 A,Structureofdimerizedinsulinreceptorincellmembrane.B,SimplifieddiagramoftheAktkinaseandMAPKpathwaysdownstreamoftheInsR.C,Summarizedactionsofinsulin/InsR-activatedAktkinase.

Insulingeneexpressionandisletcellbiogenesisaredependentonseveraltranscriptionfactorsspecifictothepancreas,liver,andkidney.Thesetranscriptionfactorsincludehepatocyte nuclear factor 4α (HNF-4α), HNF-1α, insulin promoter factor 1 (IPF-1), HNF-1β, and neurogenic differentiation 1/beta cell E-box trans-activator 2 (NeuroD1/β2). Aheterozygousnullmutationofoneofthesefactorsresultsinprogressivelyinadequateproductionofinsulinandmaturity-onset diabetes of the young (MODY) beforetheageof 25.MODYischaracterizedbynonketotichyperglycemia,oftenasymptomatic,thatbeginsinchildhoodoradolescence.Inadditiontothefivetranscriptionfactors,mutationsinglucokinase alsogiverisetoMODY.

•Fig. 39.10 Divergentproteolyticcleavagepatternsoftheproglucagonmolecule.GLUC,glucagon;GLP,glucagon-likepeptide;GRPP,glucagon-relatedpolypeptide.

domain on the cytosolic side (see

Fig. 39.10A ). Binding of insulin to the receptor induces cross-phosphorylation of the subunits. These phosphotyrosine residues are then bound by the insulin receptor substrate (IRS) proteins (i.e., IRS proteins are “recruited” to the InsR). The IRS proteins themselves are phosphorylated by the InsR on specific tyrosines, which then recruits phosphoinositide-3-kinase (PI3K) to the IRS protein bound to the InsR (see

Fig. 39.10B ). PI3K converts phosphoinositol-4,5-bisphosphate (PIP2) to phosphoinositol-3,4,5-trisphosphate (PIP3). PIP3 is an informational lipid that recruits proteins to the membrane. In this pathway, PIP3 recruits Akt protein kinase to the cell membrane where it becomes activated. This pleiotropic Akt protein kinase signaling pathway orchestrates the numerous metabolic actions of insulin in hepatocytes, skeletal muscle, and adipocytes, including (see

Fig. 39.10C 1.

translocation of the GLUT4 glucose transporter to the cell membrane, thereby allowing import of glucose into skeletal myocytes and adipocytes; 2.

activation of protein phosphatases, which in turn regulate the activity of multiple metabolic enzymes in all insulin target cells; 3.

activation of the protein complex mechanistic target of rapamycin complex 1 (mTORC1), which promotes protein synthesis and may inhibit proteosomal-mediated protein degradation in insulin target cells; 4.

activation of the transcription factor sterol response element binding protein 1 (SREBP1).

SREBP1 is especially important for insulin effects on the liver, where it orchestrates glycolysis and de novo lipogenesis (DNL) for production of phospholipids, FAs, and TGs from excessive ingested glucose and fructose. InsR/Akt signaling stimulates SREBP1 directly as well as indirectly through activation of mTORC1, which also activates SREBP1. SREBP1 also induces the enzyme that catalyzes the first reaction in the oxidative arm of the pentose phosphate pathway (PPP). This reaction generates the coenzyme NADPH, which is required for DNL.

5. inactivation of the transcription factor FOXO1. Aktmediated phosphorylation of FOXO1 promotes nuclear exclusion of FOXO1. In the absence of insulin/Akt signaling, FOXO1 induces expression of genes encoding

VLDL assembly and export. All these actions of Akt will be discussed in more detail later. The InsR also promotes proliferation/renewal of some target cells through the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway (see

Fig. 39.10B ). The MAPK pathway also participates in some metabolic regulation.

Glucagon is the primary counterregulatory hormone that increases blood glucose levels, primarily through its effects on liver glucose output. Glucagon also enhances intramitochondrial fatty acid oxidation and ketogenesis in hepatocytes.

Glucagon is a member of the secretin gene family. The precursor preproglucagon harbors the AA sequences for glucagon, GLP-1, and GLP-2 (

Fig. 39.11 ). Preproglucagon is proteolytically cleaved in the alpha cell in a cell-specific manner to produce the peptide glucagon. Glucagon circulates in an unbound form and has a short half-life of about 6 minutes. The predominant site of glucagon degradation is the liver, which degrades as much as 80% of circulating glucagon in one pass. Because glucagon enters the hepatic portal vein and is carried to the liver before reaching the systemic circulation, a large portion of the hormone never reaches the systemic circulation. The liver is the primary target organ of glucagon, with lesser effects on adipocytes. Skeletal muscle does not express the glucagon receptor.

The glucagon receptor is is a Gs-linked G protein– coupled receptor that increases adenylyl cyclase activity and thus cAMP levels. Glucagon exerts many rapid actions through PKA signaling. Glucagon also exerts some transcription effects through phosphorylation and activation of transcription factors such as CREB (cAMP response element binding) protein.

The insulin-glucagon ratio determines the net effect of metabolic pathways on blood glucose. A major stimulus for secretion of glucagon is a decline in blood glucose. Insulin inhibits glucagon secretion, so low blood glucose has an

CHAPTER 39 Hormonal Regulation of Energy Metabolism 709 indirect effect on glucagon secretion through removal of ˜ Cell Insulin inhibition by insulin (

Fig. 39.12 ). Some recent evidence also indicates that low glucose has a direct effect on alpha (+) cells to increase glucagon secretion.

Circulating catecholamines, which inhibit secretion of glucose insulin via α2-adrenergic receptors, stimulate secretion of glucagon via β2-adrenergic receptors (see

Fig. 39.12 (If low) Serum AAs promote secretion of glucagon. This means a glucose protein meal will increase postprandial levels of both insulin and glucagon (which protects against hypoglycemia), whereas a carbohydrate meal stimulates only insulin.

Catecholamines: Epinephrine  (–) and Norepinephrine

The other major counterregulatory factors are the catechol- amines epinephrine and norepinephrine. Epinephrine is the primary product of the adrenal medulla (see ), whereas norepinephrine is released from postganglionic sympathetic nerve endings (see ). Catecholamines are released in response to decreased glucose concentrations, various forms of stress, and exercise. Decreased glucose levels (i.e., hypoglycemia) are primarily • Fig. 39.11 Integrated regulation of blood glucose by insulin and sensed by neurons in the CNS, which initiate an integrated the counterregulatory factors glucagon and catecholamines (norepinephrine, epinephrine).

Fructose-1,6-bisP OA Acetyl CoA • Fig. 39.12 Metabolic pathways in hepatocytes during digestive (“D” numbers) and fasting (“F” numbers) phases. Reactions/pathways: 1D, glucokinase; 1F; G6Pase; 2D, glycogen synthesis; 2F, glycogenolysis; 3D, phosphofructokinase 1; 3F, fructose-1,6-bisphosphatase; 4D, glucose-6-phosphate dehydrogenase; 5D, pyruvate kinase; 6D, pyruvate dehydrogenase; 6F, pyruvate carboxylase; 7F, phosphoenolpyruvate carboxykinase; 8D, ATP-citrate lyase; 9D, acetyl CoA carboxylase; 9F, malonyl CoA decarboxylase; 10D fatty acid synthase; 11D, glycerol phosphate/fatty acyl CoA transferase and other enzymes involved in esterification and formation of TG; 12D, inhibition by malonyl CoA (Mal CoA) of fatty acyl CoA transporter, carnitine/palmitoyl transporter 1 (CPT1) on outer mitochondrial membrane; 13F, movement of fatty acyl CoA into mitochondrion through CPT1 and  beta-oxidation to acetyl CoA.

sympathetic response through the hypothalamus. The direct metabolic actions of catecholamines are mediated primarily by α1-, β2-, and β3-adrenergic receptors located on muscle, adipose, and liver tissue (see later). Like the glucagon receptor, β-adrenergic receptors (β2 and β3) increase intracellular cAMP. The α1-adrenergic receptor is also expressed by some organs, especially the liver.

Hormonal Regulation of Specific Metabolic Reactions and Pathways

This section discusses the main pathways in hepatocytes, skeletal myocytes, and adipocytes that contribute to integrated metabolism. For even more detailed description, the student is referred to biochemistry textbooks.

Hepatocyte Metabolism: Digestive vs. Fasting Phases

Some of the key metabolic steps regulated by insulin and glucagon (and catecholamines) in the liver are as follows (refer to

Fig. 39.13 for numbered reactions; D denotes digestive phase, F denotes fasting phase): 1. Trapping vs. releasing intracellular glucose. Although glucose enters hepatocytes through insulin-independent GLUT2 transporters, insulin increases hepatic retention and utilization of glucose by increasing expression of glucokinase (reaction 1D). Insulin increases glucokinase gene expression through increased expression and activation of the transcription factor sterol regulatory element–binding protein 1C (SREBP1C), which acts as a “master switch” in the fed state to coordinately increase levels of several enzymes involved in glucose utilization and TG synthesis. Hepatocytes also express the enzyme G6Pase (reaction 1F), which converts G6P back to glucose, which can then exit the hepatocyte via the GLUT2 transporter. Insulin prevents the futile cycle of glucose phosphorylation-dephosphorylation by repressing gene expression of the enzyme G6Pase. The transcription factor FOXO1 stimulates gene expression of G6Pase. Insulin-activated Akt kinase phosphorylates and inactivates FOXO1. During the fasting phase, FOXO1 is active and promotes G6Pase expression, whereas SREBP1C is inactive and does not stimulate glucokinase expression. The reciprocal regulation of SREBP1C and FOXO1 are thus regulated primarily by the presence or absence of insulin.

2.

Glycogen synthesis vs. breakdown. Insulin indirectly increases glycogen synthesis through increased expression of glucokinase because high levels of G6P allosterically increase glycogen synthase activity. Through stimulation of specific protein phosphatases, insulin promotes dephosphorylation and thereby activation of glycogen synthase (reaction 2D). Insulin also prevents the futile cycle of glycogen synthesis to glycogenolysis through inhibition of glycogen phosphorylase (reaction 2F). Glucagon-activated PKA phosphorylates phosphorylase kinase, which in turn phosphorylates and activates glycogen phosphorylase.

3.

Increasing glycolysis.

A. Activating phosphofructokinase 1 (PFK1) and inhibiting fructose-2,6-bisphosphatase. Insulin increases the activity of PFK1, which phosphorylates •Fig. 39.13 Insulinandcounterregulatoryhormoneregulationofphosphofructokinase1(PFK1;reaction3Din

Fig.39.12) andfructose-1,6-bisphosphatase(F1,6BPase;reaction3Fin

Fig.39.12) throughchangingtheactivityofthebifunctionalenzymephosphofructokinase2/fructose-2,6-bisphosphatase(PKFBP)andthusthelevelsoftheallostericregulatorymetabolitefructose-2,6-bisphosphate(F-2,6-bisP).Insulin/Akt-activated protein phosphatase F-2,6BPase F-1,6BPase F-1,6-bisPATP, citrate AMP PKFBP-P PFK1 (–) (–) (+) Glucagon & NE/E-activated protein kinase A (PKA) HepatocytecytoplasmPKFBP PFK2 F-2,6-bisP F6P •Fig. 39.14 Metabolisminskeletalmuscleduringdigestive(“D”reactions)vs.fasting(“F”reactions)phases.Reactions/pathways:1D,translocationofGLUT4transportertocellmembrane;1F,lossoftranslocationofGLUT4transportertocellmembrane;2D,glycogensynthesis;2F,glycogenolysis;3D,glycolysisandlactatedehydrogenase,orpyruvatedehydrogenase/TCAcycle/oxidativephosphorylation(OxPhos),dependingonmusclefibertype;3F,β-oxidationofFFAsorketolysisfollowedbytheTCAcycleandOxPhos.GLUT4 GLUT4 Transporters FFAs, KBsAcetyl CoAATP GLUT4 storage vesicle Cell membrane GlucoseGlucoseGlycogen 1D 1F 2F 2D 3D 3F GLUT4 Skeletal myocyte fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (reaction 3D). This reaction is referred to as the “commitment” reaction for glycolysis. Insulin also inhibits the reverse reaction, as catalyzed by the gluconeogenic enzyme fructose-1,6-bisphosphatase. Insulin regulates these two enzymes through an indirect two-step mechanism that is diagramed in

Fig. 39.14 . This mechanism involves the bifunctional enzyme phosphofructokinase-2/ fructose bisphosphatase (PKFBP; see

Fig. 39.14 ). Insulin/Akt-activated protein phosphatases promote dephosphorylation of PKFBP, thereby activating the kinase function and lessening the phosphatase function. This phosphorylates F6P to fructose-2,6bisphosphate (F-2,6-bisP). F-2,6-bisP in turn allosterically activates PFK1, thereby driving glycolysis. F-2,6-bisP also competitively inhibits fructose-1,6bisphosphatase, thereby blocking the futile cycle of F6P to fructose-1,6-bisphosphate to F6P.

B.	Activatingpyruvatekinase(PK).PKcatalyzestheirreversibleconversionofphosphoenolpyruvate(PEP)topyruvate(seereaction5Din

Fig.

39.12 ).Again,insulin/AktkinaseactivationofaproteinphosphatasedephosphorylatesPK,whichactivatestheenzyme.InsulinalsoincreasesPK geneexpressionthroughSREBP1C.Finally,fructose-1,6-bisphosphate(productofreaction3D)allostericallyactivatesPK.

4.

Activating pyruvate dehydrogenase (PDH) complex. PDH converts pyruvate to acetyl CoA, which can then enter the TCA cycle upon condensation with oxaloacetate (OA) to form citrate. Insulin increases PDH activity through Akt kinase activation of PDH phosphatase, which in turn dephosphorylates and activates PDH (reaction 6D).

5.

Increasing synthesis of intrahepatic TG. During the digestive phase, some acetyl CoA is transferred from the mitochondria to the cytosol in the form of citrate, which is then converted back to acetyl CoA and oxaloacetate by the cytosolic enzyme ATP-citrate lyase (reaction 8D). Insulin increases ATP-citrate lyase gene expression through transcription factor SREBP1C. Once in the cytoplasm, acetyl CoA can enter fatty acid synthesis. The first step involves conversion of acetyl CoA to malonyl CoA by the enzyme acetyl-CoA carboxylase (reaction 9D). Insulin stimulates acetyl-CoA carboxylase gene expression through the transcription factor SREBP1C. Insulin also promotes dephosphorylation of acetyl-CoA carboxylase, which activates the enzyme. Malonyl CoA is converted to the 16-carbon fatty acid palmitoyl CoA by repetitive additions of acetyl groups (contributed by malonyl CoA) by the fatty acid synthase (FASN) complex (reaction 10D). FASN gene expression is enhanced by insulin through the transcription factor SREBP1C. Insulin also stimulates palmitoyl-CoA desaturase, which produces unsaturated fatty acids, and glycerol phosphate–fatty acyl transferases that esterify FFAs to G3P to form intrahepatic TG.

Palmitate synthesis requires the coenzyme NADPH. A major source of NADPH is the pentose phosphate pathway (PPP; see

Fig. 39.12 ). The first reaction converts G6P to 6-phosphogluconolactone by the enzyme glucose-6-phosphate dehydrogenase (G6PD; reaction 4D). Insulin increases G6PD gene expression through the transcription factor SREBP1C.

By activating steps that lead to generation of malonyl CoA, insulin indirectly inhibits oxidation of FFAs. Malonyl CoA inhibits the activity of CPT-I, which transports FFAs from the cytosol into the mitochondria (reaction 12D). As a result, FFAs that are synthesized by DNL cannot be transported into mitochondria, where they undergo β-oxidation (reaction 13F). Thus increased malonyl CoA prevents the futile cycle of FFA synthesis to FFA oxidation.

FFAs are converted to TGs by the liver (reaction 11D) and are either stored in the liver or transported to adipose tissue and muscle in the form of VLDL (see later). Insulin acutely promotes degradation of the VLDL apoprotein apoB-100. This keeps the liver from secreting VLDL during a meal when the blood is rich with chylomicrons. Thus the lipid made in response to insulin during a meal is released as VLDL during the interdigestive and fasting phases and provides an important source of energy to skeletal and cardiac muscle.

6. Activation vs. inhibition of the gluconeogenic enzymes pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK). Pyruvate can also be converted to OA by PC (reaction 6F). However, this reaction is indirectly inhibited by insulin in several ways. First, insulin activates PDH as just discussed, thereby diverting pyruvate away from the PC reaction. Additionally, PC is allosterically activated by high levels of intramitochondrial acetyl CoA. Insulin keeps intramitochondrial levels of acetyl CoA low by activation of cytosolic DNL, which promotes removal of acetyl CoA via citrate from the mitochondria. Another key mechanism is to prevent β-oxidation of FFAs within the mitochondria, which generates high levels of acetyl CoA. By stimulating DNL, insulin also increases levels of cytosolic malonyl CoA, which inhibits transport of FFAs into the mitochondria (reaction 12D). Also, inhibitory actions of insulin on glucagon secretion and on lipolysis of TG within adipocytes prevents release of FFAs by adipose tissue and their import into hepatocytes.

In contrast, during the fasting phase, low insulin coupled with high glucagon and/or catecholamines stimulate release of FFAs from adipocytes (see later), which increases the flow of FFAs into hepatocytes. Glucagon also phosphorylates and activates the enzyme malonyl decarboxylase, which converts malonyl CoA back to acetyl CoA (reaction 9F). Enhanced malonyl CoA decarboxylase, along with generally low DNL due to low insulin, reduces malonyl CoA levels and thus removes the inhibition on the CPT1 transporter. This allows FFAs to enter the mitochondria and undergo β-oxidation (reaction 13F), generating high levels of intramitochondrial acetyl CoA, activating PC (reaction 6F) and also allosterically inhibiting PDH (reaction 6D).). The enzymes involved in β-oxidation are activated by PKA signaling. Glucagon also activates the transcription factor PPARα, which further induces expression of enzymes involved in β-oxidation. Fibrate drugs activate PPARα, promoting oxidation of intrahepatic TG and ameliorating insulin resistance.

Insulin also represses gene expression of the gluconeogenic enzyme PEPCK (reaction 7F), which converts pyruvate (by way of malate transferase out of the mitochondria) to phosphoenolpyruvate. PEPCK is primarily regulated at the level of transcription. Similar to its actions on G6Pase, FOXO1 stimulates transcription of PEPCK during the fasting phase, and insulin/Akt kinase signaling inactivates FOXO1 during the digestive phase. Glucagon and catecholamines also increase PEPCK gene expression through PKA-CREB signaling during the fasting phase.

Skeletal Muscle and Adipose Tissue Metabolism: Digestive vs. Fasting Phases 1.

Fig. 39.15 ). Glucose tolerance refers to the ability of an individual to minimize the increase in blood glucose concentration after a meal. A primary way by which insulin promotes glucose tolerance is activation of glucose transporters in skeletal muscle. Insulin stimulates translocation of preexisting GLUT4 transporters to the cell membrane (reaction 1D). Insulin also promotes storage of glucose in muscle as glycogen (reaction 2D) and promotes oxidation of glucose through glycolysis (reaction 3D). During the fasting phase, low insulin results in a low number of GLUT4 transporters at the membrane (reaction 1F), so these cells consume less glucose (glucose sparing). Skeletal muscle fibers with mitochondria switch to the use of FFAs from adipocytes and KBs from hepatocytes (reaction 3F). Skeletal myocytes do not express glucagon receptors. Uptake of FFAs and KBs and their oxidation for ATP is largely upregulated by intracellular Ca++ levels and a high AMP:ATP ratio. Exercise also activates these pathways, as does glycogenolysis (reaction 2F), through adrenergic receptor stimulation.

2.

(Fig. 39.16A ). Insulin also stimulates GLUT4-dependent uptake of glucose and subsequent glycolysis in adipose tissue (reactions 1D and 2D). Adipose tissue uses glycolysis for energy needs but also for generating G3P (reaction 3D), which is required for esterification of FFAs into TGs (reaction 4D). During the fasting phase, insulin is low, so GLUT4 movement to the cell membrane is blocked (reaction 1F).

2. Adipocytes → FFAs and TG (see

Fig. 39.16B ). Insulin stimulates expression of lipoprotein lipase (LPL) •Fig. 39.15 A, Glucose metabolism in an adipocyte during digestive (“D” reactions) and fasting (“F” reactions) phases. Reactions/pathways:1Dand1F(alsosee

Fig.39.14 legend);2D,glycolysis,pyruvatedehydrogenase/TCAcycle/OxPhos;3D,glycerol-3-phosphatedehydrogenase;4D,esterificationofFFAstoG3Ptoformtriglyceride.B, Lipid metabolism in adipocyte during digestive (“D” reactions) and fasting (“F” reactions) phases. Reactions/pathways:1D,synthesisoflipoproteinlipase(LPL)andLPLsecretionintosubcapillaryspace,bindingtoGPI-anchoredprotein(red box), andmigrationtoluminalsideofcapillaryendothelialcell;2D,lipolysisofchylomicronTGandreleasingfreeFFA(afterdigestion,chylomicronremnantisclearedfromcirculationbyliver);3D,activationofimportedFFAsbytransfertoacetylCoAtoformfattyacylCoA;4D,esterificationoffattyacylCoAstoG3PtoformTG(TGdropletsarecoveredandstabilizedbyperilipins[PL]);5D,dephosphorylationandinactivationofhormone-sensitivelipase(HSL),whichpromotesstorageofTG;1F,phosphorylationandactivationofHSL,whichcontributestocompletelipolysisofTG(ATGL,adipocyteTGlipase;HSL,diacylglyceridelipase;MGL,monoacylglyceridelipase);2F,finalstepinTGlipasebyMGLreleasesFFAandglycerol.

714 SECTION8Berne & Levy Physiology •Fig. 39.16 Roleoflipoproteinsinenergymetabolism.A,Digestivephase.B,Fastingphase.Dietary fats Adipose capillaries Dietary sugar & carbs Glucose fructose CM Adipose capillaries LPL CR Cardiac muscle capillaries LPL FFAs FFAs A B FFAs FFAs Cardiac & skel muscle capillaries LPL Cardiac & skeletal muscle Steroidogenic cells Growing cells FFAs TG TG Adipocyte Adipocyte TG Liver VLDL Glucose fructose Cardiac muscle ATP Skeletal muscle ATP KBs TG Liver ATP CHOL ATP IDL LDL IDL within adipocytes and its migration to the apical side of endothelia in adipose capillaries (reaction 1D). This action of insulin allows LPL to extract FFAs from chylomicrons within adipose tissue capillary beds (reaction 2D). The chylomicron remnants (discussed later) are removed by the liver. Insulin also stimulates activation of imported FFAs by their conversion to fatty acyl CoAs (reaction 3D). Insulin stimulates glycolysis in adipocytes, which generates the G3P required for reesterification of FFAs into TGs (reaction 4D). Insulin directly inhibits hormone-sensitive lipase (HSL; reaction 5D), thereby promoting storage of FFAs as opposed to their release. During the fasting phase, glucagon and catecholamines phosphorylate and activate HSL (reaction 1F), thereby promoting release of FFAs and glycerol from stored TG (reaction 2F). In the absence of insulin these two products of lipolysis are exported into the blood.

Protein Metabolism in All Hormone Target Cells: Digestive vs. Fasting Phases

Insulin promotes protein synthesis in muscle and adipose tissue by stimulating AA uptake and mRNA translation. Insulin also inhibits proteolysis. Although the liver uses AAs for ATP synthesis, insulin also promotes synthesis of proteins during the digestive phase and attenuates the activity of urea cycle enzymes in the liver. Glucagon and catecholamines activate proteosomal degradation of proteins and release of AAs during the fasting phase.

Metabolic Roles of Lipoproteins: Digestive vs. Fasting Phases

This section deals with lipoproteins in some detail. For more details, consult a biochemistry textbook.

FFAs circulate in the blood primarily bound to albumin. However, TG, free cholesterol, cholesterol esters, phospholipids and some lipid-soluble vitamins, all of which are hydrophobic and would partition into the membranes of endothelial cells instead of circulating, are transported through blood within lipid aggregations (i.e., a mix of the above) bound by specific apoproteins. These lipid-protein complexes are referred to as lipoproteins. The TG-rich lipoproteins are chylomicrons and VLDL and primarily function to deliver FFAs (as TG) to skeletal and cardiac muscle for energy and to adipocytes for storage. The cholesterol-rich lipoproteins include low-density lipoprotein (LDL) and high-density lipoprotein (HDL), which deliver cholesterol to proliferating cells, steroidogenic cells, and bile-producing hepatocytes. HDL also removes excess cholesterol (i.e., from macrophage-engulfed dead cells) from the periphery. There are also “remnants” of lipoproteins that have their lipid cargo partially digested and then cleared from the circulation by the liver.

Digestive Phase: Chylomicrons and Chylomicron Remnants

TGs in a meal are enzymatically digested to FFAs and 2-monoglycerides within the lumen of the intestine.

•Fig. 39.17 Leptinandhypothalamiccentersinvolvedinregulationofappetite.Seetextforexplanationsofabbreviations.

Intestinal enterocytes import both of these lipids and reesterify them to form TGs. TGs, along with fat-soluble vitamins, cholesterol, cholesterol esters, and phospholipids, are complexed with the protein ApoB48 to form chylomicrons. Chylomicrons are secreted, move into lymphatics, and then ultimately enter the circulation. While in the blood, other apoproteins such as ApoE and ApoC2 are transferred to the chylomicrons from HDL particles (one function of HDL is to provide a circulating reservoir of various apoproteins). This converts nascent chylomicrons into mature chylomicrons.

When chylomicrons enter the capillaries of adipose tissue during the digestive phase, they are partially digested by lipoprotein lipase (LPL). LPL is synthesized by adipocytes and secreted into the subendothelial space. LPL then binds to the endothelial membrane GPI-anchored protein GPIHBP1, which transports LPL to the luminal (apical) surface of the capillary endothelial cell. Once in this position, LPL molecules come into contact with chylomicrons. ApoC2 within chylomicrons is an activator of LPL dimerization and activity. FFAs are released from chylomicrons by LPL-mediated lipolysis of TG. (See earlier discussion and

Fig. 39.16B for an explanation of the processing of FFAs to stored TG within adipocytes.

LPL is also expressed in cardiac and skeletal muscle. Cardiac muscle preferentially uses FFA for energy and obtains most FFAs from lipoprotein particles (

Fig. 39.17 ). Thus cardiac muscle also extracts FFA from chylomicrons during the digestive phase. The activity of LPL in cardiomyocytes is highly regulated by local factors such as the local concentration of FFAs within the coronary capillary beds. LPL activity in skeletal muscle is relatively low during the digestive phase.

After lipolytic digestion within adipose tissue capillary beds, chylomicrons are converted to smaller, denser chylomicron remnants (CR) that now have reduced TG content. CR particles are able to penetrate the tunica intima of blood vessels at sites with endothelial dysfunction and thus are atherogenic. Because they still have ApoE protein associated with them, they can bind to one of several membrane receptors that recognize ApoE. CRs can also bind to specific glycolipids. Bound CRs are then endocytosed by hepatocytes (see

Fig. 39.17 ). Remaining FFAs that are released after endocytosis of CRs are reesterified into intrahepatic TG.

Fasting Phase: VLDL, IDL, and LDL

The source of circulating TG during the fasting phase is primarily the liver (see

Fig. 39.17 ). During the digestive phase, intrahepatic TGs accumulate from de novo lipogenesis using the carbons in cytosolic citrate and from endocytosed CRs. Intrahepatic TG, along with other lipids including cholesterol and cholesterol esters, is exported by hepatocytes as VLDL. VLDL particles are assembled as lipids complexed to the ApoB100 protein. Expression of ApoB100, along with other components involved in VLDL assembly, is stimulated by transcription factor FOXO1. FOXO1 in turn is inhibited by the insulin signaling pathway. This means that hepatic VLDL production is minimal during the time when blood is rich in chylomicrons. During the fasting phase, insulin levels are low, so FOXO1 activity is high, and VLDL assembly and secretion resumes. Once VLDL particles enter the circulation, they accept other apoproteins (e.g., ApoE, ApoC2) and become mature VLDL.

Adipocytes display low LPL activity during the fasting phase, in part owing to low insulin levels. However, cardiomyocytes and skeletal myocytes express LPL, which digests VLDL and provides FFA to these muscle cell types during the fasting phase. Lipolytic extraction of some FFAs from VLDL generates a remnant particle called intermediate-density lipoprotein (IDL). IDL circulates to the liver, where it s processed in one of two ways (see

Fig. 39.17 ). About half of the IDL binds to one of several ApoErecognizing receptors, undergoes receptor-mediated endocytosis, and is digested in endolysosomes. Released lipids can be reassembled into VLDL particles and returned to the circulation to provide fuel for cardiac and skeletal muscle as the fasting phase progresses. The other half of the IDL undergoes further digestion from the hepatocyte-specific LPL-related enzyme hepatic lipase. Hepatic lipase extracts most of the remaining TG in the IDL, forming the final remnant of VLDL, LDL. LDL is TG poor but cholesterol rich. It should be noted that both mature chylomicrons and VLDL can receive additional cholesterol from HDL while in the circulation through the action of cholesterol ester transport protein (CETP), so cholesterol content of the remnant particles (ChyR, IDL, and LDL) can vary. In any case the LDL particle is a small, dense, cholesterol-rich particle that is potentially very atherogenic in the face of endothelial damage. LDL particles are safely imported into cells through the LDL receptor. It should be noted that in the conversion of IDL to LDL, the ApoE protein disassociates from the particle. This means that only receptors for which ApoB100 is a ligand can remove LDL from the blood. In contrast to the multiple ApoE receptors, only one receptor, the LDL receptor, can recognize and bind ApoB100. Thus loss or decrease of a functional LDL receptor has significant clinical consequences (see

At the Cellular box). LDL receptor is expressed on proliferating cells, including some cancer cells, which need to synthesize new cell membranes. LDL receptor is also expressed on steroidogenic cells, which use cholesterol to make steroid hormones. The major site of LDL uptake is the liver, which secretes cholesterol as well as cholesterol-based bile acids, as bile into the biliary tree. Some cholesterol is excreted by the intestines. Other cholesterol byproducts (e.g., steroid hormones) are excreted primarily at the kidney.

White adipose tissue (WAT) is composed of several cell types. The TG-storing cell is called the adipocyte. These cells develop from preadipocytes during gestation in humans. This process of adipocyte differentiation, which may continue throughout life, is promoted by several transcription factors. One of these factors is SREBP1C, which is activated by lipids as well as insulin and several growth factors and cytokines. Another important transcription factor in WAT is PPARγ. Activated PPARγ promotes expression of genes involved in TG storage. Thus an increase in food consumption leads to activation of SREBP1C and PPARγ, which increase the differentiation of preadipocytes into small adipocytes and upregulation of enzymes within these cells to allow storage of excess fat.

Adipose tissue produces paracrine and endocrine factors, including adiponectin, TNF-α, resistin, interleukin-6, angiotensinogen, and acylation-stimulating protein.

Leptin is an adipocyte-derived protein that signals information to the hypothalamus about the degree of adiposity and nutrition, which in turn controls eating behavior and energy expenditure. Leptin-deficient mice and humans become morbidly obese. These findings originally raised hope that leptin therapy could be used to combat morbid obesity. However, administration of leptin to individuals who suffer from diet-induced obesity does not have a significant anorectic or energy-consuming effect. In fact, obese individuals already have elevated endogenous circulating levels of leptin and appear to have developed leptin resistance.

Leptin has an important role in liporegulation in peripheral tissues. Leptin protects peripheral tissues (e.g., liver, skeletal muscle, cardiac muscle, beta cells) from accumulation of too much lipid by directing storage of excess caloric intake into adipose tissue. This action of leptin, though opposing the lipogenic actions of insulin, contributes significantly to maintenance of insulin sensitivity (as defined by insulin-dependent glucose uptake) in peripheral tissues. Leptin also acts as a signal that the body has sufficient energy stores to allow reproduction and to enhance erythropoiesis, lymphopoiesis, and myelopoiesis. For example, in women suffering from anorexia nervosa, leptin levels are extremely low and result in low ovarian steroids, amenorrhea (lack of menstrual bleeding), anemia from low red blood cell production, and immune dysfunction.

Structure, Synthesis, and Secretion

Leptin, a 16-kDa protein secreted by mature adipocytes, is structurally related to cytokines. Thus it is sometimes

SREBP2 wasdiscoveredasatranscriptionfactorthatresidesinthemembraneoftheendoplasmicreticulum(ER).Inthepresenceofhighintracellularcholesterol,SREBP2isheldintheERbyalipid-sensingproteincalledSCAP (SREBPcleavage–activatingprotein).Inresponsetodepletedsterols,SCAPescortsSREBP2totheGolgi,whereSREBPiscleavedsequentiallybyproteasesandreleasedintothecytoplasm.SREBP2thentranslocatestothenucleusandincreasesthetranscriptionofgenesinvolvedinsynthesisanduptakeofcholesterol.AmorerecentlydiscoveredmemberofthistranscriptionfactorfamilyisSREBP1C, whichishighlyexpressedinadiposeandliver.IncontrasttoSREBP2,SREBP1CstimulatesgenesinvolvedinsynthesisofFAandTG.RegulationofSREBP1CoccursatthetranscriptionalleveloftheSREBP1C gene,withcleavageinducedbypolyunsaturatedfattyacidsandactivationbytheMAPKpathway.

Peroxisome proliferation activator receptors (PPARs) belongtothenuclearhormonereceptorsuperfamilythatalsoincludessteroidhormonereceptorsandthyroidhormonereceptors.PPARsheterodimerizewiththeretinoid X receptors (RXRs). Unlikesteroidandthyroidhormonereceptors,PPARsbindtoligandsinthemicromolarrange(i.e.,withloweraffinity).PPARsbindsaturatedandunsaturatedfattyacidsaswellasnaturalandsyntheticprostanoids.PPARγ ishighlyexpressedinadiposetissueandatalowerlevelinskeletalmuscleandliver.Itsnaturalligandsincludeseveralpolyunsaturatedfattyacids.PPARγ regulatesgenesthatpromotefatstorage.ItalsosynergizeswithSREBP1Ctopromotedifferentiationofadipocytesfrompreadipocytes.Tissue-specificknockoutofPPARγ inmiceandPPARγ-dominantnegativemutationsinhumansgiverisetolipodystrophy (i.e.,lackofwhiteadiposetissue),whichleadstodepositsofTGinmuscleandliver(calledsteatosis),insulinresistance,diabetes,andhypertension.

referred to as an adipocytokine. Circulating levels of leptin have a direct relationship with adiposity and nutritional status. Leptin output is increased by insulin, which prepares the body for correct partitioning of incoming nutrients. Leptin is inhibited by fasting and weight loss and by lipolytic signals (e.g., increased cAMP and β3-agonists).

Diet-induced obesity, advanced age, and T2DM are associated with leptin resistance. Thus mechanisms that turn off leptin signaling are potential therapeutic targets.

The amount of energy stored by an individual is determined by caloric intake and calories expended as energy per day. In many individuals, input and output are in balance, so weight remains relatively constant. However, the abundance of inexpensive high-fat, high-carbohydrate food, along with more sedentary lifestyles, is currently contributing to a pandemic of obesity and the pathological sequelae of obesity, including T2DM and cardiovascular disease.

The preponderance of stored energy consists of fat, and individuals vary greatly in the amount and percentage

Thethiazolidinediones areexogenousligandsforPPARγ.Althoughtheypromoteweightgain,moderatelevelsofthiazolidinedionessignificantlyimproveinsulinsensitivity.PPARγ alsostimulatessecretionofadiponectin, whichpromotesoxidationoflipidsinmuscleandfatandtherebyimprovesinsulinsensitivity.PPARα isabundantlyexpressedinliverandtoalesserextentinskeletalandcardiacmuscleandkidney.PPARα promotesuptakeandoxidationofFFAs.ThusPPARα isanantisteatoticmolecule.Thefibrates areexogenousligandsofPPARα andareusedtoreduceTGdepositsinmuscleandliver,therebyimprovinginsulinsensitivity.Athirdmember,PPARδ, similarlypromotesfattyacidoxidationinadiposeandmuscletissue.PPARδ promotesdevelopmentofslow-twitchoxidativemusclefibersandincreasesmusclestamina.PPARδ hasapositiveeffectonlipoproteinmetabolismbyincreasingproductionofApoAapoproteinsandthenumberofHDLparticles.

Anotherfamilyoflipid-sensingtranscriptionfactorsistheliver X receptor (LXR) family,whichiscomposedofLXRα andLXRβ.LXRα isexpressedprimarilyinadiposetissue,liver,intestine,andkidney,whereasLXRβ isubiquitouslyexpressed.LXRsarerelatedtoPPARsinthattheyaremembersofthenuclearhormonereceptorfamilyandheterodimerizewithRXR.LXRsarecholesterolsensors.Inhigh-cholesterolconditions,LXRsupregulateexpressionofATP-bindingcassette(ABC)proteins.Inthefaceofexcesscholesterol,LXRsalsoincreaseABCproteinexpressioninthegastrointestinaltract,whichpromoteseffluxofcholesterolfromenterocytestothelumenforexcretion.Mutationsinthesetransporters(ABCG5andABCG8)causesitosterolemia, characterizedbyexcessiveabsorptionofcholesterolandplantsterols.Intheliver,LXRspromoteconversionofcholesteroltobileacidsforexcretionortocholesterolestersforstorage.Inthelatteraction,LXRsincreaseSREBP1Cexpression,therebyincreasingthefattyacylCoAsneededforesterification.

of body weight that is accounted for by adipose tissue. About 25% of the variance in total body fat appears to be due to genetic factors. A genetic influence on fat mass is supported by (1) the tendency for the body mass of adopted children to correlate better with that of their biological parents than with that of their adoptive parents; (2) the greater similarity of adipose stores in identical (monozygotic) twins, whether reared together or apart, than in fraternal (dizygotic) twins; (3) the greater correlation between gains in body weight and abdominal fat in identical twins than in fraternal twins when they are fed a caloric excess; and (4) the discovery of several genes that cause obesity.

In addition, the gestational environment has a profound effect on body mass of the adult. The effect of maternal diet on the weight and body composition of offspring is called fetal programming. Low birth weights correlate with increased risk for obesity, cardiovascular disease, and diabetes. These findings suggest that the efficiency of fetal metabolism has plasticity and can be altered by the in utero environment. The development of a “thrifty” metabolism would be advantageous to an individual born to a mother

Diabetes mellitus isadiseaseinwhichinsulinlevelsorresponsivenessoftissuestoinsulin(orboth)isinsufficienttomaintainnormallevelsofplasmaglucose.Althoughthediagnosisofdiabetesisbasedprimarilyonplasmaglucose,diabetesalsopromotesimbalancesincirculatinglevelsoflipidsandlipoproteins(i.e.,dyslipidemia).Majorsymptomsofdiabetesmellitusincludehyperglycemia,polyuria,polydipsia,polyphagia,musclewasting,electrolytedepletion,andketoacidosis(inT1DM).Withnormalfasting(i.e.,nocaloricintakeforatleast8hours),plasmaglucoselevelsshouldbebelow110mg/dL.Apatientisconsideredtohaveimpairedglucosecontroliffastingplasmaglucoselevelsarebetween110and126mg/dL,andthediagnosisofdiabetesismadeiffastingplasmaglucoseexceeds126mg/dLon2successivedays.Anotherapproachtothediagnosisofdiabetesistheoralglucosetolerancetest.Afterovernightfastingthepatientisgivenabolusofglucose(usually75g)orally,andbloodglucoselevelsaremeasuredat2hours.A2-hourplasmaglucoseconcentrationgreaterthan200mg/dLon2consecutivedaysissufficienttomakethediagnosisofdiabetes.Thediagnosisofdiabetesisalsoindicatedifthepatienthassymptomsassociatedwithdiabetesandhasanonfastingplasmaglucoselevelgreaterthan200mg/dL.

Diabetesmellitusiscurrentlyclassifiedastype 1 (T1DM) ortype 2 (T2DM). T2DMisbyfarthemorecommonformandaccountsfor90%ofdiagnosedcases.However,T2DMisusuallyaprogressivediseasethatremainsundiagnosedinasignificantpercentageofpatientsforseveralyears.T2DMisoftenassociatedwithvisceralobesityandlackofexercise—indeed,obesity-relatedT2DMisreachingepidemicproportionsworldwide.UsuallytherearemultiplecausesforthedevelopmentofT2DMinagivenindividualthatareassociatedwithdefectsintheabilityoftargetorganstorespondtoinsulin(i.e.,insulin resistance),alongwithsomedegreeofbeta cell deficiency. InsulinsensitivitycanbecompromisedattheleveloftheInsRoratthelevelofpostreceptorsignaling.T2DMappearstobetheconsequenceofinsulinresistance,followedbyreactivehyperinsulinemia,butultimatelybyrelative hypoinsulinemia (i.e.,inadequatereleaseofinsulintocompensatefortheend-organresistance)andbeta cell failure.

Theunderlyingcausesofinsulinresistancedifferamongpatients.Threemajorunderlyingcausesofobesity-inducedinsulinresistanceare: 1.Decreased ability of insulin to increase GLUT4-mediated uptake of glucose, especially by skeletal muscle. Thisfunction,whichisspecificallyapartofglucometabolic regulation by insulin, maybeduetoexcessiveaccumulationofTGinmuscleinobeseindividuals.Excessivecaloricintakeinduceshyperinsulinemia.Initiallythisleadstoexcessiveglucoseuptakeintoskeletalmuscle.Justasintheliver,excessivecaloriesintheformofglucosepromotelipogenesisand,throughgenerationofmalonylCoA,repressionoffattyacylCoAoxidation.ByproductsoffattyacidandTGsynthesis(e.g.,diacylglycerol,ceramide)mayaccumulateandstimulatesignalingpathways(e.g.,proteinkinaseC–dependentpathways)thatantagonizesignalingfromtheInsRorIRSproteins,orboth.Thusinsulinresistanceintheskeletalmuscleofobeseindividualsmaybeduetolipotoxicity.

2.

Decreased ability of insulin to repress hepatic glucose production. Thelivermakesglucosebyglycogenolysisintheshorttermandbygluconeogenesisinthelongterm.Theabilityofinsulintorepresskeyhepaticenzymesinboththesepathwaysisattenuatedininsulin-resistantindividuals.Insulinresistanceinthelivermayalsobeduetolipotoxicityinobeseindividuals(e.g.,fatty liver orhepatic steatosis).Visceraladiposetissueislikelytoaffectinsulinsignalingattheliverinseveralways,inadditiontotheeffectsoflipotoxicity.Forexample,visceraladiposetissuereleasesthecytokinetumor necrosis factor (TNF)-α, whichhasbeenshowntoantagonizeinsulinsignalingpathways.Also,TGinvisceraladiposetissuehasahighrateofturnover(possiblybecauseofrichsympatheticinnervation),sotheliverisexposedtohighlevelsofFFAs,whichfurtherexacerbateshepaticlipotoxicity.

3.

Inability of insulin to repress hormone-sensitive lipase or increase LPL in adipose tissue (or both). HighHSLandlowLPLaremajorfactorsinthedyslipidemiaassociatedwithinsulinresistanceanddiabetes.AlthoughthefactorsthatresisttheactionsofinsulinonHSLandLPLarenotcompletelyunderstood,thereisevidenceforincreasedproductionofparacrinediabetogenicfactorsinadiposetissue,suchasTNF-α.ThedyslipidemiaischaracterizedashypertriglyceridemiawithlargeTG-richVLDLparticlesproducedbytheliver.BecauseoftheirhighTGcontent,largeVLDLsandIDLsaredigestedveryefficiently,therebygivingrisetosmall,denseLDLparticlesthatareveryatherogenic.Inaddition,HDLtakesonexcessTGinexchangeforcholesterolesters,whichappearstoshortenthecirculatinghalf-lifeofHDLandApoAproteins.ThustherearelowerlevelsofHDLparticles,whichnormallyplayaprotectiveroleagainstvasculardisease.T1DM ischaracterizedbydestructionofbetacells,almost alwaysbyanautoimmunemechanism.T1DMisalsotermedinsulin-dependent diabetes mellitus. CharacteristicsofT1DMare: 1.

PeoplewithT1DMneedexogenousinsulintomaintainlifeandpreventketosis;virtuallynopancreaticinsulinisproduced.

2.

Thereispathologicaldamagetothepancreaticbetacells.Insulinitiswithpancreaticmononuclearcellinfiltrationisacharacteristicfeatureattheonsetofthedisorder.Cytokinesmaybeinvolvedintheearlydestructionofthepancreas.

3.

PeoplewithT1DMarepronetoketoacidosis.

4.

Ninetypercentofcasesbegininchildhood,mostlybetween10and14yearsofage.Thiscommonobservationledtoapplicationofthetermjuvenile diabetes tothedisorder.ThistermisnolongerusedbecauseT1DMcanariseatanytimeoflife,althoughjuvenileonsetisthetypicalpattern.

5.

Isletcellautoantibodiesarefrequentlypresentaroundthetimeofonset.IfT1DMisinducedbyavirus,theautoantibodiesaretransient.Occasionallyantibodieswillpersistlongterm,particularlyiftheyareassociatedwithotherautoimmunedisorders.About50%ofT1DMisrelatedtoproblemswiththemajorhistocompatibilitycomplexonchromosome6.Itiscorrelatedwithanincreasedfrequencyofcertainhumanleukocyteantigen(HLA)alleles.TheHLAtypesDR3andDR4aremostcommonlyassociatedwithdiabetes.

who received poor nutrition and into a life that meant chronic undernourishment.

A measure of adiposity is the body mass index (BMI). The BMI of an individual is calculated as:

Equation 38.1

The BMI of healthy lean individuals ranges from 20 to 25. A BMI greater than 25 indicates that the individual is overweight, whereas a BMI higher than 30 indicates obesity. The condition of being overweight or obese is a risk factor for multiple pathologies, including insulin resistance, dyslipidemia, diabetes, cardiovascular disease, and hypertension.

WAT tissue is divided into subcutaneous and intraabdominal (visceral) depots. Intraabdominal WAT refers primarily to omental and mesenteric fat and is the smaller of the two depots. These depots receive different blood supplies that are drained in a fundamentally different way in that venous return from intraabdominal fat leads into the hepatic portal system. Thus intraabdominally derived FFAs are mostly cleared by the liver, whereas subcutaneous fat is the primary site for providing FFAs to muscle during exercise or fasting. Regulation of intraabdominal and subcutaneous adipose tissue also differs. Abdominal fat is highly innervated by autonomic neurons and has a greater turnover rate. Furthermore, these two depots display differences in hormone production and enzyme activity.

Men tend to gain fat in the intraabdominal depot (android [apple-shaped] adiposity), whereas women tend to gain fat in the subcutaneous depot, particularly in the thighs and buttocks (gynecoid [pear-shaped] adiposity). Clearly an excess of abdominal fat poses a greater risk factor for the pathologies mentioned earlier. Thus another indicator of body composition is circumference of the waist (measured in inches around the narrowest point between the ribs and hips when viewed from the front after exhaling) divided by the circumference of the hips (measured at the point where the buttocks are largest when viewed from the side). This waist-hip ratio may be a better indicator of body fat than BMI, especially as it relates to risk for development of diseases. A waist-hip ratio of greater than 0.95 in men or 0.85 in women is linked to a significantly higher risk for development of diabetes and cardiovascular disease.

In recent years, numerous hormones and neuropeptides have been implicated in both chronic and acute regulation of appetite, satiety, and energy expenditure in humans. One

CHAPTER 39 Hormonal Regulation of Energy Metabolism simplified model involves two peptide hormones, leptin

Fig. 39.17 ), already discussed. Leptin acts on at least two neuron types in the arcuate nucleus of the hypothalamus. In the first, leptin represses production of neuropeptide Y (NPY), a very potent stimulator of food- seeking behavior (energy intake) and an inhibitor of energy expenditure. Norepinephrine, another appetite stimulator, co-localizes with NPY in some of these neurons. At the same time, leptin represses production of agouti-related peptide (AGRP), an endogenous antagonist that acts on

MC4R, a hypothalamic receptor for the anorexigenic peptide α-melanocyte–stimulating hormone (α-MSH), which inhibits food intake. In another type of arcuate neuron, leptin stimulates production of proopiomelano cortin (POMC) products, one of which is α-MSH, and production of cocaine-amphetamine–regulated transcript (CART), both of which inhibit food intake. Thus leptin diture by simultaneously inhibiting NPY and the α-MSH

Fig. 39.17 ). These second-order neuropeptides are transmitted to and interact with receptors in neurons of the and lateral hypothalamic nucleus (“hunger” neurons). In activity (especially sympathetic outflow) with diverse endo crine actions on thyroid gland function, reproduction, and growth.

Another regulator of food intake and body energy stores is melanin-concentrating hormone (MCH). This antagonizing the satiety effect of α-MSH downstream from the interaction of α-MSH with its MC4R receptor. The probable importance of this molecule is demonstrated by the fact that it is the only regulator whose ablation by gene knockout actually results in leanness.

To maintain overall energy homeostasis, the system must example, carbohydrate intake with carbohydrate oxidation.

neurotransmitter responses to meals. Serotonin produces satiety after ingestion of glucose. Gastrointestinal hormones such as cholecystokinin and GLP-1 produce satiety by humoral effects, but their local production in the brain may participate in nutrient and caloric regulation. The recently discovered hormone ghrelin is an acylated peptide with potent orexigenic activity that arises in cells of the oxyntic glands in the stomach. Plasma levels of ghrelin rise in humans in the 1 to 2 hours that precede their normal meals.

Plasma levels of ghrelin fall drastically to minimum values about 1 hour after eating. Ghrelin appears to stimulate food intake by reacting with its receptor in hypothalamic neurons that express NPY.

1.

Cells make ATP to meet their energy needs. ATP is made by glycolysis and by the TCA cycle coupled to oxidative phosphorylation.

2.

Cells can oxidize carbohydrate (primarily in the form of glucose), AAs, and FFAs to make ATP. Additionally the liver makes KBs for other tissues to oxidize for energy in times of fasting.

3.

Some cell types are limited in the energy substrates they can oxidize for energy. The brain is normally exclusively dependent on glucose for energy. Thus blood glucose must be maintained above 60 mg/ dL for normal autonomic and CNS function. Conversely, inappropriately high levels of glucose (i.e., fasting glucose > 100 mg/dL) promote glucotoxicity and thereby lead to the long-term complications of diabetes.

4.

The endocrine pancreas produces the hormones insulin, glucagon, somatostatin, gastrin, and pancreatic polypeptide.

5.

Insulin is an anabolic hormone that is secreted in times of excess nutrient availability. It allows the body to use carbohydrates as energy sources and store nutrients.

6.

Major stimuli for insulin secretion include increased serum glucose and some AAs. Activation of cholinergic (muscarinic) receptors also increases insulin secretion, whereas activation of α2-adrenergic receptors inhibits insulin secretion. The gastrointestinal tract releases incretin hormones that stimulate pancreatic insulin secretion. GLP-1 is particularly potent in augmenting glucose-dependent stimulation of insulin secretion (GSIS). GLP-1 is degraded by dipeptidyl peptidase (DPP)-4. DPP-4– resistant GLP-1 analogues and inhibitors of DPP-4 are currently used to increase GSIS in patients with type 2 diabetes.

7.

Insulin binds to the insulin receptor (InsR), which is linked to multiple pathways that mediate the metabolic (Akt kinase) and growth effects (MAPK) of insulin.

8.

During the digestive phase, insulin acts on the liver to promote trapping of glucose as G6P. Insulin also increases glycogenesis, glycolysis, and de novo lipogenesis (DNL) in the liver. Insulin inhibits gluconeogenesis, glycogenolysis, and assembly of lipids into VLDL.

9.

Insulin increases GLUT4-mediated glucose uptake in muscle and adipose tissue.

10.

Insulin increases glycogenesis, glycolysis, and in the presence of caloric excess, lipogenesis in muscle.

11.

Insulin increases glycolysis and generation of G3P in adipocytes. Insulin induces expression of LPL and its transport to the luminal side of capillary endothelial cells. Insulin promotes uptake and activation of FFAs and esterification of fatty acyl CoAs to G3P to form

TG, and it decreases hormone-sensitive lipase activity in adipocytes.

12.

Insulin increases AA uptake and protein synthesis in skeletal muscle but also essentially all insulin target cells. Insulin/Akt kinase signaling activates mTORC1 and S6K to promote synthesis of ribosomal proteins and proteins involved in mRNA translation, as well as other types of proteins. Insulin inhibits proteosomal degradation of protein.

13.

Glucagon is a catabolic counterregulatory hormone. Its secretion increases during periods of food deprivation, and it acts to mobilize nutrient reserves. It also mobilizes glycogen, fat, and even protein.

14.

Glucagon is released in response to decreased serum glucose (and therefore decreased insulin) and increased serum AA levels and β-adrenergic signaling.

15.

Glucagon binds to the glucagon receptor, which is linked to PKA-dependent pathways. The primary target organ for glucagon is the liver. Glucagon increases liver glucose output by increasing glycogenolysis and gluconeogenesis. It increases β-oxidation of fatty acids and ketogenesis.

16.

Glucagon regulates hepatic metabolism both by regulation of gene expression and through posttranslational PKA-dependent pathways.

17.

The major counterregulatory factors in muscle and adipose tissue are the adrenal hormone epinephrine and the sympathetic neurotransmitter norepinephrine. These two factors act through β2-and β3-adrenergic receptors to increase cAMP levels. Epinephrine and norepinephrine enhance glycogenolysis and fatty acyl oxidation in muscle and increase hormone-sensitive lipase in adipose tissue.

18.

Diabetes mellitus is classified as type 1 (T1DM) and type 2 (T2DM). T1DM is characterized by destruction of pancreatic beta cells, and exogenous insulin is required for treatment. T2DM can be due to numerous factors but is usually characterized as insulin resistance coupled to some degree of beta cell deficiency. Patients with T2DM may require exogenous insulin at some point to maintain blood glucose levels.

19. Obesity-associated T2DM is currently at epidemic proportions worldwide and is characterized by insulin resistance due to lipotoxicity, hyperinsulinemia, and inflammatory cytokines produced by adipose tissue. T2DM is often associated with obesity, insulin resistance, hypertension, and coronary artery disease. This constellation of risk factors is referred to as the metabolic syndrome.

20. Major symptoms of diabetes mellitus include hyperglycemia, polyuria, polydipsia, polyphagia, muscle wasting, electrolyte depletion, and ketoacidosis (in T1DM).

21. The long-term complications of poorly controlled diabetes are due to excess intracellular glucose (glucotoxicity), especially in the retina, kidney, and peripheral nerves. This leads to retinopathy, nephropathy, and neuropathy.

Bailey CJ, et al. Future glucose-lowering drugs for type 2 diabetes. Lancet Diabetes Endocrinol. 2016;4:350-359.

Davidson JA, et al. Glucagon therapeutics: dawn of a new era for diabetes care. Diabetes Metab Res Rev. 2016;doi:10.1002/dmrr.2773; [Epub ahead of print].

Font-Burgada J, et al. Obesity and cancer: the oil that feeds the flame. Cell Metab. 2016;23:48-62.

22. Adipose tissue has an endocrine function, especially in terms of energy homeostasis. Hormones produced by adipose tissue include leptin and adiponectin. Leptin acts on the hypothalamus to promote satiety.

Klein MS, Shearer J. Metabolomics and type 2 diabetes: translating basic research into clinical application. J Diabetes Res. 2016;2016:3898502. doi:10.1155/2016/3898502; [Epub 2015 Nov 9].

Marion-Letellier R, et al. Fatty acids, eicosanoids and PPAR gamma. Eur J Pharmacol. 2016;785:44-49.

Pawlak M, et al. Molecular mechanism of PPARα action and its impact on lipid metabolism, inflammation and fibrosis in nonalcoholic fatty liver disease. J Hepatol. 2015;62:720-733.

Upon completion of this chapter the student should be able to answer the following questions: 1.

Describe the pool of serum calcium and phosphate, including ionized, complexed, and protein bound. Describe the normal concentration ranges of these ions and the major routes of influx and efflux.

2.

Discuss the role of the parathyroid gland in the regulation of serum calcium and explain the role of the calcium-sensing receptor in the regulation of parathyroid hormone (PTH) secretion.

3.

Describe the production of 1,25-dihydroxyvitamin D, including sources of vitamin D precursor, sites and key regulators of vitamin D hydroxylation, and transport of vitamin D metabolites in the blood.

4.

List the target organs of PTH and describe its effects on calcium and phosphate mobilization or handling at each of these sites.

5.

List the target organs and key actions of 1,25-dihydroxyvitamin D.

6.

Discuss the regulation of phosphate metabolism by FGF23.

7.

Predict the hormone responses that would be triggered by perturbations of serum calcium and phosphate or by vitamin D deficiency, and discuss the consequences of these compensatory hormone actions.

alcium (Ca) and phosphate are essential to human life because they play important structural roles in hard tissues (i.e., bones and teeth) and important regulatory roles in metabolic and signaling pathways. In biological systems, inorganic phosphate (Pi) consists of a mixture of dihydrogen phosphate (H2PO4 −) and hydrogen phosphate (HPO4 −). The two primary sources of circulating Ca and Pi are the diet and the skeleton (

Fig. 40.1 ). Two hormones, 1,25-dihydroxyvitamin D (also called calcitriol) and parathyroid hormone (PTH), regulate intestinal absorption of Ca and Pi and release of Ca and Pi into the circulation after bone resorption. The primary processes for removal of Ca and Pi from blood are renal excretion and bone mineralization (see

Fig. 40.1 ). 1,25-Dihydroxyvitamin D and PTH regulate both processes. Other hormones and paracrine growth factors also regulate Ca and Pi homeostasis.

Crucial Roles of Calcium and Phosphate in Cellular Physiology

Ca is an essential dietary element. In addition to obtaining Ca from the diet, humans contain a vast store (i.e., >1 kg) of Ca in bone mineral, which can be called upon to maintain normal circulating levels of Ca in times of dietary restriction and during the increased demands of pregnancy and nursing. Circulating Ca exists in three forms (

Table 40.1): free ionized Ca++ , protein-bound Ca, and Ca complexed with anions (e.g., phosphates, HCO3 − , citrate). The ionized form represents about 50% of circulating Ca. Since it is critical to so many cellular functions, [Ca++] in both the extracellular and intracellular compartments is tightly controlled. Circulating Ca++ is under direct hormonal control and normally maintained within a relatively narrow range. Either too little calcium (hypocalcemia; total serum calcium < 8.7 mg/dL [2.2 mM]) or too much Ca (hypercalcemia; total serum Ca > 10.4 mg/dL [2.6 mM]) in blood can lead to a broad range of pathophysiological changes, including neuromuscular dysfunction, central nervous system dysfunction, renal insufficiency, calcification of soft tissue, and skeletal pathology.

Pi is also an essential dietary element, and it is stored in large quantities in mineral. Most circulating Pi is in the free ionized form, but some Pi (<20%) circulates as a protein-bound form or complexed with cations (see

Table 40.1 ). Because soft tissues contain 10-fold more Pi than Ca, tissue damage (e.g., crush injury with massive muscle cell death) can result in hyperphosphatemia, whereupon the increased Pi complexes with Ca++ to cause acute hypocalcemia.

Pi is a key intracellular component. Indeed, it forms the high-energy phosphate bonds of adenosine triphosphate (ATP) that maintain life. Phosphorylation and dephosphorylation of proteins, lipids, second messengers, and cofactors represent key regulatory steps in numerous metabolic and signaling pathways, and phosphate also serves as the backbone for nucleic acids.

•Fig. 40.1 DailyCa++ andPiflux.

Ca8.5–10.250%45%5%Pi3–4.584%10%6%

Ca++ isbound(i.e.,complexed)tovariousanionsinplasma,includingHCO3− ,citrate,andSO42− .Piiscomplexedtovariouscations,includingNa+ andK+ .FromKoeppenBM,StantonBA.Renal Physiology. 4thed.Philadelphia:Mosby;2007.

Physiological Regulation of Calcium and Phosphate: Parathyroid Hormone and 1,25-Dihydroxyvitamin D C

PTH and 1,25-dihydroxyvitamin D are the two physiologically most important hormones dedicated to mainte- nance of normal blood Ca and Pi in humans. As such they are referred to as calciotropic hormones. The structure, synthesis, and secretion of these two hormones and their receptors will be discussed first. In the following section, the detailed actions of PTH and 1,25-dihydroxyvitamin D on the three key sites of Ca/Pi homeostasis (i.e., gut, bone, and kidney) are discussed.

The predominant parenchymal cell type in the parathyroid B gland is the principal (also called chief) cell (

Fig. 40.2

PTH is the primary hormone that protects against hypocalcemia. The primary targets of PTH are bone and the kidneys. PTH also functions in a positive feed-forward loop by stimulating production of 1,25-dihydroxyvitamin D.

•Fig. 40.2 AandB,Histologyofparathyroidglands.A,adiposetissuewithinparathyroidglands;C,capillaries;O,oxyphilcells;P,principalorchiefcells.(FromYoungBetal.Wheater’s Functional Histology. 5thed.Philadelphia:ChurchillLivingstone;2006.)

Structure, Synthesis, and Secretion

PTH is secreted as an 84–amino acid polypeptide and is synthesized as a prepro-PTH, which is proteolytically processed to pro-PTH in the endoplasmic reticulum and then to PTH in the Golgi and secretory vesicles. PTH has a short half-life in the circulation (2 minutes), consistent with its role in minute-to-minute regulation of plasma calcium.

Because the PTH receptor also binds PTH-related peptide (PTHrP), it is usually referred to as the PTH/PTHrP receptor. The PTH/PTHrP receptor is expressed on osteoblasts in bone and in the proximal and distal tubules of the kidney, and it is the receptor that mediates the systemic actions of PTH. However, the PTH/PTHrP receptor is also expressed in many developing organs in which PTHrP has important paracrine functions. One such example is regulation of chondrocyte proliferation in the growth plate during endochondral bone growth.

Vitamin D is a prohormone that must undergo two successive hydroxylation reactions to become the active form known as 1,25-dihydroxyvitamin D or calcitriol (

Fig.

40.5 ). This hormone plays a critical role in Ca absorption

Extracellular[Ca++]issensedbytheparathyroidchiefcellthroughaplasmamembranecalcium-sensing receptor (CaSR). TheprimarysignalthatstimulatesPTHsecretionisadecreaseincirculating[Ca++](

Fig.40.3 ).Conversely,increasingamountsofextracellularCa++ bindtotheCaSRandstimulatesignalingpathwaysthatrepressPTHsecretion.AlthoughtheCaSRbindstoextracellularCa++ withrelativelylowaffinity,theCaSRisextremelysensitivetominutechangesinextracellular[Ca++].Therelationshipbetween[Ca++]andtherateofPTHsecretionisdescribedbyasteepinversesigmoidalcurve.A0.2-mMdifferenceinblood[Ca++]spansthefullrangeofthecurve,alteringPTHsecretionfrombasal(5%ofmaximum)tomaximumlevels(

Fig.40.4).Thesteady-state“setpoint” willvarybetweenindividualsbuttypicallyresidesbelowthemidpointofthecurve(i.e.,half-maximalPTHsecretion).ThustheCaSRisarapid,robust,andcontinuousregulatorofPTHoutputinresponsetosubtle[Ca++]fluctuations.

InadditiontoinhibitingPTHsecretion,activationoftheCaSRalsopromotesdegradationofstoredPTHintheparathyroidchiefcell.Asaresult,biologicallyinactivecarboxyterminalPTHfragmentsaresecretedfromtheparathyroidglandandarealsoproducedbyperipheralmetabolismofPTHbytheliverandkidney.ThereforecurrentPTHassaysusetwoantibodiesthatrecognizeepitopesfrombothendsofthemoleculetoaccuratelymeasureintactPTH[1-84].

Overalongertimeframe,PTHproductionisalsoregulatedatthelevelofmRNAstabilityandgenetranscription(see

Fig.

40.3 ).Decreased[Ca++]leadstoproductionofproteinsthatbindthe3-untranslatedregionofPTHmRNAandstabilizeit,leadingtoincreasedPTHtranslation.PTH genetranscriptionisrepressedby1,25-dihydroxyvitaminDinanegativefeedbackloop(actingthroughvitaminDresponseelements—seelater).Theabilityof1,25-dihydroxyvitaminDtoholdPTH geneexpressionincheckisreinforcedbythecoordinatedupregulationofCASR geneexpressionbypositivevitaminDresponseelementsinthepromoterregionoftheCASR gene(see

Fig.40.3 ).Itshouldbenoted,however,thatduringahypocalcemicchallenge,thedecreasein[Ca++]overridestheinhibitoryeffectof1,25-dihydroxyvitaminDonPTHtranscription,allowingbothofthesehormonestobeelevatedsimultaneously.

•Fig. 40.3 RegulationofPTH geneexpressionandsecretion.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)Gq + Gi PTH PTH CaSR Downstream signaling pathway 1,25 Vitamin D CaSR Ca++ proPTH preproPTH preCaSR CaSR mRNA (+) (–) (–) (–) PTH mRNA Parathyroid cell membrane Nucleus CaSR gene PTH gene •Fig. 40.4 Sigmoidalrelationshipbetweenserum[Ca++]andserumPTH,whichreflectstherateofPTHsecretion.(ModifiedfromPor-terfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)100 50 5 1.00 1.301.201.10 1.40 Serum PTH (% of maximum) Free Ca++ in blood (mM)

Patientswithbenign familial hypocalciuric hypercalcemia (FHH) areheterozygousforinactivatingmutationsoftheCaSR.Inthesepatients,becauseofcompleteorpartiallossofoneCaSRallele,higherlevelsof[Ca++]arerequiredtosuppressPTHsecretion.Thisresultsinanelevated[Ca++]setpointforPTHsecretion,accountingforthehypercalcemia.TheCaSRisalsoexpressedinthethickascendinglimboftherenaltubule,whereitnormallyinhibitsCa++ reabsorptionwhenbloodCa++ rises.ThehypocalciuriainthefaceofhypercalcemiainFHHisduetothereducedabilityoftheCaSRinthekidneytosenseandrespondtoelevatedblood[Ca++]byincreasingCaexcretion.

Parathyroid hormone–related peptide (PTHrP) isapeptideparacrinehormoneproducedbyseveraladulttissues(skin,hair,breast),whereitmayregulateproliferationanddifferentiation.Italsoplaysaroleinrelaxationofsmoothmuscleinresponsetostretchinbloodvessels,uterus,andbladder.Duringlactation,PTHrPpromotesmaternalboneresorptionandthetransportofcalciumintomilk.Duringdevelopment,PTHrPregulatescalciumtransportacrosstheplacentaandisakeyregulatorofchondrocyteproliferationanddifferentiationinthegrowthplateoflongbones.The30aminoacidsattheN-terminusofPTHrPhavesignificantstructuralhomologywithPTH.PTHrPisnotregulatedbycirculatingCa++ andnormallydoesnotplayaroleinCa/Pihomeostasisinadults.However,certaintumorssecretehighlevelsofPTHrP,whichcauseshypercalcemia of malignancy andsymptomsthatresemblehyperparathyroidism.

Hormonal Regulation of Calcium and Phosphate Metabolism 1,25-(OH)2D3 24,25-(OH)2D3 •Fig. 40.5 Biosynthesisof1,25-dihydroxyvitaminD.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.) and to a lesser extent Pi absorption by the small intestine. It also regulates bone remodeling and renal reabsorption of Ca and Pi.

Structure, Synthesis, and Transport of Active Vitamin D Metabolites

Vitamin D3 (also called cholecalciferol) is synthesized via conversion of 7-dehydrocholesterol by ultraviolet B •Fig. 40.6 VitaminDmetabolism.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)7-Dehydrocholesterol Vit D-DBP and Vit D-chylomicron 25(OH) Vit D-DBP Vit D-chylomicron Lymphatic Vit D-DBP 1,25-Dihydroxyvitamin D Portal blood Caval blood Peripheral blood Vitamin D3 Enterocyte Dietary vitamin D3 and vitamin D2 Vit D UV-B Hepatocyte Vit D 25-Hydroxyvitamin D 25-Hydroxylase 24-Hydroxylase 24-Hydroxylase25-Hydroxyvitamin D 24,25-Dihydroxyvitamin D 1, 25-Dihydroxyvitamin D 1,24,25-Trihydroxyvitamin D Biological effects at gut, bone, kidney, etc. 1˜-Hydroxylase (UVB) light in the more basal layers of the skin (Fig. 40.6 ). Chemically, vitamin D3 is a secosteroid in which one of the cholesterol rings is opened (see

Fig. 40.5 ). Vitamin D2 (ergocalciferol) is produced in plants. Vitamin D3 and to a lesser extent vitamin D2 are absorbed from the diet and are equally effective after conversion to active hydroxylated forms. The balance between UVB-dependent endogenously synthesized vitamin D3 and absorption of the dietary forms of vitamin D becomes important in certain situations. Individuals with higher melanin content in skin who live at higher latitudes convert less 7-dehydrocholesterol to vitamin D3 and thus are more dependent on vitamin supplements or dietary sources of vitamin D (natural or fortified, e.g., milk). Institutionalized elderly patients who stay indoors and avoid dairy products are particularly at risk for development of vitamin D deficiency.

Vitamin D is transported in blood from the skin to the liver. Dietary vitamin D reaches the liver directly via transport in the portal circulation and indirectly via chylomicrons (see

Fig. 40.6 ). In the liver, vitamin D is hydroxylated at the 25-carbon position to yield 25-hydroxyvitamin D. The hepatic 25-hydroxylase is constitutively expressed and unregulated, so circulating levels of 25-hydroxyvitamin D reflect the amount of precursor available for 25-hydroxylation. For this reason, and because of its relatively long half-life in the circulation (2–3 weeks), measurement of 25-hydroxyvitamin D levels is used to assess vitamin D status.

25-Hydroxyvitamin D undergoes further hydroxylation in the proximal tubule of the kidney (see

Figs.

40.5 40.6).

Hydroxylation at the 1α position generates 1,25-dihydroxyvitamin D, the most active form of vitamin D. Hydroxylation at the 24 position generates 24,25-dihydroxyvitamin D, which does not play a major biological role and serves as an inactivation pathway.

Renal 1α-hydroxylase is tightly regulated by a number of factors (

Fig. 40.7). PTH and hypophosphatemia are the primary inducers of 1α-hydroxylase activity, resulting in 25(OH)D3 1˜-hydroxylase 1,25(OH)2D3 25(OH)D3 CYP27B1 FGFRKIotho •Fig. 40.7 Regulationof1α-hydroxylasegene(CYP27B1) expressionintheproximaltubule,showingstimulationbyPTHandinhibitionbyFGF23and1,25-dihydroxyvitaminD.Hypophosphatemiaprobablystimulates1α-hydroxylasebyreducingFGF23levelsatleastinpart.

increased levels of 1,25-dihydroxyvitamin D. Conversely, [Ca++] and 1,25-dihydroxyvitamin D, the enzyme product, inhibit it. Fibroblast growth factor (FGF)23, a major regulator of Pi metabolism (see later), also represses 1α-hydroxylase activity; a reduction of FGF23 levels likely mediates the effect of hypophosphatemia on 1,25-dihydroxyvitamin D production at least in part.

Vitamin D and its metabolites circulate in blood primarily bound to vitamin D–binding protein (DBP). DBP is a serum glycoprotein that is synthesized by the liver. DBP binds more than 85% of 1,25-hydroxyvitamin D and 24,25-dihydroxyvitamin D. Because of binding to other proteins, only 0.4% of 1,25-dihydroxyvitamin D circulates as free hormone. DBP transports the highly lipophilic vitamin D in blood and provides a reservoir of vitamin D that protects against vitamin D deficiency.

1,25-Dihydroxyvitamin D Receptor 1,25-Dihydroxyvitamin D exerts its actions primarily through binding to the nuclear vitamin D receptor (VDR), which is a member of the nuclear hormone receptor family. The VDR is a ligand-dependent transcription factor that binds to cognate DNA sequences (vitamin D response elements) as a heterodimer with the retinoid X receptor (RXR). Thus the primary action of 1,25-dihydroxyvitamin D is to regulate gene expression in its target tissues, including the small intestine, bone, kidneys, and parathyroid gland.

The genomic actions of 1,25-dihydroxyvitamin D mediated by the VDR occur over a period of hours to days. 1,25-Dihydroxyvitamin D also has rapid effects (seconds to minutes). For example, 1,25-dihydroxyvitamin D rapidly induces absorption of Ca++ by the duodenum. The VDR is also expressed in the plasma membrane of cells and is linked to rapid signaling pathways (e.g., G proteins, phosphatidylinositol-3′-kinase). Current molecular modeling has led to development of ligands that specifically bind

Hormonal Regulation of Calcium and Phosphate Metabolism to the nuclearversus the membrane-localized VDR, paving the way for selective treatment of disorders related to the rapid versus slow actions of 1,25-dihydroxyvitamin D with synthetic vitamin D analogues.

Regulation of [Ca++] and [Pi] by Small Intestine and Bone

An overview of the regulation of [Ca++] and Pi] by the action of PTH and 1,25-dihydroxyvitamin D on the small intestine, bone, and parathyroid glands is summarized in

Table 40.2 and in the following paragraphs. For details on renal handling of Ca++ , consult

Chapter 36.

Calcitonin isapeptidehormoneproducedbythemedullarycells,orC-cells,ofthethyroidgland.Calcitoninsecretionispositivelyregulatedbyserum[Ca++]viatheCaSR.Thecalcitoninreceptorisexpressedinosteoclasts,wherecalcitoninactsrapidlyanddirectlytoinhibitboneresorption.However,inhumans,calcitonindoesnotappeartoplayamajorroleinregulatingserumCa.Insupportofthisview,productionofexcesscalcitoninorcompleteabsenceofcalcitonin(e.g.,followingthyroidectomy)doesnotperturbserumCalevels.Morepotentformsofthehormone(e.g.,salmoncalcitonin)havebeenusedtherapeuticallyasanantiresorptiveinthetreatmentofPaget’s disease (characterizedbyexcessiveosteoclasticboneresorption)andinosteoporosis.Calcitoninisalsoausefulhistochemicalmarkerofmedullarythyroidcancer.

Dietary intake of Ca can vary widely among individuals and from day to day. Assuming an intake of 1000 mg (the RDA for ages 19–50), 350 mg would typically be absorbed, counterbalanced by 150 mg secreted by the intestine, for a net intake of 200 mg. Most Ca++ absorption takes place in the proximal small intestine. Importantly, absorption of Ca++ is stimulated by 1,25-dihydroxyvitamin D, so absorption is more efficient in the face of declining dietary Ca++ .

Ca++ is absorbed from the duodenum and jejunum by both a Ca++-regulated and a hormonally regulated transcellular route and by a passive paracellular route. The transcellular route of Ca++ absorption is summarized in

Fig.

40.8 . Movement of Ca++ from the gastrointestinal lumen into the enterocyte, which is driven by both chemical and electrical gradients, occurs via apical calcium channels called TRPV5 and TRPV6. Once inside the cell, Ca++ ions bind to calbindin-D9K, which maintains a low cytoplasmic [Ca++], preserving the favorable transluminal membrane Ca++ gradient. Calbindin-D9K also plays a role in apical-to-basolateral shuttling of Ca++, which is transported across the basolateral membrane against an electrochemical gradient by plasma membrane calcium ATPase (PMCA). The Na+/Ca++ exchanger (NCX) also contributes to the

CASR,calcium-sensingreceptor;M-CSF,monocytecolony-stimulatingfactor;NPT2,Na+/Picotransporter;OPG,osteoprotegerin;PTH,parathyroidhormone;

RANKL,receptoractivatorofnuclearfactorκ-B.

•Fig. 40.8 IntestinalabsorptionofCa++viathetranscellularroute.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)LuminalsideTRPV5/6SerosalsideCa++Ca++Ca++Ca++-cal indin9KCal indin9KPMCA transport of Ca++ out of enterocyte. 1,25-Dihydroxyvitamin D stimulates expression of all the components involved in absorption of Ca++ by the small intestine.

The fraction of dietary Pi absorbed by the jejunum remains relatively constant at about 70% and is under minor hormonal control by 1,25-dihydroxyvitamin D. The limiting process in transcellular Pi absorption is transport across the apical brush border, which is mediated by the Na+/Pi cotransporter (NPT2).

Bone stores vast amounts of Ca and Pi. Once peak bone mass has been achieved in an adult, the skeleton is constantly remodeled through the concerted activities of bone cells. The processes of bone formation and bone resorption are in balance in a healthy, physically active, and well-nourished individual. Of the 1 kg of Ca immobilized in bone, about 500 mg (i.e., 0.5%) is mobilized from and deposited into bone each day. However, the process of bone remodeling can be modulated to provide a net gain or loss of Ca++ and Pi into blood and is responsive to physical activity (loading), diet, age, and hormonal regulation. Because the integrity of bone is absolutely dependent on Ca and Pi, chronic dysregulation of these ions or the hormones that regulate them lead to pathological changes in bone.

Physiology of Bone

The processes of pattern formation, growth, and remodeling of the skeleton is complex and beyond the scope of this chapter. The key elements required for understanding the role of adult bone in the hormonal regulation of Ca and Pi metabolism are discussed.

In adults, bone remodeling involves (1) destruction of fatigued or microdamaged bone with the release of Ca++, Pi, and hydrolyzed fragments of bone matrix into blood and (2) synthesis of osteoid (yet to be mineralized bone matrix) at the site of resorption, followed by controlled mineralization of the osteoid by Ca++ and Pi to form new bone. Bone remodeling occurs continually at about 2 million discrete sites throughout the skeleton by packets of bone cells referred to as the basic multicellular units (BMU).

The cells involved in bone remodeling fall into two major classes: cells that form bone (osteoblasts) and cells that destroy or resorb bone (osteoclasts). The process of bone remodeling is a highly integrated process (

Fig. 40.9 ). Osteoblast-lineage cells express factors that induce differentiation of osteoclasts from progenitors of the monocyte/ macrophage lineage and also promote mature osteoclast function. Osteoblasts release monocyte colony-stimulating factor (M-CSF), which expands and differentiates early hematopoietic progenitors (CFU-GM) into preosteoclasts that express a cell surface receptor called RANK (receptor •Fig. 40.9 Osteoblastregulationofosteoclastdifferentiationandfunction.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.) activator of nuclear factor [NF]-κB). Osteoblast-lineage cells display RANK ligand (RANKL) on their cell surface. RANKL then binds to RANK on preosteoclasts and induces osteoclastogenesis. This process involves fusion of several osteoclast precursors, giving rise to a large multinucleated osteoclast. The perimeter of the osteoclast membrane facing mineralized bone adheres tightly to the bone and seals off the area of osteoclast-bone contact (see

Fig. 40.9 ). The region within the sealed zone forms a highly invaginated membrane called the ruffled border, from which HCl and hydrolytic lysosomal enzymes are secreted. The acidic enzyme-rich microenvironment beneath the osteoclast dissolves the bone mineral, thereby releasing Ca++ and Pi into blood, and also degrades the bone matrix. There is an additional inhibitory component of the RANK/RANKL system. Osteoblast-lineage cells can also produce a soluble factor called osteoprotegerin (OPG), which acts as a decoy receptor for RANKL and inhibits osteoclast differentiation and function (see

Fig. 40.9 ). Therefore the balance between RANKL and OPG expression by osteoblasts determines how much osteoclast differentiation and bone resorption will occur.

Following bone resorption in the BMU, there is a brief reversal phase, then adjacent osteoblasts migrate into the resorbed area and begin to lay down osteoid. Several components within osteoid (pyrophosphate, alkaline phosphatase, specific glycoproteins) promote slow, controlled mineralization, a process that removes Ca++ and Pi from blood. As the osteoblasts become surrounded by and entrapped within bone, they become osteocytes that sit within small spaces called lacunae. Osteocytes remain interconnected through cell processes that run within canaliculi and form communicating junctions with adjacent cell processes. The new concentric layers of bone, along with the interconnected osteocytes and the central canal, are referred to collectively as a Haversian system or osteon. Emerging evidence indicates that osteocytes are able to sense mechanical stress in bone and signal that additional local bone formation is needed. They can also detect microdamage in bone that serves to initiate remodeling at that location.

Asacalciotropichormone,PTHisapotentregulatorofboneresorptioninadults.ThePTH/PTHrPreceptorisexpressedonosteoblastsbutnotonosteoclasts.ThereforePTHactsonosteoblaststoincreaseexpressionofosteoblastparacrinefactors(i.e.,M-CSF,RANKL)thatupregulateosteoclastdifferentiationandboneresorption.1,25-DihydroxyvitaminDalsostimulatesboneresorptionbyupregulatingRANKLexpressioninosteoblasts.

ItisimportanttorecognizethatPTH(alongwith1,25-dihydroxyvitaminD)willpromoteboneresorptionwhenPTHlevelsarehigh(i.e.,duringahypocalcemicchallenge).WhenPTHlevelsarenormal,however,boneremodelingisalocallycontrolledprocessbywhicholddamagedboneisreplaced.Interestinglyithasbeenshownthatintermittentadministrationoflow-dosePTHpromotesosteoblastsurvivalandboneanabolicfunctions,increasesbonedensity,andreducestheriskoffractureinhumans.

DiscoveryoftheRANK/RANKL/OPGsystemhaspresentednewtherapeuticopportunitiesfortreatingosteoporosis.AbiologicalantiresorptivedrugbasedonahumanizedantibodydirectedagainstRANKLisnowavailablefortreatmentofpostmenopausalosteoporosis.Thishasproventobeaneffectivetreatmentthatimprovesbonedensityandreducestheriskoffracture.

Fig.40.10C )producesahypocalcemicchallengebydecreasinggastrointestinalabsorptionofCa++ andPi.Adropinserum[Ca++]increasescompensatoryPTH geneexpression,PTHsecretion,parathyroidcellproliferation,andPTH-mediatedupregulationofrenal1-hydroxylase.Intheabsenceofsufficient25-hydroxyvitaminDprecursor,however,1,25-dihydroxyvitaminDlevelsfall.ThesecondaryelevationofPTHmobilizesCa++ fromboneandkidneybutpromotesrenalexcretionofPi,causinghypophosphatemia.BecausetheCa++ × Piproductinserumislow,bonemineralizationisimpaired.Inchildrenthisleadstorickets, inwhichthegrowthoflongbonesisabnormalandimpaired.Theribcage,wrists,andanklesshowcharacteristicbonedeformities,andtheimpairedmineralizationcausesbowingofthelegs.Inadults,vitaminDdeficiencyleadstoosteomalacia, whichischaracterizedbypoormineralizationofnewlyformedosteoid,visibleonradiographsaspseudofractures.Inseverecases,osteomalaciaresultsinweakness,bonepain,andincreasedriskoffracture.

°PTH °1,25(OH)2D3 ˜1,25(OH)2D3

Regulation of Serum Phosphate by FGF23

Study of hypophosphatemic disorders has led to the discovery that FGF23, a peptide hormone produced by osteocytes, is a regulator of Pi metabolism. FGF23 binds to a receptor complex in proximal tubule cells and, like PTH, inhibits NPT2 to promote Pi excretion. Several diseases are associated with excess production of FGF23, including rickets in children and osteomalacia secondary to hypophosphatemia in adults. In autosomal dominant hypophosphatemic rickets (ADHR), a mutation in FGF23 prevents its cleavage and inactivation. X-linked hypophosphatemic rickets (XLHR) is caused by a mutation of the PHEX gene (protein with homology to endopeptidases on the X chromosome), which also causes overproduction of FGF23. Finally, FGF23 is sometimes ectopically produced by slow-growing occult mesenchymal tumors, causing a hypophosphatemic paraneoplastic syndrome. In addition to inhibition of Pi reabsorption, FGF23 also inhibits expression of 1α-hydroxylase in the proximal tubule, thereby inhibiting production of 1,25-dihydroxyvitamin D and exacerbating hypophosphatemia. The physiological role of this pathway is not completely understood, and many questions remain, °GI °1,25(OH)2D3 absorption •Fig. 40.10 IntegratedhormoneresponsestoperturbationsofCa++ (A),Pi(B),andvitaminD(C).

including how and where Pi levels are sensed. Pi is not as tightly regulated as calcium, either temporally or with respect to concentration range, but recent evidence suggest that long-term elevation of Pi is associated with increased production of FGF23. In what appears to be an emerging negative feedback loop, 1,25-dihydroxyvitamin D decreases production of FGF23 by osteocytes.

Gonadal and adrenal steroid hormones have profound effects on bone. 17β-Estradiol (E2; see has important anabolic effects on bone and is a potent regulator of osteoblast and osteoclast function. Estrogen promotes survival of osteoblasts and apoptosis of osteoclasts, thereby favoring bone formation over resorption. Androgens also have bone anabolic effects, although some of these effects are due to local conversion of testosterone to E2 in men (see ). The combined effects of testosterone and E2 account for the higher peak bone mass observed in men. In postmenopausal women, estrogen deficiency results in an initial phase of rapid bone loss that lasts about 5 years, followed by a second phase of slower age-related bone loss that is similar in both sexes. For this reason, women are susceptible to postmenopausal osteoporosis.

Glucocorticoids at high therapeutic doses promote bone resorption and inhibit intestinal Ca absorption. However, the most critical adverse effect is inhibition of osteoblast differentiation, which impairs bone formation. Therefore patients treated with high levels of a glucocorticoid as an antiinflammatory or immunosuppressive drug are at risk for glucocorticoid-induced osteoporosis and should be monitored carefully.

Integrated Physiological Regulation of Ca++/Pi Metabolism

The integrated response of PTH and 1,25-dihydroxyvitamin D to a hypocalcemic challenge is shown in

Fig. 40.10A . A decrease in serum [Ca++] detected by the CaSR on parathyroid chief cells stimulates secretion of PTH. In the kidney, PTH increases Ca++ reabsorption in the distal tubule and to a lesser extent in the distal thick ascending limb of the loop of Henle. In bone, elevated PTH stimulates osteoblast lineage cells to express RANKL, which increases osteoclast activity and leads to increased bone resorption and release of Ca++ and Pi into blood. PTH stimulates 1α-hydroxylase expression in the proximal renal tubule, thereby increasing 1,25-dihydroxyvitamin D levels. 1,25-Dihydroxyvitamin D stimulates absorption of Ca and Pi in the small intestine and upregulates osteoblast expression of RANKL, thereby amplifying the effect of PTH on bone resorption. In the kidney, PTH inhibits NPT2 in the proximal tubule to lower Pi reabsorption and increase Pi clearance, thereby counterbalancing Pi mobilized from the bone and gut.

Although not as tightly regulated as Ca++ , perturbations in serum Pi will also elicit hormonal responses (see

Fig. 40.10B ). Low serum Pi stimulates production of 1,25-dihydroxyvitamin D in the kidney, which in turn will mobilize Ca and Pi from the intestine. The rise in Ca++ will suppress PTH secretion to prevent hypercalcemia. This drop in PTH will enhance Pi reabsorption in the proximal tubule to help restore serum Pi. Over a longer time course, a decrease in serum Pi will inhibit FGF23 production, which will favor Pi reabsorption in the proximal tubule. These integrated responses will allow correction of hypophosphatemia while maintaining normocalcemia. For hormonal responses to vitamin D deficiency, see the In the Clinic box and

Fig. 40.10C

Primary hyperparathyroidism iscausedbyexcessiveproductionofPTHbytheparathyroidglands.Itismostfrequentlycausedbyasingleadenoma confinedtooneoftheparathyroids.OwingtoelevatedPTH,patientswithprimaryhyperparathyroidismhavehighserum[Ca++]and,inmostcases,lowserum[Pi].Hypercalcemia isaresultofboneresorption,increasedgastrointestinalCaabsorption(mediatedby1,25-dihydroxyvitaminD),andincreasedrenalCa++ reabsorption.Themajorsymptomsofthedisorderarerelatedtoincreasedboneresorption,hypercalcemia,andhypercalciuria. Theseincluderadiographicmanifestationsofexcessiveboneresorptionandpsychologicaldisorders,particularlydepression.Progressiveneurologicalsymptomsincludefatigue,mentalconfusion,andatveryhighlevels(>15mg/dL),coma.Kidneystones(nephrolithiasis) composedofcalciumphosphatearecommonbecausehypercalcemialeadstohypercalciuria,andincreasedPiclearancecausesphosphaturia. Fortunately,routinebloodchemistryscreeningoverthepastseveraldecadeshasresultedinearlierdetectionofprimaryhyperparathyroidism,precludingdevelopmentofseveresymptomsinmostcases.

1. Serum [Ca++] is determined by the rate of Ca 2. Serum [Pi] is determined by the rate of Pi absorption absorption by the gastrointestinal tract, bone by the gastrointestinal tract, soft tissue influx and formation and resorption, and renal excretion. Serum efflux, bone formation and resorption, and renal [Ca++] is normally maintained within a very narrow excretion. Serum [Pi] normally fluctuates over a range. relatively wider range.

3.

The major physiological hormones regulating serum [Ca++] and [Pi] are PTH, 1,25-dihydroxyvitamin D (calcitriol), and FGF23.

4.

Vitamin D is synthesized from 7-dehydrocholesterol in skin in the presence of UVB light or acquired in the diet. It is hydroxylated to 25-hydroxycholecalciferol in the liver and activated by renal 1α-hydroxylase to 1,25-dihydroxyvitamin D.

5.

1,25-Dihydroxyvitamin D promotes intestinal Ca++ absorption and modestly increases Pi absorption.

6.

The flux of Ca++ and Pi into and out of bone is determined by the relative rates of osteoblastic

Bhattacharyya N, et al. Fibroblast growth factor 23: state of the field and future directions. Trends Endocrinol Metab. 2012;23:610-618.

Boyce BF. Advances in the regulation of osteoclasts and osteoclast functions. J Dent Res. 2013;92:860-867.

Christakos S, et al. Vitamin D: metabolism, molecular mechanism of action, and pleiotropic effects. Physiol Rev. 2016;96:365-408.

bone formation and osteoclastic bone resorption.

7.

The PTH/PTHrP receptor is expressed on osteoblasts, not on osteoclasts. PTH has both anabolic and catabolic actions in bone depending on the dose and timing of administration. PTH promotes bone resorption by upregulation of M-CSF and RANKL in osteoblasts.

8.

1,25-Dihydroxyvitamin D binds to the VDR in osteoblasts to support osteoclast differentiation via RANKL and promotes bone mineralization by maintaining appropriate serum [Ca++] and [Pi].

Upon completion of this chapter the student should be able to answer the following questions: 1.

Describe the structure and composition of the pituitary gland and its structural and functional relationship to magnocellular and parvicellular hypothalamic neurons.

2.

Discuss the mechanisms by which the neurohormones antidiuretic hormone (ADH) and oxytocin are synthesized, transported, and released by magnocellular neurons.

3.

Diagram a basic scheme illustrating the components and feedback loops of a typical endocrine axis, including central input, hypothalamic releasing factors, pituitary hormones, and a peripheral endocrine gland. Explain the concept of a set point.

4.

List the endocrine cell types of the adenohypophysis and the tropic hormones they produce, noting hormones that share a common subunit.

5.

Contrast the axes of somatotropes and lactotropes with the classic endocrine axes and explain how they differ.

6.

Discuss the actions of growth hormone (GH) and insulinlike growth factor I (IGF-I) inthe regulation of growth, and the role of growth hormone in the fasted state.

7.

Describe the role of prolactin in the initiation and maintenance of lactation.

he pituitary gland (also called the hypophysis) is a small (≈0.5 g in weight) yet complex endocrine structure at the base of the forebrain (

Fig. 41.1 ). It is composed of an epithelial component called the adenohypophysis and a neural structure called the neurohypophysis. The adenohypophysis is composed of five cell types that secrete six hormones. The neurohypophysis releases several neurohormones. All endocrine functions of the pituitary gland are regulated by the hypothalamus and by negativeand positive-feedback loops.

Microscopic examination of the pituitary reveals two distinct types of tissue: epithelial and neural (

Fig. 41.2 ). The epithelial portion of the human pituitary gland is called the adenohypophysis. The adenohypophysis makes up the anterior portion of the pituitary and is often referred to as the anterior lobe of the pituitary, and its hormones are referred to as anterior pituitary hormones. The adenohypophysis is composed of three parts: (1) the pars distalis, which makes up about 90% of the adenohypophysis, (2) the pars tuberalis, which wraps around the stalk, and (3) the pars intermedia, which regresses and is absent in adult humans.

The neural portion of the pituitary is called the neurohypophysis and represents a downgrowth of the hypothalamus. The most inferior portion of the neurohypophysis is called the pars nervosa, also called the posterior lobe of the pituitary (or simply posterior pituitary). At the superior end of the neurohypophysis, a funnel-shaped swelling called the median eminence develops. The portion of the neurohypophysis that extends from the median eminence down to the pars nervosa is called the infundibulum. The infundibulum and the pars tuberalis make up the pituitary stalk—a physical connection between the hypothalamus and pituitary gland (see

Fig. 41.2).

The pituitary gland (anterior and posterior lobes) is situated within a depression of the sphenoid bone called the sella turcica. Generally, cancers of the pituitary have only one way to expand, which is up into the brain and against the optic chiasma. Thus any increase in size of the pituitary is commonly associated with visual field or visual acuity abnormalities and headaches. The sella turcica is sealed off from the brain by a membrane called the diaphragma sellae.

The Neurohypophysis

The pars nervosa is a neurovascular structure that is the site of neurohormone release adjacent to a rich capillary bed. The peptide hormones that are released are antidiuretic hormone (ADH, or arginine vasopressin) and oxytocin. The cell bodies of the neurons that project to the pars nervosa are located in the supraoptic nuclei (SON) and paraventricular nuclei (PVN) of the hypothalamus (a nucleus refers to a collection of neuronal cell bodies residing within the central nervous system (CNS); a ganglion is a collection of neuronal cell bodies residing outside the CNS). The large cell bodies of these neurons are described as magnocellular, and they project axons down the infundibular stalk as the hypothalamohypophyseal tracts. Individual magnocellular neurons are hormone specific, producing •Fig. 41.1 Cross-sectionalimageoftheheaddemonstratingtheproximityofthehypothalamusandpituitaryglandandtheirconnectionbyaneurohypophyseal(pituitary)stalk.

either ADH or oxytocin. These axons terminate in the pars nervosa (

Fig. 41.3 ). In addition to axonal processes and termini from the SON and PVN, there are glial-like support cells called pituicytes. The posterior pituitary is extensively vascularized and the capillaries are fenestrated, thereby facilitating diffusion of hormones into the systemic circulation.

Synthesis of ADH and Oxytocin

ADH and oxytocin are small peptides (nine amino acids) that differ in only two amino acids, yet they have limited overlapping activity. ADH and oxytocin are synthesized as preprohormones (

Fig. 41.4 ). Each prohormone harbors the structure of oxytocin or ADH and a co-secreted peptide, either neurophysin I (associated with ADH) or neurophysin II (associated with oxytocin). These preprohormones are called preprovasophysin and preprooxyphysin. The N-terminal signal peptide is cleaved as the peptide is transported into the endoplasmic reticulum. In cell bodies within the SON and PVN, the prohormones are packaged in the endoplasmic reticulum and Golgi apparatus in membrane-bound secretory granules (

Fig. 41.5 ). The secretory granules are conveyed through a “fast” (i.e., millimeters per hour) adenosine triphosphate (ATP)-dependent axonal transport mechanism down the infundibular stalk to axonal termini in the pars nervosa. During transit of the secretory granule, the prohormones are proteolytically cleaved to produce equimolar amounts of hormone and neurophysin. Secretory granules containing fully processed peptides are stored in the axonal termini. Expansions of the termini due to the presence of stored secretory granules can be observed by light microscopy and are termed Herring bodies.

ADH and oxytocin are released from the pars nervosa in response to stimuli that are primarily detected at the cell body and its dendrites in the SON and PVN. These stimuli are mainly in the form of neurotransmitters released from hypothalamic interneurons. With sufficient stimulus the neurons will depolarize and propagate an action potential down the axon. At the axonal termini the action potential increases intracellular [Ca++] and results in a stimulus-secretion response, with exocytosis of ADH or oxytocin along with neurophysins into the extracellular fluid of the pars nervosa (see

Fig. 41.5). Hormones and neurophysins enter the peripheral circulation, and both can be measured in blood.

Actions and Regulation of ADH and Oxytocin

ADH acts primarily at the kidney to retain water (antidiuresis). The actions of ADH and regulation of ADH secretion were described in . Oxytocin primarily acts on the pregnant uterus to induce labor and on myoepithelial cells of the breast to promote milk letdown during nursing. The actions and regulation of oxytocin are discussed in

The Adenohypophysis

The pars distalis is composed of five endocrine cell types that produce six hormones (

Table 41.1 ). Because of the histological staining properties of the cell types, the corticotropes, thyrotropes, and gonadotropes are referred to as pituitary basophils, whereas the somatotropes and lactotropes are referred to as pituitary acidophils (see

Fig. 41.2B

Before discussing the individual hormones of the adenohypophysis, it is important to understand the structural •Fig. 41.2 A,Grossstructureofthepituitarygland.Thepituitaryglandisbelowthehypothalamusandisconnectedtoitbythepituitarystalk.Theglandsitswithinthesellaturcica,afossawithinthesphenoidbone,andiscoveredbyaduralreflection,thediaphragmasellae.Theparsdistalismakesupmostoftheanteriorpituitary.B,Theparsdistalisisderivedfromepithelialtissuethatiscomposedofacidophils (A)(somatotropesandlactotropes)andbasophils(B)(thyrotropes,gonadotropes,andcorticotropes).C,Theposteriorpituitaryisderivedfromneuraltissueandhasahistologicalappearanceofnonmyelinatednerves.Cp,chromophobes;H,Herringbodies.(A,ModifiedfromStevensA.In:LoweJS[ed].Human Histology. 3rded.Philadelphia:Elsevier;2005.BandC,FromYoungBetal[eds].Wheater’s Functional Histology. 5thed.Philadelphia:ChurchillLivingstone;2006.) •Fig. 41.3 Magnocellularneuronsofthehypothalamus(paraven-tricularandsupraopticnuclei)projecttheiraxonsdowntheinfundibularprocessandterminateintheparsnervosa(posteriorlobe),wheretheyreleasetheirhormones(eitherADHoroxytocin)intoacapillarybed.(ModifiedfromLarsenPRetal[eds].Williams Textbook of Endocrinol-ogy. 10thed.Philadelphia:Saunders;2003.)Capilary bed Anterior lobe of pituitary Optic chiasm Hypothalamus Posterior lobe of pituitary Magnocellular neurosecretory cells

Becauseposteriorpituitaryhormonesaresynthesizedinthehypothalamusratherthanthepituitary,hypophysectomy (pituitaryremoval)doesnotnecessarilypermanentlydisruptsynthesisandsecretionofthesehormones.Immediatelyafterhypophysectomy,secretionofthehormonesdecreases.However,overaperiodofweekstheseveredproximalendofthetractwillshowhistologicalmodificationandpituicyteswillformaroundtheneuronterminals.Secretoryvacuolesareseen,andsecretionofhormoneresumesfromthisproximalend.Secretionofhormonecanevenpotentiallyreturntonormallevels.Incontrast,alesionhigheruponthepituitarystalkcanleadtolossofneuronalcellbodiesinthePVNandSON.

and functional organization of the adenohypophysis in the context of the endocrine axes (

Fig. 41.6 41.1 ). Each endocrine axis is composed of three levels of endocrine cells: (1) hypothalamic neurons, (2) anterior pituitary cells, and (3) peripheral endocrine glands. Hypothalamic neurons release specific hypothalamic releasing hormones (designated XRH in this generic scheme) that stimulate secretion of specific pituitary tropic hormones (XTH). In some cases, production of a pituitary tropic hormone is secondarily regulated by a release-inhibiting hormone (XIH). Pituitary tropic hormones then act on specific peripheral target endocrine glands and stimulate them to release peripheral hormones (X). The peripheral hormone X has two general functions: it regulates several aspects of human physiology, and it negatively feeds back on the pituitary gland and hypothalamus to inhibit production and secretion of tropic hormones and releasing hormones, respectively (see

Fig. 41.6

Significantprogresshasbeenmadeinunderstandingthedifferentiationofthefiveendocrinecellsoftheparsdistalisfromoneprecursorcell.ThehomeodomaintranscriptionfactorPROP-1 isexpressedsoonafterRathke’spouch(embryologicalprecursoroftheadenohypophysis)formsandpromotesthecelllineagesofsomatotropes,lactotropes,thyrotropes,andgonadotropes.Inhumans,raremutationsinthePROP1 generesultinatypeofcombined pituitary hormone deficiency. TheseindividualsdisplaydwarfismduetolackofGH,cognitivedeficitssecondarytohypothyroidism,andinfertilityduetolackofgonadotropins.Asubsequentlyexpressedpituitary-specifichomeodomaintranscriptionfactorcalledPOU1F1 (formerlyknownasPit-1)isrequiredfordifferentiationofthyrotropes,somatotropes,andlactotropes,anditdirectlystimulatestranscriptionandexpressionofTSH,GH,andprolactin.AffectedindividualswithPOU1F1 mutationshavedwarfismandintellectualdisability.Thenuclearhormonereceptor–relatedtranscriptionfactorsteroidogenic factor-1 (SF-1) wasoriginallyidentifiedintheadrenalcortexandgonadsasaregulatorofsteroidogenicenzymegeneexpression.SF-1isalsoexpressedinGnRHneuronsinthehypothalamusandinpituitarygonadotropes,whereitregulatestranscriptionofLHandFSH.MutationsintheSF1 genedisruptadrenalandgonadalfunction,includinglossofgonadotropesinthepituitarygland.TPIT isatranscriptionfactorinvolvedinthedifferentiationofcorticotropes.TPIT,actingwithothertranscriptionfactors,promotesdifferentiationofcorticotropesandexpressionofthePOMC gene(seeCorticotropessection).MutationsinthehumanTPIT generesultinisolated ACTH deficiency. Thisresultsinaformofsecondary adrenal insufficiency thatrequireslifelongreplacementwithglucocorticoids(see

The hypothalamic regulation of anterior pituitary function is neurohormonal. An area of the hypothalamus collectively referred to as the hypophysiotropic (i.e., stimulatory to the hypophysis) region contains nuclei composed of small, or parvocellular, cell bodies that project axons to the

Transcription, excision, splicing •Fig. 41.4 Synthesisandprocessingofpreprovasopressinorpreprooxytocin.

median eminence. They are distinct from the magnocellular neurons of the PVN and SON that project to the pars nervosa. Parvocellular neurons secrete releasing hormones from their axonal termini at the median eminence (Fig.

41.7 ). The releasing hormones enter a primary plexus of fenestrated capillaries and are then conveyed to a second capillary plexus located in the pars distalis by the hypothalamohypophyseal portal vessels (a portal vessel is defined as a vessel that begins and ends in capillaries without going through the heart). At the secondary capillary plexus, the releasing hormones diffuse out of the vasculature and bind to their cognate receptors on specific cell types within the pars distalis. The neurovascular link (i.e., pituitary stalk) between the hypothalamus and pituitary is somewhat fragile and can be disrupted by physical trauma, surgery, or hypothalamic disease. Damage to the stalk and subsequent functional isolation of the anterior pituitary result in a decline in all anterior pituitary tropic hormones except prolactin (discussed later).

The cells of the adenohypophysis make up the intermediate level of the endocrine axes. The adenohypophysis secretes protein hormones that are referred to as tropic hormones—adrenocorticotropic hormone (ACTH, also called corticotropin), thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), growth hormone (GH), and prolactin (PRL) (see

Table 41.1). With a few exceptions, tropic hormones bind to their cognate receptors on peripheral endocrine glands. Because of this arrangement, pituitary tropic hormones generally do not directly regulate physiological responses (see

Chapter 38).

The endocrine axes have the following important features: 1.

The activity of a specific axis is normally maintained at a set point, which varies from individual to individual, usually within a normal range. The set point is determined by the integration of hypothalamic stimulation and peripheral hormone negative feedback. Importantly, negative feedback generally is not exerted by the physiological responses regulated by a specific endocrine axis but by the peripheral hormone itself acting on the pituitary and hypothalamus (see

Fig. 41.6 ). Thus if the level of a peripheral hormone drops, secretion of hypothalamic releasing hormones and pituitary tropic hormones will increase. As the level of peripheral hormone rises, the hypothalamus and pituitary will decrease secretion because of negative feedback. Although certain nonendocrine physiological parameters (e.g., acute hypoglycemia) can regulate some endocrine axes, the axes function semiautonomously with respect to the physiological changes they produce. This configuration means a peripheral hormone (e.g., thyroid hormone) can regulate multiple organ systems without these organ systems exerting competing negative-feedback regulation of the hormone. Clinically this partial autonomy means that multiple aspects of a patient’s physiology are at the mercy of whatever derangements might exist within a specific axis.

2.

Hypothalamic hypophysiotropic neurons are often secreted in a pulsatile manner and are entrained to daily and seasonal rhythms through CNS input. Additionally, hypothalamic nuclei receive a variety of neuronal inputs from higher and lower levels of the brain. These can be • Fig. 41.5 Synthesis, processing, and transport of preprovasopressin. Human ADH (also called arginine vasopressin [AVP]) is synthesized in the hypothalamic magnocellular cell bodies and packaged into neurosecretory granules. During intraaxonal transport of the granules down the infundibular process to the pars nervosa, provasopressin is proteolytically cleaved into the active hormone (AVP = ADH), neurophysin (NP), and a C-terminal glycoprotein (GP). NP arranges into tetramers that bind five AVP molecules. All three fragments are secreted from axonal termini in the pars nervosa (posterior pituitary) and enter the systemic blood. Only AVP (ADH) is biologically active. (Modified from Larsen PR et al [eds]. Williams Textbook of Endocrinology. 10th ed. Philadelphia: Saunders; 2003.)

X Primary Endocrine Disease (Thyroid, adrenal cortex, gonads, liver) •Fig. 41.6 Negative-feedbackloopsregulatinghormonesecretioninatypicalhypothalamus-pituitaryperipheralglandaxis.X,peripheralglandhormone;XIH,hypothalamic-inhibitinghormone;XRH,hypothalamic-releasinghormone;XTH,pituitarytropichormone.

short-term (e.g., various stress/infections) or long-term (e.g., onset of reproductive function at puberty) inputs. Thus inclusion of the hypothalamus in an endocrine axis allows integration of a considerable amount of information for setting or changing the set point of that axis. Clinically this means a broad range of complex neurogenic states can alter pituitary function. Psychosocial dwarfism is a striking example in which children subject to abuse or intense emotional stress have lower growth rates as a result of decreased growth hormone secretion by the pituitary gland.

3. Abnormally low or high levels of a peripheral hormone (e.g., thyroid hormone) may be due to a defect at the level of the peripheral endocrine gland (e.g., thyroid), the pituitary gland, or the hypothalamus. Such lesions are referred to as primary, secondary, and tertiary endocrine disorders, respectively (see

Fig. 41.6 ). A thorough understanding of the feedback relationships within an axis allows the physician to determine where the defect lies. Primary endocrine deficiencies tend to be the most severe because they often involve complete absence of the peripheral hormone.

Endocrine Function of the Adenohypophysis

The adenohypophysis consists of the following endocrine cell types: corticotropes, thyrotropes, gonadotropes, somatotropes, and lactotropes (see

Table 41.1

Corticotropes stimulate the adrenal cortex as part of the hypothalamic-pituitary-adrenal (HPA) axis. Corticotropes produce the hormone ACTH (corticotropin), which stimulates two zones of the adrenal cortex (see ). ACTH is a 39–amino acid peptide that is synthesized as part of a larger prohormone called proopiomelanocortin (POMC). Thus corticotropes are also referred to as POMC cells. POMC harbors the peptide sequence for ACTH, two isoforms of melanocyte-stimulating hormone (MSH), endorphins (endogenous opioids), and enkephalins (

Fig.

41.8 ). However, the human corticotrope expresses only pro-hormone convertase-1, which produces ACTH as the sole active hormone secreted by these cells. The other fragments that are cleaved from POMC are the N-terminal fragment and β-lipotropic hormone (β-LPH), neither of which plays a physiological role in humans.

740 SECTION8Berne & Levy Physiology •Fig. 41.7 Neurovascularlinkbetweenthehypothalamusandtheanteriorlobe(parsdistalis)ofthepitu-itary.Parvicellular“hypophysiotropic”neurosecretoryneuronswithinvarioushypothalamicnucleiprojectaxonstothemedianeminence,wheretheysecretereleasinghormones(RHs).RHsflowdownthepituitarystalkinthehypothalamohypophysealportalvesselstotheanteriorpituitary.RHs(andrelease-inhibitinghormones[seetext])regulatesecretionoftropichormonesfromthefivecelltypesoftheanteriorpituitary.(FromLarsenPRetal[eds].Williams Textbook of Endocrinology. 10thed.Philadelphia:Saunders;2003.)Hormone-secreting cells Anterior lobe of pituitary Capillary beds Hypothalamohypophyseal portal vessels Hypothalamus Median eminence Parvocellular neurosecretory cells Hypophysiotropic hormones released Hormone transport in axons Hormone transport in blood Hormone transport in blood Stimulation or inhibition of anterior pituitary hormone release Action on endocrine glands

ACTH circulates as an unbound hormone and has a short half-life of about 10 minutes. It binds to the melanocortin-2 receptor (MC2R) on cells in the adrenal cortex (

Fig. 41.9 ). ACTH acutely increases cortisol and adrenal androgen production by increasing expression of steroidogenic enzyme genes. In the long term, ACTH promotes growth and survival of two zones within the adrenal cortex (see

ACTH is under stimulatory control by the hypothalamus. A subset of parvocellular hypothalamic neurons expresses the peptide procorticotropin-releasing hormone (pro-CRH) (see

Table 41.1).

Pro-CRH is processed to CRH, an amidated 41–amino acid peptide. CRH acutely stimulates ACTH secretion and increases transcription of the POMC gene. The parvocellular neurons that express CRH also express ADH, which potentiates the action of CRH on corticotropes. ACTH secretion has a pronounced diurnal pattern, with a peak in early morning and a nadir

Atsupraphysiologicallevels,ACTH causesdarkeningoftheskin(e.g.,inCushing’sdisease).KeratinocytesinthebasallayeroftheepidermisalsoexpressthePOMCgenebutprocessittoα-MSH insteadofACTH.Keratinocytessecreteα-MSHinresponsetoultravioletlight,andα-MSHactsasaparacrinefactoronneighboringmelanocytestodarkentheskin.α-MSHbindstotheMC1R onmelanocytes.Atveryhighlevels,ACTHcancross-reactwiththeMC1Rreceptoronskinmelanocytes(see

Fig.41.9 ).ThusincreasedskinpigmentationisoneindicatorofexcesscirculatingACTH.

Fig. 41.10).

In addition, secretion of CRH—and hence secretion of ACTH—is pulsatile.

There are multiple regulators of the HPA axis, and many of them are mediated through the CNS (

Fig. 41.11). Many types of stress, both neurogenic (e.g., fear) and systemic

TABLE 41.1

Adenohypophysis Cell Types: Hormonal Production and Action, Hypothalamic Regulation, and Feedback Regulation •Fig. 41.8 Theoriginalgenetranscriptofproopiomelanocortincontainsstructuresofmultiplebioactivecompounds.ACTH,adrenocorticotropichormone;CLIP,corticotropin-likeintermediatepeptide;MSH,melanocyte-stimulatinghormone.NotethatACTHistheonlybioactivepeptidereleasedbythehumancorticotrope.

•Fig. 41.9 NormallevelsofACTHactontheMC2Rtoincreasecortisol.SupraphysiologicallevelsofACTHduetodecreasedcortisolproductionactonboththeMC2RandtheMC1Ronmelanocytesandcauseskindarkening.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)UV light Keratinocytes in skin Melanocytes in skin PKA Pituitary corticotrope POMC expression ACTH secretion ACTH (physiological levels) Cortisol Diverse physiological effects ACTH (supraphysiological levels) In particular ° Blood glucose ˛ Inflammatory response High affinity binding to MC2R Low affinity binding to MC1R • Loss of feedback in Addison’s disease Physiological, high affinity PKA Steroidogenesis Cell growth ˝-MSH ° POMC expression Skin darkening Release of CRH at median eminence CRH neurons Cortisol (−) Cortisol (−) CRH CRH-R1 Adrenal cortex PKA ° Melanin synthesis and dispersal (e.g., infection), stimulate ACTH. The stress effects are mediated through CRH and ADH via the CNS. The response to many forms of severe stress can persist despite negative feedback from high cortisol levels. This means the hypothalamus has the ability to alter the set point of the HPA axis in response to stress. Severe chronic depression can reset the HPA axis as a result of hypersecretion of CRH and is a factor in the development of tertiary hypercortisolism.

Cortisol exerts negative feedback on the pituitary, where it suppresses POMC gene expression and ACTH secretion, and on the hypothalamus, where it decreases pro-CRH gene expression and release of CRH. Because cortisol has profound effects on the immune system (see ), the HPA axis and the immune system are closely coupled. Moreover, cytokines—particularly interleukin (IL)-1, IL-2, and IL-6—stimulate the HPA axis.

Thyrotropes regulate thyroid function by secreting the hormone TSH (thyrotropin) as part of the hypothalamic-pituitary-thyroid axis. TSH is one of three pituitary glycoprotein hormones (see

Table 41.1) that also include

FSH and LH (discussed later). TSH is a heterodimer composed of an α subunit, called the α-glycoprotein subunit (α-GSU), and a β subunit (β-TSH) (

Fig. 41.12 ). The α-GSU is common to TSH, FSH, and LH, whereas the β subunit is hormone specific (i.e., β-TSH, β-FSH, and β-LH are all unique). Glycosylation of the subunits increases their stability in circulation and enhances the affinity and specificity of the hormones for their receptors. The half-lives of TSH, FSH, and LH (and an LH-like placental glycoprotein hormone, human chorionic gonadotropin [hCG]) are relatively long, ranging from tens of minutes to several hours.

TSH binds to the TSH receptor on thyroid follicle cells (see

Chapter 42). As discussed in

Chapter 42, production of thyroid hormones is a complex multistep process, and TSH stimulates essentially every aspect of thyroid function. TSH also has a strong tropic effect and stimulates hypertrophy, hyperplasia, and survival of thyroid epithelial cells. In geographical regions where the availability of iodide is limited (iodide is required for the synthesis of thyroid hormone), TSH levels are elevated because of reduced negative feedback. Elevated TSH levels can produce striking growth of the thyroid, producing a bulge in the neck called a goiter.

•Fig. 41.10 DiurnalpatternofserumACTH.(ModifiedfromPor-terfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)Awake Sleep Serum ACTH level

The pituitary thyrotrope is stimulated by the releasing hormone thyrotropin-releasing hormone (TRH) (see

Table 41.1). TRH, produced by a subset of parvocellular •Fig. 41.11 Hypothalamic-pituitary-adrenalaxisillustratingfactorsregulatingsecretionofcorticotropin-releasinghormone(CRH).ACTH,adrenocorticotropichormone.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)CRH Physical Diurnal rhythms Emotional Others Set point ACTH Corticotroph Long feedback (cortisol) Short feedback (ACTH) Chemical (hypoglycemia) •Fig. 41.12 Pituitaryglycoproteinhormones.hCGismadebytheplacenta(see )andbindstotheLHreceptor.FSH,follicle-stimulatinghormone;hCG,humanchorionicgonadotropin;LH,luteinizinghormone;TSH,thyroid-stimulatinghormone.˜-TSH subunit ˜-FSH subunit ˜-LH subunit ˜-hCG subunit TSH FSH LH hCG TSH receptor FSH receptor LH receptor LH receptor Plus °-glycoprotein subunit (°-GSU)

Ca++ and PKC TSH synthesis TSH secretion TSH PKA • All aspects of thyroid  hormone synthesis and secretion • Cell growth CNS inputs Diurnal rhythm T3 (−) T4, T 3 T3 (−) TRH TRH receptor TSH receptor Pituitary thyrotrope Thyroid epithelial cell •Fig. 41.13 Hypothalamic-pituitary-thyroidaxis.PKA,proteinkinaseA;PKC,proteinkinaseC;T3,triiodothyronine(activeformofthyroidhormone);T4,thyroxine;TRH,thyrotropin-releasinghormone;TSH,thyroid-stimulatinghormone.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.) hypothalamic neurons, is a tripeptide with cyclization of a glutamine at its N-terminus (pyro-Glu) and an amidated C-terminus. TRH is synthesized as a larger prohormone that contains six copies of TRH within its sequence. It binds to the TRH receptor on thyrotropes (Fig. 41.13 ). TRH neurons are regulated by numerous CNS-mediated stimuli, and TRH is released according to a diurnal rhythm (highest during overnight hours, lowest around dinner time). TRH secretion is also regulated by stress, but in contrast to CRH, stress inhibits secretion of TRH. This includes physical stress, starvation, and infection. Triiodothyronine (T3) and thyroxine (T4) (the latter via type 2 deiodinase-mediated conversion to T3; see ) negatively feed back on both pituitary thyrotropes and TRH-producing neurons. Thyroid hormone represses both β-TSH expression and the sensitivity of pituitary thyrotropes to TRH while also inhibiting TRH production and secretion by parvocellular neurons.

Duringembryonicdevelopment,GnRHneuronsmigratetothemediobasalhypothalamusfromthenasalplacode.PatientswithKallmann syndrome havetertiary hypogonadotropic hypogonadism, oftenassociatedwithlossofthesenseofsmell(anosmia).ThisisduetoamutationintheKAL gene, whichresultsinfailureoftheGnRHneuronalprecursorstoproperlymigratetothehypothalamusandestablishaneurovascularlinktotheparsdistalis.

The Gonadotrope

The gonadotrope secretes FSH and LH (collectively called gonadotropins) and regulates the function of gonads in both sexes. As such the gonadotrope plays an integral role in the hypothalamic-pituitary-testis axis and the hypothalamic-pituitary-ovarian axis (

Fig. 41.14

FSH and LH are segregated into different secretory granules and are not co-secreted in equimolar amounts (in contrast to ADH and neurophysin, for example). This allows independent regulation and secretion of FSH/LH by gonadotropes. The actions of FSH and LH on gonadal function are complex, especially in women, and will be discussed in detail in . In general, gonadotropins promote testosterone secretion in men and estrogen and progesterone secretion in women. FSH also increases secretion of a transforming growth factor (TGF)-β–related protein hormone called inhibin in both sexes.

FSH and LH secretion are regulated by one hypothalamic releasing hormone, gonadotropin-releasing hormone (GnRH; formerly called LHRH). GnRH is a 10–amino acid peptide produced by a subset of parvocellular hypothalamic GnRH neurons (see

Fig. 41.14 ). GnRH is produced as a larger prohormone and, as part of its processing to a decapeptide, is modified with a cyclized glutamine (pyro-Glu) at its N-terminus and an amidated C-terminus.

GnRH is released in a pulsatile manner (

Fig. 41.15 ), and both the pulsatile secretion and the frequency of the pulses have important effects on the gonadotrope. Continuous infusion of GnRH downregulates the GnRH receptor, thereby resulting in a decrease in FSH and LH secretion. In contrast, pulsatile secretion does not desensitize the gonadotrope to GnRH, and FSH and LH secretion is normal. At a frequency of one pulse per hour, GnRH preferentially increases LH secretion (

Fig. 41.16 ). At a slower frequency of one pulse per 3 hours, GnRH preferentially increases FSH secretion. Gonadotropins increase sex steroid synthesis (see

Fig. 41.14 ). In men, testosterone and estrogen negatively feed back at the level of the pituitary and the hypothalamus.

Ca++, PKC, other pathways? SlowFast GnRH (+) pulses GnRH pulses LH synthesis and secretion FSH synthesis and secretion FSHLH PKA Inhibin Pulsatile release of GnRH at median eminence PubertyCNS Opioids Stress Prolactin (−)Sex steroids GnRH neurons inputs (+) (−) (−) GnRH Sex steroids GnRH receptor FSH receptorLH receptor Pituitary gonadotrope Gonadal cell types Steroidogenesis Gametogenesis •Fig. 41.14 Hypothalamic-pituitary-gonadalaxis.FSH,follicle-stimulatinghormone;GnRH,gonadotropin-releasinghormone;LH,luteinizinghormone.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine

Physiology. 3rded.Philadelphia:Mosby;2007.)

Exogenous progesterone also inhibits gonadotropin function in men and has been considered as a possible component of a male contraceptive pill. Additionally, inhibin negatively feeds back selectively on FSH secretion in men and women. In women, progesterone and testosterone negatively feed back on gonadotropic function at the level of the hypothalamus and pituitary. At low doses, estrogen also exerts negative feedback on FSH and LH secretion. However, high estrogen levels maintained for 3 days cause a surge in LH and to a lesser extent FSH secretion. This positive feedback, which is critical in promoting ovulation, is observed at the hypothalamus and pituitary. At the hypothalamus, GnRH pulse amplitude and frequency increase. At the pituitary, high estrogen levels greatly increase the sensitivity of the gonadotrope to GnRH, both by increasing GnRH receptor levels and by enhancing postreceptor signaling (see

The Somatotrope

The somatotrope produces GH (somatotropin) and is part of the hypothalamic-pituitary-liver axis (

Fig. 41.17 ). A major target of GH is the liver, where it stimulates production of insulinlike growth factor (IGF)-I. GH is a 191–amino acid protein that is similar to PRL and human placental lactogen (hPL); accordingly there is some overlap in activity among these hormones. Multiple forms of GH 746 SECTION8Berne & Levy Physiology 1.8 1.2 0.6 0 12 10 8 6 1200 1300 1400 1500 1600 Hours GnRH(pg/10 min) LH(ng/mL)

Fig. 41.15 FluctuationofperipheralveinplasmaLHlevelsandportalveinplasmaGnRHlevelsinunanesthetized,ovariectomizedfemalesheep.EachpulseofLHiscoordinatedwithapulseofGnRH.ThissupportstheviewthatpulsatilityofLHreleaseisdependentonpulsatilestimulationofthepituitarybyGnRH.(FromLevineJetal.Endocrinology 1982;111:1449.)

Fig. 41.16 Frequency-encodedregulationofFSHandLHsecretionfromgonadotropes.AhighfrequencyofGnRH(1pulse/hr)preferentiallystimulatesLHsecretion,whereasaslowerfrequencyofGnRHpromotesFSHsecretion.(FromLarsenPRetal[eds].Williams Textbook of Endocrinology. 10th ed.Philadelphia:Saunders;2003.) are present in serum, with the 191–amino acid (22-kDa) form representing approximately 75% of circulating GH. The GH receptor is a member of the cytokine/GH/PRL/ erythropoietin receptor family and as such is linked to the JAK/STAT signaling pathway (see ). Human GH can also act as an agonist of the PRL receptor. About 50% of the 22-kDa form of GH in serum is bound to GH-binding protein (GHBP), which is derived from the N-terminal portion (the extracellular domain) of the GH receptor. Individuals with Laron syndrome, who lack normal GH receptors but have normal GH secretion, do not have detectable GHBP in their serum. GHBP reduces renal clearance and thus increases the biological half-life of GH, which is about 20 minutes. The liver and kidney are major sites of GH degradation.

GH secretion is under dual positive/negative control by the hypothalamus (see

Fig. 41.17 ). The hypothalamus predominantly stimulates GH secretion via the peptide growth •Fig. 41.17 Hypothalamic-pituitary-liveraxis.ALS,acidlabilesubunit;GHBP,growthhormone–bindingprotein;GHRH,growthhormone–releasinghormone;IGFBP,insulinlikegrowthfactor–bindingprotein;IGF-I,insulinlikegrowthfactorI;SS,somatostatin.(FromPorterfieldSP,WhiteBA.Endocrine Physiology.

3rded.Philadelphia:Mosby;2007.) hormone–releasing hormone (GHRH). This hormone is a member of the vasoactive intestinal polypeptide (VIP)/ secretin/glucagon family and is processed from a larger prohormone into a 44–amino acid peptide with an amidated C-terminus. GHRH enhances GH secretion and GH gene expression. The hypothalamus inhibits pituitary GH synthesis and release via the peptide somatostatin. In the anterior pituitary, somatostatin inhibits release of GH and TSH. GH secretion is also stimulated by ghrelin, which acts through the GH secretagogue receptor on somatotropes. Ghrelin is primarily produced by the stomach but is also expressed in the hypothalamus. Ghrelin increases appetite and may serve as a signal to coordinate nutrient acquisition with growth.

The primary negative feedback on the somatotrope is exerted by IGF-I (see

Fig. 41.17). GH stimulates IGF-I pro duction by the liver, and IGF-I then inhibits GH synthesis and secretion by the pituitary and hypothalamus in a classic “long feedback” loop. In addition, GH itself exerts negative feedback on release of GHRH through a “short feedback” loop. GH also increases somatostatin release.

GH secretion, like ACTH, shows prominent diurnal rhythms, with peak secretion occurring in the early morning just before awakening. Its secretion is stimulated during deep slow-wave sleep (stages III and IV). GH secretion is lowest during the day. This rhythm is entrained to sleep-wakefulness patterns rather than light-dark patterns, so a phase shift occurs in people who work night shifts. As is typical of anterior pituitary hormones, GH secretion is pulsatile. Levels of GH in serum vary widely (0–30 ng/ mL, with most values usually falling between 0 and 3). Because of this marked variation and the heterogeneity of circulating GH, measurement of serum GH levels is of limited clinical utility. Since IGF-I secretion is regulated by GH and possesses a longer half-life that buffers pulsatile and diurnal changes in GH secretion, it may be used to assess the status of the GH axis, especially in young patients.

GH secretion is differentially regulated depending on the physiological state. GH is classified as one of the “stress hormones” and is increased by neurogenic and physical stress. It promotes lipolysis, increases protein synthesis, and antagonizes the ability of insulin to reduce blood glucose levels. It is not surprising, therefore, that acute hypoglycemia is a stimulus for GH secretion and GH is classified as a hyperglycemic hormone. A rise in the serum concentration of some amino acids also stimulates GH secretion; administration of arginine is used for provocative testing of GH secretion. In contrast, an increase in blood glucose or free fatty acids inhibits secretion of GH. Obesity also inhibits GH secretion, in part because of insulin resistance (relative hyperglycemia) and increased circulating free fatty acids. Conversely, exercise and starvation stimulate GH secretion.

The lifetime pattern of GH secretion is shown in

Fig.

41.18 . GH secretion increases in the neonatal period as growth becomes GH and IGF-I dependent. It remains high throughout childhood and peaks during puberty, when estrogen (in females and also males via aromatization) promotes even higher rates of GH secretion. Thyroid hormone also enhances GH and IGF-I secretion to support bone growth and maturation. Adults continue to produce GH, consistent with its role in metabolism, before levels fall during senescence.

IGFs are multifunctional hormones that regulate cellular proliferation, differentiation, and metabolism. These protein hormones resemble insulin in structure and function. The two hormones in this family, IGF-I and IGF-II, are produced in many tissues and have autocrine, paracrine, and endocrine actions. IGF-I is the major form produced in most adult tissues. IGF-II is the major form produced in the fetus, where it regulates growth of both the fetus and the placenta in a GH-independent manner. Both hormones are structurally similar to proinsulin, with IGF-I exhibiting 42% structural homology with proinsulin. IGFs and insulin show receptor cross-reactivity; IGFs in high concentration mimic the metabolic actions of insulin. Both IGF-I and IGF-II act through type I IGF receptors, which are similar to insulin and epidermal growth factor (EGF) receptors and contain intrinsic tyrosine kinase activity. However, IGF-II also binds to the type II IGF/mannose-6-phosphate receptor. This receptor does not resemble the insulin receptor, does not have intrinsic tyrosine kinase activity, and probably functions to limit IGF-II signaling through the type I receptor. IGFs stimulate glucose and amino acid uptake and protein and DNA synthesis. They were initially called somatomedins because of their growth-mediating actions on cartilage, bone, and other organs. It was originally proposed that IGF-I is produced exclusively in the liver upon GH stimulation. During puberty, when GH levels increase (

Fig. 41.19 ), IGF-I levels increase in parallel. However, it is now known that IGFs are produced in many extrahepatic tissues, exhibiting both autocrine and paracrine actions. Some of these are under the control of GH, whereas others are not. In bone, for example, IGF-I has both endocrine and paracrine effects on linear growth, some of which are GH independent. Hormones such as parathyroid hormone (PTH) and estradiol are also effective stimuli for IGF-I production by osteoblasts. At the same time, GH exerts stimulatory effects on the growth plate that are independent of IGF-I. The liver appears to be the predominant source of the circulating pool of IGF-I (see

Fig. 41.19 •Fig. 41.18 LifetimepatternofGHsecretion.GHlevelsarehigherinchildrenthaninadults,withapeakperiodduringpuberty.GHsecretiondeclineswithaging.

Essentially all circulating IGFs are transported in serum bound to IGF binding proteins (IGFBP). IGFBP-3 binds to IGF then associates with another protein called the acid labile subunit (ALS) (see

Fig. 41.19 ). GH stimulates hepatic production of IGF-I, IGFBP-3, and ALS. The IGFBP-3/ALS/IGF-I complex mediates transport and bioavailability of IGF-I. Although IGFBPs generally inhibit IGF action, they greatly increase the biological half-life of IGFs (up to 12 hours). IGFBP proteases degrade IGFBP and play a role in locally generating free (i.e., active) IGFs. This is of interest in the context of IGF-responsive cancers (e.g., prostate cancer), which may overexpress one or more IGFBP proteases.

GH plays a dual role in metabolism that is highly dependent on physiological context. At the risk of oversimplification, its dual roles are to: (1) promote growth and protein anabolism when nutritional status is favorable, and (2) switch fuel consumption to lipids, sparing glucose in the fasted state.

•Fig. 41.19 RelationshipofGHandIGF-I.GHhasdirectendocrineactionsongrowthandstimulatestheproductionofIGF-I,IGFBP-3andALSintheliver.CirculatingIGF-Iexertsendocrineactionsontargetorgans.IGF-1isalsoproducedlocallyinbone,whereitexertsparacrineeffects.Part,butnotallofthislocalIGF-IproductionisGH-dependent.

GH acts through a specific GH receptor (GHR) that is a member of the cytokine receptor family. A GHR dimer binds to GH, which triggers activation of the JAK/STAT signaling pathway (see

Fig. 41.17 ). This results in phosphorylation of STAT5b, which translocates to the nucleus to stimulate transcription of GH-responsive genes. Additional signaling pathways activated by GH include MAPK and PI3K among others.

In the fed state, GH is a protein anabolic hormone that increases cellular amino acid uptake and incorporation into protein. Consequently it produces nitrogen retention (positive nitrogen balance) and decreases urea production. The muscle wasting that occurs concomitant with aging has been proposed to be caused at least in part by the decrease in GH secretion that occurs during senescence. In children, GH increases skeletal, muscular, and visceral growth; children without GH show growth stunting or dwarfism. GH promotes cartilage growth and both linear and appositional growth of long bones (

Fig. 41.20 , green arrowheads).

Although GH is an effective stimulator of IGF production, this response requires insulin, which supports GH receptor expression and signaling in hepatocytes. When a balanced supply of nutrients is available, high serum glucose levels stimulate insulin secretion and high serum amino acid levels promote GH secretion (

Fig. 41.21, top ). These conditions are appropriate for growth, and GH in turn stimulates IGF-I production by the liver. IGFs are mitogenic and have profound anabolic effects on many organs and tissues, including muscle, cartilage, and bone. Together, GH and IGF-I promote chondrocyte proliferation, differentiation, and hypertrophy during the process of endochondral ossification (see

Fig. 41.20, green arrowheads). After closure of the epiphyses, longitudinal growth ceases but appositional growth of long bones continues. IGF-I stimulates osteoblast replication and synthesis of collagen and bone matrix. Not surprisingly, serum IGF levels correlate well with growth in children. The role of GH changes with alteration of nutritional status. If the diet is high in calories but low in amino acids, for example, high carbohydrate availability promotes insulin secretion but low serum amino acid levels inhibit GH and IGF production (see

Fig. 41.21, middle). These responses allow dietary carbohydrates and fats to be stored, but conditions are unfavorable for growth.

In the fasted state, on the other hand, when nutrient availability wanes, serum GH levels rise and serum insulin levels fall in response to hypoglycemia (see

Fig. 41.21, bottom). In the absence of insulin, peripheral glucose utilization decreases, thereby conserving glucose for essential tissues such as the brain. In these circumstances the rise in 750 SECTION8Berne & Levy Physiology •Fig. 41.20 BiologicaleffectsofGHandIGF-I.Anabolicgrowth-promotingeffectsthatoccurwhennutritionalstatusisfavorableareindicatedbygreenarrowheads.MetaboliceffectsofGHthatmobilizefatwhilesparingglucoseandproteinduringfastingaredenotedbyredarrowheads.IGFBP,insulinlikegrowthfactor–bindingprotein.Adipose tissue GH Liver Muscle ˜ Lean body mass ˛ Adiposity ˛ Glucose uptake ˜ Lipolysis ˜ RNA synthesis ˜ Protein synthesis ˜ Gluconeogenesis ˜ IGFBP ˜ IGFs ˛ Glucose uptake ˜ Amino acid uptake ˜ Protein synthesis IGF-I fed state fasted state Kidney Pancreas Intestine Islets Parathyroids Skin Connective tissue Bone, heart, lung ˜ Protein synthesis ˜ RNA synthesis ˜ DNA synthesis ˜ Cell size and number ˜ Amino acid uptake ˜ Protein synthesis ˜ RNA synthesis ˜ DNA synthesis ˜ Collagen ˜ Chondroitin sulfate ˜ Cell size and number ˜ Linear growth Chondrocytes ˜ Organ size ˜ Organ function ˜ Caloric mobilization (e.g., lipids) •Fig. 41.21 DifferentialregulationofGH,insulin,andIGF-Isecretioncoordinatesavailabilityofnutrientswithgrowthandproteinanabolism,caloricstorage,orcaloricmobilization(primarilylipids).

GH secretion is beneficial because it shifts metabolism to lipid as an energy source, thereby conserving carbohydrate and protein. This involves coordinated direct actions of GH on the liver, muscle, and adipose tissue (see

Fig. 41.20 , red arrowheads).

GH is a lipolytic hormone. In adipocytes it mobilizes fatty acids and glycerol from triacylglycerol by combined direct and indirect activation of adipocyte lipases. An important indirect action of GH is sensitization of adipocytes to the lipolytic actions of catecholamines, which are also elevated during fasting. Serum fatty acid levels rise as a result of GH action, and more fats are used for energy production. Fatty acid uptake and β-oxidation increase in skeletal muscle and liver. GH can be ketogenic as a result of the increase in fatty acid oxidation when insulin is absent. GH also alters carbohydrate metabolism, causing blood glucose levels to rise. Many of its actions may be secondary to increased fat mobilization and oxidation. For example, an increase in serum free fatty acids inhibits uptake of glucose in skeletal muscle and adipose tissue. The hyperglycemic effects of GH are mild and slower than those of glucagon and epinephrine. Liver glucose output increases, but this is not an effect of GH on glycogenolysis. The increase in fatty acid oxidation and hence the rise in liver acetyl CoA stimulate gluconeogenesis. GH also directly stimulates expression of the gluconeogenic enzyme PEPCK through activation of STAT5b. These actions increase glucose production by the liver from substrates such as lactate and glycerol. The latter is released into the circulation as a result of GH-induced lipolysis in adipocytes.

GH antagonizes the action of insulin at the postreceptor level in skeletal muscle and adipose tissue (but not the liver). Hypophysectomy (removal of the pituitary gland) can improve diabetic management because GH, like cortisol, decreases insulin sensitivity. Because GH produces insulin insensitivity, it is considered a diabetogenic hormone. Therefore when secreted in excess (e.g., in acromegaly), GH can cause diabetes mellitus, and the insulin levels necessary to maintain normal metabolism increase. Excessive insulin secretion resulting from an excess of GH can cause damage to pancreatic beta cells. In the absence of GH, insulin secretion declines. Thus normal levels of GH are required for normal pancreatic function and insulin secretion.

GH deficiency in adults is becoming recognized as a pathological syndrome. If GH deficiency occurs after the epiphyses close, growth is not impaired. GH deficiency is one of many possible causes of hypoglycemia. Recent studies have shown that extended deficiencies of GH lead to changes in body composition. Fat as a percentage of body weight increases, whereas lean body mass declines. In addition, muscle weakness and early exhaustion are symptoms of GH deficiency. There has been interest in using GH in elderly populations to reverse age-related physical decline and body composition, but studies to date have shown small changes in body composition, no functional benefits, and increased risk of adverse events.

GHisnecessaryforgrowthbeforeadulthood.GHdeficienciesresultinseveregrowthdeficits,andexcessesresultingigantism.ExcessGHinadulthoodafterepiphysealclosurecausesacromegaly, characterizedbyinsidiousenlargementofthehandsandfeet,coarseningoffacialfeatures,insulinresistance,anddiabetes.GeneticdisordersoftheGH–IGF-Iaxiscauseseveregrowthimpairment.IdentifiedmutationscausingisolatedGHdeficiencymostcommonlyoccurintheGHandGHRHreceptorgenes.ThesepatientscanbetreatedwithrecombinanthGHtorestorefunctionofthedownstreamaxis.InLaronsyndrome,amutationoftheGHreceptorcausesGHresistance.InthisinstancetheliverdoesnotproduceIGF-I;thisisduetoalackofresponsivenesstoGH.ThesepatientscanbetreatedwithIGF-I,butwithoutthedirectactionsofGH,theeffectivenessofthetreatmentislimited.OtherdownstreamgeneticmutationsthathavebeenreportedincludethoseinSTAT5B,IGF-I,andALS.

The Lactotrope

The lactotrope produces the hormone prolactin, which is a 199–amino acid single-chain protein. PRL is structurally related to GH and hPL (see ). Like GH, the PRL receptor is a member of the cytokine family coupled to the JAK/STAT signaling pathways. Because the primary action of PRL in humans is related to breast development and function during pregnancy and lactation, the regulation and actions of prolactin will be discussed in detail in

In the context of the pituitary gland, it should be appreciated that the lactotrope differs from the other endocrine cell types of the adenohypophysis in two major ways: 1.

The lactotrope is not part of an endocrine axis. This means PRL acts directly on nonendocrine cells (primarily of the breast) to induce physiological changes.

2.

Production and secretion of PRL are predominantly under inhibitory control by the hypothalamus. Thus disruption of the pituitary stalk and the hypothalamohypophyseal portal vessels (e.g., secondary to surgery or physical trauma) results in an increase in PRL levels but a decrease in ACTH, TSH, FSH, LH, and GH. PRL circulates unbound to serum proteins and thus has a relatively short half-life of about 20 minutes. Normal basal serum concentrations are similar in men and women. Release of PRL is normally under tonic inhibition by the hypothalamus. This is exerted by dopaminergic tracts that secrete dopamine in the median eminence. There is also evidence for existence of a prolactin-releasing factor (PRF). The exact nature of this compound is not known, although many factors, including TRH and hormones in the glucagon family (secretin, glucagon, VIP, and gastric inhibitory polypeptide [GIP]) can stimulate release of PRL.

PRL is one of the many hormones released in response to stress. Surgery, fear, stimuli causing arousal, and exercise are

Upon completion of this chapter the student should be able to answer the following questions: 1.

Describe the anatomy and histology of the thyroid gland, including the structure of the thyroid follicle.

2.

Explain how thyroid hormones are synthesized within the thyroid gland, including the processes of iodine uptake, iodination of tyrosine residues in thyroglobulin by thyroid peroxidase/dual oxidase, and coupling to form T4 and T3.

3.

Describe the process of endocytosis by which thyroglobulin is retrieved from the follicle lumen and processed to yield T3 and T4, which are secreted into the circulation.

4.

Diagram the hypothalamic-pituitary-thyroid axis to show how TSH regulates thyroid function and how thyroid hormones feed back to regulate the axis. List examples of how central input can alter the set point of the axis.

5.

Discuss the role of thyroid-binding proteins in the transport and stability of thyroid hormones, and the role of peripheral deiodinases in the activation of T4 to T3 or inactivation to reverse T3. Contrast the cellular location and function of the D1 and D2 deiodinases.

6.

Describe the mechanisms of thyroid hormone action, including the nature and location of the thyroid hormone receptor and its ability to either repress or activate target gene transcription.

7.

Discuss the actions of thyroid hormone during development, especially on the central nervous system (CNS) and skeleton, including the consequences of severe hypothyroidism.

8.

Describe the effects of thyroid hormone on basal metabolic rate and thermogenesis, on the cardiovascular system (heart rate, cardiac output, systemic vascular resistance), and on other organ systems (skin, skeletal muscle, digestive tract).

he thyroid gland produces the prohormone tetraiodothyronine (T4, also called thyroxine) and the active hormone triiodothyronine (T3). Synthesis of T4 and T3 requires iodine, which can be a limiting factor in some parts of the world. Much of T3 is also made by peripheral conversion of T4 to T3. Thyroid hormone acts primarily through a nuclear receptor that regulates gene transcription. T3 is critical for normal brain and bone development and has broad effects on metabolism and cardiovascular function in adults.

Anatomy and Histology of the Thyroid Gland

The thyroid gland is composed of right and left lobes that sit anterolateral to the trachea (

Fig. 42.1). Typically the two lobes are connected by a midventral isthmus. The thyroid gland receives a rich blood supply. It is drained by three sets of veins on each side: the superior, middle, and inferior thyroid veins. The thyroid gland receives sympathetic innervation that is vasomotor but not secretomotor.

The functional unit of the thyroid gland is the thyroid follicle, a spherical structure about 200 to 300 µm in diameter that is surrounded by a single layer of thyroid epithelial cells (

Fig. 42.2 ). The epithelium sits on a basal lamina, the outermost structure of the follicle, and is surrounded by a rich capillary supply. The apical side of the follicular epithelium faces the lumen of the follicle. The follicular lumen itself is filled with colloid, which is composed of thyroglobulin. This large (660 kDa) protein is secreted into the lumen and iodinated by the thyroid epithelial cells, serving as a scaffold for production of thyroid hormones. The size of the epithelial cells and the amount of colloid are dynamic features that change with activity of the gland. The thyroid gland contains another type of cell in addition to follicular cells. Scattered within the gland are parafollicular cells, or C cells, which are the source of the polypeptide hormone calcitonin (see

Production of Thyroid Hormones

The secretory products of the thyroid gland are iodothyronines (

Fig. 42.3 ), a class of hormones formed by the coupling of two iodinated tyrosine molecules. Approximately 90% of the thyroid output is 3,5,3′,5′-tetraiodothyronine (thyroxine, or T4). T4 is primarily a prohormone. About 10% is 3,5,3′-triiodothyronine (T3), which is the active form of thyroid hormone. Less than 1% of thyroid output is 3,3′,5′-triiodothyronine (reverse T3, or rT3), which is inactive. Normally these three products are secreted in the same proportions at which they are stored in the gland.

Because the primary product of the thyroid gland is T4, yet the active form of thyroid hormone is T3, the thyroid axis relies heavily on peripheral conversion through the action of thyronine-specific deiodinases (see

Fig. 42.3 ). Most • Fig. 42.1 A and B, Anatomy of the thyroid gland. C, Image of pertechnetate uptake by a normal thyroid gland. (Modified from Drake RL et al. Gray’s Anatomy for Students. Philadelphia: Churchill Livingstone; 2005.) conversion of T4 to T3 by type 1 deiodinase (D1) occurs in tissues with high blood flow and rapid exchange with plasma, such as the liver and kidneys. This process supplies basal circulating T3 for uptake by other tissues in which local T3 generation is low or absent. D1 is also expressed in the thyroid (again, where T4 is abundant) and has relatively low affinity (i.e., a Km of 1 µM) for T4. Levels of D1 are paradoxically increased in hyperthyroidism and contribute to the elevated circulating T3 levels in this disease.

The brain maintains constant intracellular levels of T3 by a high-affinity deiodinase called type 2 deiodinase (D2) that is expressed in glial cells of the CNS. D2 has a Km of 1 nM and maintains intracellular concentrations of T3 even when circulating T4 falls to low levels. D2 is also present in pituitary thyrotropes. There, D2 acts as a “thyroid axis sensor” that mediates the ability of circulating T4 to feed back on secretion of thyroid stimulating hormone (TSH). Expression of D2 is increased during hypothyroidism, which helps maintain constant T3 levels in the brain.

There is also an “inactivating” deiodinase called type 3 deiodinase (D3). D3 is a high-affinity inner ring deiodinase that converts T4 to the inactive rT3. Type 3 deiodinase is increased during hyperthyroidism, which helps blunt overproduction of T4. All forms of iodothyronines are eventually further deiodinated to noniodinated thyronine.

Because iodide plays a unique role in thyroid physiology, a description of thyroid hormone synthesis requires some understanding of iodide turnover (

Fig. 42.4 ). An average of 400 µg of iodide per person is ingested daily in the United States, versus a minimum daily requirement of 150 µg for adults, 90 to 120 µg for children, and 200 µg for pregnant women. In the steady state, the same amount, 400 µg, is excreted in urine. Iodide is actively concentrated in the thyroid gland, salivary glands, gastric glands, lacrimal glands, mammary glands, and choroid plexus. About 70 to 80 µg of iodide is taken up daily by the thyroid gland from a circulating pool that contains approximately 250 to 750 µg of iodide. The total iodide content of the thyroid gland averages 7500 µg, virtually all of which is in the form of stored iodothyronine in colloid thyroglobulin. In the steady state, 70 to 80 µg of iodide, or about 1% of the total, is released from the gland daily. Of this amount, 75% is secreted as thyroid hormone and the remainder as free iodide. The large ratio (100:1) of iodide stored in the form of hormone to the amount turned over daily protects against iodide deficiency for about 2 months. Iodide is also conserved by a marked reduction in renal excretion of iodide as its concentration in serum falls.

•Fig. 42.2 Histologyofthethyroidglandatlow(upper panel) andhigh(lower panel) magnification.C,colloid;F,thyroidfollicles;S,connectivetissuesepta.(FromYoungBetal.Wheater’s Functional Histology. 5thed.Philadelphia:ChurchillLivingstone;2006.)

CHAPTER 42 The Thyroid Gland

Overview of Thyroid Hormone Synthesis

To understand thyroid hormone synthesis and secretion, one must appreciate the directionality of each process as it relates to the polarized thyroid epithelial cell (

Fig. 42.5 ). Synthesis of thyroid hormone requires two precursors: iodide and thyroglobulin. Iodide is transported across cells from the basal (vascular) side to the apical (follicular luminal) side of the thyroid epithelium. Thyroglobulin is synthesized and secreted across the apical membrane into the follicular lumen. Thus synthesis involves a basal-to-apical movement of these precursors into the follicular lumen (see

Fig. 42.5 ). Actual synthesis of iodothyronines occurs enzymatically within the follicular lumen close to the apical membrane of the epithelial cells (see

Synthesis of Iodothyronines on ). Secretion of thyroid hormone involves endocytosis of iodinated thyroglobulin and apicalto-basal movement of the endocytotic vesicles, which fuse with lysosomes. Thyroglobulin is enzymatically degraded by lysosomal enzymes, resulting in release of thyroid hormones from the thyroglobulin backbone. Finally, thyroid hormones move across the basolateral membrane, probably through a specific transporter, and ultimately into the blood. Thus secretion involves apical-to-basal movement (see

Fig. 42.5

Synthesis of Iodothyronines on a Thyroglobulin Backbone

Iodide is actively transported into the gland against chemical and electrical gradients by a sodium-iodide symporter (NIS) located in the basolateral membrane of thyroid epithelial cells (see

Fig. 42.5 ). NIS is highly expressed in the thyroid gland, but it is also expressed at lower levels in the placenta, salivary glands, and actively lactating breast. One iodide ion is transported uphill against an iodide gradient while two sodium ions move down their electrochemical gradient from extracellular fluid into the thyroid cell. The driving force for this secondary active transporter is provided by plasma membrane Na+,K+-ATPase. Expression of the NIS gene is inhibited by iodide and stimulated by TSH. A reduction in dietary iodide intake depletes the circulating iodide pool and greatly enhances the activity of the iodide trap. When dietary iodide intake is low, the percentage of thyroid uptake of iodide can reach 80% to 90%.

The steps in thyroid hormone synthesis are shown in

Fig. 42.6 . After entering the gland, iodide rapidly moves to the apical plasma membrane of epithelial cells. From there, iodide is transported into the lumen of the follicles by a sodium-independent iodide/chloride transporter called pendrin. Iodide is immediately oxidized and incorporated into tyrosine residues within thyroglobulin (see

Fig. 42.5 ). A single iodination forms a monoiodotyrosine (MIT); a second iodination of the same residue produces diiodotyrosine (DIT) (see

Fig. 42.6 ). After iodination, two DIT molecules are coupled to form T4; one MIT and one DIT are coupled to form T3. Coupling occurs between iodinated tyrosines that remain part of the primary structure of

I I 3,5,3¢ 5¢ -Tetraiodothyronine (thyroxine, or T4) NH2 HO O CH2CHCOOH Outer ring deiodination (activation) Inner ring deiodination Deiodinases type 1 and 2 I I Deiodinases type 3 I 3,5,3¢ -Triiodothyronine (T3) I NH2 CH2CHCOOHO (inactivation) I I 3,3¢ 5¢ -Triiodothyronine (reverse T3) NH2 CH2CHCOOHHO O •Fig. 42.3 StructureoftheiodothyroninesT4,T3,andreverseT3.

•Fig. 42.4 Iodinedistributionandturnoverinhumans.HI,hormone-associatediodine.Stool 10 µg HIUrine 390 µg ITissues Thyroid Extracellular fluid 10 µg HI60 µg HI80 µg I400 µg IDiet 320 µg I20 µg I50 µg I thyroglobulin. This entire sequence of reactions is catalyzed by thyroid peroxidase (TPO), an enzyme complex that spans the apical membrane. The immediate oxidant (electron acceptor) for the reaction is hydrogen peroxide (H2O2). Generation of H2O2 in the follicular lumen is catalyzed by dual oxidases (DUOX1, DUOX2) that are also localized in the apical plasma membrane.

When iodide availability is restricted, formation of T3 is favored. Because T3 is three times as potent as T4, this response provides more active hormone per molecule of organified iodide. The proportion of T3 also increases when the thyroid gland is hyperstimulated by TSH or other activators.

Secretion of Thyroid Hormones

Once thyroglobulin has been iodinated, it is stored in the lumen of the follicle as colloid (see

Fig. 42.2 ). Release of T4 and T3 into the bloodstream is initiated by endocytosis of colloid from the follicular lumen by the processes of macroand micropinocytosis. Endocytotic vesicles then fuse with lysosomes and thyroglobulin is degraded (

Fig.

42.7

Fig. 42.5 ). MIT and DIT molecules, which also are released during proteolysis of thyroglobulin, are rapidly deiodinated within the follicular cell by the enzyme iodotyrosine deiodinase (see

Fig. 42.5 ). This deiodinase is specific for MIT and DIT and cannot use T4 and T3 as substrates. The iodide is then recycled into synthesis of T4 and T3. Amino acids from the digestion of thyroglobulin reenter the intrathyroidal amino acid pool and can be reused for protein synthesis. Only minor amounts of intact thyroglobulin leave the follicular cell under normal circumstances. Enzymatically released T4 and T3 are transported across the basal side of the cell and enter the blood.

•Fig. 42.5 Synthesisandsecretionofthyroidhormonesbythethyroidepithelialcell.

I I I I 3,5,3¢ 5¢ -Tetraiodothyronine (thyroxine, or T4) NH2 CH2CHCOOHHO O I I NH2 CH2CHCOOHHO O I NH2 CH2CHCOOHHO I NH2 CH2CHCOOH + I I DIT DIT NH2 CH2CHCOOH + I I NH2 CH2CHCOOHHO I Diiodotyrosine (DIT) or NH2 CH2CHCOOHHO Monoiodotyrosine (MIT) NH2 CH2CHCOOHHO Tyrosine NH2 CH2CHCOOHI2 + HO

DIT MIT 3,5,3¢ -Triiodothyronine (T3) •Fig. 42.6 Reactionsinvolvedinthegenerationofiodide,MIT,DIT,T3,andT4.

Transport and Metabolism tissues in addition to exerting negative feedback on the pituitary and hypothalamus. The major binding protein is thyroxine-binding globulin (TBG), which is synthesized Secreted T4 and T3 circulate in the bloodstream almost in the liver and binds one molecule of T4 or T3. About 70% entirely bound to proteins. Normally only about 0.03% of circulating T4 and T3 is bound to TBG; 10% to 15% of total plasma T4 and 0.3% of total plasma T3 exist in is bound to another specific thyroid-binding protein called the free state (

Fig. 42.8 ). Free T3 is biologically active and transthyretin (TTR). Albumin binds 15% to 20%, and mediates the effects of thyroid hormone on peripheral 3% is bound to lipoproteins. Ordinarily only alterations in ).CirculatingthyroidhormonesfeedbackonthepituitaryglandtodecreaseTSHsecretion,primarilybyrepressingTSHβ subunitgeneexpression.Thepituitaryglandexpressesthehigh-affinityD2,whichconvertsT4enteringthesecellstoT3.ThusfeedbackinthyrotropesmediatedbyintracellularT3representsanintegratedmeasureofcirculatingfreeT4andT3(see ).BecausethediurnalvariationinTSHsecretionissmall,thyroidhormonesecretionandplasmaconcentrationsarerelativelyconstant.OnlysmallnocturnalincreasesinsecretionofTSHandreleaseofT4occur.Thyroidhormonesalsofeedbackonhypothalamicthyroid-releasinghormone(TRH)secretingneurons.Intheseneurons,T3inhibitsexpressionoftheprepro-TRHgene.

Autoregulationofthyroidglandfunctioniscausedbyiodideitself,whichhasabiphasicaction.Atrelativelylowlevelsofiodideintake,therateofthyroidhormonesynthesisisdirectlyrelatedtotheavailabilityofiodide.However,iftheintakeofiodideexceeds2mg/day,theintraglandularconcentrationofiodidereachesalevelthatparadoxicallysuppressesTPOactivity,blockinghormonebiosynthesis.ThisphenomenonisknownastheWolff-Chaikoff effect. AdaptationtohighiodideintakenormallyoccursbyreducingexpressionofNIS,whichcausestheintrathyroidaliodideleveltofall.TPOactivitythenreturnstonormal,andthyroidhormonesynthesisresumeswithindaystoweeks.Inunusualinstances,failureofNIStodownregulateleadstoprolongedinhibitionofhormonesynthesisbyiodideandresultanthypothyroidism.Thetemporaryreductioninhormonesynthesisbyexcessiodidehasalsobeenusedtherapeuticallyinhyperthyroidism.

Becauseofitsabilitytotrap andincorporateiodineintothyroglobulin(calledorganification),theactivityofthethyroidcanbeassessedbyradioactive iodine uptake (RAIU). Inthistestatracerdoseof123IisadministeredandRAIUismeasuredbyplacingagammadetectorontheneckat4to6hoursandat24hours.IntheUnitedStates,wherethedietisrelativelyrichiniodine,RAIUistypicallyaround15%after6hoursand25%after24hours(

Fig.42.9 ).AbnormallyhighRAIU(>60%)after24hoursindicateshyperthyroidism.AbnormallylowRAIU(<5%)after24hoursindicateshypothyroidism.Inindividualswithextremechronicstimulationofthethyroid(Gravesdisease–associatedthyrotoxicosis),iodideistrapped,organified,andreleasedashormoneveryrapidly.Inthesecasesofelevatedturnover,6-hourRAIUwillbeveryhighbut24-hourRAIUwillbelower(see

Fig.42.8 ).Anumberofanions,suchasthiocyanate(CNS−),perchlorate(HClO4−),andpertechnetate(TcO4−),arecompetitiveornoncompetitiveinhibitorsofiodidetransportviaNIS.Ifiodidecannotberapidlyincorporatedintotyrosine(organification defect) afteritsuptakebythecell,administrationofoneoftheseanionswill,byblockingfurtheriodideuptake,causerapidreleaseofiodidefromthegland(see

Fig.42.9

Thisreleaseoccursasaresultofthehighthyroid-plasmaconcentrationgradient.

Thethyroidcanbeimagedwitharectilinearscannerorgammacameraafteradministrationofatracer,123I,131I,ortheiodine-mimicpertechnetate(99mTc).Imagingcandisplaythesizeandshapeofthethyroid(see

Fig.42.1C )aswellasheterogeneitiesofactiveversusinactivetissuewithinthethyroidgland.Suchheterogeneitiesareoftenduetothedevelopmentofthyroid nodules, whichareregionsofenlargedfollicleswithevidenceofregressivechangesduetocyclesofstimulationandinvolution.“Hot” nodules (i.e.,nodulesthatdisplayhighRAIUonimaging)arenotusuallycancerousbutmayleadtothyrotoxicosis(hyperthyroidism).“Cold” nodules are10timesmorelikelytobecancerous.Suchnodulescanbesampledforpathologicalanalysisbyfine-needle aspiration biopsy.

Thethyroidcanalsobeimagedbyultrasonography, whichissuperiorinresolutiontoRAIUimaging.Ultrasonographyisusedtoguidethephysicianduringfine-needleaspirationbiopsyofanodule.Thehighestresolutionofthethyroidisachievedwithmagnetic resonance imaging (MRI).

A B Colloid in endocytic vesicles Colloid in lumen of thyroid follicle •Fig. 42.7 Before(A)andminutesafter(B)rapidinductionofthy-roglobulinendocytosisbyTSH.(FromWollmanSHetal.J Cell Biol 1964;21:191.)

TBG concentration significantly affect total plasma T4 and T3 levels. Two important biological functions have been ascribed to TBG. First, it maintains a large circulating reservoir of T4 that buffers any acute changes in thyroid gland function. Second, binding of plasma T4 and T3 to proteins prevents loss of these relatively small hormone molecules in urine and thereby helps conserve iodide. TTR transports T4 in cerebrospinal fluid and provides thyroid hormones to the CNS.

Regulation of Thyroid Function

The most important regulator of thyroid gland function and growth is the hypothalamic-pituitary thyroid releasing •Fig. 42.8 TransportofT4andT3inserumbytransportproteinsandpercentagesofboundandfreehormone.Thyroid secretion Protein-Bound T4 (99.98%) & T3 (99.7%) Thyroid-binding globulin (TBG) Transthyretin (TTR) Albumin • Feedback • Activation to T3 • Inactivation • Excretion • Tissue actions • Feedback • Inactivation • Excretion Free T4 (0.02%) & T3 (0.3%) Total T4 8 µg/dL Total T3 0.14 µg/dL Free T4 1.6 ng/dl Free T3 – 0.42 ng/dl T3 T4 123I Uptake (% of dose) •Fig. 42.9 Thyroidglandiodothyronineuptakedefectiveorganificationstates.

). TSH stimulates every aspect of thyroid function. TSH has immediate, intermediate, and long-term actions on the thyroid epithelium. Rapid actions of TSH include pinocytosis of colloid droplets in the cytoplasm, which represent thyroglobulin within endocytic vesicles (see

Fig. 42.7 ). TSH stimulates the proteolysis of thyroglobulin and release of T4 and T3 from the gland. Iodide uptake and TPO activity increase. TSH also stimulates entry of glucose into the hexose monophosphate shunt pathway, which generates the reduced nicotinamide adenine dinucleotide phosphate (NADPH) needed for the peroxidase reaction. Intermediate effects of TSH on the thyroid gland occur after hours to days and involve protein synthesis and expression of numerous genes, including those encoding NIS, thyroglobulin, and TPO. Sustained TSH stimulation leads to the long-term effects of hypertrophy and hyperplasia of follicular cells. Capillaries proliferate and thyroid blood flow increases. These actions, which underlie the growth-promoting effects of TSH on the gland, are supported by local production of growth factors. A noticeably enlarged thyroid gland is called a goiter (

Fig. 42.10 ). Endemic goiter is due to lack of adequate iodine in the diet, which results in low thyroid hormone and elevated TSH levels.

Physiological Effects of Thyroid Hormone

Thyroid hormone acts on essentially all cells and tissues, and imbalances in thyroid function constitute some of the most common endocrine diseases. Thyroid hormone has many direct actions, but it also acts in more subtle ways to optimize the actions of several other hormones and neurotransmitters.

curvesfornormal,hypothyroid,hyperthyroid,and •Fig. 42.10 Thethyroidglandislocatedintheanterioraspectoftheneck,whereitiseasilyvisualizedwhenenlarged(goiter).

˜ Cardiac muscle Myosin heavy chain ˝/˙ ratio Na+, K+-ATPase Sarcoplasma Ca-ATPase ˙-Adrenergic signaling G-protein stimulatory/ •Fig. 42.11 Mechanismsbywhichthyroidhormoneincreasescardiacoutput.Theindirectmechanismsareprobablyquantitativelymoreimportant.

Graves disease isthemostcommonformofhyperthyroidism. Itoccursmostfrequentlybetweentheagesof20and50andis10timesmorecommoninwomenthaninmen.GravesdiseaseisanautoimmunedisorderinwhichactivatingautoantibodiesareproducedagainsttheTSHreceptor.Hyperthyroidismdrivenbytheantibodyisoftenaccompaniedbyadiffusegoiterasaresultofhyperplasiaandhypertrophyofthegland.Thefollicularepithelialcellsbecometallcolumnarcells,andthecolloidshowsascallopedperipheryindicativeofrapidturnover.

TheprimaryclinicalstatefoundinGravesdiseaseisthyrotoxicosis—thestateofexcessivethyroidhormoneinbloodandtissues.Apatientwiththyrotoxicosispresentsoneofthemoststrikingpicturesinclinicalmedicine.Thelargeincreaseinmetabolicrateismanifestedasweightlossdespiteincreasedfoodintake.Excessheatproductioncausesdiscomfortinwarmenvironments,sweating,andgreaterintakeofwater.Theincreaseinadrenergicactivityproducesarapidheartrate,hyperkinesis,tremor,nervousness,andawide-eyedstare.Weaknessiscausedbyalossofmusclemassaswellasimpairedmusclefunction.Othersymptomsincludealabileemotionalstate,breathlessnessduringexercise,anddifficultyswallowingorbreathingbecauseofcompressionoftheesophagusortracheabytheenlargedthyroidgland.Themostcommoncardiovascularsignissinustachycardia.There

Perhaps the most clinically important actions of thyroid hormone are those on cardiovascular physiology. T3 increases cardiac output, thereby ensuring sufficient O2 delivery to tissues (

Fig. 42.11). The resting heart rate and stroke volume are increased. The speed and force of myocardial contractions are enhanced (positive chronotropic and inotropic effects, respectively), and the diastolic relaxation isincreasedcardiacoutputassociatedwithawidenedpulsepressuresecondarytoapositiveinotropiceffectcoupledwithdecreasedsystemicvascularresistance.AmajorclinicalsigninGravesdiseaseisexophthalmos (abnormalprotrusionoftheeyeball)andperiorbital edema. ThisiscausedbyautoantibodybindingtotheTSHreceptorexpressedonorbitalfibrocytes,leadingtoproductionofinflammatorycytokines.

GravesdiseaseisusuallydiagnosedbyelevatedserumfreeandtotalT4andT3andtheclinicalsignsofdiffusegoiterandophthalmopathy.SerumTSHlevelsarelowbecausethehypothalamusandpituitaryareinhibitedbythehighlevelsofT4andT3.Inmostcases,radioiodineuptakebythethyroidisexcessiveanddiffuse.AssayofTSHlevelsandthepresenceofcirculatingthyroidstimulatingimmunoglobulinwilldistinguishGravesdisease(aprimarydisorder)fromarareadenomaofpituitarythyrotrophs(asecondarydisorder)thatproduceshighlevelsofTSH.

TreatmentofGravesdiseaseisusuallyremovalofthethyroidtissue,followedbylifelongreplacementtherapywithT4.Thyroidtissuecanberemovedeitherbyradioablationwith131Iorbysurgery.Withsurgicalremovalofthegland,precautionsmustbetakentoavoidamassive,potentiallylife-threateningreleaseofthyroidhormonesknownasthyroid storm. Analternativetoremovalofthyroidtissueisadministrationofantithyroid drugs thatinhibitTPOactivity.

time is shortened (positive lusitropic effect). Systolic blood pressure is modestly augmented and diastolic blood pressure is decreased. The resultant widened pulse pressure reflects the combined effects of the increased stroke volume and the reduction in systemic vascular resistance secondary to blood vessel dilation in skin, muscle, and heart. These effects in turn are partly due to the increase in tissue production of heat and CO2 that thyroid hormone induces (see on Basal Metabolic Rate and Thermogenesis). In addition, however, thyroid hormone decreases systemic resistance by dilating arterioles in the peripheral circulation. Total blood volume is increased by activation of the renin-angiotensinaldosterone axis, thereby increasing renal tubular sodium reabsorption (see

The cardiac inotropic effects of T3 are both direct and indirect. The latter are due primarily to enhanced responsiveness to catecholamines (see ). Direct inotropic effects (see

Fig. 42.11 ) involve regulation of multiple proteins that enhance contractility, including increased α-myosin heavy chain expression and inhibition of the plasma membrane Na+/Ca++ exchanger. Sarcoplasmic reticulum Ca++-ATPase (SERCA) is increased by T3, whereas phospholamban is decreased. As a result, sequestration of calcium during diastole is enhanced and the relaxation time is shortened. Increased ryanodine Ca++ channels in the sarcoplasmic reticulum promote release of Ca++ from the sarcoplasmic reticulum during systole.

Thyroidhormonelevelsinthenormalrangearenecessaryforoptimumcardiacperformance.Adeficiencyofthyroidhormoneinhumansreducesstrokevolume,leftventricularejectionfraction,cardiacoutput,andtheefficiencyofcardiacfunction.Thelatterdefectisshownbythefactthatthestrokeworkindex[(strokevolume/leftventricularmass)× peaksystolicbloodpressure]isdecreasedtoagreaterextentthanmyocardialoxidativemetabolism.Theriseinsystemicvascularresistancemaycontributetothiscardiacdebility.Incontrast,excessthyroidhormoneenhancescardiacoutputbyincreasingbothheartrateandstrokevolume.Pulsepressureiswidenedbyincreasedsystolicpressureanddecreaseddiastolicpressureduetodecreasedsystemicvascularresistance.Thyrotoxicosisisassociatedwithpalpitations,atrialfibrillation,andmitralvalveprolapse(see

Increased O2 use ultimately depends on an increased supply of substrates for oxidation. T3 augments glucose absorption from the gastrointestinal tract and increases glucose turnover (glucose uptake, oxidation, and synthesis). In adipose tissue, thyroid hormone induces enzymes for the synthesis of fatty acids, including acetyl-CoA carboxylase and fatty acid synthase, and enhances lipolysis by increasing the number of β-adrenergic receptors (see the Autonomic Nervous System ). Thyroid hormone also enhances the clearance of chylomicrons. Thus lipid turnover (free fatty acid release from adipose tissue and oxidation) is augmented.

Protein turnover (release of muscle amino acids, protein degradation, and to a lesser extent protein synthesis and urea formation) is also increased. T3 potentiates the respective stimulatory effects of epinephrine, norepinephrine, glucagon, cortisol, and growth hormone on gluconeogenesis,

CHAPTER 42 The Thyroid Gland lipolysis, ketogenesis, and proteolysis of the labile protein pool. The overall metabolic effect of thyroid hormone has been aptly described as accelerating the physiological response to starvation. In addition, thyroid hormone stimulates synthesis of bile acids from cholesterol and promotes biliary secretion. The net effect is a decrease in the body pool and plasma levels of total and low-density lipoprotein cholesterol. Metabolic clearance of adrenal and gonadal steroid hormones, some B vitamins, and certain administered drugs is also increased by thyroid hormone.

Thyroid hormones stimulate thermogenesis by affecting both adenosine triphosphate (ATP) utilization and the efficiency of ATP synthesis. ATP utilization is enhanced by upregulation of several energy-dependent processes, including Na+,K+-ATPase and SERCA, particularly in skeletal muscle, where calcium cycling between the cytoplasm and sarcoplasmic reticulum uses ATP and generates heat. Recently it has been demonstrated that brown fat in humans, once thought to be important only in neonates, appears to play a role in facultative thermogenesis in adults. Imaging studies have demonstrated the presence of brown fat in the mediastinum, particularly in lean individuals, and metabolic activity in brown fat is enhanced by exposure to cold. Brown fat expresses uncoupling protein-1 (UCP1), also called thermogenin, which causes the proton gradient across the inner mitochondrial membrane to be dissipated as heat, which is then disseminated to the rest of the body by the circulation. UCP1 is regulated by thyroid hormone, and brown fat expresses D2, providing intracellular conversion of T4 to T3. Brown fat thermogenesis involves a synergistic interaction between thyroid hormones and the sympathetic nervous system. Catecholamines promote lipolysis and upregulate expression of D2. T3 in turn upregulates adrenergic receptors and enhances catecholamine responsiveness. Hyperthyroidism is accompanied by heat intolerance, whereas hypothyroidism is accompanied by cold intolerance.

Thyroid hormone stimulates O2 utilization and enhances O2 delivery. Appropriately, T3 increases the resting respiratory rate, minute ventilation, and the ventilatory response to hypercapnia and hypoxia. These actions maintain a normal arterial PO2 when O2 utilization is increased and a normal PCO2 when CO2 production is increased. Additionally the hematocrit increases slightly to enhance O2-carrying capacity. This increase results from stimulation of erythropoietin production by the kidney.

Normal function of skeletal muscles also requires optimal amounts of thyroid hormone. This requirement may be related to regulation of energy production and storage. Glycolysis and glycogenolysis are increased, whereas glycogen and creatine phosphate are reduced by thyroid hormone excess. The inability of muscle to take up and phosphorylate creatine leads to its increased urinary excretion.

Effects on the Autonomic Nervous System and Catecholamine Action

As already mentioned, there is important synergism between catecholamines and thyroid hormones. Thyroid hormones are synergistic with catecholamines in increasing the metabolic rate, heat production, heart rate, motor activity, and excitation of the CNS. T3 may enhance sympathetic nervous system activity by increasing the number of β-adrenergic receptors in heart muscle and the generation of intracellular second messengers such as cyclic adenosine monophosphate (cAMP).

A major effect of thyroid hormone is to promote growth and maturation. A small but crucial amount of thyroid hormone crosses the placenta, and the fetal thyroid axis becomes functional at midgestation. Thyroid hormone is extremely important for normal neurological development and proper bone formation in the fetus. In infants, insufficient fetal thyroid hormone causes congenital hypothyroidism, characterized by irreversible intellectual disability and short stature (see In the Clinic box).

Effects on Bone, Hard Tissue, and Dermis

Thyroid hormone promotes endochondral ossification, linear bone growth, and maturation of the epiphyseal bone centers. T3 enhances maturation and activity of chondrocytes in the cartilage growth plate, in part by increasing local growth factor production and action. During linear postnatal growth, T3 supports the actions of growth hormone, insulinlike growth factor (IGF)-I, and other growth factors. T3 also supports normal adult bone remodeling.

The progression of tooth development and eruption depends on thyroid hormone, as does the normal cycle of growth and maturation of the epidermis, its hair follicles, and nails. The normal degradative processes in these structural and integumentary tissues are stimulated by thyroid hormone. Thus either too much or too little thyroid hormone can lead to hair loss and abnormal nail formation. Thyroid hormone regulates the structure of subcutaneous tissue by inhibiting synthesis and increasing degradation of mucopolysaccharides (glycosaminoglycans) and fibronectin in the extracellular connective tissue (see later description of myxedema).

Effects on the Nervous System

Thyroid hormone regulates the timing and pace of development of the CNS. Thyroid hormone deficiency in utero and in early infancy inhibits growth of the cerebral and cerebellar cortex, proliferation of axons and branching of dendrites, synaptogenesis, myelination, and cell migration. Irreversible CNS impairment results when neonatal thyroid hormone deficiency is not recognized and treated promptly. These morphological defects are paralleled by biochemical abnormalities. Decreased thyroid hormone levels reduce cell size, RNA and protein content, tubulinand microtubuleassociated protein, protein and lipid content of myelin, local production of critical growth factors, and rates of protein synthesis.

Thyroid hormone also enhances wakefulness, alertness, responsiveness to various stimuli, auditory sense, awareness of hunger, memory, and learning capacity. In addition, normal emotional tone depends on proper thyroid hormone availability. Furthermore the speed and amplitude of peripheral nerve reflexes are increased by thyroid hormone, as is motility of the gastrointestinal tract.

In both women and men, thyroid hormone plays an important permissive role in regulation of reproductive function. The normal ovarian cycle of follicular development, maturation, and ovulation, the homologous testicular process of spermatogenesis, and maintenance of the healthy pregnant state are all disrupted by significant deviations in thyroid hormone levels from the normal range. In part these deleterious effects may be caused by alterations in the metabolism or availability of steroid hormones. For example, thyroid hormone stimulates hepatic synthesis and release of sex steroid–binding globulin.

Hypothyroidism referstoinsufficientproductionofthyroidhormonesandcanoccurasprimary,secondary,ortertiaryendocrinedisease(see ).Inprimaryhypothyroidism,T4andT3levelsareabnormallylowandTSHishigh.Insecondaryandtertiaryhypothyroidism,boththyroidhormonesandTSHarelow.TheresponseofTSHlevelstosyntheticTRHcanbeusedtodistinguishbetweenpituitaryandhypothalamicdisease.

Fig.42.12 ]).Affectedindividualshavesevereintellectualdisability,shortstaturewithincompleteskeletaldevelopment,coarsefacialfeatures,andaprotrudingtongue.Themostcommoncauseofhypothyroidisminchildrenworldwideisiodidedeficiency.Historically,iodidedeficiencywasviewedasamajorcauseofhypothyroidismincertainmountainousregionsofSouthAmerica,Africa,andAsia,butrecentevidencesuggeststhattheproblemisevenmorewidespread.Thistragicformofendemic hypothyroidism canbepreventedbypublichealthprogramsthataddiodidetotablesaltorprovideyearlyinjectionsofaslowlyabsorbediodidepreparation.Congenital defects arealesscommoncauseofneonatal/childhypothyroidism.Inmostcasesthethyroidglandsimplydoesnotdevelop(thyroid gland dysgenesis). Lessfrequentcausesofchildhood •Fig. 42.12 A,Normal6-year-oldchild(left) andacongenitallyhypothyroid17-year-old(right) fromthesamevillageinanareaofendemichypothyroidism.Notetheshortstature,obesity,malformedlegs,anddullexpressionoftheintellectuallydisabledhypothyroidchild.Otherfeaturesareaprominentabdomen,aflatbroadnose,ahypoplasticmandible,dryscalyskin,delayedpuberty,andmuscleweakness.Radiographsofthehandcomparinganormal13-year-old(B)tothatofa13-year-oldsufferingfromhypothyroidism(C).Notethatthepatientwithhypothyroidismhasamarkeddelayindevelopmentofthesmallbonesofthehands,ingrowthplatesateitherendofthefingers,andinthegrowthplateofthedistalradius.(A,FromDelangeFM.In:BravermanLE,UtigerRD[eds].Werner and Ingbar’s The Thyroid. 7thed.Philadelphia:Lippincott-Raven;1996.B,FromTannerJMetal.

Assessment of Skeletal Maturity and Prediction of Adult Height (TW2 Method). NewYork:AcademicPress;1975.C,FromAndersenHJ.In:GardnerLI[ed].Endocrine and Genetic Diseases of Childhood and Adolescence. Philadelphia:Saunders;1975.) hypothyroidismaremutationsingenesinvolvedinthyroidhormoneproduction(e.g.NIS,TPO,thyroglobulin,pendrin)orblockingantibodiestotheTSHreceptor.Theseverityoftheneurologicalandskeletaldefectsiscloselylinkedtothetimingofdiagnosisandthyroidhormone(T4)replacement,withearlytreatmentresultinginanormalcognitiveabilityandsubtleneurologicaldeficits.Ontheotherhand,ifhypothyroidismatbirthremainsuntreatedforonly2to4weeks,theCNSwillnotmaturenormallyinthefirstyearoflife.Developmentalmilestonessuchassitting,standing,andwalkingwillbelate,andsevereirreversiblecognitivedeficitscanresult.Hypothyroidbabiesusuallyappearnormalatbirthbecauseofprotectionbymaternalthyroidhormones.Neonatal screening (T4andTSHlevels)hasthereforeplayedacriticalroleindiagnosisandpreventionofcongenitalhypothyroidism.

HypothyroidisminadultswhoarenotiodidedeficientmostoftenresultsfromanotherautoimmunedisorderknownasHashimoto’s disease (formerlycalledlymphocytic thyroiditis).IncontrasttothestimulatoryeffectofautoantibodiesseeninGravesdisease,thyroidautoantibodiesinHashimoto’sdisease(againstTPO,thyroglobulin,orTSHreceptor)causeapoptosisofthyroidcellsanddestructionofthyroidfollicles.Theseantibodiesfixcomplementandpromotelysisofthyroidcells,causingreleaseofthyroglobulinintothecirculation.ThethyroidglandbecomesinfiltratedbybothBandTlymphocytes,whichmaycauseenlargementofthegland.

Othercausesofhypothyroidismincludeiatrogeniccauses(e.g.,radiochemicaldamageorsurgicalremovalfortreatmentofhyperthyroidism),nodulargoiters,andpituitaryorhypothalamicdisease.Treatmentofpatientswiththeantiarrhythmicdrugamiodarone,whichcontainsalargeamountofiodine,maycauseeitherhypo-orhyperthyroidism.Thyroidfunctionmustbecarefullymonitoredinpatientstakingthismedication.

Theclinicalpictureofhypothyroidisminadultsisinmanyrespectstheexactoppositeofthatseeninhyperthyroidism.Thelower-than-normalmetabolicrateleadstoweightgainwithoutanappreciableincreaseincaloricintake.Thedecreasedthermogenesislowersbodytemperatureandcausesintolerancetocold,decreasedsweating,anddryskin.Adrenergicactivityisdecreased,andthereforebradycardiamayoccur.Movement,speech,andthoughtareallslowed,andlethargy,sleepiness,andloweringoftheuppereyelids(ptosis)occur.Accumulationofnegativelychargedmucopolysaccharidesinconnectivetissuesattractssodiumandfluid.Theresultingnonpittingmyxedema producespuffyfeatures,anenlargedtongue,hoarseness,jointstiffness,effusionsinthepleural,pericardial,andperitonealspaces,andpressureonperipheralandcranialnervesentrappedbyexcessgroundsubstance.Constipation,lossofhair,menstrualdysfunction,andanemiaareothersigns.Inadultslackingthyroidhormone,positronemissiontomographydemonstratesageneralizedreductionincerebralbloodflowandglucosemetabolism.Thisabnormalitymayexplainthepsychomotorimpairmentanddepressedaffectofhypothyroidindividuals.

ReplacementtherapywithadailydoseofT4thatnormalizesTSHlevelsisusuallycurativeinadults.Inmostpatients,T3isnotneededbecauseitisgeneratedasneededbyperipheralD1andD2.Furthermore,administrationofT3iscomplicatedbyitshighpotencyandshorthalf-life,requiringfrequentdosingandcausingdifficultyinmaintainingconsistentphysiologicallevelsofT3.

Thyroidhormonealsohassignificanteffectsonotherpartsoftheendocrinesystem.Pituitaryproductionofgrowthhormoneisincreasedbythyroidhormone,whereasthatofprolactinisdecreased.Adrenocorticalsecretionofcortisol(see )aswellasmetabolicclearanceofthishormoneisstimulated,butplasmafreecortisollevelsremainnormal.Theratioofestrogenstoandrogens(see )isincreasedinmen(inwhombreastenlargementmayoccurwithhyperthyroidism).Decreasesinbothparathyroidhormoneand1,25-(OH)2-vitaminDproductionarecompensatoryconsequencesoftheeffectsofthyroidhormoneonboneresorption(see

Chapter40).Kidneysize, renalplasmaflow,glomerularfiltrationrate,andtransportratesforanumberofsubstancesarealsoincreasedbythyroidhormone.

Non-thyroidal illness syndrome (NTIS), also known as euthyroid sick syndrome, occurs frequently in severely ill patients who require hospitalization. NTIS is characterized by decreased levels of both circulating thyroid hormone and TSH caused by CNS-mediated suppression of the hypothalamic-pituitary-thyroid axis. In addition, peripheral metabolism of T4 to inactive rT3 is also increased. A similar pattern is seen upon prolonged fasting. Although it remains incompletely understood, NTIS has been proposed to represent a physiological energy-sparing adaptation to chronic illness or starvation.

Mechanism of Thyroid Hormone Action

Formanyyearsitwasthoughtthatthyroidhormonesdiffusepassivelyacrosscellmembranes,butthisisnowknowntorequiretransportproteins.TheseincludethemonocarboxylatetransportersMCT8andMCT10,whicharecapableoftransportingbothT4andT3acrosstheplasmamembrane(

Fig.

42.13

Fig.42.5 ).Recently,mutationsinMCT8havebeenshowntocauseanX-linkeddevelopmentalsyndromeinhumanscharacterizedbyelevatedT3levels,musclehypoplasia,andsevereneurologicalimpairment.Anothertransporter,OATP1C1,appearstoplayaroleintransportofT4acrosstheblood-brainbarrier.

ManybutnotallT3actionsaremediatedthroughitsbindingtooneofthemembersofthethyroid hormone receptor (TR) family. TheTRfamilybelongstothenuclearhormonereceptorsuperfamilyoftranscriptionfactors(seealso ).Inhumanstherearetwothyroidhormonereceptorgenes,THRA andTHRB, locatedonchromosomes17and3,respectively,thatencodethenuclearthyroidhormonereceptors.THRAencodesTRα, whichisalternativelysplicedtoformtwomainisoforms.TRα1isabonafideTR,whereastheotherisoformdoesnotbindT3.THRBencodesTRβ1 andTRβ2, whicharehigh-affinityreceptorsforT3.ThetissuedistributionofTRα1andTRβ1iswidespread.TRα1isstronglyexpressedincardiacandskeletalmuscle.TRα1istheprimarymediatorofthyroidhormoneactionontheheart.Incontrast,TRβ1isexpressedmainlyinbrain,liver,andkidney.TRβ2expressionisrestrictedtothepituitaryandcriticalareasofthehypothalamus,aswellasthecochleaandretina.T3actingviaTRβ2isresponsibleforinhibitingexpressionoftheprepro-TRHgeneintheparaventricularneuronsofthehypothalamusandtheβ subunitTSHgeneinpituitarythyrotropes.Thusthenegative-feedbackeffectsofthyroidhormoneonbothTRHandTSHsecretionarelargelymediatedbyTRβ2.

•Fig. 42.13 Mechanismsofthyroidhormoneaction,includingtheroleofMCTtransporters,D2deiodinase,andTR-RXRheterodimers.CoA,coactivator;CoR,co-repressor;RXR,retinoidXreceptor.

Fig.42.13 ).UnligandedTR-RXRbindstothyroidresponseelementsintargetgenesandrecruitsco-repressorsthatinhibitgenetranscription.UponT3binding,theco-repressorsarereleased,andcoactivatorsarerecruitedtothehormone-receptorcomplex,inducinggenetranscription.

AnunderstandingofTRsubtypesisimportantbecauseinactivatingTRmutationshavebeenfoundtocausethyroid hormone resistance syndromes.ThemostcommonmutationsoccurintheTRβ2subtype,resultinginincompletenegativefeedbackatthehypothalamic-pituitarylevel.ThusT4levelsareelevated,butTSHisnotsuppressed.Whenresistanceispredominantlyatthehypothalamic-pituitarylevel,thepatientmayexhibitsignsofhyperthyroidismduetoeffectsofelevatedthyroidhormonelevelsonperipheraltissues,particularlyontheheart,mediatedbyTRα1.TRisoformsmayalsoofferpotentialtherapeutictargets.Forexample,researchisunderwaytodevelopTRβ-specificagoniststhathavebeneficialeffectsonlipidandcholesterolmetabolismwithouttheriskofadversecardiovascularsideeffects.

ThereisemergingevidencefornongenomicactionsofT3andT4thataremediatedbyreceptorsactingintheplasmamembrane,mitochondria,orcytoplasm.Insomecasesthesearemodifiedversionsofthenuclearthyroidreceptors.Forexample,truncatedisoformsofTRα1havebeenreportedthatbindT3intheplasmamembranetomediatenongenomiceffectsinboneorthatbindT4inthecytoplasmtoregulatemicrofilamentorganization.Ithasalsobeenreportedthatanintegrin,αvβ3,canactasaT4receptoratthecellsurfacetoregulatecellularproliferationandangiogenesisbyanongenomicmechanism.Theinterplaybetweentheclassicalgenomicandnongenomicactionsofthyroidhormonesislikelytobeanotheractiveareaoffutureresearch.

1.

The thyroid gland is situated in the ventral aspect of the neck and is composed of right and left lobes anterolateral to the trachea and connected by an isthmus.

2.

The thyroid gland is the source of tetraiodothyronine (thyroxine, T4) and triiodothyronine (T3).

3.

The basic endocrine unit in the gland is a follicle that consists of a single spherical layer of epithelial cells surrounding a central lumen that contains colloid or stored hormone.

4.

Iodide is taken up into thyroid cells by a sodium-iodide symporter in the basolateral plasma membrane.

5.

T4 and T3 are synthesized from tyrosine and iodide by the enzyme complex of dual oxidase and thyroid peroxidase. Tyrosine residues in thyroglobulin undergo iodination, after which two iodotyrosine molecules are coupled to yield the iodothyronines.

6.

Secretion of stored T4 and T3 requires retrieval of thyroglobulin from the follicle lumen by endocytosis. Thyroglobulin is then degraded in endolysosomes to liberate T4 and T3. Iodide is conserved by recycling any iodotyrosine molecules that did not undergo coupling within thyroglobulin.

7.

TSH acts on the thyroid gland via its plasma membrane receptor to stimulate all steps in the production of T4 and T3. These steps include iodide uptake, iodination and coupling, and retrieval from thyroglobulin. TSH also stimulates glucose oxidation, protein synthesis, and growth of epithelial cells.

8.

More than 99.5% of T4 and T3 circulates bound to the following proteins: thyroid-binding globulin,

Bernal J, et al. Thyroid hormone transporters–functions and clinical implications. Nat Rev Endocrinol. 2015;11:406-417.

Bianco AC. Minireview: cracking the metabolic code for thyroid hormone signaling. Endocrinology. 2011;152:3306-3311.

transthyretin, and albumin. Only the free fractions of T4 and T3 are biologically active.

9.

T4 functions largely as a prohormone whose disposition is regulated by three types of deiodinases. Monodeiodination of the outer ring yields 75% of the daily production of T3, which is the principal active hormone. Alternatively, monodeiodination of the inner ring yields reverse T3, which is biologically inactive. Proportioning of T4 between T3 and reverse T3 regulates the availability of active thyroid hormone.

10.

Thyroid hormone is a major positive regulator of the basal metabolic rate and thermogenesis. Other important actions of thyroid hormone are increased heart rate, cardiac output, and ventilation and decreased systemic vascular resistance. Substrate mobilization and disposal of metabolic products are enhanced.

11.

Thyroid hormone action on the CNS and skeleton are crucial for normal growth and development. Absence of the hormone causes congenital hypothyroidism, characterized by poor brain development, short stature, and immature skeletal development. In adults, thyroid hormone supports bone remodeling and degradation of skin and hair.

12.

T3 binds to thyroid hormone receptor subtypes responsible for the various actions of thyroid hormone. The thyroid hormone receptor heterodimerizes with RXR to regulate thyroid response elements on target genes, resulting in induction or repression in the presence or absence of T3, respectively.

Brent GA. Mechanisms of thyroid hormone action. J Clin Invest. 2012;122:3035-3043.

de Vries EM, et al. The molecular basis of the non-thyroidal illness syndrome. J Endocrinol. 2015;225:R67-R81.

Krude H, et al. Treatment of congenital thyroid dysfunction: achievements and challenges. Best Pract Res Clin Endocrinol Metab. 2015;29:399-413.

Upon completion of this chapter the student should be able to answer the following questions: 1.

Describe the anatomy and microscopic anatomy of the adrenal gland, including the chromaffin cells of the adrenal medulla and the three zones of the adrenal cortex.

2.

Explain the enzymatic reactions involved in generating norepinephrine and epinephrine and integrate those reactions with the regulation of epinephrine synthesis and secretion by the adrenal medulla.

3.

Utilize the specific actions of catecholamines to explain an overall sympathetic response to a stress imposed on the body.

4.

Describe the first two common reactions of the steroidogenic pathway, and their subcellular locations, and the function of StAR protein in the first reaction.

5.

Compare the steroidogenic pathways within the zona glomerulosa, zona fasciculata, and zona reticularis with respect to common and zona-specific reactions.

6.

Describe the mechanism of action of glucocorticoids and mineralocorticoids, including the cross-reactivity of cortisol with the mineralocorticoid receptor, and the mechanism to prevent this.

7.

Integrate the multiple actions of cortisol throughout the body to explain the hormone’s role during normal development and physiology, and to describe the multiple aspects of the pathophysiology of Addison’s disease and Cushing’s syndrome.

8.

Map out the hypothalamic-pituitary-adrenal axis, including the “loophole” in the feedback mechanisms that leads to excessive androgen production (e.g., in congenital adrenal hyperplasia) in the face of an enzyme deficiency specific to the zona fasciculata and cortisol synthesis.

9.

Review the regulation and actions of aldosterone.

n adults the adrenal glands emerge as fairly complex endocrine structures that produce two structurally distinct classes of hormones: steroids and catecholamines. The catecholamine hormone epinephrine acts as a rapid responder to stresses such as hypoglycemia and exercise to regulate multiple parameters of physiology, including energy metabolism and cardiac output. Stress is also a major secretagogue of the longer-acting steroid hormone cortisol, which regulates glucose utilization, immune and inflammatory homeostasis, and numerous other processes. In addition the adrenal glands regulate salt and volume homeostasis through the steroid hormone aldosterone. Finally, the adrenal gland secretes large amounts of the androgen precursor dehydroepiandrosterone sulfate (DHEAS), which plays a major role in fetoplacental estrogen synthesis and as a substrate for peripheral androgen synthesis in women.

The adrenal glands are bilateral structures located immediately above the kidneys (ad, near; renal, kidney) (

Fig.

43.1 ). In humans they are also referred to as the suprarenal glands because they sit on the superior pole of each kidney. The adrenal glands are similar to the pituitary in that they are derived from both neuronal tissue and epithelial (or epithelial-like) tissue. The outer portion of the adrenal gland, called the adrenal cortex (

Fig. 43.2 ), develops from mesodermal cells in the vicinity of the superior pole of the developing kidney. These cells form cords of epithelial endocrine cells. The cells of the cortex develop into steroidogenic cells (see ). In adults the adrenal cortex is composed of three zones—the zona glomerulosa, the zona fasciculata, and the zona reticularis—that produce mineralocorticoids, glucocorticoids, and adrenal androgens, respectively (see

Fig. 43.2B

Soon after the cortex forms, neural crest–derived cells associated with the sympathetic ganglia, called chromaffin cells, migrate into the cortex and become encapsulated by cortical cells. Thus the chromaffin cells establish the inner portion of the adrenal gland, which is called the adrenal medulla (see

Fig. 43.2). The chromaffin cells of the adrenal medulla have the potential to develop into postganglionic sympathetic neurons. They are innervated by cholinergic preganglionic sympathetic neurons and can synthesize the catecholamine neurotransmitter norepinephrine from tyrosine. However, high levels of cortisol that drain into the medulla from the adrenal cortex induce expression of the enzyme phenylethanolamine N-methyl transferase (PNMT), which transfers a methyl group onto norepinephrine to produce the catecholamine hormone epinephrine, the primary hormonal product of the adrenal medulla (see

Fig. 43.2B).

CHAPTER 43 The Adrenal Gland 767 • Fig. 43.1 The adrenal glands sit on the superior poles of the kidneys and receive a rich arterial supply from the inferior, middle, and superior suprarenal arteries. The adrenals are drained by a single suprarenal vein. (Modified from Drake RL et al. Gray’s Anatomy for Students. Philadelphia: Churchill Livingstone; 2005.)

Instead of being secreted near a target organ and acting as neurotransmitters, adrenomedullary catecholamines are secreted into blood and act as hormones. About 80% of the cells of the adrenal medulla secrete epinephrine, and the remaining 20% secrete norepinephrine. Although circulating epinephrine is derived entirely from the adrenal medulla, only about 30% of the circulating norepinephrine comes from the medulla. The remaining 70% is released from postganglionic sympathetic nerve terminals and diffuses into the vascular system. Because the adrenal medulla is not the sole source of catecholamine production, this tissue is not essential for life.

Synthesis of Epinephrine

The enzymatic steps in epinephrine synthesis are shown in

Fig. 43.4 . Synthesis begins with transport of the amino acid tyrosine into the chromaffin cell cytoplasm and subsequent hydroxylation of tyrosine by the rate-limiting enzyme tyrosine hydroxylase to produce dihydroxyphenylalanine (DOPA). DOPA is converted to dopamine by a cytoplasmic enzyme, aromatic amino acid decarboxylase, and is then transported into the secretory vesicle (also called the chromaffin granule). Within the granule, all dopamine is completely converted to norepinephrine by the enzyme dopamine β-hydroxylase. In most adrenomedullary cells, essentially all of the norepinephrine diffuses out of the chromaffin granule by facilitated transport and is methylated by the cytoplasmic enzyme PNMT to form epinephrine.

The high local concentration of cortisol in the medulla is maintained by the vascular configuration within the adrenal gland. The outer connective tissue capsule of the adrenal gland is penetrated by a rich arterial supply coming from three main arterial branches: the inferior, middle, and superior suprarenal arteries (see

Fig. 43.1 ). These give rise to two types of blood vessels that carry blood from the cortex to the medulla (Fig. 43.3 ): (1) relatively few medullary arterioles, which provide high oxygen- and nutrient-laden blood directly to the medullary chromaffin cells, and (2) relatively numerous cortical sinusoids into which cortical cells secrete steroid hormones (including cortisol). Both vessel types fuse to give rise to the medullary plexus of vessels that ultimately drains into a single suprarenal vein. Thus secretions of the adrenal cortex percolate through the chromaffin cells and bathe them in high concentrations of cortisol before leaving the gland and entering the inferior vena cava. Cortisol inhibits neuronal differentiation of the medullary cells, so they fail to form dendrites and axons. Additionally, cortisol induces expression of the enzyme PNMT, which converts norepinephrine to epinephrine (

Fig. 43.4 ). Glucocorticoid receptor–knockout mice have an enlarged cortex, but the size of the medulla is decreased and PNMT activity is undetectable.

Epinephrine is then transported back into the granule for storage and to undergo regulated exocytosis.

Secretion of epinephrine and norepinephrine from the adrenal medulla is regulated primarily by descending sympathetic signals in response to various forms of stress, including exercise, hypoglycemia, and hemorrhagic hypovolemia •Fig. 43.2 Histologyoftheadrenalgland.A,Lowmagnificationillustratingtheoutercortex(C)andinnermedulla(M;notethecentralvein[V]).B,Highermagnificationclearlyillustratingthezonationofthecortex.Thecorrespondingendocrinefunctionandthedifferentzonesofthecortexandthemedullaarenoted.(FromYoungBetal.Wheater’s Functional Histology. 5thed.Philadelphia:ChurchillLivingstone;2006.)

Fig. 43.5 ). The primary autonomic centers that initiate sympathetic responses reside in the hypothalamus and brainstem, and they receive input from the cerebral cortex, the limbic system, and other regions of the hypothalamus and brainstem.

The chemical signal for secretion of catecholamine from the adrenal medulla is acetylcholine (ACh), which is secreted from preganglionic sympathetic neurons and binds to nicotinic receptors on chromaffin cells (see

Fig.

43.5 ). ACh increases the activity of the rate-limiting enzyme tyrosine hydroxylase in chromaffin cells (see

Fig. 43.4 ). It also increases the activity of dopamine β-hydroxylase and stimulates exocytosis of the chromaffin granules. Synthesis of epinephrine and norepinephrine is closely coupled to secretion so that levels of intracellular catecholamines do not change significantly even in the face of changing sympathetic activity.

Mechanism of Action of Catecholamines

Adrenergic receptors are generally classified as α-and β-adrenergic receptors, with the α-adrenergic receptors • Fig. 43.3 Blood flow through the adrenal gland. Capsular arteries give rise to sinusoidal vessels that carry blood centripetally through the cortex to the medulla. (Modified from Young B et al. Wheater’s Functional Histology. 5th ed. Philadelphia: Churchill Livingstone; 2006.) further divided into α1 and α2 receptors and the β-adrenergic receptors divided into β1, β2, and β3 receptors (

Table 43.1 ). These receptors can be characterized according to: 1.

Relative potency of endogenous and pharmacological agonists and antagonists. Epinephrine and norepinephrine are potent agonists for α receptors and for β1 and β3 receptors, whereas epinephrine is more potent than norepinephrine for β2 receptors. A large number of synthetic selective and nonselective adrenergic agonists and antagonists now exist.

2.

Downstream signaling pathways.

Table 43.1 shows the primary pathways that are coupled to the different adrenergic receptors. This is an oversimplification, because differences in signaling pathways for a given receptor have been linked to the duration of agonist exposure and cell type.

3.

Location and relative density of receptors. Importantly, different receptor types predominate in different tissues. For example, although both α and β receptors are expressed by pancreatic islet beta cells, the predominant response to a sympathetic discharge is mediated by α2 receptors.

Physiological Actions  of Adrenomedullary Catecholamines

Because the adrenal medulla is directly innervated by the autonomic nervous system, adrenomedullary responses are very rapid. Furthermore, because of the involvement of several centers in the central nervous system (CNS), most notably the cerebral cortex, adrenomedullary responses can precede onset of the actual stress (i.e., they can be anticipated) (see

Fig. 43.5 ). In many cases the adrenomedullary output, which is primarily epinephrine, is coordinated with sympathetic nervous activity as determined by the release of norepinephrine from postganglionic sympathetic neurons. However, some stimuli (e.g., hypoglycemia) evoke a stronger adrenomedullary than sympathetic nervous response and vice versa.

Many organs and tissues are affected by a sympathoadrenal response (

Table 43.2). An informative example of the major physiological roles of catecholamines is the sympathoadrenal response to exercise. Exercise is similar to the “fight-or-flight” response but without the subjective element of fear, and it involves a greater adrenomedullary response (i.e., endocrine role of epinephrine) than a sympathetic nervous response (i.e., neurotransmitter role of norepinephrine). The overall goal of the sympathoadrenal system during exercise is to meet the increased energy demands of skeletal and cardiac muscle while maintaining sufficient oxygen and glucose supply to the brain. The response to exercise includes the following major physiological actions of epinephrine (

Fig. 43.6): 1. Increased blood flow to muscles is achieved by the integrated action of norepinephrine and epinephrine on the heart, veins and lymphatics, and nonmuscular (e.g., splanchnic) and muscular arteriolar beds.

•Fig. 43.4 Stepsinsynthesisandsecretionofcatecholaminesfromadrenalmedullarychromaffincells.

2. Epinephrine promotes glycogenolysis in muscle. Exercising muscle can also utilize free fatty acids (FFAs), and epinephrine and norepinephrine promote lipolysis in adipose tissue. Epinephrine increases blood glucose by increasing hepatic glycogenolysis and gluconeogenesis. The promotion of lipolysis in adipose tissue is also coordinated with an epinephrine-induced increase in hepatic ketogenesis. Finally, the effects of catecholamines on metabolism are reinforced by the fact that they stimulate glucagon secretion (β2 receptors) and inhibit insulin secretion (α2 receptors). Efficient production of adenosine triphosphate (ATP) during normal exercise •Fig. 43.5 Stimulithatenhancecatecholaminesecretion.

cAMP,cyclicadenosinemonophosphate;DAG,diacylglycerol.

Arteriolardilation:↓ BP↑ Arteriolarvasoconstriction; (β2)(muscle)↑ BP(α1)(splanchnic,renal,cutaneous,genital)

BP,bloodpressure.

(i.e., a 1-hour workout) also requires efficient exchange of gases with an adequate supply of oxygen to exercising muscle. Catecholamines promote this by relaxation of bronchiolar smooth muscle.

3. Catecholamines decrease energy demand by visceral smooth muscle. In general a sympathoadrenal response decreases overall motility of the smooth muscle in the gastrointestinal (GI) and urinary tracts, thereby conserving energy where it is not immediately needed.

Metabolism of Catecholamines

Two primary enzymes are involved in the degradation of catecholamines: monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). The neurotransmitter norepinephrine is degraded by MAO and COMT after uptake into the presynaptic terminal. This mechanism is also involved in the catabolism of circulating adrenal catecholamines. However, the predominant fate of adrenal catecholamines is methylation by COMT in nonneuronal tissues such as the liver and kidney. Urinary vanillylmandelic acid (VMA) and metanephrine are sometimes used clinically to assess the level of catecholamine production in a patient. Much of the urinary VMA and metanephrine is derived from neuronal rather than adrenal catecholamines.

Pheochromocytoma isatumorofchromaffintissuethatproducesexcessivequantitiesofcatecholamines.Thesearecommonlyadrenalmedullarytumors,buttheycanoccurinotherchromaffincellsoftheautonomicnervoussystem.Althoughpheochromocytomasarenotcommontumors,theyarethemostcommoncauseofhyperfunctioningoftheadrenalmedullary.Thecatecholaminemostfrequentlyelevatedinpheochromocytomaisnorepinephrine.Forunknownreasonsthesymptomsofexcessivecatecholaminesecretionareoftensporadicratherthancontinuous.Symptomsincludehypertension,headaches(fromhypertension),sweating,anxiety,palpitations,andchestpain.Inaddition,patientswiththisdisordermayshoworthostatichypotension(despitethetendencyforhypertension).Thisoccursbecausehypersecretionofcatecholaminescandecreasethepostsynapticresponsetonorepinephrineasaresultofdownregulationofthereceptors(see ).Consequentlythebaroreceptorresponsetobloodshiftsthatoccursonstandingisblunted.

The zona fasciculata produces the glucocorticoid hormone cortisol. This zone is an actively steroidogenic tissue composed of straight cords of large cells. These cells have a “foamy” cytoplasm because they are filled with lipid droplets that represent stored cholesterol esters (CEs). These cells make some cholesterol de novo but import a significant amount of cholesterol from the blood in the form of low-density lipoprotein (LDL). LDL particles bind to their receptor (LDLR) and are endocytosed. Within endolysosomes, free cholesterol (FC) is released from CEs by a lysosomal lipase, and the FC is transported out of the endolysosome by Niemann-Pick C (NPC) proteins. Free cholesterol is stored in lipid droplets in the cytoplasm after esterification by acyl CoA-cholesterol acyltransferase (ACAT) (

Fig. 43.7 ). The stored cholesterol is continually turned back into free cholesterol by hormone-sensitive lipase (HSL), a process that is increased in response to adrenocorticotropic hormone (ACTH; see

Regulation of

All steroid hormone synthesis begins in the mitochondria, where the first enzyme, CYP11A1, is attached to the inner mitochondrial membrane. Although several proteins appear to be involved in the transfer of FC into the inner mitochondrial matrix, one protein called steroidogenic acute regulatory protein (StAR protein) is indispensable in this process (see

Fig. 43.7). StAR protein is short-lived and rapidly activated posttranslationally (phosphorylation) and transcriptionally by pituitary tropic hormones. In patients with inactivating mutations in StAR protein, cells of the zona fasciculata become excessively laden with lipid (“lipoid”) because cholesterol cannot be accessed by CYP11A1 within the mitochondria and used for cortisol •Fig. 43.6 Someoftheindividualactionsofcatecholaminesthatcontributetotheintegratedsympa-thoadrenalresponsetoexercise.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.)Bronchiole (˜2) Dilation ° Exchange of O2 and CO2 ° Cardiac output ° Venous return GI and urinary tracts (˜2) ˝ Motility ˝ Energy usage ° Blood glucose ° Blood ketones ° Blood FFAs ° Blood lactate ° Blood glycerol Adipose (˜2, ˜3) ° Lipolysis ˝ Glucose uptake Liver (˜2) ° Glycogenolysis ° Gluconeogenesis ° Ketogenesis Hormonal reinforcement Skeletal muscle (˜2) ° Glycogenolysis ˝ Glucose uptake ˜ Cells (˙2) ˝ Insulin secretion ˙ Cells (˜2) ° Glucagon secretion ° Blood glucagon/insulin ratio ° Nutrient supply to muscle and adequate supply of oxygen and glucose to brain ˝ Blood flow to GI tract ° Blood flow to skeletal muscle Splanchnic arterioles (˙) Vasoconstriction Skeletal muscle arterioles (˜2) Vasodilation Veins and lymphatics (˙) Vasoconstriction Heart (˜1) (+)ˆve Inotropic (+)ˆve Chronotropic (+)ˆve Lusitropic •Fig. 43.7 Eventsinvolvedinthefirsttworeactionsinthesteroidogenicpathway:conversionofcholesteroltopregnenolone;conversionofpregnenolone(P5)toprogesterone(P4)inzonafasciculatacells.ACAT,acylCoA:cholesterolacyltransferase;3β HSD,3β hydroxysteroiddehydrogenase;CE,cholesterolesters;CYP11A1alsocalledP450 side-chain cleavage enzyme; FC,freecholesterol;HSL,hormone-sensitivelipase;LAL,lysosomalacidhydrolase;LDL,low-densitylipoprotein;LDLR,low-densitylipoproteinreceptor;P5,pregnenolone;StAR,steroidogenicacuteregulatoryprotein.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.) •Fig. 43.8 Summaryofthesteroidogenicpathwaysforeachofthethreezonesoftheadrenalcortex.Theenzymaticreactionsarecolorcodedacrosszones.sER,smoothendoplasmicreticulum.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.) synthesis. Moreover, these individuals cannot synthesize gonadal steroid hormones. The placenta does not express StAR, so these individuals have normal placental steroid production in utero.

Inthezonafasciculata,cholesterolisconvertedsequentially to pregnenolone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, and cortisol (Figs. 43.8 43.9 ). A parallel pathway in the zona fasciculata involves a pathway that bypasses 17-hydroxylation, in which progesterone is converted to 11-deoxycorticosterone (DOC) and then to corticosterone (see

Fig. 43.9C ). This pathway is minor in humans, but in the absence of active CYP11B1 (11-hydroxylase activity), the production of DOC is significant. Because DOC acts as a weak mineralocorticoid ( 43.3 ), elevated levels of DOC cause hypertension.

Transport and Metabolism of Cortisol

Cortisol is transported in blood predominantly bound to corticosteroid-binding globulin [CBG] (also called transcortin), which binds about 90% of cortisol, and albumin, which binds 5% to 7% of cortisol. The liver is the predominant site of steroid inactivation. It both inactivates cortisol and conjugates active and inactive steroids with glucuronide or sulfate so that they can be excreted more readily by the kidney. The circulating half-life of cortisol is about 70 minutes.

Cortisol is reversibly inactivated by conversion to cortisone. This action is catalyzed by the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Inactivation of cortisol by 11β-HSD2 occurs in cells that also express the mineralocorticoid receptor (MR)and are

Steroidogenicenzymesfallintotwosuperfamilies.Mostbelongtothecytochrome P-450 monooxidase gene family andarethusreferredtoasCYPs. Theseenzymesarelocatedeitherintheinnermitochondrialmatrix,wheretheyusemolecularoxygenandaflavoproteinelectrondonor,orinthesmoothendoplasmicreticulum,wheretheyuseadifferentflavoproteinforelectrontransfer.DifferentCYPenzymesactashydroxylases,lyases(desmolases),oxidases,oraromatases.Twooftheseenzymeshavemultiplefunctions.CYP17hasbotha17-hydroxylasefunctionanda17,20-lyase(desmolase)function.CYP11B2,alsocalledaldosterone synthase, hasthreefunctions:11-hydroxylase,18-hydroxylase,and18-oxidase.

Theotherenzymesinvolvedinsteroidogenesisbelongtothreehydroxysteroid dehydrogenase (HSD)families.3β-HSDs havetwoisoformsthatconvertthehydroxylgrouponcarbon3ofthecholesterolringtoaketoneandshiftthedoublebondfromthe5-6(Δ5) positiontothe4-5(Δ4) position.AllactivesteroidhormonesmustbeconvertedtoΔ4structuresby3β-HSD.The17β-HSDs haveatleastfivemembersandcanactaseitheroxidasesorreductases.17β-HSDsprimarilyactonsexsteroidsandcanbeactivatingordeactivating.Finally,the11β-HSDs havetwoisoformsthatcatalyzetheinterchangebetweencortisol(active)andcortisone(inactive).

•Fig. 43.9 A,Reaction1,catalyzedbyCYP11A1,inmakingcortisol.B,Reactions2a/bandreactions3a/b,involvingCYP17(17-hydroxylasefunction)and3β-hydroxysteroiddehydrogenase(3β-HSD),inmakingcortisol.ThisfigureshowstheΔ5versusΔ4pathway.

•Fig. 43.9, cont’d C,Reactions4and5,involvingCYP21BandCYP11B1,inwhichthelasttwostepsincortisolsynthesisarecarriedout.Alsoshownistheminorpathwayleadingtocorticosteronesynthesisinthezonafasciculata.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology.

4thed.Philadelphia:Mosby;2013.) target cells of aldosterone (see below). The conversion of cortisol to cortisone prevents the binding of cortisol to the MR and having inappropriate mineralocorticoid actions on these cells. Inactivation of cortisol by 11β-HSD2 is reversible in that another enzyme, 11β-HSD1, converts cortisone back to cortisol. This conversion occurs in tissues expressing the glucocorticoid receptor (GR), including liver, adipose tissue, and the CNS, as well as in skin.

Mechanism of Action of Cortisol

Cortisol acts primarily through the glucocorticoid receptor, which regulates gene transcription (see ). In the absence of hormone, the GR resides in the cytoplasm in a stable complex with several molecular chaperones, including heat shock proteins and cyclophilins. Cortisol-GR binding promotes dissociation of the chaperone proteins, followed by: aAllvaluesarerelativetotheglucocorticoidandmineralocorticoidpotenciesofcortisol,whichhaveeachbeenarbitrarilysetat1.0.Cortisolactuallyhasonly1/500thepotencyofthenaturalmineralocorticoidaldosterone.

1.

rapid translocation of the cortisol-GR complex into the nucleus, 2.

dimerization and binding to glucocorticoid response elements (GREs, both “positive” GREs and “negative” GREs) near the basal promoters of cortisol-regulated genes, and 3.

recruitment of coactivator proteins and assembly of general transcription factors leading to increased or decreased transcription of the targeted genes.

In some cases the GR interacts with other transcription factors, such as the proinflammatory nuclear factor (NF)κB transcription factor, and interferes with their ability to activate gene expression.

Physiological Actions of Cortisol

Cortisol has a broad range of actions and is often characterized as a “stress hormone.” In general, cortisol maintains blood glucose levels, CNS function, and cardiovascular function during fasting and increases blood glucose during stress at the expense of muscle protein. Cortisol protects the body against the self-injurious effects of unbridled inflammatory and immune responses. Cortisol also partitions energy to cope with stress by inhibiting reproductive function. As stated later, cortisol has several other effects on bone, skin, connective tissue, the GI tract, and the developing fetus that are independent of its stress-related functions.

As the term glucocorticoid implies, cortisol is a steroid hormone from the adrenal cortex that regulates blood glucose. It increases blood glucose by stimulating gluconeogenesis (

Fig. 43.10 ). Cortisol enhances gene expression of the key hepatic gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Cortisol also decreases GLUT4-mediated glucose uptake in skeletal muscle and adipose tissue. During the interdigestive period (low insulin-glucagon ratio), cortisol promotes glucose sparing by potentiating the effects of catecholamines on lipolysis, thereby making FFAs available as energy sources. Cortisol inhibits protein synthesis and increases proteolysis, especially in skeletal muscle, thereby providing a rich source of carbon for hepatic gluconeogenesis.

Fig. 43.10 also contrasts the normal role of cortisol in response to stress and the effects of chronically elevated cortisol as a result of pathological conditions. As discussed later, there are important differences in the overall metabolic effects of cortisol between these two states, particularly with respect to lipid metabolism. During stress, cortisol synergizes with catecholamines and glucagon to promote a lipolytic, gluconeogenic, ketogenic, and glycogenolytic metabolic response while synergizing with catecholamines to promote an appropriate cardiovascular response. During chronically elevated cortisol secondary to pathological overproduction, cortisol synergizes with insulin in the context of elevated levels of glucose (from increased appetite) and hyperinsulinemia (from elevated glucose and glucose intolerance) to promote lipogenesis and truncal (abdominal/ visceral) adiposity.

Cortisol reinforces its effects on blood glucose by its positive effects on the cardiovascular system. Cortisol has permissive actions on catecholamines by increasing adrenergic receptor expression and thereby contributes to cardiac output and blood pressure. Cortisol stimulates erythropoietin synthesis and hence increases red blood cell production. Anemia occurs when cortisol is deficient, and polycythemia occurs when cortisol levels are excessive.

Inflammation and immune responses are often part of the response to stress. However, inflammation and immune responses have the potential for significant harm and may cause death if they are not held in homeostatic balance. As a stress hormone, cortisol plays an important role in maintaining immune homeostasis. Cortisol, along with epinephrine and norepinephrine, represses production of proinflammatory cytokines and stimulates production of antiinflammatory cytokines.

Stress during interdigestive period (e.g., elderly person slips on ice and dislocates hip) Liver ˜ Glycogenolysis ˜ Gluconeogenesis

Metabolic responses to stress ensure sufficient energy to meet increased demand on body, and will maintain adequate blood glucose levels necessary for conscious, deliberate action by the individual. The metabolic responses are supported by cardiovascular ones, first by those induced by the sympathoadrenal output (see Fig.

43.6) but also by cortisol, which optimizes adrenergic receptor function. Cortisol will also contribute to providing energy for the incipient inflammatory and immune response to this stress, but will also protect the individual from the potential damage of unregulated inflammation.

Metabolic responses to chronically elevated cortisol in patients with Cushing’s disease who are otherwise healthy tend to promote localized obesity (abdominal, neck, and face), with muscle wasting and weakness, especially at the extremities. Note that the elevated cortisol in this case usually occurs in the context of an elevated insulin/glucagon ratio. Elevated cortisol stimulates appetite, which increases the insulin/glucagon ratio. High levels of cortisol antagonize insulin’s effect on GLUT-4mediated glucose uptake into skeletal muscle and adipose tissue, so that glucose intolerance is frequently a symptom in patients with Cushing’s disease. This further contributes to hyperglycemia and hyperinsulinemia. Both cortisol and insulin promote the differentiation of preadipocytes into adipocytes, and lipogenesis.

Chronically elevated levels of cortisol in otherwise well-fed person (e.g., Cushing’s disease) •Fig. 43.10 Metabolicactionsofcortisol(integratedwithcatecholaminesandglucagon)inresponsetostress(upper panel) andcontrastedtotheactionsofchronicallyelevatedcortisol(integratedwithinsulin)inanotherwisehealthyindividual(lower panel). (ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.)

The inflammatory response to injury consists of local dilation of capillaries and increased capillary permeability, with resultant local edema and accumulation of white blood cells. These steps are mediated by prostaglandins, thromboxanes, and leukotrienes. Cortisol inhibits phospholipase A2, a key enzyme in prostaglandin, leukotriene, and thromboxane synthesis. Cortisol also stabilizes lysosomal membranes, thereby decreasing release of the proteolytic enzymes that augment local swelling. In response to injury, leukocytes normally leave the vascular system and migrate to the site of injury. This complex process is generally inhibited by cortisol, as is the phagocytic activity of neutrophils, although release of neutrophils from bone marrow is stimulated. Analogues of glucocorticoid are frequently used pharmacologically because of their antiinflammatory properties.

Cortisol inhibits the immune response, and for this reason glucocorticoid analogues have been used as immunosuppressants in organ transplants. High cortisol levels decrease the number of circulating T lymphocytes (particularly helper T lymphocytes) and reduce their ability to migrate to the site of antigenic stimulation. Glucocorticoids promote atrophy of the thymus and other lymphoid tissue. Although corticosteroids inhibit cellular-mediated immunity, antibody production by B lymphocytes is not impaired.

Effects of Cortisol on the Reproductive Systems

Reproduction exacts a considerable anabolic cost on the organism. In humans, reproductive behavior and function are dampened in response to stress. Cortisol decreases the function of the reproductive axis at the hypothalamic, pituitary, and gonadal levels.

Effects of Cortisol on Bone

Glucocorticoids increase bone resorption. They have multiple actions that alter bone metabolism. Glucocorticoids decrease intestinal Ca++ absorption and renal Ca++ reabsorption. Both mechanisms serve to lower serum [Ca++]. As serum [Ca++] drops, secretion of parathyroid hormone (PTH) increases, and PTH mobilizes Ca++ from bone by stimulating resorption of bone. In addition to this action, glucocorticoids directly inhibit osteoblast bone-forming functions (see ). Although glucocorticoids are useful for treating the inflammation associated with arthritis, excessive use will result in bone loss (osteoporosis).

Actions of Cortisol on Connective Tissue

Cortisol inhibits fibroblast proliferation and collagen formation. In the presence of excessive amounts of cortisol, the skin thins and is more readily damaged. The connective tissue support of capillaries is impaired, and capillary injury, or bruising, is increased.

Actions of Cortisol on the Kidney

Cortisol inhibits the secretion and action of antidiuretic hormone (ADH), and thus it is an ADH antagonist. In the absence of cortisol, the action of ADH is potentiated, which makes it difficult to increase free water clearance in response to a water load and increases the likelihood of water intoxication. Although cortisol binds to the mineralocorticoid receptor with high affinity, this action is normally blocked by inactivation of cortisol to cortisone by the enzyme 11βHSD2. However, the mineralocorticoid activity (i.e., renal Na+ and H2O retention, K+ and H+ excretion) of cortisol depends on the relative amount of cortisol (or synthetic glucocorticoids) and the activity of 11β-HSD2. Certain agents (e.g., compounds in black licorice) inhibit 11βHSD2 and thereby increase the mineralocorticoid activity of cortisol. Cortisol increases the glomerular filtration rate by both increasing cardiac output and acting directly on the kidney.

Actions of Cortisol on Muscle

When cortisol levels are excessive, muscle weakness and pain are common symptoms. The weakness has multiple origins. In part it is a result of the excessive proteolysis cortisol produces. High cortisol levels can result in hypokalemia (via mineralocorticoid actions), which can produce muscle weakness because it hyperpolarizes and stabilizes the muscle cell membrane and thus makes stimulation more difficult.

Actions of Cortisol on the Gastrointestinal Tract

Cortisol exerts a trophic effect on the GI mucosa. In the absence of cortisol, GI motility decreases, GI mucosa degenerates, and GI acid and enzyme production decreases. Because cortisol stimulates appetite, hypercortisolism is frequently associated with weight gain. The cortisol-mediated stimulation of gastric acid and pepsin secretion increases the risk for development of ulcers.

Psychological Effects of Cortisol

Psychiatric disturbances are associated with either excessive or deficient levels of corticosteroids. Excessive corticosteroids can initially produce a feeling of well-being, but continued excessive exposure eventually leads to emotional lability and depression. Frank psychosis can occur with either excessive or deficient hormone. Cortisol increases the tendency for insomnia and decreases rapid eye movement (REM) sleep. People who are deficient in corticosteroids tend to be depressed, apathetic, and irritable.

Effects of Cortisol During Fetal Development

Cortisol is required for normal development of the CNS, retina, skin, GI tract, and lungs. The best studied system is the lungs, in which cortisol induces differentiation and maturation of type II alveolar cells. During late gestation these cells produce surfactant, which reduces surface tension in the lungs and thus allows the onset of breathing at birth.

Regulation of Cortisol Production

Cortisol production by the zona fasciculata is regulated by a standard hypothalamic-pituitary-adrenal axis involving corticotropin-releasing hormone (CRH), ACTH, and cortisol (see ). The hypothalamus and pituitary stimulate cortisol production, and cortisol negatively feeds back on the hypothalamus and pituitary to maintain its set point. Both neurogenic (e.g., fear) and systemic (e.g., hypoglycemia, hemorrhage, cytokines) forms of stress stimulate release of CRH. CRH is also under strong diurnal rhythmic regulation emerging from the suprachiasmatic nucleus, such that cortisol levels surge during the early predawn and morning hours and then continually decline throughout the day and evening. CRH acutely stimulates release of ACTH and chronically increases proopiomelanocortin (POMC) gene expression and corticotrope hypertrophy and proliferation. Some parvicellular neurons coexpress CRH and ADH, which potentiates the actions of CRH.

ACTH binds to the melanocortin 2 receptor (MC2R) located on cells in the zona fasciculata (

Fig. 43.11 ). The effects of ACTH can be subdivided into three phases: 1.

The acute effects of ACTH occur within minutes. Cholesterol is rapidly mobilized from lipid droplets by posttranslational activation of cholesterol ester hydrolase and transported to the outer mitochondrial membrane. ACTH both rapidly increases StAR protein gene expression and activates StAR protein through protein kinase A (PKA)-dependent phosphorylation. Collectively, these acute actions of ACTH increase pregnenolone levels.

2.

The chronic effects of ACTH occur over a period of several hours. These effects involve increasing transcription of the genes encoding the steroidogenic enzymes and their coenzymes. ACTH also increases expression of the LDL receptor and scavenger receptor BI (SR-BI; the HDL receptor).

3.

The trophic actions of ACTH on the zona fasciculata and zona reticularis occur over a period of weeks and •Fig. 43.11 OverviewoftheactionsofACTHontargetadrenocorticalcells.Notethatthemajorsecondmessenger,cAMP,activatesimmediateproteinmediatorsandalsoinducesproductionoflaterproteinmediators.HDL,high-densitylipoprotein;LDL,low-densitylipoprotein.

months. This effect is exemplified by atrophy of the zona fasciculata in patients receiving therapeutic (i.e., supraphysiological) levels of glucocorticoid analogues for at least 3 weeks. Under these conditions the exogenous corticosteroids completely repress CRH and ACTH production, thereby resulting in atrophy of the zona fasciculata and a decline in endogenous cortisol production (

Fig. 43.12 ). At the end of therapy, these patients need to be slowly weaned off exogenous glucocorticoids to allow the hypothalamic-pituitary-adrenal axis to reestablish itself and the zona fasciculata to enlarge and produce adequate amounts of cortisol. Cortisol inhibits both POMC gene expression at the corticotropes and pro-CRH gene expression at the hypothalamus. However, intense stress can override the negative-feedback effects of cortisol at the hypothalamus and reset the “set point” at a higher level.

The innermost zone, the zona reticularis, begins to appear after birth at about 5 years of age. Adrenal androgens, especially DHEAS, the main product of the zona reticularis, become detectable in the circulation at about 6 years of age. This onset of adrenal androgen production is called adrenarche, and it contributes to the appearance of axillary and pubic hair at about age 8. DHEAS levels continue to increase, peak during the mid-20s, and then progressively decline with age.

Androgen Synthesis by the Zona Reticularis

The zona reticularis differs from the zona fasciculata in several important ways with respect to steroidogenic enzyme activity (see

Fig. 43.8 ). First, 3β-HSD is expressed at very low levels in the zona reticularis; thus the Δ5 pathway predominates in the zona reticularis. Second, the zona reticularis expresses cofactors or conditions that enhance the 17,20-lyase function of CYP17, thereby generating the 19-carbon androgen precursor molecule dehydroepiandrosterone (DHEA) from 17-hydroxypregnenolone. Additionally the zona reticularis expresses DHEA sulfotransferase (SULT2A1 gene), which converts DHEA into DHEAS (

Fig. 43.13 ). A limited amount of the Δ4 androgen androstenedione is also made in the zona reticularis. Although small amounts of potent androgens (e.g., testosterone) or 18-carbon estrogens are normally produced by the human adrenal cortex, most active sex steroids are produced primarily from peripheral conversion of DHEAS and androstenedione.

Metabolism and Fate of DHEAS and DHEA

DHEAS can be converted back to DHEA by peripheral sulfatases, and DHEA and androstenedione can be converted to active androgens (testosterone, dihydrotestosterone) peripherally in both sexes. DHEA binds to albumin and other globulins in blood with low affinity, so it is excreted efficiently by the kidney. The half-life of DHEA is 15 to 30 minutes. In contrast, DHEAS binds to albumin with very high affinity and has a half-life of 7 to 10 hours.

Quiescent HPA axis in patient undergoing long-term (>3 wk) Normal HPA axis treatment with glucocorticoid

Hypothalamus CRH ACTH Pituitary Zona fasciculata Atrophied zona fasciculata Cortisol Exogenous corticosteroid (supraphysiological levels) Biological actions Biological actions and therapeutic actions (e.g., antiinflammatory or immunosuppressive) Pituitary Hypothalamus (–) (–) (–) (–) •Fig. 43.12 Comparisonofanormalhypothalamic-pituitary-adrenal(HPA)axistoaquiescentHPAaxisinindividualreceivingexogenousglucocorticoidtherapy.Thelattercausesthezonafasciculatatoatrophyafter3weeks,thusrequiringacarefulwithdrawalregimentoallowrebuildingoftheadrenaltissuebeforetotalcessationofexogenouscorticosteroidadministration.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.)

Physiological Actions of Adrenal Androgens

In men the contribution of adrenal androgens to active androgens is negligible. However, in women the adrenal contributes to about 50% of circulating active androgens, which are required for the growth of axillary and pubic hair and for libido.

Apart from providing androgen precursors, it is not clear what other role or roles if any the zona reticularis plays in adult humans. DHEAS is the most abundant circulating hormone in young adults. It increases steadily until it peaks in the mid-20s and then steadily declines thereafter. Thus there has been considerable interest in the possible role of DHEAS in the aging process. However, the function of this abundant steroid in young adults and the potential impact of its gradual disappearance on aging are still poorly understood. It should be noted that the age-related decline in DHEA and DHEAS has led to the popular use of these steroids as dietary supplements, even though recent studies indicate no beneficial effects.

Duringadrenalandrogenexcess(e.g.,adrenaltumor,Cushing’ssyndrome,congenitaladrenalhyperplasia),masculinization of women canoccur.Thisinvolvesmasculinizationoftheexternalgenitalia(e.g.,enlargedclitoris)inuteroandexcessivefacialandbodyhair(calledhirsutism)andacneinadultwomen.Excessiveadrenalandrogensalsoappeartoplayaroleinovariandysovulation(i.e.,polycysticovariansyndrome).

Acrucialclinicalaspectofregulationofthezonareticularisisthatneitheradrenalandrogensnortheirmorepotentmetabolites(e.g.,testosterone,dihydrotestosterone,estradiol-17β)negativelyfeedbackonACTHorCRH(

Fig.

43.14 ).Thismeansthatanenzymaticdefectassociatedwiththesynthesisofcortisol(e.g.,CYP21Bdeficiency)isassociatedwithadramaticincreaseinbothACTH(nonegativefeedbackfromcortisol)andadrenalandrogens(becauseoftheelevatedACTH).Itisthis“loophole”inthehypothalamic-pituitary-adrenalaxisthatgivesriseto congenital adrenal hyperplasia (CAH).

Regulation of Zona Reticularis Function

ACTH is the primary regulator of the zona reticularis. Both DHEA and androstenedione display the same diurnal rhythm as cortisol (DHEAS does not because of its long circulating half-life). Moreover, the zona reticularis shows the same atrophic changes as the zona fasciculata in conditions typified by little or no ACTH. However, other factors must regulate adrenal androgen function. Adrenarche occurs in the face of constant ACTH and cortisol levels, and the rise and decline of DHEAS is not associated with a similar pattern of ACTH or cortisol production. However, the other factors, whether extraadrenal or intraadrenal, remain unknown.

The thin outermost zone of the adrenal, the zona glomerulosa, produces the mineralocorticoid aldosterone, which regulates salt and volume homeostasis (see ). The zona glomerulosa is minimally influenced by ACTH. Rather it is regulated primarily by the reninangiotensin system, plasma [K+], and atrial natriuretic peptide (ANP).

An important feature in the steroidogenic capacity of the zona glomerulosa is that it does not express CYP17. Therefore zona glomerulosa cells never make cortisol, nor do they make adrenal androgens in any form. Pregnenolone is converted to progesterone and DOC by 3β-HSD and •Fig. 43.13 Steroidogenicpathwaysinthezonareticularis.Thefirstcommonreactioninthepathway,conversionofcholesteroltopregnenolonebyCYP11A1,isnotshown.Expressionof3β-hydroxysteroiddehydrogenase(3β-HSD)isrelativelylowinthezonareticularis,soandrostenedioneisaminorproductincomparisontoDHEAandDHEAS.Thezonareticularisalsomakesasmallamountoftestosteroneandestrogens(notshown).(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.)

CYP21, respectively (

Fig. 43.15 ). A completely unique last three reactions from DOC to aldosterone within the feature of the zona glomerulosa among the steroidogenic zona glomerulosa. These reactions are 11-hydroxylation of glands is its expression of CYP11B2, which is regulated DOC to form corticosterone, 18-hydroxylation to form by different signaling pathways. Furthermore the enzyme 18-hydroxycorticosterone, and 18-oxidation to form aldocoded by CYP11B2, aldosterone synthase, catalyzes the sterone (see

Figs. 43.8 43.15).

•Fig. 43.14 The“loophole”inthehypothalamic-pituitary-adrenalaxis.ACTHstimulatesproductionofbothcortisolandadrenalandrogens,butonlycortisolnegativelyfeedsbackonACTHandCRH.Thusifcortisolproductionisblocked(i.e.,CYP11B1deficiency),ACTHlevelsincreasealongwithadrenalandrogens.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.)

CYP11B1(expressedonlyinthezonafasciculata)andCYP11B2(expressedonlyinthezonaglomerulosa)arelocatedonchromosome8inhumans,display95%similarity,andareseparatedfromeachotherbyonlyabout50kilobases.Thisincreasesthepossibilityofunevencrossingoverduringgametogenesis,withtheformationofhybridgenes.Inonecasethepromoterregionand5′ endoftheCYP11B1 geneisfusedtothe3′ endoftheCYP11B2 gene.ThisarrangementleadstoaldosteronesynthasebeingexpressedinthezonaefasciculataandreticularisunderthecontrolofACTH.Becausealdosteroneisnolongerunderfeedbackcontrolbytherenin-angiotensinsystem(see ),aldosteronelevelsarehighandhypertensionensues.Thisformofprimaryaldosteronismiscalledglucocorticoid-remediable aldosteronism, anditisinheritedinanautosomaldominantmanner.Thisdiseasecanbeconfirmedbythepolymerasechainreactiontechniqueandbymeasurementof18-hydroxycortisoland18-oxicortisolina24-hoururinesample.Thediseaseistreatedbytheadministration of glucocorticoid, whichsuppressesACTHandthusexpressionofthehybridgene.

Transport and Metabolism of Aldosterone

Aldosterone binds to albumin and corticosteroid-binding protein in blood with low affinity and therefore has a biological half-life of about 20 minutes. Almost all aldosterone is inactivated by the liver in one pass, conjugated to a glucuronide group, and excreted by the kidney.

Mechanism of Aldosterone Action

Aldosterone acts much like cortisol (and other steroid hormones) in that its primary mechanism of action is mediated by binding to a specific intracellular receptor (i.e., mineralocorticoid receptor [MR]). After dissociation of chaperone proteins, nuclear translocation, dimerization, and binding to the mineralocorticoid response element (MRE), the aldosterone-MR complex regulates expression of specific genes (see ). As discussed earlier, cortisol binds to the MR with significant affinity. However, as also discussed, cells that express MR also express 11β-HSD2, which converts cortisol to the inactive steroid cortisone (

Fig. 43.16 ). Cortisone can be converted back to cortisol by 11β-HSD1, which is expressed in several glucocorticoidresponsive tissues, including the liver and skin.

Clinicalstudiesinhumanshaverevealedadeleteriouseffectofaldosteroneoncardiovascularfunctionindependentofitseffectsonrenalsodiumandwaterreabsorption.Aldosteronehasaproinflammatory, profibrotic effect onthecardiovascularsystemandcausesleftventricularhypertrophyandremodeling.Thiseffectofaldosteroneisassociatedwithincreasedmorbidityandmortalityinpatientswithessentialhypertension.

Physiological Actions of Aldosterone

The actions and regulation of aldosterone are discussed in

CHAPTER 43 The Adrenal Gland •Fig. 43.15 Steroidogenicpathwaysinthezonaglomerulosa.Thefirstcommonreactioninthepathway,conversionofcholesteroltopregnenolonebyCYP11A1,isnotshown.NotethatthelastthreereactionsarecatalyzedbyCYP11B2.(ModifiedfromWhiteBA,PorterfieldSP.EndocrineandReproductivePhysiol-ogy.4thed.Philadelphia:Mosby;2013.)CH3 CH3 CH3 Pregnenolone C O 3˜-HSD CH3 CH3 CH3 Progesterone C O CYP21B HO CH3 CH3 CH2OH 11-Deoxycorticosterone (DOC) C O CYP11B2 CH3 CH3 CH2OH Corticosterone C O CYP11B2 O HO HO CH3 CH3 CH2OH 18(OH) Corticosterone C O CYP11B2 O HO CH3 CH2OH Aldosterone C O O O CH

Addison’s disease isdefinedbyprimary adrenal insufficiency, withbothmineralocorticoidsandglucocorticoidsusuallybeingdeficient.InNorthAmericaandEurope,themostprevalentcauseofAddison’sdiseaseisautoimmune destruction of the adrenal cortex. Becauseofthecortisoldeficiency,ACTHsecretionincreases.ElevatedlevelsofACTHcancompeteforMC1Rinmelanocytesandcauseanincreaseinskinpigmentation,particularlyinskincreases,scars,andgums(see

Fig.43.14 ).Thelossofmineralocorticoidsresultsincontractionofextracellularvolume,whichproducescirculatoryhypovolemiaandthereforeadropinbloodpressure.Becauselossofcortisoldecreasesthevasopressiveresponsetocatecholamines,peripheralvascularresistancedrops,therebyfacilitatingthedevelopmentofhypotension.IndividualswithAddison’sdiseasearealsopronetohypoglycemiawhenstressedorfasting,andwaterintoxicationcandevelopifexcesswaterisingested.Becausecortisolisimportantformusclefunction,muscleweaknessalsooccursincortisoldeficiency.Lossofcortisolresultsinanemia,decreasedGImotilityandsecretion,andreducedironandvitaminB12absorption.Appetitedecreaseswithcortisoldeficiency,andthisdecreasedappetitecoupledwiththeGIdysfunctionpredisposestheseindividualstoweightloss.Thesepatientsoftenhavedisturbancesinmoodandbehaviorandaremoresusceptibletodepression.

•Fig. 43.16 Themineralocorticoidreceptor(MR)isprotectedfromactivationbycortisolbytheenzyme11β-hydroxysteroiddehydrogenasetype2(11β-HSD2),whichconvertscortisoltoinactivecortisone.Cortisonecanbeconvertedbacktocortisolinglucocorticoidtargetcellsbytheenzyme11β-HSDtype1.GRE,glucocorticoidresponseelement;GTF,generaltranscriptionfactors;MRE,mineralocorticoidresponseelement.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.)Aldosterone Aldosterone 11˜-HSD2 MR-chaperone complex Dissociated chaperones Dissociated chaperonesMR-aldosterone complex Mineralocorticoid-specific biological response Glucocorticoid-specific biological response Mineralocorticoid target cell 11˜-HSD1 Glucocorticoid target cell • Dimerization • Nuclear transport • Binding to MREs • Recruitment of coactivator proteins and GTFs • Altered transcription of aldosterone-regulated genes • Dimerization • Nuclear transport • Binding to GREs • Recruitment of coactivator proteins and GTFs • Altered transcription of cortisol-regulated genes Cortisol Cortisol Cortisol GR-chaperone complex GR-cortisol complex Cortisone Cortisone Cortisone Blood

Adrenocortical hormone excess istermedCushing’s syndrome. Pharmacological useofexogenouscorticosteroidsisnowthemostcommoncauseofCushing’ssyndrome.ThenextmostprevalentcauseisACTH-secreting tumors. TheformofCushing’ssyndromecausedbyafunctionalpituitaryadenomaiscalledCushing’s disease. ThefourthmostcommoncauseofCushing’ssyndromeisprimary hypercortisolism resultingfromafunctionaladrenaltumor.Ifthedisorderisprimaryorifitisaresultofcorticosteroidtreatment,secretionofACTHwillbesuppressedandincreasedskinpigmentationwillnotoccur.However,ifhypersecretionoftheadrenalistheresultofanACTH-secretingnonpituitarytumor,ACTHlevelssometimesbecomehighenoughtoincreaseskinpigmentation.

Increasedcortisolsecretioncausesweightgainwithacharacteristiccentripetalfatdistributionanda“buffalohump.”

Thefaceappearsrounded(fatdeposition),andthecheeksmaybereddened,inpartbecauseofthepolycythemia.Thelimbsarethinowingtoskeletalmusclewasting(fromincreasedproteolysis),andmuscleweaknessisevident(frommuscleproteolysisandhypokalemia).Proximalmuscleweaknessisapparent,sothepatientmayhavedifficultyclimbingstairsorrisingfromasittingposition.Theabdominalfataccumulationcoupledwithatrophyoftheabdominalmusclesandthinningoftheskinproducesalargeprotrudingabdomen.Purpleabdominalstriaeareseenasaresultofdamagetotheskinbytheprolongedproteolysis,increasedintraabdominalfat,andlossofabdominalmuscletone.Capillaryfragilityoccursbecauseofdamagetotheconnectivetissuesupportingthecapillaries.Patientsarelikelytoshowsignsofosteoporosisandpoorwoundhealing.Theyhavemetabolicdisturbancesthatincludeglucoseintolerance,hyperglycemia,andinsulinresistance(see

Fig.43.10 ).Prolongedhypercortisolismcanleadtomanifestationsofdiabetesmellitus.Becauseofsuppressionoftheimmunesystemcausedbyglucocorticoids,patientsaremoresusceptibletoinfection.Themineralocorticoidactivitiesofglucocorticoidsandthepossibleincreaseinaldosteronesecretionproducesaltretentionandsubsequentwaterretentionthatresultinhypertension.Excessiveandrogensecretioninwomencanproducehirsutism,malepatternbaldness,andclitoralenlargement(adrenogenitalsyndrome).

AnyenzymeblockagethatdecreasescortisolsynthesiswillincreaseACTHsecretionandproduceadrenalhyperplasia.Themostcommonformofcongenitaladrenalhyperplasiaisduetodeficiencyoftheenzyme21-hydroxylase (CYP21). Theseindividualscannotproducenormalquantitiesofcortisol,deoxycortisol, DOC,corticosterone,oraldosterone(see

Figs.

43.8 43.10C ).BecauseofimpairedcortisolproductionandresultantelevatedACTHlevels,steroidogenesisisstimulated,therebyincreasingthesynthesisproducts“upstream”ofthemissingenzyme,aswellasproductsofthezonareticularis.Becausethelatterincludetheadrenalandrogens,afemale 1.

The adrenal gland is composed of a cortex that is of mesodermal origin and a medulla that is of neuroectodermal origin. The cortex produces steroid hormones, and the medulla produces catecholamines.

2.

The rate-limiting enzymes in medullary catecholamine synthesis are tyrosine hydroxylase and dopamine β-hydroxylase, which are induced by sympathetic stimulation, and phenylethanolamine-Nmethyltransferase, which is induced by cortisol.

3.

Catecholamines increase serum glucose and fatty acid levels. They stimulate gluconeogenesis, glycogenolysis, and lipolysis. Catecholamines increase cardiac output but have selective effects on blood flow to different organs.

4.

Pheochromocytoma is a tumor of chromaffin tissue that produces excessive quantities of catecholamines. Symptoms of pheochromocytoma are often sporadic and include hypertension, headaches, sweating, anxiety, palpitations, chest pain, and orthostatic hypotension.

5.

The adrenal cortex displays clear structural and functional zonation: the zona glomerulosa produces the mineralocorticoid aldosterone, the zona fasciculata produces the glucocorticoid cortisol, and the zona reticularis produces the weak androgens DHEA and DHEAS.

6.

Cortisol binds to the glucocorticoid receptor. During stress, cortisol increases blood glucose by increasing gluconeogenesis in the liver and breaking muscle protein down to supply gluconeogenic precursors. Cortisol also decreases glucose uptake by muscle and adipose tissue and has permissive actions on glucagon and catecholamines. Cortisol has multiple effects on other tissue. From a pharmacological point of view, the most important is the immunosuppressive/ antiinflammatory effect.

7.

Cortisol is regulated by the CRH-ACTH-cortisol axis. Cortisol negatively feeds back at the hypothalamus on both CRH-producing neurons and pituitary corticotropes. CRH is regulated by several fetuswillbemasculinized.Becausetheyareunabletoproducethemineralocorticoids,aldosterone,DOC,andcorticosterone,patientswiththisdisorderhavedifficultyretainingsaltandmaintainingextracellularvolume.Consequentlytheyarelikelytobehypotensive.Iftheblockageisatthenextstep,11β-hydroxylase (CYP11B1),DOC willbeformedandlevelsofDOCwillaccumulate(see

Figs.43.8 43.9C ).BecauseDOChassignificantmineralocorticoid activity anditslevelsbecomehigh,theseindividualstendtoretainsaltandwaterandbecomehypertensive.

forms of stress, including proinflammatory cytokines, hypoglycemia, neurogenic stress, and hemorrhage, and by diurnal input.

8.

The adrenal androgens DHEA, DHEAS, and androstenedione are androgen precursors. They can be converted to active androgens peripherally and provide about 50% of circulating androgens in women. In men the role of adrenal androgens if any remains obscure. In women, adrenal androgens promote pubic and axillary hair growth and libido. Excessive adrenal androgens in women can lead to various degrees of virilization and ovarian dysfunction.

9.

The zona glomerulosa of the adrenal cortex is the site of aldosterone production. Aldosterone is the strongest naturally occurring mineralocorticoid in humans. It promotes Na+ and water reabsorption by the distal tubule and collecting duct while promoting renal K+ and H+ secretion. Aldosterone promotes Na+ and water absorption in the colon and salivary glands. It also has a proinflammatory, profibrotic effect on the cardiovascular system and causes left ventricular hypertrophy and remodeling.

10.

Major actions of angiotensin II on the adrenal cortex are increased growth and vascularity of the zona glomerulosa, increased StAR and CYP11B2 enzyme activity, and increased aldosterone synthesis.

11.

Major stimuli for aldosterone production are a rise in angiotensin II and a rise in serum [K+]. The major inhibitory signal is ANP.

12.

Addison’s disease is adrenocortical insufficiency. Common symptoms include hypotension, hyperpigmentation, muscle weakness, anorexia, hypoglycemia, and hyperkalemic acidosis.

13.

Cushing’s syndrome results from hypercortisolemia. If the basis of the disorder is increased pituitary adrenocorticotropin secretion, the disorder is called Cushing’s disease. Common symptoms of Cushing’s syndrome include centripetal fat distribution, muscle wasting, proximal muscle weakness, thin skin with abdominal striae, capillary fragility, insulin resistance, and polycythemia.

14. Congenital adrenal hyperplasia is caused by a congenital enzyme deficiency that blocks production of cortisol. The enzyme blockage results in elevated

De Bosscher K, et al. Activation of the glucocorticoid receptor in acute inflammation: the SEDIGRAM concept. Trends Pharmacol Sci. 2016;37:4-16.

Guerineau NC, et al. Functional chromaffin cell plasticity in response to stress: focus on nicotinic, gap junction, and voltage-gated Ca2+ channels. J Mol Neurosci. 2012;48:368-386.

Namsolleck P, Unger T. Aldosterone synthase inhibitors in cardiovascular and renal diseases. Nephrol Dial Transplant. 2014;29(suppl 1): i62-i68.

ACTH secretion, which stimulates adrenal cortical growth and secretion of precursors produced before the block. 21-Hydroxylase (CYP21B) deficiency is the most common form.

Rafacho A, et al. Glucocorticoid treatment and endocrine pancreas function: implications for glucose homeostasis, insulin resistance and diabetes. J Endocrinol. 2014;223:R49-R62.

Sundahl N, et al. Selective glucocorticoid receptor modulation: new directions with non-steroidal scaffolds. Pharmacol Ther. 2015;152:28-41.

Turcu AF, Auchus RJ. Adrenal steroidogenesis and congenital adrenal hyperplasia. Endocrinol Metab Clin North Am. 2015;44: 275-296.

Whittier X, Saag KG. Glucocorticoid-induced osteoporosis. Rheum Dis Clin North Am. 2016;42:177-189.

Upon completion of this chapter the student should be able to answer the following questions: 1.

Describe the general anatomical components of the male and female reproductive system.

2.

Map out the organization of the testis, with the Sertoli cells and developing sperm cells within the intralobular compartment, and the Leydig cells and capillary plexus within the interlobular/interstitial compartment.

3.

Describe the processes of spermatogenesis and spermiogenesis.

4.

List the functions of the Sertoli cell.

5.

Diagram the process of testosterone synthesis within the Leydig cells, and the peripheral conversion of testosterone to estradiol or dihydrotestosterone.

6.

Diagram the male hypothalamus/pituitary/testis axis, including all cell types and hormones involved.

7.

Map out the organization of the ovary, and describe the various stages of follicular development, ovulation and corpus luteum formation.

8.

List the stages and control of female germ cell progression from oogonia to egg.

9.

Map out the steroidogenic pathways in the corresponding cell types that lead to androgen, estrogen and progesterone synthesis.

10.

Diagram the female hypothalamus/pituitary/ovarian axis during the menstrual cycle, including all cell types and hormones involved.

11.

Explain changes in the female tract, with emphasis on the uterine endometrium, during the menstrual cycle.

12.

List the events involved in fertilization.

13.

Describe the development and function of the placenta.

14.

Describe the development and function of the mammary glands he two most basic components of the reproductive system are the gonads and the reproductive tract. The gonads (testes and ovaries) perform an endocrine function that is regulated within a hypothalamic-pituitary-gonadal axis. The gonads are distinct from other endocrine glands in that they also perform gametogenesis. The reproductive tract is involved in several aspects of gamete development, function, and transport and in women allows fertilization, implantation, and gestation. Normal gametogenesis in the gonads and development and physiology of the reproductive tract are absolutely dependent on the endocrine function of the gonads. The clinical ramifications of this hormonal dependence include infertility in the face of low sex hormone production, ambiguous genitalia in dysregulated hormone or receptor expression, and hormone-responsive cancers, especially uterine and breast cancer in women and prostate cancer in men.

The male reproductive system has evolved for continuous lifelong gametogenesis coupled with occasional internal insemination with a high density of sperm (>60 × 106/ mL in 3–5 mL of semen). In adult men the basic roles of gonadal hormones are (1) support of gametogenesis (spermatogenesis), (2) maintenance of the male reproductive tract and production of semen, and (3) maintenance of secondary sex characteristics and libido. There is no overall cyclicity of this activity in men.

The Testis

Unlike the ovaries, the testes reside outside the abdominal cavity in the scrotum (

Fig. 44.1). This location maintains testicular temperature at about 2°C lower than body temperature, which is crucial for optimal sperm development. The human testis is covered by a connective tissue capsule and divided into about 300 lobules by fibrous septa (

Fig.

44.2 ). Within each lobule are two to four loops of seminiferous tubules. Each loop empties into an anastomosing network of tubules called the rete testis. The rete testis is continuous with small ducts, the efferent ductules, that lead the sperm out of the testis into the head of the epididymis on the superior pole of the testis (see

Fig. 44.2 ). Once in the epididymis, the sperm pass from the head, to the body, to the tail of the epididymis and then to the vas (ductus) deferens. Viable sperm can be stored in the tail of the epididymis and vas deferens for several months.

• Fig. 44.1 Anatomy of the male reproductive system. (Modified from Drake RL et al. Gray’s Anatomy for Students. Philadelphia: Churchill Livingstone; 2005.)

The presence of the seminiferous tubules creates two compartments within each lobule: an intratubular compartment, which is composed of the avascular seminiferous epithelium of the seminiferous tubule, and a peritubular compartment, which is composed of neurovascular elements, connective tissue cells, immune cells, and the interstitial cells of Leydig, whose main function is to produce testosterone (

Fig. 44.3).

The seminiferous tubule is lined by a complex seminiferous epithelium composed of two cell types: sperm cells in various stages of spermatogenesis and the Sertoli cell, which is a “nurse cell” in intimate contact with all sperm cells (Fig. 44.4

Spermatogenesis involves the processes of mitosis and meiosis. Stem cells called spermatogonia reside at the basal level of the seminiferous epithelium (see

Fig. 44.4,

SA and SB). Spermatogonia divide mitotically to generate daughter spermatogonia (spermatocytogenesis). One or more spermatogonia remain within the stem cell population, firmly adherent to the basal lamina. However, the majority of these daughter spermatogonia enter meiotic division, which results in haploid spermatozoa on completion of meiosis. These divisions are accompanied by incomplete cytokinesis such that all daughter cells remain interconnected by a cytoplasmic bridge. This configuration contributes to the synchrony of development of a clonal population of sperm cells. Spermatogonia migrate apically away from the basal lamina as they enter the first meiotic prophase. At this time they are called primary spermatocytes (see

Fig. 44.4 , S1). During the first meiotic prophase the hallmark processes of sexual reproduction involving chromosomal reduplication, synapsis, crossing over, and homologous recombination take place. Completion of the first meiotic division gives rise to secondary spermatocytes, which quickly (i.e., within 20 minutes) complete the second meiotic division. The initial products of meiosis are haploid spermatids (see

Fig. 44.4,

S3).

Spermatids are small round cells that undergo a remarkable metamorphosis called spermiogenesis (

Fig. 44.5). The products of spermiogenesis are the streamlined spermatozoa see

Fig. 44.4 , S4). As the spermatid matures into a spermatozoon the size of the nucleus decreases and a prominent tail is formed. The tail contains microtubular structures that propel sperm, similar to a flagellum. The chromatin material in the sperm nucleus condenses, and most of the cytoplasm is lost. The acrosome is a membrane-enclosed structure on the head of the sperm that acts as a lysosome

CHAPTER 44 The Male and Female Reproductive Systems 789

Ligamentous remnant Vas deferens of processus vaginalis

Fibrous septum Head of epididymis

Body of epididymis

Tail of epididymis • Fig. 44.3 Histology of a testicular lobule. (From Young B etal. Wheater’s Functional Histology. A Text and Colour Atlas. 5th ed. London:ChurchillLivingstone;2006.) and contains hydrolytic enzymes that are important for fertilization. These enzymes remain inactive until the acrosomal reaction occurs (see

Fig. 44.4 , S4) are found at the luminal surface of the seminiferous tubule. Release of sperm, or spermiation, is controlled by Sertoli cells. The process of • Fig. 44.4 Histology of a seminiferous tubule. M, myoid cell just outside the basal lamina; S1, primary spermatocyte; S3, spermatid; S4, mature spermatid or spermatozoon; SB and SA, spermatogonia; St,Sertolicell.(FromYoungBetal.Wheater’s Functional Histology. A Text and Colour Atlas.5thed.London:ChurchillLivingstone;2006.) spermatogenesis takes about 72 days. A cohort of adjacent spermatogonia enter the process every 16 days so that the process is staggered at one point along a seminiferous tubule. In addition the process is staggered along the length of a seminiferous tubule (i.e., not all spermatogonia enter the process of spermatogenesis at the same time along the •Fig. 44.5 Structureofspermcellsduringtheprocessofspermatogenesisandspermiogenesis.

entire length of the tubule or in synchrony with every other tubule; there are about 500 seminiferous tubules per testis; see later). Because the seminiferous tubules within one testis are about 400 m in length, spermatozoa are continually being generated at many sites within the testis at any given time.

The Sertoli Cell

Sertoli cells are the true epithelial cells of the seminiferous epithelium and extend from the basal lamina to the lumen (see

Fig. 44.4 , St). Sertoli cells surround sperm cells and provide structural support within the epithelium, and they form adhering and gap junctions with all stages of sperm cells. Through formation and breakdown of these junctions, Sertoli cells guide sperm cells toward the lumen as they advance to later stages of spermatogenesis. Spermiation requires the final breakdown of Sertoli–sperm cell junctions.

Another important structural feature of Sertoli cells is the formation of tight junctions between adjacent Sertoli cells (

Fig. 44.6 ). These Sertoli-Sertoli cell occluding junctions divide the seminiferous epithelium into a basal compartment containing the spermatogonia and early-stage primary spermatocytes and an adluminal compartment containing later-stage primary spermatocytes and all subsequent stages of sperm cells. As early primary spermatocytes move apically from the basal to the adluminal compartment, the tight junctions need to be disassembled and reassembled. These tight junctions form the physical basis for the blood-testis barrier (see

Fig. 44.6 ), which creates a specialized immunologically safe microenvironment for developing sperm. By blocking paracellular diffusion the tight junctions restrict movement of substances between blood and the developing germ cells through a trans–Sertoli cell transport pathway and in this manner allow the Sertoli cell to control the availability of nutrients to germ cells.

Healthy Sertoli cell function is essential for sperm cell viability and development. In addition, spermatogenesis is absolutely dependent on testosterone produced by peritubular Leydig cells (see

The Leydig Cell ), yet it is the Sertoli cells that express the androgen receptor and respond to testosterone, not the developing sperm cells. Similarly the pituitary hormone follicle-stimulating hormone (FSH) is also required for maximal sperm production, and again it is the Sertoli cell that expresses the FSH receptor, not the developing sperm. Thus these hormones support spermatogenesis indirectly through stimulation of Sertoli cell function.

Sertoli cells have multiple additional functions. They express the enzyme CYP19 (also called aromatase), which converts Leydig cell–derived testosterone to the potent estrogen estradiol-17β (see ). This local production of estrogen may enhance spermatogenesis in humans. Sertoli cells also produce androgen-binding protein (ABP), which maintains a high androgen level within the adluminal compartment, the lumens of the seminiferous tubules, and the proximal part of the male reproductive tract. Sertoli cells also produce a large amount of fluid. This fluid provides an appropriate bathing medium for the sperm and assists in moving the immotile spermatozoa from the seminiferous tubule into the epididymis. Sertoli cells perform an important phagocytic function by engulfing residual bodies, which represent cytoplasm shed by spermatozoa during spermiogenesis.

Finally, the Sertoli cell has an important endocrine role. During development, Sertoli cells produce antimüllerian hormone (AMH; also called müllerian inhibitory substance), which induces regression of the embryonic müllerian duct that is programmed to give rise to the female reproductive tract (discussed later). The Sertoli cells also produce the hormone inhibin. Inhibin is a heterodimer protein hormone related to the transforming growth factor-β family. FSH stimulates inhibin production, which then negatively feeds back on gonadotropes to inhibit FSH production. Thus inhibin keeps FSH levels within a set point.

The peritubular compartment contains the primary endocrine cell of the testis, the Leydig cell (

Fig. 44.7 ). This compartment also contains the common cell types of loose connective tissue and an extremely rich peritubular capillary network that provides nutrients to the seminiferous tubules (by way of Sertoli cells) while conveying testosterone away from the testes to the peripheral circulation.

The Leydig Cell

Leydig cells are steroidogenic stromal cells. These cells synthesize cholesterol de novo, as well as acquire it through low-density lipoprotein (LDL) receptors and high-density lipoprotein (HDL) receptors (also called scavenger receptor BI [SR-BI]), and store cholesterol as cholesterol esters as

CHAPTER 44 The Male and Female Reproductive Systems 791 • Fig. 44.6 Placement of germ cells within seminiferous tubule as they progress through spermatogenesis (From Carlson BM. Human Embryology and Developmental Biology. Philadelphia: Mosby; 2004.) ). Free to progesterone, 17-hydroxyprogesterone, and androstenecholesterol is generated by a cholesterol ester hydrolase and dione by 3β-hydroxysteroid dehydrogenase (3β-HSD) and transferred to the outer mitochondrial membrane and then CYP17 (

Fig. 44.8).

CYP17 to the inner mitochondrial membrane in a steroidogenic is a bifunctional enzyme with 17-hydroxylase activity acute regulatory (StAR) protein–dependent manner. and 17,20-lyase activity. CYP17 displays a robust level As in all steroidogenic cells, cholesterol is converted to of both activities in the Leydig cell. In this respect the pregnenolone by CYP11A1. Pregnenolone is then processed Leydig cell is similar to the zona reticularis cell, except that it expresses a higher level of 3β-HSD, so the Δ4 pathway is ultimately favored. Another major difference is that the Leydig cell expresses a Leydig cell–specific isoform of •Fig. 44.7 Histologyoftheperitubularspace(betweenthreesemi-niferoustubules)containingLeydigcells(L)andrichlyvascularizedbyperitubularcapillaries(cap).(ModifiedfromYoungBetal.Wheater’s Functional Histology. A Text and Colour Atlas. 5thed.London:ChurchillLivingstone;2006.)capSeminiferoustubuleSeminiferoustubuleSeminiferoustubuleLcapSeminiferoustubuleSeminiferoustubuleSeminiferoustubuleL 17β-hydroxysteroid dehydrogenase (17β-HSD type 3), which efficiently converts androstenedione to testosterone (see

Fig. 44.8

Fates and Actions of Androgens

The testosterone produced by Leydig cells has several fates and multiple actions. Because of the proximity of Leydig cells to the seminiferous tubules, significant amounts of testosterone diffuse into the seminiferous tubules and become concentrated within the adluminal compartment by ABP (see

Fig. 44.8 ). Testosterone levels within the seminiferous tubules that are greater than 100 times more concentrated than circulating testosterone levels are absolutely required for normal spermatogenesis. As mentioned earlier, Sertoli cells express the enzyme CYP19 (aromatase), which converts a small amount of testosterone into the highly potent estrogen estradiol-17β. Human sperm cells express at least one isoform of the estrogen receptor, and there is some evidence from aromatase-deficient men that this locally produced estrogen optimizes spermatogenesis in humans.

Peripheral Conversion to Estrogen

In several tissues (especially adipose tissue), testosterone is converted to estrogen (see

Fig. 44.8 ). Studies involving men with aromatase deficiency have shown that an inability to produce estrogen results in tall stature because of the lack of epiphyseal closure in long bones, as well as osteoporosis. Thus peripheral estrogen plays an important role in bone maturation and biology in men. These studies also implicated estrogen in promoting insulin sensitivity, improving lipoprotein profiles (i.e., increasing HDL, decreasing triglycerides and LDL), and exerting negative feedback on gonadotropins at the pituitary and hypothalamus.

Peripheral Conversion to Dihydrotestosterone

Testosterone can also be converted into a potent nonaromatizable androgen, 5αα-dihydrotestosterone (DHT), by the enzyme 5α-reductase (see

Fig. 44.8 ). There are two isoforms of 5α-reductase, type 1 and type 2. Major sites of 5α-reductase 2 expression are the male urogenital tract, genital skin, hair follicles, and liver. 5α-Reductase 2 generates DHT, which is required for masculinization of the external genitalia in utero and for many of the changes associated with puberty, including growth and activity of the prostate gland (see ), growth of the penis, darkening and folding of the scrotum, growth of pubic and axillary hair, growth of facial and body hair, and increased muscle mass (

Fig. 44.9 ). The onset of 5α-reductase 1 expression occurs at puberty. This isozyme is expressed primarily in the skin and contributes to sebaceous gland activity and the acne associated with puberty. Because DHT has strong growth-promoting (i.e., trophic) effects on its target organs, development of selective 5α-reductase 2 inhibitors has benefited the treatment of prostatic hypertrophy and prostatic cancer.

Testosterone has a direct action (i.e., without conversion to DHT) in several cell types (see

Fig. 44.9).

As mentioned earlier, testosterone regulates Sertoli cell function. It induces development of the male tract from the mesonephric duct in the absence of 5α-reductase. Testosterone has several metabolic effects, including increasing very low-density lipoprotein (VLDL) and LDL while decreasing HDL, promoting deposition of abdominal adipose tissue, increasing red blood cell production, promoting bone growth and health, and exerting a protein anabolic effect on muscle. Testosterone is sufficient to maintain erectile function and libido.

Mechanism of Androgen Action

Testosterone and DHT act through the same androgen receptor (AR). The AR resides in the cytoplasm bound to chaperone proteins in the absence of ligand.Testosterone-AR

HO 3˜-HSD CYP17 (17-hydroxylase) CH3 CH3 CH3 C O CH3 OH Pregnenolone OH CH3 CH3 CH3 O C O CH3 CH3 CH3 O H C O Progesterone 17(OH) Progesterone Type III 17˜-HSD Into seminiferous tubule T + ABP T • ABP (predominantly as T) SHBG + T CH3 CH3 O O Androstenedione CH3 CH3 O OH Testosterone (T) CH3 CH3 O OH Dihydrotestosterone (DHT) Into peripheral circulation CYP19 (adipose tissue) 5°-Reductase (genital skin, prostate) Estradiol-17˜Leydig cell HO SHBG • T •Fig. 44.8 SteroidogenicpathwayinLeydigcells(thefirststepofconvertingcholesteroltopregnenoloneisomitted).Testosteroneissequesteredbybindingtoandrogen-bindingprotein(ABP)withintheseminiferoustubulesorcirculateswithintheperipheralcirculationboundtosexhormone–bindingglobulin(SHBG)andcanbeperipherallyconvertedtodihydrotestosterone(DHT)orestradiol-17β (E2).(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) binding or DHT-AR binding causes dissociation of the vicinity of a specific gene’s promoter. It remains unclear how chaperone proteins, followed by nuclear translocation of testosterone and DHT differ in their ability to activate the the androgen-AR complex, dimerization, binding to an AR in the context of different cell types, although the pres-androgen response element (ARE), and recruitment of ence of different coactivator proteins in different cell types coactivator proteins and general transcription factors to the is probably involved. Such coactivator proteins would have

Feedback suppression of gonadotropin secretion

Imprint male pattern of gonadotropins, sex drive, behavior • Fig. 44.9 Spectrum of effects of testosterone (T). Note that some effects result from the action of testosterone itself, whereas others are mediated by dihydrotestosterone (DHT) and estradiol (E2) after they are produced from testosterone. VLDL, LDL, HDL, very-low-density, low-density, and high-density lipoproteins, respectively.

different affinities for the testosterone-induced configuration of the AR versus the DHT-induced configuration of the AR.

Transport and Metabolism of Androgens

As testosterone enters the peripheral circulation, it binds to and quickly reaches equilibrium with serum proteins. About 60% of circulating testosterone is bound to sex hormone–binding globulin (SHBG), 38% is bound to albumin, and about 2% remains as “free” hormone. Testosterone and its metabolites are primarily excreted in urine. Approximately 50% of excreted androgens are found as urinary 17-ketosteroids, with most of the remainder being conjugated androgens or diol or triol derivatives. Only about 30% of the 17-ketosteroids in urine are from the testis; the rest are produced from adrenal androgens. Androgens are conjugated with glucuronate or sulfate in the liver, and these conjugated steroids are excreted in urine.

The testis is regulated by an endocrine axis (Fig. 44.10 ) involving parvicellular hypothalamic gonadotropin-releasing hormone (GnRH) neurons and pituitary gonadotropes that produce both luteinizing hormone (LH) and follicle-stimulating hormone (FSH).

Regulation of Leydig Cell Function

The Leydig cell expresses the LH receptor, which acts on Leydig cells much like adrenocorticotropic hormone (ACTH, or corticotropin) does on zona fasciculata cells in the adrenal cortex (see ). Rapid effects include hydrolysis of cholesterol esters and new expression of StAR protein. Less acute effects include an increase in steroidogenic enzyme gene expression and expression of the LDL. Over the long term, LH promotes Leydig cell growth and proliferation.

•Fig. 44.10 Thehypothalamic-pituitary-testicularaxis.Abbreviationsasinotherfigures.Kisspeptin hypothalamic neurons GnRH hypothalamic neurons GnRH Pituitary gonadotropes LH FSH Sertoli cell Sperm cells E2 T DHT Leydig cell T (–) (–) (–)(–) (–) Inhibin B

Testosterone, and estradiol produced from peripheral conversion of testosterone, negatively feed back on GnRH hypothalamic neurons indirectly through inhibition of kisspeptin-producing neurons (see

Fig. 44.10 ). Testosterone and estradiol also negatively feed back on pituitary gonadotropes. DHT, the other major product of peripheral conversion of testosterone, has little effect on LH or FSH levels.

Regulation of Sertoli Cell Function

The Sertoli cell is stimulated by both testosterone and FSH. In addition to stimulating synthesis of proteins involved in the “nurse cell” aspect of Sertoli cell function (e.g., ABP), FSH stimulates synthesis of the dimeric protein inhibin. Inhibin is induced by FSH and negatively feeds back on the gonadotrope to selectively inhibit FSH production (see

Fig. 44.10

Once spermatozoa emerge from the efferent ductules, they leave the gonad and enter the male reproductive tract (see

Fig. 44.1 ). The segments of the tract are the: epididymis (head, body, and tail), vas deferens, ejaculatory duct, prostatic urethra, membranous urethra, and penile urethra. Unlike the female tract, there is a contiguous lumen from the seminiferous tubule to the end of the male tract (i.e., the tip of the penile urethra), and the male reproductive tract connects to the distal urinary tract (i.e., male urethra). In addition to conveying sperm, the primary functions of the male reproductive tract are: 1. Sperm maturation. Sperm spend about a month in the epididymis, where they undergo further maturation.

Thereisanimportant“loophole”inthemalereproductiveaxisthatisbasedonthefactthatintratesticular levels of testosterone needtobegreaterthan100-foldhigherthancirculatinglevelsofthehormonetomaintainnormalratesofspermatogenesis,yetitisthecirculating levels of testosterone (and estradiol) thatprovidethenegativefeedbacktothepituitaryandhypothalamus.ThismeansthatexogenousadministrationoftestosteronecanraisecirculatinglevelsoftestosteroneandestradiolsufficienttoinhibitLHbutnotsufficienttoaccumulateinthetestisattherequiredconcentrationfornormalspermatogenesis.However,thedecreasedLHlevelswilldiminishintratesticularproductionoftestosteronebyLeydigcells,whichresultsinreducedlevels ofspermatogenesis(Fig.44.11 ).This“loophole”iscurrentlybeinginvestigatedasapossiblestrategyfordevelopingamale oral contraceptive. Itisalsothebasisforsterility insomecasesofsteroid abuse inmen.

The epithelium of the epididymis is secretory and adds numerous components to the seminal fluid. Spermatozoa that enter the head of the epididymis are weakly motile but are strongly unidirectionally motile by the time they exit the tail. Spermatozoa also undergo the process of decapacitation, which involves changes in the cell membrane to prevent spermatozoa from undergoing the acrosome reaction before contact with an egg (see later). Sperm become capacitated by the female reproductive tract within the oviduct. The function of the epididymis is dependent on luminal testosterone-ABP complexes that come from the seminiferous tubules and on testosterone from blood.

Normal set point determined by relatively low levels of circulating T, not intratesticular T •Fig. 44.11 Thedifferenceinintratesticulartestosteroneversuscirculatingtestosteroneconcentrationsanditsimportanceinthehypothalamic-pituitary-testisaxis.Upper panel, Feedbackloopinanormaladultman.Lower panel, Administrationoftestosterone(oranandrogenicanalogue)increasescirculatingtestosterone(androgen)levels,whichinturnincreasenegativefeedbackonreleaseofLH.DecreasedLHlevelsdiminishLeydigcellactivityandintratesticularproductionofandrogen.Loweredintratesticulartestosteronelevelsresultinreducedspermproductionandcancauseinfertility.(Theinhibinfeedbackloophasbeenomittedfromthisdiagram.)(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) 2.

Sperm storage and emission. Sperm are stored in the tail The vas deferens has a very thick muscularis that is richly of the epididymis and vas deferens for several months innervated by sympathetic nerves. Normally in response without loss of viability. The primary function of the vas to repeated tactile stimulation of the penis during coitus, deferens, besides providing a storage site, is to propel the muscularis of the vas deferens receives bursts of symsperm during sexual intercourse into the male urethra. pathetic stimulation that cause peristaltic contractions.

Emptying of the contents of the vas deferens into the prostatic urethra is called emission. Emission immediately precedes ejaculation, which is the propulsion of semen out of the male urethra.

3.

Production and mixing of sperm with seminal contents. During emission, contraction of the vas deferens coincides with contraction of the muscular coats of the two accessory sex glands, the seminal vesicles (right and left) and the prostate gland (which surrounds the prostatic urethra). At this point, sperm become mixed with all the components of semen. The seminal vesicles secrete approximately 60% of the volume. These glands are the primary source of fructose, a critical nutrient for sperm. The seminal vesicles also secrete semenogelins, which induce coagulation of semen immediately after ejaculation. The alkaline secretions of the prostate, which make up about 30% of the volume, are high in citrate, zinc, spermine, and acid phosphatase. Prostate-specific antigen (PSA) is a serine protease that liquefies coagulated semen after a few minutes. PSA can be detected in blood under conditions of prostatic infection, benign prostatic hypertrophy, and prostatic carcinoma and is currently used as one indicator of prostatic health. The predominant buffers in semen are phosphate and bicarbonate. A third accessory gland, the bulbourethral glands (also called Cowper’s glands), empty into the penile urethra in response to sexual excitement before emission and ejaculation. This secretion is high in mucus, which lubricates, cleanses, and buffers the urethra. Average sperm counts are between 60 and 100 million/mL semen. Men with sperm counts below 20 million/mL, less than 50% motile sperm, or less than 60% normally conformed sperm are usually infertile.

4.

Erection, penetration, and ejaculation. Emission and ejaculation occur during coitus in response to a reflex arc that involves sensory stimulation from the penis (via the pudendal nerve) followed by sympathetic motor stimulation to the smooth muscle of the male tract and somatic motor stimulation to the musculature associated with the base of the penis. However, for sexual intercourse to occur in the first place, the man has to achieve and maintain an erection of the penis. The penis has evolved as an intromittent organ designed to separate the walls of the vagina, pass through the potential space of the vaginal lumen, and deposit semen at the distal end of the vaginal lumen near the cervix. This process of internal insemination can be performed only if the penis is stiffened from the process of erection. Erection is a neurovascular event. The penis is composed of three erectile bodies: two corpora cavernosa and one corpus spongiosum (Fig. 44.12A ). The penile urethra runs through the corpus spongiosum. These three bodies are composed of erectile tissue—an anastomosing network of potential cavernous vascular spaces lined with continuous endothelia within a loose connective tissue support. During the flaccid state, blood flow to the cavernous spaces is minimal (see

Fig. 44.12A). This is due

CHAPTER 44 The Male and Female Reproductive Systems of the vasculature (called the helicine arteries) and shunting of blood flow away from the cavernous spaces. In response to sexual arousal the parasympathetic cavernous nerves innervating the vascular smooth muscle of the helicine arteries release nitric oxide (NO). NO activates guanylyl cyclase, thereby increasing cyclic guanosine monophosphate (cGMP), which decreases intracellular [Ca++] and causes muscular relaxation (see

Fig. 44.12B ). Vasodilation allows blood to flow into the cavernous spaces to induce engorgement and erection. It also presses on veins in the penis and reduces venous drainage (see

Fig. 44.12B

Inabilitytoachieveormaintainanerectionistermederectile dysfunction (ED) andisonecauseofinfertilityinmen.MultiplefactorscanleadtoED,includinginsufficientandrogenproduction;neurovasculardamage(e.g.,fromdiabetesmellitus,spinalcordinjury);structuraldamagetothepenis,perineum,orpelvis;psychogenicfactors(e.g.,depression,performanceanxiety);andprescribedmedicationsandrecreationaldrugs,includingalcoholandtobacco.AmajordevelopmentinthetreatmentofsomeformsoferectiledysfunctionisuseofselectivecGMPphosphodiesteraseinhibitorsthatassistinmaintaininganerection(see

Fig.44.12B

There is no distinct andropause in men. However, as men age, gonadal sensitivity to LH decreases and androgen production drops. As this occurs, serum LH and FSH levels rise. Although sperm production typically begins to decline after age 50, many men can maintain reproductive function and spermatogenesis throughout life.

The female reproductive system is composed of the gonads, called ovaries, and the female reproductive tract, which includes the oviducts, uterus, cervix, vagina, and external genitalia.

The Ovary

The ovary is located within a fold of peritoneum called the broad ligament, usually close to the lateral wall of the pelvic cavity (

Fig. 44.13 ). Because the ovary extends into the peritoneal cavity, ovulated eggs briefly reside within the peritoneal cavity before they are captured by the oviducts.

The ovary is divided into an outer cortex and inner medulla (

Fig. 44.14 ). Neurovascular elements innervate the medulla of the ovary. The cortex of the ovary is composed of a densely cellular stroma. Within this stroma reside the ovarian follicles (see

Fig. 44.14, F ), which contain a primary oocyte surrounded by follicle cells. The cortex is covered by a connective tissue capsule, the tunica albuginea, (tactile, visual, auditory, psychic)

Collapse of venous return from cavernous spaces

Contraction of muscles around base of penis • Fig. 44.12 A, Arrangement of the vasculature and cavernous tissue within the penis. During the flaccid state, blood flow into the cavernous spaces is limited by contraction of the helicine arteries. B, Outline of neurovascular events leading to penile erection. (A, From Bhasun S et al. In: Larsen P et al [eds]. Williams Textbook of Endocrinology. 10th ed. Philadelphia: Saunders; 2003.)

CHAPTER 44 The Male and Female Reproductive Systems 799

Labia minora Recto-uterine pouch of douglas • Fig. 44.13 Anatomy of the female reproductive system. (Modified from Drake RL et al. Gray’s Anatomy for Students. Philadelphia: Churchill Livingstone; 2005.) • Fig. 44.14 Histology of the ovary. CL, corpus luteum; F, follicle. (Modified from Young B et al. Wheater’s Functional Histology. A Text and Colour Atlas. 5th ed. London: Churchill Livingstone; 2006.) and a layer of simple epithelium consisting of ovarian surface epithelial cells. There are no ducts emerging from the ovary to convey its gametes to the reproductive tract. Thus the process of ovulation involves an inflammatory event that erodes the wall of the ovary. After ovulation the ovarian surface epithelial cells rapidly divide to repair the wall. The majority of ovarian cancer originates from this highly proliferative epithelium.

Growth, Development, and Function  of the Ovarian Follicle

The ovarian follicle is the functional unit of the ovary, and it performs both gametogenic and endocrine functions. A histological section of the ovary from a premenopausal cycling woman contains follicular structures at many different stages of development. The life history of a follicle can be divided into the following stages: 1.

2.

3.

4.

dominant (preovulatory, graafian) follicle 5.

dominant follicle within the periovulatory period 6.

corpus luteum (of menstruation or of pregnancy) 7.

Fig. 44.15 ) represent the earliest and simplest follicular structure in the ovary. Primordial follicles appear during midgestation through the interaction of gametes and somatic cells. Primordial germ cells that have migrated to the gonad continue to divide mitotically as oogonia until the fifth month of gestation in humans. At this point the approximately 7 million oogonia enter the process of meiosis and become primary oocytes. During this time the primary oocytes become surrounded by a simple epithelium of somatic follicle cells, thereby creating the primordial follicles (see

Fig. 44.15 ). The follicle cells establish gap junctions with each other and the oocyte. The follicle cells themselves represent a true avascular epithelium surrounded by a basal lamina. Similar to Sertoli cell–sperm interactions, a subpopulation of granulosa cells remains intimately attached to the oocytes throughout their development. Granulosa cells provide nutrients such as amino acids, nucleic acids, and pyruvate to support oocyte maturation.

•Fig. 44.15 Developmentofaprimordialfollicleuptoasecondarypreantralfollicle.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)Primordial follicle Primary oocyte Basal lamina Pregranulosa cells Germinal vesicle with nucleolus Zona pellucida Cuboidal granulosa cells Primary follicle Secondary preantral follicle Theca Stratified granulosa cells

The primordial follicles represent the ovarian reserve of follicles (

Fig. 44.16). This reserve is reduced from a starting number of about 7 million to less than 300,000 follicles at reproductive maturity. Of these, a woman will ovulate about 450 between menarche (first menstrual cycle) and menopause (cessation of menstrual cycles). At menopause, less than 1000 primordial follicles are left in the ovary. Primordial follicles are lost primarily from death as a result of follicular atresia. However, a small subset of primordial follicles will enter follicular growth in waves. Because the ovarian follicular reserve represents a fixed finite number, the rate at which resting primordial follicles die or begin to develop (or both) will determine the reproductive life span of a woman. Age at the onset of menopause has a strong genetic component but is also influenced by environmental factors. For example, cigarette smoking significantly depletes the ovarian reserve. An overly rapid rate of atresia or development will deplete the reserve and give rise to premature ovarian insufficiency.

Pituitary gonadotropins maintain a normal ovarian reserve by promoting the general health of the ovary. However, the rate at which resting primordial follicles enter the growth process appears to be independent of pituitary gonadotropins. The decision of a resting follicle to enter the early growth phase is primarily dependent on intraovarian paracrine factors produced by both the follicle cells and oocytes.

The Gamete

In primordial follicles the gamete is derived from oogonia that have entered the first meiotic division; such oogonia are referred to as primary oocytes. Primary oocytes progress through most of prophase of the first meiotic division

Ovarian reserve ~300,000 primordial follicles at menarche Atresia ~270,000 primordial follicles Growth ~30,000 primordial follicles Atresia About 30,000 primary, secondary, or tertiary follicles Ovulation ~450 dominant follicles •Fig. 44.16 Fateofovarianfollicles.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) (termed prophase I) over a 2-week period and then arrest in the diplotene stage. This stage is characterized by the decondensation of chromatin, which supports the transcription needed for oocyte maturation. Meiotic arrest at this stage, which may last for up to 50 years, appears to be due to “maturational incompetence,” or lack of the cell cycle proteins needed to support the completion of meiosis. The nucleus of the oocyte, called the germinal vesicle, remains intact at this stage.

The first stage of follicular growth is preantral, which refers to the development that occurs before the formation of a fluid-filled antral cavity. One of the first visible signs of follicle growth is the appearance of cuboidal granulosa cells. At this point the follicle is referred to as a primary follicle (see

Fig. 44.15 ). As granulosa cells proliferate, they form a multilayered (i.e., stratified) epithelium around the oocyte. At this stage the follicle is referred to as a secondary follicle (see

Fig. 44.15

Once a secondary follicle acquires three to six layers of granulosa cells, it secretes paracrine factors that induce nearby stromal cells to differentiate into epithelioid thecal cells. Thecal cells form a flattened layer of cells around the follicle. Once a thecal layer forms, the follicle is referred to as a mature preantral follicle (see

Fig. 44.15 ). In humans it takes several months for a primary follicle to reach the mature preantral stage.

Follicular development is associated with an inward movement of the follicle from the outer cortex to the inner cortex, closer to the vasculature of the ovarian medulla. Follicles release angiogenic factors that induce development of one to two arterioles that form a vascular wreath around the follicle.

The Gamete

During the preantral stage, the oocyte begins to grow and produce cellular and secreted proteins. The oocyte initiates secretion of extracellular matrix glycoproteins called ZP1, ZP2, and ZP3 that form the zona pellucida (see

Fig. 44.15 ). The zona pellucida increases in thickness and provides a species-specific binding site for sperm during fertilization (see ). Importantly, granulosa cells and the oocyte maintain gap junctional contact via cellular projections through the zona pellucida. The oocyte also continues to secrete paracrine factors that regulate follicle cell growth and differentiation.

Granulosa cells express the FSH receptor during this period, but they are primarily dependent on factors from the oocyte to grow. They do not produce ovarian hormones at this early stage of follicular development. The newly acquired thecal cells are analogous to testicular Leydig cells in that they reside outside the epithelial “nurse” cells, express the LH receptor, and produce androgens. The main difference •Fig. 44.17 Developmentofanearlyantralfollicletoamaturepreovulatoryfollicle.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) between Leydig cells and thecal cells is that thecal cells do not express high levels of 17β-HSD. Thus the major product of theca cells is androstenedione as opposed to testosterone. Androstenedione production at this stage is minimal.

Fig. 44.17 ) over a period of about 25 days, during which they grow from a diameter of about 0.1 mm to a diameter of 0.2 mm. Once the granulosa epithelium increases to six to seven layers, fluid-filled spaces appear between cells and coalesce into the antrum. Over a period of about 45 days, this wave of small antral follicles will continue to grow to large recruitable antral follicles that are 2 to 5mm in diameter. This period of growth is characterized by about a 100-fold increase in granulosa cells (from about 10,000 to 1,000,000 cells). It is also characterized by swelling of the antral cavity, which increasingly divides the granulosa cells into two discrete populations: mural granulosa cells and cumulus cells (see

Fig. 44.17

Mural granulosa cells (also called the stratum granulosum) form the outer wall of the follicle. The basal layer is adherent to the basal lamina and in close proximity to 802 SECTION8Berne & Levy Physiology Oogonium Primary oocyte arrested at prophase I Meiosis begins, but levels of proteins required for completion of meiosis are too low – oocyte arrests at prophase I.

As oocyte grows, it synthesizes enough proteins (e.g., CDK1, cyclin B) to complete meiosis (i.e., meiotic competence), but high cAMP levels generated by GPR3 actively maintain arrest.

A few hours before ovulation, the oocyte completes meiosis I and extrudes the first polar body. It has synthesized enough MAPK pathway proteins to arrest at metaphase II.

The secondary oocyte completes meiosis at fertilization and extrudes second polar body. Haploid ovum •Fig. 44.18 Eventsinvolvedinmeioticarrestandmaturationoftheoocyte.MAPK,mitogen-activatedproteinkinase.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) the outer-lying thecal layers. Mural granulosa cells become highly steroidogenic and remain in the ovary after ovulation to differentiate into the corpus luteum.

Cumulus cells are the inner cells that surround the oocyte (they are also referred to as the cumulus oophorus and corona radiata). The innermost layer of cumulus cells maintains gap and adhesion junctions with the oocyte. Cumulus cells are released with the oocyte (collectively referred to as the cumulus-oocyte complex) during the process of ovulation. Cumulus cells are crucial for the ability of the fimbriated end of the oviduct to “capture” and move the oocyte by a ciliary transport mechanism along the length of the oviduct to the site of fertilization (see

Early antral follicles are dependent on pituitary FSH for normal growth. Large antral follicles become highly dependent on pituitary FSH for their growth and sustained viability. As discussed later, 2to 5-mm follicles are recruited to enter a rapid growth phase via the transient increase in FSH that occurs toward the end of the previous menstrual cycle.

The Gamete

The oocyte grows rapidly in the early stages of antral follicles; growth then slows in larger follicles. During the antral stage the oocyte synthesizes sufficient amounts of cell cycle components so it becomes competent to complete meiosis I at ovulation. (Note that the human egg arrests after ovulation at a second point, metaphase II, until it is fertilized by sperm.) Thus in early primary and secondary follicles, the oocyte fails to complete meiosis I because of a dearth of specific meiosis-associated proteins. However, larger antral follicles gain meiotic competence but still maintain meiotic arrest until the midcycle LH surge. Meiotic arrest is achieved by maintenance of elevated cyclic adenosine monophosphate (cAMP) levels in the mature oocyte (

Figs.

•Fig. 44.19 ModelofhowLHsurgeleadstoresumptionofmeiosis.PDE,phosphodiesterase.Granulosa & cumulus cells ZP GPR3 AMP cAMP PKA PDE 3A MEIOTIC ARREST (–) (–) LH SURGE LHSURGEcGMPcAMPMEIOTICRESUMPTIONGsACPDE3AcGMP cGMP cGMP 44.18 44.19 ). The constitutively active (i.e., not requiring a ligand) Gs protein-coupled receptor GPR3 maintains high cAMP. The oocyte-specific phosphodiesterase PDE3A degrades cAMP to inactive AMP. Before the LH surge, PDE3A is inhibited by cGMP, which is produced within cumulus and granulosa cells and enters the oocyte via gap junctions.

•Fig. 44.20 Two-cellmodelforsteroidogenesisinthedominantfollicle.Top panel: MG,muralgranulosa;T,theca.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)LHreceptor LH LDLR and HDLR-mediated uptake FSH receptor Expression of LH receptor Expression of CYP19 (aromatase) LH receptor FSH Expression and activation of StAR protein Expression of steroidogenic enzymes Acetate Cholesterol Pregnenolone Progesterone 17 (OH) Progesterone Androstenedione Testosterone Thecal cell Granulosa cell StAR protein CYP11A1 CYP17 CYP17 CYP19 CYP19 EstroneAndrostenedione Testosterone Estradiol-17˜3˜-HSD 17˜-HSD 17˜-HSD 17˜-HSD Blood vessel Basal lamina MG A a C C T

The thecal cells of large antral follicles produce significant amounts of androstenedione and less testosterone. Androgens are converted to estradiol-17β by the granulosa cells (

Fig. 44.20 ). At this stage, FSH stimulates proliferation of granulosa cells and induces the expression of CYP19 (aromatase) required for estrogen synthesis. Additionally the mural granulosa cells of the large antral follicles produce increasing amounts of inhibin during the early follicular phase. Low levels of estrogen and inhibin negatively feed back on FSH secretion, thereby contributing to selection of the follicle with the most FSH-responsive cells.

As already discussed, at the end of a previous menstrual cycle, a crop of large (2–5 mm) antral follicles (see

Fig.

44.17 ) are recruited by a rise in FSH to begin rapid gonadotropin-dependent development. The total number of recruited follicles in both ovaries can be as high as 20 in a younger woman (<33 years old) but rapidly declines at older ages. The number of recruited follicles is reduced to the ovulation quota (one in humans) by the process of selection. As FSH levels decline the rapidly growing follicles progressively undergo atresia until one follicle is left. Generally the largest follicle with the most FSH receptors of the recruited crop becomes the dominant follicle. Selection occurs during the early follicular phase. By midcycle the dominant follicle becomes a large preovulatory follicle that is 20 mm in diameter and contains about 50 million granulosa cells by the midcycle gonadotropin surge.

The Gamete

The oocyte is competent to complete meiosis I but remains arrested in the dominant follicle until the LH surge. Growth of the oocyte continues but at a slower rate until the oocyte reaches a diameter of about 140 µm by ovulation (i.e., ≈20 times the diameter of an erythrocyte).

The newly selected follicle emerges as a significant steroidogenic “gland.” Ovarian steroidogenesis requires both theca and granulosa cells. As discussed earlier, thecal cells (see

Fig. 44.20, T ) express LH receptors and produce primarily androstenedione. Basal levels of LH stimulate expression of steroidogenic enzymes in thecal cells. The thecal cells are richly vascularized and thus have access to cholesterol within the lipoprotein articles LDL and HDL. LH promotes expression of the LDL receptor and HDL receptor (SR-B1), which import cholesterol. LH also promotes robust expression of CYP11A1 (side chain cleavage enzyme), 3β-HSD, and CYP17 with both 17-hydroxylase activity and 17,20-lyase activity. Androgens (primarily androstenedione but also some testosterone) released from the theca diffuse into the mural granulosa cells or enter the vasculature surrounding the follicle.

The mural granulosa cells (see

Fig. 44.20, MG) of the selected follicle have a high number of FSH receptors and are very sensitive to FSH, which upregulates CYP19 (aromatase) gene expression and activity. CYP19 converts androstenedione to the weak estrogen estrone and converts testosterone to the potent estrogen estradiol-17β. Granulosa cells express activating isoforms of 17β-HSD, which converts the less active estrone to highly active estradiol17β. In addition, FSH induces expression of inhibin B during the follicular phase.

Importantly, FSH also induces expression of LH receptors in mural granulosa cells during the second half of the follicular phase (see

Fig. 44.20). Thus mural granulosa cells acquire the ability to respond to LH, which allows these cells to maintain high levels of CYP19 in the face of declining FSH levels. Acquisition of LH receptors also ensures that mural granulosa cells respond to the LH surge.

The Dominant Follicle During the Periovulatory Period

The periovulatory period is defined as the time from the onset of the LH surge to expulsion of the cumulus-oocyte complex out of the ovary (i.e., ovulation). This process lasts for 32 to 36 hours in women. Starting at the same time and superimposed on the process of ovulation is a change in the steroidogenic function of theca and mural granulosa cells. This process is called luteinization and culminates in formation of a corpus luteum that is capable of producing high amounts of progesterone, along with estrogen, within a few days after ovulation. Thus the LH surge induces the onset of complex processes during the periovulatory period that complete the gametogenic function of the ovary for a given month and switches the endocrine function to prepare the female reproductive tract for implantation and gestation.

The LH surge induces dramatic structural changes in the dominant follicle that involves its rupture, ovulation of the cumulus-oocyte complex, and biogenesis of a new structure called the corpus luteum from the remaining thecal and mural granulosa cells. Major structural changes occur during this transition: 1. Before ovulation the large preovulatory follicle presses against the ovarian surface and generates a poorly vascularized bulge of the ovarian wall called the stigma. The LH surge induces release of inflammatory cytokines and hydrolytic enzymes from the theca and granulosa cells. These secreted components lead to breakdown of the follicle wall, tunica albuginea, and surface epithelium in the vicinity of the stigma (

Fig. 44.21 ). At the end of this process the antral cavity becomes continuous with the peritoneal cavity.

•Fig. 44.21 Ovulation.GVBD,germinalvesiclebreakdown.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadel-phia:Mosby;2013.)Surface epithelium Tunica albugineaLuteinizing theca Blood vessels invading granulosa Luteinizing mural granulosa without basal lamina Peritoneal cavity Ruptured antrum Expanded cumulus Zona pellucida First polar body 2° oocyte after GVDB 2.

The attachment of the cumulus cells to the mural granulosa cells degenerates, and the cumulus-oocyte complex becomes free-floating within the antral cavity (see

Fig. 44.21 ). Cumulus cells also respond to the LH surge by secreting hyaluronic acid and other extracellular matrix components. These substances enlarge the entire cumulus-oocyte complex, a process called cumulus expansion (see

Fig. 44.21 ). This enlarged cumulusoocyte complex is more easily captured and transported by the oviduct. The expanded cumulus also makes the cumulus-oocyte complex easier for spermatozoa to find. Sperm express a membrane hyaluronidase that allows them to penetrate the expanded cumulus. The cumulusoocyte complex is released through the ruptured stigma through a relatively slow process.

3.

The basal lamina of mural granulosa cells is broken down so that blood vessels and outer-lying theca cells can push into the granulosa cells. Granulosa cells secrete angiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietin 2, and basic fibroblast growth factor (bFGF), which significantly increase the blood supply to the new corpus luteum.

The Gamete

Before ovulation the primary oocyte is competent to complete meiosis, but it is arrested in prophase I (see

Fig.

44.18 ). The LH surge inhibits production of cGMP by granulosa and cumulus cells, thereby removing inhibition of the oocyte-specific PDE3A. PDE3A proceeds to degrade cAMP to inactive AMP, thereby removing the brake on meiotic progression. The oocyte then progresses to meta-phase II and subsequently arrests at metaphase II until fertilization.

Both theca and mural granulosa cells express LH receptors at the time of the LH surge. The LH surge induces differentiation of the granulosa cells—a process that continues for several days after ovulation. During the periovulatory period, the LH surge induces the following shifts in steroidogenic activity of the mural granulosa cells (now turning into granulosa lutein cells).

1.

Transient inhibition of CYP19 expression and consequently estrogen production. The rapid decline in estrogen helps turn off the positive feedback on LH secretion.

2.

Breakdown of the basal lamina and vascularization of the granulosa cells. This makes LDL and HDL cholesterol accessible to these cells for steroidogenesis. The LH surge also increases expression of the LDL receptor and HDL receptor (SR-BI) in granulosa cells.

3.

Onset of expression of StAR protein, CYP11A1 (side chain cleavage enzyme), and 3β-HSD (

Fig. 44.22 ). Expression of these enzymes are key to the onset of production of high levels of progesterone by these cells. As discussed later, high progesterone synthesis is absolutely necessary for maintenance of pregnancy. Because CYP17 activity, especially its 17,20-lyase function, is largely absent in granulosa lutein cells, progesterone is not further metabolized to another steroid but instead exits the cells and enters the circulation.

•Fig. 44.22 Steroidogenicpathwaysinthecorpusluteum(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)LH LH receptor LDLR and HDLR-mediated uptake Acetate Cholesterol Stimulation of steroidogenesis LH LH receptor LDLR and HDLR-mediated uptake Cholesterol Pregnenolone Stimulation of steroidogenesis StAR protein CYP11A1 Androstenedione Androstenedione Progesterone Estradiol-17˜3˜-HSD

The Corpus Luteum

After ovulation the remnant of the antral cavity fills with blood from damaged blood vessels in the vicinity of the stigma. This gives rise to a corpus hemorrhagicum. Within a few days, red blood cells and debris are removed by macrophages, and fibroblasts fill in the antral cavity with a hyaline-like extracellular matrix. In the mature corpus luteum the granulosa cells, now called granulosa lutein cells, enlarge and become filled with lipid (cholesterol esters). The enlarged granulosa lutein cells collapse into and partially fill in the old antral cavity. The theca along with blood vessels, mast cells, macrophages, leukocytes, and other resident connective tissue cells infiltrate the granulosa layer at multiple sites.

The human corpus luteum is programmed to live for 14 days, plus or minus 2 days (corpus luteum of menstruation), unless “rescued” by the LH-like hormone human chorionic gonadotropin (hCG), which originates from an implanting embryo. If rescued, the corpus luteum of pregnancy will remain viable during the pregnancy (usually ≈ 9 months). The mechanism by which the corpus luteum of menstruation regresses in 14 days is not fully understood. The corpus luteum appears to become progressively less sensitive to basal levels of LH, so that hCG binding to the LH receptor is needed for continued health and function of the corpus luteum. Regression appears to involve release of the prostaglandin PGF2α from both granulosa lutein cells and the uterus in response to declining levels of progesterone during the second week of the luteal phase. Several paracrine factors (endothelin, monocyte chemotactic protein-1) from immune and vascular cells are likely to play a role in the demise and removal of granulosa lutein cells. The corpus luteum is ultimately turned into a scarlike body called the corpus albicans, which sinks into the medulla of the ovary and is slowly absorbed.

The Gamete

The LH surge induces two parallel events, ovulation and luteinization. If ovulation occurs normally, the corpus luteum is devoid of a gamete.

Before the LH surge, the granulosa cells have very low capacity to convert cholesterol into a steroid hormone. The LH surge induces the onset of expression of CYP11A1, 3β-HSD and StAR protein, allowing granulosa lutein cells to convert cholesterol into progesterone. Because CYP17 expression is extremely low, progesterone accumulates and moves out of the granulosa lutein cells and enters the vasculature. Progesterone production by the corpus luteum (see

Fig. 44.22 ) increases steadily from the onset of the LH surge and peaks during the midluteal phase. The main purpose of this timing is to transform the uterine lining into an adhesive and supportive structure for implantation and early pregnancy. As discussed later the midluteal phase is synchronized with early embryogenesis so the uterus is optimally primed when a blastocyst tumbles into the uterus around day 22 of the menstrual cycle. Estradiol continues to be produced by theca lutein cells and granulosa lutein cells. Estrogen production transiently decreases in response to the LH surge but then rebounds and peaks at the midluteal phase. Estradiol induces the progesterone receptor in progesterone target cells, such as the uterine endometrium, and ensures a full response to progesterone.

Luteal hormonal output is absolutely dependent on basal LH levels (see

Fig. 44.22 ). In fact, progesterone output is closely correlated with the pulsatile pattern of LH release in women. Both FSH and LH are reduced to basal levels during the luteal phase by negative feedback from progesterone and estrogen. In addition, granulosa lutein cells secrete inhibin, which selectively represses FSH secretion.

The corpus luteum must generate large amounts of progesterone to support implantation and early pregnancy. Accordingly the life of the corpus luteum is very regular, and a shortened luteal phase typically leads to infertility. The quality of the corpus luteum is largely dependent on the size and health of the dominant follicle from which it developed, which in turn is dependent on normal hypothalamic and pituitary stimulation during the follicular phase. Numerous factors that perturb hypothalamic and pituitary output during the follicular phase, including heavy exercise, starvation, high prolactin levels, and abnormal thyroid function, can lead to luteal phase deficiency and infertility.

Follicular atresia refers to the demise of an ovarian follicle. During atresia the granulosa cells and oocytes undergo apoptosis. The thecal cells typically persist and repopulate the cellular stroma of the ovary. The thecal cells retain LH receptors and the ability to produce androgens and are collectively referred to as the “interstitial gland” of the ovary. Follicles can undergo atresia at any time during development.

Follicular Development With Respect to the Monthly Menstrual Cycle

The human menstrual cycle strictly refers to the monthly discharge of discarded uterine lining as menstrual blood or menstrual flow (a period referred to as menses) through the process of menstruation (see later). In fact it is the onset or lack thereof of menses, as detected by the woman herself, that is the primary evidence for the cessation of menstruation (e.g., due to pregnancy or menopause) or a change in the duration and/or frequency of the menstrual cycle. However, it is useful from an endocrinological perspective to consider the human menstrual cycle as having an ovarian cycle and a uterine cycle, with the latter being driven by the former. As discussed later, there are also hypothalamic, pituitary, oviductal, and vaginal components of the human menstrual cycle. The reproductive function of the menstrual cycle is the collective orchestration by ovarian hormones of the functions of the hypothalamus, pituitary, uterus, oviduct, cervix, and vagina—and even the ovary itself—to: (1) produce a fertilizable gamete (egg), (2) provide a supportive environment for intercourse, reception of sperm, fertilization of egg and early embryogenesis, (3) prepare the uterine lining for implantation, placentation and pregnancy, and (4) minimize the possibility of a superimplantation (i.e., a second implantation) from occurring, and/or prevent an ascending infection from moving up to the uterus from the vagina.

The first half of the monthly menstrual cycle is referred to as the follicular phase of the ovary and is characterized by recruitment and growth of a large antral follicle, selection of the dominant follicle, and growth of the dominant follicle until ovulation. The dominant follicle must contain a fully developed oocyte and somatic follicle cells that secrete high levels of estrogen.

The second half of the monthly menstrual cycle is referred to as the luteal phase of the ovary and is dominated by hormonal secretions of the corpus luteum. The corpus luteum must secrete both progesterone and estradiol for progression of the normal cycle.

Regulation of Late Stages of Follicular Development, Ovulation, and Luteinization: The Human Menstrual Cycle

As stated earlier, late stages of follicular development and luteal function are absolutely dependent on normal hypothalamic and pituitary function. As in the male, hypothalamic neurons secrete GnRH in a pulsatile manner. GnRH in turn stimulates LH and FSH production by pituitary gonadotropes. A high frequency of GnRH pulses (1 pulse per 60–90 minutes) selectively promotes LH production, whereas a slow frequency promotes FSH production. A major difference between the male and female reproductive axes is the midcycle gonadotropin surge, which is dependent on a constant high level of estrogen coming from the dominant follicle.

A highly dynamic “conversation” occurs among the ovary, pituitary, and hypothalamus in which the events of the menstrual cycle are orchestrated, beginning with the ovary at the end of the luteal phase of a previous nonfertile cycle (

Fig. 44.23 ). Events that follow are numbered according to

Fig. 44.24 : Event 1: In the absence of fertilization and implantation, the corpus luteum regresses and dies (called luteolysis). This leads to a dramatic decline in levels of progesterone, estrogen, and inhibin by day 24 of the menstrual cycle.

Event 2: The pituitary gonadotrope perceives the end of luteal function as a release from negative feedback (see

Fig. 44.23B , Late Luteal Phase). This leads to an increase in FSH about 2 days before the onset of menstruation. The basis for the selective increase in FSH is incompletely understood, but it may be due to the slow frequency of GnRH pulses during the luteal phase, which in turn is due to high progesterone levels.

CHAPTER 44 The Male and Female Reproductive Systems

Event 3: The rise in FSH levels recruits a crop of large (2–5 mm in diameter) antral follicles to begin rapid, highly gonadotropin-dependent growth. These follicles produce low levels of estrogen and inhibin B.

Event 4: The gonadotrope responds to the slowly rising levels of estrogen and inhibin B by decreasing FSH secretion (see

Fig. 44.23A , Early Follicular Phase). The absence of progesterone promotes an increase in the frequency of GnRH pulses, thereby selectively increasing LH synthesis and secretion by the gonadotrope. Thus the LH:FSH ratio slowly increases throughout the follicular phase.

Event 5: The ovary’s response to declining FSH levels is follicular atresia of all of the recruited follicles except for one dominant follicle (see

Fig. 44.23A , Early Follicular Phase). Thus the process of selection is driven by an extreme dependency of follicles on FSH in the face of declining FSH secretion. Usually only the largest follicle with the most FSH receptors and best blood supply can survive. This follicle produces increasing amounts of estradiol-17β and inhibin B. A critical action of FSH at this time is induction of the expression of LH receptors in the mural granulosa cells of the dominant follicle (see

Fig. 44.23A , Late Follicular Phase).

Event 6: Once the dominant follicle causes circulating estrogen levels to exceed 200 pg/mL for about 50 hours in women, estrogen exerts a positive feedback on the gonadotrope to produce the midcycle LH surge. This is enhanced by the small amount of progesterone secreted at midcycle. The exact mechanism of the positive feedback is unknown, but it occurs largely at the level of the pituitary. GnRH receptors and the sensitivity to GnRH signaling increase dramatically in the gonadotropes. The hypothalamus contributes to the gonadotropin surge by increasing the frequency of GnRH pulses.

Event 7: The LH surge drives meiotic maturation, ovulation, and differentiation of granulosa cells into progesterone-producing cells (see

Fig. 44.23A , Late Follicular Phase).

Event 8: Rising levels of progesterone, estrogen, and inhibin A by the mature corpus luteum negatively feed back on pituitary gonadotropes. Even though estradiol levels exceed the 200-pg/mL threshold for positive feedback, the high progesterone levels now produced by the corpus luteum block any positive feedback of estradiol. Consequently both FSH and LH levels decline to basal levels (see

Fig. 44.23B , Mid-Luteal Phase).

Event 9: Basal levels of LH (but not FSH) are absolutely required for normal corpus luteum function. However, the corpus luteum becomes progressively insensitive to LH signaling and will die unless LH-like activity (i.e., hCG from an implanted embryo) increases. In a nonfertile cycle the corpus luteum of menstruation will regress in 14 days, and progesterone and estrogen levels will start to decline by about 10 days, thereby cycling back to event 1 (see

Fig. 44.23B , Late Luteal Phase).

808 SECTION8Berne & Levy Physiology •Fig. 44.23 A,Endocrinesignalingleadingtotheovulationofadominantfollicleattheendofthefollicularphaseofthemenstrualcycle.B,Endocrinesignalingduringthelutealphaseofanon-pregnantmenstrualcycleleadingtothedeathofthecorpusluteumandrecruitmentoffolliclestobeginnextcycle.LATE FOLLICULAR d8-14 EARLY FOLLICULAR d3-7 Dominant follicle COC Ruptured follicle Growing large antral follicles recruited by FSH Decreasing FSH basal LH (–)(–)(+)(+)High E2 no P4 Gonado-tropes GnRH neurons GnRH neurons Kisspeptin neurons Kisspeptin neurons Moderate E2 & inhibin no P4 LH surge @ midcycle GnRH rapid pulses Gonado-tropes GnRH slow pulses • Increasing (–) feedback causing lower FSH • Follicular atresia • Selection of dominant follicle Switch to (+) feedback-especially at level of pituitary sensitivity to rapid GnRH pulses • Meiotic maturation • Luteinization • Ovulation of cumulus-oocyte complex (COC) A. Follicular Phase LATE LUTEAL (d25-28) MID-LUTEAL (d17-24) Corpus luteum from ovulated dominant follicle Large antral recruitable follicles Dying corpus luteum (corpus albicans) If no implantation no rescue by hCG Maintains active corpus luteum FSH recruits large antral follicles new cycle starts Loss of negative feedack from ovary after corpus luteum dies Gonado-tropes GnRH neurons Kisspeptin neurons GnRH rapid pulses Gonado-tropes GnRH neurons Kisspeptin neurons GnRH slow pulses (–)(–)High P4 moderate & inhibition FSH Peak Basal LH & FSH No E2 inhibin or P4 B. Luteal Phase

CHAPTER 44 The Male and Female Reproductive Systems 809 1. Corpus luteum dies, E and P levels fall. 5. Declining FSH levels progressively cause atresia of all but 1 follicle – leading to selection of dominant follicle, which produces high levels of E. 9. The corpus luteum progressively becomes less sensitive to basal LH – dies if levels of LH-like activity (i.e., hCG) do not increase. 3. FSH recruits a cohort of large antral follicles to enter rapid growth phase. Follicles secrete low amounts of E and inhibin. 7. LH surge induces meiotic maturation, ovulation and luteinization. The corpus luteum produces high P, along with E and inhibin.

4. E and inhibin negatively feed back on FSH. FSH LH Pituitary

Progesterone OvaryEstradiol-17˜2. Pituitary responds to falling E and P by increasing FSH secretion. 6. High E has positive feedback on gonadotropes— LH (and some FSH) surges. 8. High P, E and inhibin negatively feed back on LH and FSH, returning them to basal levels.

•Fig. 44.24 Thehumanmenstrualcycle,withemphasisonthe“dialogue”betweenovaryandpituitarygonadotropes.NotethattherelativechangesinthelevelsofE2andinhibinareshownbythesameline.

From this sequence of events it is evident that the ovary is the primary clock for the menstrual cycle. The timing of the two main pituitary-based events—the transient rise in FSH that recruits large antral follicles and the LH surge that induces ovulation—is determined by two ovarian events. These are respectively the highly regular life span of the corpus luteum and its demise after 14 days, and growth of the dominant follicle to the point at which it can maintain a sustained high production of estrogen that induces a switch to positive feedback at the pituitary. In essence the dominant follicle tells the pituitary it is ready to undergo ovulation and luteinization.

The Oviduct

The oviducts (also called the uterine tubes and the fallopian tubes) are muscular tubes with the distal ends close to the surface of each ovary and the proximal ends traversing the wall of the uterus. The oviducts are divided into four sections (going from distal to proximal): the infundibulum, or open end of the oviduct, which has fingerlike projections called fimbriae that sweep over the surface of the ovary; the ampulla, which has a relatively wide lumen and extensive folding of the mucosa; the isthmus, which has a relatively narrow lumen and less mucosal folding; and the intramural or uterine segment, which extends through the uterine wall at the superior corners of the uterus (

Fig. 44.25

The main functions of the oviducts are to: 1.

Capture the cumulus-oocyte complex at ovulation and transfer the complex to a midway point (the ampullary-isthmus junction), where fertilization takes place. Oviductal secretions coat and infuse the cumulusoocyte complex and are likely required for viability and fertilizability.

2.

Provide a site for sperm storage. Women who ovulate up to 5 days after sexual intercourse can get pregnant. Sperm remain viable by adhering to the epithelial cells lining the isthmus. The secretions of the oviduct also induce capacitation and hyperactivity of sperm.

3.

Secrete fluids that provide nutritional support to the preimplantation embryo.

The timing of movement of the embryo into the uterus is critical because the uterus has an implantation window of about 3 days. The oviduct needs to hold the early embryo until it reaches the blastocyst stage (5 days after fertilization) and then allow it to pass into the uterine cavity.

810 SECTION8Berne & Levy Physiology •Fig. 44.25 Schematicofthefemalereproductivesystem.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)Fundus of uterusIsthmusAmpulla Infundibulum with fimbriae Uterine (fallopian) tube Ovary Body (corpus) of uterus Internal genitalia External genitalia Cervix of uterus Vagina Clitoris Vaginal introitus Opening of female urethra Mons pubis Labia majora Labia minora

The wall of the oviduct is composed of a mucosa (called the endosalpinx), a two-layered muscularis (called the myosalpinx), and an outer-lying connective tissue (the perisalpinx). The endosalpinx is thrown into many folds, almost to the extent that the lumen is obliterated, and is lined by a simple epithelium made up of two cell types: ciliated cells and secretory cells. The cilia are most numerous at the infundibular end and propel the cumulus-oocyte complex toward the uterus. The cilia on the fimbriae are the sole mechanism for transport of the ovulated cumulus-oocyte complex. Once the complex passes through the ostium of the oviduct and enters the ampulla, it is moved by both cilia and peristaltic contractions of the muscularis.

The secretory cells produce a protein-rich mucus that is conveyed along the oviduct to the uterus by the cilia. This ciliary-mucus escalator maintains a healthy epithelium, moves the cumulus-oocyte complex toward the uterus, and may provide directional cues for swimming sperm. Movement of the cumulus-oocyte complex slows at the ampullary-isthmus junction, where fertilization normally takes place. This appears to be due in part to thick mucus produced by the isthmus and increased tone of the muscularis of the isthmus. The composition of oviductal secretions is complex and includes growth factors, enzymes, and oviduct-specific glycoproteins. Note that the clinical process of in vitro fertilization has shown that secretions of the oviduct are not absolutely necessary for fertility. However, normal oviductal function is absolutely required for both fertilization and implantation after in vivo insemination (i.e., natural sexual intercourse). Normal oviductal function also minimizes the risk of ectopic implantation and ectopic pregnancy, which occurs most often within the oviduct.

Hormonal Regulation During the Menstrual Cycle

In general, estrogen secreted during the follicular phase increases epithelial cell size and height in the endosalpinx. Estrogen increases blood flow to the lamina propria of the oviducts, promotes production of oviduct-specific glycoproteins (whose functions are poorly understood), and increases ciliogenesis throughout the oviduct. Estrogen promotes secretion of thick mucus in the isthmus and increases the tone of the muscularis of the isthmus, thereby keeping the cumulus-oocyte complex at the ampullary-isthmus junction for fertilization. High progesterone, along with estrogen, during the early luteal to midluteal phase decreases epithelial cell size and function. Progesterone promotes deciliation. It also decreases the secretion of thick mucus and relaxes the tone in the isthmus. In addition it should be noted that oviductal epithelial cells express the LH receptor, which may synergize with estrogen to optimize oviductal function during the periovulatory period.

The Uterus

The uterus is a single organ that sits in the midline of the pelvic cavity between the bladder and the rectum. The mucosa of the uterus is called the endometrium, the three-layered thick muscularis is called the myometrium, and the outer connective tissue and serosa are called the perimetrium. The parts of the uterus are (1) the fundus, which is the portion that rises superiorly from the entrance of the oviducts, (2) the body of the uterus, which makes up most of the uterus, (3) the isthmus, a short narrowed part of the body at its inferior end, and (4) the cervix, which extends into the vagina (see Figs. 44.13 and 43.25). Because the cervical mucosa is distinct from the rest of the uterus and does not undergo the process of menstruation, it will be discussed separately later.

The established functions of the uterus are all related to fertilization and pregnancy (discussed later). The main functions of the uterus are to: 1.

assist movement of sperm from the vagina to the oviducts 2.

provide a suitable site for attachment and implantation of the blastocyst, including a thick nutrient-rich stroma 3.

limit the invasiveness of the implanting embryo so it stays in the endometrium and does not reach the myometrium 811 •Fig. 44.26 Diagramoftheorganizationofglandsandbloodflowwithintheuterineendometrium.(FromStrausIII.In:YenSSCetal[eds].Reproductive Endocrinology. 4thed.Philadelphia:Saunders;1999.)Radial branch Spiral artery Uterine gland ENDOMETRIUM MYOMETRIUM Uterine artery Functional Zone Basal Zone Venous lakes Arcuate

CHAPTER 44 The Male and Female Reproductive Systems 4.

provide a maternal side of the mature placental architecture, including the basal plate to which the fetal side attaches, and large intervillous spaces that become filled with maternal blood after the first trimester 5.

grow and expand with the growing fetus so it develops within an aqueous nonadhesive environment 6.

provide strong muscular contractions to expel the fetus and placenta at term To understand the function of the uterus and uterine changes during nonfertile menstrual cycles, the fine structure of the endometrium and the relationship of uterine blood supply to the endometrium will be reviewed (Fig. 44.26). The luminal surface of the endometrium is covered with a simple cuboidal/columnar epithelium. The epithelium is •Fig. 44.27 Themenstrualcycleoftheuterineendometrium.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) continuous with mucosal glands (called uterine glands) that extend deep into the endometrium. The mucosa is vascularized by spiral arteries, which are branches of the uterine artery that run through the myometrium. The terminal arterioles of the spiral arteries project just beneath the surface epithelium. These arterioles give rise to a subepithelial plexus of capillaries and venules that have ballooned thin-walled segments called venous lakes or lacunae. The lamina propria itself is densely cellular. The stromal cells of the lamina propria play important roles during both pregnancy and menstruation.

About two-thirds of the luminal side of the endometrium is lost during menstruation and is called the functional zone (also called the stratum functionalis) (see

Fig.

44.26 ). The basal third of the endometrium that remains after menstruation is called the basal zone (also called the stratum basale). The basal zone is fed by straight arteries that are separate from the spiral arteries, and it contains all the cell types of the endometrium (i.e., epithelial cells from the remaining tips of glands, stromal cells, and endothelial cells).

Hormonal Regulation of the Uterine Endometrium During the Menstrual Cycle

Monthly oscillations in ovarian steroids induce the uterine endometrium to enter different stages. At the time of selection of the dominant follicle and its elevating production of estradiol, the uterine endometrium is just ending menstruation. The stratum functionalis has been shed and only the stratum basale remains (

Fig. 44.27).

The rising levels of estrogen during the mid to late follicular phase of the ovary induce the proliferative phase of the uterine endometrium. Estrogen induces all cell types in the stratum basale to grow and divide. In fact the definition of an “estrogenic” compound has historically been one that is “uterotropic.” Estrogen increases cell proliferation directly through its cognate receptors (ER-α and ER-β), which regulate gene expression (

Fig. 44.28 ). Estrogen also controls uterine growth indirectly through local production of growth factors. In addition, estrogen induces expression of progesterone receptors, thereby “priming” the uterine endometrium so it can respond to progesterone during the luteal phase of the ovary.

By ovulation, the thickness of the stratum functionalis has been reestablished under the proliferative actions of estradiol-17β (see

Fig. 44.27 ). After ovulation the corpus luteum produces high levels of progesterone along with estradiol-17β. The luteal phase of the ovary switches the proliferative phase of the uterine endometrium to the secretory phase. In general, progesterone inhibits further endometrial growth and induces differentiation of epithelial and stromal cells. Progesterone induces the uterine glands to secrete a nutrient-rich product that supports blastocyst viability. As the secretory phase proceeds the mucosal uterine glands become corkscrewed and sacculated (see

Fig.

44.27 ). Progesterone also induces changes in adhesivity of the surface epithelium, thereby generating the “window of receptivity” for implantation of an embryo (see nancy). Additionally, progesterone promotes differentiation of stromal cells into “predecidual cells,” which must be prepared to form the decidua of pregnancy or to orchestrate menstruation in the absence of pregnancy.

•Fig. 44.28 Molecularmechanismbywhichtheestrogenreceptor(ER)regulatesgeneexpression.Left, Estradiol-17β bindstotheERandchangesitsconformationsothatitbindsasadimertoestrogen-responseelement(ERE)andrecruitscoactivatorproteins(Co-Act),whichleadstostimulationofgeneexpression.Right, Selectiveestrogenreceptormodulators(SERMs),suchastamoxifeninthebreast,alterERconformationsothatitrecruitsco-repressorproteins(Co-Rep),therebyinhibitinggeneexpression.InthiscasetheSERMactsasanERantagonist,butinsometissuesthesameSERMcanactasanERagonist.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)

Progesteroneopposestheproliferativeactionsofestradiol-17β anddownregulatestheestrogenreceptor(ER).Progesteronealsoinducesinactivating isoforms of 17β-HSD, therebyconvertingtheactiveestradiol-17β intotheinactiveestrone.Thisoppositionofthemitogenicactionsofestradiol-17β byprogesteroneisimportanttoprotecttheuterineendometriumfromestrogen-induceduterinecancer.Incontrast,administrationof“unopposed estrogen” towomensignificantlyincreasestheriskforuterinecancer.

Fig.44.28 ).Forexample,theSERMtamoxifen isusedasanERantagonistforthetreatmentofbreastcancer(whoseearlyprogressionispromotedbyestrogen).BindingofSERMtotheERinducesconformationalchangesthatallowco-repressorstobindtotheERorpromotedegradationoftheER(orboth;see

Fig.

44.28 ).Becausetamoxifenhassomeuterotropicactivity(i.e.,makesuterineendometrialtissuegrow),newerSERMssuchasraloxifene havebeendevelopedtohaveERantagonistactivityonthebreast,beneficialERagonistactivityonbone(seelater),andnoactivityorERantagonisticactivityontheuterineendometrium.

In a nonfertile cycle, death of the corpus luteum results in sudden withdrawal of progesterone, which leads to changes in the uterine endometrium that result in loss of the lamina functionalis (see Fig. 43.27). Menstruation normally lasts for 4 to 5 days (called a period), and the volume of blood loss ranges from 25 to 35 mL. Menstruation coincides with the early follicular phase of the ovary.

Hormonal Regulation of the Myometrium

The smooth muscle cells of the myometrium are also responsive to changes in steroid hormones. Peristaltic contractions of the myometrium favor movement of the luminal contents from the cervix to the fundus at ovulation, and these contractions probably play a role in rapid bulk transport of ejaculated sperm from the cervix to the oviducts. During menstruation, contractions propagate from the fundus to the cervix, thereby promoting expulsion of sloughed stratum functionalis. The size and number of smooth muscle cells are determined by estrogen and progesterone. Healthy cycling women maintain a robust myometrium, whereas the myometrium progressively thins in postmenopausal women. The most drastic changes are seen during pregnancy, when the smooth muscle cells increase from 50 to 500 µm in length. The pregnant myometrium also has a greater number of smooth muscle cells and more extracellular matrix.

Disordersofmenstruationarerelativelycommonandincludemenorrhagia (heavymenstrualflowleadingtolossofmorethan80mLofblood),metrorrhagia irregularandsometimesprolongedmenstrualflowbetweennormalperiods),anddysmenorrhea (painfulperiods).Theexistenceofafewirregularperiods,calledoligomenorrhea, andtheabsenceofperiods,calledamenorrhea, areoftenduetodysfunctionofthehypothalamic-pituitary-ovarianaxisasopposedtolocalpelvicpathophysiology.

Because endometrial tissue is naturally sloughed in fragments that contain viable cells, endometrial tissue occasionally gains access to other parts of the female tract (e.g., oviducts, ovary), as well as the lower part of the abdomen and associated structures (e.g., rectouterine pouch of Douglas, as shown in

Fig. 44.13 ). These implants give rise to endometriosis—a foci of hormonally responsive endometrial tissue outside the uterus. The spread of endometriosis may be due to reflux of menstrual tissue into the oviducts or movement of tissue through lymphatics, or both. Endometriosis frequently exhibits cyclic bleeding and is associated with infertility, pain on defecation, pain on urination, pain with sexual intercourse, or generalized pelvic pain.

The Cervix

The cervix is the inferior extension of the uterus that projects into the vagina (see

Figs. 44.13 44.25 ). It has a mucosa that lines the endocervical canal, which has a highly elastic lamina propria and a muscularis that is continuous with the myometrium. The part of the cervix that extends into the vaginal vault is called the ectocervix, whereas the part surrounding the endocervical canal is called the endocervix. The openings of the endocervical canal at the uterus and vagina are called the internal cervical os and the external cervical os, respectively. The cervix acts as a gateway to the upper female tract—at midcycle the endocervical canal facilitates sperm viability and entry. During the luteal phase the endocervical canal impedes passage of sperm and microbes, thereby inhibiting superimplantation of a second embryo or ascending infection into the placenta, fetal membranes, and fetus. The cervix physically supports the weight of the growing fetus. At term, cervical softening and dilation allow passage of the newborn and placenta from the uterus into the vagina.

Hormonal Regulation of Cervical Mucus During the Menstrual Cycle

The endocervical canal is lined by simple columnar epithelium that secretes cervical mucus in a hormonally responsive manner. Estrogen stimulates production of a copious quantity of thin, watery, slightly alkaline mucus that is an ideal environment for sperm. Progesterone stimulates production of a scant, viscous, slightly acidic mucus that is hostile to sperm. During the normal menstrual cycle the conditions of the cervical mucus are ideal for sperm penetration and viability at the time of ovulation.

The Vagina

The vagina is one of the copulatory structures in women and acts as the birth canal (see

Figs. 44.13 44.25 ). Its mucosa is lined by a nonkeratinized stratified squamous epithelium. The mucosa has a thick lamina propria enriched with elastic fibers and is well vascularized. There are no glands in the vagina, so lubrication during intercourse comes from (1) cervical mucus (especially with intercourse that occurs midcycle), (2) a transudate (i.e., ultrafiltrate) from the blood vessels of the lamina propria, and (3) the vestibular glands. The mucosa is surrounded by a relatively thin (i.e., relative to the uterus and cervix) two-layered muscularis and an outer connective tissue. The vaginal wall is innervated by branches of the pudendal nerve, which contribute to sexual pleasure and orgasm during intercourse.

Hormonal Regulation During the Menstrual Cycle

The superficial cells of the vaginal epithelium are continually desquamating, and the nature of these cells is influenced by the hormonal environment. Estrogen stimulates proliferation of the vaginal epithelium and increases its glycogen content (referred to as “cornification”—but in humans, true cornification or keratinization does not occur). The glycogen is metabolized to lactic acid by commensal lactobacilli, thereby maintaining an acidic environment. This inhibits infection by noncommensal bacteria and fungi. Progesterone increases the desquamation of epithelial cells.

The External Genitalia

The female external genitalia are surrounded by the labia majora (homologues of the scrotum) laterally and the mons pubis anteriorly (see

Fig. 44.25 ). The vulva collectively refers to an area that includes the labia majora and mons pubis plus the labia minora, the clitoris, the vestibule of the vagina, the vestibular bulbs (glands), and the external urethral orifice. The vulva is also referred to as the pudendum by clinicians. The structures of the vulva serve the functions of sexual arousal and climax, directing the flow of urine, and partially covering the opening of the vagina, thereby inhibiting entry of pathogens.

The clitoris is the embryological homologue of the penis and is composed of two corpora cavernosa, which attach the clitoris to the ischiopubic rami, and a glans. These structures are composed of erectile tissue and undergo the process of erection in essentially the same manner as the penis. Unlike the penis, clitoral tissue is completely separate from the urethra. Thus the clitoris is involved in sexual arousal and climax at orgasm. The vagina is likewise involved in sexual satisfaction but also serves as the copulatory organ and birth canal.

Hormonal Regulation During the Menstrual Cycle

The structures of the vulva do not show marked changes during the menstrual cycle. However, the health and function of these structures are dependent on hormonal support. The external genitalia and vagina are responsive to androgens (testosterone and dihydrotestosterone) and estrogen. Androgens also act on the central nervous system (CNS) to increase libido in women.

Biology of Estradiol-17β and Progesterone

Biological Effects of Estrogen and Progesterone

Estradiol-17β and progesterone fluctuate during the menstrual cycle, and they have multiple effects that can be categorized according to whether they are directly related to the reproductive system or not. Both hormones have profound effects on the ovary, oviduct, uterus, cervix, vagina, and external genitalia and on the hypothalamus and pituitary. Estrogen and progesterone also have important effects on nonreproductive tissues: Bone: Estrogen is required for closure of the epiphyseal plates of long bones in both sexes. Estradiol-17β has a bone anabolic and calciotropic effect (see ). It stimulates intestinal Ca++ absorption. Estradiol-17β is also one of the most potent regulators of osteoblast and osteoclast function. Estrogen promotes survival of osteoblasts and apoptosis of osteoclasts, thereby favoring bone formation over resorption. Low estrogen levels associated with menopause leads to bone loss and osteoporosis.

Liver: The overall effect of estradiol-17β on the liver is to improve circulating lipoprotein profiles. Estrogen increases expression of the LDL receptor, thereby increasing clearance of cholesterol-rich LDL particles by the liver. Estrogen also increases circulating levels of HDL. Estrogen regulates hepatic production of several transport proteins, including cortisol-binding protein, thyroid hormone–binding protein, and SHBG.

CHAPTER 44 The Male and Female Reproductive Systems

Cardiovascular organs: Premenopausal women have significantly less cardiovascular disease than men or post-menopausal women do. Estrogen promotes vasodilation through increased production of nitric oxide, which relaxes vascular smooth muscle and inhibits platelet activation. Single-nucleotide polymorphisms in the estrogen receptor have been associated with increased cardiovascular disease.

Integument: Estrogen and progesterone maintain healthy smooth skin with normal epidermal and dermal thickness. Estrogen stimulates proliferation and inhibits apoptosis of keratinocytes. In the dermis, estrogen and progesterone increase collagen synthesis and inhibit breakdown of collagen by suppressing matrix metalloproteinases. Estrogen also increases glycosaminoglycan production and deposition in the dermis and promotes wound healing.

CNS: Estrogen is neuroprotective—that is, it inhibits neuronal cell death in response to hypoxia or other insults. Estrogen’s positive effects on angiogenesis may account for some of the beneficial and stimulant-like actions of estrogen on the CNS. Progesterone acts on the hypothalamus to increase the set point for thermoregulation, thereby elevating body temperature approximately 0.5°F. This is the basis for using body temperature measurements to determine whether ovulation has occurred. Progesterone is a CNS depressant. Loss of progesterone on demise of the corpus luteum of menstruation is the basis for premenstrual dysphoria (premenstrual syndrome [PMS]). Progesterone also acts on the brainstem to sensitize the ventilatory response to PCO2 so that ventilation increases and PCO2 decreases.

Adipose tissue: Estrogen decreases adipose tissue by decreasing lipoprotein lipase activity and increasing hormone-sensitive lipase (i.e., it has a lipolytic effect). Loss of estrogen results in accumulation of adipose tissue, especially in the abdomen.

Transport and Metabolism of Ovarian Steroids

Steroid hormones are slightly soluble in blood and are bound to plasma proteins. Approximately 60% of the estrogen is transported bound to SHBG, 20% is bound to albumin, and 20% is in the free form. Progesterone binds primarily to cortisol-binding globulin (transcortin) and albumin. Because it has relatively low binding affinity for these proteins, its circulating half-life is about 5 minutes.

Although the ovary is the primary site of estrogen production, peripheral aromatization of androgens to estrogens can generate locally high levels of estradiol-17β in some tissues. Peripheral conversion of adrenal and ovarian androgens serves as an important source of estrogen after menopause (discussed later). The fact that CYP19 (aromatase) is expressed in the breast is the basis for the use of aromatase inhibitors in the treatment of estrogen-dependent breast cancer in postmenopausal women.

•Fig. 44.29 Patternofgonadotropinsecretionthroughoutlife.Notethetransientpeaksduringgestationandearlyinfancyandthelowlevelsthereafterinchildhood.Womensubsequentlyhavemonthlycyclicbursts,withluteinizinghormone(LH)exceedingfollicle-stimulatinghormone(FSH);mendonot.Bothgendersshowincreasedgonadotropinproductionafterage50,withFSHexceedingLH.

Estrogens and progestins are degraded in the liver to inactive metabolites, conjugated with sulfate or glucuronide, and excreted in urine. Major metabolites of estradiol include estrone, estriol, and catecholestrogens (2-hydroxyestrone and 2-methoxyestrone). The major metabolite of progesterone is pregnanediol, which is conjugated with glucuronide and excreted in urine.

Ontogeny of the Reproductive Systems

Unlike most other organ systems, the reproductive systems undergo significant changes in their activity during the life span of a man or woman (

Fig. 44.29 ). Development of the reproductive systems occurs in utero and results in female or male fetuses. After birth and during infancy, the reproductive systems are largely quiescent. At puberty the hypothalamic-pituitary-gonadal axes “wake up” and the gonads begin producing sex steroids, which in turn induce the sexually dimorphic changes in appearance and behavior associated with men and women. The reproductive life span of women is set by their ovarian reserve and degree of follicular development (see earlier) and ends at menopause, usually in the fifth decade of life. Loss of estrogen production by the ovaries has a clear clinical impact on many postmenopausal women. Men continue to produce sperm throughout life but can experience a decline in androgen production (andropause), which is associated with its own clinical sequelae.

The reproductive system of women undergoes dramatic changes during pregnancy. Production of gonadotropin and gonadal steroids is switched from the maternal hypothalamic-pituitary-ovarian axis, which is strongly repressed during pregnancy, to the fetal placenta. Indeed, it is the endocrine function of fetal placental tissue that maintains a quiescent gravid uterus, (2) alters maternal physiology to ensure fetal nutrition in utero, (3) alters maternal pituitary function and mammary gland development to ensure ongoing fetal nutrition after birth, and determines the time of labor and delivery (also called parturition). The placenta also plays an important role in fetal testosterone production and male differentiation of the reproductive system before the fetal hypothalamus and pituitary develop into a functional axis.

Fertilization, Early Embryogenesis, Implantation, and Placentation

Fertilization, early embryogenesis, implantation, and early gestation are all synchronized with the human menstrual cycle (

Fig. 44.30 ). Just before ovulation the ovary is in the late follicular stage and produces high levels of estrogen. Estrogen promotes growth of the uterine endometrium and induces expression of the progesterone receptor. Estrogen ultimately induces the LH surge, which in turn induces meiotic maturation of the oocyte and ovulation of the cumulus-oocyte complex.

The events between fertilization and implantation take about 6 days to complete, so implantation occurs at about day 22 of the menstrual cycle. At this time the ovary is in the midluteal phase and secreting large amounts of progesterone. Progesterone stimulates secretion from the uterine glands, which provide nutrients to the embryo. This is referred to as histiotropic nutrition and is an important

CHAPTER 44 The Male and Female Reproductive Systems 817 •Fig. 44.30 Synchronizationofeventsofthemenstrualcycle(ovaryandendometrium)withfertiliza-tion,earlyembryonicdevelopmentwithintheoviduct,andimplantationofembryo(blastocyst)intotheuterineendometrium.E2,estradiol;P4,progesterone.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)d13 Peak E2 levels Onset of LH surge Dominant follicle-high E2 output d7 Ovary and pituitary Late follicular phase d13 Resumption of meiosis d20 Blastocyst in uterus d14 d15 Early luteal phase d15 First polar body and arrest at metaphase II 35 hours after onset of LH surge d21 Late luteal phase Growth of functional zone Induction of P4 receptors P4 induces secretion from E2-primed glands, and opposition of E2 mitogenic effects Receptive period Decreased antiadhesion protein expression Increased adhesion protein expression Uterine endometrium Arrested primary oocyte Egg and embryo Late proliferative phase Early secretory phase Late secretory phase d28 Rescue of CL by hCG d20 d24 d16 Fertilization pronuclear stage d23 Blastocyst hatches from zona pellucida and implantation begins on day 22–23 (6–7 days after fertilization) d22d17 2-cell embryo d26 Completion of interstitial implantation mode of maternal-to-fetal transfer of nutrients for about the first trimester of pregnancy, after which it is replaced by hemotropic nutrition (see later). Progesterone inhibits myometrial contraction and prevents release of paracrine factors (e.g., cytokines, prostaglandins, chemokines, and vasoconstrictors) that lead to menstruation. Progesterone induces the “window of receptivity” in the uterine endometrium, which exists from about day 20 to day 24 of the menstrual cycle. This receptive phase is associated with increased adhesivity of the endometrial epithelium and involves formation of cellular extensions called pinopodes on the apical surface of endometrial epithelia, along with increased expression of adhesive proteins (e.g., integrins, cadherins) and decreased expression of antiadhesive proteins (e.g., mucins) in the apical cell membrane.

When a fertilized egg implants in the uterus, the uterine endometrium is at its full thickness, is actively secreting, and is capable of tightly adhering to the implanting embryo.

Fertilization accomplishes both recombination of genetic material to form a new genetically distinct organism and initiation of events that begin embryonic development. Several steps must occur to achieve successful (unassisted) fertilization (Fig. 44.31 3,4 ˜Ca++ Zona Egg plasma pellucida ZP2 membrane 3 • Fig. 44.31 Events involved in fertilization (see text for details). (Modified from White BA, Porterfield SP. Endocrine Physiology. 4th ed. Philadelphia: Mosby; 2013.)

Step 1: Penetration of the expanded cumulus by the sperm. This involves digestion of the extracellular matrix of the cumulus by a membrane hyaluronidase, PH-20.

Step 2: Penetration of the zona pellucida by the sperm. This involves binding of the sperm to the zona protein ZP3 (step 2a), which induces release of acrosomal enzymes (called the acrosomal reaction (step 2b). The sperm secondarily bind to another zona protein, ZP2 (step 2c), as the zona pellucida is digested and the sperm swims through to the egg (step 2d).

Step 3: Fusion of the sperm and egg membrane takes place.

Step 4: A Ca++ signaling cascade (see occurs.

Step 5: The signaling cascade activates the exocytosis of enzyme-filled vesicles called cortical granules that reside in the outermost, or cortical, region of the unfertilized egg. The enzymes contained in the cortical granules are released to the outside of the egg upon exocytosis. These enzymes modify both ZP2 and ZP3 of the zona pellucida such that ZP2 can no longer bind acrosomereacted sperm, and ZP3 can no longer bind capacitated acrosome-intact sperm. Thus only one sperm usually enters the egg. Occasionally, more than one sperm does enter the egg. This results in a triploid cell that is unable to develop further. Therefore prevention of polyspermy is critical for normal development of the fertilized egg.

Step 6: The entire sperm enters the egg during fusion. The flagellum and mitochondria disintegrate, so most of the mitochondrial DNA in cells is maternally derived. Once inside the egg, decondensation of the sperm DNA occurs. A membrane called the pronucleus forms around the sperm DNA as the newly activated egg completes the second meiotic division. In mammalian eggs a large initial release of Ca++ is fol lowed by a series of subsequent smaller Ca++ oscillations that can last for hours. A major consequence of this signaling pathway is that it “wakes up” the metabolically quiescent •Fig. 44.32 Overviewofgeneticeventsafterfertilizationuptothefirstembryoniccleavage.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) egg so it can resume meiosis and begin embryonic development. This process is called egg activation.

The activated egg completes the second meiotic division as the sperm DNA decondenses and a pronucleus forms around it (

Fig. 44.32). Once the egg has completed meiosis,

CHAPTER 44 The Male and Female Reproductive Systems a pronucleus forms around the female chromosomes as well. A centrosome contributed by the sperm becomes a microtubule organizing center from which microtubules extend until they contact the female pronucleus. The male and female DNA replicate as the two pronuclei are pulled together. Once the pronuclei contact each other, the nuclear membranes break down, the chromosomes align on a common metaphase plate, and the first cleavage occurs.

Fertilization typically occurs on day 16 to 17 of the men strual cycle, and implantation occurs about 6 days later.

Thus the first week of embryogenesis takes place within the lumens of the oviduct and uterus. For most of this time the embryo remains encapsulated by the zona pellucida.

The first two cleavages take about 2 days, and the embryo reaches a 16-cell morula by 3 days. The outer cells of the morula become tightly adhesive with each other and begin transporting fluid into the embryonic mass. During days 4 and 5 the transport of fluid generates a cavity called the blastocyst cavity, and the embryo is now called a blas

Fig. 44.33 ). The blastocyst is composed of two subpopulations of cells: an eccentric inner cell mass and an outer epithelial-like layer of trophoblasts. The region of the trophoblast layer immediately adjacent to the inner cell mass is referred to as the embryonic pole, and it is this region that attaches to the uterine endometrium at

Fig. 44.33

The embryo resides within the oviduct during the first 3 days and then enters the uterus. By 5 to 6 days of develop ment the trophoblasts of the blastocyst secrete proteases that digest the outer-lying zona pellucida. At this point, corresponding to about day 22 of the menstrual cycle, the “hatched” blastocyst is able to adhere to and implant into the receptive uterine endometrium (see

Fig. 44.33

At the time of attachment and implantation the tro phoblasts differentiate into two cell types: an inner layer of cytotrophoblasts and an outer layer of multinuclear/

Fig. 44.33).

The cytotrophoblasts initially provide a feeder layer of continu ously dividing cells. Syncytiotrophoblasts initially perform three general types of function: adhesive, invasive, and endocrine. Syncytiotrophoblasts express adhesive surface proteins (i.e., cadherins and integrins) that bind to uterine surface epithelia and, as the embryo implants, to compo nents of the uterine extracellular matrix. In humans the embryo completely burrows into the superficial layer of the endometrium (see

Fig. 44.33). This mode of implanta tion, called interstitial implantation, is the most invasive among placental mammals. Invasive implantation involves adhesion-supported migration of syncytiotrophoblasts into the endometrium, along with the breakdown of extracel lular matrix by secretion of matrix metalloproteinases and other hydrolytic enzymes.

The endocrine function begins with the onset of implantation, when syncytiotrophoblasts start secreting the LH-like protein hCG, which maintains the viability of

Extraembryonic Primary Secondary Extraembryonic Remains of primary mesoderm yolk sac yolk sac forming coelom yolk sac •Fig. 44.33 A,Beginningofimplantation.Thetrophoblasthasdifferentiatedintocytotrophoblastand syncytiotrophoblast layers. B, As the syncytiotrophoblast layer increases in size and invades deeper, this layer begins to surround and erode maternal vessels, forming lacunae filled with maternal blood. C, Interstitial implantation is almost complete. Extensions of cytotrophoblasts have formed that will becomecoveredbyalayerofsyncytiotrophoblast.Atthispoint,theyarecalled“primaryvilli”.D,Interstitial implantationiscomplete.Extraembryonicmesodermalhasdevelopedfromtheepitheliallayers(amnion, primaryyolksac),andwillformaninnerlayerofthevilli,forming“secondaryvilli”.Ultimately,themesoderm willgiverise toumbilicalbloodvesselswithinthecoreofthe villus,therebyformingtertiaryvilli.(From CarlsonBM.Human Embryology and Developmental Biology.Philadelphia:Mosby;2004.) the corpus luteum and thus progesterone secretion. Syn-As implantation and placentation progress, syncytiotrocytiotrophoblasts also become highly steroidogenic. By 10 phoblasts take on the important functions of phagocytosis weeks the syncytiotrophoblasts acquire the ability to make (during histiotropic nutrition) and bidirectional placental progesterone at sufficient levels to maintain pregnancy transfer of gases, nutrients, and wastes. Exchange across independently of a corpus luteum. Syncytiotrophoblasts the syncytiotrophoblasts involves diffusion (e.g., gases), produce several other hormones as well as enzymes that facilitated transport (e.g., GLUT1-mediated transfer of modify hormones. glucose), active transport (e.g., amino acids by specific transporters), and pinocytosis/transcytosis (e.g., of irontransferrin complexes).

There is also a maternal response to implantation that involves transformation of the endometrial stroma. This response, called decidualization, involves an enlargement of stromal cells as they become lipid-and glycogen-filled decidual cells (at this time the endometrium is referred to as the decidua). The decidua forms an epithelial-like sheet with adhesive junctions that inhibit migration of the implanting embryo. The decidua also secretes factors such as tissue inhibitors of metalloproteinases (TIMPs) that moderate the activity of syncytiotrophoblast-derived hydrolytic enzymes in the endometrial matrix. Consequently, decidualization allows regulated invasion during implantation. Normally the implanting embryo and placenta do not extend to and involve the myometrium.

Placenta accreta istheburrowingoftheembryocompletelythroughtheendometriumandadhesionoftheplacentatothemyometrium,aconditionassociatedwithpotentiallylife-threateningpostpartum hemorrhage. Thedecidualresponseoccursonlyintheuterus.Thusthehighlyinvasivenatureofthehumanembryoposesconsiderablerisktothemotherinthecaseofectopic implantation. Ectopic implantation referstoimplantationofanembryoatasiteotherthantheuterus,andectopic pregnancy referstoadevelopingembryoatasiteofectopicimplantation.Mostectopicpregnancies(>90%)occurwithintheoviducts(calledtubal pregnancies),buttheycanalsooccurintheovaryandabdominalcavity.Implantationintheoviductsisoftenassociatedwithlong-terminfectionandinflammation(calledpelvic inflammatory disease)andobstructionofthetube.Inatubalpregnancythehighlyinvasivenatureofthehumansyncytiotrophoblast,whichisnormallymoderatedbytheuterinedecidualresponse,usuallyleadstoburrowingoftheimplantedembryothroughthewalloftheoviduct.Althoughabdominalpregnanciescanproceedtoterm,undetectedoviductalpregnanciesusuallyleadtoruptureoftheoviductalwall.Theresultinginternalhemorrhagecanbecatastrophictothemotherandrequiresimmediatesurgicalintervention.

The first hormone produced by syncytiotrophoblasts is hCG, which is structurally related to the pituitary glycoprotein hormones (see ). As such, hCG is composed of a common α-glycoprotein subunit (α-GSU) and a hormone-specific β subunit (β-hCG). Antibodies used to detect hCG (i.e., in laboratory assays and over-the-counter pregnancy tests) are designed to specifically detect the β subunit. hCG is most similar to LH and binds with high affinity to the LH receptor. The β subunit of hCG is longer than that of LH and contains more sites for glycosylation, which greatly increases the half-life of hCG to 24 to 30 hours. The stability of hCG allows it to rapidly accumulate in the maternal circulation such that hCG is detectable within maternal serum within 24 hours of implantation. Serum hCG levels double every 2 days for the first 6 weeks and peak at about 10 weeks. Serum hCG then declines to a constant level at about 50% of the peak value (Fig. 44.34A •Fig. 44.34 CirculatinglevelsofhCGandhPLinmaternalbloodduringpregnancy.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)100 80 60Human chorionic gonadotropin(mIU/mL)40 20 10 0 10 20 30 40 Weeks of gestationA B 10 8Human placental lactogen(µg/mL)6 4 2 0 10 20 30 40 Weeks of gestation

The primary action of hCG is to stimulate LH receptors on the corpus luteum. This prevents luteolysis and maintains a high level of luteal-derived progesterone production during the first 10 weeks. The rapid increase in hCG is responsible for the nausea of “morning sickness” associated with early pregnancy. A small amount (i.e., 1%–10%) of hCG enters the fetal circulation. hCG stimulates fetal Leydig cells to produce testosterone before the fetal gonadotropic axis is fully mature. hCG also stimulates the fetal adrenal cortex (see later) during the first trimester.

The placenta produces a high amount of progesterone, which is absolutely required to maintain a quiescent myometrium and a pregnant uterus. Progesterone production by the placenta is largely unregulated—the placenta produces as much progesterone as the supply of cholesterol and the levels of CYP11A1 and 3β-HSD allow (

Fig. 44.35 ). Notably, placental steroidogenesis differs from that in the adrenal cortex, ovaries, and testis in that cholesterol is transported into the placental mitochondria by a mechanism that is independent of StAR protein. Thus this first step in steroidogenesis is not a regulated rate-limiting step in the placenta as it is in other steroidogenic glands. This means that fetuses with an inactivating mutation in StAR protein will develop lipoid congenital adrenal hyperplasia (see ) and hypogonadism but will have normal progesterone levels produced by their placenta. Progesterone production by the placenta does not require fetal tissue. Consequently, progesterone levels are largely independent of fetal health and cannot be used as a measure of fetal health. Maternal progesterone levels continue to increase throughout pregnancy.

•Fig. 44.35 Productionofprogesteronebysyncytiotrophoblastandestrogensbyfetoplacentalunit.Intervillous space (Maternal) Sycytiotrophoblast Cholesterol pregnenolone Fetal zone adrenal cortex Pregnenolone Pregnenolone-s 17OH-Pregnenolone-s Sulfo-Transferase CYP11A1 Star-related protein CYP11A1 Star protein CYP17 (Hydroxylase) CYP17 (17/20 lyase) ACTH Cholesterol Fetal pituitary CYP19 Aromatase E3 E2 E1 CYP19 Aromatase Sulfatase 16OH DHEA 16OH A'DIONE 16OH T Progesterone 16OH DHEAS DHEAS DHEAS DHEAS DHEAS16OH DHEAS Fetal liver DHEA A'DIONE T 3˜HSD 3˜HSD 17˜HSD Actions in maternal compartment Actions in maternal compartment Peripheral conversion to E2 MC2R

Progesterone is released primarily into the maternal circulation and is required for implantation and maintenance of pregnancy. Progesterone also has several effects on maternal physiology and induces breast growth and differentiation. The switch from corpus luteum–derived progesterone to placental-derived progesterone (referred to as the luteal-placental shift) is complete at about the eighth week of pregnancy. Progesterone (and pregnenolone) are used by the transitional zone of the fetal cortex to make cortisol late in pregnancy.

Estrogens are also produced by syncytiotrophoblasts. Syncytiotrophoblasts are similar to ovarian granulosa cells in that they lack CYP17 and are dependent on another cell type to provide 19-carbon androgens for aromatization (see

Fig.

44.35 ). The ancillary androgen-producing cells reside in the fetal adrenal cortex.

The fetal adrenal cortex contains an outer definitive zone, a middle transitional zone, and an inner fetal zone. The definitive and transitional zones give rise to the zona glomerulosa and zona fasciculata, respectively. Aldosterone synthesis is initiated close to parturition. Synthesis of cortisol begins at about 6 months and increases during late gestation. The fetal zone is the predominant portion of the adrenal cortex in the fetus; it constitutes as much as 80% of the bulk of the large fetal adrenal and is the site of most fetal adrenal steroidogenesis. The fetal zone strongly resembles the zona reticularis in that it expresses little or no 3β-HSD (see

Fig. 44.35 ). The fetal zone primarily releases the sulfated form of the inactive androgen dehydroepiandrosterone sulfate (DHEAS) throughout most of gestation. Production of DHEAS from the fetal adrenal is absolutely dependent on fetal ACTH from the fetal pituitary by the end of the first trimester.

The DHEAS released from the fetal zone has two fates. First, DHEAS can go directly to the syncytiotrophoblast, where it is desulfated by a placental steroid sulfatase and used as a 19-carbon substrate for the synthesis of estradiol-17β and estrone (see

Fig. 44.35 ). The second fate of DHEAS is 16-hydroxylation in the fetal liver by the enzyme CYP3A7. 16-Hydroxyl-DHEAS is then converted by syncytiotrophoblasts to the major estrogen of pregnancy, estriol (see

Fig. 44.35 ). In X-linked ichthyosis, the steroid sulfatase is low or missing, resulting in loss of active (i.e., desulfated) estrogen production by the fetoplacental unit. Pregnancy is normal, but because estrogens promote parturition, the pregnancy is prolonged and usually ends with physician-induced labor. The baby boy has a skin disorder of varying degrees of severity that is called ichthyosis (scaly skin), owing to buildup of layers of shed cells within the stratum corneum. This form of ichthyosis is readily treatable by topical creams.

Maternal estrogen levels increase throughout pregnancy. Because estrogen production is dependent on a healthy fetus, estriol levels can be used as one measure of fetal health. The collective term used for the placental syncytiotrophoblasts and fetal organs in the context of estrogen production is the fetoplacental unit. Estrogens increase uteroplacental blood flow, enhance LDL receptor expression in syncytiotrophoblasts, and induce several components (e.g., prostaglandins, oxytocin receptors) involved in parturition. Estrogens increase breast growth directly and indirectly through stimulation of maternal pituitary prolactin production. Estrogens also increase lactotrope size and number, thereby increasing overall pituitary mass by more than twofold by term. Estrogens also affect several other aspects of maternal physiology.

Human placental lactogen (hPL), also called human chorionic somatomammotropin (hCS), is a 191–amino acid protein hormone produced in the syncytiotrophoblast that is structurally similar to growth hormone (GH) and prolactin (PRL). Its function overlaps those of both GH and PRL. It can be detected within the syncytiotrophoblast by 10 days after conception and in maternal serum by 3 weeks’ gestation (see

Fig. 44.34 ). Maternal serum levels rise progressively throughout the remainder of the pregnancy. The quantity of hormone produced is directly related to the size of the placenta, such that as the placenta grows during gestation, hPL secretion increases. As much as 1 g/day of hPL can be secreted late in gestation.

Like GH, hPL is protein anabolic and lipolytic. Its antagonistic action to insulin is the major basis for the diabetogenicity of pregnancy. Like PRL, it stimulates mammary gland growth and development. Mammary gland development in pregnancy results from the actions of hPL, PRL, estrogens, and progestins. hPL inhibits maternal glucose uptake and use, thereby increasing serum glucose levels. Glucose is a major energy substrate for the fetus, and hPL increases fetal glucose availability.

CHAPTER 44 The Male and Female Reproductive Systems

As with hCG, far less hPL is found in the fetal circulation than in the maternal circulation. This suggests that the hormones may play a more important role in the mother than in the fetus. hPL is not essential for the pregnancy.

Both hPL and PRL act as fetal growth hormones and stimulate production of the fetal growth-promoting hormones insulinlike growth factor (IGF)-I and IGF-II.

Ironically, fetal GH does not appear to regulate growth, and have normal birth weight.

Diabetogenicity of Pregnancy

Fig.

44.36 ). During the last half of pregnancy when hPL levels are highest, maternal energy metabolism shifts from an anabolic state in which nutrients are stored to a catabolic state sometimes described as accelerated starvation, in which maternal energy metabolism shifts toward fat utilization with sparing of glucose. As maternal use of glucose for energy decreases, lipolysis increases and fatty acids become major energy sources. Peripheral responsiveness to insulin decreases and pancreatic insulin secretion increases. Beta cell hyperplasia occurs in pregnancy. Although this does not usually lead to a clinical condition, pregnancy aggravates existing diabetes mellitus, and diabetes can develop for the first time in pregnancy. If the diabetes resolves spontaneously with delivery, the condition is referred to as gestational diabetes. Other hormones contributing to the diabetogenicity of pregnancy are estrogens and progestins, because both these hormones decrease insulin sensitivity.

Human pregnancy lasts an average of 40 weeks from the beginning of the last menstrual period (gestational age).

This corresponds to an average fetal age of 38 weeks. Par turition is the process whereby uterine contractions lead to childbirth. Labor consists of three stages: strong uterine contractions that force the fetus against the cervix, with dilation and thinning of the cervix (several hours); delivery of the fetus (<1 hour); and delivery of the placenta, along with contractions of the myometrium to halt bleeding (<10 minutes).

Control of parturition in humans is complex, and the exact mechanisms underlying its control are not well understood.

Placental CRH and the Fetal Adrenal Axis

The placenta produces corticotropin-releasing hormone (CRH), which is identical to the 41–amino acid peptide produced by the hypothalamus. Placental CRH production and maternal serum CRH levels increase rapidly during late pregnancy and labor. Moreover, circulating CRH is either in the form of free CRH, which is bioactive, or complexed to a CRH-binding protein. Maternal levels of CRH-binding protein plummet during late pregnancy and labor, so free CRH levels increase. Placental CRH also accumulates in the fetal circulation and stimulates fetal ACTH secretion. ACTH stimulates both fetal adrenal cortisol production and fetoplacental estrogen production. In contrast to the inhibitory effect of cortisol on hypothalamic CRH production, cortisol stimulates placental CRH production. This establishes a self-amplifying positive feedback. CRH itself promotes myometrial contractions by sensitizing the uterus to oxytocin and prostaglandins (see ). Estrogens also directly and indirectly stimulate myometrial contractility. Moreover, this model correlates the onset of parturition with cortisol-induced maturation of fetal systems, including the lungs and gastrointestinal tract.

824 SECTION8Berne & Levy Physiology •Fig. 44.36 Overviewofenergyusebythematernalandfetalcompartments.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)Decreased maternal glucose use Antiinsulin action hPL Placenta Glucose Amino acids Fetus Lipolysis Proteolysis Amino acidsFatty acids for maternal use

Although a rise in maternal serum estrogen and a drop in progesterone levels occur late in gestation in some species, no change in the ratio of the two hormones is seen in human serum. However, “functional” progesterone withdrawal involving changes in uterine progesterone receptor and progesterone metabolism has been proposed.

Oxytocin is secreted from the pars nervosa (posterior pituitary) (see ). Oxytocin, which stimulates powerful uterine contractions, plays a major role in progression and completion of parturition. Oxytocin is released in response to stretch of the cervix through a neuroendocrine reflex; it stimulates uterine contractions and thereby facilitates delivery. Oxytocin can be used to induce parturition, and uterine sensitivity to oxytocin increases before parturition. Because maternal serum oxytocin levels do not increase until after parturition has begun, oxytocin is not thought to initiate parturition. However, progesterone inhibits and estrogen stimulates synthesis of oxytocin receptors, and although maternal serum progesterone levels do not decrease immediately before human parturition, estrogen levels rise and oxytocin receptor synthesis increases.

Prostaglandins and other cytokines increase uterine motility, and levels of these compounds increase during parturition, thereby facilitating delivery. Their exact role in initiating parturition is not known. Prostaglandin levels in amniotic fluid, fetal membranes, and uterine decidua increase before the onset of labor. Prostaglandin F2α and prostaglandin E2 increase uterine motility. Large doses of these compounds have been used to induce labor. Because estrogens stimulate prostaglandin synthesis in the uterus, amnion, and chorion, the rising estrogen levels late in gestation can increase uterine prostaglandin formation before parturition.

Uterine size is thought to be a factor regulating parturition because stretch of smooth muscle, including the uterus, increases muscle contraction. In addition, uterine stretch stimulates uterine prostaglandin production. Multiple births generally occur prematurely. The tendency for early delivery can be a result of increased uterine size, increased fetal production of chemicals stimulating delivery, or both.

CHAPTER 44 The Male and Female Reproductive Systems 825 • Fig. 44.37 Diagram of the structure of the breast, along with some pathological conditions of the breast and where they occur. (From Crum CP et al. In: Kumar V et al [eds]. Robbins Basic Pathology. 7th ed. Philadelphia: Saunders; 2003.)

Structure of the Mammary Gland

The mammary gland is composed of 15 to 20 lobes, each with an excretory lactiferous duct that opens at the nipple (

Fig. 44.37 ). The lobes in turn are composed of several lobules that contain secretory structures called alveoli and the terminal portions of the ducts. The epithelium of the alveoli and ducts is composed of two cell layers: apical luminal epithelial cells and basal myoepithelial cells. There is strong evidence for the presence of adult mammary stem cells within this epithelium. The luminal epithelial cells of the alveoli are the producers of milk, and the luminal cells of the ducts convey and modify the secreted milk. Myoepithelial cells are stellate smooth muscle–like cells, and contraction of these cells in response to a stimulus (i.e., milk let-down) expels milk from the lumens of the alveoli and ducts. Lobes and lobules are supported within a connective tissue matrix. The other major tissue component of the breast is adipose tissue. The lactiferous ducts empty at the nipple, a highly innervated hairless protrusion of the breast designed for suckling by an infant. The nipple is surrounded by a pigmented hairless areola that is lubricated by sebaceous glands. Protrusion of the nipple, called erection, is mediated by sympathetic stimulation of smooth muscle fibers in response to suckling and other mechanical stimulation, erotic stimulation, and cold.

Hormonal Regulation of Mammary  Gland Development

At puberty, estrogen increases ductal growth and branching. With onset of the luteal phases of the ovary, progesterone and estrogen induce ductal growth and formation of rudimentary alveoli. During nonpregnant cycles the breasts develop somewhat and then regress. Estrogen also increases deposition of adipose tissue, which makes a major contribution to breast size and overall form. Adipose tissue expresses CYP19/aromatase, so accumulation of this tissue in the breast increases local production of estrogens from circulating androgens.

Breast development is facilitated by pregnancy, during which extensive ductal growth and branching and lobuloalveolar development occur. The parenchymal growth of the breast during development occurs at the expense of stroma, which is degraded to make room for enlarging lobuloalveolar structures. Several placental hormones stimulate breast development, including estrogen, progesterone, placental lactogen, and a growth hormone variant (GH-V). Estrogen acts on the breast both directly and indirectly through increasing maternal pituitary PRL. Estrogen increases PRL secretion from pituitary lactotropes. Estrogen also stimulates lactotrope hypertrophy and proliferation, which accounts for the twofold increase in pituitary volume during pregnancy in humans. Although epithelial cells express genes encoding milk protein and enzymes involved in milk production, progesterone inhibits the onset of milk production and secretion 826 SECTION8Berne & Levy Physiology •Fig. 44.38 Neuroendocrinereflexcausedbysucklingatthenippleandleadingtosecretionofoxytocinandprolactin.Inturnthesehormonesinducecontinuedmilkproduction(galactopoiesis)andmilklet-down.Prolactinalsoinduceslactationalamenorrhea.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)Magnocellular neurons Parvicellular neurons Lactotropes Gonadotropes Lactational amenorrhea Parvicellular neurons Hypothalamus Breast Ovary Pars distalis Pars nervosa ˜ GnRH˜ Dopamine ˜ LH and FSH ° PRF?? ° Prolactin GalactopoiesisConcentration of myoepithelial cells Milk let-down Sucking at nipple ° Oxytocin (lactogenesis).

After parturition, the human breast produces colostrum, which is enriched with antimicrobial and antiinflammatory proteins. In the absence of placental progesterone, normal breast milk production occurs within a few days. The lobuloalveolar structures produce milk, which is subsequently modified by the ductal epithelium. Lactogenesis and maintenance of milk production (galactopoiesis) require stimulation by pituitary PRL in the presence of normal levels of other hormones, including insulin, cortisol, and thyroid hormone. Although placental estrogen stimulates PRL secretion during pregnancy, the stimulus for PRL secretion during the nursing period is suckling by the infant (

Fig. 44.38 ). Levels of PRL are directly correlated with frequency and duration of suckling at the nipple. The link between suckling at the nipple and PRL secretion involves a neuroendocrine reflex in which dopamine secretion at the median eminence is inhibited (the PRL release inhibitory factor; see ). It is also possible suckling increases secretion of unidentified PRL-releasing hormones.

PRL also inhibits release of GnRH, and consequently, nursing can be associated with lactational amenorrhea (see

Fig. 44.38 ). This effect of prolactin has been called “nature’s contraceptive,” and it may play a role in spacing out pregnancies. However, only regular nursing over a 24-hour period is sufficient to induce a PRL-induced anovulatory state in the mother. Thus lactational amenorrhea is not an effective or reliable form of birth control for most women. Inhibition of GnRH by high levels of PRL is important clinically. A prolactinoma is the most common form of hormone-secreting pituitary tumor, and hyperprolactinemia is a significant cause of infertility in both sexes. Hyperprolactinemia can likewise be associated with galactorrhea (inappropriate flow of breast milk) in men and women.

Suckling at the nipple also stimulates release of oxytocin from the pars nervosa (see

Fig. 44.38). Contraction of myoepithelial cells induces milk let-down, or expulsion of milk from the

Invasive breast cancer (IBC) isamajorcancerinwomenandcanbeclassifiedintoseveralcategories.MostnewlydiagnosedIBCisclassifiedasluminal A, whichisusuallyderivedfromluminalcellsofterminalductsoralveoli.LuminalAIBCdisplayssomeepithelialorganization,includingEcadherin–mediatedcell-cellcontacts,andispoorlymotileandpoorlyaggressive.Thisformalsoexpressestheestrogen receptor α (ERα) andisdependentonestrogenicstimulationforgrowth.EarlydiagnosisofluminalAIBCconfersagoodprognosis.Treatmentforearlysmalltumorsthatare“node-negative”(i.e.,havenotspreadtonearbylymphnodes)typicallyinvolvessurgicalremoval(“lumpectomy”),followedbyradiationtreatment,followedby5yearsofdailytamoxifentreatment.TamoxifenisaSERMthatopposesestrogeninthebreast.

Therearemultiplebehavioralmethodsofcontraception. Totalabstinenceisthebestwaytoavoidgettingpregnant.Othermethodsincludetherhythmmethod,whichreliesonabstinencefromsexualintercourseduringfertileperiodsaroundthetimeofovulation.Thefertileperiodextendsfrom3to4daysbeforethetimeofovulationuntil3to4daysafterward.Asecondmethodiswithdrawalbeforeejaculation,coitus interruptus. Boththesemethodshavehigherfailurerates(20%–30%)thanbarrier methods (2%–12%),intrauterine devices (IUDs) (<2%),andoral contraceptives (<1%)do.Barrierssuchascondoms ordiaphragms aremoreeffectivewhenusedwithspermicidal jellies. Ofallmethods,onlycondomsprovideeffectiveprotectionfromsexuallytransmitteddiseasesinsexuallyactiveindividuals.IUDsarerelativelyeffective.Theypreventimplantationbylocallyproducinganinflammatoryresponseintheendometrium.SomeformsofIUDscontaincopper,zinc,orprogestins,whichinhibitspermtransportorviabilityinthefemalereproductivetract.

OralcontraceptiveshavebeenmarketedintheUnitedStatessincetheearly1960s.Thedosesofsteroidsusedtodayaresignificantlylowerthanthoseused35yearsago.Properlyused,oralcontraceptiveshavealowfailurerate.Manyformsoforalcontraceptivesaremarketedtoday.Thetrendovertheyearshasbeentodecreasethedosageofsteroidsusedbecausethesideeffectsaredosedependent.Alloralsteroidalcontraceptivescontaineitheracombinationofanestrogenandaprogestinoraprogestinalone.Oralcontraceptivesworkthroughmultiplemechanisms.MostblocktheLHsurgethattriggersovulation.However,somepills(e.g.,progestin-onlyminipill)donotpreventLHsurges.Fertilityisalsoblockedbychangingthenatureofcervicalmucus,alteringendometrialdevelopment,orregulatingfallopiantubemotility.BecausethesecontraceptivessuppressFSH,theyimpairearlyfolliculardevelopment.

Emergency contraception involveshormonaltreatmentdesignedtoinhibitordelayovulation,inhibitcorpusluteumfunction,disruptthefunctionoftheoviductsanduterus,oranycombinationofthesemechanisms.Forexample,candidatesforemergencycontraceptionincludewomenwhoaresexuallyassaultedorwhoexperiencedfailureofabarriermethod(e.g.,rupturedcondom).Therearemorethan20typesofcommerciallyavailable“morning-after”pills.Thecurrentlypreferredmedicationislevonorgestrel (Plan B), whichisasyntheticprogestin-onlypill.Theefficacyofthepillisinverselycorrelatedwiththetimeitistakenafterintercourse.Theexactmechanismofactionisnotknown.Treatmenthasnoeffectifimplantationhasoccurred.

Medical(hormonal) termination ofpregnancy(abortion)canbeachievedupto49days’gestationbyadministrationofmifepristone (RU-486), aprogesteronereceptorantagonistthatinducescollapseofthepregnantendometrium.Mifepristoneisfollowed48hourslaterbyingestionorvaginalinsertionofasynthetic prostaglandin E (e.g.,misoprostol),whichinducesmyometrialcontractions.

alveolar and ductal lumens. Thus the nursing infant does not gain milk by applying negative pressure to the breast from suckling. Rather, milk is actively ejected through a neuroendocrine reflex. Oxytocin release and milk let-down can be induced by psychogenic stimuli such as the mother hearing a baby crying on television or thinking about her baby. Such psychogenic stimuli do not affect PRL release.

Though related to depletion of ovarian follicles, the causes and process of menopause are poorly understood. Age-related changes in the CNS, including critical patterns of GnRH secretion, precede follicular depletion and may play an important role in menopause. Because follicles do not develop in response to LH and FSH secretion, estrogen and progesterone levels drop. Loss of the negative-feedback inhibition of estrogen on GnRH and LH/FSH results in a marked rise in serum LH and FSH. FSH levels rise more than LH levels. This could result from loss of ovarian inhibin.

Menopause typically occurs between 45 and 55 years of age. It extends over a period of several years. Initially the cycles become irregular and are periodically anovulatory. The cycles tend to shorten, primarily in the follicular phase. Eventually the woman ceases to cycle altogether. Serum estradiol levels drop to about a sixth the mean levels for younger cycling women, and progesterone levels drop to about a third those in the follicular phase of younger women. Production of these hormones does not cease entirely, but the primary source of these hormones in postmenopausal women becomes the adrenal, although interstitial cells of the ovarian stroma continue to produce some steroids. Most circulating estrogens are now produced peripherally from androgens. Because estrone is the primary estrogen produced in adipose tissue, it becomes the predominant estrogen in postmenopausal women.

Most symptoms associated with menopause result from estrogen deficiency. The vaginal epithelium atrophies and becomes dry, and bone loss is accelerated and may lead to osteoporosis. The incidence of coronary artery disease increases markedly after menopause. Hot flashes result from periodic increases in core temperature, which produces peripheral vasodilation and sweating. Hot flashes are thought to be linked to increases in LH release and are probably associated not with the pulsatile rise in LH secretion but rather with central mechanisms controlling GnRH release. Hot flashes typically subside within 1 to 5 years of the onset of menopausal symptoms.

1.

The reproductive systems are composed of gonads, an internal reproductive tract with associated glands, and external genitalia. Mammary glands are accessory reproductive glands in women.

2.

Gonads have two main functions: production of gametes and production of hormones. Hormones (primarily sex steroids) are absolutely necessary for normal function of the reproductive system, and their production is regulated by a hypothalamic-pituitarygonadal axis.

3.

Seminiferous tubules in the testis contain Sertoli cells and developing sperm cells.

4.

Spermatogenesis refers to the progression of sperm cells from spermatogonia through the processes of meiosis and spermiogenesis to form mature spermatozoa.

5.

Testosterone and pituitary FSH are required for normal sperm production. Only Sertoli cells express the androgen receptor and the FSH receptor, so these hormones regulate spermatogenesis indirectly through their actions on Sertoli cells. Sertoli cells produce the hormone inhibin, which negatively feeds back on pituitary FSH production.

6.

Sertoli cells have many functions, including production of androgen-binding protein (ABP) and fluid and creation of the blood-testis barrier.

7.

Leydig cells are stromal cells that reside outside the seminiferous tubules. They respond to LH by producing testosterone.

8.

Testosterone is an active androgen. It can be converted peripherally to DHT, which is more active in certain tissues (e.g., prostate), or to estradiol.

9.

Leydig cells are regulated within a hypothalamicpituitary-testicular axis. The hypothalamus produces GnRH, which stimulates pituitary gonadotropes to secrete LH and FSH. Testosterone, DHT, and estradiol negatively feed back at the pituitary and hypothalamus and inhibit LH more than FSH secretion. Inhibin from the Sertoli cells selectively inhibits FSH.

10.

Testosterone, DHT, and estradiol have numerous actions on the male reproductive tract, external genitalia, and male secondary sex characteristics, as well as on other organ systems (e.g., blood cell production, lipoprotein production, bone maturation).

11.

The male tract includes tubal structures (epididymis, ductus deferens, and male urethra), accessory sex glands (seminal vesicles, prostate), and the penis. The seminal vesicles and the prostate produce most of the ejaculate, which nourishes, buffers, and protects sperm.

12.

Penile erection involves a complex neurovascular response leading to engorgement of the erectile tissue within the penis base and shaft with blood.

13.

The follicle is the functional unit of the ovary. Follicles contain epithelial cells (granulosa and cumulus) and outer stromal cells (thecal). All these cells surround a primary oocyte that remains arrested in the first meiotic prophase until just before ovulation.

14.

Follicles develop from the smallest (primordial) to a large antral follicle over a period of months. The latter part of follicular development requires gonadotropins.

15.

The menstrual cycle refers to an approximately 28-day cycle that is driven by the following ovarian events: development of one large antral follicle to a preovulatory follicle (follicular phase), ovulation, and formation and death of a corpus luteum of menstruation (luteal phase).

16.

The follicular phase of the ovary corresponds to the menstrual and proliferative phases of the uterine endometrium. The luteal phase of the ovary corresponds to the secretory phase of the uterine endometrium.

17.

One dominant follicle is selected per menstrual cycle—usually the largest follicle with the most FSH receptors.

18.

High levels of estradiol occur around midcycle and exert positive feedback on gonadotropin secretion. This induces the LH (and a smaller FSH) surge. The midcycle gonadotropin surge induces (a) meiotic maturation of the primary oocyte so that it progresses to a secondary oocyte (with one polar body) arrested at metaphase of the second meiotic division, (b) breakdown of the ovarian and follicular wall so that the oocyte-cumulus complex is extruded (called ovulation), and (c) differentiation of the remaining follicular cells into a corpus luteum. The corpus luteum produces high levels of progesterone, estradiol, and inhibin.

19.

If pregnancy does not occur, the corpus luteum will die in 14 days. This constitutes the luteal phase of the menstrual cycle.

20.

The oviducts capture the ovulated cumulus-oocyte complex and transport it medially into the oviduct and toward the uterus. Estrogen promotes ciliation and transport; progesterone inhibits transport.

21.

The uterine mucosa, called the endometrium, is the normal site of embryonic implantation. The mucosa is increased in thickness in preparation for implantation and is sloughed away if no pregnancy occurs.

22.

During the mid to late follicular phase (days 6–14 of the menstrual cycle), the ovary produces estradiol, which induces all cells of the endometrium to proliferate (called the proliferative phase of the uterus).

23.

After ovulation the ovary enters the luteal phase (days 16–28) and produces progesterone. Progesterone stimulates secretion from the uterine glands (called the secretory phase of the uterus).

24.

In the absence of an implanting embryo the corpus luteum dies, progesterone production ceases, and the uterine endometrium is sloughed (called the menstrual phase, or period, of the uterus—this corresponds to days 1 to 5 of the follicular phase of the ovary).

25.

The cervix is the lower portion of the uterus. Cervical mucus is hormonally regulated so that at midcycle in response to estrogen, cervical mucus promotes entry of sperm into the uterus from the vagina. During the luteal phase in response to progesterone, cervical mucus becomes thick and poses a barrier to entry of sperm and microbes into the uterus.

We wish to thank Dr. Lisa Mehlmann for her advice on this chapter, and especially for help in drawing

Figures 44.31 44.32

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26.

Fertilization is a complex series of events that occur in the oviduct and lead to penetration of the oocyte by sperm.

27.

Early embryogenesis (up to day 6 after fertilization) occurs in the oviduct and gives rise to a blastocyst that hatches from the zona pellucida.

28.

The placenta develops from the outer extraembryonic trophoblast. The endocrine function of the placenta includes production of hCG, progesterone, estrogens, and placental lactogen. Estrogen production requires placental cells (syncytiotrophoblasts) as well as the fetal adrenal and liver—collectively called the fetoplacental unit.

29.

Pregnancy and the hormones of pregnancy induce major changes in maternal physiology, including an increase in insulin resistance, an increase in the use of free fatty acids by the mother, and development of the mammary glands. Mammary gland development (but not lactation) is promoted by estrogen, progesterone, and placental lactogen but also by maternal pituitary prolactin, whose secretion is stimulated by placental estrogens.

30.

Oxytocin is a pituitary hormone that promotes contraction of certain smooth muscles, including myometrial contractions during labor and myoepithelial contractions in the breasts that lead to let-down of milk in response to suckling.

31.

Menopause results from exhaustion of the ovarian reserve and is characterized by low ovarian hormone and elevated gonadotropin levels.

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