Pathophysiology

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THE CLINICAL CONTEXT

This chapter continues a theme of providing the building block concepts underlying physiology,

pathophysiology, and pharmacology. Disorders of membrane transport, cell signaling, and cell death include conditions such as cystic fi brosis (altered ion channel), familial hypercholesterolemia (receptor-mediated endocytosis), receptor-targeting autoimmune disorders (myasthenia gravis and Graves disease), and cancer (failure of apoptosis), among many others. Of at least equal signifi cance for students preparing for independent practice is the fact that many commonly prescribed and over- the-counter drugs target the proteins and mech- anisms described in this chapter. For example, common medications to reduce heartburn block histamine receptors or active transport of hydro- gen ions (proton pumps), some cardiovascular dis- orders are treated with calcium channel blockers (ion channels), and selective serotonin reuptake inhibitors used to manage depression and anxiety block a secondary active transport protein. This foundational content on cell biology thus provides the context in which many disease processes can be understood and appropriately managed.

OVERVIEW

As you read this book, what are some of the cells of your body doing?

• Photoreceptor cells in your retinas are detecting pat- terns on the page or screen.

CELL PHYSIOLOGY AND PATHOPHYSIOLOGY

Nancy C. Tkacs, Fruzsina K. Johnson, Robert A. Johnson, and Spencer A. Rhodes

• Additional neurons are relaying the information to neurons of the cortex that interpret the information as words that add up to concepts and (we hope!) new learning.

• Muscle cells, bones, and joints create movements of holding the book or e-reader, occasionally turning a page.

• Neurons in your brainstem are initiating rhythmic contractions of your diaphragm for regular breathing.

• Red blood cells are carrying oxygen throughout your body.

• White blood cells are producing immune mediators to protect you from infections.

• Cells in the sinoatrial node of the heart are initiating regular heartbeats.

• Gastrointestinal and liver cells are processing the contents of your last meal.

• Kidney cells are processing fi ltered plasma and pro- ducing urine.

• Myriad endocrine cells are synthesizing and secret- ing hormones that regulate body functions.

• And you are not consciously aware of all of this cel- lular activity that is keeping you alive and maintain- ing homeostasis!

The basic unit of life is the cell, and the vast major- ity of physiological processes are carried out within cells. Pathophysiological alterations at the cellular level underlie many diseases, and medications to treat those diseases often target specifi c cell proteins and functions. Unique functions of the organs and systems of the body are based on the differentiated structures and functions of the cells making up those organs and systems. This brief review of cell biology builds on the earlier reviews of chemistry, biochemistry, and molec- ular biology in Chapters 2, Chemical and Biochemical Foundations, and 3, Molecular Biology, Genetics, and

74 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

Genetic Diseases, to provide the foundation for the remainder of the book. The organelles and proteins described here make up the “cast of characters” acting out physiology at the cellular level. You will encounter membrane transporters, receptors, contractile mecha- nisms, and cell death pathways linked to pathophysiol- ogy and pharmacology in many different chapters, so this chapter provides a general introduction to prepare you for their roles in specific organs.

CELL COMPONENTS

CELL MEMBRANE The boundary between intracellular and extracellular fluids is the cell membrane, often referred to as the plasma membrane. The cell membrane is a phospho- lipid bilayer in which the outer leaflet phospholipids are oriented with their polar head groups facing out- ward toward the extracellular fluid, and the inner leaf- let phospholipids are oriented with their polar head groups facing the intracellular fluid. The nonpolar fatty acid tails of both leaflets make up the hydrophobic core of the membrane, providing the critical barrier that separates intracellular from extracellular fluid.

Molecules of cholesterol are interspersed with the phospholipids, controlling membrane fluidity. One can picture the cell membrane as being like a grilled cheese sandwich, with the outermost bread layers able to mix with water and polar solutes and the melted inner core acting like a barrier, impenetrable to water and to polar and charged solutes.

Integral proteins cross the membrane; some of these function as receptors, others as transporters or channels. Peripheral proteins are often attached to the inner leaflet and work with integral proteins to alter cell function. The outer leaflet is rich in glycoproteins and glycolipids, with complex branching carbohydrates attached. The layer of sugars projecting outward from the cell is referred to as its glycocalyx, and it functions in cell–cell recognition. The inner leaflet of the membrane is attached to proteins of the cytoskeleton. The cell membrane functions as a barrier between intracellular and extracellular fluids, as an electrical insulator that allows separation of electri- cal charges so that a potential difference or membrane voltage can exist between intracellular and extracellular compartments, and as an interface between extracel- lular messengers and intracellular changes. Within the cell, similar phospholipid membranes form the outer coats of many of the organelles that carry out the work of the cells (Box 4.1 and Figure 4.1).

BOX 4.1 Characteristics of the Cell Membrane

GPI-anchored protein

Lipid raft

Carbohydrate

Hydrophilic region

(Hydrophobic region)

Phospholipid

Glycoprotein Extracellular face

Intracellular face

Integral protein

Peripheral protein

Cholesterol

FIGURE 4.1 The cell membrane.

(continued)

Chapter 4 • Cell Physiology and Pathophysiology 75

CYTOPLASM The cytoplasm refers to the entire contents of a cell. It is made up of cytosol (intracellular fl uid), organ- elles, and other structural components of the cell ( Figure 4.2 ). The aqueous intracellular fl uid contains

dissolved electrolytes, small biomolecules and metab- olites, high-energy phosphate compounds, and key intracellular proteins. In muscle cells, the cytoplasm also contains the movement proteins myosin and actin. Protein synthesis and many biochemical processes

Mitochondria

Plasma membrane

Microtubule

Centrosome

Microfilament

Lysosome Smooth endoplasmic

reticulum

Secretory vesicle

Peroxisome

Vacuole

Cytoplasm

Golgi vesicle

Golgi apparatus

Chromatin

Nucleolus

Nucleus

Rough endoplasmic

reticulum

Ribosomes

Intermediate filament

FIGURE 4.2 The cell and organelles. A typical eukaryotic cell with organelles. Note the plasma membrane boundary separating intracellular fl uid (cytoplasm) from extracellular fl uid. Membrane- bounded organelles include the nucleus and mitochondria (each with two outer membranes), rough and smooth endoplasmic reticulum, Golgi apparatus and vesicle budding from Golgi, lyso somes, peroxisomes, and vacuoles. Ribosomes, centrosome, and protein fi laments do not have membranes.

BOX 4.1 (continued) Characteristics of the Cell Membrane

• The cell membrane (also called the plasma membrane) is a complex phospholipid/cholesterol bilayer with hydrophilic/polar regions of phos- pholipids and cholesterol facing the extra cellular and intracellular fl uids, and a central fatty acid and cholesterol ring core that is hydrophobic and restricts passage of polar/hydrophilic solutes.

• The membrane is studded with proteins—both integral proteins that span the membrane and proteins attached to the inner or outer membrane.

• The membrane is dynamic, moving with motile cells such as white blood cells, conducting

exocytosis and endocytosis, and enlarging with cell hypertrophy. Membrane proteins can be internalized and recycled, and new membrane proteins can be inserted from vesicles.

• The clustered lipids shown in red in the fi gure represent a lipid raft—a region of tighter lipid and cholesterol packing that tends to move as a cohesive unit. A protein is linked to the lipid raft by a GPI anchor, allowing the protein to be associated with the plasma membrane without actual insertion through the membrane.

GPI, glycosylphosphatidylinositol.

76 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

occur in the cytoplasm, while some biochemical reac- tions occur in specific organelles like the mitochondria or smooth endoplasmic reticulum (SER).

NUCLEUS The nucleus holds most of a cell’s DNA, with the remain- der found in the mitochondria. The nucleus also contains a nucleolus that functions in synthesis of ribosomes. Unless a cell is preparing to divide by mitosis, the nucleus contains DNA organized in chromosomes that are wound around histone proteins. The DNA is dispersed through- out the nucleus with regions that are tightly compacted and others that are more loosely arranged. Regions of DNA upstream of genes have binding sites for transcrip- tion factors that regulate gene expression. Patterns of gene expression are determined by cell type and differ- entiation, giving each cell type a unique set of proteins that relate to the functions of that cell within its tissue and organ niche. For example, liver cells produce many proteins for metabolism of glucose and lipids, as the liver is the major organ of energy homeostasis. On the other hand, neurons produce ion channels appropriate to gen- erating action potentials and enzymes to synthesize neu- rotransmitters, needed for neuronal signaling.

The nuclear membrane has two layers with relatively low permeability; however, nuclear pores allow move- ment of proteins, signaling molecules, and messenger RNA (mRNA) molecules between nucleus and cyto- plasm. Red blood cells do not contain nuclei, and skeletal muscle cells contain multiple nuclei. With these excep- tions, each of the body’s cells contains one nucleus.

RIBOSOMES AND ROUGH ENDOPLASMIC RETICULUM Ribosomes are manufactured in the nucleus from RNA and proteins, and then migrate to the cytoplasm. A ribo- some is made of large and small subunits that attach to mRNA for the purpose of protein synthesis, as noted in Chapter 3, Molecular Biology, Genetics, and Genetic Diseases. Ribosomes can be found free within the cyto- plasm; others are attached to endoplasmic reticulum, forming the rough endoplasmic reticulum, so named for its bumpy appearance. Endoplasmic reticulum is contiguous with the nucleus and is the site of synthe- sis of proteins that will ultimately be secreted from the cell or moved to a specific cellular location. Within the rough endoplasmic reticulum, proteins fold to assume their secondary, tertiary, and (if applicable) quaternary structures. Rough endoplasmic reticulum is the site of chaperone proteins that detect the progress of protein folding and that mark misfolded proteins for destruc- tion, through the unfolded protein response.

SMOOTH ENDOPLASMIC RETICULUM SER is an intracellular membrane system connected to rough endoplasmic reticulum and containing enzymes

for metabolic processes. It is particularly abundant in liver cells, which do a great deal of metabolic process- ing of lipid molecules as well as conducting biotransfor- mation of drugs and toxins. SER is the location of some steps in steroid hormone synthesis in certain endocrine gland cells. It is also the site of synthesis of new mem- branes that can refresh the cell membrane or organelle membranes. SER is a major site of cellular calcium homeostasis, sequestering calcium to keep cytoplasmic free calcium levels low most of the time, and releasing calcium in response to membrane receptor signaling, described later in this chapter. In muscle cells, the spe- cialized SER, called the sarcoplasmic reticulum, func- tions to release calcium ions that initiate muscle cell contraction, subsequently taking up calcium ions and causing muscle cell relaxation.

GOLGI APPARATUS The Golgi apparatus is a stack of membranes orga- nized in flattened sacks. It functions to sort, modify, and package proteins and lipids that are delivered in vesicles from the nearby endoplasmic reticulum. After proceeding through the Golgi stacks, these substances may leave the cell by exocytosis or be delivered to dif- ferent intracellular locations. The Golgi apparatus also forms other organelles that are vesicle-type organelles, including lysosomes and other secretory vesicles, as well as being a source of plasma membrane.

MITOCHONDRIA The mitochondria are the energy powerhouses of the cell, producing much of the cell’s adenosine triphos- phate (ATP). ATP is the energy source for cellular chemical reactions and ion transport, as well as being the source of phosphate for phosphorylation reactions. Energy-producing reactions of the Krebs cycle and oxi- dative phosphorylation take place in the mitochondria; thus, they are the site of cellular respiration, consum- ing oxygen and generating carbon dioxide. Cells with the highest energy requirements have a correspond- ingly higher number of mitochondria. Other signifi- cant aspects of mitochondrial function include the following:

• Mitochondria have two membranes—an outer mem- brane and a highly folded inner membrane. Some mitochondrial functions and proteins occur within the mitochondrial matrix, while others are localized to the inner membrane.

• Mitochondria contain their own DNA that codes for several mitochondrial proteins.

• Some steps of steroid hormone synthesis and of fatty acid catabolism take place in mitochondria.

• Mitochondria are able to sequester calcium and also play a role in initiating apoptosis (programmed cell death).

Chapter 4 • Cell Physiology and Pathophysiology 77

LYSOSOMES Lysosomes contain degradative enzymes that break down aging cell organelles and molecules. The deg- radative enzymes are in the class called acid hydro- lases , which depend on low pH for their function. Examples include proteases, which degrade proteins, and phospholipases, which degrade phospholipids and membranes. In phagocytic cells, such as neutrophils, phagocytosis of bacteria is followed by fusion with lyso- somes, forming a phagolysosome. The lysosomal deg- radative enzymes then contribute to bacterial killing. Lysosomes can also contribute to cell death pathways.

PEROXISOMES Peroxisomes are enriched in enzymes that use oxygen for their reactions. An example is β-oxidation of fatty acids, a pathway of energy generation that is conducted in both peroxisomes and mitochondria. The chemical reactions within peroxisomes often generate hydrogen peroxide (H

2 O

2 ) as a byproduct, and these organelles

contain the enzyme catalase to detoxify the H 2 O

2 .

CYTOSKELETON The cytoskeleton is made up of protein fi laments that extend through the cytoplasm, many of which are anchored to membrane proteins giving the cell its shape. These fi laments can be organized by size from small to large: microfi laments (made of actin), inter- mediate fi laments (composed of several proteins), and microtubules (made of tubulin). Organelles and ves- icles can move along these fi laments, and in mobile cells (such as white blood cells), they are responsible for cell movement. Microtubules contribute to chro- mosome alignment and movement during mitosis, and drugs that interfere with microtubule functions are used to treat cancer and other diseases. In muscle cells, contractile proteins are anchored to the cytoskeleton and membrane so that the whole cell shortens when actin and myosin cross-links are formed.

MECHANISMS OF MEMBRANE TRANSPORT

As previously noted, the cell membrane provides a lipid barrier between two aqueous solutions of differing composition: the intracellular fl uid and the extracellu- lar fl uid. Oxygen, carbon dioxide, and steroid hormones and other lipid-soluble substances are able to freely dif- fuse across the membrane, whereas ions and biomole- cules such as glucose and amino acids require specifi c membrane proteins and mechanisms to cross it. The type of membrane transport mechanism depends on:

• The chemical nature of the substance—primarily whether it is hydrophobic or hydrophilic

• The molecular size of the substance • The concentration gradient of the substance across

the membrane • The direction of movement: Is the substance mov-

ing down its concentration gradient or uphill from the side with lower concentration to the side with higher concentration? In the latter case, energy input is required for transport to occur ( Figure 4.3 ).

The extracellular and intracellular concentrations of several solutes of interest are listed in Table 4.1 , along with corresponding pH differences. Note the striking difference in sodium and potassium concentrations— sodium is high extracellularly and potassium is high intracellularly. The concentration gradients for sodium and potassium drive many transport processes and also shape the membrane electrical activity of excitable cells. There is a striking concentration gradient for cal- cium as well, with extracellular calcium approximately 10,000 times greater than calcium in the intracellular fl uid. We will return to the physiological importance of calcium movements several times in the book. Also note the concentration gradient for glucose. Many cells use glucose as their main energy source, keeping intracellular glucose concentration low and favoring glucose movement into the cell. The major classes of membrane transport mechanisms for these and other solutes are described next, with examples.

DIFFUSION Sometimes called simple diffusion , this term describes movement across a barrier without a specifi c trans- port protein. Diffusion occurs down a concentration gradient; that is, from an area of high concentration to an area of low concentration ( Figure 4.4 ). To diffuse through the cell membrane’s lipid core requires a sub- stance that is relatively small, with a hydrophobic, non- polar chemical structure.

Net diffusion from one side to another is in the downhill direction, in response to the concentration gradient. Oxygen and carbon dioxide are examples, with oxygen entering tissues from capillaries, dif- fusing into cells and being continually used for cell metabolic processes, lowering its intracellular con- centration, and favoring continual diffusion into the cell. Carbon dioxide is produced in many of these metabolic processes, increasing its intracellular con- centration and favoring diffusion out of the cell and into capillary blood. In the lungs, thin alveolar walls and a minimal diffusion barrier to pulmonary capil- lary blood facilitate movement of oxygen and carbon dioxide by diffusion. In alveolar air, partial pressure of oxygen is high and partial pressure of carbon dioxide is low, favoring diffusion of oxygen into pulmonary capillary blood and diffusion of carbon dioxide from pulmonary capillary blood to the alveolus for removal

78 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

(a)

(b) (c)

High concentration Low concentration Low concentration

Selectively permeable membrane

Movement along a concentration gradient

Movement against a concentration gradient

High concentration

FIGURE 4.3 Concentration gradients. (a) Concentration gradient in a solution—no barrier. (b) Concentration gradient across a barrier permitting movement from high to low concentration (downhill movement). (c) Concentration gradient across a barrier—solute needs energy to be moved from region of low to high concentration (uphill movement, requires energy source).

by respiration. Rate of diffusion for a substance fol- lows Fick’s first law of diffusion:

= − ∆ ∆

J DA c

where

J = flux, the amount of substance that moves across the barrier from a region of higher con- centration to a region of lower concentration

D = the diffusion constant for the substance A = the surface area for diffusion

Dc = concentration gradient for the substance across a barrier

D x = thickness of the diffusion barrier Pathophysiologically, some lung diseases thicken the

barrier between alveolar air and capillary blood, reduc- ing the rate of oxygen and carbon dioxide diffusion. In

these cases, low levels of oxygen supplementation can increase the partial pressure difference (analogous to the concentration difference, Dc) to compensate for the increased barrier thickness (Dx).

Steroid hormones are relatively hydrophobic and diffuse across cell membranes, binding to intracellu- lar receptors to exert their biological effects. Fatty acids enter cells and can be used as an energy source through oxidation in mitochondria and peroxisomes. Many drugs are hydrophobic and diffuse into cells to produce their biological effects. In summary, sub- stances that are small, uncharged, and nonpolar, and substances that are larger, uncharged, hydrophobic, and nonpolar are all able to cross cell membranes by diffusing through the lipid bilayer. All other substances require other means of transport across this barrier, whether moving from extracellular to intracellular fluid or in the reverse direction.

Chapter 4 • Cell Physiology and Pathophysiology 79

ENDOCYTOSIS AND EXOCYTOSIS Large molecules such as proteins can be brought into cells or secreted by cells by endocytosis and exocyto- sis, respectively.

Endocytosis In endocytosis, a portion of extracellular fl uid is engulfed by the cell membrane into a vesicle that pinches off and

enters the cell ( Figure 4.5 ). Cells are able to conduct surveillance of molecules in their environment through this process. Endocytosis of small amounts of extra- cellular fl uid is referred to as pinocytosis . Professional phagocytic cells, such as macrophages and neutrophils, use a similar mechanism to engulf large particles, such as bacteria, internalizing them and destroying them as part of their role in host defense. This process is

TABLE 4.1 pH and Concentration Gradients for Physiological Solutes in a Typical Cell

Solutes and pH Extracellular Concentration Intracellular Concentration

pH 7.4 7.2

Solutes

Sodium 135–147 mEq/L 10–15 mEq/L

Potassium 3.5–5.0 mEq/L 120–150 mEq/L

Calcium 2.1–2.8 mmol/L (total) 1.1–1.4 mmol/L (free, ionized)

~10 −7 mmol/L (ionized)

Chloride 95–105 mEq/L 20–30 mEq/L

Phosphate 1.0–1.4 mmol/L (total) 0.5–0.7 mmol/L (ionized)

0.5–0.7 mmol/L (ionized)

Bicarbonate 22–28 mEq/L 12–16 mEq/L

Magnesium 0.6 mmol/L (ionized) 1 mmol/L (ionized) 18 mmol/L (total)

Glucose 5.5 mmol/L Very low

Sources: Data from Aronson et al. (2017) ; Koeppen BM, Stanton BA, eds. Berne & Levy Physiology. 7th ed. Philadelphia, PA: Elsevier; 2018.

Extracellular fluid

Cytoplasm

Oxygen molecules

Time

Lipid bilayer (plasma membrane)

FIGURE 4.4 Simple diffusion. This method of transmembrane movement is used by small, nonpolar molecules, such as oxygen and carbon dioxide, and by lipids such as fatty acids and steroid hormones.

80 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

referred to as phagocytosis to differentiate it as a pro- cess most common to phagocytes.

Cells can take up specific extracellular particles such as low-density lipoproteins (LDLs) through interactions between membrane receptors and membrane regions where the cytoskeletal protein clathrin is attached. In this process, termed receptor-mediated endocytosis, binding of LDLs, for example, to their receptors sig- nals the underlying membrane and clathrin protein to form an endocytic vesicle. The clathrin protein then folds to enclose the endocytic vesicle, which enters the cell to deliver its contents. This is a selective process enhanced by the presence on a given cell membrane of receptors for a given solute that cell needs to function. The most common form of familial hypercholesterol- emia is due to malfunction of LDL receptors that do not trigger endocytosis—this disorder manifests with extremely high cholesterol levels, tissue cholesterol accumulation, and early-onset atherosclerosis.

Exocytosis The process of exocytosis is essentially the reverse of endocytosis. A membrane-coated vesicle moves to the cell membrane and fuses with it, releasing its con- tents to the extracellular fluid (Figure 4.6). As with endocytosis, there are two prominent modes of exo- cytosis: general (also referred to as constitutive) and signal-generated (also referred to as regulated). General exocytosis is an ongoing process of cells that primarily

function to synthesize secreted proteins—often pro- teins that will be carried in the blood plasma. Examples include many of the proteins synthesized by the liver (albumin, coagulation proteins, and carrier proteins) or by immune plasma cells (immunoglobulins). In these types of cells, protein synthesis may be upregulated or downregulated, but proteins are generally secreted soon after they are synthesized (Figure 4.6a).

Regulated exocytosis (see Figure 4.6b) is a process in which a molecule or protein destined for secretion is held in intracellular vesicles until a specific event trig- gers exocytotic release. For neurons, this would involve an action potential reaching an axon terminal, opening of voltage-dependent calcium channels, and release of neurotransmitter vesicles into a synaptic cleft. For β cells of the pancreatic islets, depolarization can occur in response to increased cellular glucose metabolism similarly opening voltage-gated calcium channels, stim- ulating regulated exocytosis of vesicles containing the hormone insulin. Thus, endocytosis and exocytosis are mechanisms for bulk movement of large numbers of solutes or large solutes such as proteins across the cell membrane by means of membrane-bounded vesicles.

FACILITATED DIFFUSION Many of the substances that move across cell mem- branes are polar and hydrophilic, and are thus unable to move by simple diffusion across the membrane’s

Phagocytosis

Pseudopodium

Phagosome

Extracellular fluid

Pinocytosis

Large particle

Receptor-mediated endocytosis

Coated pit

Coat protein

Coated vesicle

Vesicle

Cytoplasm

Receptor

FIGURE 4.5 Endocytosis. Extracellular fluid and particulate matter are pinched off into a vesicle that invaginates into the cell. Phagocytosis is used for larger particles, even cells, while pinocytosis is the internalization of small amounts of fluid and solutes. Receptor-mediated endocytosis is initiated by a specific extracellular particle (such as a low-density lipoprotein) binding to its receptor in the region of a coated pit and signaling for membrane internalization.

Chapter 4 • Cell Physiology and Pathophysiology 81

hydrophobic lipid core. Movement of these substances down their concentration gradient is facilitated by membrane protein transporters, which bind a mole- cule of the substance on the side of higher concentra- tion and release it on the side of lower concentration. Because the direction of net movement is from higher to lower concentration, this process is referred to as diffusion, and because the movement requires a pro- tein carrier, it is referred to as facilitated ( Figure 4.7 ).

Glucose is an example of a biomolecule that com- monly enters cells through facilitated diffusion pro- teins, because it is highly hydrophilic and polar. Many cell types use glucose as their primary fuel, and the continual consumption of glucose keeps intracellu- lar glucose concentration lower than extracellular glucose concentration. Glucose transporters have the generic name GLUT, with the various subtypes identifi ed by number (e.g., GLUT1 through GLUT5 ).

Vesicle

Extracellular fluid

Cytoplasm(a)

Ca2+

Intracellular

Extracellular Plasma membrane

Fusion

Priming

Docking

Transport(b)

FIGURE 4.6 Exocytosis. (a) Constitutive exocytosis. This process basically reverses endocytosis— a substance made in the cell is packaged in a vesicle by the Golgi apparatus, moves to the membrane, and is released to the extracellular fl uid. Exocytosis can occur continuously; an example is seen in proteins that must be constantly renewed, as in liver production of albumin and many clotting proteins. (b) Regulated exocytosis. Secretion of hormones and neurotransmitters does not occur constantly; rather, a signal is required before they can be released. This is a calcium-dependent process in which vesicles of hormone or transmitter are located near membrane release sites. When intracellular calcium increases, vesicles can dock and fuse with the membrane, followed by exocytosis of vesicle contents.

82 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

The GLUT2 protein is particularly associated with moving glucose at high rates in specific sites. Of these, GLUT2 will be highlighted again in Chapters 14, Liver, and 17, Endocrine System, describing the metabolic role of the liver and control of circulating glucose. Glucose can enter liver cells via GLUT2 after a meal, when glucose and insulin levels are high and the liver is metabolizing glucose and also storing it in the form of glycogen. Then, during fasting, liver cells produce glucose through glycogen breakdown and new glucose synthesis (glycogenolysis and glucone- ogenesis, respectively). In that state, glucose leaves the liver cells, also via GLUT2, maintaining fasting blood glucose levels in the normal range. GLUT4, on the other hand, is found in vesicles inside muscle and fat cells. When insulin is present, GLUT4 trans- porters move to the cell membrane and facilitate glu- cose entry into these cells. This process is discussed further in Chapter 17, Endocrine System, in the context of insulin actions in the body. In sum- mary, facilitated diffusion is a mechanism by which hydrophilic polar and charged substances can move across cell membranes down their concentration gradients.

ACTIVE TRANSPORT Active transport is a mechanism for moving a solute against its concentration gradient, which requires the input of energy. There are two types of active trans- port: primary and secondary.

Primary Active Transport The most abundant primary active transporter of the body is the sodium–potassium pump (Na+/K+ pump), also referred to as the sodium–potassium ATPase (adenosine triphosphatase). This protein, found on all cells of the body, splits ATP and, with the energy provided, transports sodium out of the cell and trans- ports potassium into the cell (Figure 4.8). Both of these ions are moving against their concentration gra- dients, and the ability to maintain normal intracellular and extracellular fluid homeostasis is dependent on these transport activities. The concentration gradient for sodium created by the Na+/K+ pump is a source of potential energy in that other solutes can be trans- ported against their concentration gradients by trans- porters that link their movement to sodium influx into cells. (This process is secondary active transport, described later.)

The Na+/K+ pump is active continuously. Beginning with the step of sodium binding, three cytoplasmic sodium ions bind to sites facing the intracellular fluid. ATP then binds to the transport protein and is split, donating a high-energy phosphate group and changing the pump’s configuration. The protein shifts its orienta- tion and releases the sodium ions to the extracellular fluid, subsequently binding two potassium ions. This is followed by release of potassium to the intracellu- lar fluid and release of the bound phosphate. The cycle is repeated with the next round of sodium binding and ATP hydrolysis.

Two additional ATP-requiring active transporters are referred to throughout the book (Figure 4.9). The first of these is the calcium ATPase, or calcium pump. Another name for this protein is the sarcoendoplas- mic reticulum calcium ATPase (SERCA). This pump is found on cell membranes and also on the membrane of the endoplasmic reticulum (or sarcoplasmic retic- ulum in muscle cells; Figure 4.9a). The large calcium gradient between extracellular fluid and intracellular fluid is maintained by these pumps that move calcium across the cell membrane or into the endoplasmic reticulum. Certain cell signaling pathways involved in muscle contraction or receptor activation can release endoplasmic/sarcoplasmic reticulum calcium in brief bursts that cause a cascade of physiological responses. Calcium ATPase activity then restores the low intracellular calcium level and terminates the sig- nal. Finally, the proton pump, or potassium–hydrogen ATPase, is involved in secretion of hydrochloric acid

FIGURE 4.7 Facilitated diffusion is used to move polar or charged solutes down a concentration gradient. Thus, the direction is the same as that of simple diffusion, from a region of higher concentration to a region of lower concentration. A protein carrier is needed because the plasma membrane’s hydrophobic core blocks passage of the solute and its shell of water. This is the most common way for glucose and amino acids to enter cells.

Chapter 4 • Cell Physiology and Pathophysiology 83

ATP

ADP

Sodium Potassium

Extracellular fluid

Intracellular fluid

FIGURE 4.8 Active transport: sodium–potassium (Na + /K + ) pump. Every cell of the body has Na + / K + pumps that maintain the proper ionic gradients of high intracellular K + and low intracellular Na + . These conditions allow the electrophysiological operation of electrically active cells like the cardiac myocytes and the neurons of the brain, and drive transport processes in all tissues. Much of the body’s oxygen consumption at rest and during physical activity provides the ATP needed for this continual movement of Na + and K + ions against their concentration gradients. When tissues become hypoxic, cells swell and eventually die because of inability to maintain ionic homeostasis. ADP, adenosine diphosphate; ATP, adenosine triphosphate; P, phosphate; Pi, inorganic phosphate.

in the stomach and in renal acid–base regulation. The proton pump is the target of drugs that inhibit gastric acid secretion ( Figure 4.9b ).

Secondary Active Transport Based on the sodium gradient established by the sodium–potassium pump, cells have many transporters that link downhill sodium movement into the cell to drive the uphill movement of other substances against their concentration gradient. This process is called sec- ondary active transport because it does not use ATP, but it does use the favorable energy of allowing sodium to sneak back into the cell after it is actively removed by the Na + /K + pump. There are numerous examples of secondary active transporters contributing to solute movements in specialized tissues.

Cells of the renal tubules and gastrointestinal tract function to completely remove glucose molecules from one side of an epithelial barrier to the other, against its concentration gradient. The secondary active transport proteins called SGLT1 and SGLT2 (sodium–glucose transporter) accomplish this function in the gastroin- testinal tract and renal tubule, respectively. This is an example of cotransport (also referred to as symport ), in that the sodium and glucose are both moving in the

H+–K+ ATPaseCalcium pump/ calcium ATPase

Extracellular fluid or ER lumen

Extracellular fluid

Intracellular fluid Cytoplasm

H�

K�Ca2+

)a( )b(

FIGURE 4.9 Active transport. (a) Active transport of calcium (Ca 2+ ) and (b) hydrogen (H + ) and potassium (K+) ions. Uphill movement of the ions is accomplished, in part, by these ATPases that maintain cell ionic and acid–base homeostasis. ATPase, adenosine triphosphatase; ER, endoplasmic reticulum.

84 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

same direction (Figure 4.10). SGLT2 is a target of a class of drugs (SGLT2 inhibitors) used in management of type 2 diabetes.

Other secondary active transporters move sodium in the opposite direction of the paired solute. For example, in muscle cells, some of the calcium that enters the cell to cause muscle contraction is removed by a transporter that links entry of three sodium ions to exit of one cal- cium ion. This opposite movement of ions is referred to as counter transport (also known as antiport, for example, Na+/Ca2+ exchange [NCX]). Other second- ary active transporters described in this book are the sodium, potassium, and chloride cotransporters found in the tubular cells in the kidney that are the target of loop diuretic drugs. In the brain, neurotransmitters are removed from synapses by secondary active transport- ers selective for given transmitters, such as selective serotonin reuptake transporters. Table 4.2 lists the transporters that are described elsewhere in this book.

AQUAPORINS Although water molecules are polar, it was once thought that they were small enough to be able to freely diffuse across cell membranes. Aquaporins were later identified as channels that serve as the channels that serve as major transport mechanism for water to move into and out of cells, under the influence of osmotic pressure (Figure 4.11). Although aquaporin proteins are present in all cells, aquaporin movements are regu- lated in kidney cells in the distal nephron, where water absorption is controlled by the hormone vasopressin.

ION CHANNELS Ion channels are transmembrane proteins with a cen- tral pore that allow movement of an ion down its con- centration gradient. When they are open, ions freely diffuse through until the pores close, or until the ions reach electrochemical equilibrium based on the con- centration difference of the ion and the membrane voltage that develops as a consequence of ion move- ment. Ion channels, by and large, are selective for one ion only. They are named according to the ion that they are selective for, such as sodium channels, potassium channels, and calcium channels (Figure 4.12). Ion channels are found in all body cells, but they have a particularly prominent role in electrically excitable cells such as neurons and muscle cells.

The presence of ion channels in the cell membrane contributes to the ability of cells to develop a resting membrane potential—a voltage difference across the cell membrane. Most cells have this potential differ- ence as the inside of the cell is electrically negative with respect to the extracellular fluid, with the cell membrane able to separate the charges, maintaining the voltage difference.

The resting membrane potential develops, in part, based on the differential distribution of ions across the plasma membrane (sodium high outside, potas- sium high inside), as described earlier. In addition, the cell membrane has limited permeability to ions under resting conditions, but is more permeable to potassium than to any other ion. Finally a portion of the resting membrane potential is determined by the Na+/K+ pump activity that moves three sodium ions out of the cell for every two potassium ions that enter. How do these factors combine to create the resting mem- brane potential?

The role of membrane ion channels in produc- ing a resting membrane potential is illustrated in Figure 4.13. Study Figure 4.13a—a diagram of a barrier separating two fluids of differing composition. As with extracellular fluid, the compartment on the left is high in sodium and low in potassium. As with intra- cellular fluid, the compartment on the right is high in potassium and low in sodium. Not shown in this figure

Na+

Glucose

FIGURE 4.10 Secondary active transport. Secondary active transport links the movement of a solute down its concentration gradient (sodium being the most common) to the movement of a second solute against its concentration gradient. This is favorable when cells can benefit from maintaining a concentration gradient of a solute preferentially on one side of the membrane, while sparing ATP. The consumption of ATP by the sodium–potassium pump sustains the sodium concentration gradient so that sodium entry via these transporters is energetically favored. Sodium–glucose transporters are one example of secondary active transport. ATP, adenosine triphosphate.

Chapter 4 • Cell Physiology and Pathophysiology 85

are the accompanying anions that give electrical neu- trality to each of these solutions. Given an imperme- able membrane, none of these ions can move, and no potential difference can develop across the membrane.

Now refer to Figure 4.13b . All conditions are the same as in panel (a), but potassium channels are now inserted in the membrane. Now that the potassium ions have a pathway to leave the cell, they fl ow through the potassium channels, down their concentration gradient, as shown. However, without accompanying anion channels, negative charges are left behind, and

are attracted to the membrane and the greater num- ber of positive charges now on the other side of the membrane. At this point, there is a potential difference across the membrane, with the right side compart- ment negative with respect to the left side compart- ment. The negative charge will build up until it attracts potassium ions back through their channels at a rate equal to their movement away. This electrical charge can be calculated based on the concentrations of potassium on each side of the membrane and is known as the Nernst potential. Most cells of the body have a

TABLE 4.2 Transport Mechanisms Described in This Book

Mode of Transport Examples Transporter Names and Locations

Facilitated diff usion Glucose GLUT1—red blood cells, blood–brain barrier, many other cells

GLUT2—liver, pancreatic β cells, renal tubules

GLUT3—neurons

GLUT4—muscle and adipose cells (insulin-sensitive)

Fructose GLUT5—gastrointestinal tract

Amino acids Several transporter types found on most cells, enriched in small intestine and kidney tubules

Active transport (primary active transport)

Sodium–potassium (Na + /K + ) pump Membranes of all cells

Calcium pump Cell and endoplasmic reticulum membranes

Potassium–hydrogen pump Renal tubules, stomach parietal cells

Secondary active transport

Sodium–glucose cotransport SGLT1 (gastrointestinal)

SGLT2 (renal)

Sodium–calcium exchange NCX

Sodium–potassium–2 chloride cotransport

NKCC

Sodium–chloride cotransport NCC

Sodium–hydrogen exchange NHE

Sodium–serotonin cotransport SERT

Sodium–norepinephrine cotransport NET

Sodium–dopamine cotransport DAT

Ion channels Potassium leak channels Many cells—set resting membrane potential

Delayed potassium channels Electrically excitable cells—action potential repolarization

Fast sodium channels Electrically excitable cells—action potential initiation and propagation

Slow calcium channels Muscle cells—some action potential initiation, provide calcium for contraction

86 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

resting membrane potential that is negative, owing to these factors:

• Potassium leak channels outnumber other open ion channels at rest, so potassium movements are the greatest factor setting the electrical equilibrium.

• The composition of the extracellular fluid and intracellular fluid differs regarding distribution of sodium, potassium, chloride, and calcium, so that each of these has different gradients for movement when ion channels open.

• Constant activity of the Na+/K+ pump maintains the proper ion composition of extracellular and intracel- lular fluids.

Na+

ECF ICF ECF ICF

Na+

+ + +

+ + + + +

+ +

– – –

– – – – –

– –

K+ K+

(a) (b)

FIGURE 4.13 Potassium leak channels contribute to a resting membrane potential. Potassium (K+) is high in the ICF and low in the ECF. (a) If fluids with this composition are separated by a solid membrane, no ion movement will occur. (b) If potassium channels are inserted in the membrane, K+ will move from ICF to ECF, down its concentration gradient. This will generate a membrane potential difference, with the inside negative with respect to the outside. ECF, extracellular fluid; ICF, intracellular fluid; Na+, sodium.

FIGURE 4.12 Ion channels. An ion channel is a pore that, when open, permits a flow of ions in a stream, down its concentration gradient. Most ion channels are selective for one ion, from which they derive their name.

Water molecules

Lipid bilayer

Aquaporin

FIGURE 4.11 Aquaporins (water channels). Although some water molecules are able to move across plasma membranes by simple diffusion, the rate of water movement is greatly enhanced by the presence of aquaporin proteins in membranes of most cells. The movement is from a region of lesser osmotic strength to a region of greater osmotic strength, and the water movement is termed osmosis.

Chapter 4 • Cell Physiology and Pathophysiology 87

diffuse through the cell membrane, binding to nuclear receptors and altering cell transcription and translation for long-term effects on cell function. Many of these reactions are reversible once the ligand diffuses away from the receptor, and there are several other mecha- nisms that stop or reverse the actions of receptor acti- vation, allowing compensatory adjustments to return to normal after a time of altered activity. One can think of these as the brakes that slow a system down after a period of applying a gas pedal .

AUTONOMIC NERVOUS SYSTEM AND SIGNALING BY G PROTEIN–COUPLED RECEPTORS The autonomic nervous system is the major rapid regulatory system of the body. Autonomic neurons located within the central nervous system consist of preganglionic neurons that use the neurotransmitter acetylcholine. Parasympathetic preganglionic neurons are located in brainstem cranial nerve nuclei and in the sacral spinal cord, while sympathetic pregangli- onic neurons are located in thoracic and lumbar spinal segments. These preganglionic neurons have synaptic connections with autonomic postganglionic neurons in peripheral autonomic ganglia, with the parasym- pathetic ganglia generally located within or close to organs and sympathetic ganglia located lateral to the vertebral column or in front of the vertebral column. Parasympathetic postganglionic neurons are choliner- gic , releasing the neurotransmitter acetylcholine, and most sympathetic postganglionic neurons are adren- ergic , releasing the neurotransmitter norepinephrine. Sympathetic postganglionic neurons innervating sweat glands and certain vascular beds are cholinergic.

Much of our knowledge of GPCR mechanisms derives from biochemical, physiological, and pharma- cological studies of cholinergic and adrenergic effects on target organs, as these end-organ responses to para- sympathetic and sympathetic stimulation are mediated by these receptors. Both autonomic branches have par- ticularly profound effects on cardiovascular control. In addition to autonomic transmitters, many neurotrans- mitters and hormones act via GPCRs. In this mode of cell signaling, an extracellular hydrophilic ligand binds to a recognition site on its membrane receptor, and this binding initiates a cascade of intracellular reactions in and near the membrane, generating intracellular sec- ond messengers that alter target cell activity.

Box 4.2 summarizes characteristics of GPCRs. The GPCRs have certain common structural ele- ments, the chief one being that each is a long protein chain that crosses the plasma membrane seven times ( Figure 4.14 ). When the appropriate ligand binds on the extracellular side of the receptor, the receptor’s shape changes and it binds to and activates a three- part, guanosine triphosphate (GTP)-binding protein or G protein located at the intracellular face of the

A consequence of having a resting membrane potential is that, for excitable cells, inputs that lead to depolariza- tion (decreased or less negative membrane potential) can open voltage-gated channels, some of which pro- duce action potentials—large electrical spikes of depo- larization that can be propagated for long distances along axons and ultimately release neurotransmitter molecules into synapses. Action potentials of neurons are the basis of brain activity, and action potentials of muscle cells are the basis of contraction of skeletal and cardiac muscles. Action potentials can also produce contractions of smooth muscle cells, and depolariza- tion can contribute to hormone release from endocrine cells. In summary, ion channels play many roles in cell and organ function and are critical for the function of excitable neurons and muscle cells. Mutations in ion channel genes are the source of many disorders of neu- ron and muscle function, including epilepsy.

Thought Questions

1. What properties of a solute are relevant in determining the type of transport that moves the solute into and out of cells?

2. Explain the role of the Na + /K+ pump in enabling the function of secondary active transport proteins.

3. What role do potassium leak channels play in creating a negative resting membrane potential?

MECHANISMS OF CELL SIGNALING

OVERVIEW OF SIGNAL TRANSDUCTION Many rapid homeostatic adjustments are made pos- sible by the autonomic transmitters norepinephrine (sympathetic nervous system) and acetylcholine (para- sympathetic nervous system). Adaptations including cardiovascular regulation, airway diameter, gastroin- testinal tone and secretions, and kidney functions are modulated by these autonomic mediators. In addition, hormones such as epinephrine, vasopressin, glucagon, and pituitary trophic hormones also have rapid actions to modulate activity of their target cells, glands, and organs. The actions of all of these ligands are mediated by binding to membrane G protein–coupled receptors (GPCRs). Growth and development signals, as well as other types of homeostatic adjustments requiring cell division and new protein synthesis, usually involve receptors working through tyrosine kinase mecha- nisms. Finally, steroid and thyroid hormones are able to

88 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

membrane. The G protein then dissociates, and its active α subunit activates a membrane-bound enzyme that begins to generate intracellular second messenger molecules.

G Protein–Coupled Receptors Linked to Adenylyl Cyclase The first type of GPCRs discovered were those that stimulate the enzyme adenylyl cyclase and generate the intracellular second messenger cyclic adenos- ine monophosphate (cAMP). Many hormones and

neurotransmitters exert their effects through increas- ing cAMP levels. The principles of the system are illus- trated here by focusing on one of these receptors, the β-adrenergic receptor that responds to the hormone epinephrine and the sympathetic neurotransmitter norepinephrine.

When norepinephrine binds to the β-adrenergic receptor, the receptor undergoes a shape change and increases its affinity for a three-part G

s protein

on the inner leaflet of the plasma membrane. The G protein, made up of α

s , β, and γ subunits, dissociates

P 21 7 3 6 4 5

G Helix 8

C-terminus

N-terminus

+Agonist

C-terminus

Helix 8

N-terminus

Active GPCRInactive GPCR

Signaling

Extracellular

Intracellular

FIGURE 4.14 General features of GPCR structure. These receptors are often described as serpentine, because of the number of bends in the protein as they cross the membrane. Ligand- binding sites for hormones or neurotransmitters face the extracellular fluid, while receptor sites of G protein interactions face the intracellular fluid and inner membrane. GPCR, G protein–coupled receptor.

BOX 4.2 Characteristics of G Protein–Coupled Receptors

• Found on all cells, GPCRs are the most common mechanism by which hormones and neurotransmitters influence cell and organ function.

• Signaling is amplified as a small group of receptors can activate a larger group of G proteins, ultimately stimulating membrane- bound enzyme production of many molecules of second messengers.

• Second messengers act by turning on protein kinase enzymes that phosphorylate intracellular proteins to change their function to meet the body’s changing needs.

• Phosphorylation of some proteins increases their activity, while phosphorylation of other proteins

decreases their activity, but in both cases a change in the protein’s activity produces the final effect of hormone/receptor signaling.

• The strength and duration of GPCR effects are limited by various inhibitory and off-switches, including inhibitory receptors themselves, an intrinsic timing function of the GTPase activity of G proteins, and protein phosphatase enzymes that remove phosphate groups from proteins.

• Finally, the second messengers can be metabolized, terminating their action. For example, a phosphodiesterase cleaves cAMP and converts it into 5¢-AMP, which is inactive.

5¢-AMP, 5¢-adenosine monophosphate; cAMP, cyclic adenosine monophosphate; GPCR, G protein–coupled receptor; GTPase, guanosine triphosphatase.

Chapter 4 • Cell Physiology and Pathophysiology 89

into α s , and a βγ pair ( Figure 4.15 ). The α

s moves

to the nearby enzyme adenylyl cyclase and increases its activity. Activated adenylyl cyclase converts many molecules of ATP to cAMP, amplifying the signal ( Figure 4.16 ). In the next step of the cascade, cAMP molecules diffuse throughout the cytoplasm, activat- ing the enzyme protein kinase A (PKA; Figure 4.17 ). A protein kinase is an enzyme that adds a phosphate group onto target proteins. This process of phosphor- ylation occurs on the hydroxyl-containing amino acids serine, threonine, and tyrosine. Many of these protein kinases, including PKA, are serine/threonine kinases, phosphorylating target proteins at sites of the amino acids serine and threonine, thus altering target protein activity level. Each activated PKA can phosphorylate many copies of target protein mole- cules, further amplifying the signal to quickly change cell function. The end result is that an extracellular signal, in this case norepinephrine, can quickly alter cell and organ function through its GPCRs. β-Adren- ergic receptor activation is the mechanism by which the sympathetic nervous system increases heart rate and force of contraction, for example.

The cAMP system is also the target of inhibitory receptors. An example of these is the M

2 subtype of mus-

carinic acetylcholine receptor. This receptor is found on sinoatrial node cells in the heart and is central to the mechanism used in slowing of heart rate by the para- sympathetic transmitter acetylcholine. Activation of M

receptors activates a G i protein that dissociates into α

and a βγ pair. The α i protein inhibits adenylyl cyclase,

decreasing cAMP levels, and the βγ pair moves within the membrane to increase the activity of inhibitory potassium channels, in this case slowing the heart rate. Again, at each step of this cascade, there is amplifi cation of the signal, so activation of several M 2 receptors can rapidly decrease cell cAMP levels and open many potas- sium channels, resulting in inhibition. The mechanism of α

i is depicted in Figure 4.16 . The presence of both stim-

ulatory and inhibitory types of receptors for the adenylyl

Receptor G protein Receptor

Activated ACAC

s s

FIGURE 4.15 AC activation by GPCRs. Ligand binding to a stimulatory AC-linked receptor causes the dissociation of the attached G protein, allowing the α

s subunit to move to and activate

the enzyme AC. The βγ subunits remain bound together, but can move within the membrane to modulate the activity of other membrane proteins. AC, adenylyl cyclase; GPCRs, G protein–coupled receptors.

cAMP R-inh R-stim

GiGs

FIGURE 4.16 Stimulatory and inhibitory AC–linked receptors. AC can be stimulated or inhibited, depending on the type of receptor activated. Stimulatory receptors (R-stim) work through G

s to increase AC activity and cyclic adenosine monophosphate

(cAMP) production; inhibitory receptors (R-inh) work through G

i to decrease AC activity and cAMP production.

AC, adenylyl cyclase.

90 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

cyclase system provides for homeostatic adjustments that can upregulate or downregulate adaptive changes.

G Protein–Coupled Receptors Linked to Phospholipase C The second GPCR system has an additional layer of complexity relative to the adenylyl cyclase system. In the phospholipase C–linked signaling cascade, recep- tors work through the G

q type of G protein, with α

stimulating the membrane-bound enzyme phospholi- pase C. Activated phospholipase C splits the membrane phospholipid phosphatidylinositol bisphosphate (PIP

2 )

into diacylglycerol (DAG) and inositol trisphosphate (IP

3 ), with DAG remaining in the membrane and IP

diffusing into the cytoplasm and serving as an intra- cellular second messenger (Figures 4.18 and 4.19). DAG activates protein kinase C, which then proceeds to phosphorylate target proteins. IP

3 binds to recep-

tors on the endoplasmic reticulum, causing the endo- plasmic reticulum to release stored calcium in a burst, transiently increasing cytoplasmic calcium concen- trations. The calcium can have several downstream effects, including initiating muscle contraction, binding to calmodulin to regulate many cell activities, and acti- vating other protein kinases. An example of a phospho- lipase C–linked receptor is the α

1 -adrenergic receptor

on vascular smooth muscle. When sympathetic nerves innervating blood vessels release norepinephrine, it binds with high affinity to these receptors, increasing intracellular levels of IP

3 and releasing calcium from

the endoplasmic reticulum. Calcium elevations lead

Receptor Receptor

PLC

FIGURE 4.18 PLC-activating receptor. In the PLC system, the G q protein stimulates enzyme

activity, and there are no known inhibitory receptors. When a ligand binds to the receptor, the G protein dissociates into an α

q subunit that stimulates PLC and a βγ pair that may have other

physiological actions. PLC, phospholipase C.

ATP cAMP

Phosphorylation of target proteins: Enzymes Ion channels DNA-binding proteins

Active protein

kinase A

Inactive protein

kinase A

FIGURE 4.17 Downstream effects of cAMP. As an intra- cellular second messenger, cAMP binds to inactive PKA, releasing the active enzyme. Active PKA, in turn, conducts phosphorylation of intracellular proteins, changing their shape and thus changing their activity. These allosteric modifications depend on addition of phosphate groups to certain serine and threonine amino acids within the target proteins; thus, PKA is called a serine/threonine kinase. Examples of PKA targets are shown. These are specific to the organ and cell sites where receptor stimulation is occurring. AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; ATP, adenosine triphosphate; PKA, protein kinase A.

Chapter 4 • Cell Physiology and Pathophysiology 91

to calcium/calmodulin binding and activation of an enzyme that causes cell contraction, ultimately produc- ing vasoconstriction and increasing blood pressure.

An interesting note is that there is also a calcium/ calmodulin-activated phosphatase enzyme that reverses phosphorylation, stripping the phosphate away from phosphorylated proteins. This phosphatase, calcineurin, provides a reversal signal, allowing for downregulation of a function once homeostatic adapta- tion is complete. The major clinical use for calcineurin inhibitors is post-transplantation as these drugs are immunosuppressive.

G Protein GTPase Activity Is a Timing Mechanism One characteristic held in common by the various G proteins that link to GPCRs is that the α subunits are GTPase enzymes. They are capable of splitting GTP to release guanosine diphosphate (GDP) and a free phos- phate group. Before a ligand binds to its GPCR, the associated G protein is initially GDP-bound and inac- tive. After the ligand binds and activates the receptor, the G protein α subunit GDP is replaced by GTP; the α subunit dissociates from the βγ pair and activates or inhibits the relevant enzyme (adenylyl cyclase or phospholipase C). As the second messenger is gener- ated, the α subunit is also breaking down the bound

GTP, leaving GDP bound to the α protein, returning the βγ proteins to their initial state of binding to α , and turning off second messenger production. If suffi cient ligand is present in the extracellular space, it can bind to another receptor and initiate more second messen- ger production; however, in the absence of additional ligand, the GTPase mechanism will terminate the intra- cellular signaling cascade ( Figure 4.20 ).

CELL SIGNALING BY CYCLIC GUANOSINE MONOPHOSPHATE On the spectrum of cell signaling from GPCR/second messenger systems and enzyme-linked receptors, sig- naling by cyclic guanosine monophosphate (cGMP) is something of a hybrid and a novelty, with two major physiologically defi ned roles. First, cGMP is synthe- sized in retinal photoreceptor cells and plays a critical role in vision. Second, cGMP acts in vascular smooth muscle as a potent vasodilator. The enzyme that pro- duces cGMP, guanylyl cyclase, occurs in two forms in vascular smooth muscle cells: particulate guanylyl cyclase that is part of membrane-bound receptors for natriuretic hormones, and soluble guanylyl cyclase found in the cytoplasm. Soluble guanylyl cyclase is activated by endothelium-produced nitric oxide that

PLC

DAG

Protein kinase C

Ca 2+

PIP2

IP3

FIGURE 4.19 Consequences of PLC activation. Downstream effects of PLC activation leads to splitting of a membrane lipid phospholipid PIP

2 , with the release of DAG, which remains in the

membrane and activates protein kinase C, and IP 3 , which diffuses to the ER, causing a burst of ER

calcium release. Most of the intracellular changes occurring after PLC activation are caused by the increase in intracellular calcium. DAG, diacylglycerol; ER, endoplasmic reticulum; IP

3 , inositol trisphosphate; PIP

2 , phosphatidylinositol

bisphosphate; PLC, phospholipase C.

92 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

diffuses to the blood vessel’s smooth muscle layer. The resulting increase in cGMP produces vasodilation and lowers blood pressure.

CELL SIGNALING BY ENZYME-LINKED RECEPTORS Many of the receptors used for growth, development, and rapid cell proliferation have intrinsic enzyme activity, rather than requiring a G protein to activate an enzyme. In addition, most of these enzyme-contain- ing or enzyme-linked receptors have tyrosine kinase activity—they phosphorylate target proteins at tyro- sine sites. As a general pattern, ligand binding to these receptors is followed by dimerization, in which two copies of the same receptor (homodimerization) or two different tyrosine kinase receptors (heterodimeriza- tion) are drawn together in the membrane. This is fol- lowed by autophosphorylation, in which each chain phosphorylates the other. After this self-activation, the tyrosine kinase portions of the receptor begin to phos- phorylate additional proteins, some of which attach and build up a complex of proteins along the intracellular portion of the receptor (Figures 4.21 and 4.22). Most of these receptors have their greatest activity during

periods of rapid growth and cell proliferation, embry- onic and fetal stages, and during childhood. In adult- hood, their activity is brief and situation specific, for example, during wound healing or immune responses. Dysregulation of tyrosine kinase signaling pathways is common in cancer, fibrosis, chronic inflammation, and disorders of immune hyper-responses.

The receptors for insulin and insulin-like growth factor (a mediator of growth hormone signaling) do not need to dimerize; they consist of two identical or similar protein subunits bonded together. On the other hand, the epidermal growth factor receptor (EGFR) in its unstimulated form exists as a monomer and requires dimerization for activity. The extracellular portion of both of these receptors is the site of ligand binding, while the intracellular portion contains the tyrosine kinase domain that becomes activated by ligand binding.

One mechanism of dimerization after ligand bind- ing is illustrated in Figure 4.22. The ligand has two receptor binding sites, so binding leads to reversible cross-linking of two receptors. This draws the recep- tors close enough to allow interaction of the intracel- lular enzyme regions, initiating autophosphorylation.

(a)

(b) (d)

(c)

GTP

GDP

Hormone or neurotransmitter

GTPase

GTP

GDP

GTP GDP

Ion channels Adenylyl cyclase Phospholipase C Other proteins

FIGURE 4.20 Hydrolysis of GTP by G proteins limits the duration of hormone-receptor (R) actions. This can be thought of as a timing switch. (a) No ligand binds receptor, receptor is inactive and G protein is bound to GDP. (b) Hormone binds to the receptor, GDP is replaced by GTP, and the G protein dissociates into α and βγ subunits. (c) α subunit activates target enzymes and proteins. (d) α subunit intrinsic GTPase activity splits GTP, binding GDP and the βγ subunits, and returning to the inactive state. GDP, guanosine diphosphate; GTP, guanosine triphosphate; GTPase, guanosine triphosphatase.

Chapter 4 • Cell Physiology and Pathophysiology 93

Cysteine- rich domain

Tyrosine kinase domain

ss ss

EGF receptor

Insulin receptor, IGF-1 receptor

FIGURE 4.21 Enzyme-linked receptors. This type of receptor has an extracellular ligand-binding site, crosses the membrane once, and has an intracellular enzyme site, often consisting of a tyrosine kinase enzyme. The insulin receptor has two identical subunits linked by a disulfi de bond, with each subunit consisting of two protein chains also linked together by disulfi de bonds (SS). Once two insulin molecules have bound to the insulin receptor, the intracellular portion of the receptor becomes phosphorylated, and its own tyrosine kinase activity begins to phosphorylate nearby proteins. EGF, epidermal growth factor; IGF-1, insulin-like growth factor 1.

P P PPP P

Tyrosine kinase domain

(a) (b) (c) (d)

Extracellular space

Cytosol1

2 3

Signal protein

FIGURE 4.22 Intracellular signaling initiated by receptors using tyrosine kinase mechanisms. (a) These receptors are present in monomeric form (single, noninteracting chains) and are inactive when no extracellular signal is present. (b) Binding of ligands causes the formation of a dimer with two receptor molecules coming to close physical proximity and the tyrosine kinase regions of each phosphorylating the other chain. (c) Once phosphorylated, the intracellular portion of each receptor adds more phosphates outside the tyrosine kinase region. (d) The newly phosphorylated sites become scaffolds where other signaling proteins (numbered 1–3 in the fi gure) can bind, be phosphorylated, and exert their own enzyme activities. Some of the tyrosine kinase–type receptors stimulate cell proliferation and are implicated in wound repair and in cancerous transformation of cells. P, phosphate group.

94 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

Once phosphorylated, intracellular downstream signal- ing proteins (many of which are also kinase enzymes) bind and become activated.

The EGFR is particularly well studied, because it is mutated in many cancers, particularly those that involve epithelial tissues, such as colon and breast. In addition, some of the downstream proteins activated by EGFR and other growth-related proteins are also mutated in cancers. The Ras protein was one of the first identified oncoproteins (mutated forms of it are found in many cancers; Figure 4.23). EGFR activa- tion turns on several signaling pathways that ultimately alter gene expression, transcription, and translation to increase rates of cell division. While this growth- promoting action of epidermal growth factor is needed during embryonic and fetal development, when it is initiated in an unregulated fashion in adulthood, it promotes cancer formation. A close relative of EGFR encoded by the HER2/neu gene is ErbB2—this form of the EGFR is commonly mutated in breast cancer and has been targeted by specific drugs used in treating these cancers.

Cytokines are protein messages of the immune system. Immune responses to foreign proteins

include rapid proliferation of clones of immune cells (monocytes and lymphocytes) to fight off invaders. Cytokine receptors do not have intrinsic tyrosine kinase activity; rather, when they are activated by extracellular ligand binding, their intracellular por- tion changes shape to bind Janus kinase (JAK) pro- teins—tyrosine kinases that bind to the receptor and begin to phosphorylate target proteins. Many JAK targets are signal transducer and activator of tran- scription (STAT) proteins (Figure 4.24). Although classically associated with cytokine signaling and immune responses, JAK-STAT proteins are also mutated in some cancers and are the target of some antineoplastic drugs.

Transforming growth factor beta (TGF-β) is required for embryonic and fetal development of many tissues. In adulthood, TGF-β can normally inhibit cell proliferation, but its actions reverse to promote pro- liferation in the context of immune activation, inflam- mation, and cancer. The TGF-β receptor differs from other receptors in this class in that it has serine/ threonine kinase activity rather than tyrosine kinase activity. However, the structure and function of the TGF-β receptor is similar to the tyrosine kinase–type

Cytokine receptor

activation

Janus kinase (JAK)

Nuclear effects

STAT

FIGURE 4.24 Cytokine receptor intracellular signaling pathway. The Janus kinase–signal transducer and activator of transcription (JAK-STAT) sequence of activation is strongly associated with cytokine actions in target cells, including leukocyte proliferation and immune cell signaling.

EGFR activation

PLC Ras PI3K

DAG Raf AKT

PKC Mek Nuclear effects

Erk

Nuclear effects

FIGURE 4.23 EGFR intracellular signaling pathways. The nuclear actions of these pathways converge to promote new protein synthesis and to stimulate cell mitosis and proliferation. AKT, AKR mouse tumor 8 kinase (an oncogene also known as PKB, protein kinase B); DAG, diacylglycerol; EGFR, epidermal growth factor receptor; PI3K, phosphoinositide-3- kinase; PKC, protein kinase C; PLC, phosopholipase C.

Chapter 4 • Cell Physiology and Pathophysiology 95

TGF-ß receptor

activation

Smad cascade

Nuclear effects

FIGURE 4.25 TGF-β receptor intracellular signaling pathway. The TGF-β receptor is similar to tyrosine kinase–linked receptors in having a single transmembrane protein chain, but it differs in having serine/threonine kinase activity, rather than tyrosine kinase actions. TGF-β, transforming growth factor beta.

receptors in having a single transmembrane chain with an intracellular kinase domain, and phosphory- lating a cascade of intracellular proteins—as shown in Figure 4.25 , proteins of the Smad family. TGF-β is implicated in pathophysiological induction of fi brosis in a variety of diseases of the lung, liver, blood vessels, and other tissues.

CELL SIGNALING BY CYTOPLASMIC AND NUCLEAR RECEPTORS Steroid and thyroid hormones are relatively nonpolar and hydrophilic, and are able to move across the cell membrane by simple diffusion. Once inside the cell, one of two pathways is followed: (1) Some steroids bind to receptors in the cytoplasm and are then trans- ported into the nucleus. (2) Other steroid hormones and thyroid hormones diffuse across nuclear mem- branes to bind to receptors already in the nucleus. In either case, the hormone/receptor complex ultimately binds to regulatory elements on DNA, altering transcription and translation within that cell ( Figure 4.26 ). The onset of hormone effects is slower with steroid and thyroid hormones, as they have fewer direct, rapid actions on cells. However,

Ligand

mRNA

Peptides

Nucleus

Cytoplasm

Ligand-NR complex

HREs DNA

Transcription

Translation

FIGURE 4.26 Signaling by an NR or cytoplasmic receptor. Steroid and thyroid hormone receptors can be found in the nucleus, binding to DNA. Hormone binding activates transcription and translation of target cell proteins. This is a slower and long-lasting mechanism of cell signaling than that associated with G protein–coupled receptors. For some hormones, the receptors are initially located in the cytoplasm, and binding of their ligand fi rst stimulates transport into the nucleus before the DNA binding step occurs. HRE, hormone response element; mRNA, messenger RNA; NR, nuclear receptor.

96 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

by altering cell protein levels, they can induce pro- found and relatively long-lasting changes in function as well as structure.

SUMMARY OF CELL SIGNALING Maintaining physiological homeostasis requires the ability to make short- and long-term adaptations to the changing environment within the body and exter- nal to the organism. Short-term adaptations mediated by autonomic and endocrine signals often act by way of GPCRs, with rapid onset and several mechanisms of signal termination. Intermediate and longer-term adap- tations can be mediated by steroid and thyroid hormone receptors that alter gene expression and protein trans- lation. Finally, some normal and pathophysiological adaptations alter the activity of receptors involved in growth and development, promoting cell proliferation that can be helpful (as in immune responses) or patho- physiological (as in immune hyperreactivity or cancer).

Thought Questions

4. Trace the events involved in signaling by GPCRs that act through the adenylyl cyclase system.

5. List three of the brakes that terminate downstream actions of GPCRs.

6. What are the major points of diff erence between tyrosine kinase–linked intracellular actions and downstream actions of GPCR signaling?

MitochondrionNucleus

Muscle fiber

Myofibril

Sarcolemma

Sarcomere

Thick (myosin) filament

Sarcoplasmic reticulum Thin (actin)

filament

Z disc

I band I band M lineA band

H zone Z disc

Dark A band

Light I band

FIGURE 4.27 Sarcomere structure. Skeletal and cardiac muscle cells shorten by forming cross-bridges between actin and myosin fi laments highly organized into sarcomeres that shorten as a unit.

MECHANISMS OF CONTRACTILE CELLS

OVERVIEW OF CONTRACTILE CELL STRUCTURE AND FUNCTION There are three types of muscle cells in the body— skeletal muscle, cardiac muscle, and smooth mus- cle—with further specializations in these types. Historically, muscle cell structures have a unique nomenclature, with the prefi x sarco- , from the Greek meaning fl esh . In this fashion, the sarcolemma refers to the cell membrane, sarcoplasm to the cytoplasm, and sarcoplasmic reticulum to the endoplasmic reticulum. There are common elements of force development and cell shortening in each type of muscle, involving crossbridge formation between the proteins myosin and actin, the two strands ratch- eting past each other, and a requirement for cal- cium and ATP. Yet each type of muscle has unique aspects of function and regulation. General mech- anisms involved are described here, while other chapters provide additional details regarding cer- tain organ-specifi c aspects of muscle function and regulation.

THE SARCOMERE OF SKELETAL AND CARDIAC MUSCLE The microscopic appearance of skeletal and car- diac muscle cells shows a striated appearance, dark bands alternating with light bands ( Figure 4.27 ). The repeating unit is the sarcomere , the functional

Chapter 4 • Cell Physiology and Pathophysiology 97

unit of muscle contraction. The sarcomeres span from Z disc to Z disc, with darker A bands fl anked by lighter I bands, and an M line at the middle of the sarcomere. The fi ne structure responsible for this appearance includes hundreds of copies each of two linear proteins that reversibly bind together to cre- ate muscle contractions. Actin is a protein made up of repeated subunits joined together to form a thin strand, which is further surrounded in a spiral fash- ion with regulatory proteins troponin and tropomy- osin (described later in this section). Myosin is a protein containing two heavy chains and two pairs of light chains. Myosin’s heavy chain tail region forms thick interwoven strands with other myosin mole- cules, while the movable head region contains bind- ing sites for actin, ATP, and regulatory light chains ( Figure 4.28 ). Together, these proteins are referred to as myofi brils .

MECHANISM OF MYOFIBRIL CROSSBRIDGE FORMATION AND CONTRACTION During muscle contraction, myosin heads bind to recognition sites on actin strands and pull the actin strands toward the center of the sarcomere, causing sarcomere shortening and ultimately shortening the whole muscle. Other proteins are involved in holding the actin and myosin strands in alignment and linking sarcomeres together. Actin strands are held in align- ment by binding to the Z disks. Myosin strands are held in alignment by binding to a large, elastic protein called titin, which is also anchored to the Z disks. Additional protein complexes connect the sarcomeres to the cell membrane and cytoskeletal proteins so the long mus- cle fi bers shorten as a unit. Genetic mutations of some of these connecting proteins are responsible for some of the hereditary muscular dystrophy syndromes.

At rest, myosin and actin strands are lined up across from each other but are unable to form crossbridges because of the presence of regulatory proteins that block myosin–actin interactions ( Figure 4.29 ). Myosin head groups are binding ADP and free phosphate from a previous hydrolysis of ATP. Tropomyosin is a linear protein that physically blocks the sites on actin where myosin binds. A complex of three troponins—troponin I,troponin C, and troponin T—sit at regular intervals along the tropomyosin fi lament, keeping it in place. Troponin C is the site of calcium binding under condi- tions of muscle contraction. As sarcoplasmic calcium levels increase, calcium binding shifts the troponin/ tropomyosin complex, exposing actin’s binding sites and enabling crossbridge formation. As myosin head proteins bind to actin, creating crossbridges, the heads swivel, pulling the actin strands inward, and shorten- ing the sarcomere ( Figure 4.30 ). ATP then replaces the ADP, the crossbridges break, and new crossbridges form, ratcheting the actin strands further. This cycle is repeated many times, until the cytoplasmic calcium drops and the troponin/tropomyosin complex returns to its resting state.

As noted earlier, intracellular calcium levels are held very low in comparison to extracellular calcium. One mechanism of calcium entry into muscle cells is by action potentials that depolarize the membrane. Membrane voltage-gated calcium channels allow some calcium entry to initiate contraction. A much bigger source of calcium for contraction comes from the sarcoplasmic reticulum. The structures of the sarco- lemma and sarcoplasmic reticulum interact to rapidly increase intracellular calcium concentrations through a structure termed the triad . Invaginations of the sar- colemma, the T-tubules , propagate the action potential deep into the muscle fi ber, near terminal cisterns of

Actin Tropomyosin

Heavy chains

Essential light chains

Regulatory light chains

Troponin

Myosin filament

FIGURE 4.28 Proteins of muscle contraction. Each single myosin molecule consists of a heavy chain (divided between a longer tail region and a shorter, angled head region) that binds two light chains near the head region. The tail regions of several myosin molecules twine around each other, forming a thick, straight bundle, with heads projecting off at angles. Within muscle cells, chains of actin molecules line up in parallel, with the accessory proteins tropomyosin (a fi lamentous protein positioned over actin binding sites) and the tripartite troponin complex holding the tropomyosin molecule in place.

98 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

the sarcoplasmic reticulum (Figures 4.31 and 4.32). As the action potential depolarizes the membrane, a dihydropyridine receptor signals this voltage change to ryanodine receptors on the terminal cisterns, sig- naling for rapid release of stored calcium. The calcium released from the sarcoplasmic reticulum quickly reaches the troponin proteins and binds to troponin C, initiating muscle fiber contraction.

COMPARISON OF SKELETAL MUSCLE FIBERS AND CARDIAC MUSCLE CELLS Sarcomere structure and the striated appearance are quite similar between skeletal muscles and cardiac cells. However, there are significant differences in structure, organization, and control. Skeletal muscles carry out involuntary, reflex, and voluntary activity directed by the nervous system, and their contractions are initiated by motor neurons in the spinal cord and brainstem, as described in Chapter 15, Nervous System. The heart con- tracts autonomously, in response to pacemaker cells of the sinoatrial node, as described in Chapter 10, Heart. Even when denervated, after a heart transplant proce- dure, for example, the heart will continuously generate rhythmic contractions at a rate of about 60 to 70 beats each minute. Skeletal muscle fibers are elongated and are organized into progressively larger bundles that ulti- mately transition to tendons that join muscle to bone, allowing purposeful movement. Cardiac cells are short and linked mechanically and electrically to neighboring cells that surround the cardiac chambers. Contraction compresses and squeezes the chambers, ultimately lead- ing to ejection of blood into the vasculature of the body or the lungs. Skeletal muscles are activated in sequence to create a series of movements, whereas the cells in the atria and the ventricles of the heart contract almost simultaneously for efficient ejection.

The strength of contraction of both types of striated muscle fibers is modulated by their length as the con- traction starts and by the load the muscle experiences as it contracts. The first property, the length–tension relationship, is demonstrated when a muscle is length- ened prior to contraction. After this initial stretching, the muscle generates greater force when it contracts. Picture a baseball batter or a golfer, stretching his or her arm flexors with a backswing prior to the forward movement of the bat or golf club. A hypothesis for this mechanism is that prior lengthening brings more sar- comeres to an optimal length to conduct crossbridge cycling. The second property, the force–velocity rela- tionship, is demonstrated when the muscle is exposed to a load against which it must work as it is contracting. The greater that load, the slower will be the speed of

ADP Pi

Actin

(a)

Calcium Tropomyosin Troponin

Actin

Calcium binding Myosin head

(b)

(c)

(d)

Crossbridge

ADP

ATP

ADP

FIGURE 4.29 Myosin–actin crossbridge form ation. (a) Myosin is bound to ADP and inorganic phosphate (P

i ). A spike in cell

Ca2+ concentration facilitates calcium binding to troponin C, shifting the troponin complex and tropomyosin. This exposes actin binding sites, and the myosin head attaches to myosin. (b) The myosin head swivels, pulling myosin and actin in opposite directions in a move called the power stroke. ADP and P

i are released. (c) Binding of ATP breaks

the crossbridge between myosin and actin. The myosin head returns to its original angle, splits the ATP, and reattaches to an adjacent actin binding site, continuing to pull the actin strand inward toward the center of the sarcomere. (d). When calcium levels drop, tropomyosin shifts back to its original position, myosin–actin crossbridges are released, and the muscle relaxes. ADP, adenosine diphosphate; ATP, adenosine triphosphate.

Chapter 4 • Cell Physiology and Pathophysiology 99

H zone

H zoneZ zone Z zone

FIGURE 4.30 Sarcomere shortening due to myosin–actin crossbridge formation. The action of many strands of myosin and actin forming crossbridges adds up to the shortening of all sarcomeres in a muscle fi ber, producing contractile force and movement.

Sarcolemma Terminal cisternae

T-tubule

Triad

Sarcoplasmic reticulum

FIGURE 4.31 The triad of structures regulating muscle calcium release. Invaginations of the plasma membrane, T-tubules align next to terminal cisterns of sarcoplasmic reticulum. When the muscle action potential is propagated along the membrane and down the T-tubules, the signal is conveyed to the sarcoplasmic reticulum to release its calcium stores.

100 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

contraction. To imagine how this works, picture your- self trying to lift a box of tissues quickly and contrast that with how you would feel lifting a box of weights. These relationships are similar in the heart as well, and are discussed in that context under the topics of pre- load and afterload. Characteristics of these two muscle types are compared in Table 4.3.

Levels of muscle protein expression are dynamic, with turnover of actin, myosin, and other proteins replacing damaged myofibrils. Increasing the workload on a skeletal or cardiac muscle will increase protein synthesis relative to protein degradation, resulting in hypertrophy. This is a common consequence of strength training for skeletal muscle or hypertension for cardiac muscle. On the other hand, decrease in workload after an acute limb injury (fracture) can result in skeletal muscle atrophy (disuse atrophy), with protein degrada- tion at a greater rate than protein synthesis. A relative increase of muscle protein breakdown over synthesis is

common in aging, resulting in the sarcopenia that is a component of frailty in older adults.

MECHANISMS OF SMOOTH MUSCLE CONTRACTION Smooth muscle cells are found in the walls of hollow organs such as the gastrointestinal tract, bladder, and uterus. They also make up contractile layers of the blood vessels and airways. Contraction and relaxation of vascular and organ smooth muscle layers are primar- ily regulated by the autonomic nervous system and by local and circulating chemical mediators. Smooth mus- cles do not have striations or sarcomeres; rather, the actin and myosin filaments are more loosely arranged within the cytoplasm (Figure 4.33). Actin and myosin filaments are anchored to dense bodies in the cyto- plasm and to the cell membrane, so crossbridge for- mation and movement of the filaments by crossbridge

T-tubule

RyR

DHP SR cistern

Sarcoplasmic reticulum

Ca2

(a)

(b)

FIGURE 4.32 Close-up view of T-tubule–SR triad at rest and during activation. (a) DHP receptors of the T-tubule are adjacent to RyR of the SR cistern. (b) Membrane depolarization due to the muscle action potential causes the DHP to activate the RyR, and the SR releases its stored calcium. The wave of calcium quickly diffuses around the actin filaments, binding to troponin, shifting tropomyosin, and allowing myosin–actin crossbridges to form. DHP, dihydropyridine; RyR, ryanodine receptor; SR, sarcoplasmic reticulum.

Chapter 4 • Cell Physiology and Pathophysiology 101

cycling shortens the cell. In most cases, the force gen- erated by smooth muscle cells is weaker than force produced by striated muscles, and ATP requirements of smooth muscle cells are lower. Some smooth muscle cells are able to maintain a state of crossbridge bind- ing without cycling (called a latch-bridge mechanism), holding a degree of muscle tone that supports organ or blood vessel function with very low energy cost.

The role of calcium in smooth muscle contraction differs from that in striated muscle ( Figure 4.34 ). When intracellular calcium increases in smooth muscle cells, it binds to the protein calmodulin (step 1). This activates calmodulin, and the calcium/calmodulin com- plex can then bind to the enzyme myosin light chain kinase , activating the kinase function (step 2). Myosin light chain kinase then phosphorylates the regulatory light chain of myosin (step 3), causing the head to move closer to the nearby actin fi lament (step 4), resulting in crossbridge formation and cell contraction (step 5). There are two main mechanisms producing the increase in intracellular calcium in smooth muscle cells ( Figure 4.35 ). First, there are voltage-gated calcium

channels that can be opened by smooth muscle cell depolarization. Second, many smooth muscle cells have receptors linked to G

q and signaling via the phos-

pholipase C system. As previously noted, a prominent consequence of this pathway is generation of IP

3 ,

which causes the release of calcium from the sarco- plasmic reticulum. For example, in the circulatory sys- tem, vasoconstriction by norepinephrine and several other circulating substances (angiotensin II, vasopres- sin) is due to activation of G

q -linked receptors and IP

3 -

mediated calcium release. Relaxation of smooth muscle cells occurs when

receptor stimulation or depolarization stops, and the sarcoplasmic reticulum calcium pump reduces intra- cellular calcium, ultimately reducing myosin light chain kinase activity. Cyclic nucleotides cAMP and cGMP produced by adenylyl or guanylyl cyclase–linked receptors also mediate smooth muscle cell relaxation. An additional mechanism of smooth muscle relax- ation is through myosin light chain phosphatase—an enzyme that removes the phosphate from the myosin light chain, restoring myosin to its original position

TABLE 4.3 Comparison of Skeletal Muscle Fibers and Cardiac Muscle Cells

Characteristic Skeletal Muscle Fibers Cardiac Muscle Cells

Cell morphology Long fi bers formed by fusion of precursor cells Contain many nuclei Connect to tendons to generate force across joints

Short cells linked to neighboring cells both mechanically (desmosomes) and electrically (gap junctions)

Contain one nucleus

Action potential generation

Acetylcholine released from motor neuron terminals at the neuromuscular junction generates action potentials that propagate along the membrane

Pacemaker cells in the sinoatrial node generate action potentials that propagate along cardiac conduction pathways

Action potential type Brief twitch in response to one or a few action potentials, or

Fused contractions (tetany) in response to prolonged fi ring of motor neurons can sustain muscle contraction for long periods of time, if necessary

Action potentials are discrete; shaped by specialized sodium, calcium, and potassium channels; always ending with repolarization and relaxation

Cardiac muscle cannot have tetanic contractions

Mechanism of calcium release from the SR

Depolarization of T-tubules is relayed via the dihydropyridine receptor, activating calcium release via the ryanodine receptor

Calcium entry through the T-tubule dihydropyridine receptor initiates SR calcium release via ryanodine receptor

Metabolism Anaerobic and aerobic Primarily aerobic, contain greater numbers of mitochondria

Length–tension relationship

The longer the muscle fi ber at the start of contraction, the greater will be the tension it can develop

The greater the fi lling of the cardiac chamber at the start of contraction, the greater will be the tension it can develop

SR, sarcoplasmic reticulum.

102 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

(see Figure 4.34 , step 6). Certain vasodilating medi- ators work by increasing myosin light chain phospha- tase activity in vascular smooth muscle.

SUMMARY OF MUSCLE CELL FUNCTION Muscle cells are contractile cells that shorten upon stimulation, providing force for body movements, heart pumping, and contractions of the walls of blood ves- sels, airways, and internal organs. Actin–myosin cross- linking and crossbridge cycling are responsible for muscle contractions, with crossbridge formation and cycling also requiring calcium ions and ATP. Smooth muscle is regulated differently in the various organs and blood vessels, dependent on the receptors present

on the cell membranes. Throughout this book, specifi c examples are provided in their organ- or system-specifi c context.

Thought Questions

7. How do the mechanisms of muscle contraction diff er between striated and smooth muscle cells?

8. What are examples of each of these cell types and their function?

Actin

Actin-myosin filaments

Actin filament

Myosin filament Nucleus

Tropomyosin

Dense body

Intermediate filament (desmin, vimentin)

-Actin—containing cytoplasmic densities (dense bodies)

-Actin

(a) (b)

FIGURE 4.33 Contraction of smooth muscle. (a) Relaxed. (b) Contracted. In smooth muscle cells, actin–myosin strands form a loose meshwork crossing the cell. The strands are anchored by attachments to the plasma membrane and to dense bodies within the cytoplasm. When calcium increases, the cell shortens as shown.

Chapter 4 • Cell Physiology and Pathophysiology 103

MLCP

ATP

Active calcium–CaM

MLCK

Inactive MLCK

Active Ca2+– CaM

Inactive CaM

Ca2+

ADP

Relaxation

PO4

Contraction

FIGURE 4.34 Activation of smooth muscle contraction by calcium (Ca 2+ ). (See text for description of the numbered steps.) ADP, adenosine diphosphate; ATP, adenosine triphosphate; CaM, calmodulin; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; PO

4 , phosphate.

Membrane depolarization

Vasoconstrictor agonist

Phospholipase C Sarcolemma

Contraction

DAGIP3 PIP2

Ca 2+ Sarcoplasmic

reticulum

R G G

Ca2+

FIGURE 4.35 General mechanisms for activation of the vascular smooth muscle. Electromechanical coupling is shown on the left; a voltage-operated calcium (Ca 2+ ) channel allows calcium entry upon cell depolarization. Pharmacomechanical coupling is shown on the right; a receptor-operated Ca 2+

channel allows calcium entry upon activation of certain receptors. Other receptors increase [Ca2+] i

through the phospholipase C system and IP 3 .

DAG, diacylglycerol; G, GTP-binding protein; IP 3 , inositol trisphosphate; PIP

2 , phospha tidylinositol

bisphosphate; R, agonist-specifi c receptor; [Ca2+] i , intracellular calcium concentration.

104 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

CELL RENEWAL, STRESS, AND CELL DEATH

OVERVIEW OF CELL RENEWAL, MAINTENANCE, AND ADAPTATION Many cells have finite and short or intermediate life spans, including white and red blood cells, skin cells, and the cells lining the gastrointestinal tract. As these cells die, they must be replaced by stem cells that are self-renewing and give rise to daughter cells that dif- ferentiate into the appropriate cell type. In addition to whole cells, individual cells undergo renewal by removal of damaged or malfunctioning proteins and new protein synthesis. Cells that have sustained dam- age due to injury, infection, or malnutrition can attempt repair pathways, and if not successful they can undergo various patterns of cell death.

Cells exposed to physiological or pathophysiologi- cal challenges can also adapt (Figure 4.36). Common adaptations include cell enlargement (hypertrophy) by addition of new cell components. As previously described, this is commonly seen in the cells of muscles after strength training. Muscle fibers are nonrenew- ing cells, so their mode of adaptation is to synthesize additional myosin and actin molecules, increasing fiber

diameter and overall muscle size. For organs contain- ing self-renewing cells, excessive hormone stimula- tion or workload increases organ mass and volume by hyperplasia—production of greater numbers of cells. This is seen in the uterus during pregnancy or in the thyroid gland during Graves disease, in which there is excessive stimulation of the thyroid-stimulating hor- mone receptor.

Denervation of muscles, disuse, or withdrawal of hormone stimulation results in cell atrophy—loss of cell volume or number. These losses are observable on visual inspection of muscle or imaging of organs as an overall decrease in size. Mild organ atrophy is common with aging, for reasons described in the section on Gerontological Considerations. Chronic mechanical or chemical stressors can cause cells to change their tissue-specific characteristics and develop metaplasia or an epithelial–mesenchymal transition—unstable cell forms with abnormal func- tion and the potential to be transformed to cancer. Dysplasia is another form of cell transition in which proper quality control measures of cell division are lost, producing cells that are abnormal in appearance and are also precancerous.

Dysplasia

Metaplasia Hypertrophy and hyperplasia

Atrophy

Normal

Hypertrophy Hyperplasia

FIGURE 4.36 Cell adaptations to stressors. Adaptations to increased workload can produce cell hypertrophy, hyperplasia, or both. A variety of pathophysiological processes (nutrient deficits, decreased perfusion, neuromuscular disease, growth factor/trophic factor deprivation) can cause cells to atrophy. Chronic injury and inflammation can cause metaplasia and dysplasia, and can proceed to cancerous transformation.

Chapter 4 • Cell Physiology and Pathophysiology 105

OVERVIEW OF CELL INJURY AND DEATH The study of pathophysiology and loss of cell func- tion due to disease inevitably connects with the topic of cell injury and death. Cells, tissues, and organs can sustain injury from thromboembolic ischemia, trauma, burns, infections, exaggerated infl ammation, chemi- cals and toxins, neoplastic processes, and radiation. Tissues that lack signifi cant stem cell populations and self- renewing properties, such as brain and heart, may suffer irreversible loss of structure and function. Other tissues that have greater regenerative capacity, such as the liver and the kidney, may rebound from injury and recover close to normal function, depending on the magnitude of the insult. There is ever- increasing knowl- edge of cell-damaging mediators, cell death pathways, and the overlap between some of these systems with normal healthy aging processes. Specifi c examples of three cell death pathways are described here.

Tissue Responses to Acute, Severe Ischemia— Necrotic Cell Death Some of the most devastating health events are those resulting from a blood clot occluding a blood vessel, either in the heart (myocardial infarction) or in the brain (stroke). Although angioplasty to reopen vessels and thrombectomy procedures to remove cerebral clots have successfully limited damage and salvaged function, these events still cause significant organ damage, morbidity, and mortality. In this scenario, many cells will die by the pathway of necrosis , creating inflammation and ultimately a scar containing noncontractile tissue (after a myocardial infarction), or a region of reactive glio- sis and neuronal loss and atrophy (after a stroke). In stroke, for example, imaging studies aim to iden- tify the region of the core (the center of the area of ischemia that has little or no collateral blood flow) and the region of the penumbra (the outer zone that may be somewhat protected by collateral blood flow).

Cells in the stroke core have the worst hypoxic/ ischemic insult, with the following characteristics that lead to cell death by necrosis:

• Rapid loss of ATP production, resulting in dimin- ished membrane potential

• Depressed Na + /K + pump activity • Intracellular calcium overload • Cell swelling • Membrane rupture • Loss of antioxidant function • Accumulation of oxygen-derived free radicals (also

known as reactive oxygen species [ROS]) • Acidosis • Activation of degradative enzymes that contribute

to membrane rupture and spread of the damage to adjacent cells

Paradoxically, restoring blood flow (reperfusion) may actually worsen cell death by providing oxygen to fuel production of ROS and by recruiting white blood cells that perpetuate the infl ammatory response ( Figure 4.37 ). Cells in the penumbra vary in their out- comes, with some of the cells proceeding to necrotic cell death, others to apoptotic cell death (a slower pathway with less infl ammation), and yet others grad- ually recovering normal function. Membrane rupture and leakage of cell contents during necrotic cell death can be detected by laboratory evaluation. For example, death of cardiomyocytes as a myocardial infarction evolves is associated with release of the enzyme cre- atine phosphokinase (MB type) and the cardiac-specifi c troponin T and I proteins, among others. Acute liver injury resulting from toxic doses of the drug acetamin- ophen causes hepatocyte death and elevates blood lev- els of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

Apoptotic Cell Death Apoptosis is a very common mechanism of cell death that contributes to normal cell turnover, and there are many examples of the utility of apoptotic cell death. During early-life brain development, many more neurons are born than are ultimately retained in the mature brain. The remaining cells die by apoptosis and are removed by macrophages without any pathological remnants. Similarly, devel- opment of T lymphocytes in the thymus involves local scrutiny for cells with the potential to become autore- active and create autoimmune disease. These cells are removed by apoptosis. Cytotoxic T lymphocytes can recognize self -cells that are infected with viruses and bacteria, initiating cell death pathways, including death by apoptosis that helps to limit the infection. Cytotoxic T lymphocytes also protect against cancer by recogniz- ing transformed cells (those conducting abnormal cell division and expressing abnormal proteins), and killing them by pathways that include apoptosis. In the con- text of cell damage due to these various agents, apop- tosis is another pathway of cell death, particularly for damage that is limited or lasts a relatively short period of time.

Apoptosis is also known as programmed cell death because the steps follow an orderly sequence (a death program ) and tissue disruption, organ dysfunction, and inflammation are minimized. Apoptosis can be initiated by extracellular signals (extrinsic pathway) that bind to cell membrane tumor necrosis factor (TNF) receptors, causing assembly of intracellular proteins that can activate the apoptotic enzymes. Apoptosis can also be ini- tiated by intracellular signals (intrinsic pathway) of abnormal cell function that results in release of the enzyme cytochrome C from mitochondria— cytochrome C then triggers a cascade of increases

106 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

in pro-apoptotic proteins that activate the apop- totic enzymes. The apoptotic enzymes are caspases, and once activated, they begin to degrade cell components. The steps of apoptosis are outlined in Box 4.3, and characteristics of necrosis and apopto- sis are compared in Table 4.4.

Autophagy Cell adaptation to a nutrient lack, as in malnutrition, starvation, or a local region of poor circulation, can initiate the protective mechanism of autophagy. In this pathway, a vacuole forms within the cell and begins to take up some of the cell components—proteins and organelles. That vacuole then fuses with a cell lyso- some, and the contents are digested back to small

building blocks—amino acids, sugars, lipids—that can be used to nourish the remaining cells. If deprivation is severe enough, some of the affected cells will also die by autophagy, so this response is on a continuum from mild to severe, with severe cases leading to cell death.

Thought Questions

9. What are the hallmarks of necrotic cell death?

10. Why is it important for cell death programs to exist? How do they contribute to normal physiology and prevention of disease?

Obstruction or cessation of blood flow

Damage to electron transport chain

Ischemia

Detachment of ribosomes

↓ Protein synthesis

↓ Na pump

↓ ATP

↑ Intracellular Na ↑ Extracellular K ↑ Intracellular Ca2

↑ Glycolysis

↓ Glycogen

↓ pH

↑ Lactate

↓ Ca2 pump

↑ Intracellular

Loss of phospholipids

Damage to cell and organelle membranes

Damage to mitochondria Mitochondrial pore transition

Calcium phosphate deposition

Possible reperfusion injury Production of reactive

oxygen species by damaged mitochondria

Reperfusion

Calpain activation Phospholipase A2 activation↑ H2O

↑ Acute cellular swelling ↑ Release of

lysosomal enzymes

(hydrolases)

Cellular digestion (autodigestion)

Dilation of endoplasmic

reticulum

Ca2

FIGURE 4.37 Cascade of events in ischemic cell injury and death. Source: From Reisner HM. Pathology: A Modern Case Study. McGraw-Hill Education; 2015.

Chapter 4 • Cell Physiology and Pathophysiology 107

BOX 4.3 Steps in Apoptosis

1. Nuclear condensation and clumping of DNA

2. Enzymatic digestion of DNA into segments of uniform length

3. Nuclear membrane fragmentation

4. Cell membrane shrinkage—removal of small pieces of membrane and cytoplasm by blebbing

5. Blebbing—proceeds until the entire cell has been replaced by small fragments termed apoptotic bodies

6. Cellular debris and apoptotic bodies are phagocytosed by tissue macrophages

TABLE 4.4 Characteristics of Necrotic and Apoptotic Cell Death Pathways

Characteristic Necrosis Apoptosis

Cell size Cells swell Cells shrink

Fate of nucleus Ruptures Small packages containing fragmented DNA

DNA Fragment size varies Fragment size uniform

Cell membrane Ruptures Removed in small fragments by blebbing

Cellular contents Enzymatic digestion and leakage into surrounding tissue

Enzymatic digestion, then packaged into apoptotic bodies and released

Adjacent inflammation Frequent No

Physiological or pathological role

Invariably pathological, resulting from severe tissue injury

Often physiological as a means of eliminating unneeded or diseased cells

108

GERONTOLOGICAL CONSIDERATIONS Lori St. John

CELLULAR THEORIES OF AGING

Normal healthy aging is associated with decreased numbers and function of cells, in some organ systems more than others. Why do we age? Humankind has tried to answer this question for centuries, and yet the answer remains elusive. Theologists, philosophers, and scientists alike have sought to figure out why we age, how we age, and whether anything can prevent the consequences of aging. Over the years, scientists have put forth theories of what drives aging that range from an evolutionary to a molecular-level focus, although no one theory is sufficient to encompass all aspects of this process.1 Several of these theories are summarized here.

GLOBAL THEORIES OF AGING The programmed theory states that aging is a genetic program that has developed to direct senescence and death in order to benefit future generations of a species. A variety of genes regulate the aging process, suggest- ing that aging is innate to our genetic program. Current research, however, has failed to find evidence of any specific gene that has evolved for the sole purpose of aging. Similarly, the evolutionary theory argues that the forces of natural selection decline with aging due to accumulation of mutations that are not harmful to fitness early in life and during reproductive years, but are harmful later in life when selection is inefficient to remove them. The disposable soma theory postulates that organisms have limited resources that have to be divided for maintenance and reproduction. Because cells cannot use all of their resources for maintenance, protection against insults is not as efficient as it could be, leaving cells vulnerable to accumulating damage and accelerating aging. The hyperfunction theory sug- gests that pathological processes that lead to senes- cence are a result of gene overactivity rather than damage, breakdown, or failure. Aging can be seen as an increasing mass of pathological events with differ- ent causes.1

FREE RADICAL THEORY OF AGING The free radical theory of aging was one of the first to focus on the accumulated biochemical effects of constant exposure to these chemically reactive mole- cules. Free radicals, and more specifically ROS, have been implicated in numerous pathological pathways, so deeper understanding of the pathological nature of

these molecules is important to understand this theory and other pathophysiological processes. A free radical is a molecule that has an unpaired electron in its outer orbit that makes it highly chemically reactive. ROS are free radicals that are commonly produced from oxy- gen as a byproduct of mitochondrial respiration and energy generation. Cells have antioxidant molecules and enzymes that neutralize ROS before they cause molecular damage to membranes, proteins, and DNA. However, if there is a disruption in the removal process or there is an increase in ROS production, cellular dam- age occurs.

There are three principal cellular targets of ROS- mediated damage. The first target is cell membranes that undergo lipid peroxidation. This process occurs when double bonds in unsaturated fatty acids of the cell membrane lipids are attacked by ROS, leading to the formation of unstable peroxide molecules, caus- ing an autocatalytic chain reaction that can result in extensive cell membrane damage. The next target is proteins that undergo oxidative modification. ROS promote oxidation of amino acid side chains, accel- erating protein cross-linking and oxidation of the pro- tein backbone. These molecular changes alter enzyme active sites, disrupt the conformation of structural proteins, and increase degradation of unfolded or mis- folded proteins. The third target is DNA. ROS cause single- and double-strand breaks in DNA, DNA strand cross-linking, and adduct formation.2 This mechanism has been implicated in cell aging and malignant trans- formation. Whether or not the damage leads to cellular aging or cancer formation is dependent on the amount and location of damage, specific repair, checkpoint, and effector systems involved.3

NINE HALLMARKS OF AGING

Aging itself is an abstract concept that is hard to quan- tify. When does aging start? And how can we tell that something is aging? It is important to identify these concepts as they can guide research to better under- stand how we age and understand the mechanisms behind age-related pathology. Researchers have pos- ited nine hallmarks that are considered to lead to the aging process and determine the aging phenotype. These processes were chosen because they are all found in normal aging. Also, under experimental condi- tions, exacerbation or mitigation of these factors could hasten death or increase life span, respectively.4

Chapter 4 • Cell Physiology And Pathophysiology 109

CELLULAR SENESCENCE Senescence is a phenomenon that only occurs in cells that have the ability to replicate. 5 As cells sustain dam- age, senescence prevents further propagation of dam- aged cells and alerts the immune system to degrade and clear them. Senescence is driven by two mecha- nisms: telomere attrition (discussed later) and activa- tion of tumor suppressor genes. Activation of specifi c tumor suppressor genes also contributes to replicative senescence. Expression of tumor suppressor protein p16 is most commonly correlated with aging. 4 p16 pro- tects the cells from uncontrolled proliferative signals and pushes cells along the senescence pathway. 2

Accumulation of senescent cells with aging is poten- tially a refl ection of an increase in the conversion of cells to a senescent state or a decrease in the rate of the death and clearance of these cells. Although pre- venting the replication of potentially harmful cells is a protection against diseases such as cancer, this mech- anism relies on an effi cient cell replacement system to clear the senescent cells and signal progenitors to replace the old cells. In older organisms, this system may become ineffi cient or may exhaust the regenera- tive capacity of progenitor cells, eventually resulting in the accumulation of senescent cells that may aggravate the damage and contribute to aging. 4

TELOMERE ATTRITION Telomere attrition is the mechanism of replicative senescence involving progressive shortening of telo- meres, thus resulting in cell cycle arrest. Telomeres are DNA sequences found at the ends of chromosomes and are required for complete replication during the DNA synthesis phase of the cell cycle. With each round of replication, the telomeres grow shorter. As telomeres become shorter, the chromosome ends are less well protected; eventually they are fl agged as damaged DNA, signaling cell cycle arrest. Telomere length is extended by telomerase, which is not found in most somatic tissues. Consequently, as somatic cells age, their telo- meres become shorter and they eventually cannot pro- duce new cells to replace damaged cells. 2 Since cells are unable to restore telomere length, DNA damage at telomeres is notably persistent and highly effi cient in inducing senescence, apoptosis, or both. 4

GENOMIC INSTABILITY DNA integrity is threatened by a variety of exogenous challenges (e.g., chemicals or infectious agents), as well as by internal factors such as replication errors and free radicals. DNA alterations can potentially affect important genes and transcriptional pathways, result- ing in dysfunctional cells that, if not eliminated, may prevent an organism from carrying out necessary func- tions of survival. In aging organisms, this is particularly important in stem cells, because damaged progenitor

cells can no longer participate in tissue renewal, thus contributing to the overall aging phenotype.

LOSS OF PROTEOSTASIS Aging is linked to impaired protein homeostasis, or proteostasis . Proteostasis has two mechanisms: main- taining proteins in correctly folded configurations (mediated by chaperones) and degrading proteins that have been misfolded. 2 As cells age, the formation of cytosolic and organelle-specifi c chaperones is signifi - cantly impaired, resulting in an increased number of misfolded proteins. Also, the activities of the two main protein degradation systems that assist with protein quality control (the ubiquitin–proteasome system and the autophagy–lysosomal system) decline with aging. This can lead to an accumulation of misfolded proteins, triggering apoptosis and age-associated diseases such as Alzheimer disease. 4

DEREGULATED NUTRIENT SENSING Two major pathways that regulate metabolism are the insulin and insulin-like growth factor 1 (IGF-1) signaling pathway and sirtuins. Insulin is secreted from the pan- creas, and IGF-1 is produced by the liver in response to growth hormone secreted by the pituitary gland. IGF-1 receptor activation has similar downstream effects as intracellular signaling by insulin, promoting an ana- bolic state as well as cell growth and replication.

Sirtuins are part of a group of nicotinamide adenine dinucleotide (NAD)–dependent protein deacetylases (enzymes that remove acetyl groups from proteins, including histone proteins that package DNA). They are designed to help the body adapt to environmental stressors, such as nutrient deprivation and DNA dam- age, and may also promote the expression of genes that increase longevity. These genes have roles in decreas- ing metabolic activity, reducing apoptosis, stimulating protein folding, and reducing the harmful effects of ROS. They also increase insulin sensitivity and glucose metabolism. 2 Caloric restriction is the only intervention that has been shown to extend a healthy life span in animal models, and it is hypothesized that increased sirtuin levels and reduced insulin signaling are at least somewhat responsible for this extension.

MITOCHONDRIAL DYSFUNCTION Mitochondrial dysfunction has a major impact on the aging process. As cells age, the effi cacy of the respi- ratory chain tends to decrease, which increases elec- tron leakage and reduces the production of ATP. Mitochondrial defi ciencies may affect apoptotic sig- naling by increasing the mitochondrial permeability changes that precipitate apoptosis. The combination of increased damage and reduced turnover in mitochon- dria, due to lower biogenesis and reduced clearance, may contribute to the aging process. 4

110 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

STEM CELL EXHAUSTION As tissues age, their ability to regenerate is signifi cantly reduced due to the inability of stem cells to reproduce. While a decrease in stem cell proliferation clearly impairs the functionality of tissues, excessive prolifera- tion of stem cells can also be dangerous by accelerating the exhaustion of stem cell reserves and leading to pre- mature aging. 4

EPIGENETIC ALTERATIONS A variety of epigenetic factors affect cells throughout the life span, including DNA methylation patterns, post-translational modifi cation of histones, and chro- matin remodeling. Epigenetic alterations can directly affect regulation of telomere length, one of the other hallmarks of aging discussed earlier. There is also evi- dence of age-related transcriptional changes in genes that encode key components of infl ammatory, mito- chondrial, and lysosomal degradation pathways that can contribute to the aging phenotype. 4

ALTERED CELLULAR COMMUNICATION Aging does not happen in a vacuum, and there is compel- ling evidence to suggest that some age-related changes occur with intercellular signaling. Neurohormonal sig- naling becomes deregulated in the setting of increased inflammatory reactions, decreased immunosurveil- lance against infectious agents and premalignant cells, and peri- and extracellular environment changes. Evidence also suggests that aging can have a domino effect on other tissues, causing an interorgan aging phenotype. So-called contagious aging effects include senescent cells that cause senescence in neighboring cells through gap junction contacts and ROS-mediated processes. 4

FRAILTY

This book covers the pathological processes of various disease states, but is there a state of disease associated with old age itself? Age is a risk factor in a variety of diseases, such as cancer, cardiovascular disease, and dementia; however, aging can also be pathological in and of itself under the right conditions.

Frailty is a multisystem syndrome that marks a loss of reserve that leaves an individual at an increased risk of death, loss of independence, and decreased mobility. 6

It is a unique state that indicates multisystem physiologi- cal changes. 7 Although many older individuals who meet criteria for frailty typically have multiple comorbidities, people can still be classifi ed as frail without having a life-threatening disease process. Frailty is associated with reduced functional reserves and decreased adapt- ability to both extrinsic and intrinsic stressors.

Clinically, frailty in older adults is often measured by the Fried Phenotype Frailty Index. The fi ve criteria of this index are self-reported exhaustion, reduced physi- cal activity, slow walking speed, reduced grip strength, and unintentional weight loss. A person must meet three or more of the fi ve criteria in order to be classi- fi ed as frail. 6 , 7

INFLAMMATION An important marker of frailty is infl ammation. Chronic levels of infl ammation can be a consequence of a vari- ety of different mechanisms. Aging leads to decreased levels of sex steroids, growth hormones, and vitamin D levels, and these declines, in turn, are associated with higher levels of infl ammation. Older adults also typically have other comorbid conditions that promote baseline proinfl ammatory states, such as atheroscle- rosis or subacute infections. Interleukin 6 (IL-6) is a cytokine that is produced by leukocytes in response to noxious stimuli. High levels of IL-6 are often associated with poor functional status and can predict onset of disability in older adults. Tumor necrosis factor alpha (TNF-α ) is a cytokine associated with acute infl am- mation. TNF-α is strongly associated with death in community-dwelling older adults. 6

Circulating infl ammatory signals such as TNF-α stimulate production of IL-6 and C-reactive protein, which in turn have a catabolic effect on muscles. Muscles store energy in the form of glycogen and pro- teins that can be used for energy in times of extreme stress or malnutrition. Hormones are produced and catabolized in muscle tissue. Stored amino acids can be used in acute infections to produce antibodies. Loss of muscle can contribute to a reduced response to immunological insults and lower metabolic adaptation. 8

Despite the many hypotheses of cellular aging, there is much that has not been confi rmed by preclinical or clinical research. Active research continues in this fi eld, particularly in individuals and groups who have extraordinary longevity. The intersection of genetic infl uences with environmental infl uences on life span is beginning to give clues as to mechanisms of human aging.

Thought Questions

11. What are the top three classes of cellular biomolecules that are vulnerable to ROS- mediated damage?

12. Explain the role of telomeres in maintaining chromosomal integrity over repeated cycles of mitosis.

Chapter 4 • Cell Physiology and Pathophysiology 111

KEY POINTS

• Human cells are bounded by a cell membrane consisting of a phospholipid bilayer with polar components facing the outside, and a lipid core made up of fatty acids and cholesterol. The membrane is a hydrophobic barrier between intracellular fl uid and extracellular fl uid.

• The organelles—nucleus, endoplasmic retic- ulum, Golgi apparatus, ribosomes, mitochon- dria, lysosomes, and peroxisomes—carry out the work of the cell, with many reactions being compartmentalized within one or more compartments.

• The membrane’s lipid barrier allows movement of small nonpolar substances such as oxygen and carbon dioxide between extracellular and intracellular fl uid, but most other molecules require transporters.

• The main factors involved in predicting the mechanism by which a solute will cross the membrane include the degree to which it is polar and hydrophilic (as opposed to nonpo- lar and hydrophobic); the concentration gra- dient for the solute across the cell membrane; and the direction of movement relative to that gradient—either downhill (moving from a region of high concentration to a region of low concentration) or uphill (moving from low to high concentration).

• Modes of transport across the cell membrane are diffusion, exocytosis and endocytosis, facilitated diffusion, active transport, sec- ondary active transport, aquaporins, and ion channels.

• Active transport by the sodium–potassium (Na + / K + ) pump generates and maintains the charac- teristic ionic composition of extracellular fl uid (low potassium, high sodium) and intracellular fl uid (high potassium, low sodium).

• In secondary active transport, the movement of one solute down its concentration gradient (often sodium) is coupled to the movement of another solute against its concentration gradi- ent. This mechanism requires ongoing activity of the Na + /K + pump to generate the sodium concentration gradient.

• Ion channels allow ions to move down their concentration gradients. Potassium leak chan- nels are common components of cell mem- branes and set up a resting membrane potential that is particularly signifi cant in electrically excitable cells such as muscle and nerve cells.

• Cells respond to neurotransmitters and hor- mones by receptors found in the cell mem- brane or intracellularly.

• The sympathetic and parasympathetic branches of the autonomic nervous system are the major rapidly acting regulators of organ system adaptations to environmental and inter- nal challenges to homeostasis.

• Receptors for the autonomic neurotransmit- ters norepinephrine and acetylcholine are examples of GPCRs. Many hormones and other neurotransmitters work by way of GPCRs.

• Cell signaling by GPCRs initiates an amplifying cascade of activated proteins. Following ligand binding to its receptor, a three-part G protein is activated and splits into α and βγ subunits. The α subunit activates an enzyme within the membrane, and the βγ subunit may have addi- tional effects. The activated enzyme produces second messengers that diffuse throughout the cytoplasm to initiate changes in intracellular activity.

• Adenylyl cyclase is stimulated by receptors linking to G

s proteins; their activation increases

intracellular concentrations of cAMP. The result of increasing cAMP is the activation of PKA, an enzyme that phosphorylates intra- cellular target proteins to alter their activity, either upregulating or downregulating activity to adapt to homeostatic challenges. PKA is a serine/threonine kinase, adding phosphate groups to proteins specifi cally at sites of the amino acids serine or threonine.

• Adenylyl cyclase is inhibited by receptors link- ing to G

i proteins; this inhibition reverses the

actions of stimulatory receptors and reduces cAMP levels and PKA activity.

• Receptors linked to G q activate phospholi-

pase C. This enzyme splits a membrane lipid, PIP

2 , producing two second messengers—

membrane-bound DAG and intracellular IP 3 .

IP 3 diffuses to the endoplasmic reticulum and

releases calcium from its storage, producing a brief increase of intracellular calcium with a variety of downstream consequences for cell function.

• Several mechanisms terminate the actions of GPCRs: diffusion of neurotransmitter or hor- mone away from the receptor ends G protein stimulation; the intrinsic GTPase activity of G proteins leads to their inactivation; and intra- cellular phosphatase enzymes can reverse the actions of protein kinases.

112 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

• Certain receptors have an extracellular ligand-binding site and an intracellular enzyme site or binding sites for enzymes. Many of these types of receptors are tyrosine kinases, phos- phorylating target proteins on tyrosine sites.

• Tyrosine kinase downstream activities often include activation of cell prolifera- tion, and ligands for tyrosine kinase–linked receptors promote body growth and devel- opment and proliferation of immune cells. When dysregulated, many of the receptors and signaling cascade proteins of this class are also oncoproteins—having cancer- promoting activity.

• Steroid and thyroid hormones cross cell mem- branes and ultimately bind to receptors on DNA, altering transcription and translation for their biological effects.

• Muscle cells produce skeletal movement or organ contractions by interactions between the contractile proteins actin and myosin. Skeletal and cardiac muscle cells are striated muscle, having highly organized bundles of actin and myosin within the functional unit of the sar- comere. Smooth muscle cells found in blood vessels and organs have more loosely arranged bundles of actin and myosin.

• Regulation of striated muscle contraction by calcium depends on troponins and tropomyo- sin. Regulation of smooth muscle contraction depends on calmodulin and myosin light chain kinase.

• Cells can adapt to a variety of homeostatic challenges by hypertrophy, atrophy, prolifera- tion, dysplasia, or death.

• Necrotic cell death is caused by severe traumatic or ischemic injury, whereas apoptotic cell death is sometimes physiological rather than patholog- ical. Autophagic cell death is slower, and auto- phagy itself may be a protective mechanism that maintains the cell and prevents cell death.

• The biological cause of aging has not been defin- itively determined, but humans and other organ- isms experience gradual losses of cell and organ function that end in death, even in the absence of a specific disease process. Different path- ways can contribute to aging, including lifelong exposure to injurious chemicals such as ROS and inflammatory cytokines, and gradual dimin- ishment of cellular quality control systems for integrity of DNA, proteins, and mitochondria.

• Nutrient signaling cascades appear to play a role in aging, and caloric restriction extends life in many animal models.

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