Why is it important for cell death programs to exist? How do they contribute to normal physiology and the prevention of disease? What are the characteristics of Necr

 73

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

4

• 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

Copyright Springer Publishing Company. All Rights Reserved. From: Advanced Physiology and Pathophysiology: Essentials for Clinical Practice DOI: 10.1891/9780826177087.0004

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

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

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

x

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

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