Physiology (Linda) chapter 1.docx

VOLUME AND COMPOSITION OF BODY FLUIDS Distribution of Water in the Body Fluid Compartments In the human body, water constitutes a high proportion of body weight. The total amount of fluid or water is called total body water, which accounts for 50% to 70% of body weight. For example, a 70-kilogram (k...

VOLUME AND COMPOSITION OF BODY FLUIDS Distribution of Water in the Body Fluid Compartments In the human body, water constitutes a high proportion of body weight. The total amount of fluid or water is called total body water, which accounts for 50% to 70% of body weight. For example, a 70-kilogram (kg) man whose total body water is 65% of his body weight has 45.5 kg or 45.5 liters (L) of water (1 kg water ≈ 1 L water). In general, total body water correlates inversely with body fat. Thus total body water is a higher percentage of body weight when body fat is low and a lower percentage when body fat is high. Because females have a higher percentage of adipose tissue than males, they tend to have less body water. The distribution of water among body fluid compart- ments is described briefly in this chapter and in greater detail in Chapter 6. Total body water is distributed between two major body fluid compartments: intracel- lular fluid (ICF) and extracellular fluid (ECF) (Fig. 1.1). The ICF is contained within the cells and is two-thirds of total body water; the ECF is outside the cells and is one-third of total body water. ICF and ECF are separated by the cell membranes. ECF is further divided into two compartments: plasma and interstitial fluid. Plasma is the fluid circulating in the blood vessels and is the smaller of the two ECF 1 2 Physiology TOTAL BODY WATER Intracellular fluid Extracellular fluid Cell membrane Capillary wall Fig. 1.1 Body fluid compartments. subcompartments. Interstitial fluid is the fluid that actually bathes the cells and is the larger of the two subcompartments. Plasma and interstitial fluid are separated by the capillary wall. Interstitial fluid is an ultrafiltrate of plasma, formed by filtration processes across the capillary wall. Because the capillary wall is virtually impermeable to large molecules such as plasma proteins, interstitial fluid contains little, if any, protein. The method for estimating the volume of the body fluid compartments is presented in Chapter 6. Composition of Body Fluid Compartments The composition of the body fluids is not uniform. ICF and ECF have vastly different concentrations of various solutes. There are also certain predictable differences in solute concentrations between plasma and interstitial fluid that occur as a result of the exclusion of protein from interstitial fluid. Units for Measuring Solute Concentrations Typically, amounts of solute are expressed in moles, equivalents, or osmoles. Likewise, concentrations of solutes are expressed in moles per liter (mol/L), equivalents per liter (Eq/L), or osmoles per liter (Osm/L). In biologic solutions, concentrations of solutes are usually quite low and are expressed in millimoles per liter (mmol/L), milliequivalents per liter (mEq/L), or milliosmoles per liter (mOsm/L). One mole is 6 × 1023 molecules of a substance. One millimole is 1/1000 or 10−3 moles. A glucose concentra- tion of 1 mmol/L has 1 × 10−3 moles of glucose in 1 L of solution. An equivalent is used to describe the amount of charged (ionized) solute and is the number of moles of the solute multiplied by its valence. For example, one mole of potassium chloride (KCl) in solution dis- sociates into one equivalent of potassium (K+) and one equivalent of chloride (Cl−). Likewise, one mole of calcium chloride (CaCl2) in solution dissociates into two equivalents of calcium (Ca2+) and two equivalents of chloride (Cl−); accordingly, a Ca2+ concentration of 1 mmol/L corresponds to 2 mEq/L. One osmole is the number of particles into which a solute dissociates in solution. Osmolarity is the con- centration of particles in solution expressed as osmoles per liter. If a solute does not dissociate in solution (e.g., glucose), then its osmolarity is equal to its molarity. If a solute dissociates into more than one particle in solution (e.g., NaCl), then its osmolarity equals the molarity multiplied by the number of particles in solu- tion. For example, a solution containing 1 mmol/L NaCl is 2 mOsm/L because NaCl dissociates into two particles. pH is a logarithmic term that is used to express hydrogen (H+) concentration. Because the H+ concen- tration of body fluids is very low (e.g., 40 × 10−9 Eq/L in arterial blood), it is more conveniently expressed as a logarithmic term, pH. The negative sign means that pH decreases as the concentration of H+ increases, and pH increases as the concentration of H+ decreases. Thus pH = − log10[H+ ] SAMPLE PROBLEM. Two men, Subject A and Subject B, have disorders that cause excessive acid production in the body. The laboratory reports the acidity of Subject A’s blood in terms of [H+] and the acidity of Subject B’s blood in terms of pH. Subject A has an arterial [H+] of 65 × 10−9 Eq/L, and Subject B has an arterial pH of 7.3. Which subject has the higher concentration of H+ in his blood? SOLUTION. To compare the acidity of the blood of each subject, convert the [H+] for Subject A to pH as follows: pH = − log10[H+ ] = − log10(65 × 10−9 Eq/L) = − log10(6.5 × 10−8 Eq/L) log100 6.5 = 0.81 log10 10−8 = −8.0 log10 6.5 × 10−8 = 0.81 + (−8.0) = −7.19 pH = −(−7.19) = 7.19 Thus Subject A has a blood pH of 7.19 computed from the [H+], and Subject B has a reported blood pH of 7.3. Subject A has a lower blood pH, reflecting a higher [H+] and a more acidic condition. Electroneutrality of Body Fluid Compartments Each body fluid compartment must obey the principle of macroscopic electroneutrality; that is, each 1—Cellular Physiology 3 compartment must have the same concentration, in mEq/L, of positive charges (cations) as of negative charges (anions). There can be no more cations than anions, or vice versa. Even when there is a potential difference across the cell membrane, charge balance still is maintained in the bulk (macroscopic) solutions. (Because potential differences are created by the sepa- ration of just a few charges adjacent to the membrane, this small separation of charges is not enough to measurably change bulk concentrations.) Composition of Intracellular Fluid and Extracellular Fluid The compositions of ICF and ECF are strikingly differ- ent, as shown in Table 1.1. The major cation in ECF is sodium (Na+), and the balancing anions are chloride (Cl−) and bicarbonate (HCO −). The major cations in ICF are potassium (K+) and magnesium (Mg2+), and the balancing anions are proteins and organic phosphates. Other notable differences in composition involve Ca2+ and pH. Typically, ICF has a very low concentration of ionized Ca2+ (≈10−7 mol/L), whereas the Ca2+ concentra- tion in ECF is higher by approximately four orders of magnitude. ICF is more acidic (has a lower pH) than ECF. Thus substances found in high concentration in ECF are found in low concentration in ICF, and vice versa. Remarkably, given all of the concentration differ- ences for individual solutes, the total solute concentra- tion (osmolarity) is the same in ICF and ECF. This equality is achieved because water flows freely across cell membranes. Any transient differences in osmolar- ity that occur between ICF and ECF are quickly dissi- pated by water movement into or out of cells to reestablish the equality. TABLE 1.1 Approximate Compositions of Extracellular and Intracellular Fluids Creation of Concentration Differences Across Cell Membranes The differences in solute concentration across cell membranes are created and maintained by energy- consuming transport mechanisms in the cell membranes. The best known of these transport mechanisms is the Na+-K+ ATPase (Na+-K+ pump), which transports Na+ from ICF to ECF and simultaneously transports K+ from ECF to ICF. Both Na+ and K+ are transported against their respective electrochemical gradients; therefore an energy source, adenosine triphosphate (ATP), is required. The Na+-K+ ATPase is responsible for creating the large concentration gradients for Na+ and K+ that exist across cell membranes (i.e., the low intracellular Na+ concentration and the high intracel- lular K+ concentration). Similarly, the intracellular Ca2+ concentration is maintained at a level much lower than the extracellular Ca2+ concentration. This concentration difference is established, in part, by a cell membrane Ca2+ ATPase that pumps Ca2+ against its electrochemical gradient. Like the Na+-K+ ATPase, the Ca2+ ATPase uses ATP as a direct energy source. In addition to the transporters that use ATP directly, other transporters establish concentration differences across the cell membrane by utilizing the transmem- brane Na+ concentration gradient (established by the Na+-K+ ATPase) as an energy source. These transporters create concentration gradients for glucose, amino acids, Ca2+, and H+ without the direct utilization of ATP. Clearly, cell membranes have the machinery to establish large concentration gradients. However, if cell membranes were freely permeable to all solutes, these gradients would quickly dissipate. Thus it is critically important that cell membranes are not freely permeable to all substances but, rather, have selec- tive permeabilities that maintain the concentration gradients established by energy-consuming transport processes. Directly or indirectly, the differences in composition between ICF and ECF underlie every important physi- ologic function, as the following examples illustrate: (1) The resting membrane potential of nerve and muscle critically depends on the difference in concentration of K+ across the cell membrane; (2) The upstroke of the action potential of these same excitable cells depends + on the differences in Na concentration across the cell aThe major anions of intracellular fluid are proteins and organic phosphates. bThe corresponding total [Ca2+] in extracellular fluid is 5 mEq/L or 10 mg/dL. cpH is −log10 of the [H+]; pH 7.4 corresponds to [H+] of 40 × 10−9 Eq/L. membrane; (3) Excitation-contraction coupling in muscle cells depends on the differences in Ca2+ concen- tration across the cell membrane and the membrane of the sarcoplasmic reticulum (SR); and (4) Absorption of essential nutrients depends on the transmembrane Na+ concentration gradient (e.g., glucose absorption in the small intestine or glucose reabsorption in the renal proximal tubule). Physiology Concentration Differences Between Plasma and Interstitial Fluids As previously discussed, ECF consists of two subcom- partments: interstitial fluid and plasma. The most sig- nificant difference in composition between these two compartments is the presence of proteins (e.g., albumin) in the plasma compartment. Plasma proteins do not readily cross capillary walls because of their large molecular size and therefore are excluded from inter- stitial fluid. The exclusion of proteins from interstitial fluid has secondary consequences. The plasma proteins are negatively charged, and this negative charge causes a redistribution of small, permeant cations and anions across the capillary wall, called a Gibbs-Donnan equil- ibrium. The redistribution can be explained as follows: The plasma compartment contains the impermeant, negatively charged proteins. Because of the requirement for electroneutrality, the plasma compartment must have a slightly lower concentration of small anions (e.g., Cl−) and a slightly higher concentration of small cations (e.g., Na+ and K+) than that of interstitial fluid. The small concentration difference for permeant ions is expressed in the Gibbs-Donnan ratio, which gives the plasma concentration relative to the interstitial fluid concentration for anions and interstitial fluid relative to plasma for cations. For example, the Cl− concentration in plasma is slightly less than the Cl− concentration in interstitial fluid (due to the effect of the impermeant plasma proteins); the Gibbs-Donnan ratio for Cl− is 0.95, meaning that [Cl−]plasma/[Cl−]interstitial fluid equals 0.95. For Na+, the Gibbs-Donnan ratio is also 0.95, but Na+, being positively charged, is oriented the opposite way, and [Na+]interstitial fluid/[Na+]plasma equals 0.95. Generally, these minor differences in concentration for small cations and anions between plasma and interstitial fluid are ignored. CHARACTERISTICS OF CELL MEMBRANES Cell membranes are composed primarily of lipids and proteins. The lipid component consists of phospholip- ids, cholesterol, and glycolipids and is responsible for the high permeability of cell membranes to lipid-soluble substances such as carbon dioxide, oxygen, fatty acids, and steroid hormones. The lipid component of cell membranes is also responsible for the low permeability of cell membranes to water-soluble substances such as ions, glucose, and amino acids. The protein component of the membrane consists of transporters, enzymes, hormone receptors, cell-surface antigens, and ion and water channels. Water Oil Water A Water B Fig. 1.2 Orientation of phospholipid molecules at oil and water interfaces. Depicted are the orientation of phospholipid at an oil-water interface (A) and the orientation of phospholipid in a bilayer, as occurs in the cell membrane (B). Phospholipid Component of Cell Membranes Phospholipids consist of a phosphorylated glycerol backbone (“head”) and two fatty acid “tails” (Fig. 1.2). The glycerol backbone is hydrophilic (water soluble), and the fatty acid tails are hydrophobic (water insolu- ble). Thus phospholipid molecules have both hydro- philic and hydrophobic properties and are called amphipathic. At an oil-water interface (see Fig. 1.2A), molecules of phospholipids form a monolayer and orient themselves so that the glycerol backbone dis- solves in the water phase and the fatty acid tails dis- solve in the oil phase. In cell membranes (see Fig. 1.2B), phospholipids orient so that the lipid-soluble fatty acid tails face each other and the water-soluble glycerol heads point away from each other, dissolving in the aqueous solutions of the ICF or ECF. This orienta- tion creates a lipid bilayer. Protein Component of Cell Membranes Proteins in cell membranes may be either integral or peripheral, depending on whether they span the mem- brane or whether they are present on only one side. The distribution of proteins in a phospholipid bilayer is illustrated in the fluid mosaic model, shown in Figure 1.3. Integral membrane proteins are embedded in, and anchored to, the cell membrane by hydrophobic interactions. To remove an integral protein from the cell membrane, its attachments to the lipid bilayer must be disrupted (e.g., by detergents). Some inte- gral proteins are transmembrane proteins, meaning they span the lipid bilayer one or more times; thus 1—Cellular Physiology 5 Intracellular fluid Lipid bilayer Peripheral Integral Gated ion protein protein channel Extracellular fluid Fig. 1.3 Fluid mosaic model for cell membranes. TABLE 1.2 Summary of Membrane Transport Type of Transport Active or Passive Carrier- Mediated Uses Metabolic Energy Dependent on Na+ Gradient Simple diffusion Passive; downhill No No No Facilitated diffusion Passive; downhill Yes No No Primary active transport Active; uphill Yes Yes; direct No Cotransport Secondary activea Yes Yes; indirect Yes (solutes move in same direction as Na+ across cell membrane) Countertransport Secondary activea Yes Yes; indirect Yes (solutes move in opposite direction as Na+ across cell membrane) aNa+ is transported downhill, and one or more solutes are transported uphill. transmembrane proteins are in contact with both ECF and ICF. Examples of transmembrane integral proteins are ligand-binding receptors (e.g., for hor- mones or neurotransmitters), transport proteins hydrogen bonds. One example of a peripheral mem- brane protein is ankyrin, which “anchors” the cytoskeleton of red blood cells to an integral mem- brane transport protein, the Cl−-HCO − exchanger (e.g., Na+-K+ ATPase), pores, ion channels, cell adhesion molecules, and GTP-binding proteins (G proteins). A second category of integral proteins is embedded in the lipid bilayer of the membrane but does not span it. A third category of integral proteins is associated with membrane proteins but is not embedded in the lipid bilayer. Peripheral membrane proteins are not embedded in the membrane and are not covalently bound to cell membrane components. They are loosely attached to either the intracellular or extracellular side of the cell membrane by electrostatic interac- tions (e.g., with integral proteins) and can be removed with mild treatments that disrupt ionic or (also called band 3 protein). TRANSPORT ACROSS CELL MEMBRANES Several types of mechanisms are responsible for trans- port of substances across cell membranes (Table 1.2). Substances may be transported down an electro- chemical gradient (downhill) or against an electro- chemical gradient (uphill). Downhill transport occurs by diffusion, either simple or facilitated, and requires no input of metabolic energy. Uphill transport occurs by active transport, which may be primary or second- ary. Primary and secondary active transport processes 6 Physiology Membrane A B Fig. 1.4 Kinetics of carrier-mediated transport. Tm, Trans- port maximum. are distinguished by their energy source. Primary active transport requires a direct input of metabolic energy; secondary active transport utilizes an indirect input of metabolic energy. Further distinctions among transport mechanisms are based on whether the process involves a protein carrier. Simple diffusion is the only form of transport that is not carrier mediated. Facilitated diffusion, primary active transport, and secondary active trans- port all involve integral membrane proteins and are called carrier-mediated transport. All forms of carrier- mediated transport share the following three features: saturation, stereospecificity, and competition. Saturation. Saturability is based on the concept that carrier proteins have a limited number of binding sites for the solute. Figure 1.4 shows the relationship between the rate of carrier-mediated transport and solute concentration. At low solute concentrations, Fig. 1.5 Simple diffusion. The two solutions, A and B, are separated by a membrane, which is permeable to the solute (circles). Solution A initially contains a higher concentration of the solute than does Solution B. Stereospecificity. The binding sites for solute on the transport proteins are stereospecific. For example, the transporter for glucose in the renal proximal tubule recognizes and transports the natural isomer D-glucose, but it does not recognize or transport the unnatural isomer L-glucose. In contrast, simple dif- fusion does not distinguish between the two glucose isomers because no protein carrier is involved. Competition. Although the binding sites for trans- ported solutes are quite specific, they may recognize, bind, and even transport chemically related solutes. For example, the transporter for glucose is specific for D-glucose, but it also recognizes and transports a closely related sugar, D-galactose. Therefore the presence of D-galactose inhibits the transport of D-glucose by occupying some of the binding sites and making them unavailable for glucose. Simple Diffusion Diffusion of Nonelectrolytes Simple diffusion occurs as a result of the random thermal motion of molecules, as shown in Figure 1.5. many binding sites are available and the rate of transport increases steeply as the concentration increases. However, at high solute concentrations, the available binding sites become scarce and the rate of transport levels off. Finally, when all of the binding sites are occupied, saturation is achieved at a point called the transport maximum, or Tm. The kinetics of carrier-mediated transport are similar to Michaelis-Menten enzyme kinetics—both involve proteins with a limited number of binding sites. (The Tm is analogous to the Vmax of enzyme kinetics.) Tm-limited glucose transport in the proximal tubule of the kidney is an example of saturable transport. Two solutions, A and B, are separated by a membrane that is permeable to the solute. The solute concentra- tion in A is initially twice that of B. The solute molecules are in constant motion, with equal probability that a given molecule will cross the membrane to the other solution. However, because there are twice as many solute molecules in Solution A as in Solution B, there will be greater movement of molecules from A to B than from B to A. In other words, there will be net diffusion of the solute from A to B, which will continue until the solute concentrations of the two solutions become equal (although the random movement of molecules will go on forever). Net diffusion of the solute is called flux, or flow (J), and depends on the following variables: size of the concentration gradient, partition coefficient, diffusion coefficient, thickness of the membrane, and surface area available for diffusion. CONCENTRATION GRADIENT (CA − CB) The concentration gradient across the membrane is the driving force for net diffusion. The larger the difference in solute concentration between Solution A and Solu- tion B, the greater the driving force and the greater the net diffusion. It also follows that, if the concentrations in the two solutions are equal, there is no driving force and no net diffusion. PARTITION COEFFICIENT (K) The partition coefficient, by definition, describes the solubility of a solute in oil relative to its solubility in water. The greater the relative solubility in oil, the higher the partition coefficient and the more easily the solute can dissolve in the cell membrane’s lipid bilayer. Nonpolar solutes tend to be soluble in oil and have high values for partition coefficient, whereas polar solutes tend to be insoluble in oil and have low values for partition coefficient. The partition coefficient can be measured by adding the solute to a mixture of olive oil and water and then measuring its concentration in the oil phase relative to its concentration in the water phase. Thus 1—Cellular Physiology 7 THICKNESS OF THE MEMBRANE (AX) The thicker the cell membrane, the greater the distance the solute must diffuse and the lower the rate of diffusion. SURFACE AREA (A) The greater the surface area of membrane available, the higher the rate of diffusion. For example, lipid-soluble gases such as oxygen and carbon dioxide have particu- larly high rates of diffusion across cell membranes. These high rates can be attributed to the large surface area for diffusion provided by the lipid component of the membrane. To simplify the description of diffusion, several of the previously cited characteristics can be combined into a single term called permeability (P). Permeability includes the partition coefficient, the diffusion coeffi- cient, and the membrane thickness. Thus P = KD ∆x By combining several variables into permeability, the rate of net diffusion is simplified to the following expression: J = PA(CA − CB ) where K = Concentration in olive oil Concentration in water DIFFUSION COEFFICIENT (D) The diffusion coefficient depends on such characteris- tics as size of the solute molecule and the viscosity of the medium. It is defined by the Stokes-Einstein equa- tion (see later). The diffusion coefficient correlates inversely with the molecular radius of the solute and the viscosity of the medium. Thus small solutes in nonviscous solutions have the largest diffusion coeffi- cients and diffuse most readily; large solutes in viscous solutions have the smallest diffusion coefficients and diffuse least readily. Thus D = KT 6πrη J = Net rate of diffusion (mmol/s) P = Permeability (cm/s) A = Surface area for diffusion (cm2 ) CA = Concentration in Solution A (mmol/L) CB = Concentration in Solution B (mmol/L) where D = Diffusion coefficient K = Boltzmann constant T = Absolute temperature (K) r = Molecular radius η = Viscosity of the medium 8 Physiology Diffusion of Electrolytes Thus far, the discussion concerning diffusion has assumed that the solute is a nonelectrolyte (i.e., it is uncharged). However, if the diffusing solute is an ion or an electrolyte, there are two additional consequences of the presence of charge on the solute. First, if there is a potential difference across the membrane, that potential difference will alter the net rate of diffusion of a charged solute. (A potential dif- ference does not alter the rate of diffusion of a nonelec- trolyte.) For example, the diffusion of K+ ions will be slowed if K+ is diffusing into an area of positive charge, and it will be accelerated if K+ is diffusing into an area of negative charge. This effect of potential difference can either add to or negate the effects of differences in concentrations, depending on the orientation of the potential difference and the charge on the diffusing ion. If the concentration gradient and the charge effect are oriented in the same direction across the membrane, they will combine; if they are oriented in opposite directions, they may cancel each other out. Second, when a charged solute diffuses down a concentration gradient, that diffusion can itself gener- ate a potential difference across a membrane called a diffusion potential. The concept of diffusion potential will be discussed more fully in a following section. Facilitated Diffusion Like simple diffusion, facilitated diffusion occurs down an electrochemical potential gradient; thus it requires no input of metabolic energy. Unlike simple diffusion, however, facilitated diffusion uses a membrane carrier and exhibits all the characteristics of carrier-mediated transport: saturation, stereospecificity, and competi- tion. At low solute concentration, facilitated diffusion typically proceeds faster than simple diffusion (i.e., is facilitated) because of the function of the carrier. However, at higher concentrations, the carriers will become saturated and facilitated diffusion will level off. (In contrast, simple diffusion will proceed as long as there is a concentration gradient for the solute.) An excellent example of facilitated diffusion is the transport of D-glucose into skeletal muscle and adipose cells by the GLUT4 transporter. Glucose transport can proceed as long as the blood concentration of glucose is higher than the intracellular concentration of glucose and as long as the carriers are not saturated. Other monosaccharides such as D-galactose, 3-O-methyl glucose, and phlorizin competitively inhibit the trans- port of glucose because they bind to transport sites on the carrier. The competitive solute may itself be trans- ported (e.g., D-galactose), or it may simply occupy the binding sites and prevent the attachment of glucose (e.g., phlorizin). As noted previously, the nonphysio- logic stereoisomer, L-glucose, is not recognized by the carrier for facilitated diffusion and therefore is not bound or transported. Primary Active Transport In active transport, one or more solutes are moved against an electrochemical potential gradient (uphill). In other words, solute is moved from an area of low concentration (or low electrochemical potential) to an area of high concentration (or high electrochemical potential). Because movement of a solute uphill is work, metabolic energy in the form of ATP must be provided. In the process, ATP is hydrolyzed to adenos- ine diphosphate (ADP) and inorganic phosphate (Pi), releasing energy from the terminal high-energy phos- phate bond of ATP. When the terminal phosphate is released, it is transferred to the transport protein, initi- ating a cycle of phosphorylation and dephosphoryla- tion. When the ATP energy source is directly coupled to the transport process, it is called primary active transport. Three examples of primary active transport in physiologic systems are the Na+-K+ ATPase present in all cell membranes, the Ca2+ ATPase present in SR and endoplasmic reticulum, and the H+-K+ ATPase present in gastric parietal cells and renal α-intercalated cells. Na+-K+ ATPase (Na+-K+ Pump) Na+-K+ ATPase is present in the membranes of all cells. It pumps Na+ from ICF to ECF and K+ from ECF to ICF (Fig. 1.6). Each ion moves against its respective elec- trochemical gradient. The stoichiometry can vary but, typically, for every three Na+ ions pumped out of the cell, two K+ ions are pumped into the cell. This stoichi- ometry of three Na+ ions per two K+ ions means that, for each cycle of the Na+-K+ ATPase, more positive charge is pumped out of the cell than is pumped into the cell. Thus the transport process is termed electro- genic because it creates a charge separation and a potential difference. The Na+-K+ ATPase is responsible 1—Cellular Physiology 9 Intracellular fluid Extracellular fluid Intracellular fluid Extracellular fluid 3Na+ E1~P E2~P Na+ ADP + Pi ATP 3Na+ 2K+ 2K+ Cardiac ATP Cardiac glycosides K+ E1 E2 glycosides Fig. 1.6 Na+-K+ pump of cell membranes. ADP, Adenosine diphosphate; ATP, adenosine tri- phosphate; E, Na+-K+ ATPase; E~P, phosphorylated Na+-K+ ATPase; Pi, inorganic phosphate. for maintaining concentration gradients for both Na+ and K+ across cell membranes, keeping the intracellular Na+ concentration low and the intracellular K+ concen- tration high. The Na+-K+ ATPase consists of α and β subunits. The α subunit contains the ATPase activity, as well as the binding sites for the transported ions, Na+ and K+. The Na+-K+ ATPase switches between two major con- formational states, E1 and E2. In the E1 state, the binding sites for Na+ and K+ face the ICF and the enzyme has a high affinity for Na+. In the E2 state, the binding sites for Na+ and K+ face the ECF and the enzyme has a high affinity for K+. The enzyme’s ion-transporting function (i.e., pumping Na+ out of the cell and K+ into the cell) is based on cycling between the E1 and E2 states and is powered by ATP hydrolysis. The transport cycle is illustrated in Figure 1.6. The cycle begins with the enzyme in the E1 state, bound to ATP. In the E1 state, the ion-binding sites face the ICF, and the enzyme has a high affinity for Na+; three Na+ ions bind, ATP is hydrolyzed, and the terminal phos- phate of ATP is transferred to the enzyme, producing a high-energy state, E1~P. Now, a major conformational change occurs, and the enzyme switches from E1~P to E2~P. In the E2 state, the ion-binding sites face the ECF, the affinity for Na+ is low, and the affinity for K+ is high. The three Na+ ions are released from the enzyme to ECF, two K+ ions are bound, and inorganic phosphate is released from E2. The enzyme now binds intracellular ATP, and another major conformational change occurs that returns the enzyme to the E1 state; the two K+ ions are released to ICF, and the enzyme is ready for another cycle. Cardiac glycosides (e.g., ouabain and digitalis) are a class of drugs that inhibits Na+-K+ ATPase. Treat- ment with this class of drugs causes certain predict- able changes in intracellular ionic concentration: The intracellular Na+ concentration will increase, and the intracellular K+ concentration will decrease. Cardiac glycosides inhibit the Na+-K+ ATPase by binding to the E2~P form near the K+-binding site on the extracellular side, thereby preventing the conversion of E2~P back to E1. By disrupting the cycle of phosphorylation- dephosphorylation, these drugs disrupt the entire enzyme cycle and its transport functions. Ca2+ ATPase (Ca2+ Pump) Most cell (plasma) membranes contain a Ca2+ ATPase, or plasma-membrane Ca2+ ATPase (PMCA), whose function is to extrude Ca2+ from the cell against an electrochemical gradient; one Ca2+ ion is extruded for each ATP hydrolyzed. PMCA is responsible, in part, for maintaining the very low intracellular Ca2+ concentra- tion. In addition, the sarcoplasmic reticulum (SR) of muscle cells and the endoplasmic reticulum of other cells contain variants of Ca2+ ATPase that pump two Ca2+ ions (for each ATP hydrolyzed) from ICF into the interior of the SR or endoplasmic reticulum (i.e., Ca2+ sequestration). These variants are called SR and endo- plasmic reticulum Ca2+ ATPase (SERCA). Ca2+ ATPase functions similarly to Na+-K+ ATPase, with E1 and E2 states that have, respectively, high and low affinities for Ca2+. For PMCA, the E1 state binds Ca2+ on the intracellular side, a conformational change to the E2 state occurs, and the E2 state releases Ca2+ to ECF. For SERCA, the E1 state binds Ca2+ on the intracellular side and the E2 state releases Ca2+ to the lumen of the SR or endoplasmic reticulum. H+-K+ ATPase (H+-K+ Pump) H+-K+ ATPase is found in the parietal cells of the gastric mucosa and in the α-intercalated cells of the renal collecting duct. In the stomach, it pumps H+ from the ICF of the parietal cells into the lumen of the stomach, where it acidifies the gastric contents. Omeprazole, an inhibitor of gastric H+-K+ ATPase, can be used thera- peutically to reduce the secretion of H+ in the treatment of some types of peptic ulcer disease. 10 Physiology Secondary Active Transport Secondary active transport processes are those in which the transport of two or more solutes is coupled. One of the solutes, usually Na+, moves down its electro- chemical gradient (downhill), and the other solute moves against its electrochemical gradient (uphill). The downhill movement of Na+ provides energy for the uphill movement of the other solute. Thus metabolic energy, as ATP, is not used directly, but it is supplied indirectly in the Na+ concentration gradient across the cell membrane. (The Na+-K+ ATPase, utilizing ATP, creates and maintains this Na+ gradient.) The name secondary active transport therefore refers to the indi- rect utilization of ATP as an energy source. Inhibition of the Na+-K+ ATPase (e.g., by treatment with ouabain) diminishes the transport of Na+ from ICF to ECF, causing the intracellular Na+ concentration to increase and thereby decreasing the size of the trans- membrane Na+ gradient. Thus indirectly, all secondary active transport processes are diminished by inhibitors of the Na+-K+ ATPase because their energy source, the Na+ gradient, is diminished. There are two types of secondary active transport, distinguishable by the direction of movement of the uphill solute. If the uphill solute moves in the same direction as Na+, it is called cotransport, or symport. If the uphill solute moves in the opposite direction of Na+, it is called countertransport, antiport, or exchange. Cotransport Cotransport (symport) is a form of secondary active transport in which all solutes are transported in the same direction across the cell membrane. Na+ moves into the cell on the carrier down its electrochemical gradient; the solutes, cotransported with Na+, also move into the cell. Cotransport is involved in several critical physiologic processes, particularly in the absorbing epithelia of the small intestine and the renal tubule. For example, Na+-glucose cotransport (SGLT) and Na+-amino acid cotransport are present in the luminal membranes of the epithelial cells of both small intestine and renal proximal tubule. Another example of cotransport involving the renal tubule is Na+-K+-2Cl− cotransport, which is present in the luminal membrane of epithelial cells of the thick ascending limb. In each example, the Na+ gradient established by the Na+-K+ ATPase is used to transport solutes such as glucose, amino acids, K+, or Cl− against electrochemical gradients. Figure 1.7 illustrates the principles of cotransport using the example of Na+-glucose cotransport (SGLT1, or Na+-glucose transport protein 1) in intestinal epithe- lial cells. The cotransporter is present in the luminal membrane of these cells and can be visualized as having Lumen Intestinal epithelial cell Blood 3Na+ Na+ ATP SGLT1 2K+ Glucose Glucose Luminal or Basolateral apical membrane membrane Fig. 1.7 Na+-glucose cotransport in an intestinal epithelial cell. ATP, Adenosine triphosphate; SGLT1, Na+-glucose transport protein 1. two specific recognition sites, one for Na+ ions and the other for glucose. When both Na+ and glucose are present in the lumen of the small intestine, they bind to the transporter. In this configuration, the cotransport protein rotates and releases both Na+ and glucose to the interior of the cell. (Subsequently, both solutes are transported out of the cell across the basolateral membrane—Na+ by the Na+-K+ ATPase and glucose by facilitated diffusion.) If either Na+ or glucose is missing from the intestinal lumen, the cotransporter cannot rotate. Thus both solutes are required, and neither can be transported in the absence of the other (Box 1.1). Finally, the role of the intestinal Na+-glucose cotrans- port process can be understood in the context of overall intestinal absorption of carbohydrates. Dietary carbo- hydrates are digested by gastrointestinal enzymes to an absorbable form, the monosaccharides. One of these monosaccharides is glucose, which is absorbed across the intestinal epithelial cells by a combination of Na+- glucose cotransport in the luminal membrane and facilitated diffusion of glucose in the basolateral mem- brane. Na+-glucose cotransport is the active step, allow- ing glucose to be absorbed into the blood against an electrochemical gradient. Countertransport Countertransport (antiport or exchange) is a form of secondary active transport in which solutes move in opposite directions across the cell membrane. Na+ moves into the cell on the carrier down its electrochemical gradient; the solutes that are countertransported or exchanged for Na+ move out of the cell. Countertrans- port is illustrated by Ca2+-Na+ exchange (Fig. 1.8) and by Na+-H+ exchange. As with cotransport, each process 1—Cellular Physiology 11 Muscle cell 3Na+ 3Na+ ATP Ca2+ 2K+ Fig. 1.8 Ca2+-Na+ countertransport (exchange) in a muscle cell. ATP, Adenosine triphosphate. uses the Na+ gradient established by the Na+-K+ ATPase as an energy source; Na+ moves downhill and Ca2+ or H+ moves uphill. Ca2+-Na+ exchange is one of the transport mecha- nisms, along with the Ca2+ ATPase, that helps maintain the intracellular Ca2+ concentration at very low levels (≈10−7 molar). To accomplish Ca2+-Na+ exchange, active transport must be involved because Ca2+ moves out of the cell against its electrochemical gradient. Figure 1.8 illustrates the concept of Ca2+-Na+ exchange in a muscle cell membrane. The exchange protein has recognition sites for both Ca2+ and Na+. The protein must bind Ca2+ on the intracellular side of the membrane and, simul- taneously, bind Na+ on the extracellular side. In this configuration, the exchange protein rotates and delivers Ca2+ to the exterior of the cell and Na+ to the interior of the cell. The stoichiometry of Ca2+-Na+ exchange varies between different cell types and may even vary for a single cell type under different conditions. Usually, however, three Na+ ions enter the cell for each Ca2+ ion extruded from the cell. With this stoichiometry of three Na+ ions per one Ca2+ ion, three positive charges move into the cell in exchange for two positive charges leaving the cell, making the Ca2+-Na+ exchanger electrogenic. Osmosis Osmosis is the flow of water across a semipermeable membrane because of differences in solute concentra- tion. Concentration differences of impermeant solutes establish osmotic pressure differences, and this osmotic pressure difference causes water to flow by osmosis. Osmosis of water is not diffusion of water: Osmosis occurs because of a pressure difference, whereas diffu- sion occurs because of a concentration (or activity) difference of water. 12 Physiology Osmolarity The osmolarity of a solution is its concentration of osmotically active particles, expressed as osmoles per liter or milliosmoles per liter. To calculate osmolarity, it is necessary to know the concentration of solute and whether the solute dissociates in solution. For example, glucose does not dissociate in solution; theoretically, NaCl dissociates into two particles and CaCl2 dissoci- ates into three particles. The symbol “g” gives the number of particles in solution and also takes into account whether there is complete or only partial dis- sociation. Thus if NaCl is completely dissociated into two particles, g equals 2.0; if NaCl dissociates only partially, then g falls between 1.0 and 2.0. Osmolarity is calculated as follows: Osmolarity = g C where Osmolarity = Concentration of particles (mOsm/L) g = Number of particles per mole in solution (Osm/mol) C = Concentration (mmol/L) If two solutions have the same calculated osmolarity, they are called isosmotic. If two solutions have differ- ent calculated osmolarities, the solution with the higher osmolarity is called hyperosmotic and the solution with the lower osmolarity is called hyposmotic. Osmolality Osmolality is similar to osmolarity, except that it is the concentration of osmotically active particles, expressed as osmoles (or milliosmoles) per kilogram of water. Because 1 kg of water is approximately equivalent to 1 L of water, osmolarity and osmolality will have essentially the same numerical value. Osmotic Pressure Osmosis is the flow of water across a semipermeable membrane due to a difference in solute concentration. The difference in solute concentration creates an osmotic pressure difference across the membrane and that pressure difference is the driving force for osmotic water flow. Figure 1.9 illustrates the concept of osmosis. Two aqueous solutions, open to the atmosphere, are shown in Figure 1.9A. The membrane separating the solutions is permeable to water but is impermeable to the solute. Initially, solute is present only in Solution 1. The solute in Solution 1 produces an osmotic pressure and causes, by the interaction of solute with pores in the membrane, a reduction in hydrostatic pressure of Solution 1. The resulting hydrostatic pressure difference across the membrane then causes water to flow from Solution 2 into Solution 1. With time, water flow causes the volume of Solution 1 to increase and the volume of Solution 2 to decrease. Figure 1.9B shows a similar pair of solutions; however, the preparation has been modified so that water flow into Solution 1 is prevented by applying pressure to a piston. The pressure required to stop the flow of water is the osmotic pressure of Solution 1. The osmotic pressure (π) of Solution 1 depends on two factors: the concentration of osmotically active particles and whether the solute remains in Solution 1 (i.e., whether the solute can cross the membrane or not). Osmotic pressure is calculated by the van’t Hoff equation (as follows), which converts the concentra- tion of particles to a pressure, taking into account whether the solute is retained in the original solution. Thus where π = g C σ R T π = Osmotic pressure (atm or mm Hg) g = Number of particles per mole in solution (Osm/mol) C = Concentration (mmol/L) σ = Reflection coefficient (varies from 0 to 1) R = Gas constant (0.082 L − atm/mol − K) T = Absolute temperature (K) The reflection coefficient (σ) is a dimensionless number ranging between 0 and 1 that describes the 1—Cellular Physiology 13 Semipermeable membrane Time 1 2 1 2 A atm Time 1 2 Piston applies 1 2 pressure to stop water flow B Fig. 1.9 Osmosis across a semipermeable membrane. A, Solute (circles) is present on one side of a semipermeable membrane; with time, the osmotic pressure created by the solute causes water to flow from Solution 2 to Solution 1. The resulting volume changes are shown. B, The solutions are closed to the atmosphere, and a piston is applied to stop the flow of water into Solution 1. The pressure needed to stop the flow of water is the effective osmotic pressure of Solution 1. atm, Atmosphere. ease with which a solute crosses a membrane. Reflec- tion coefficients can be described for the following three conditions (Fig. 1.10): σ = 1.0 (see Fig. 1.10A). If the membrane is imper- meable to the solute, σ is 1.0, and the solute will be retained in the original solution and exert its full osmotic effect. In this case, the effective osmotic pressure will be maximal and will cause maximal water flow. For example, serum albumin and intra- cellular proteins are solutes where σ = 1. σ = 0 (see Fig. 1.10C). If the membrane is freely permeable to the solute, σ is 0, and the solute will diffuse across the membrane down its concentration gradient until the solute concentrations of the two solutions are equal. In other words, the solute behaves as if it were water. In this case, there will be no effective osmotic pressure difference across the membrane and therefore no driving force for osmotic water flow. Refer again to the van’t Hoff equation and notice that, when σ = 0, the calculated effective osmotic pressure becomes zero. Urea is an example of a solute where σ = 0 (or nearly 0). σ = a value between 0 and 1 (see Fig. 1.10B). Most solutes are neither impermeant (σ = 1) nor freely permeant (σ = 0) across membranes, but the reflec- tion coefficient falls somewhere between 0 and 1. In such cases, the effective osmotic pressure lies between its maximal possible value (when the solute is completely impermeable) and zero (when the solute is freely permeable). Refer once again to the van’t Hoff equation and notice that, when σ is between 0 and 1, the calculated effective osmotic pressure will be less than its maximal possible value but greater than zero. When two solutions separated by a semipermeable membrane have the same effective osmotic pressure, 14 Physiology σ = 1 σ = between 0 and 1 σ = 0 Membrane A B C Fig. 1.10 Reflection coefficient (σ). they are isotonic; that is, no water will flow between them because there is no effective osmotic pressure difference across the membrane. When two solutions have different effective osmotic pressures, the solution with the lower effective osmotic pressure is hypotonic and the solution with the higher effective osmotic pres- sure is hypertonic. Water will flow from the hypotonic solution into the hypertonic solution (Box 1.2). DIFFUSION POTENTIALS AND EQUILIBRIUM POTENTIALS Ion Channels Ion channels are integral, membrane-spanning proteins that, when open, permit the passage of certain ions. Thus ion channels are selective and allow ions with specific characteristics to move through them. This selectivity is based on both the size of the channel and the charges lining it. For example, channels lined with negative charges typically permit the passage of cations but exclude anions; channels lined with positive charges permit the passage of anions but exclude cations. Chan- nels also discriminate on the basis of size. For example, a cation-selective channel lined with negative charges might permit the passage of Na+ but exclude K+; another 1—Cellular Physiology 15 The gates on ion channels are controlled by three types of sensors. One type of gate has sensors that respond to changes in membrane potential (i.e., voltage-gated channels); a second type of gate responds to changes in signaling molecules (i.e., second messenger–gated channels); and a third type of gate responds to changes in ligands such as hormones or neurotransmitters (i.e., ligand-gated channels). Voltage-gated channels have gates that are con- trolled by changes in membrane potential. For example, the activation gate on the nerve Na+ channel is opened by depolarization of the nerve cell membrane; opening of this channel is responsible for the upstroke of the action potential. Interestingly, another gate on the Na+ channel, an inactivation gate, is closed by depolarization. Because the activa- tion gate responds more rapidly to depolarization than the inactivation gate, the Na+ channel first opens and then closes. This difference in response times of the two gates accounts for the shape and time course of the action potential. Second messenger–gated channels have gates that are controlled by changes in levels of intracellular signaling molecules such as cyclic adenosine mono- phosphate (cAMP) or inositol 1,4,5-triphosphate (IP3). Thus the sensors for these gates are on the intracellular side of the ion channel. For example, the gates on Na+ channels in cardiac sinoatrial node are opened by increased intracellular cAMP. Ligand-gated channels have gates that are controlled by hormones and neurotransmitters. The sensors for these gates are located on the extracellular side of the ion channel. For example, the nicotinic receptor on the motor end plate is actually an ion channel that opens when acetylcholine (ACh) binds to it; when open, it is permeable to Na+ and K+ ions. cation-selective channel (e.g., nicotinic receptor on the motor end plate) might have less selectivity and permit the passage of several different small cations. Ion channels are controlled by gates, and, depend- ing on the position of the gates, the channels may be open or closed. When a channel is open, the ions for which it is selective can flow through it by passive diffusion, down the existing electrochemical gradient. In the open state, there is a continuous path between ECF and ICF, through which ions can flow. When the channel is closed, the ions cannot flow through it, no matter what the size of the electrochemical gradient. The conductance of a channel depends on the probabil- ity that it is open. The higher the probability that the channel is open, the higher is its conductance or permeability. Diffusion Potentials A diffusion potential is the potential difference gener- ated across a membrane when a charged solute (an ion) diffuses down its concentration gradient. Therefore a diffusion potential is caused by diffusion of ions. It follows, then, that a diffusion potential can be gener- ated only if the membrane is permeable to that ion. Furthermore, if the membrane is not permeable to the ion, no diffusion potential will be generated no matter how large a concentration gradient is present. The magnitude of a diffusion potential, measured in millivolts (mV), depends on the size of the concen- tration gradient, where the concentration gradient is the driving force. The sign of the diffusion potential depends on the charge of the diffusing ion. Finally, as noted, diffusion potentials are created by the 16 Physiology movement of only a few ions, and they do not cause changes in the concentration of ions in bulk solution. Equilibrium Potentials The concept of equilibrium potential is simply an extension of the concept of diffusion potential. If there is a concentration difference for an ion across a mem- brane and the membrane is permeable to that ion, a potential difference (the diffusion potential) is created. Eventually, net diffusion of the ion slows and then stops because of that potential difference. In other words, if a cation diffuses down its concentration gradi- ent, it carries a positive charge across the membrane, which will retard and eventually stop further diffusion of the cation. If an anion diffuses down its concentra- tion gradient, it carries a negative charge, which will retard and then stop further diffusion of the anion. The equilibrium potential is the diffusion potential that exactly balances or opposes the tendency for diffusion down the concentration difference. At electrochemical equilibrium, the chemical and electrical driving forces acting on an ion are equal and opposite, and no further net diffusion occurs. The following examples of a diffusing cation and a diffusing anion illustrate the concepts of equilibrium potential and electrochemical equilibrium. Example of Na+ Equilibrium Potential Figure 1.11 shows two solutions separated by a theoreti- cal membrane that is permeable to Na+ but not to Cl−. The NaCl concentration is higher in Solution 1 than in Solution 2. The permeant ion, Na+, will diffuse down its concentration gradient from Solution 1 to Solution 2, but the impermeant ion, Cl−, will not accompany it. As a result of the net movement of positive charge to Solution 2, an Na+ diffusion potential develops and Solution 2 becomes positive with respect to Solution 1. The positivity in Solution 2 opposes further diffusion of Na+, and eventually it is large enough to prevent further net diffusion. The potential difference that exactly balances the tendency of Na+ to diffuse down its concentration gradient is the Na+ equilibrium potential. When the chemical and electrical driving forces on Na+ are equal and opposite, Na+ is said to be at electrochemical equilibrium. This diffusion of a few Na+ ions, sufficient to create the diffusion potential, does not produce any change in Na+ concentration in the bulk solutions. Example of Cl− Equilibrium Potential Figure 1.12 shows the same pair of solutions as in Figure 1.11; however, in Figure 1.12, the theoretical membrane is permeable to Cl− rather than to Na+. Cl− will diffuse from Solution 1 to Solution 2 down its concentration gradient, but Na+ will not accompany it. A diffusion potential will be established, and Solution 2 will become negative relative to Solution 1. The potential difference that exactly balances the tendency of Cl− to diffuse down its concentration gradient is the Cl− equilibrium potential. When the chemical and electrical driving forces on Cl− are equal and opposite, then Cl− is at electrochemical equilibrium. Again, diffusion of these few Cl− ions will not change the Cl− concentration in the bulk solutions. Nernst Equation The Nernst equation is used to calculate the equilibrium potential for an ion at a given concentration difference across a membrane, assuming that the membrane is permeable to that ion. By definition, the equilibrium potential is calculated for one ion at a time. Thus E = −2.3RT log [Ci ] x zF 10 [C ] Na+-selective membrane Na+ Na+ Time – Na+ – Na+ Cl Cl – + – + Cl– – + Cl– – + 1 2 1 2 Fig. 1.11 Generation of an Na+ diffusion potential. 1—Cellular Physiology 17 Cl–-selective membrane Na+ Na+ Time – Na+ – Na+ Cl Cl + – + – Cl– + – Cl– + – 1 2 1 2 Fig. 1.12 Generation of a Cl− diffusion potential. where EX = Equilibrium potential (mV) for a given ion, X 2.3RT = Constant (60 mV at 37°C) F z = Charge on the ion (+1 for Na+; + 2 for Ca2+; − 1 for Cl− ) Ci = Intracellular concentration of X (mmol/L) Ce = Extracellular concentration of X (mmol/L) In words, the Nernst equation converts a concentra- tion difference for an ion into a voltage. This conversion is accomplished by the various constants: R is the gas constant, T is the absolute temperature, and F is Faraday constant; multiplying by 2.3 converts natural logarithm to log10. By convention, membrane potential is expressed as intracellular potential relative to extracellular potential. Hence, a transmembrane potential difference of −70 mV means 70 mV, cell interior negative. Typical values for equilibrium potential for common ions in skeletal muscle, calculated as previously described and assuming typical concentration gradients across cell membranes, are as follows: ENa+ = +65 mV ECa2+ = +120 mV EK+ = −95 mV ECl− = −90 mV It is useful to keep these values in mind when considering the concepts of resting membrane potential and action potentials. 18 Physiology Driving Force When dealing with uncharged solutes, the driving force for net diffusion is simply the concentration difference of the solute across the cell membrane. However, when dealing with charged solutes (i.e., ions), the driving force for net diffusion must consider both concentra- tion difference and electrical potential difference across the cell membrane. The driving force on a given ion is the difference where IX = GX(Em − EX ) IX = ionic current (mAmp) GX = ionic conductance ( /ohm) where conductance is the reciprocal of resistance Em − EX = driving force on ion X (mV) between the actual, measured membrane potential (Em) and the ion’s calculated equilibrium potential (EX). In other words, it is the difference between the actual Em and the value the ion would “like” the membrane potential to be. (The ion would “like” the membrane potential to be its equilibrium potential, as calculated by the Nernst equation.) The driving force on a given ion, X, is therefore calculated as: Net driving force (mV) = Em − Ex where Driving force = Driving force (mV) Em = Actual membrane potential (mV) EX = Equilibrium potential for X (mV) When the driving force is negative (i.e., Em is more negative than the ion’s equilibrium potential), that ion X will enter the cell if it is a cation and will leave the cell if it is an anion. In other words, ion X “thinks” the membrane potential is too negative and tries to bring the membrane potential toward its equilibrium poten- tial by diffusing in the appropriate direction across the cell membrane. Conversely, if the driving force is posi- tive (Em is more positive than the ion’s equilibrium potential), then ion X will leave the cell if it is a cation and will enter the cell if it is an anion; in this case, ion X “thinks” the membrane potential is too positive and tries to bring the membrane potential toward its equi- librium potential by diffusing in the appropriate direc- tion across the cell membrane. Finally, if Em is equal to the ion’s equilibrium potential, then the driving force on the ion is zero, and the ion is, by definition, at electrochemical equilibrium; since there is no driving force, there will be no net movement of the ion in either direction. Ionic Current Ionic current (IX), or current flow, occurs when there is movement of an ion across the cell membrane. Ions will move across the cell membrane through ion chan- nels when two conditions are met: (1) there is a driving force on the ion, and (2) the membrane has a conduc- tance to that ion (i.e., its ion channels are open). Thus You will notice that the equation for ionic current is simply a rearrangement of Ohm’s law, where V = IR or I = V/R (where V is the same thing as E). Because conductance (G) is the reciprocal of resistance (R), I = G × V. The direction of ionic current is determined by the direction of the driving force, as described in the previ- ous section. The magnitude of ionic current is deter- mined by the size of the driving force and the conductance of the ion. For a given conductance, the greater the driving force, the greater the current flow. For a given driving force, the greater the conductance, the greater the current flow. Lastly, if either the driving force or the conductance of an ion is zero, there can be no net diffusion of that ion across the cell membrane and no current flow. RESTING MEMBRANE POTENTIAL The resting membrane potential is the potential differ- ence that exists across the membrane of excitable cells such as nerve and muscle in the period between action potentials (i.e., at rest). As stated previously, in expressing the membrane potential, it is conventional to refer the intracellular potential to the extracellular potential. The resting membrane potential is established by diffusion potentials, which result from the concentra- tion differences for various ions across the cell mem- brane. (Recall that these concentration differences have been established by primary and secondary active transport mechanisms.) Each permeant ion attempts to drive the membrane potential toward its own equilib- rium potential. Ions with the highest permeabilities or conductances at rest will make the greatest contribu- tions to the resting membrane potential, and those with the lowest permeabilities will make little or no contribution. The resting membrane potential of most excitable cells falls in the range of −70 to −80 mV. These values can best be explained by the concept of relative perme- abilities of the cell membrane. Thus the resting mem- brane potential is close to the equilibrium potentials for K+ and Cl− because the permeability to these ions at rest is high. The resting membrane potential is far from 1—Cellular Physiology 19 the equilibrium potentials for Na+ and Ca2+ because the permeability to these ions at rest is low. One way of evaluating the contribution each ion makes to the membrane potential is by using the chord conductance equation, which weights the equilibrium potential for each ion (calculated by the Nernst equa- tion) by its relative conductance. Ions with the highest conductance drive the membrane potential toward their equilibrium potentials, whereas those with low conduc- tance have little influence on the membrane potential. (An alternative approach to the same question applies the Goldman equation, which considers the contribu- tion of each ion by its relative permeability rather than by its conductance.) The chord conductance equation is written as follows: the membrane potential. Action potentials are the basic mechanism for transmission of information in the nervous system and in all types of muscle. Terminology The following terminology will be used for discussion of the action potential, the refractory periods, and the propagation of action potentials: Depolarization is the process of making the mem- brane potential less negative. As noted, the usual resting membrane potential of excitable cells is ori- ented with the cell interior negative. Depolarization makes the interior of the cell less negative, or it may even cause the cell interior to become positive. Such g + g + g − g 2+ a change in membrane potential should not be Em = K E + + Na E gT K gT Na where + + Cl E gT Cl − + Ca E 2+ gT Ca described as “increasing” or “decreasing” because those terms are ambiguous. (For example, when the membrane potential depolarizes, or becomes less negative, has the membrane potential increased or Em = Membrane potential (mV) gK+ etc. = K+ conductance etc.(mho, reciprocal of resistance) gT = Total conductance (mho) EK+ etc. = K+ equilibrium potential etc.(mV) At rest, the membranes of excitable cells are far more permeable to K+ and Cl− than to Na+ and Ca2+. These differences in permeability account for the resting membrane potential. What role, if any, does the Na+-K+ ATPase play in creating the resting membrane potential? The answer has two parts. First, there is a small direct electrogenic contribution of the Na+-K+ ATPase, which is based on the stoichiometry of three Na+ ions pumped out of the cell for every two K+ ions pumped into the cell. Second, the more important indirect contribution is in maintain- ing the concentration gradient for K+ across the cell membrane, which then is responsible for the K+ diffu- sion potential that drives the membrane potential toward the K+ equilibrium potential. Thus the Na+-K+ ATPase is necessary to create and maintain the K+ concentration gradient, which establishes the resting membrane potential. (A similar argument can be made for the role of the Na+-K+ ATPase in the upstroke of the action potential, where it maintains the ionic gradient for Na+ across the cell membrane.) ACTION POTENTIALS The action potential is a phenomenon of excitable cells such as nerve and muscle and consists of a rapid depolarization (upstroke) followed by repolarization of decreased?) Hyperpolarization is the process of making the membrane potential more negative. As with depolar- ization, the terms “increasing” or “decreasing” should not be used to describe a change that makes the membrane potential more negative. Inward current is the flow of positive charge into the cell. Thus inward currents depolarize the mem- brane potential. An example of an inward current is the flow of Na+ into the cell during the upstroke of the action potential. Outward current is the flow of positive charge out of the cell. Outward currents hyperpolarize the membrane potential. An example of an outward current is the flow of K+ out of the cell during the repolarization phase of the action potential. Threshold potential is the membrane potential at which occurrence of the action potential is inevitable. Because the threshold potential is less negative than the resting membrane potential, an inward current is required to depolarize the membrane potential to threshold. At threshold potential, net inward current (e.g., inward Na+ current) becomes larger than net outward current (e.g., outward K+ current), and the resulting depolarization becomes self-sustaining, giving rise to the upstroke of the action potential. If net inward current is less than net outward current, the membrane will not be depolarized to threshold and no action potential will occur (see all-or-none response). Overshoot is that portion of the action potential where the membrane potential is positive (cell interior positive). 20 Physiology Undershoot, or hyperpolarizing afterpotential, is that portion of the action potential, following repo- larization, where the membrane potential is actually more negative than it is at rest. Refractory period is a period during which another normal action potential cannot be elicited in an excitable cell. Refractory periods can be absolute or relative. (In cardiac muscle cells, there is an addi- tional category called effective refractory period.) Characteristics of Action Potentials Action potentials have three basic characteristics: ste- reotypical size and shape, propagation, and all-or-none response. Stereotypical size and shape. Each normal action potential for a given cell type looks identical, depo- larizes to the same potential, and repolarizes back to the same resting potential. Propagation. An action potential at one site causes depolarization at adjacent sites, bringing those adjacent sites to threshold. Propagation of action potentials from one site to the next is nondecremental. All-or-none response. An action potential either occurs or does not occur. If an excitable cell is depolarized to threshold in a normal manner, then the occurrence of an action potential is inevitable. On the other hand, if the membrane is not depolar- ized to threshold, no action potential can occur. Indeed, if the stimulus is applied during the refrac- tory period, then either no action potential occurs, or the action potential will occur but not have the stereotypical size and shape. Ionic Basis of the Action Potential The action potential is a fast depolarization (the upstroke), followed by repolarization back to the resting membrane potential. Figure 1.13 illustrates the events of the action potential in nerve and skeletal muscle, which occur in the following steps: Resting membrane potential. At rest, the membrane potential is approximately −70 mV (cell interior Fig. 1.13 Time course of voltage and conductance changes during the action potential of nerve. 1—Cellular Physiology 21 negative). The K+ conductance or permeability is high and K+ channels are almost fully open, allowing K+ ions to diffuse out of the cell down the existing concentration gradient. This diffusion creates a K+ diffusion potential, which drives the membrane potential toward the K+ equilibrium potential. The conductance to Cl− (not shown) also is high, and, at rest, Cl− also is near electrochemical equilibrium. At rest, the Na+ conductance is low, and thus the resting membrane potential is far from the Na+ equilibrium potential, and Na+ is far from electro- chemical equilibrium. Upstroke of the action potential. An inward current, usually the result of current spread from action potentials at neighboring sites, causes depolarization of the nerve cell membrane to threshold, which occurs at approximately −60 mV. This initial depo- larization causes rapid opening of the activation gates of the Na+ channel, and the Na+ conductance promptly increases and becomes even higher than the K+ conductance (Fig. 1.14). The increase in Na+ conductance results in an inward Na+ current; the membrane potential is further depolarized toward, but does not quite reach, the Na+ equilibrium poten- tial of +65 mV. Tetrodotoxin (a toxin from the Japa- nese puffer fish) and the local anesthetic lidocaine block these voltage-sensitive Na+ channels and prevent the occurrence of nerve action potentials. Repolarization of the action potential. The upstroke is terminated, and the membrane potential repolar- izes to the resting level as a result of two events. First, the inactivation gates on the Na+ channels respond to depolarization by closing, but their response is slower than the opening of the activation gates. Thus after a delay, the inactivation gates close, which closes the Na+ channels and terminates the upstroke. Second, depolarization opens K+ chan- nels and increases K+ conductance to a value even higher than occurs at rest. The combined effect of closing of the Na+ channels and greater opening of the K+ channels makes the K+ conductance much higher than the Na+ conductance. Thus an outward K+ current results, and the membrane is repolarized. Tetraethylammonium (TEA) blocks these voltage- gated K+ channels, the outward K+ current, and repolarization. Fig. 1.14 States of activation and inactivation gates on the nerve Na+ channel. 1, In the closed but available state, at the resting membrane potential, the activation gate is closed, the inactivation gate is open, and the channel is closed (but available, if depolarization occurs). 2, In the open state, during the upstroke of the action potential, both the activation and inactivation gates are open and the channel is open. 3, In the inactivated state, at the peak of the action potential, the activation gate is open, the inactivation gate is closed, and the channel is closed. 22 Physiology Hyperpolarizing afterpotential (undershoot). For a brief period following repolarization, the K+ conduc- tance is higher than at rest and the membrane potential is driven even closer to the K+ equilibrium potential (hyperpolarizing afterpotential). Eventu- ally, the K+ conductance returns to the resting level, and the membrane potential depolarizes slightly, back to the resting membrane potential. The mem- brane is now ready, if stimulated, to generate another action potential. The Nerve Na+ Channel A voltage-gated Na+ channel is responsible for the upstroke of the action potential in nerve and skeletal muscle. This channel is an integral membrane protein, consisting of a large α subunit and two β subunits. The α subunit has four domains, each of which has six transmembrane α-helices. The repeats of transmem- brane α-helices surround a central pore, through which Na+ ions can flow (if the channel’s gates are open). A conceptual model of the Na+ channel demonstrating the function of the activation and inactivation gates is shown in Figure 1.14. The basic assumption of this model is that in order for Na+ to move through the channel, both gates on the channel must be open. Recall how these gates respond to changes in voltage. The activation gates open quickly in response to depolariza- tion. The inactivation gates close in response to depo- larization, but slowly, after a time delay. Thus when depolarization occurs, the activation gates open quickly, followed by slower closing of the inactivation gates. The figure shows three combinations of the gates’ posi- tions and the resulting effect on Na+ channel opening. Closed, but available. At the resting membrane potential, the activation gates are closed and the inactivation gates are open. Thus the Na+ channels are closed. However, they are “available” to fire an action potential if depolarization occurs. (Depolar- ization would open the activation gates and, because the inactivation gates are already open, the Na+ channels would then be open.) Open. During the upstroke of the action potential, depolarization quickly opens the activation gates and both the activation and inactivation gates are briefly open. Na+ can flow through the channels into the cell, causing further depolarization. Inactivated. At the peak of the action potential, the slow inactivation gates finally close in response to depolarization; now the Na+ channels are closed, the upstroke is terminated, and repolarization begins. How do the Na+ channels return to the closed, but available state? In other words, how do they recover, so that they are ready to fire another action potential? Repolarization back to the resting membrane potential causes the inactivation gates to open. The Na+ channels now return to the closed, but available state and are ready and “available” to fire another action potential if depolarization occurs. Refractory Periods During the refractory periods, excitable cells are inca- pable of producing normal action potentials (see Fig. 1.13). The refractory period includes an absolute refrac- tory period and a relative refractory period. Absolute Refractory Period The absolute refractory period overlaps with almost the entire duration of the action potential. During this period, no matter how great the stimulus, another action potential cannot be elicited. The basis for the absolute refractory period is closure of the inactivation gates of the Na+ channel in response to depolarization. These inactivation gates are in the closed position until the cell is repolarized back to the resting membrane potential and the Na+ channels have recovered to the “closed, but available” state (see Fig. 1.14). Relative Refractory Period The relative refractory period begins at the end of the absolute refractory period and overlaps primarily with the period of the hyperpolarizing afterpotential. During this period, an action potential can be elicited, but only if a greater than usual depolarizing (inward) current is applied. The basis for the relative refractory period is the higher K+ conductance than is present at rest. Because the membrane potential is closer to the K+ equilibrium potential, more inward current is needed to bring the membrane to threshold for the next action potential to be initiated. Accommodation When a nerve or muscle cell is depolarized slowly or is held at a depolarized level, the usual threshold potential may pass without an action potential having been fired. This process, called accommodation, occurs because depolarization closes inactivation gates on the Na+ channels. If depolarization occurs slowly enough, the Na+ channels close and remain closed. The upstroke of the action potential cannot occur because there are insufficient available Na+ channels to carry inward current. An example of accommodation is seen in persons who have an elevated serum K+ concentration, or hyperkalemia. At rest, nerve and muscle cell mem- branes are very permeable to K+; an increase in extra- cellular K+ concentration causes depolarization of the resting membrane (as dictated by the Nernst equation). This depolarization brings the cell membrane closer to 1—Cellular Physiology 23 BOX 1.3 Clinical Physiology: Hyperkalemia With Muscle Weakness DESCRIPTION OF CASE. A 48-year-old woman with determined by the concentration gradient for K+ across insulin-dependent diabetes mellitus reports to her the cell membrane (Nernst equation). At rest, the cell physician that she is experiencing severe muscle weak- membrane is very permeable to K+, and K+ diffuses out ness. She is being treated for hypertension with pro- of the cell down its concentration gradient, creating a pranolol, a β-adrenergic blocking agent. Her physician K+ diffusion potential. This K+ diffusion potential is immediately orders blood studies, which reveal a serum responsible for the resting membrane potential, which [K+] of 6.5 mEq/L (normal, 4.5 mEq/L) and elevated is cell interior negative. The larger the K+ concentration BUN (blood urea nitrogen). The physician tapers off gradient, the greater the negativity in the cell. When the dosage of propranolol, with eventual discontinua- the blood [K+] is elevated, the concentration gradient tion of the drug. He adjusts her insulin dosage. Within across the cell membrane is less than normal; resting a few days, the patient’s serum [K+] has decreased to membrane potential will therefore be less negative (i.e., 4.7 mEq/L, and she reports that her muscle strength depolarized). has returned to normal. It might be expected that this depolarization would EXPLANATION OF CASE. This diabetic patient has make it easier to generate action potentials in the severe hyperkalemia caused by several factors: (1) muscle because the resting membrane potential would Because her insulin dosage is insufficient, the lack of be closer to threshold. A more important effect of adequate insulin has caused a shift of K+ out of cells depolarization, however, is that it closes the inactiva- into blood (insulin promotes K+ uptake into cells). (2) tion gates on Na+ channels. When these inactivation Propranolol, the β-blocking agent used to treat the gates are closed, no action potentials can be generated, woman’s hypertension, also shifts K+ out of cells into even if the activation gates are open. Without action blood. (3) Elevated BUN suggests that the woman is potentials in the muscle, there can be no contraction. developing renal failure; her failing kidneys are unable TREATMENT. Treatment of this patient is based on to excrete the extra K+ that is accumulating in her shifting K+ back into the cells by increasing the woman’s blood. These mechanisms involve concepts related to insulin dosages and by discontinuing propranolol. By renal physiology and endocrine physiology. reducing the woman’s blood [K+] to normal levels, the It is important to understand that this woman has a resting membrane potential of her skeletal muscle cells severely elevated blood [K+] (hyperkalemia) and that will return to normal, the inactivation gates on the Na+ her muscle weakness results from this hyperkalemia. channels will be open at the resting membrane poten- The basis for this weakness can be explained as follows: tial (as they should be), and normal action potentials The resting membrane potential of muscle cells is can occur. threshold and would seem to make it more likely to fire an action potential. However, the cell is actually less likely to fire an action potential because this sustained depolarization closes the inactivation gates on the Na+ channels (Box 1.3). Propagation of Action Potentials Propagation of action potentials down a nerve or muscle fiber occurs by the spread of local currents from active regions to adjacent inactive regions. Figure 1.15 shows a nerve cell body with its dendritic tree and an axon. At rest, the entire nerve axon is at the resting membrane potential, with the cell interior negative. Action potentials are initiated in the initial segment of the axon, nearest the nerve cell body. They propagate down the axon by spread of local currents, as illustrated in the figure. In Figure 1.15A the initial segment of the nerve axon is depolarized to threshold and fires an action potential (the active region). As the result of an inward Na+ current, at the peak of the action potential, the polarity of the membrane potential is reversed and the cell interior becomes positive. The adjacent region of the axon remains inactive, with its cell interior negative. Figure 1.15B illustrates the spread of local current from the depolarized active region to the adjacent inac- tive region. At the active site, positive charges inside the cell flow toward negative charges at the adjacent inactive site. This current flow causes the adjacent region to depolarize to threshold. In Figure 1.15C the adjacent region of the nerve axon, having been depolarized to threshold, now fires an action potential. The polarity of its membrane potential is reversed, and the cell interior becomes positive. At this time, the original active region has been repolarized back to the resting membrane poten- tial and restored to its inside-negative polarity. The process continues, transmitting the action potential sequentially down the axon. Conduction Velocity The speed at which action potentials are conducted along a nerve or muscle fiber is the conduction velocity. This property is of great physiologic importance because 24 Physiology Fig. 1.15 Spread of depolarization down a nerve fiber by local currents. A, The initial segment of the axon has fired an action potential, and the potential difference across the cell membrane has reversed to become inside positive. The adjacent area is inactive and remains at the resting membrane potential, inside negative. B, At the active site, positive charges inside the nerve flow to the adjacent inactive area. C, Local current flow causes the adjacent area to be depolarized to threshold and to fire action potentials; the original active region has repolarized back to the resting membrane potential. it determines the speed at which information can be transmitted in the nervous system. To understand conduction velocity in excitable tissues, two major concepts must be explained: the time constant and the length constant. These concepts, called cable proper- ties, explain how nerves and muscles act as cables to conduct electrical activity. The time constant (τ) is the amount of time it takes following the injection of current for the potential to change to 63% of its final value. In other words, the time constant indicates how quickly a cell membrane depolarizes in response to an inward current or how quickly it hyperpolarizes in response to an outward current. Thus membrane capacitance (Cm), is the ability of the cell membrane to store charge. When Cm is high, the time constant is increased because injected current first must discharge the membrane capacitor before it can depolarize the membrane. Thus the time constant is greatest (i.e., takes longest) when Rm and Cm are high. The length constant (λ) is the distance from the site of current injection where the potential has fallen by 63% of its original value. The length constant indicates how far a depolarizing current will spread along a nerve. In other words, the longer the length constant, the farther the current spreads down the nerve fiber. Thus λ ∝ where τ = RmCm τ = Time constant Rm = Membrane resistance Cm = Membrane capacitance where λ = Length constant Rm = Membrane resistance Ri = Internal resistance Two factors affect the time constant. The first factor is membrane resistance (Rm). When Rm is high, current does not readily flow across the cell membrane, which makes it difficult to change the membrane potential, thus increasing the time constant. The second factor, Again, Rm represents membrane resistance. Internal resistance, Ri, is inversely related to the ease of current flow in the cytoplasm of the nerve fiber. Therefore the length constant will be greatest (i.e., current will travel the farthest) when the diameter of the nerve is large, when membrane resistance is high, and when internal 1—Cellular Physiology 25 resistance is low. In other words, current flows along the path of least resistance. Changes in Conduction Velocity There are two mechanisms that increase conduction velocity along a nerve: increasing the size of the nerve fiber and myelinating the nerve fiber. These mecha- nisms can best be understood in terms of the cable properties of time constant and length constant. Increasing nerve diameter. Increasing the size of a nerve fiber increases conduction velocity, a relation- ship that can be explained as follows: Internal resistance, Ri, is inversely proportional to the cross- sectional area (A = πr2). Therefore the larger the fiber, the lower the internal resistance. The length constant is inversely proportional to the square root of Ri (refer to the equation for length constant). Thus the length constant (λ) will be large when internal resistance (Ri) is small (i.e., fiber size is large). The largest nerves have the longest length constants, and current spreads farthest from the active region to propagate action potentials. Increasing nerve fiber size is certainly an important mechanism for increas- ing conduction velocity in the nervous system, but anatomic constraints limit how large nerves can become. Therefore a second mechanism, myelina- tion, is invoked to increase conduction velocity. Myelination. Myelin is a lipid insulator of nerve axons that increases membrane resistance and decreases membrane capacitance. The increased membrane resistance forces current to flow along the path of least resistance of the axon interior rather than across the high resistance path of the axonal membrane. The decreased membrane capacitance produces a decrease in time constant; thus at breaks in the myelin sheath (see following), the axonal membrane depolarizes faster in response to inward current. Together, the effects of increased membrane resistance and decreased membrane capacitance result in increased conduction velocity (Box 1.4). BOX 1.4 Clinical Physiology: Multiple Sclerosis DESCRIPTION OF CASE. A 32-year-old woman had Myelin is an insulator of axons that increases mem- her first episode of blurred vision 5 years ago. She had brane resistance and decreases membrane capacitance. trouble reading the newspaper and the fine print on By increasing membrane resistance, current is forced labels. Her vision returned to normal on its own, but to flow down the axon interior and less current is lost 10 months later, the blurred vision recurred, this time across the cell membrane (increasing length constant); with other symptoms including double vision, and a because more current flows down the axon, conduction “pins and needles” feeling and severe weakness in her velocity is increased. By decreasing membrane capaci- legs. She was too weak to walk even a single flight of tance, local currents depolarize the membrane more stairs. She was referred to a neurologist, who ordered rapidly, which also increases conduction velocity. In a series of tests. Magnetic Resonance Imaging (MRI) of order for action potentials to be conducted in myelin- the brain showed lesions typical of multiple sclerosis. ated nerves, there must be periodic breaks in the myelin Visual evoked potentials had a prolonged latency that sheath (at the nodes of Ranvier), where there is a was consistent with decreased nerve conduction veloc- concentration of Na+ and K+ channels. Thus at the ity. Since the diagnosis, she has had two relapses and nodes, the ionic currents necessary for the action she is currently being treated with interferon beta. potential can flow across the membrane (e.g., the EXPLANATION OF CASE. Action potentials are propa- inward Na+ current necessary for the upstroke of the gated along nerve fibers by spread of local currents as action potential). Between nodes, membrane resistance follows: When an action potential occurs, the inward is very high and current is forced to flow rapidly down current of the upstroke of the action potential depolar- the nerve axon to the next node, where the next action izes the membrane at that site and reverses the polarity potential can be generated. Thus the action potential (i.e., that site briefly becomes inside positive). The appears to “jump” from one node of Ranvier to the depolarization then spreads to adjacent sites along the next. This is called saltatory conduction. nerve fiber by local current flow. Importantly, if these Multiple sclerosis is the most common demyelinat- local currents depolarize an adjacent region to thresh- ing disease of the central nervous system. Loss of the old, it will fire an action potential (i.e., the action myelin sheath around nerves causes a decrease in potential will be propagated). The speed of propagation membrane resistance, which means that current “leaks of the action potential is called conduction velocity. out” across the membrane during conduction of local The further local currents can spread without decay currents. For this reason, local currents decay more (expressed as the length constant), the faster the con- rapidly as they flow down the axon (decreased length duction velocity. There are two main factors that constant) and, because of this decay, may be insuffi- increase length constant and therefore increase conduc- cient to generate an action potential when they reach tion velocity in nerves: increased nerve diameter and the next node of Ranvier. myelination. 26 Physiology If the entire nerve were coated with the lipid myelin sheath, however, no action potentials could occur because there would be no low resistance breaks in the membrane across which depolarizing current could flow. Therefore it is important to note that at intervals of 1 to 2 mm, there are breaks in the myelin sheath, at the nodes of Ranvier. At the nodes, membrane resis- tance is low, current can flow across the membrane, and action potentials can occur. Thus conduction of action potentials is faster in myelinated nerves than in unmyelinated nerves because action potentials “jump” long distances from one node to the next, a process called saltatory conduction. SYNAPTIC AND NEUROMUSCULAR TRANSMISSION A synapse is a site where information is transmitted from one cell to another. The information can be trans- mitted either electrically (electrical synapse) or via a chemical transmitter (chemical synapse). Types of Synapses Electrical Synapses Electrical synapses allow current to flow from one excitable cell to the next via low resistance pathways between the cells called gap junctions. Gap junctions are found in cardiac muscle and in some types of smooth muscle and account for the very fast conduc- tion in these tissues. For example, rapid cell-to-cell conduction occurs in cardiac ventricular muscle, in the uterus, and in the bladder, allowing cells in these tissues to be activated simultaneously and ensuring that contraction occurs in a coordinated manner. Chemical Synapses In chemical synapses, there is a gap between the pre- synaptic cell membrane and the postsynaptic cell membrane, known as the synaptic cleft. Information is transmitted across the synaptic cleft via a neurotrans- mitter, a substance that is released from the presynaptic terminal and binds to receptors on the postsynaptic terminal. The following sequence of events occurs at chemical synapses: An action potential in the presynaptic cell causes Ca2+ channels to open. An influx of Ca2+ into the presynaptic terminal causes the neurotransmitter, which is stored in synaptic vesicles, to be released by exocytosis. The neurotransmitter diffuses across the synaptic cleft, binds to receptors on the postsynaptic membrane, and produces a change in membrane poten- tial on the postsynaptic cell. The change in membrane potential on the postsyn- aptic cell membrane can be either excitatory or inhibi- tory, depending on the nature of the neurotransmitter released from the presynaptic nerve terminal. If the neurotransmitter is excitatory, it causes depolarization of the postsynaptic cell; if the neurotransmitter is inhibitory, it causes hyperpolarization of the postsyn- aptic cell. In contrast to electrical synapses, neurotransmission across chemical synapses is unidirectional (from pre- synaptic cell to postsynaptic cell). The synaptic delay is the time required for the multiple steps in chemical neurotransmission to occur. Neuromuscular Junction—Example of a Chemical Synapse Motor Units Motoneurons are the nerves that innervate muscle fibers. A motor unit comprises a single motoneuron and the muscle fibers it innervates. Motor units vary considerably in size: A single motoneuron may activate a few muscle fibers or thousands of muscle fibers. Predictably, small motor units are involved in fine motor activities (e.g., facial expressions), and large motor units are involved in gross muscular activities (e.g., quadriceps muscles used in running). Sequence of Events at the Neuromuscular Junction The synapse between a motoneuron and a muscle fiber is called the neuromuscular junction (Fig. 1.16). An action potential in the motoneuron produces an action potential in the muscle fibers it innervates by the fol- lowing sequence of events: The numbered steps cor- relate with the circled numbers in Figure 1.16. Action potentials are propagated down the motoneu- ron, as described previously. Local currents depolar- ize each adjacent region to threshold. Finally, the presynaptic terminal is depolarized, and this depo- larization causes voltage-gated Ca2+ channels in the presynaptic membrane to open. When these Ca2+ channels open, the Ca2+ permeabil- ity of the presynaptic terminal increases, and Ca2+ flows into the terminal down its electrochemical gradient. Ca2+ uptake into the terminal causes release of the neurotransmitter acetylcholine (ACh), which has been previously synthesized and stored in synaptic vesicles. To release ACh, the synaptic vesicles fuse with the plasma membrane and empty their contents into the synaptic cleft by exocytosis. ACh is formed from acetyl coenzyme A (acetyl CoA) and choline by the action of the enzyme choline acetyltransferase (Fig. 1.17). ACh is stored in vesicles with ATP and proteoglycan for subsequent 1—Cellular Physiology 27 MOTONEURON MUSCLE Na+ Choline Acetate 7 AChE ACh ACh Depolarization Action potential 1 3 6 of motor end plate in nerve ACh 4 Action potential ACh in muscle ACh Ca2+ Na+ K+ 2 5 Presynaptic nerve ACh Motor terminal end plate Fig. 1.16 Sequence of events in neuromuscular transmission. 1, Action potential travels down the motoneuron to the presynaptic terminal. 2, Depolarization of the presynaptic terminal opens Ca2+ channels, and Ca2+ flows into the terminal. 3, Acetylcholine (ACh) is extruded into the synapse by exocytosis. 4, ACh binds to its receptor on the motor end plate. 5, Channels for Na+ and K+ are opened in the motor end plate. 6, Depolarization of the motor end plate causes action potentials to be generated in the adjacent muscle tissue. 7, ACh is degraded to choline and acetate by acetylcholinesterase (AChE); choline is taken back into the presynaptic terminal on an Na+- choline cotransporter. Choline + Acetyl CoA Synthesis choline acetyltransferase Reuptake into nerve Acetylcholine terminal acetylcholinesterase Degradation Choline + Acetate Fig. 1.17 Synthesis and degradation of acetylcholine. Acetyl CoA, Acetyl coenzyme A. release. On stimulation, the entire content of a synaptic vesicle is released into the synaptic cleft. The smallest possible amount of ACh that can be released is the content of one synaptic vesicle (one quantum), and for this reason, the release of ACh is said to be quantal. ACh diffuses across the synaptic cleft to the postsyn- aptic membrane. This specialized region of the muscle fiber is called the motor end plate, which contains nicotinic receptors for ACh. ACh binds to the α subunits of the nicotinic receptor and causes a conformational change. It is important to note that the nicotinic receptor for ACh is an example of a ligand-gated ion channel: It also is an Na+ and K+ channel. When the conformational change occurs, the central core of the channel opens, and the per- meability of the motor end plate to both Na+ and K+ increases. When these channels open, both Na+ and K+ flow down their respective electrochemical gradients, Na+ moving into the end plate and K+ moving out, each ion attempting to drive the motor end plate potential (EPP) to its equilibrium potential. Indeed, if there were no other ion channels in the motor end plate, the end plate would depolarize to a value about halfway between the equilibrium potentials for Na+ and K+, or approximately 0 mV. (In this case, zero is not a “magic number”—it simply happens to be the value about halfway between the two equilib- rium potentials.) In practice, however, because other ion channels that influence membrane potential are present in the end plate, the motor end plate only depolarizes to about −50 mV, which is the EPP. The EPP is not an action potential but is simply a local depolarization of the specialized motor end plate. The content of a single synaptic vesicle produces the smallest possible change in membrane potential of the motor end plate, the miniature end plate 28 Physiology potential (MEPP). MEPPs summate to produce the full-fledged EPP. The spontaneous appearance of MEPPs proves the quantal nature of ACh release at the neuromuscular junction. Each MEPP, which represents the content of one synaptic vesicle, depolarizes the motor end plate by about 0.4 mV. An EPP is a multiple of these 0.4-mV units of depolarization. How many such quanta are required to depolarize the motor end plate to the EPP? Because the motor end plate must be depolarized from its resting potential of −90 mV to the threshold potential of −50 mV, it must therefore depolarize by 40 mV. Depolarization by 40 mV requires 100 quanta (because each quantum or vesicle depolarizes the motor end plate by 0.4 mV). Depolarization of the motor end plate (the EPP) then spreads by local currents to adjacent muscle fibers, which are depolarized to threshold and fire action potentials. Although the motor end plate itself cannot fire action potentials, it depolarizes sufficiently to initiate the process in the neighboring “regular” muscle cell membranes. Action potentials are propa- gated down the muscle fiber by a continuation of this process. The EPP at the motor end plate is terminated when ACh is degraded to choline and acetate by acetyl- cholinesterase (AChE) on the motor end plate. Approximately 50% of the choline is returned to the presynaptic terminal by Na+-choline cotransport, to be used again in the synthesis of new ACh. Agents That Alter Neuromuscular Function Several agents interfere with normal activity at the neuromuscular junction, and their mechanisms of action can be readily understood by considering the steps involved in neuromuscular transmission (Table 1.3; see Fig. 1.16). Botulinus toxin blocks the release of ACh from presynaptic terminals, causing total blockade of neuromuscular transmission, paralysis of skeletal TABLE 1.3 Agents Affecting Neuromuscular Transmission muscle, and, eventually, death from respiratory failure. Curare competes with ACh for the nicotinic recep- tors on the motor end plate, decreasing the size of the EPP. When administered in maximal doses, curare causes paralysis and death. D-Tubocurarine, a form of curare, is used therapeutically to cause relaxation of skeletal muscle during anesthesia. A related substance, α-bungarotoxin, binds irre- versibly to ACh receptors. Binding of radioactive α-bungarotoxin has provided an experimental tool for measuring the density of ACh receptors on the motor end plate. AChE inhibitors (anticholinesterases) such as neo- stigmine prevent degradation of ACh in the synaptic cleft, and they prolong and enhance the action of ACh at the motor end plate. AChE inhibitors can be used in the treatment of myasthenia gravis, a disease characterized by skeletal muscle weakness and fatigability, in which ACh receptors are blocked by antibodies (Box 1.5). Hemicholinium blocks choline reuptake into pre- synaptic terminals, thus depleting choline stores from the motoneuron terminal and decreasing the synthesis of ACh. Types of Synaptic Arrangements There are several types of relationships between the input to a synapse (the presynaptic element) and the output (the postsynaptic element): one-to-one, one-to- many, or many-to-one. One-to-one synapses. The one-to-one synapse is illustrated by the neuromuscular junction (see Fig. 1.16). A single action potential in the presynaptic cell, the motoneuron, causes a single action potential in the postsynaptic cell, the muscle fiber. One-to-many synapses. The one-to-many synapse is uncommon, but it is found, for example, at the Example Action Effect on Neuromuscular Transmission Botulinus toxin Blocks ACh release from presynaptic Total blockade, paralysis of respiratory muscles, and death terminals Curare Competes with ACh for receptors on Decreases siz