PHYSIOLOGY IS THE THEORETICAL BASES OF MEDICINE.
HISTORY LANDMARKS OF PHYSIOLOGICAL SCIENCE.
BIOELECTRICAL PHENOMENA IN NERVE CELLS.
EXCERPT FROM INSTRUCTION OF FIRE SAFETY MEASURES IN THE CASE OF BUILDING EXPLUATATION IN TERNOPIL STATE MEDICAL UNIVERSITY NAMED BY I. Ya. GORBACHEVSKY
General safety rules at laboratory use
1. Students are obligated to dress a doctor’s smock before entering the laboretory room.
2. Every student are obligated to work at constantly fixed by him (her) place. Matching another work place is strictly forbidden without instructors’ permission.
3. Students are forbidden to work at the laboratory in instructor or laboratory assistant absence.
4. Students are categorically forbidden to make experimental jobs irrelevant to education process execution.
5. Students are permitted to set at each work only being instructed in protection of labor and fire safety, after instructors’ permission. Instruction fixes in periodical briefing journal.
6. Tytors, laborators and students are obligated to know the fire danger of the labs lodgment, liquids and materials used in, also stick to fire safety rules.
7. All jobs concerned with isolation of noxious substances fire-explode-dangerous vapors and gases have to be hold at functional draft hoods only. Draft hoods have to be made of incombustible materials. The use of out of order draft hoods is strictly forbidden.
8. FORBIDDEN to leave work place out of observation, alighted burners and other heating supplies.
9. Collaborators and students are obligated to know the location of fire-extinguishing means and be able to use them practically in case of fire origin.
– to obstruct the passages, and also passages leading to fire-extinguishing means, to create passages between the equipment less then 1meter.
– to leave tarred pieces of cloth and paper at work place.
– to keep in work lodgments any kind of substances with unknown fire dangerous properties.
The goal of physiology is to explain the physical and chemical factors that are responsible for the origin, development, and progression of life. Each type of life, from the very simple virus to the largest tree or to the complicated human being, has its own functional characteristics. Therefore, the vast field of physiology can be divided into viral physiology, bacterial physiology, cellular physiology, plant physiology, human physiology, and many more subdivisions.
CELLS AS THE LIVING UNITS OF THE BODY
The basic living unit of the body is the cell, and each organ is an aggregate of many different ceils held together by intercellular supporting structures. Each type of cell is specially adapted to perform one or a few particular functions. For instance, the red blood ceils, 25 trillion in each human being, transport oxygen from the lungs to the tissues. Although this type of cell is perhaps the most abundant in the body, there are about another 75 trillion cell. The entire body, then, contains about 100 trillion ceils. Although the many cells of the body often differ markedly from each other, all of them have certain basic characteristics that are alike. For instance, in all ceils, oxygen combines with breakdown products of carbohydrate, fat, or protein to release the energy required for cell function. Furthermore, the general chemical mechanisms for changing nutrients into energy are basically the same in all cells, and all the cells also deliver the end products of their chemical reactions into the surrounding fluids.
The Body: an Open System with an Internal Environment
The existence of unicellular organisms is the epitome of life in its simplest form. Even simple protists must meet two basic but essentially conflicting demands in order to survive. A unicellular organism must, on the one hand, isolate itself from the seeming disorder of its inanimate surroundings, yet, as an “open system”, it is dependent on its environment for the exchange of heat, oxygen, nutrients, waste materials, and information. “Isolation” is mainly ensured by the cell membrane, the hydrophobic properties of which prevent the potentially fatal mixing of hydrophilic components in watery solutions inside and outside the cell. Protein molecules within the cell membrane ensure the permeability of the membrane barrier. They may exist in the form of pores (channels) or as more complex transport proteins known as carriers. Both types are selective for certain substances, and their activity is usually regulated. The cell membrane is relativelywell permeable to hydrophobic molecules such as gases. This is useful for the exchange of O2 and CO2 and for the uptake of lipophilic signal substances, yet exposes the cell to poisonous gases such as carbon monoxide (CO) and lipophilic noxae such as organic solvents. The cell membrane also contains other proteins—namely, receptors and enzymes. Receptors receive signals from the external environment and convey the information to the interior of the cell (signal transduction), and enzymes enable the cell to metabolize extracellular substrates. Let us imagine the primordial sea as the external environment of the unicellular organism. This milieu remains more or less constant, although the organism absorbs nutrients from it and excretes waste into it. In spite of its simple structure, the unicellular organism is capable of eliciting motor responses to signals from the environment. This is achieved by moving its pseudopodia or flagella, for example, in response to changes in the food concentration.
The evolution from unicellular organisms to multicellular organisms, the transition from specialized cell groups to organs, the emergence of the two sexes, the coexistence of individuals in social groups, and the transition from water to land have tremendously increased the efficiency, survival, radius of action, and independence of living organisms.
This process required the simultaneous development of a complex infrastructure within the organism. Nonetheless, the individual cells of the body still need a milieu like that of the primordial sea for life and survival. Today, the extracellular fluid is responsible for providing constant environmental conditions, but the volume of the fluid is no longer infinite. In fact, it is even smaller than the intracellular volume. Because of their metabolic activity, the cells would quickly deplete the oxygen and nutrient stores within the fluids and flood their surroundings with waste products if organs capable of maintaining a stable internal environment had not developed. This is achieved through homeostasis, a process by which physiologic self-regulatory mechanisms maintain steady states in the body through coordinated physiological activity.
The term homeostasis is used by physiologists to mean maintenance of static or constant conditions in the internal environment. Essentially all of the organs and tissues of the body perform functions that help to maintain these constant conditions. For instance, the lungs provide oxygen to the extracellular fluid to continually replenish the oxygen that is being used by the ceils, the kidneys maintain constant ion concentrations, and the gastrointestinal system provides nutrients. A large segment of this text is concerned with the manner in which each organ or tissue contributes to homeostasis. To begin this discussion, in this chapter, the different functional systems of the body and their contributions to homeostasis are outlined; then we also outline briefly the basic theory of the control systems that cause the functional systems to operate in harmony with one another.
Specialized organs ensure the continuous absorption of nutrients, electrolytes and water and the excretion of waste products via the urine and feces. The circulating blood connects the organs to every inch of the body, and the exchange of materials between the blood and the intercellular spaces (interstices) creates a stable environment for the cells. Organs such as the digestive tract and liver absorb nutrients and make them available by processing, metabolizing and distributing them throughout the body.
The lung is responsible for the exchange of gases (O2 intake, CO2 elimination), the liver and kidney for the excretion of waste and foreign substances, and the skin for the release of heat. The kidney and lungs also play an important role in regulating the internal environment, e.g., water content, osmolality, ion concentrations, pH (kidney, lungs) and O2 and CO2 pressure (lungs). The specialization of cells and organs for specific tasks naturally requires integration, which is achieved by convective transport over long distances (circulation, respiratory tract), humoral transfer of information (hormones), and transmission of electrical signals in the nervous system, to name a few examples.
These mechanisms are responsible for supply and disposal and thereby maintain a stable internal environment, even under conditions of extremely high demand and stress. Moreover, they control and regulate functions that ensure survival in the sense of preservation of the species. Important factors in this process include not only the timely development of reproductive organs and the availability of fertilizable gametes at sexual maturity, but also the control of erection, ejaculation, fertilization, and nidation. Others include the coordination of functions in the mother and fetus during pregnancy and regulation of the birth process and the lactation period.
The central nervous system(CNS) processes signals from peripheral sensors (single sensory cells or sensory organs), activates outwardly directed effectors (e.g., skeletal muscles), and influences the endocrine glands. The CNS is the focus of attention when studying human or animal behavior. It helps us to locate food and water and protects us from heat or cold. The central nervous systemalso plays a role in partner selection, concern for offspring even long after their birth, and integration into social systems. The CNS is also involved in the development, expression, and processing of emotions such as desire, listlessness, curiosity, wishfulness, happiness, anger, wrath, and envy and of traits such as creativeness, inquisitiveness, self-awareness, and responsibility. This goes far beyond the scope of physiology— which in the narrower sense is the study of the functions of the body—and, hence, of this book. Although behavioral science, sociology, and psychology are disciplines that border on physiology, true bridges between them and physiology have been established only in exceptional cases.
Nervous System. The nervous system is composed of three major parts: the sensory input portion, the central nervous system (or integrative portion), and the motor output portion. Sensory receptors detect the state of the body or the state of the surroundings. For instance, receptors present everywhere in the skin apprise one every time an object touches the skin at any point. The eyes are sensory organs that give one a visual image of the surrounding area. The ears also are sensory organs.
The central nervous system is composed of the brain and spinal cord. The brain can store information, generate thoughts, create ambition, and determine reactions that the body performs in response to the sensations. Appropriate signals are then transmitted through the motor output portion of the nervous system to carry out one’s desires. A large segment of the nervous system is called the autonomic system. It operates at a subconscious level and controls many functions of the internal organs, including the level of pumping activity by the heart, movements of the gastrointestinal tract, and glandular secretion.
Hormonal System of Regulation. Located in the body are eight major endocrine glands that secrete chemical substances called hormones. Hormones are transported in the extracellular fluid to all parts of the body to help regulate cellular function. For instance, thyroid hormone increases the rates of most chemical reactions in all ceils. In this way, thyroid hormone helps to set the tempo of bodily activity. Insulin controls glucose metabolism; adrenocortical hormones control sodium ion, potassium ion, and protein metabolism: and parathyroid hormone controls bone calcium and phosphate. Thus, the hormones are a system of regulation that complements the nervous system. The nervous system regulates mainly muscular and secretory activities of the body, whereas the hormonal system regulates mainly metabolic functions.
Most control systems of the body act by negative feedback, which can best be explained by reviewing some of the homeostatic control systems mentioned previously. In the regulation of carbon dioxide concentration, a high concentration of carbon dioxide in the extracellular fluid increases pulmonary ventilation. This in turn decreases the extracellular fluid carbon dioxide concentration because the lungs then excrete greater amounts of carbon dioxide from the body. In other words, the high concentration causes a decreased concentration, which is negative to the initiating stimulus. Conversely, if the carbon dioxide concentration falls too low, this causes a feedback increase in the concentration. This response also is negative to the initiating stimulus. In the arterial pressure-regulating mechanisms, a high pressure causes a series of reactions that promote a lowered pressure, or a low pressure causes a series of reactions that promote an elevated pressure. In both instances, these effects are negative with respect to the initiating stimulus.
Therefore, in general, if some factor becomes excessive or deficient, a control system initiates negative feedback, which consists of a series of changes that return the factor toward a certain mean value, thus maintaining homeostasis.
Control and Regulation. In order to have useful cooperation between the specialized organs of the body, their functions must be adjusted to meet specific needs. In other words, the organs must be subject to control and regulation. Control implies that a controlled variable such as the blood pressure is subject to selective external modification, for example, through alteration of the heart rate. Because many other factors also affect the blood pressure and heart rate, the controlled variable can only be kept constant by continuously measuring the current blood pressure, comparing it with the reference signal (set point), and continuously correcting any deviations. If the blood pressure drops—due, for example, to rapidly standing up from a recumbent position—the heart rate will increase until the blood pressure has been reasonably adjusted. Once the blood pressure has risen above a certain limit, the heart rate will decrease again and the blood pressure will normalize. This type of closed-loop control is called a negative feedback control system or a control circuit. It consists of a controller with a programmed set-point value (target value) and control elements (effectors) that can adjust the controlled variable to the set point.
The system also includes sensors that continuously measure the actual value of the controlled variable of interest and report it (feedback) to the controller, which compares the actual value of the controlled variable with the set-point value and makes the necessary adjustments if disturbance-related discrepancies have occurred. The control system operates either fromwithin the organ itself (autoregulation) or via a superordinate organ such as the central nervous system or hormone glands.
Unlike simple control, the elements of a control circuit can work rather imprecisely without causing a deviation from the set point (at least on average). Moreover, control circuits are capable of responding to unexpected dis turbances.
In the case of blood pressure regulation, for example, the system can respond to events such as orthostasis or sudden blood loss. The type of control circuits described above keep the controlled variables constant when disturbance variables cause the controlled variable to deviate from the set point. Within the body, the set point is rarely invariable, but can be “shifted” when requirements of higher priority make such a change necessary. In this case, it is the variation of the set point that creates the discrepancy between the nominal and actual values, thus leading to the activation of regulatory elements.
Since the regulatory process is then triggered by variation of the set point (and not by disturbance variables), this is called servocontrol or servomechanism. Fever and the adjustment of muscle length by muscle spindles and motor neurons are examples of servocontrol. In addition to relatively simple variables such as blood pressure, cellular pH, muscle length, body weight and the plasma glucose concentration, the body also regulates complex sequences of events such as fertilization, pregnancy, growth and organ differentiation, as well as sensory stimulus processing and the motor activity of skeletal muscles, e.g., to maintain equilibrium while running. The regulatory process may take parts of a second (e.g., purposeful movement) to several years (e.g., the growth process).
In the control circuits described above, the controlled variables are kept constant on average, with variably large, wave-like deviations. The sudden emergence of a disturbance variable causes larger deviations that quickly normalize in a stable control circuit (!E, test subject no. 1). The degree of deviation may be slight in some cases but substantial in others. The latter is true, for example, for the blood glucose concentration, which nearly doubles after meals. This type of regulation obviously functions only to prevent extreme rises and falls (e.g., hyper- or hypoglycemia) or chronic deviation of the controlled variable. More precise maintenance of the controlled variable requires a higher level of regulatory sensitivity (high amplification factor). However, this extends the settling time and can lead to regulatory instability, i.e., a situation where the actual value oscillates back and forth between extremes.
Oscillation of a controlled variable in response to a disturbance variable can be attenuated by either of two mechanisms. First, sensors with differential characteristics ensure that the intensity of the sensor signal increases in proportion with the rate of deviation of the controlled variable from the set point. Second, feedforward control ensures that information regarding the expected intensity of disturbance is reported to the controller before the value of the controlled variable has changed at all.
Feedforward control can be explained by example of physiologic thermoregulation, a process in which cold receptors on the skin trigger counterregulation before a change in the controlled value (core temperature of the body) has actuallyoccurred. The disadvantage of having only D sensors in the control circuit can be demonstrated by example of arterial pressosensors (pressoreceptors) in acute blood pressure regulation. Very slow but steady changes, as observed in the development of arterial hypertension, then escape regulation. In fact, a rapid drop in the blood pressure of a hypertensive patient will even cause a counterregulatory increase in blood pressure. Therefore, other control systems are needed to ensure proper long-term blood pressure regulation.
The Cell. The cell is the smallest functional unit of a living organism. In other words, a cell (and no smaller unit) is able to perform essential vital functions such as metabolism, growth, movement, reproduction, and hereditary transmission. Growth, reproduction, and hereditary transmission can be achieved by cell division.
All cells consist of a cell membrane, cytosol or cytoplasm (ca. 50 vol.%), and membrane-bound subcellular structures known as organelles. The organelles of eukaryotic cells are highly specialized. For instance, the genetic material of the cell is concentrated in the cell nucleus, whereas “digestive” enzymes are located in the lysosomes. Oxidative ATP production takes place in the mitochondria.
The cell nucleus contains a liquid known as karyolymph, a nucleolus, and chromatin. Chromatin contains deoxyribonucleic acids (DNA), the carriers of genetic information. Two strands of DNA forming a double helix (up to 7cm in length) are twisted and folded to form chromosomes 10 μm in length. Humans normally have 46 chromosomes, consisting of 22 autosomal pairs and the chromosomes that determine the sex (XX in females, XY in males). DNA is made up of a strand of three-part molecules called nucleotides, each of which consists of a pentose (deoxyribose) molecule, a phosphate group, and a base. Each sugar molecule of the monotonic sugar–phosphate backbone of the strands (. . .deoxyribose – phosphate–deoxyribose. . .) is attached to one of four different bases. The sequence of bases represents the genetic code for each of the roughly 100 000 different proteins that a cell produces during its lifetime (gene expression). In a DNA double helix, each base in one strand of DNA is bonded to its complementary base in the other strand according to the rule: adenine (A) with thymine (T) and guanine (G) with cytosine (C).
The nuclear envelope consists of two membranes (two phospholipid bilayers) tha tmerge at the nuclear pores. The two membranes consist of different materials. The external membrane is continuous with the membrane of the endoplasmic reticulum.
Transport In, Through and Between Cells. The lipophilic cell membrane protects the cell interior from the extracellular fluid, which has a completely different composition. This is imperative for the creation and maintenance of a cell’s internal environment by means of metabolic energy expenditure. Channels (pores), carriers, ion pumps and the process of cytosis allow transmembrane transport of selected substances. This includes the import and export of metabolic substrates and metabolites and the selective transport of ions used to create or modify the cell potential, which plays an essential role in excitability of nerve and muscle cells. In addition, the effects of substances that readily penetrate the cell membrane in most cases (e.g.,water and CO2) can be mitigated by selectively transporting certain other substances.
This allows the cell to compensate for undesirable changes in the cell volume or pH of the cell interior. Intracellular Transport The cell interior is divided into different compartments by the organelle membranes. In some cases, very broad intracellular spaces must be crossed during transport. For this purpose, a variety of specific intracellular transport mechanisms exist, for example: Nuclear pores in the nuclear envelope provide the channels for RNA export out of the nucleus and protein import into it; Protein transport from the rough endoplasmic reticulum to the Golgi complex; Axonal transport in the nerve fibers, in which distances of up to 1meter can be crossed. These transport processes mainly take place along the filaments of the cytoskeleton. Example: while expending ATP, the microtubules set dynein-bound vesicles in motion in the one direction, and kinesinbound vesicles in the other.
Intracellular Transmembrane Transport Main sites:
Lysosomes: Uptake of H+ ions from the cytosol and release of metabolites such as amino acids into the cytosol; ! Endoplasmic reticulum (ER): In addition to a translocator protein (!p. 10), the ER has two other proteins that transport Ca2+. Ca2+
can be pumped from the cytosol into the ER by a Ca2+-ATPase called SERCA (sarcoplasmic endoplasmic reticulum Ca2+-transporting ATPase). The resulting Ca2+ stores can be released into the cytosol via a Ca2+ channel (ryanodine receptor, RyR) in response to a triggering signal.
Mitochondria: The outer membrane contains large pores called porins that render it permeable to small molecules (5 kDa), and the inner membrane has high concentrations of specific carriers and enzymes. Enzyme complexes of the respiratory chain transfer electrons from high to low energy levels, thereby pumping H+ ions from the matrix space into the intermembrane space, resulting in the formation of an H+ ion gradient directed into the matrix. This not only drives ATP synthetase (ATP production), but also promotes the inflow of pyruvate – and anorganic phosphate (symport). Ca2+ ions that regulate Ca2+-sensitive mitochondrial enzymes in muscle tissue can be pumped into the matrix space with ATP expenditure, thereby allowing the mitochondria to form a sort of Ca2+ buffer space for protection against dangerously high concentrations of Ca2+ in the cytosol. The insidenegative membrane potential (caused by H+ release) drives the uptake of ADP3 – in exchange for ATP4 – (potential-driven transport). Transport between Adjacent Cells In the body, transport between adjacent cells occurs either via diffusion through the extracellular space (e.g., paracrine hormone effects) or through channel-like connecting structures (connexons) located within a so-called gap junction or nexus (!C). A connexon is a hemichannel formed by six connexin molecules. One connexon docks with another connexon on an adjacent cell, thereby forming a common channel through which substances with molecular masses of up to around 1 kDa can pass. Since this applies not only for ions such as Ca2+, but also for a number of organic substances such as ATP, these types of cells are united to form a close electrical and metabolic unit (syncytium), as is present in the epithelium, many smooth muscles (singleunit type), the myocardium, and the glia of the central nervous system.
Electric coupling permits the transfer of excitation, e.g., from excited muscle cells to their adjacent cells, making it possible to trigger awave of excitation across wide regions of an organ, such as the stomach, intestine, biliary tract, uterus, ureter, atrium, and ventricles of the heart. Certain neurons of the retina and CNS also communicate in this manner (electric synapses). Gap junctions in the glia and epithelia help to distribute the stresses that occur in the course of transport and barrier activities (see below) throughout the entire cell community. However, the connexons close when the concentration of Ca2+ (in an extreme case, due to a hole in cell membrane) or H+ concentration increases too rapidly. In other words, the individual (defective) cell is left to deal with its own problems wheecessary to preserve the functionality of the cell community.
Transport through Cell Layers Multicellular organisms have cell layers that are responsible for separating the “interior” from the “exterior” of the organism and its larger compartments. The epithelia of skin and gastrointestinal, urogenital and respiratory tracts, the endothelia of blood vessels, and neuroglia are examples of this type of extensive barrier. They separate the immediate extracellular space from other spaces that are greatly different in composition, e.g., those filled with air (skin, bronchial epithelia), gastrointestinal contents, urine or bile (tubules, urinary bladder, gallbladder), aqueous humor of the eye, blood (endothelia) and cerebrospinal fluid (blood–cerebrospinal fluid barrier), and from the extracellular space of the CNS (blood–brain barrier). Nonetheless, certain substances must be able to pass through these cell layers. This requires selective transcellular transport with import into the cell followed by export from the cell. Unlike cells with a completely uniform plasma membrane (e.g., blood cells), epi- and endothelial cells are polar cells, as defined by their structure and transport function. Hence, the apical membrane (facing exterior) of an epithelial cell has a different set of transport proteins from the basolateral membrane (facing the blood). Tight junctions (described below) at which the outer phospholipid layer of the membrane folds over, prevent lateral mixing of the two membranes.
Whereas the apical and basolateral membranes permit transcellular transport, paracellular transport takes place between cells. Certain epithelia (e.g., in the small intestinal and proximal renal tubules) are relatively permeable to small molecules (leaky), whereas others are less leaky (e.g., distal nephron, colon). The degree of permeability depends on the strength of the tight junctions (zonulae ” occludentes) holding the cells together. The paracellular pathway and the extent of its permeability (sometimes cation-specific) are essential functional elements of the various epithelia. Macromolecules can cross the barrier formed by the endothelium of the vessel wall by transcytosis, yet paracellular transport also plays an essential role, especially in the fenestrated endothelium. Anionic macromolecules like albumin, which must remain in the bloodstream because of its colloid osmotic action, are held back by the wall charges at the intercellular spaces and, in some cases, at the fenestra.
Long-distance transport between the various organs of the body and between the body and the outside world is also necessary. Convection is the most important transport mechanism involved in long-distance transport.
Passive Transport by Means of Diffusion. Diffusion is movement of a substance owing to the random thermal motion (brownian movement) of its molecules or ions (!A1) in all directions throughout a solvent. Net diffusion or selective transport can occur only when the solute concentration at the starting point is higher than at the target site. (Note: unidirectional fluxes also occur in absence of a concentration gradient—i.e., at equilibrium— but net diffusion is zero because there is equal flux in both directions.) The driving force of diffusion is, therefore, a concentration gradient. Hence, diffusion equalizes concentration differences and requires a driving force: passive transport (= downhill transport). Example: When a layer of O2 gas is placed onwater, the O2 quickly diffuses into thewater along the initially high gas pressure gradient. As a result, the partial pressure of O2 (Po2) rises, and O2 can diffuse further downward into the next O2-poor layer ofwater (!A1). (Note: with gases, partial pressure is used in lieu of concentration.) However, the steepness of the Po2 profile or gradient (dPo2/ dx) decreases (exponentially) in each subsequent layer situated at distance x from the O2 source. Therefore, diffusion is only feasible for transport across short distances within the body. Diffusion in liquids is slower than in gases. The diffusion rate is the amount of substance that diffuses per unit of time. It is proportional to the area available for diffusion (A) and the absolute temperature (T) and is inversely proportional to the viscosity of the solvent and the radius (r) of the diffused particles.
Since the driving “force”—i.e., the concentration gradient (dC/dx)—decreases with distance, as was explained above, the time required for diffusion increases exponentially with the distance traveled (t#x2). If, for example, a molecule travels the first μm in 0.5 ms, it will require 5 s to travel 100 μmand a whopping 14 h for
Osmosis, Filtration and Convection Water flow or volume flow (JV) across a membrane, in living organisms is achieved through osmosis (diffusion of water) or filtration. They can occur only if the membrane is water-permeable. This allows osmotic and hydrostatic pressure differences (!” and !P) across the membrane to drive the fluids through it.
Active Transport. Active transport occurs in many parts of the body when solutes are transported against their concentration gradient (uphill transport) and/or, in the case of ions, against an electrical potential. All in all, active transport occurs against the electrochemical gradient or potential of the solute. Since passive transport mechanisms represent “downhill” transport, they are not appropriate for this task. Active transport requires the expenditure of energy. A large portion of chemical energy provided by foodstuffs is utilized for active transport once it has been made readily available in the form of ATP. The energy created by ATP hydrolysis is used to drive the transmembrane transport of numerous ions, metabolites, and waste products. According to the laws of thermodynamics, the energy expended in these reactions produces order in cells and organelles—a prerequisite for survival and normal function of cells and, therefore, for the whole organism. In primary active transport, the energy produced by hydrolysis of ATP goes directly into ion transport through an ion pump. This type of ion pump is called an ATPase. They establish the electrochemical gradients rather slowly, e.g., at a rate of around 1 μmol s–1 m–2 of membrane surface area in the case of Na+-K+- ATPase. The gradient can be exploited to achieve rapid ionic currents in the opposite direction after the permeability of ion channels has been increased. Na+ can, for example, be driven into a nerve cell at a rate of up to 1000 μmol s–1 m–2 during an action potential. ATPases occur ubiquitously in cell membranes (Na+-K+-ATPase) and in the endoplasmic reticulum and plasma membrane (Ca2+-ATPase), renal collecting duct and stomach glands (H+,K+ -ATPase), and in lysosomes (H+-ATPase). They transport Na+, K+, Ca2+ and H+, respectively, by primarily active mechanisms. All except H+-ATPase consist of 2 !-subunits and 2 “-subunits (P-type ATPases). The !-subunits are phosphorylated and form the ion transport channel. Na+-K+-ATPase is responsible for maintenance of intracellular Na+ and K+ homeostasis and, thus, for maintenance of the cell membrane potential. During each transport cycle (!A1, A2), 3 Na+ and 2 K+ are “pumped” out of and into the cell, respectively, while 1 ATP molecule is used to phosphorylate the carrier protein (!A2b). Phosphorylation first changes the conformation of the protein and subsequently alters the affinities of the Na+ and K+ binding sites. The conformational change is the actual ion transport step since it moves the binding sites to the opposite side of the membrane. Dephosphorylation restores the pump to its original state (!A2e–f). The Na+/K+ pumping rate increases when the cytosolic Na+ concentration rises— due, for instance, to increased Na+ influx, or when the extracellular K+ rises. Therefore, Na+,K+-activatable ATPase is the full name of the pump.Na-+K+-ATPase is inhibited by ouabain and cardiac glycosides. Secondary active transport occurs when uphill transport of a compound (e.g., glucose) via a carrier protein (e.g., sodium glucose transporter type 2, SGLT2) is coupled with the passive (downhill) transport of an ion (in this example Na+; !B1). In this case, the electrochemical Na+ gradient into the cell (created by Na+-K+-ATPase at another site on the cell membrane; provides the driving force needed for secondary active uptake of glucose into the cell. Coupling of the transport of two compounds across a membrane is called cotransport, which may be in the form of symport or antiport. Symport occurs when the two compounds (i.e., compound and driving ion) are transported across the membrane in the same direction. Antiport (countertransport) occurs when they are transported in opposite directions. Antiport occurs, for example, when an electrochemical Na+ gradient drives H+ in the opposite direction by secondary active transport. The resultingH+ gradient can then be exploited for tertiary active symport of molecules such as peptides. Electroneutral transport occurs when the net electrical charge remains balanced during transport, e.g., during Na+/H+ antiportand Na+-Cl– symport. Small charge separation occurs in electrogenic (rheogenic) transport, e.g., in Na+-glucose0 symport (!B1), Na+-amino acid0 symport, 2Na+-amino acid– symport, or H+-peptide symport.
The chemical Na+ gradient provides the sole driving force for electroneutral transport (e.g., Na+/H+ antiport), whereas the negative membrane potential provides an additional driving force for electrogenication–coupled cotransport into the cell. When secondary active transport (e.g., of glucose) is coupled with the influx of not one but two Na+ ions (e.g., SGLT1 symporter), the driving force is doubled. The aid of ATPases is necessary, however, if the required “uphill” concentration ratio is several decimal powers large, e.g.,
Variable quantities of the similar substances are transported at a given concentration, i.e., each has a different affinity (~1/KM) to the transport system. Active transport is inhibited when the energy supply to the cell is disrupted. All of these characteristics except the last apply to passive carriers, that is, to uniportermediated (facilitated) diffusion. Cytosis is a completely different type of active transport involving the formation of membrane-bound vesicles with a diameter of 50–400 nm. Vesicles are either pinched off from the plasma membrane (exocytosis) or incorporated into it by invagination (endocytosis) in conjunction with the expenditure of ATP. In cytosis, the uptake and release of macromolecules such as proteins, lipoproteins, polynucleotides, and polysaccharides into and out of a cell occurs by specific mechanisms similar to those involved in intracellular transport. Endocytosis can be broken down into different types, including pinocytosis, receptor-mediated endocytosis, and phagocytosis. Pinocytosis is characterized by the continuous unspecific uptake of extracellular fluid and molecules dissolved in it through relatively small vesicles. Receptor-mediated endocytosis involves the selective uptake of specific macromolecules with the aid of receptors. This usually begins at small depressions (pits) on the plasma membrane surface. Since the insides of the pits are often densely covered with the protein clathrin, they are called clathrin-coated pits. The receptors involved are integral cell membrane proteins such as those for low-density lipoprotein (LPL; e.g., in hepatocytes) or intrinsic factor-bound cobalamin (e.g., in ileal epithelial cells). Thousands of the same receptor type or of different receptors can converge at coated pits, yielding a tremendous increase in the efficacy of ligand uptake. The endocytosed vesicles are initially coated with clathrin, which is later released.
The vesicles then transform into early endosomes, and most of the associated receptors circulate back to the cell membrane. The endocytosed ligand is either exocytosed on the opposite side of the cell (transcytosis, see below), or is digested by lysosomes. Phagocytosis involves the endocytosis of particulate matter, such as microorganisms or cell debris, by phagocytes in conjunction with lysosomes. Small digestion products, such as amino acids, sugars and nucleotides, are transported out of the lysosomes into the cytosol, where they can be used for cellular metabolism or secreted into the extracellular fluid. When certain hormones such as insulin bind to receptors on the surface of target cells, hormonereceptor complexes can also enter the coated pits and are endocytosed (internalized) and digested by lysosomes. This reduces the density of receptors available for hormone bind ing.
In other words, an increased hormone supply down-regulates the receptor density. Exocytosis is a method for selective export of macromolecules out of the cell (e.g., pancreatic enzymes) and for release of many hormones (e.g., posterior pituitary hormone) or neurotransmitters. These substances are kept “packed” and readily available in (clathrin-coated) secretory vesicles, waiting to be released when a certain signal is received (increase in cytosolic Ca2+). The “packing material” (vesicle membrane) is later re-endocytosed and recycled. Exocytotic membrane fusion also helps to insert vesicle-bound proteins into the plasma membrane.The liquid contents of the vesicle then are automatically emptied in a process called constitutive exocytosis. In constitutive exocytosis, the protein complex coatomer (coat assembly protomer) takes on the role of clathrin (see above). Within the Golgi membrane, GNRP (guanine nucleotide-releasing protein) phosphorylates the GDP of the ADP-ribosylation factor (ARF) to GTP ), resulting in the dispatch of vesicles from the trans-Golgi network. ARF-GTP complexes then anchor on the membrane and bind with coatomer (!D2), thereby producing coatomer-coated vesicles. The membranes of the vesicles contain v-SNAREs (vesicle synaptosome-associated protein receptors), which recognize t-SNAREs (target-SNAREs) in the target membrane (the plasma membrane, in this case). This results in cleavage of ARF-GTP, dissociation of ARF-GDP and coatomer molecules and, ultimately, to membrane fusion and exocytosis (!D4, D5) to the extracellular space (ECS).
Transcytosis is the uptake of macromolecules such as proteins and hormones by endocytosis on one side of the cell, and their release on the opposite side. This is useful for transcellular transport of the macromolecules across cell layers such as endothelia.
Cell Migration. Most cells in the body are theoretically able to move from one place to another or migrate, but only a fewcell species actually do so. The sperm are probably the only cells with a special propulsion mechanism. By waving their whip-like tail, the sperm can travel at speeds of up to around 2000 μm/min. Other cells also migrate, but at much slower rates. Fibroblasts, for example,move at a rate of around 1.2 μm/min. When an injury occurs, fibroblasts migrate to the wound and aid in the formation of scar tissue. Cell migration also plays a role in embryonal development. Chemotactically attracted neutrophil granulocytes and macrophages can even migrate through vessel walls to attack invading bacteria. Cells of some tumors can also migrate to various tissues of the body or metastasize, thereby spreading their harmful effects. Cells migrate by “crawling” on a stable surface. The following activities occur during cell migration:
Back end of the cell: (a) Depolymerization of actin and tubulin in the cytoskeleton; (b) endocytosis of parts of the cell membrane, which are then propelled forward as endocytotic vesicles to the front of the cell, and (c) release of ions and fluids from the cell.
Front end of the cell (lamellipodia): (a) Polymerization of actin monomers is achieved with the aid of profilin. The monomers are propelled forward with the help of plasma membrane-based myosin I (fueled by ATP); (b) reinsertion of the vesicles in the cell membrane; (c) uptake of ions and fluids from the environment.
Parts of the cell membrane that are not involved in cytosis are conveyed from front to back, as on a track chain. Since the cell membrane is attached to the stable surface (primarily fibronectin of the extracellular matrix in the case of fibroblasts), the cell moves forward relative to the surface. This is achieved with the aid of specific receptors, such as fibronectin receptors in the case of fibroblasts. Electrical Membrane Potentials
Ion Channels. An electrical potential difference occurs due to the net movement of charge during ion transport. A diffusion potential develops for instance, when ions (e.g., K+) diffuse (down a chemical gradient) out of a cell, making the cell interior negative relative to the outside. The rising diffusion potential then drives the ions back into the cell (potentialdriven ion transport). Outward K+ diffusion persists until equilibrium is reached. At equilibrium, the two opposing forces become equal and opposite. In other words, the sum of the two or the electrochemical gradient (and thereby the electrochemical potential) equals zero, and there is no further net movement of ions (equilibrium concentration) at a certain voltage (equilibrium potential).
At equilibrium potential, the chemical gradient will drive just as many ions of species X in the one direction as the electrical potential does in the opposite direction. The electrochemicalpotential (Em– Ex) or so-called electrochemical driving “force”, will equal zero, and the sum of ionic inflow and outflow or the net flux (Ix) will also equal zero. Membrane conductance (gx), a concentration- dependent variable, is generally used to describe the permeability of a cell membrane to a given ion instead of the permeability coefficient P (see Eq. 1.5 on p. 22 for conversion).
Realistic values in resting nerve cells are: fK = 0.90, fNa = 0.03, fCl = 0.07; EK = – 90mV, ENa =+ 70mV, ECl = – 83mV. Inserting these values into equation 1.21 results in an Em of – 85mV. Thus, the driving forces (= electrochemical potentials = Em–Ex), equal + 5mV for K+, – 145mV for Na+, and – 2mV for Cl–. The driving force for K+ efflux is therefore low, though gK is high.
Despite a high driving force for Na+, Na+ influx is lowbecause the gNa and fNa of resting cells are relatively small. Nonetheless, the sodium current, INa, can rise tremendously when large numbers of Na+ channels open during an action potential.
Electrodiffusion. The potential produced by the transport of one ion species can also drive other cations or anions through the cell membrane, provided it is permeable to them. The K+diffusion potential leads to the efflux of Cl–, for example, which continues until ECl = Em. According to Equation 1.18, this means that the cytosolic Cl– concentration is reduced to 1/25 th of the extracellular concentration (passive distribution of Cl– between cytosol and extracellular fluid). In the above example, therewas a small electrochemical Cl– potentialdriving Cl– out of the cell (Em – ECl = – 2 mV). This means that the cytosolic Cl– concentration is higher than in passive Cl– distribution (ECl = Em). Therefore, Cl– ions must have been actively taken up by the cell, e.g., by a Na+- Cl– symport carrier: active distribution of Cl– . To achieve ion transport, membranes have a variable number of channels (pores) specific for different ion species (Na+, Ca2+, K+, Cl–, etc.). The conductance of the cell membrane is therefore determined by the type and number of ion channels that are momentarily open. Patch–clamp techniques permit the direct measurement of ionic currents through single ion channels. Patch–clamp studies have shown that the conductance of a membrane does not depend on the change of the pore diameter of its ion channels, but on their average frequency of opening. The ion permeability of a membrane is therefore related to the open-probability of the channels in question. Ion channels open in frequent bursts.
Several ten thousands of ions pass through the channel during each individual burst, which lasts for only a few milliseconds. During a patch–clamp recording, the opening (0.3–3 μm in diameter) of a glass electrode is placed over a cell membrane in such a way that the opening covers only a small part of the membrane (patch) containing only one or a small number of ion channels. The whole cell can either be left intact, or a membrane patch can excised for isolated study. In singlechannel recording, the membrane potential is kept at a preset value (voltage clamp). This permits the measurement of ionic current in a single channel. The measurements are plotted as current (I) over voltage (V). The slope of the I/V curve corresponds to the conductance of the channel for the respective ion species. The zero-current potential is defined as the voltage at which the I/V curve intercepts the x-axis of the curve (I = 0). The ion species producing current I can be deducedfrom the zero-current potential. In example B, the zero-current potential equals – 90mV. Under the conditions of this experiment, an electrochemical gradient exists only for Na+ and K+, but not for Cl– (!B). At these gradients, EK = – 90mVand ENa = + 90mV. As EK equals the zero-current potential, the channel is exclusively permeable to K+ and does not allow other ions like Na+ to pass. The channel type can also be determined by adding specific channel blockers to the system. Control of ion channels (!C). Channel open-probability is controlled by five main factors:
Membrane potential, especially in Na+, Ca2+ and K+ channels ierve and muscle fibers.
External ligands that bind with the channel. This includes acetylcholine on the postsynaptic membrane of nicotinic synapses (cation channels), glutamate (cation channels), and glycine or GABA (Cl– channels).
Intracellular messenger substances such as: — cAMP (e.g., in Ca2+ channels in myocardial cells and Cl– channels in epithelial cells); — cGMP (plays a role in muscarinergic effects of acetylcholine and in excitation of the retinal rods); — IP3 (opening of Ca2+ channels of intracellular Ca2+ stores); — Small G-proteins (Ca2+ channels of the cell membrane); — Tyrosine kinases (Cl– and K+ channels during apoptosis); — Ca2+ (affects K+ channels and degree of activation of rapid Na+ channels).
Role of Ca2+ in Cell Regulation The cytosolic Ca2+ concentration, [Ca2+]i, (ca. 0.1 to 0.01 μmol/L) is several decimal powers lower than the extracellular Ca2+ concentration [Ca2+]o (ca. 1.3 mmol/L). This is because Ca2+ is continuously pumped from the cytosol into intracellular Ca2+ stores such as the endoplasmic and sarcoplasmic reticulum (! p.
A rise in [Ca2+]i is a signal for many important cell functions, including myocyte contraction, exocytosis of neurotransmitters in presynaptic nerve endings, endocrine and exocrine hormone secretion, the excitation of certain sensory cells, the closure of gap junctions in various cells, the opening of other types of ion channels, and the migration of leukocytes and tumor cells as well as thrombocyte activation and sperm mobilization.Some of these activities are mediated by calmodulin. A calmodulin molecule can bind up to 4 Ca2+ ions when the [Ca2+]i rises. The Ca2+-calmodulin complexes activate a number of different enzymes, including calmodulin-dependent protein kinase II (CaMkinase II) and myosin light chain kinase (MLCK), which is involved in smooth muscle contraction. [Ca2+]i oscillation is characterized by multiple brief and regular [Ca2+]i increases (Ca2+ spikes) in response to certain stimuli or hormones (!B). The frequency, not amplitude, of[Ca2+]i oscillation is the quantitative signal for cell response. When low-frequency [Ca2+]i oscillation occurs, CaM-kinase II, for example, is activated and phosphorylates only its target proteins, but is quickly and completely deactivated. High-frequency [Ca2+]i oscillation results in an increasing degree of autophosphorylation and progressively delays thedeactivation of the enzyme. As a result, the activity of the enzyme decays more andmore slowly between [Ca2+]i signals, and each additional [Ca2+]i signal leads to a summation of enzyme activity. As with action potentials, this frequency-borne, digital all-or-none type of signal transmission provides a much clearer message than the [Ca2+]i amplitude, which is influenced by a number of factors. Ca2+ sensors. The extracellular Ca2+ concentration [Ca2+]o plays an important role in blood coagulation and bone formation as well as ierve and muscle excitation. [Ca2+]o is tightly controlled by hormones such as PTH, calcitriol and calcitonin, and represents the feedback signal in this control circuit. The involved Ca2+sensors are membrane proteins that detect high [Ca2+]o levels on the cell surface and dispatch IP3 and DAG (diacylglycerol) as intracellular second messengers with the aid of a Gq protein. IP3 triggers an increase in the [Ca2+]i of parafollicular C cells of the thyroid gland. This induces the exocytosis of calcitonin, a substance that reduces [Ca2+]o. In parathyroid cells, on the other hand, a high [Ca2+]o reduces the secretion of PTH, a hormone that increases the [Ca2+]o. This activity is mediated by DAG and PKC (protein kinase C) and, perhaps, by a (Gi protein-mediated) reduction in the cAMP concentration. Ca2+ sensors are also located on osteoclasts as well as on renal and intestinal epithelial cells.
The protein channels are believed to provide watery pathways through the interstices of the protein molecules. In fact, computerized three-dimensional reconstructions of some of these proteins have demonstrated actual curving tube-shaped channels from the extracellular to the intracellular ends. Therefore, substances can diffuse by simple diffusion directly through these channels from one side of the membrane to the other. The protein channels are distinguished by two important characteristics: (1) they are often selectively permeable to certain substances and (2) many of the channels can be opened or closed by gates.
Selective Permeability of Many Protein Channels. Many of the protein channels are highly selective for the transport of one or more specific ions or molecules. This results from the characteristics of the channel itself, such as its diameter, its shape, and the nature of the electrical charges along its inside surfaces. To give an example, one of the most important of the protein channels, the so-called sodium channels, calculates to be only 0.3 by 0.5 nanometer in size, but more important, the inner surfaces of these channels are strongly negatively charged. These strong negative charges pull small dehydrated sodium ions into these channels, actually pulling the sodium ions away from their hydrating water molecules. Once in the channel, the sodium ions then diffuse in either direction according to the usual laws of diffusion. Thus, the sodium channel is specifically selective for the passage of sodium ions. Conversely, another set of protein channels is selective for potassium transport. These channels calculate to be slightly smaller than the sodium channels, only 0.3 by 0.3 nanometer, but they are not negatively charged. Therefore, no strong attractive force is pulling ions into the channels, and the ions are not pulled away from the water molecules that hydrate them. The hydrated form of the potassium ion is considerably smaller than the hydrated form of sodium because the sodium ion attracts far more water molecules than does the potassium. Therefore, the smaller hydrated potassium ions can pass easily through this small channel, whereas sodium ions are mainly rejected, thus once again providing selective permeability for a specific ion.
Gating of Protein Channels. Gating of protein channels provides a means for controlling ion permeability of the channels. This is shown in both the upper and the lower panels of Figure 4-4 for selective gating of sodium and potassium ions. It is believed that the gates are actual gatelike extensions of the transport protein molecule, which can close over the opening of the channel or can be lifted away from the opening by a conformational change in the shape of the protein molecule itself. In the case of the sodium channels, this gate opens and closes at the end of the channel on the outside of the cell membrane, whereas for the potassium channels, it opens and closes at the intracellular end of the channel.
The opening and closing of gates are controlled in two principal ways:
1. Voltage gating. In this instance, the molecular conformation of the gate responds to the electrical potential across the cell membrane. For instance, there is a strong negative charge on the inside of the cell membrane, and this causes the outside sodium gates to remain tightly closed: conversely, when the inside of the membrane
loses its negative charge, these gates open suddenly and allow tremendous quantities of sodium to pass inward through the sodium pores. This is the basic cause of action potentials ierves that are responsible for nerve signals. These gates are on the intracellular ends of the potassium channels, and they open when the inside of the cell membrane becomes positively charged. The opening of these gates is partly responsible for terminating the action potential.
2. Chemical gating (“ligand” gating). Some protein channel gates are opened by the binding of a chemical substance (a “ligand”) with the protein; this causes a conformational change in the protein molecule that opens or closes the gate. This is called chemical gating or ligand gating. One of the most important instances of chemical gating is the effect of acetylcholine on the so-called acetylcholine channel. This opens the gate of this channel, providing a negatively charged pore about 0.65 nanometer in diameter that allows all uncharged molecules and positive ions smaller than this diameter to pass through. This gate is exceedingly important for the transmission of nerve signals from one nerve cell to another and from nerve cells to muscle cells to cause muscle contraction.
Open-State, Closed-State of Gated Channels. The gate of the channel snaps open and then snaps closed, each open state lasting only a fraction of a millisecond up to several milliseconds. This demonstrates the rapidity with which conformational changes can occur during the opening and closing of the protein molecular gates. At one voltage potential, the channel may remain closed all the time or almost all the time, whereas at another voltage level it may remain open either all or most of the time. At in-between voltages, as shown in the figure, the gates tend to snap open and closed intermittently, giving an average current flow somewhere between the minimum and the maximum.
Patch-Clamp Method for Recording Ion Current Flow Through Single Channels. One might wonder how it is technically possible to record ion current flow through single protein channels. This has been achieved by using the “‘patch-clamp” method. Very simply, a micropipette, having a tip diameter of only 1 or 2 micrometers, is abutted against the outside of a cell membrane. Then suction is applied inside the pipette to pull the membrane slightly into the tip of the pipette. This creates a seal where the edges of the pipette touch the cell membrane. The result is a minute membrane “patch” at the tip of the pipette through which electrical current flow can be recorded. Alternatively, the small cell membrane patch at the end of the pipette can be torn away from the cell. The pipette with its sealed patch is then inserted into a free solution. This allows the concentra- tions of the ions both inside the micropipette and in the out- side solution to be altered as desired. Also, the voltage be- tween the two sides of the membrane can be set at will–that is, “clamped” to a given voltage. It has been possible to make such patches small enough that one often finds only a single channel protein in the mem- brane patch that is being studied. By varying the concentra- tions of different ions as well as the voltage across the mem- brane, one can determine the transport characteristics of the single channel and also its gating properties.
Basic physics of membrane potentials. The potassium concentration is great inside the membrane, whereas that outside the membrane is very low. Let us assume that the membrane in this instance is permeable to the potassium ions but not to any other ions. Because of the large potassium concentration gradient from the inside toward the outside, there is a strong tendency for extra numbers of potassium ions to diffuse outward. As they do so, they carry positive charges to the outside, thus creating a state of electropositivity outside the membrane but electronegativity on the inside because of negative anions that remain behind and do not diffuse outward along with the potassium. Within a millisecond or so, the potential becomes great enough to block further net potassium diffusion to the exterior despite the high potassium ion concentration gradient. In the normal large mammaliaerve fiber, the potential difference required is about 94 millivolts, with negativity inside the fiber membrane. These ions are also positively charged, and this time the membrane is highly permeable to the sodium ions but impermeable to all other ions. Diffusion of the positively charged sodium ions to the inside creates a membrane potential now of opposite polarity,with negativity outside and positivity inside. Again, the membrane potential rises high enough within milliseconds to block further net diffusion of sodium ions to the inside; however, this time, in the large mammaliaerve fiber, the potential is about 61 millivolts positivity inside the fiber.
Thus, we see that a concentration difference of ions across a selectively permeable membrane can, under appropriate conditions, create a membrane potential. In later sections of this chapter, we will see that many of the rapid changes in membrane potentials observed during nerve and muscle impulse transmission result from the occurrence of such rapidly changing diffusion potentials.
Relation of the Diffusion Potential to the Concentration Difference–the Nernst Equation. The potential level across the membrane that exactly opposes net diffusion of a particular ion through the membrane is called the Nernst potential for that ion, a term. The magnitude of this Nernst potential is determined by the ratio of the concentrations of that specific ion on the two sides of the membrane. The greater this ratio, the greater the tendency for the ion to diffuge in one direction, and therefore the greater the Nernst potential required to prevent the diffusion. The following equation, called the Nernst equation, can be used to calculate the Nernst potential for any univalent ion at normal body temperature of
Thus, when the concentration of positive potassium ions on the inside is 10 times that on the outside, the log of 10 is 1, so that the Nernst potential c’,dculates to be -61 millivolts inside the membrane.
Measuring the Membrane Potential. The method for measuring the membrane potential is simple in theory but often difficult in practice because of the small sizes of most of the fibers. Figure 5-2 shows a small filled pipette containing an electrolyte solution that is impaled through the cell membrane to the interior of the fiber. Then another electrode, called the “indifferent electrode,” is placed in the extracellular fluid, and the potential difference between the inside and outside of the fiber is measured using an appropriate voltmeter. This voltmeter is a highly sophisticated electronic apparatus that is capable of measuring very small voltages despite extremely high resistance to electrical flow through the tip of the micropipette, which has a lumen diameter usually less than 1 micrometer and a resistance often as great as I billion ohms. For recording rapid changes in the membrane potential during transmission of nerve impulses, the microelectrode is connected to an oscilloscope, as explained later in the chapter.
The electrical potential that is measured at each point in or near the nerve fiber membrane, beginning at the left side of the figure and passing to the ,right. As long as the electrode is outside the nerve membrane, the potential that is recorded is zero, which is the potential of the extracellular fluid. Then, as the recording electrode passes through the voltage charge area at the cell membrane (called the electrical dipole layer), the potential decreases abruptly to -90 millivolts. Moving across the center of the fiber, the potential remains at a steady -90-millivolt level but reverses back to zero the instant it passes through the membrane on the opposite side of the cell. To create a negative potential inside the membrane, only enough positive ions must be transported outward to develop the electrical dipole layer at the membrane itself. All the remaining ions inside the nerve fiber still can be both positive and negative ions. Therefore, an incredibly small number of ions need to be transferred through the membrane to establish the normal potential of -90 millivolts inside the nerve fiber;. Also, an equally small number of positive ions moving from outside to the inside of the fiber can reverse the potential from -90 millivolts to as much as +35 millivolts within as little as јo,o00 of a second. Rapid shifting of ions in this manner causes the nerve signals that we discuss in subsequent sections of this chapter.
Neuron Structure and Function. An excitable cell reacts to stimuli by altering its membrane characteristics. There are two types of excitable cells: nerve cells, which transmit and modify impulses within the nervous system, and muscle cells, which contract either in response to nerve stimuli or autonomously. The humaervous system consists of more than 1010 nerve cells or neurons. The neuron is the structural and functional unit of the nervous system. A typical neuron (motor neuron) consists of the soma or cell body and two types of processes: the axon and dendrites. Apart from the usual intracellular organelles (!p. 8ff.), such as a nucleus and mitochondria, the neuron contains neurofibrils and neurotubules. The neuron receives afferent signals (excitatory and inhibitory) from a few to sometimes several thousands of other neurons via its dendrites (usually arborescent) and sums the signals along the cell membrane of the soma (summation). The axon arises from the axon hillock of the soma and is responsible for the transmission of efferent neural signals to nearby or distant effectors (muscle and glandular cells) and adjacent neurons. Axons often have branches (collaterals) that further divide and terminate in swellings called synaptic knobs or terminal buttons. If the summed value of potentials at the axon hillock exceeds a certain threshold, an action potential is generated and sent down the axon, where it reaches the next synapse via the terminal buttons described below.
Vesicles containing materials such as proteins, lipids, sugars, and transmitter substances are conveyed from the Golgi complex of the soma to the terminal buttons and the tips of the dendrites by rapid axonal transport (40 cm/day). This type of anterograde transport along the neurotubules is promoted by kinesin, a myosin-like protein, and the energy required for it is supplied by ATP. Endogenous and exogenous substances such as nerve growth factor (NGF), herpes virus, poliomyelitis virus, and tetanus toxin are conveyed by retrograde transport from the peripheral regions to the soma at a rate of ca. 25 cm/day. Slow axon transport (1 mm/day) plays a role in the regeneration of severed neurites. Along the axon, the plasma membrane of the soma continues as the axolemma. The axolemma is surrounded by oligodendrocytes in the central nervous system (CNS), and by Schwann cells in the peripheral nervous system. A nerve fiber consists of an axon plus its sheath. In some neurons, Schwann cells form multiple concentric double phospholipid layers around an axon,comprising themyelin sheath that insulates the axon from ion currents. The sheath is interrupted every 1.5mm or so at the nodesof Ranvier. The conduction velocity of myelinated nerve fibers is much higher thanthat of unmyelinated nerve fibers and increases with the diameter of the nerve fiber. A synapse is the site where the axon of a neuron communicates with effectors or other neurons. With very few exceptions, synaptic transmissions in mammals aremediated by chemicals, not by electrical signals. In response to an electrical signal in the axon, vesicles on the presynaptic membrane release transmitter substances (neurotransmitters) by exocytosis. The transmitter diffuses across the synaptic cleft (10–40 nm) to the postsynaptic membrane, where it binds to receptors effecting new electrical changes. Depending on the type of neurotransmitter and postsynaptic receptor involved, the transmitter will either have an excitatory effect (e.g., acetylcholine in skeletal muscle) or inhibitory effect (e.g., glycine in the CNS) on the postsynaptic membrane. Since the postsynaptic membrane normally does not release neurotransmitters (with only few exceptions), nerve impulses can pass the synapse in one direction only. The synapse therefore acts like a valve that ensures the orderly transmission of signals. Synapses are also the sites at which neuronal signal transmissions can be modified by other (excitatory or inhibitory) neurons.
Resting Membrane Potential. An electrical potential difference, or membrane potential (Em), can be recorded across the plasma membrane of living cells. The potential of unstimulated muscle and nerve cells, or resting potential, amounts to – 50 to – 100mV (cell interior is negative). A resting potential is caused by a slightly unbalanced distribution of ions between the intracellular fluid (ICF) and extracellular fluid (ECF).The following factors are involved in establishing the membrane potential. Maintenance of an unequal distribution of ions: The Na+-K+-ATPase continuously “pumps” Na+ out of the cell and K+ into it. As a result, the intracellular K+ concentration is around 35 times higher and the intracellular Na+ concentration is roughly 20 times lower than the extracellular concentration. As in any active transport, this process requires energy, which is supplied by ATP. Lack of energy or inhibition of the Na+-K+-ATPase results in flattening of the ion gradient andbreakdown of the membrane potential. Because anionic proteins and phosphates present in high concentrations in the cytosol are virtually unable to leave the cell, purely passive mechanisms (Gibbs–Donnan distribution) could, to a slight extent, contribute to the unequal distribution of diffusable ions.
However, this has practically no effect on the development of resting potentials. Low resting Na+ and Ca2+ conductance, gNa, gCa: The membrane of a resting cell is only very slightly permeable to Na+ and Ca2+, and the resting gNa comprises only a small percentage of the total conductance. Hence, the Na+ concentration difference cannot be eliminated by immediate passive diffusion of Na+ back into the cell. ! High K+conductance, gK: It is relatively easy for K+ ions to diffuse across the cell membrane (gK 90% of total conductance). Because of the steep concentration gradient, K+ ions diffuse from the ICF to the ECF. Because of their positive charge, the diffusion of even small amounts of K+ ions leads to an electrical potential (diffusion potential) across the membrane. This (inside negative) diffusion potential drives K+ back into the cell and rises until large enough to almost completely compensate for the K+ concentration gradient driving the K+-ions out of the cell (!A4). As a result, the membrane potential, Em, is approximately equal to the K+ equilibrium potential EK. Cl– distribution: Since the cell membrane is also conductive to Cl– (gCl greater in muscle cells than ierve cells), the membrane potential (electrical driving “force”) expels Cl– ions from the cell until the Cl– concentration gradient (chemical driving “force”) drives them back into the cell at the same rate. The intracellular Cl– concentration, [Cl–]i, then continues to rise until the Cl– equilibrium potential equals Em. [Cl–]i can be calculated using the Nernst equation.
Such a “passive” distribution of Cl– between the intra- and extracellular spaces exists only as long as there is no active Cl– uptake into thecell. Why is Em less negative than EK? Although the conductances of Na+ and Ca2+ are very low in resting cells, a few Na+ and Ca2+ ions constantly enter the cell. This occurs because the equilibrium potential for both typesof ions extends far into the positive range, resulting in a high outside-to-inside electrical and chemical driving “force” for these ions. This cation influx depolarizes the cell, thereby driving K+ ions out of the cell (1 K+ for each positive charge that enters). If Na+-K+-ATPase did not restore these gradients continuously (Ca2+ indirectly via the 3Na+/Ca2+ exchanger), the intracellular Na+ and Ca2+ concentrations would increase continuously, whereas [K+]i would decrease, and EK and Em would become less negative. All living cells have a (resting) membrane potential, but only excitable cells such as nerve and muscle cells are able to greatly change the ion conductance of their membrane in response to a stimulus, as in an action potential.
Skeletal muscle constitutes 40% of muscle mass. Derangement of muscle function can have profound systemic effects.
Physiological skeletal muscle contraction requires generation and spread of a membrane action potential, transduction of the electrical energy into an intracellular chemical signal that, in turn, triggers myofilament interaction.
Intracellular cytoskeletal proteins, cell membrane structures and the associated glycoprotein extracellular matrix are important for maintenance of cell architecture and force transmission.
Smooth and graded changes in force of contraction are achieved through summation of responses to successive stimuli and recruitment of motor units.
Sustained muscle contraction requires de novo synthesis of ATP, which is principally aerobic or anaerobic depending on muscle fibre type.
The skeletal muscles are the effector organs of the locomotor system. They are under voluntary control, although much of their activity is subconsciously regulated. Skeletal muscle and cardiac muscle are both described as striated muscle because of their striped microscopic appearance. This appearance results from the ordered and regular arrangement of the sub-cellular contractile elements. Unlike cardiac muscle, skeletal muscle has no intrinsic spontaneous activity because it lacks the ion channels responsible for spontaneous membrane depolarization. Therefore, the stimulus for physiological skeletal muscle activity is always derived from a nerve impulse. The great majority of skeletal muscle fibres receive their nerve inputs at single central swellings of the fibres known as motor endplates. A few muscles, notably some of the facial muscles, are more diffusely innervated along the length of their fibres; such multifocal innervation may explain why these muscles respond with a more pronounced initial increase in tension after administration of succinylcholine.
However, irrespective of the type of innervation, the charge density arriving at the motor nerve terminal is insufficient to directly activate the much larger muscle fibres. The electrical neuronal impulse is amplified at the neuromuscular junction, the mechanism of which is beyond the remit of this review. The resulting generation of the endplate potential is the first step in muscle contraction.
Electrical events in muscle contraction
Muscle fibres are excitable cells. The cell membrane (sarcolemma) contains the ion channels and pumps necessary to maintain a very negative resting membrane potential and the voltage gated ion channels necessary for generation of an action potential. As with all excitable cells, the membrane potential of muscle cells at any time is a function of the net electrochemical gradients of ions that the membrane is permeable to at that time. This is largely determined by the instantaneous permeability of the sarcolemma to ions that are unevenly distributed across it, which in turn is dependent on which ion-selective membrane channels are open. The potential difference between intra-cellular and extra-cellular compartments when concentration and electrochemical gradients of a permeable ion are balanced is known as the equilibrium potential, which can be
calculated for an ion, X, using the Nernst equation: where
EX = equilibrium or Nernst potential for ion X;
[X]out = extracellular concentration of X;
[X]in = intracellular concentration of X;
R = universal gas constant;
T = absolute temperature;
z = valency of ion; and
F = Faraday constant.
Determinants of force of contraction
For a single muscle fibre the force of contraction is proportional to the number of actin–myosin bonds formed. This will be optimal when the initial sarcomere length is such that all myosin heads are overlapped by thin filaments. If the sarcomere is stretched too far, the central myosin heads will be redundant. If the sarcomere is too short, the distance between the actin and myosin binding sites increases and their alignment may also be distorted, both of which will reduce the efficiency of contraction.
However, unlike in cardiac muscle, skeletal muscle fibres are maintained near their optimal length in their working range; therefore, the Frank-Starling length–tension relationship is not a major factor in skeletal muscle physiology. Consequently, the force generated by a single skeletal muscle fibre will be a function of both the cross-sectional area and the length of the fibre. The same relationship applies to the muscle as a whole. Of course, it would not be very useful if each muscle could contract only at its maximum force. Graded muscular contraction is achieved through two main mechanisms: summation and recruitment.
Summation
Summation of skeletal muscle fibre contractions is possible because the absolute refractory period of the sarcolemma is considerably less than the duration of raised cytoplasmic Ca2+ concentration and subsequent tension generation. If a second stimulus is applied to the muscle before it has fully relaxed from the first, the response to the second stimulus will add to the residual response of the first stimulus. This summation reaches a peak when the second stimulus occurs ∼20 ms after the first, corresponding to a stimulus frequency of 50 Hz. Between stimulus frequencies of 20–50 Hz the summed responses form a smooth ramped increase in tension, or tetanic response. The usual firing frequency of vertebrate motor neurons is within the tetanic range.
Recruitment
Single motor neurons innervate multiple muscle fibres. A motor neuron and the muscle fibres it innervates are collectively called a motor unit. The number of muscle fibres within a motor unit varies within and between muscles. The smallest motor units, containing as few as 3–10 muscle fibres, are found in muscles used for fine intricate movements. Much larger motor units, containing up to several hundred muscle fibres, are predominant in muscles used for gross vigorous movements.
When a muscle is required to produce a progressive increase in tension, initially, when the load applied to the muscle is small, the smallest motor units within the muscle are used. As the load increases larger and larger motor units are recruited, so that when the load is the maximum attainable by that muscle, all its motor units will be operating.
Energy for contraction
We have seen that ATP is required for significant tension to develop and it is also crucial for muscle relaxation. However, ATP is a relatively unstable compound and the instantaneously available ATP is able to maintain contraction for <1 s. Muscle has a specialized means of storing high-energy phosphate in the form of creatine phosphate. ATP is derived from creatine phosphate by the action of creatine kinase but the so-derived ATP is sufficient to maintain contraction for only a further 5–8 s. More prolonged contractile activity requires synthesis of ATP by intermediary metabolism. Aerobic metabolism of 1 mole of glucose produces 38 moles of ATP but even maximal oxygen delivery is insufficient to meet the demands of vigorous muscle activity. Anaerobic metabolism is less efficient in that 1 mole of glucose produces only 2 moles of ATP, but the ATP produced is more readily available. However, this is at the expense of a build-up of lactate, which is an important factor in the development of muscle fatigue.
Muscle fibre-types
The diameter of muscle fibres varies from 10 to 100 μm. Most human muscles contain a mixture of fibres within this range. The thinner fibres are type I fibres and they are adapted for sustained activity requiring submaximal tension generation. The thickest fibres (type IIb) are adapted for short bursts of near-maximal activity. Muscles containing a predominance of type I fibres appear a deeper red colour than those with few type I fibres because type I fibres have a high myoglobin content. Myoglobin is pigmented because of a haem moiety that is responsible for its oxygen-binding capability. Myoglobin provides a storage capacity for oxygen within muscle cells; its affinity for oxygen is greater than that of haemoglobin, which aids oxygen delivery to muscle, but is such that oxygen is released for aerobic metabolism when demand is increased. Type IIa fibres are intermediate in size and myoglobin content.