Chapter 1 Basic principles and cell physiology

The physiology of the body is often described in terms of the homeostatic mechanisms which control the cellular environment. Cell function also relies on an adequate energy supply and the appropriate integration of the activities of the different cells within the body through efficient signalling mechanisms, both electrical and chemical. The cell membrane is very important in many of these functions since it forms a barrier between the intracellular and extracellular environments across which important molecules must be transported. The properties of the membranes of excitable cells also account for the generation of action potentials, and many of the receptors which recognize specific chemical messengers are membrane bound.

1.1 Cells, systems and homeostasis

Physiology is the study of normal biological function. Although the ultimate goal is to understand the intact human, function is often considered in terms of individual physiological systems, each of which consists of different organs and structural elements. The different components of any system may be spread widely throughout the body but they serve a common functional purpose. Therefore, we talk of the cardiovascular system, consisting of the heart, arteries, capillaries, veins, lymphatics and related control mechanisms, all of which are involved in the circulation of the blood. The fundamental biological unit is the cell, however, and each organ’s function reflects the integrated activity of the various specialized cells within it.

Cell structure

Although cells from different organs show considerable differences in shape (morphology) and function, all cells share some general characteristics. Each is bounded by a cell membrane (or plasma membrane) which separates the aqueous solution inside the cell (the cytoplasm or intracellular fluid) from the aqueous solution outside (the extracellular fluid). These two fluids have very different ionic compositions (see Section 5.1) and these differences are more easily maintained because ions cannot readily cross the double layer (bilayer) of phospholipids in the plasma membrane (Fig. 1). These are oriented with their hydrophilic heads adjacent to the aqueous solutions on either side, while their hydrophobic tails form a fatty core. Membrane proteins move about within the two-dimensional confines of the lipid layer (the fluid mosaic model of the membrane).

Fig. 1 The plasma membrane. Membrane proteins may be exposed on either side or may cross the whole thickness of the lipid bilayer.

Membranes also delimit a number of subcellular units known as organelles. These include:

• the nucleus, which contains the genetic code for protein synthesis in the form of deoxyribonucleic acid (DNA). Since proteins are key structural and functional molecules in the cell, changes in gene expression regulate how a cell looks and behaves.

• mitochondria, which are surrounded by both an outer membrane and a highly folded inner membrane. Mitochondria are responsible for oxidative breakdown of food molecules, providing the cell with usable energy in the form of ATP.

• the endoplasmic reticulum (or sarcoplasmic reticulum in muscle cells), which is an interconnecting series of membranous channels and vesicles. In some regions this has a large number of ribosomes attached to the surface, thus leading to the distinction between rough and smooth endoplasmic reticulum. Ribosomes play a key role in protein synthesis.

• the Golgi apparatus, a system of stacked vesicles found close to the nucleus. This is involved in the formation of secretory vesicles and lysosomes.

The body contains many different aqueous solutions, which may be classified into a series of fluid compartments depending on their location (Fig. 2). The main subdivision is between intracellular and extracellular fluids. The extracellular compartment represents all fluid not inside cells, including the plasma component of blood, aqueous humour in the eye, synovial fluid in joints and cerebrospinal fluid within the central nervous system. The largest single extracellular subcompartment, however, consists of the interstitial fluid, which lies in the connective tissue matrix surrounding most body cells. It is normally this fluid which is in direct contact with the cell membrane and controlling interstitial conditions is vital for normal cell function. Much of this control is achieved by the continuous circulation of blood through the cardiovascular system. The rate of blood flow to any region and the plasma concentrations of different solutes greatly influence the cellular environment since, with the exception of plasma proteins, water and solutes move freely between plasma and the interstitium.

Fig. 2 Fluid compartments within the body showing the relative volumes occupied by intracellular fluid, extracellular fluid and plasma (part of the extracellular space).

Homeostasis

Normal cell function relies on appropriate environmental conditions since the temperature, pH, ionic concentrations, O₂ and CO₂ levels in the extracellular fluid all influence biochemical activity inside the cell. The mechanisms which control the body’s internal environment are called homeostatic mechanisms. These keep conditions inside the body relatively constant despite considerable changes in the external environment.

The following general elements are present in all homeostatic control systems (Fig. 3).

• Detectors, or receptors, sensitive to the variable to be controlled.

• A comparator, or integrating centre, which receives the afferent signal from the detectors and compares this measured value of the variable with a preferred value, or set point, for the system. If the actual value differs from the set point the integrator generates an efferent (outgoing) signal.

• Effector systems which can change the measured variable are activated by the signals from the integrator. Their effect is to bring the variable back towards the set point.

Fig. 3 Elements of a homeostatic control system. The controlled variable may oscillate, or hunt, around a mean value (the set point).

In such a system, any deviation of the variable away from its normal (set) value stimulates responses which reduce that deviation. This is referred to as negative feedback control and is an important element of homeostasis. The overall effect is to maintain a constant environment but often a phenomenon known as hunting is observed in which the controlled variable oscillates around a fixed mean value rather than remaining exactly at the set point (Fig. 3).

Biological systems may also demonstrate positive feedback in which deviations from the steady state are actually amplified, rather than diminished, by a feedback loop. Such mechanisms, however, have no part to play in homeostasis.

Homeostatic regulation of body temperature

Regulation of body temperature provides a good example of homeostasis. The body core is normally held close to 37°C, even though the environmental temperature (which affects the rate of heat loss from the body) and rate of metabolic heat production may vary widely.

Temperature detectors (thermoreceptors) monitor both the core temperature (the regulated variable) and the peripheral temperature (a separate but relevant variable). The afferent inputs go to an integrating centre (the thermoregulatory centre) in the hypothalamus of the brain, which compares core temperature with the set point of 37°C. If these differ, outgoing nerve signals activate a number of effector systems which alter the rates of heat production and heat loss.

In cold conditions:

• reflex increases in muscle tone and shivering liberate metabolic energy as heat

• in infants, there may also be increased heat production in brown fat (Section 9.6)

• heat loss is reduced by sympathetic constriction of peripheral blood vessels, which decreases skin blood flow; erection of skin hair (piloerection) also occurs but has limited significance in humans.

In hot conditions the rate of heat loss can be greatly accelerated by:

• increased sweating stimulated by sympathetic nerves which in this specific case release the neurotransmitter acetylcholine

• dilatation of cutaneous blood vessels caused by reduced activity in vasoconstrictor sympathetic nerves releasing the more typical sympathetic neurotransmitter noradrenaline (norepinephrine). There may also be active dilatation of arterioles (Section 3.7).

Behavioural aspects are important too, e.g., we dress up warmly, exercise and eat hot food when we are cold, while preferring light clothes and cool drinks in hot weather.

Box 1 Clinical note: Pyrexia

Pyrexia or fever is a temporary elevation of the core temperature commonly caused by infection. It is not due to any breakdown of the detector or effector systems responsible for temperature control but results from a change in the set point. Toxins released from bacteria and immune cells cause the hypothalamus to respond to a core temperature of 37°C as if it were abnormally low and so it increases heat production, e.g., through shivering (rigors). This raises body temperature.

1 2 Energy sources in the cell

Learning objectives

At the end of this section you should be able to:

• outline the main steps in cellular energy exchange.

Body cells require energy to carry out mechanical work (e.g., in muscle cells), to transport ions or molecules against concentration gradients (e.g., ion pumps) and for the synthesis of complex molecules. These active processes are ultimately fuelled by the energy released through the breakdown of carbohydrate, fat and protein from our diet. The initial series of reactions in the breakdown of glucose is referred to as glycolysis and does not require O₂ (anaerobic metabolism). This leads to the formation of lactic acid and produces small amounts of adenosine triphosphate (ATP), the main source of usable energy within the cell. In the presence of O₂ (aerobic conditions), however, intramitochondrial enzymes catalyse the complete catabolism of the products of glycolysis, releasing much greater amounts of ATP and generating CO₂ and water as byproducts (Section 4.7). Whereas glycolysis leads to a net production of two molecules of ATP per glucose molecule, oxidative metabolism can produce an additional 34–36 ATP molecules. Subsequent hydrolysis of the high-energy phosphate bonds in ATP by ATPase enzymes makes energy available for the active processes of the cell.

1.3 Transport across cell membranes

There is a constant traffic across cell membranes, supplying O₂ and substrate molecules for intracellular metabolism and removing CO₂, waste substances and active products. A variety of transport mechanisms are involved.

Diffusion

Diffusion can occur whenever a substance is present at a higher concentration on one side of the cell membrane than the other. It results in net movement from high to low concentration, i.e., a net flux of the ion or molecule. No energy source is required so this is referred to as a passive transport mechanism. Diffusion of materials into and out of cells is affected by the following factors.

Solute concentration gradients, i.e., not the absolute concentrations but the difference in concentration across the cell membrane.

Membrane permeability to the solute. The plasma membrane is selectively permeable to fatty and small nonpolar molecules which dissolve in the membrane lipid. Thus, fatty acids, steroid hormones, O₂ and CO₂ all diffuse readily into cells. The permeability to water-soluble (lipid-insoluble) ions and large polar molecules such as proteins, however, is generally low. Certain ions can diffuse across the cell membrane much more readily (i.e., they are more permeant) than their lipid solubility would predict. This is because of membrane proteins which bridge the lipid barrier and provide an easier route for ion diffusion. These may take the form of carrier molecules, which bind to the ion and then move it across the membrane by changing conformation, or they may provide fluid-filled channels through which the ions can pass (Section 1.4). The roles of both these types of molecule are considered in more detail later in the chapter. Specific carriers and channels are selective for different kinds of ion and so membrane permeability to a given ion may differ widely from cell to cell, depending on which proteins are present.

Transmembrane voltage gradients affect the movement of ions. If the inside of the membrane is negative with respect to the outside, cations (positively charged) will be electrostatically attracted into the cell and anions (negatively charged) will be repelled outwards. The net transmembrane flux of an ion is proportional to the combined effect of the electrical and concentration gradients acting on it, i.e., on the electrochemical gradient for that ion.

Molecular weight of the diffusing substance. Small molecules diffuse more rapidly.

Diffusion distance. Diffusion is too slow to allow effective exchange over distances of more than about 100 μm.

Membrane surface area. For a given set of conditions rate of diffusion is proportional to the surface area of membrane.

Osmosis

Osmosis depends on the passive diffusion of water across a membrane from a region of low solute concentration (effectively high water concentration) to a region of high solute concentration (low water concentration). Osmosis requires:

• a solute concentration gradient across a membrane

• a semipermeable membrane, i.e., permeable to the solvent (water) but not the solute. If the membrane is highly permeable to the solute, conditions on either side of the membrane will rapidly equilibrate by solute diffusion, thus removing the osmotic driving force.

Cell membranes are permeable to water mole-cules (because of their small size), so any solute which cannot cross the membrane (an impermeant solute) can generate an osmotic gradient. Normally cells exist in osmotic equilibrium, the osmotically active particles inside the cell being balanced by those in the extracellular fluid. Any disturbance of this balance will lead to a net movement of water across the cell membrane and a change in cell volume.

The osmotic properties of a solution can be described in several ways.

Osmolality is defined as the total number of dissolved particles per kg of solvent (H₂O), and has units of mosmol kg⁻¹. Osmolality will be used throughout this text since this determines osmotic effects and is usually reported in biochemistry tests. Since H₂O has a density of 1 kg L⁻¹, however, osmolality for dilute solutions is very similar to osmolarity, which refers to the number of particles per litre of solution (mosmol L⁻¹), and physiology and medical texts often use the terms osmolarity and osmolality interchangeably. Substances which dissociate in solution increase the number of dissolved particles, and this raises the osmolality above the molar concentration of solute. For example, 1 mmol L⁻¹ of glucose has an osmolality of 1 mosmol kg⁻¹ but a 1 mmol L⁻¹ NaCl solution has an osmolality of 2 mosmol kg⁻¹ since each NaCl molecule dissociates to produce two ions.

Tonicity is a biological term relating to the actual effect of a solution on living cells, specifically erythrocytes. A solution may be:

• Isotonic, i.e., in osmotic equilibrium with the osmotically active solutes in intracellular fluid under normal conditions. Plasma and interstitial fluid are, by definition, isotonic in healthy individuals, and intravenous fluid supplements should generally be isotonic to avoid red cell damage.

• Hypertonic, i.e., contains a higher concentration of osmotically active particles than the intracellular fluid, leading to osmotic water loss and cell shrinking (crenation). Plasma can become hypertonic in certain disease states due to an increased concentration of solutes, e.g., increased glucose in diabetes mellitus (see Section 8.7).

• Hypotonic, i.e., contains a lower concentration of osmotically active particles than the intracellular fluid, leading to osmotic cell swelling and possibly cell lysis. Body fluids become hypotonic if their relative water content is increased. This dilution may occur because of electrolyte (usually Na⁺) loss or water retention.

Tonicity depends both on the osmolality of a solution and the ease with which the solute in question can pass through the cell membrane. Readily diffusible substances have no osmotic effect on a cell even at high osmolality. Thus a 300 mosmol kg⁻¹ solution of NaCl (an effectively impermeant solute) is isotonic while a 300 mosmol kg⁻¹ solution of urea (which crosses cell membranes readily) is extremely hypotonic, leading to water absorption and almost immediate cell lysis caused by the unbalanced osmotic effect of trapped intracellular solutes (mainly K⁺ and inorganic anions).

Osmotic pressure is the hydrostatic pressure that would be necessary to exactly oppose the osmotic effect of a solution and prevent any net water movement. It is usually expressed in mmHg or kPa (kPa = 1000 N m⁻² = mmHg × 0.133). The osmotic pressure exerted on the cell membrane by isotonic fluids is over 770 kPa, i.e., over 7.5 × atmospheric pressure. Normally this is exactly balanced by the osmotic pressure resulting from impermeant intracellular solutes, i.e., the osmotic pressure gradient across the cell membrane is zero.

Carrier-mediated transport

Carrier proteins in the cell membrane bind to a specific substrate and then undergo some conformational change. As a result, the substrate is transported across the membrane and released on the other side. The rate of transport increases as the substrate concentration increases, but the maximum rate of transport (V_max) is dependent on the density of carriers in a given cell, since all transport sites will be occupied above a certain substrate concentration (Fig. 4). This is referred to as saturation. Substrate specificity and saturation are two hallmarks of carrier-mediated transport, whether it is passive (facilitated diffusion) or active.

Fig. 4 The rate of transport as a function of substrate concentration for carrier-mediated transport.

Facilitated diffusion

The energy driving facilitated diffusion is the substrate concentration gradient, so this is a passive process, i.e., no additional energy input is required. Movement across the membrane is, nevertheless, dependent on the availability of transport or carrier proteins (Fig. 5). Cellular absorption of glucose from the extracellular fluid is one example.

Fig. 5 Carrier-mediated transport mechanisms. (A) In facilitated diffusion the substrate (S) moves down a concentration gradient. (B) Primary active transport links ATP breakdown to the transport of a substrate against a concentration gradient. (C) Secondary active transport uses passive movement of one substance to drive a substrate against a concentration gradient.

Active transport

In active transport systems carrier molecules transport molecules or ions against concentration or electrical gradients. Such carriers must use energy from some other source to do the necessary work. The energy can be provided in two ways.

Primary active transport uses the energy released by hydrolysis of ATP, e.g., to transport Na⁺ out of the cell and K⁺ into the cell against their electrochemical gradients (the Na⁺/K⁺ ATPase, or Na⁺/K⁺ pump: Fig. 5).

Secondary active transport uses the energy released during the passive movement of one substance down its electrochemical gradient to transport another substance against a concentration gradient (Fig. 5). For example, sodium cotransport systems couple Na⁺ diffusion into intestinal cells with the absorption of glucose from the gut lumen (Section 7.4). Absorption can continue even if the concentration of glucose is higher inside the cell than outside. Although the cotransport step itself does not require an external energy source, such systems ultimately depend on active pumping of Na⁺ back into the extracellular space by Na⁺/K⁺ ATPase to maintain the normal Na⁺ diffusion gradient into the cell.

Vesicular transport

Vesicular transport depends on the transport of substances within membrane-bound vesicles. Limitations on transport resulting from large molecular size and low membrane permeability can be circumvented since the vesicle contents never physically cross the membrane. Endocytosis involves the invagination of a portion of plasma membrane into the cell, which forms a vesicle containing extracellular fluid and near membrane molecules. In exocytosis a vesicle from within the cell fuses with the membrane, releasing its contents to the extracellular fluid (Fig. 6). Exocytosis is particularly important in the release of glandular secretions and chemical transmitters.

Fig. 6 Fluid and large membrane-impermeant molecules can be moved in and out of cells using vesicular transport.

1.4 Electrical signals and excitable cells

Electrical signalling within the nerves and muscles of the body is a vital aspect of body function. These cells are said to be excitable because they are capable of generating self-propagating electrical signals known as action potentials.

Resting membrane potential

Electrical recordings from nerves show that there is a potential difference of about 70 mV across the cell membrane with the inside negative with respect to the outside. We say that the resting membrane potential is −70 mV. This can be explained by the fact that the cell membrane separates two solutions with different ionic concentrations and is not equally permeable to all the ions involved. As a result, a diffusion potential is generated across the membrane.

Diffusion potentials and equilibrium potentials

Suppose we start with zero electrical potential across the cell membrane (Fig. 7). The concentration of K⁺ is higher inside the cell than outside and the membrane is permeable to K⁺, so K⁺ diffuses outwards. Permeability is selective, however, and the large intracellular anions cannot follow K⁺. Consequently, an imbalance of charge builds up across the membrane producing a potential difference, with the inside negative with respect to the outside. This is a diffusion potential. The voltage gradient opposes further diffusion of the positive K⁺ ions, making it more and more difficult for them to leave the cell. The diffusion potential will increase until an equilibrium state is achieved in which the concentration gradient is exactly balanced by the opposing voltage gradient. The potential difference under these conditions is known as the equilibrium potential for K⁺ (E_K). This depends on the ratio of [K⁺] on either side of the membrane and can be calculated using the Nernst equation:

Fig. 7 A diffusion potential results from movement of K⁺ down its concentration gradient across a membrane that is impermeable to anions. At E_K, there is no net movement of K⁺.

(Eq. 3)

where R = ideal gas constant, T = absolute (or thermodynamic) temperature (°C + 273K), F = Faraday’s constant and z = ionic valency (+1 for K⁺).

If we apply this equation to a nerve cell, the concentration of K⁺ is much higher inside the cell than outside and the calculated value for E_K is approximately −90 mV. The measured value of the resting membrane potential (−70 mV) is more positive than this, so it cannot be explained solely in terms of the diffusion potential generated by K⁺. In fact other ions can also cross the membrane, particularly Na⁺. The concentration gradient for Na⁺ is in the opposite direction to K⁺ (Fig. 9, below) and the calculated value for E_Na is approximately +65 mV. The Na⁺ gradient tends to make the membrane potential more positive than it would otherwise be, but because the resting membrane is much more permeable to K⁺ than Na⁺, the resting potential is much closer to E_K than E_Na. One equation which takes account of the involvement of both K⁺ and Na⁺ in determining the resting membrane potential (RMP) is:

Fig. 9 The mechanisms underlying action potential generation. Increased Na⁺ conductance leads to the initial depolarization, while increased K⁺ conductance accounts for repolarization.

(Eq. 4)

This equation works well using α = 0.01, i.e., assuming the membrane permeability to K⁺ is 100 × permeability to Na⁺ at rest.

Electrogenic ion pumps

Any current flowing across the cell membrane will affect its potential, and one possible source of such currents is the ionic pump which maintains the normal transmembrane concentration gradients. The Na⁺/K⁺ ATPase pumps 3Na⁺ out of the cell for every 2K⁺ transported in (Fig. 5). This imbalance means that current flows from the inside to the outside of the membrane during active pumping. Such systems are said to be electrogenic and can affect the resting membrane potential. In this case, the net loss of positive charge from inside the cell makes the membrane potential more negative than it would otherwise be. Although this is probably not an important mechanism in determining the resting membrane potential in nerve and striated muscle, it may make a significant contribution in some smooth muscles.

Action potentials

If a nerve cell is stimulated by injecting (positive) electric current, the membrane potential becomes less negative (Fig. 8). We say the membrane potential has reduced (because the magnitude of the potential difference is reduced even though it is less negative) or that the membrane has been depolarized. With small stimuli (subthreshold), the membrane potential simply returns to normal after the stimulus ceases. If, however, the membrane is depolarized to a certain level, known as the threshold potential, the nerve itself generates a series of changes in the potential, known as an action potential. Action potentials are a feature of nerves and muscles, and it is the ability to generate these characteristic electrical signals which typifies excitable tissues.

Fig. 8 Intracellular recordings of membrane potential in a nerve. If depolarization reaches threshold, the nerve produces an action potential. Further stimulation is inhibited during the refractory period.

The action potential in nerve has an initial phase of rapid depolarization which reverses the potential difference across the membrane, reaching a peak at about +50 mV within a few tenths of a millisecond. The membrane then begins to repolarize, falling back to the normal resting potential about 1 ms after initiation of the action potential. The potential may actually become more negative than normal for a time (hyperpolarization), but eventually returns to the resting potential after a further 2–3 ms.

Action potential properties

Action potentials demonstrate a number of important properties.

• Action potentials obey an all-or-none law. Subthreshold stimuli do not elicit any active voltage changes, while any stimulus which exceeds threshold (suprathreshold stimuli) will lead to a full action potential (Fig. 8). Increasing the stimulus further has no effect on action potential shape or size.

• During an action potential it is either impossible or more difficult than normal to stimulate a further action potential and the cell is said to be refractory (Fig. 8). During the early part of the action potential there is an absolute refractory period when no stimulus is effective. This is followed by the relative refractory period during which an action potential can be stimulated but a larger than normal stimulus is required.

• Action potentials are self-propagating, i.e., once an action potential has been generated by an external stimulus at one site, it will automatically be conducted over the whole of the excitable membrane.