Gutter=18> 1 Introduction
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Principles of cell signaling  5.1 IntroductionIndividual cells are specialized to play a specific physiological role, such as secretion or contraction. In order to coordinate their activities they need to receive and transmit signals of various kinds. Cells communicate with each other in three different ways:
Diffusible chemical signals allow cells to communicate at a distance, while direct contact between cells is particularly important in cell—cell recognition during development. Direct cytoplasmic contact between neighboring cells via gap junctions permits the electrical coupling of cells and plays an important role in the spread of excitation between adjacent cardiac muscle cells. This chapter will focus chiefly on the ways that cells employ diffusible chemical signals. ^ Cells release a variety of chemical signals. Some are local mediators that act on neighboring cells, reaching their targets by diffusion over relatively short distances (up to a few millimeters). This is known as paracrine signaling. When the secreted chemical also acts on the cells that secreted it, the signal is said to be an autocrine signal. Frequently, substances are secreted into the blood by specialized glands—the endocrine glands—to act on various tissues around the body. The secreted chemicals themselves are called hormones. Finally, nerve cells release chemicals at their endings to affect the cells they contact. This is known as synaptic signaling. Local chemical signals—paracrine and autocrine secretions Paracrine secretions are derived from individual cells rather than from a collection of similar cells or a specific gland, and they act on cells close to the point of secretion to have a local effect (see Fig. 5.1a). The signaling molecules are destroyed rapidly by extracellular enzymes or by uptake into the target cells. Consequently very little of the secreted material enters the blood. One example of paracrine signaling is provided by mast cells. These cells are found in connective tissue all over the body and have large secretory granules that contain histamine, which is secreted in response to injury or infection. The secreted histamine dilates the local arterioles, resulting in an increase in the local blood flow. In addition, histamine increases the permeability of the nearby capillaries to proteins such as immunoglobulins. This increase in capillary permeability must be local, not widespread, otherwise there would be a significant loss of protein from plasma to the interstitial fluid, which could result in circulatory failure (Section 28.5). The mast cells also secrete small peptides that are released with the histamine to stimulate the invasion of the affected tissue by white blood cells—phagocytes and eosinophils. These actions form part of the inflammatory response and play an important role in halting the spread of infection (Chapter 14). The inflammatory response is also associated with an increase in the synthesis and secretion of a group of local chemical mediators called the prostaglandins (Section 5.5). The prostaglandins secreted by a cell act on neighboring cells to stimulate them to produce more prostaglandins. This is another example of paracrine signaling. In addition, the secreted prostaglandin stimulates further prostaglandin production by the cell that initiated the response. Here the prostaglandin is acting as an autocrine signal. This autocrine action amplifies the initial signal and may help it to spread throughout a population of cells, so ensuring a rapid mobilization of the body's defenses in response to injury or infection. Hormonal secretions provide a means of diffuse long-distance signaling to regulate the activity of distant tissues Hormones play an extensive and vital role in regulating many physiological processes and these will be discussed at length in later chapters. Here their role as cell signals will be discussed only in general terms. Endocrine cells synthesize hormones and secrete them into the extracellular space from which they are 54 5 Principles of cell signaling    Fig. 5.1 Comparison between paracrine and endocrine signaling.
able to diffuse into the blood. Once in the bloodstream a hormone will be distributed throughout the body and it is therefore able to influence the activity of tissues remote from the gland that secreted it. In this important respect hormones differ from local chemical mediators. The secretion and distribution of hormones is shown schematically in Fig. 5.1b. The endocrine glands secrete hormones in response to a variety of signals:
Since hormones are distributed throughout the body via the circulation, their actions are usually relatively slow in onset and long lasting (ranging from seconds to hours). The releasing hormones of the hypothalamus (a small region in the base of the brain) are an important exception to this rule. These hormones are secreted into the portal blood vessels in minute quantities and travel a few millimeters to the anterior pituitary where they control the secretion of the anterior pituitary hormones (Chapter 12). While cells are exposed to almost all the hormones secreted into the bloodstream, a particular cell will only respond to a hormone if it possesses receptors of the appropriate type (Section 5.3). Thus the ability of a cell to respond to a particular hormone depends on whether it has the right kind of receptor. This diffuse signaling is beautifully adapted to regulate a wide variety of cellular activities in different tissues. Finally, as the endocrine glands secrete their products into the blood, the concentrations of the various hormones in the blood are generally very low indeed—typically about 10~9 moles I"1. This means that the receptors must bind hormones very effectively. To put it another way, the individual receptors must have a high affinity for their particular hormone. The difference between endocrine and paracrine signaling depends fundamentally on the quantity of the chemical signal secreted. If sufficient quantities of a signaling molecule are secreted so that it enters the blood, it will be able to affect tissues remote from the point of secretion and is being employed as a hormone. Whereas if the amounts secreted are sufficient only to affect cells close to the point of secretion, the chemical signal is acting as paracrine signal. Consequently, a substance can be a hormone in one situation and a paracrine signal in another. For example, the peptide somatostatin is found in the hypothalamus. Neurons in the hypothalamus secrete somatostatin into the portal blood vessels which carry it to the anterior pituitary gland where it acts to inhibit the release of growth hormone (Chapter 12). As somatostatin has entered the blood to be carried to its target tissue it is acting as a hormone. Somatostatin is also found in the D-cells of the gastric mucosa. It is secreted when the hydrogen ion concentration in the stomach rises and it inhibits the secretion of gastrin by the adjacent G-cells. (Gastrin is the hormone that stimulates acid secretion by the parietal cells of the gastric mucosa—see Chapter 18). In this situation somatostatin acts on neighboring cells as a paracrine signal rather than as a hormone. Fast signaling over long distances is accomplished by nerve cells Chemical signals that are released into the extracellular space and need to diffuse some distance to reach their target cells have two disadvantages:
For many purposes these factors are not important, but there are circumstances where the signaling needs to be both rapid (occurring within milliseconds) and discrete. For example, during locomotion different sets of muscles are called into play at different times to provide coordinated movement of the limbs. This type of rapid signaling is performed by nerve cells. The contact between the nerve ending and the target cell is called a 5.2 Paracrine, endocrine, and synaptic signaling 55   synapse and the overall process is known as synaptic signaling.To perform their role, nerve cells need to make direct contact with their target cells. They do this via long, thin, hair-like extensions of the cell called axons. Each nerve cell gives rise to a single axon which may branch to contact a number of different targets. As axons may extend over considerable distances (in some cases up to 1 m), nerve cells need to be able to transmit their signals at relatively high speeds. This is achieved by means of an electrical signal (an action potential) that passes along the length of the axon from the cell body to its terminal (rhe nerve terminal). When an action potential reaches a nerve terminal it triggers the release of a small quanrity of a chemical (a neurotransmitter) which then acts on the target cell. Frequently the nerve terminal is very closely attached to its target and so the neurorransmitter released by the nerve rerminal has to diffuse a  Fig. 5.2 Synaptic signaling is performed by nerve cells. Electrical signals (action potentials) originating in the cell body pass along the axon and trigger the secretion of a signaling molecule by the nerve terminal. As the nerve terminal is closely attached to the target cell (in this case a skeletal muscle fiber), the signal is highly localized. The juncrion between the nerve cell and its rarget is called a synapse. very short distance (about 20 nm) to reach its point of action. Since the receptors for the neurotransmitter are located directly under the nerve terminal only very small quantities of neurotransmitter are required to activate the target cell and neighboring cells will not be affected. The combination of electrical signaling and very short diffusion time therefore permits both rapid and discrete activation of the targer. The essential features of synaptic signaling are summarized in Fig. 5.2. The organization and properries of nerve cells and synapses will be discussed more fully in Chaprer 6. Signaling molecules are very diverse in structure The chemical signals employed by cells are very diverse. Cells use both water-soluble molecules and hydrophobic molecules for signaling. Some, such as nitric oxide and glycine, have a small molecular mass (<100) while others are very large molecules (such as growth hormone—191 amino acids linked together and a molecular mass of 21 500). Many of the water-soluble signaling molecules are derived from amino acids—examples are peptides, proteins, and the biological amines. Hydrophobic signaling molecules include the prostaglandins (see below), the steroid hormones such as testosterone, and thyroid hormone. Many chemical signaling molecules are stored in membrane-bound vesicles prior ro secrerion by exocytosis (Section 4.5). This is the case with many hormones (e.g. epinephrine and antidiuretic hormone (ADH)) and neurorransmirters (e.g. acetylcholine). Other signaling molecules are so lipid soluble that they cannot be stored in vesicles on their own but may be stored bound ro a specific storage prorein. This is rhe case wirh thyroid hormone. Finally, some chemical mediarors are secreted as they are formed. This happens with the steroid hormones and rhe prosraglandins. Lipid-soluble hormones (thyroid hormone and the steroids) are not very soluble in water and are carried in the blood to their targer tissues bound to plasma proteins. Table 5.1 lists the principal families of signaling molecules and rheir chief modes of action. Summary
56 5 Principles of cell signaling Table 5.1 Examples of signaling molecules used in cell—cell communication
  ^ There are many different types of cell in the body, secreting a wide variety of chemical signals. Consequently, target cells must have a means of detecting the signals that are intended for them. They do this by using specific molecules known as receptors to bind individual chemical signals. Substances that activate a particular receptor are called agonists, while those drugs that block the effect of an agonist are called antagonists. All receptors are proteins and many are located in the plasma membrane where they are able to bind the water-soluble signaling molecules that are present in the extracellular fluid. Hydrophobic signaling molecules, such as the steroid hormones, cross the plasma membrane and bind to cytoplasmic receptors. Finally, some intracellular organelles possess receptors for molecules that are generated within the cell (second messengers, see below). In recent years it has become clear that an individual cell may possess many different types of receptor so that it is able to respond to a variety of extracellular signals. The response of a cell to a specific signal depends on which receptors are activated. Consequently, a particular chemical mediator can produce different responses in different cell types. For example, acetylcholine released from the nerve terminals of motor nerve fibers onto skeletal muscle causes the muscle to contract. When it is released from the endings of the vagus nerve it slows the beating of the heart (the heart rate). The same mediator has different effects in these two tissues because it acts on different receptors. The acetylcholine receptors of skeletal muscle are known as nicotinic receptors because they can also be activated by the alkaloid nicotine. Those of the heart have a different structure and are called muscarinic receptors as they can be activated by another chemical, muscarine. How do receptors control the activity of the target cells? Cells respond to chemical signals by initiating an appropriate physiological response. This process is called transduction. There are four basic ways in which activation of a receptor can alter the activity of a cell (Fig. 5.3): • first, it may open an ion channel and so modulate the membrane potential; ^ 57    Fig. 5.3 The principal ways in which chemical signals affect their target cells. Examples of each type of coupling are shown. (R, Receptor; E, enzyme; G, G-protein; +, indicates increased activity; —, decreased activity.)
Second messengers may be molecules such as cyclic AMP that are synthesized in response to receptor activation (Section 5.4), or inorganic ions such as Ca2T that may enter the cytoplasm via the plasma membrane or from intracellular stores. Second messengers work by modulating the activity of a specific set of enzymes within the cell. The series of events linking the change in the level of the second messenger to the final response is called a signaling cascade. Many receptors are directly coupled to ion channels. These receptor—channel complexes are called ligand-gated ion channels (Section 4.4) and they are employed by cells to regulate a variety of functions. In general, ligand-gated channels open for a short period of time following the binding of their specific agonist and this transiently alters the membrane potential of the target cell and thereby modulates its physiological activity. In some instances, control of the membrane potential is sufficient. This is the situation when one nerve cell inhibits the activity of another (Chapter 6 p. 78). More often, the change in membrane potential triggers some further event. Thus, in many cases, activation of ligand-gated channels causes the target cell to depolarize. This depolarization then activates voltage-gated ion channels which trigger the appropriate cellular response. In this way the activation of a ligand-gated ion channel can be used to control events within the cell. This pattern of events is illustrated by the stimulatory effect of acetylcholine on epinephrine secretion by the chromaffin cells of the adrenal gland. Acetylcholine released by the terminals of Fig. 5.4 The principal sequence of events in an adrenal chromaffin cell that link activation of a nicotinic receptor to the secretion of the hormone epinephrine. Acetylcholine binds to a nicotinic receptor on the plasma membrane of the chromaffin cell and opens an ion channel. This increases the permeability of the membrane to sodium ions and the membrane depolarizes. The depolarization activates voltage-gated calcium channels and calcium ions enter the cell to trigger the secretion of epinephrine by exocytosis. ^ 5 Principles of cell signaling the splanchnic nerve binds to nicotinic receptors on the plasma membrane and this increases the permeability of the membrane to sodium ions so that the membrane depolarizes. The depolarization results in the opening of voltage-gated calcium channels and calcium ions flow down their concentration gradient into the cell via these channels. The intracellular calcium concentration rises and triggers the secretion of epinephrine. This complex sequence of events is summarized in Fig. 5.4.^ that become activated when they bind their specific ligand. (A kinase is an enzyme that adds a phosphate group to its substrate, which can be another enzyme.) A typical example of a catalytic receptor is the insulin receptor that is found in liver, muscle, and fat cells. This receptor is activated when it binds insulin and in turn it activates other enzymes by adding a phosphate group to tyrosine residues. This results in an increase in the activity of the affected enzymes, which culminates in an increase in the rate of glucose  Fig. 5.5 Receptor activation of heterotrimeric G-proteins leads to activation of enzymes and ion channels. The top panels show a schematic representation of a receptor—G-protein interaction. The ligand binds to its receptor which is then able to associate with a G-protein. When this occurs the alpha (a) subunit exchanges bound GDP for GTP and dissociates from the beta (/3) and gamma (y) subunits (center). The free α-subunit can then either interact with adenylyl cyclase (bottom left) or with phospholipase C (bottom center) or directly open an ion channel (bottom right). uptake. Many peptide hormone and growth factor receptors are tyrosine-specific kinases. G-proteins link receptor activation directly to the control of an intracellular mediator GTP-binding regulatory proteins, or G-proteins, are a specific class of membrane-bound regulatory proteins that are activated when a receptor binds its specific ligand. The receptor-linked G-proteins have three subunits (a, /3, and y), each with a different amino acid composition. They are therefore called heterotrimeric G-proteins. When the largest subunit (the α-subunit) binds GDP the three subunits associate together. Activation of a G-protein-linked receptor results in the G-protein exchanging bound GDP for GTP and this causes the G-protein to dissociate into two parts: the α-subunit and the /3y-subunit complex. The a- and /3y-subunits can then migrate in the plasma membrane to modulate the activity of ion channels or membrane-bound enzymes (Fig. 5.5). There are many different kinds of G-protein but they either act to open an ion channel or to alter the rate of producrion of a second messenger—for example, cyclic AMP or inositol trisphosphate (IP3). In their turn, the second messengers regulate a variety of intracellular events. Changes in the level of cyclic AMP alter the activity of a variety of enzymes, while IP3 acts mainly by releasing calcium from intracellular stores. Other GTP-binding proteins are known that consist of a single polypeptide chain. These are called monomeric G-proteins and they play an important role in the control of cell growth. In this book any reference to G-proteins will usually mean the heterotrimeric G-proteins. ^ Cyclic AMP is generated when adenylyl cyclase (often incorrectly called adenylate cyclase) is activated by binding the α-subunit of a G-protein called Gs. The cyclic AMP formed as a result of receptor activation then binds to other proteins (enzymes and ion channels) within the cell and thereby alters their activity. The exact pattern of activity initiated by cyclic AMP in a particular type of cell will depend on which enzymes are expressed by that cell. Only one molecule of hormone or other chemical mediator is required to activate the membrane receptor, and activated adenylyl cyclase can produce many molecules of cyclic AMP. Consequently, the activation of adenylyl cyclase allows a cell to amplify the initial signal many times. The signal is terminated by conversion of cyclic AMP ro AMP by enzymes known as phosphodiesterases. The principal features of G-protein activation of adenylyl cyclase are shown in Fig. 5.5. The action of epinephrine on skeletal muscle highlights the role of G-protein regulation of cyclic AMP. Skeletal muscle stores glucose as glycogen, which is a large polysaccharide 5.4 Cyclic AMP: a ubiquitous second messenger 59    (Chapter 2). During exercise ATP is required to fuel muscle contraction and this necessitates the breakdown of glycogen to glucose. This change in metabolism is triggered by the hormone epinephrine, which is secreted into the blood from rhe adrenal medulla. Increased levels of circulating epinephrine activate a particular kind of adrenergic receptor on the muscle membrane, called a β-adrenergic receptor or ^-adrenoceptor. These receptors are linked ro Gs and when the α-subumt of Gs dissociates it activates adenylyl cyclase. The activation of adenylyl cyclase leads to an increase in the intracellular concentration of cyclic AMP. In turn, cyclic AMP activates another enzyme called protein kinase A, which in its turn activates another enzyme called glycogen phosphorylase, which breaks glycogen down to glucose. The whole cascade serves to amplify the initial response caused by the binding of epinephrine to its receptor and leads to Fig. 5.6 A simplified diagram to show the transduction pathway for the action of epinephrine on the glycogen stores of skeletal muscle. On the left is a key to the main stages in the signaling pathway. The details of the individual steps are shown on the right. Epinephrine binds to /^-adrenoceptors which are linked to a specific type of G-protein (Gs) that can activate adenylyl cyclase. This enzyme increases the intracellular concentration of the second messenger, cyclic AMP, which in turn leads to the activation of enzymes that break glycogen down to glucose. rapid mobilization of glucose. The main features of this cascade are summarized in Fig. 5.6. While Gs activates adenylyl cyclase and so stimulates the production of cyclic AMP, another G-protein (G;) inhibits adenylyl cyclase. Activation of receptors coupled to G, causes the intracel-lulaf level of cyclic AMP to fall. This, for example, is how somatostatin inhibits the release of gastrin by the G-cells of the gastric mucosa. Inositol trisphosphate and diacylglycerol are formed by the enzymatic breakdown of inositol lipids; both act as second messengers The inner leaf of the plasma membrane contains a small quantity of a phospholipid called phosphatidylinositol 4,5-bisphosphate. This phospholipid is the starting point for another important second-messenger cascade. Certain G-protein-linked receptors, Fig. 5.7 The transduction pathway for the formation of inositol trisphosphate (IP3) and diacylglycerol (DAG), both of which act as second messengers. The main stages in the signal transduction pathway are shown on the left of the figure while the detailed steps are shown on the right. In this example, acetylcholine acts on muscarinic receptors rhat are linked to a G-protein that can activate phospholipase C. This enzyme breaks down membrane phosphoinositides to form DAG and IP3. ^ 5 Principles of cell signaling  such as the muscarinic receptor (Section 5.3), activate an enzyme known as phospholipase C. This enzyme hydrolyzes phos-phatidylinositol 4,5-bisphosphate to produce diacylglycerol and inositol 1,4,5-trisphosphate (IP3), both of which act as intracellular mediators.IP5 is a water-soluble molecule that can mobilize calcium from the store within the endoplasmic reticulum. IP3 generation is therefore able to couple activation of a receptor in the plasma membrane to the release of calcium from an intracellular store (Fig. 5.7). Many cellular responses depend on this pathway. Examples are enzyme secretion by the pancreatic acinar cells and platelet aggregation by thrombin. A number of cytosolic proteins bind calcium and one, calmodulin, is an activator of a specific set of protein kinases. In this way activation of the IP3 signaling pathway can also regulate the pattern of enzymatic activity within the cell. The diacylglycerol that is generated by hydrolysis of phos-phatidylinositol is a hydrophobic molecule. Consequently, it is retained in the membrane when IP3 is formed. Like other membrane lipids, it is able to diffuse in the plane of the membrane, where it is able to interact with and activate another enzyme called protein kinase C. This enzyme activates other enzymes in its turn and thereby regulates a variety of cellular responses (Fig. 5.7), including DNA transcription. Diacylglycerol can also be metabolized to form prostaglandins which are themselves potent chemical mediators (see below). Summary
^ 5  .5 Prostaglandins and nitric oxide are local mediators that are synthesized as neededRecent evidence indicates that the prostaglandins and nitric oxide are important local mediators for a wide variety of physiological processes, including rhe regulation of local blood flow and the responses to injury and infecrion. The prostaglandins are members of a group of 20-carbon compounds known as the ekosanoids that are derived from the unsaturated farty acid aracbidonic acid. The prostaglandins are lipid soluble and, unlike most other paracrine secretions, they  Fig. 5.8 The pathways leading to the synthesis of prosraglandins and leukotrienes from membrane phospholipids. LTB4, leukotriene B/,; LTC4, leukotriene C4; PGE2, prosraglandin E2. are not stored in vesicles. Instead, their secretion is regulated continuously by increasing or decreasing their rate of synthesis from membrane phospholipids. Prostaglandin synthesis is initiated in response to a cell-specific stimulus. For example, platelets synthesize prostaglandins when treated with the clotting factor thrombin. The stimuli activate enzymes known as phospholipases which hydrolyze membrane phospholipids ro form diacylglycerol, which is hydrolyzed in its turn to give rise to arachidonic acid. Arachidonic acid may be metabolized by one of two pathways: via cyclo-oxygenase to generate prostaglandins, thromboxane, and prostacyclin, or via lipoxygenases to give rise ro leukorrienes (Fig. 5.8) Differenr cell types produce different kinds of prosraglandin— over 16 different types are known. Though they are local chemical mediators, the prosraglandins as a group have many effects throughour the body (Table 5.2) but the specific effect exerted by a particular prostaglandin depends on the individual tissue: prostaglandins PGEj and PGE2 relax vascular smooth muscle and are powerful vasodilators; in conrrasr, rhey cause contraction of the smooth muscle of the gut and uterus. Thromboxane A2 plays an important role in hemosrasis by causing plarelets to aggregate. The diversity of effects shown by differenr rissues to rhe same prostaglandin is explained by the presence of differenr prosraglandin receprors in differenr rissues. These receptors are locared in rhe plasma membrane of the target cells and are linked to second-messenger cascades via G-proreins. Both prostaglandins and leukotrienes play a complex role in regulating the inflammatory response ro injury and infection. When tissues become inflamed, the affected region reddens, becomes swollen, and feels hot and painful. These effects are, in part, the resulr of rhe actions of prostaglandins and leukotrienes, which cause vasodilatation in the affecred region. These substances also increase the permeability of the capillary walls to immunogloblulins and rhis leads to local accumulation of tissue fluid and swelling. Leukotriene B4 (LTB4) also attracts phagocytes. Nonsteroidal anti-inflammarory drugs such as aspirin are used when the inflammatory response becomes excessively 5.5 Prostaglandins and nitric oxide 61 Table 5.2 Some actions of eicosanoids
   painful or persistent (as in arthritis). They act as inhibitors of cyclo-oxygenase to prevent prostaglandin synthesis. The inflammatory response will be considered in greater detail in Chapter 14.Nitric oxide dilates blood vessels by increasing the production of cyclic GMP in smooth muscle Acetylcholine is able to relax the smooth muscle of the wall of certain blood vessels and this leads to vasodilatation. If the vascular endothelium (the layer of cells lining the blood vessels) is first removed, acetylcholine causes contraction of the smooth muscle rather than relaxation. Thus acetylcholine releases another substance in intact blood vessels—the highly reactive gas nitric oxide (NO). Many other vasoactive materials, including adenine nucleotides, bradykinin, and histamine, also act by releasing NO. It is now thought that the vasodilatation that occurs when the walls of blood vessels are subjected to stretch is also attributable to the release of NO by the endothelial cells and that this may play an important role in the local regulation of blood flow (see p. 302). How is NO formed by the endothelial cells and how does it cause the smooth muscle to relax? NO is derived from the amino acid arginine by an enzyme called nitric oxide synthase. This enzyme is activated when the intracellular free calcium concentration of the endothelial cells is increased by various ligands (acetylcholine, bradykinin, etc.) or by the opening of stress-activated ion channels (i.e. ion channels that are activated by stretching of the plasma membrane). As it is a gas, the newly synthesized NO diffuses readily across the plasma membrane of the endothelial cell and into neighboring smooth muscle cells. In the smooth muscle cells it binds to and activates an enzyme called guanylyl (or guanylate) cyclase. This enzyme converts GTP into cyclic GMP (guanosine monophosphate). Thus stimulation of the endothelial cells leads to an increase in cyclic GMP within the smooth muscle and this in turn activates other  Fig. 5.9 The synthesis of nitric oxide (NO) by endothelial cells and its action on vascular smooth muscle. The trigger for increased synthesis of NO is a rise in calcium in the endothelial cell. This can occur as a result of stimulation by chemical signals (acetylcholine, bradykinin, ADP, etc.) acting on receptors in the plasma membrane or as a result of the opening of ion channels by stretching of the plasma membrane (shear stress). The NO diffuses across the plasma membrane of the endothelial cell into the neighboring smooth muscle cells and converts guanylyl cyclase into its active form. The increased production of cyclic GMP leads to relaxation of the smooth muscle. enzymes to bring about muscle relaxation. This sequence of events is summarized in Fig. 5.9- The NO formed by endothelial cells depends on the presence of the enzyme nitric oxide synthase and the degree of activation is regulated by the intracellular calcium. Nitric oxide synthase is 62 5 Principles of cell signaling   not normally present in macrophages but when these cells are exposed to bacterial toxins the gene controlling the synthesis of this enzyme is switched on (a process known as induction) and the cells begin to make NO. In this case the NO is not used as a signaling molecule but as a lethal agent to kill invading organisms. Organic nitrites and nitrates such as amyl nitrite and nitroglycerine have been used for over 100 years to treat the pain that occurs when the blood flow to the heart muscle is insufficient. (This pain is called angina pectoris or angina). These compounds promote the relaxation of the smooth muscle in the walls of blood vessels. Detailed investigation revealed that this effect can be attributed to the formation of NO by enzymatic conversion of nitrite ions that are derived from the organic nitrates. This exogenous NO then acts in a similar way to that derived from normal metabolism. ^ The steroid hormones are themselves lipids and so they are able to pass through the lipid bilayer of the plasma membrane freely—unlike polar, water-soluble signaling molecules such as peptide hormones. They are thus able to bind to receptors within the cytoplasm of the target cell, although they can also bind to receptors on the cell surface. The existence of cytoplasmic receptors for steroid hormones was first shown for estradiol, which is accumulated by its specific target tissues (the uterus and vagina) but not by other tissues. The target tissues possess a specific binding protein for the hormone—a cytoplasmic receptor protein. When the estradiol receptor has bound a molecule of the hormone, there is an increase in the synthesis of those proteins that are specific to the target tissue. Other Fig. 5.10 A simplified diagram to show how steroid hormones regulate gene transcription in their target cells. Steroid hormones are lipophilic and pass through the plasma membrane to bind to specific receptor proteins in the cytoplasm of the targer cells. The hormone—receptor complex diffuses to the cell nucleus where it binds to a specific region of DNA to regulate gene transcriprion. steroid hormones, such as the glucocorticoids and aldosterone, are now known ro act in a similar way. The full sequence of events can be summarized as follows: the hormone first crosses the plasma membrane by diffusing through the lipid bilayer and then binds to its cytoplasmic receptor. The receptor—hormone complex then migrates to the nucleus where it increases the transcription of DNA into the appropriate mRNA. The new mRNA is then used as a template for protein synthesis (Chapter 2). This scheme is outlined in Fig. 5.10. A steroid receptor protein has a specific region for binding a particular hormone. When a molecule of the appropriate hormone has been bound, the receptor undergoes a conformational change to expose a DNA-binding region that is specific for a particular DNA sequence. The activated receptor protein can only bind to the appropriate sequence if the gene is active. Consequently, steroid hormones can only affect those genes that are currently being transcribed by the target cell. As steroid hormones act by regulating gene transcription, their effect on protein synthesis takes place over a period of time. These effects are long lasting and slow in onset (for example sodium retention by the kidney occurs after a delay of 2-4 hours following administration of aldosterone; Section 17.7). Thyroid hormone (T3) is also highly lipophilic and regulates gene transcription in a similar way (Section 1 2.4). Summary 1  . Prostaglandins, thromboxanes, and leukotrienes are all eicosanoidssynthesized from arachidonic acid in response to a variety of stimuli. Although they are employed as autocrine and paracrine signaling molecules, they are not stored in vesicles but are synthesized as required from membrane phospholipids. Different cell types produce different eicosanoids and their effects are specific to a particular tissue. 2. Nitric oxide is a highly reactive gas that acts as a short-lived signal ing molecule. It is a powerful vasodilator that is synthesized by the endothelial cells of blood vessels in response to a variety of stimuli. It acts by increasing the synthesis of cyclic GMP in the smooth muscle of blood vessels and this, in turn, leads to muscle relaxation. 3. Steroid and thyroid hormones are very hydrophobic and are carried in the blood bound to specific carrier proteins. They enter cells by diffusing across the plasma membrane and bind to receptors in the cytosol. The hormone—receptor complexes then migrate to the nucleus where they alter the. transcription of specific genes. This leads to the increased synthesis of specific proteins. ^ 5  .7 Cells use specific cell-surface molecules to assemble into tissuesTo form complex tissues, different cell types must aggregate together. Some cells therefore migrate from their point of origin to another part of the body during development. When they arrive at the appropriate region they must recognize their target cells and participate in the diffetentiation of the tissue. To do so, 5.8 Gap junctions 63  they must attach to other cells and to the extracellular matrix. What signals are employed by developing cells to establish their correct positions, and why do they cease their migrations when they have found their correct target?Unlike adult cells, embryonic cells do not form strong attachments to each other. Instead, when they interact, their cell membranes become closely apposed to each other, leaving a very small gap of only 10—20 nm. Exactly how cells are able to recognize their correct associations is not known, but it is likely that each type of cell has a specific marker on its surface. When cell membranes touch each other the surface marker proteins on the surfaces of the cells can interact. If the cells have complementary proteins they are then able to cross-link and the cells will adhere. This must be an early step in tissue formation. It has been shown that cells will associate only if they recognize the correct surface markers. Thus, if differentiated embryonic liver cells are dispersed by treatment with enzymes and grown in culture with cells from the retina, the two cell types aggregate with others of the same kind. Thus liver cells aggregate together and exclude the retinal cells. The various kinds of cell-cell and cell—matrix junctions have already been discussed in Chapter 3 and many of the proteins involved have been characterized. These may be grouped into several families, including the cadherins that form desmosomes, the connexins that form gap junctions (see below), the immuno-globulin-like (Ig-like) cell adhesion molecules (e.g. N-CAM), and the integrins that form hemidesmosomes which attach cells to the extracellular matrix. The integrins also play an important role in development and wound repair. The cadherins, integrins, and Ig-like cell adhesion molecules are also involved in non-junctional cell—cell adhesion, which must play an important role in the formation of integrated tissues. While the integrins are important in maintaining attachments between the majority of cells, those of platelets are not normally adhesive. If they were, blood clots would form spontaneously with disastrous consequences. During hemostasis (blood clotting), however, platelets adhere to fibrin and to the damaged wall of the blood vessel (Section 13.8). This adhesion results from a change in the properties of the platelets when a nonadhering integrin precursor present in the platelet membrane is transformed into an adhesive protein. This transformation is triggered by factors released from the walls of damaged blood vessels that activate second-messenger cascades within the platelets which, in turn, trigger modification of the structure of preformed integrins so that they act as receptors for extracellular molecules, including fibrin. The final result is an increase in platelet adhesion and clotting of the blood. ^ Some cells are joined together by a specific type of junction known as a gap junction. These junctions are formed by specific membrane proteins that associate to form doughnut-shaped structures known as connexons. When the connexons of two adjacent cells are aligned, the cells become joined by a water-filled pore. As the connexons jut out above the surface of the plasma membrane the cell membranes of the two cells forming the junction are separated by a small gap—hence the name gap junction (Section 3-4). Unlike ion channels, the pores of gap junctions are kept open most of the time so that small molecules of less than 1500 Da and inorganic ions can pass readily from one cell to another. Consequently gap junctions form a low-resistance pathway between the cells and electrical current can spread from one cell to another. The cells are thus electrically coupled. This property is exploited by the myocytes of the heart which are connected by gap junctions. Since the cells are electrically coupled, depolarization of one myocyte causes current to pass between it and its immediate neighbors, which become depolarized. In their turn, these cells cause the depolarization of their neighbors and so on. Consequently, current from a single point of excitation spreads across the whole of the heart via the gap junctions and the heart muscle behaves as a syncytium (a collection of cells fused together). By this means the electrical and contractile activity of individual myocytes is coordinated. This allows the muscle of the heart to provide the wave of contraction (the heart beat) that propels the blood around the body (Section 15.3). In the liver, the role of gap junctions between adjacent liver cells (hepatocytes) is quite different. The gap junctions allow the exchange of intracellular signals (second messengers) between cells. For example, the hormone glucagon srimulates the breakdown of glycogen to glucose by increasing the level of cyclic AMP which is able to diffuse through the water-filled pores of the gap junctions from one cell to another. Thus cells not directly activated by glucagon can be stimulated to initiate glycogen breakdown. The gap junctions provide a means of spreading the initial stimulus from one cell to another. Summary
^ 5 Principles of cell signaling Recommended readingAnswers  Biochemistry and cell biologyAlberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D. (1994). Molecular biology of the cell, (3rd edn), Chapters 15 and 19. Garland, New York. Barritt, G. J. (1992). Communication within animal cells, Chapters 1, 3—7, and 10. Oxford University Press, Oxford. Evans, W. H. and Graham, J. M. (1989). Membrane structure and function, Chapter 3- IRL Press, Oxford. Srryer, L. (1995). Biochemistry, (4th edn), Chapter 38. Freeman, New York. Pharmacology Rang, H. P., Dale, M. M., and Ritter, J. M. (1995). Pharmacology, (3rd edn), Chapters 2, 10, and 11. Churchill-Livingstone, Edinburgh. Self-test questions Each statement is either true or false. The answers are given below. 1. Hormones: a. Are chemical signals that are secreted into the blood; b. Can influence the behavior of many different cell types; c. Act only on neighboring cells; d. Are secreted by specialized glands. 2. The following are released by one type of cell specifically to regulate the activity of others: a. Nitric oxide; b. Prostaglandins; c. Insulin; d. Epinephrine; e. Ca2 + ; f. Glucose; 3. Receptors: a. Are always proteins; b. Are always located in the plasma membrane; c. May be membrane-bound enzymes; d. Can activate second-messenger cascades via G-proteins. 4. Prostaglandins: a. Are secreted by exocytosis; b. Are synthesized from phospholipids; c. Are hormones; d. Act on G-protein-linked receptors. 5. Steroid hormones: a. Are lipid soluble; b. Activate ion channels directly; c. Can alter gene transcription; d. Are secreted as they are synthesized. 1. Hormones are secreted by endocrine glands into the blood stream to act on cells at a distance. Paracrine signals act locally. a. True; b. True; c. False; d. True. 2. Nitric oxide and prostaglandins are paracrine signaling molecules. Insulin and epinephrine are hormones. Calcium and cyclic AMP are both intracellular mediators but cyclic AMP can spread from cell to cell via gap junctions. Glucose is a substrate for metabolism a. True; b. True; c. True; d. True; e. False; f. False; 3. Many receptors are membrane proteins but some are intra cellular proteins (e.g. steroid hormone receptors).Receptors may activate ion channels directly but some receptors are protein kinases that are activated when they bind their ligand. Many receptors activate G-proteins and thereby modulate the levels of second messengers. a. True; b. False; c. True; d. True. 4. Prostaglandins are metabolites of arachidonic acid that is derived from membrane phospholipids by the action of phosphohpases. They are lipid-soluble molecules that are secreted as they are formed. They are paracrine and auto crine signaling molecules which activate G-protein-linked receptors in the plasma membrane. a. False; b. True; c. False; d. True. 5. Steroid hormones are lipid signaling molecules that act mainly by altering gene transcription. As they are lipids they cannot be stored in membrane-bound vesicles and are therefore secreted as they are synthesized. a. True; b. False; c. True; d. True. |
n order to coordinate their activities, cells need to send and receive
hemical signals are detected by specific receptor molecules. When a