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CHAPTER OUTLINE Cellular Communication 3 Neural Regulation of the GI Tract 5 Peptide Hormones of the GI Tract 6 Synthesis, Post-Translational Modification, and Secretion 6 Gastrin 6 Cholecystokinin 7 Secretin 7 Vasoactive Intestinal Polypeptide 8 Glucagon 8 Glucose-Dependent Insulinotropic Polypeptide 8 Pancreatic Polypeptide Family 9 Substance P and the Tachykinins 9 Somatostatin 9 Motilin 9 Leptin 10 Ghrelin 10 Other Chemical Messengers of the Gastrointestinal Tract 10 Acetylcholine 10 Catecholamines 11 Dopamine 11 Serotonin 11 Histamine 11
Cells throughout the gastrointestinal (GI) tract receive information in many forms, including chemical messengers that emanate from other cells. The initial stimulus for hormone secretion is the ingestion of food. Food provides central neural stimulation in the form of thought (anticipation) and sight, chemical stimulation in the form of odor and taste, nutrient stimulation of the epithelial cells lining the GI tract, and mechanical stimulation. These processes all stimulate the release of peptides and other transmitters from cells of the mucosa into the nearby space, where they act locally, or into the bloodstream, where they circulate to distant target tissues. Therefore, chemical messengers from the GI tract can have far-reaching effects throughout the body.
CELLULAR COMMUNICATION
Chemical transmitters of the gut are produced by discrete cells of the GI mucosa and can be classified as endocrine, paracrine, synaptic (neurocrine), or autocrine (Fig. 1-1). Specialized signaling cells that secrete transmitters into the
blood are known as endocrine cells, and the transmitters they produce are known as hormones. Hormones bind to specific receptors on the surface of target cells at remote sites and regulate metabolic processes.1 In contrast with endocrine cells that act on distant target tissues, other signaling cells of the GI tract may produce transmitters that act on neighboring cells. This process is known as paracrine signaling and is typical of cells that produce somatostatin.2 Paracrine transmitters are secreted locally and cannot diffuse far. They bind to receptors on nearby cells to exert their biological actions. These actions are limited because they are taken up rapidly by their target cells, destroyed by extracellular enzymes, and adhere to extracellular matrix, all of which limit their ability to act at distant sites. Because paracrine signals act locally, their onset of action is generally rapid and can be terminated abruptly. By comparison, endocrine signaling takes much longer, and termination of signaling requires clearance of hormone from the circulation. A third form of signaling in the GI tract is neurotransmission. The enteric nervous system is a complex and sophisticated array of nerve cells and ganglia that is intimately involved in all aspects of GI function. When neurons of the
Endocrine
Autocrine
Paracrine
Neurocrine
Figure 1-1. Examples of cell-to-cell communication by chemical transmitters in the gastrointestinal tract. Hormones are secreted from endocrine cells into the blood, where they are carried to distant targets. Paracrine cells secrete transmitters into the paracellular space and act locally. Neurons secrete chemical transmitters or peptides into synapses or onto other cell types. Autocrine transmitters bind to receptors on the cell from which they originate.
GI tract are activated, signals in the form of neurotransmitters are released from the nerve terminals. These synapses deliver neurotransmitters to nerves, muscle cells, epithelial and secretory cells, and other specialized cells of the GI tract. Neurotransmitters are critical for the processes of digestion including the coordination of gut motility and secretion. Many of the same transmitters are produced by endocrine, paracrine, and neural cells. For example, cholecystokinin (CCK) is produced by typical endocrine cells of the upper small intestine and is secreted into the bloodstream on ingestion of a meal. However, CCK is also abundant in nerves of the GI tract and brain. In neural tissue, CCK functions as a neurotransmitter, although when secreted into the blood, CCK is a classic GI hormone. This conservation of transmitters allows the same messenger to have different physiologic actions at different locations and is made possible by the manner in which the transmitter is delivered to its target tissues. Endocrine cells secrete many different hormones into the blood, and their actions depend on the specificity of the receptor on the target tissues. In contrast, in synaptic transmission, the variety of neurotransmitters is more limited, and the specificity of action is dependent on the precise location at which the nerves synapse with the target cells. The concentration of signaling molecules can be adjusted quickly because the transmitter can be rapidly metabolized. In the synaptic cleft, transmitters are either rapidly destroyed or taken back up by the secretory neuron. Concentrations of these transmitters can be regulated rapidly by changes in their rate of synthesis, secretion, or catabolism. Many peptide transmitters have extremely short halflives (generally on the order of minutes), which allows the rapid initiation and termination of signaling. Endocrine transmitters of the GI tract consist predominantly of peptides (e.g., gastrin, secretin). Paracrine transmitters can be peptides, such as somatostatin, or nonpeptides, such as histamine, that act locally on neighboring cells. Neurotransmitters can be peptides, such as vasoactive intestinal polypeptide (VIP) and tachykinins, or small molecules, such as acetylcholine and norepinephrine, that are secreted, or nitric oxide (NO), which simply diffuses across the syn-
aptic cleft. The major transmitters and hormones of the GI tract are listed in Table 1-1. Criteria for establishing whether a candidate transmitter functions as a true hormone requires the following: (1) that the peptide be released into the circulation in response to
Serosa Circular muscle Submucosa Submucosal plexus Longitudinal muscle Myenteric plexus
Muscularis mucosa Mucosal nerves Mucosa Figure 1-2. Organization of the enteric nervous system. The enteric nervous system is composed of two major plexuses, one submucosal and one located between the circular and longitudinal smooth muscle layers. These neurons receive and coordinate neural transmission from the GI tract and central nervous system.
Prepropeptide
The expression of peptides is regulated at the level of the gene that resides on defined regions of specific chromosomes. The genes for most of the known GI peptides have now been identified. Specific gene regulatory elements determine if and when a protein is produced and the particular cell in which it will be expressed. Gut hormone gene expression is generally linked to peptide production and regulated according to the physiologic needs of the organism. For example, the production of a hormone may increase when gut endocrine cells are stimulated by food, changes in intraluminal pH, exposure to releasing factors, or other transmitters or hormones. These factors may simultaneously stimulate hormone secretion and increase gene expression. Ultimately, hormones are secreted into the circulation, where they can bind to receptors on target tissues. Once a biological response is elicited, signals may then be sent back to the endocrine cell to turn off hormone secretion. This negative feedback mechanism is common to many physiologic systems and avoids excess production and secretion of hormone. All GI peptides are synthesized via gene transcription of DNA into messenger RNA (mRNA) and subsequent translation of mRNA into precursor proteins known as preprohormones. Peptides that are to be secreted contain a signal sequence that directs the newly translated protein to the endoplasmic reticulum, where the signal sequence is cleaved and the prepropeptide product is prepared for structural modifications.9 These precursors undergo intracellular processing and are transported to the Golgi apparatus and packaged in secretory granules. Further modifications in peptide structure may occur within the Golgi apparatus (e.g., sulfation) that is important for the bioactivity of many peptide hormones, such as CCK. Secretory granules may be targeted for immediate release or stored in close proximity to the plasma membrane for release following appropriate cell stimulation. When GI endocrine cells are stimulated, mature hormone is secreted into the paracellular space and is taken up into the bloodstream. For many hormones, such as gastrin and CCK, multiple molecular forms exist in blood and tissues. Although there is only a single gene for these peptides, the different molecular forms result from differences in pretranslational or post-translational processing (Fig. 1-3). A common mechanism of pretranslational processing includes alternative splicing of mRNA, which generates unique peptides from the same gene. Post-translational changes include cleavage of precursor molecules. Enzymatic cleavage of the signal peptide produces a prohormone. Other post-translational features that result in mature GI peptides include peptide cleavage to smaller forms (e.g., somatostatin), amidation of the carboxyl terminus (e.g., gastrin), and sulfation of tyrosine residues (e.g., CCK). These processing steps are usually critical for biological activity of the hormone. For example, sulfated CCK is 100-fold more potent than its unsulfated form. The vast biochemical complexity of gastroenteropancreatic hor-
Propeptide
Peptide AB Peptide A Figure 1-3. Schematic representation of the production of gastrointestinal peptides. The genetic information is transcribed into mRNA, which is translated to a prepropeptide. Subsequent enzymatic cleavage produces peptides of various lengths. mRNA, messenger RNA.
mones is evident in the different tissues that secrete these peptides. As GI peptides are secreted from endocrine as well as nervous tissue, the distinct tissue involved often determines the processing steps for production of the peptide. Many hormone genes are capable of manufacturing alternatively spliced mRNAs or proteins that undergo different post-translational processing and ultimately produce hormones of different sizes. These modifications are important for receptor binding, signal transduction, and consequent cellular responses.10 It has become possible to express human genes in other species. By introducing specific hormone-producing genes into pigs or sheep, human hormones have been produced for medicinal use.11 With the rapid sequencing of the human genome, it is likely that novel methods of gene expression will expand the therapeutic use of human proteins. Moreover, drugs are being developed that inhibit the transcription of DNA into mRNA or that block the gene elements responsible for turning on specific hormone production (e.g., antisense oligonucleotides).12 This technology is based on the principle that nucleotide sequences bind to critical DNA regions and prevent transcription into mRNA. Similarly, oligonucleotides can be made to interact with mRNA and alter (or inhibit) translation of a protein product. These principles may be applicable to the treatment of the growing list of diseases that result from aberrant protein processing.13,14
GASTRIN
As discussed in more detail in Chapter 49, gastrin is the major hormone that stimulates gastric acid secretion. Subsequently, gastrin was found to have growth-promoting effects on the gastric mucosa and possibly some cancers.15 Human gastrin is the product of a single gene located on chromosome 17. The active hormone is generated from a precursor peptide called preprogastrin. Human preprogastrin contains 101 amino acids (AAs), including a signal peptide (21 AAs), spacer sequence (37 AAs), gastrin component (34 AAs), and a 9-AA extension at the carboxyl terminus. The enzymatic processing of preprogastrin
SECRETIN
CHOLECYSTOKININ
CCK is a peptide transmitter produced by I cells of the small intestine and is secreted into the blood following ingestion of a meal. Circulating CCK binds to specific CCK-1 receptors on the gallbladder, pancreas, smooth muscle of the stomach, and peripheral nerves to stimulate gallbladder contraction and pancreatic secretion, regulate gastric emptying and bowel motility, and induce satiety.19 These effects serve to
Pancreas
Small intestine
Glucagon
GLP-1
GLP-2
Figure 1-4. Different post-translational processing of glucagon in the pancreas and small intestine. The glucagon gene transcript is transcribed and translated into a prohormone (proglucagon) capable of producing glucagon and glucagon-like peptides (GLP-1 and GLP-2). However, only glucagon is produced in the pancreas because of specific processing. In the small intestine, GLP-1 and GLP-2 are the primary products.
glucagon-like peptides (GLPs). This precursor peptide consists of a signal peptide, a glucagon-related polypeptide, glucagon, and GLP-1 and GLP-2. Tissue-specific peptide processing occurs through prohormone convertases that produce glucagon in the pancreas and GLP-1 and GLP-2 in the intestine (Fig. 1-4).42 Glucagon and GLP-1 regulate glucose homeostasis.43 Glucagon is released from the endocrine pancreas in response to a meal and binds to G proteincoupled receptors on skeletal muscle and the liver to exert its glucoregulatory effects. GLP-1 stimulates insulin secretion and augments the insulin-releasing effects of glucose on the pancreatic beta cell (see later, Enteroinsular Axis). GLP-1 analogs have been developed for the treatment of type II diabetes mellitus. A long-acting human GLP-1 analog improves beta cell function and can lower body weight in patients with type II diabetes.44,45 GLP-2 is an intestinal growth factor and may have therapeutic implications in the maintenance of the GI mucosal mass and the reversal of villus atrophy.
GLUCAGON
Glucagon is synthesized and released from pancreatic alpha cells and from intestinal L cells of the ileum and colon. Pancreatic glucagon is a 29amino acid peptide that regulates glucose homeostasis via gluconeogenesis, glycogenolysis, and lipolysis and is counterregulatory to insulin. The gene for glucagon encodes not only preproglucagon but also
GIP was discovered based on its ability to inhibit gastric acid secretion (enterogastrone effect) and was originally termed gastric inhibitory polypeptide. It was subsequently shown that the effects on gastric acid secretion occur only at very high concentrations that are above the physiologic range. However, GIP has potent effects on insulin release that (like GLP-1) potentiates glucose-stimulated insulin secretion.46 Based on this action, GIP was redefined as glucose-dependent insulinotropic polypeptide. GIP is a 42amino acid peptide produced by K cells in the mucosa of the small intestine. GIP is released into the blood in response to ingestion of glucose or fat. In the presence of elevated blood glucose levels, GIP binds to its receptor on pancreatic beta cells, activating adenylate cyclase and other pathways that increase intracellular calcium concentrations, leading to insulin secretion. Importantly, however, the effects on insulin secretion occur only if hyperglycemia exists; GIP does not stimulate insulin release under normoglycemic conditions. GIP receptors are also expressed on adipocytes through which GIP augments triglyceride storage, which may contribute to fat accumulation. Based on the insulinotropic properties of GIP, coupled with its effects on adipocytes, it has been proposed that GIP may play a role in obesity and development of insulin resistance associated with type II diabetes mellitus.47 Consistent with this proposal was the experimental finding that mice lacking the GIP receptor do not gain weight when placed on a high-fat diet.48 It remains
Substance P belongs to the tachykinin family of peptides, which includes neurokinin A and neurokinin B. The tachykinins are found throughout the peripheral and central nervous systems, and are important mediators of neuropathic inflammation.63 Tachykinins, as a group, are encoded by two genes that produce preprotachykinin A and preprotachykinin B. Common to both is a well-conserved C-terminal pentapeptide. Transcriptional and translational processing produce substance P, neurokinin A, and/or neurokinin B, which are regulated in large part by alternative splicing. These peptides function primarily as neuropeptides. Substance P is a neurotransmitter of primary sensory afferent neurons and binds to specific receptors in lamina I of the spinal cord.64 Three receptors for this family of peptides have been identifiedNK-1, NK-2, and NK-3.65 Substance P is the primary ligand for the NK-1 receptor, neurokinin A for the NK-2 receptor, and neurokinin B for the NK-3 receptor. However, all these peptides can bind and signal through all three receptor subtypes. Substance P has been implicated as a primary mediator of neurogenic inflammation. In the intestine, Clostridium difficileinitiated experimental colitis results from toxininduced release of substance P and consequent activation of the NK-1 receptor.66 These inflammatory sequelae can be blocked by substance P receptor antagonists. Substance P receptors are more abundant in the intestine of patients with ulcerative colitis and Crohns disease.67
MOTILIN
SOMATOSTATIN
Somatostatin is a 14amino acid cyclic peptide that was initially identified as an inhibitor of growth hormone secretion. Since its discovery, it has been found in almost every organ in the body and throughout the GI tract. In the gut, somatostatin is produced by D cells in the gastric and intes-
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LEPTIN
GHRELIN
ACETYLCHOLINE
Acetylcholine is synthesized in cholinergic neurons and is the principal regulator of GI motility and pancreatic secretion. Acetylcholine is stored in nerve terminals and released by nerve depolarization. Released acetylcholine binds to postsynaptic muscarinic and/or nicotinic receptors. Nicotinic acetylcholine receptors belong to a family of ligand-gated ion channels and are homopentamers or heteropentamers composed of , , , , and subunits.106 The
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SEROTONIN
CATECHOLAMINES
The primary catecholamine neurotransmitters of the enteric nervous system include norepinephrine and dopamine. Norepinephrine is synthesized from tyrosine and released from postganglionic sympathetic nerve terminals that innervate enteric ganglia and blood vessels. Tyrosine is converted to dopa by tyrosine hydroxylase. Dopa is initially converted into dopamine by dopa decarboxylase and packaged into secretory granules. Norepinephrine is formed from dopamine by the action of dopamine -hydroxylase in the secretory granule. After an appropriate stimulus, norepinephrine-containing secretory granules are released from nerve terminals and bind to adrenergic receptors. Adrenergic receptors are G proteincoupled, have seven typical membrane-spanning domains, and are of two basic types, and . -Adrenergic receptors are further classified into 1A, 1B, 2A, 2B, 2C, and 2D. Similarly, receptors include 1, 2, and 3. Adrenergic receptors are known to signal through various G proteins, resulting in stimulation or inhibition of adenylate cyclase and other effector systems. Norepinephrine signaling is terminated by intracellular monoamine oxidase or by rapid reuptake by an amine transporter. The actions of adrenergic receptor stimulation regulate smooth muscle contraction, intestinal blood flow, and GI secretion. Dopamine is an important mediator of GI secretion, absorption, and motility and is the predominant catecholamine neurotransmitter of the central and peripheral nervous systems. In the central nervous system, dopamine regulates food intake, emotions, and endocrine responses and, peripherally, it controls hormone secretion, vascular tone, and GI motility. Characterization of dopamine in the GI tract has been challenging for several reasons. First, dopamine can produce inhibitory and excitatory effects on GI motility.107 Generally, the excitatory response, which is mediated by presynaptic receptors, occurs at a lower agonist concentration than the inhibitory effect, which is mediated by postsynaptic receptors. Second, localization of dopamine receptors has been hampered by identification of dopamine receptors in locations that appear to be species specific.108 Third, studies of dopamine in GI tract motility have often used pharmacologic amounts of this agonist. Therefore, the interpretation of results has been confounded by the ability of dopamine to activate adrenergic receptors at high doses. Classically, dopamine was thought to act via two distinct receptor subtypes, type 1 and type 2. Molecular cloning has now demonstrated five dopamine receptor subtypes, each with a unique molecular structure and gene locus.108 Dopamine receptors are integral membrane GPCRs, and each receptor subtype has a specific pharmacologic profile when exposed to agonists and antagonists. After release from the nerve terminal, dopamine is cleared from the synaptic cleft by a specific dopamine transporter.
DOPAMINE
HISTAMINE
In the GI tract, histamine is best known for its central role in regulating gastric acid secretion (see Chapter 49) and intestinal motility. Histamine is produced by enterochromaffin-like cells of the stomach and intestine as well as enteric nerves. Histamine is synthesized from L-histidine by histidine decarboxylase and activates three
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CNS Longitudinal muscle 5-HT3 Excitatory motor neuron 5-HT4 5-HT4 Inhibitory motor neuron 5-HT3
Myenteric plexus
Circular muscle
Figure 1-5. Role of serotonin in the enteric nervous system. This model illustrates the location of 5hydroxytryptamine3 (5-HT3) and 5-HT4 receptor subtypes in the GI tract. CNS, central nervous system. (Modified from Talley NJ. Serotoninergic neuroenteric modulators. Lancet 2001; 358:2061-8).
Intrinsic primary
5-HT3
Sensory neuron
5-HT4
Enterochromaffin cells
Rapid diffusion of NO
Nitric oxide Smooth muscle cell Activated neuron Figure 1-6. Nitric oxide (NO) signals smooth muscle relaxation. NO, synthesized from arginine by nitric oxide synthase, diffuses across the plasma membrane into smooth muscle cells. NO binds to and activates guanylyl cyclase, which converts GTP to cGMP. cGMP causes smooth muscle relaxation. (Modified from Alberts B, Bray D, Lewis J, et al, editors. Molecular biology of the cell. 4th ed. New York: Garland Science; 2002. p 831.)
GTP
cGMP
Relaxation
GPCR subtypes. H1 receptors are found on smooth muscle and vascular endothelial cells and are linked to phospholipase C (PLC) activation. As such, the H1 receptor mediates many of the allergic responses induced by histamine. H2 receptors are present on gastric parietal cells, smooth muscle, and cardiac myocytes. H2 receptor binding stimulates Gs (G proteins that stimulate adenylate cyclase) and activates adenylate cyclase. H3 receptors are present in the central nervous system and GI tract enterochromaffin cells. These receptors signal through Gi and inhibit adenylate cyclase.118 Histamine can also interact with the N-methyl-Daspartate (NMDA) receptor and enhance activity of NMDAbearing neurons independently of the three known histamine receptor subtypes. Unlike other neurotransmitters, there is no known transporter responsible for termination of histamines action. However, histamine is metabolized to telemethylhistamine by histamine N-methyltransferase and is then degraded to telemethylimidazoleacetic acid by monoamine oxidase B and an aldehyde dehydrogenase.
known as endothelial NOS and neuronal NOS, respectively, and are constitutively active. Small changes in NOS activity can occur through elevations in intracellular calcium. The inducible form of NOS (type II) is apparent only when cells become activated by specific inflammatory cytokines. This form of NOS is capable of producing large amounts of NO and is calcium-independent. NOS is often colocalized with VIP and PACAP in neurons of the enteric nervous system.120 NO, being an unstable gas, has a relatively short half-life. Unlike most neurotransmitters and hormones, NO does not act via a membrane-bound receptor. Instead, NO readily diffuses into adjacent cells to activate guanylate cyclase directly (Fig. 1-6). NO activity is terminated by its oxidation to nitrate and nitrite. Many enteric nerves use NO to signal neighboring cells and induce epithelial secretion, vasodilation, or muscle relaxation. NO is also produced by macrophages and neutrophils to help kill invading organisms.121
NITRIC OXIDE
ADENOSINE
NO is a unique chemical messenger produced from Larginine by the enzyme nitric oxide synthase (NOS).119 Three types of NOS are known. Types I and III are also
Adenosine is an endogenous nucleoside that acts through any of four GPCR subtypes.122 Adenosine causes relaxation of intestinal smooth muscle and stimulates intestinal secretion. Adenosine can also cause peripheral vasodilation and
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CYTOKINES
Intracellular
Figure 1-7. Molecular structure of a typical heptahelical G proteincoupled receptor. The amino terminus is extracellular and of variable length. It often contains N-linked glycosylation sites (Y) important in ligand binding. There are seven membrane-spanning domains and intracellular loops that contain sites for G protein binding and possible phosphorylation residues (orange circles).
Ligand
Extracellular
SIGNAL TRANSDUCTION
Cells live in a constantly changing milieu. The structure and biochemical nature of this environment are dynamic and, for cells to function normally, they must be able to access this changing information. The biochemical mediators of this information are cell surface receptors and transmitters. Receptors transduce signals from the extracellular space to the intracellular compartment. Each step in the process from receptor activation to receptor desensitization, internalization, and resensitization represents a potential regulatory checkpoint and possible target for therapeutic intervention. Cell surface receptors include GPCRs, ion channels, and enzyme-coupled receptors. GPCRs are seven membrane-spanning domain proteins associated with a heterotrimeric G protein (Fig. 1-7). The membrane regions consist of -helical domains with a conserved structural motif.124 GPCRs contain an extracellular amino terminus and an intracellular carboxyl terminus (see Fig. 1-7). When stimulated by the appropriate chemical messenger, the GPCR undergoes a conformational change and couples to a specific G protein. The first crystal structure of a GPCR, for rhodopsin, was elucidated in 2000.125 The three-dimensional structure of the rhodopsin receptor reveals a highly organized heptahelical transmembrane component with a portion of the C-terminus perpendicular to the seventh and final membrane-spanning domains of the protein.
Effector
Intracellular
Intracellular events Figure 1-8. Hormones (ligands) bind to specific G proteincoupled receptors at a unique location within the receptor-binding pocket. On binding, the receptor conformation is altered so that a specific G protein subunit is activated. G protein activation leads to dissociation of the subunit from the subunit and activation of effector pathways. These effectors include adenylate cyclase, ion channels, and an array of other systems.
G PROTEINCOUPLED RECEPTORS
G PROTEINS
G proteins are molecular intermediaries that initiate the intracellular communication process on ligand binding to its GPCR (Fig. 1-8).126 G proteins are composed of three
subunits, , and and are classified according to their subunit. They activate various effector systems, including adenylate cyclase, guanylate cyclase, phospholipases, and specific ion channels.127 G proteins that stimulate adenylate cyclase are classified as Gs; those that inhibit adenylate cyclase are called Gi.128 When an agonist binds to a Gs-coupled receptor, a conformational change occurs, allowing the receptor to associate with the Gs subunit. Under basal (unstimulated) conditions, Gs is bound to guanosine diphosphate (GDP); however, with agonist binding, GDP is released and replaced with guanosine triphosphate (GTP). The Gs-GTP complex then activates adenylate cyclase, resulting in the generation of cAMP from adenosine triphosphate (ATP) within the cell cytoplasm. cAMP phosphorylates effector proteins that ultimately lead to responses such as secretion, cell movement, and growth. Receptor activation also initiates
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SIGNALING
Adenylate cyclase, calcium channels Adenylate cyclase, cyclic guanosine monophosphate, phosphodiesterase, c-Src, STAT 3 Phospholipase C- Sodium-hydrogen exchange
Phosphorylation of the receptor is one of the initial events involved in turning off the signal after agonist binding and occurs through binding of arrestin-like molecules, which uncouple the receptor from the G protein.132 This uncoupling and subsequent receptor internalization (sequestration) continue the process of signal termination and eventually lead to the reestablishment of cell responsiveness.
Receptor Resensitization
the dissociation of the subunit from the subunits. However, the subunits remain tightly associated and themselves participate in a vast array of cellular signals. For example, not only can subunits activate GPCR kinases, adenylate cyclase, and ion channels, they induce receptor desensitization and stimulate Ras-mediated mitogenactivated protein (MAP) kinase.129,130 The Gs-GTP complex is gradually inactivated by guanosine triphosphatase (GTPase), which converts GTP to GDP. This enzymatic conversion occurs spontaneously by the G protein, which is itself a GTPase. The conversion of GTP to GDP terminates G protein stimulation of adenylate cyclase and is one way whereby the basal condition is restored. Certain GPCRs activate an inhibitory G protein (Gi) that inhibits cAMP accumulation and antagonizes the effects of Gs-coupled events. In this manner, GPCRs can maintain fine control of the cellular cAMP concentration and subsequent intracellular signaling. Members of this GPCR family also activate phospholipases and phosphodiesterases, and are often involved with ion channel regulation. Other GPCRs couple with Gq and G12 (see Table 1-2). The Gq family of G protein subunits regulates the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).131 Following subunit dissociation from , when the subunit reverts to the GDP-bound form, it reassociates with . With reestablishment of the heterotrimer, along with other mechanisms of desensitization, receptor signaling via the separate subunits ceases.
Internalization or sequestration of the receptor occurs within minutes of receptor occupancy. Agonist-activated receptors are phosphorylated by G proteincoupled receptor kinases at specific intracellular sites, which causes G protein uncoupling and initiates receptor endocytosis. GPCR endocytosis is followed by receptor dephosphorylation, recycling, and down-regulation. Chronic exposure of cells to high concentrations of hormones frequently leads to a decrease in cell surfacebinding sites. This reduction in surface receptor expression is termed down-regulation and is the result of receptor internalization. The mechanisms used by the cell that distinguish receptor internalization and recycling from down-regulation are not clear. However, long-term agonist exposure to some receptors has been shown to activate signaling molecules that may be important in receptor down-regulation.
Effector Systems
Following receptor occupation, G protein subunits cause activation of enzymes or other proteins, ultimately resulting in intracellular signaling events (Table 1-2). Enzymes such as adenylate cyclase or phospholipase C generate specific second messengers such as cAMP or IP3 and DAG. Some G proteins couple directly to specific ion channels, such as potassium or calcium channels, and initiate changes in ion permeability. The effector systems are not well understood for some receptors, such as those involved with cell growth and differentiation. Other G proteins such as Go may activate the phosphoinositide system. When bound to hormone, receptors that couple to Go activate PLC, which acts on inositol phospholipids found in the cell membrane. PLC can cause the hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating 1,2-DAG and IP3. DAG and IP3 can regulate cell metabolism by increasing intracellular calcium levels.
Receptor Desensitization
To ensure the rapidity of hormone signaling, shortly after receptor stimulation, a series of events is initiated that ultimately acts to turn off signaling. The principal events in this process involve receptor desensitization and internalization, which reestablish cell responsiveness.
Receptor Tyrosine Kinases Unlike GPCRs, where ligand-receptor interaction causes activation of a G protein intermediary, some ligand receptors possess intrinsic protein tyrosine kinase activity. These membrane-spanning cell surface receptors catalyze the transfer of phosphate from ATP to target proteins. Such receptors are structurally unique in that they contain glycosylated extracellular binding domains, a single transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain contains a protein tyrosine kinase region and substrate region for agonist-activated receptor phosphorylation. With activation, these receptors may phosphorylate themselves or be phosphorylated by other protein kinases.133 In general, receptor tyrosine kinases exist in the cell membrane as monomers. However, with ligand binding, these receptors dimerize, autophosphorylate, and initiate other intracellular signal transduction pathways. Most receptor tyrosine kinases couple, via ligand binding, to Ras and subsequently activate MAP kinase. MAP kinase is then able to modulate the regulation of other cellular proteins, including transcription factors. Members of the receptor tyrosine kinase family include the insulin receptor, growth factor receptors (vascular endothelial growth factor, PDGF, epidermal growth factor [EGF], fibroblast growth factor [FGF], insulin-like growth factor I [IGF] I, macrophage-CSF, nerve growth factor), and receptors involved in development.134 Receptor tyrosine kinases are discussed further in Chapter 3 in relation to cellular growth and neoplasia. Activated tyrosine kinase receptors participate in a number of intracellular signaling events that involve the phosphorylated cytoplasmic domain. Specific phosphorylated tyrosine residues serve as binding sites for Src homology regions 2 and 3 (SH2 and SH3 domains). The result of SH2 domain binding is activation or modulation of the signaling protein that contains this binding domain. In this manner, receptor tyrosine kinases activate diverse signaling pathways.135
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Growth factors regulate cellular proliferation by interacting with specific cell surface receptors. These receptors are membrane proteins that possess specific binding sites for the growth factor ligand. An unusual form of signaling occurs when the ligand interacts with its receptor within the same cell. For example, PDGF receptors present on the intracellular surface of fibroblast cell lines are activated by intracellular ligand. This process is known as intracrine signaling. Most peptide growth factors, however, interact with receptors on different cells to regulate proliferation. Growth factor receptors can be single polypeptide chains containing one membrane-spanning region, such as the receptor for EGF, or they may be composed two subunit heterodimers, with one subunit containing a transmembrane domain and the other residing intracellularly but covalently bound to the transmembrane subunit (Fig. 1-9). Heterodimers may also dimerize to form a receptor composed of four subunits (e.g., IGF receptor). Binding of the ligand to its receptor usually causes aggregation of two or more receptors and activation of intrinsic tyrosine kinase activity. Growth factor receptors also have the ability to autophosphorylate when bound to ligand. In addition, receptor tyrosine kinase activity may phosphorylate other intracellular proteins important in signal transduction. Autophosphorylation attenuates the receptors kinase activity and often leads to down-regulation and internalization of the receptor. Mutation of the receptor at its autophos-
Proteoglycan
Ion channelcoupled receptors are involved in rapid signaling between cells. Ion channel signaling is particularly important in nerve cells and other electrically excitable tissues such as muscle. In nerve cells, a relatively small number of neurotransmitters are released that act directly on ion channel proteins, causing them to open or close. Ion channels are selective to specific anions or cations and, when open, allow the flow of those particular ions across the plasma membrane according to the concentration inside and outside the cell. This flow of ions regulates the excitability of the target cell, which can trigger cellular responses such as neurotransmission, muscle contraction, electrolyte and fluid secretion, and hormone release.
Plasma membrane
Cytoplasm
EGF receptor
PDGF TGF-I TGF-II TGF-III receptor receptor receptor receptor Tyrosine kinase Serine/Threonine kinase
Figure 1-9. Growth factor receptors in the gastrointestinal tract. Schematic examples of growth factor receptor families are depicted in relation to the cell surface. Receptor regions that contain kinase activity are shown in boxes. On activation, these receptors have the ability to autophosphorylate or phosphorylate other proteins to propagate intracellular cell signaling. EGF, epidermal growth factor; IGF-I, insulin-like growth factor I; PDGF, platelet-derived growth factor; TGF, transforming growth factor (-I, -II, -III). (Modified from Podolsky DK: Peptide growth factors in the gastrointestinal tract. In Johnson LR, editor. Physiology of the gastrointestinal tract. New York: Raven Press; 1994. p 129.)
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At least seven related FGFs have been identified.148 These peptides have mitogenic effects on various cell types, including mesenchymal cells, and likely play an important role in organogenesis and neovascularization.149 Although not unique to the GI tract, PDGF is one of the most thoroughly studied growth factors. It is important for fibroblast growth, and its receptor is expressed in the liver and throughout the GI tract, where it appears to promote wound healing. Trefoil factors (pS2, spasmolysin, and intestinal trefoil factor, also known as TTF1, 2, and 3, respectively) are a family of proteins expressed throughout the GI tract.150 They share a common structure, having six cysteine residues and three disulfide bonds, creating a cloverleaf appearance that stabilizes the peptide within the gut lumen. The pS2 peptide is produced in the gastric mucosa, spasmolysin is found in the antrum and pancreas, and intestinal trefoil factor is produced throughout the small and large intestines. These peptides are produced by mucous neck cells in the stomach or goblet cells in the intestine and are secreted onto the mucosal surface of the gut. It is likely that trefoil factors act on the apical surface of the epithelial cells, where they have growth-promoting properties on the GI mucosa.
TREFOIL FACTORS
TGF- is produced by most epithelial cells of the GI tract and acts through the EGF receptor. Therefore, it shares trophic properties with EGF. It is believed to play a key role in gastric reconstitution after mucosal injury. Moreover, it
Other peptides signaling through GPCRs may also have growth-promoting effects. Three important examples include gastrin, CCK, and gastrin-releasing peptide (GRP). Gastrin stimulates the growth of enterochromaffin-like cells of the stomach and induces proliferation of the oxyntic mucosa containing parietal cells.151 Gastrin binds to CCK-2
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TASTE RECEPTORS
CCK-RF
Trypsin
Food
Monitor peptide
Pancreas
CCK Figure 1-10. Regulation of cholecystokinin (CCK) secretion by intraluminal releasing factors. Endocrine cells containing CCK are stimulated by trypsinsensitive releasing factors (CCK-RF) present in the lumen of the GI tract. Releasing factors secreted from the intestine are responsible for negative feedback regulation of pancreatic secretion. Under basal conditions, local trypsin inactivates CCK-RF; however, with ingestion of nutrients that compete as substrates for trypsin, CCK-RF is available to stimulate CCK secretion. Monitor peptide, a pancreatic releasing factor, may contribute to sustained CCK release and pancreatic secretion after a meal.
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ENTEROINSULAR AXIS
GI hormones play an important role in the regulation of insulin secretion and glucose homeostasis. These hormones control processes that facilitate the digestion and absorption of nutrients, as well as disposal of nutrients that have reached the bloodstream. In particular, gut peptides control postprandial glucose levels through three different mechanisms: (1) stimulation of insulin secretion from pancreatic beta cells; (2) inhibition of hepatic gluconeogenesis by suppression of glucagon secretion; and (3) delaying the delivery of carbohydrates to the small intestine by inhibiting gastric emptying.171 Each of these actions reduces the blood glucose excursions that normally occur after eating. Approximately 50% of the insulin released after a meal is the result of GI hormones that potentiate insulin secretion.172 This interaction is known as the enteroinsular axis and the gut peptides that stimulate insulin release are known as incretins. The major incretins are GLP-1 and GIP. GLP-1 not only stimulates insulin secretion but also increases beta cell mass, inhibits glucagon secretion, and delays gastric emptying. GIP stimulates insulin secretion when glucose levels are elevated and decreases glucagonstimulated hepatic glucose production.173 Thus, on ingestion of a meal, glucose, as it is absorbed, stimulates GLP-1 and GIP secretion. Circulating glucose then stimulates beta cell production of insulin, and this effect is substantially augmented by incretins acting in conjunction with glucose to increase insulin levels. Postprandial hyperglycemia may also be controlled by delaying the delivery of food from the stomach to the small
Table 1-3 Gastrointestinal Peptides That Regulate Satiety and Food Intake REDUCE FOOD INTAKE
Cholecystokinin (CCK) Glucagon-like peptide-1 Peptide tyrosine tyrosine (PYY3-36) Gastrin-releasing peptide Amylin Apolipoprotein A-IV Somatostatin
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of GLP-1 is preserved, indicating that the beta cell can respond normally to this incretin hormone. These observations suggest that GLP-1 administration could be a viable treatment for the hyperglycemia associated with diabetes.177 The growing evidence that beta cell failure may develop in type II diabetes supports the use of incretin hormones, such as GLP-1, or agents that delay GLP-1 degradation by the enzyme dipeptidyl peptidase-4 (DPP-4) to enhance beta cell function.178,179
KEY REFERENCES
intestine, allowing the rise in insulin to keep pace with the rate of glucose absorption. Several gut hormones that delay gastric emptying have been shown to reduce postprandial glucose excursions (Table 1-4).171 Amylin (islet amyloid polypeptide) is a 37amino acid peptide synthesized primarily in the beta cells of the pancreatic islets together with insulin. Although it was originally recognized for its ability to form amyloid deposits in association with beta cell loss, it has more recently been found to suppress glucagon secretion, delay gastric emptying, and induce satiety.174 Insulin resistance in obese patients is associated with increased levels of both insulin and amylin. Type II diabetes mellitus is characterized by high circulating insulin levels and insulin resistance. In addition, insulin levels do not increase appropriately after a meal and significant hyperglycemia occurs, which is consistent with an impaired incretin effect. GIP secretion is preserved in type II diabetes; however, the insulinotropic effect of GIP is reduced.175 Although the precise cause is unknown, the defect in GIP-stimulated insulin release is most pronounced in the late phase of insulin secretion. In contrast to GIP, GLP-1 secretion has been shown to be reduced in insulinresistant type II diabetics. The lower GLP-1 levels are caused by impaired secretion rather than increased degradation of the hormone.176 Unlike GIP, the insulin response to infusion
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Full references for this chapter can be found on www.expertconsult.com.