Current aspects of gut hormones

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602-618

(1981)

CURRENT Current IRVIN

M.

MODLIN,

Department

Aspects

M.D.,

of Surgery,

RESEARCH

A.

Submitted

of Gut Hormones

SANK,

Downstate

M.B.CH.B.,

Medical

for publication

INTRODUCTION Gastrointestinal hormones consist of a heterogenous group of biologically active substances which are involved in the regulation of gastrointestinal function. They are secreted by endocrine cells which are widely distributed throughout the gut mucosa in such profusion that the gut has been labeled the largest endocrine organ in the body [70]. In addition, a significant number of these biologically active peptides are present in nerves of the gastrointestinal tract and also in the central nervous system itself [7]. Thus, it seems reasonable to assume that while some of these peptides function as classical hormones others are paracrine or even neuracrine in their mode of action. Elucidation of the complex manner in which the gut endocrine hormones function has been facilitated in recent times by the rapid advances in technology which have enabled purification of small peptides, measurement of minute amounts of circulating substances, and the localization of peptides to specific secretory cells 1831. Historical

Perspective

Prior to 1902 when Bayliss and Starling provided evidence for the existence of a chemical messenger in duodenal mucosa, Pavlov’s view that the modification of physiological function was primarily under neural control had been accepted. They utilized the term hormone (I arouse to 0022-480418

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Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.

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activity) to describe a substance released into blood with an action on a distant organ. In 1953, Feyrter described a system of clear cells which was diffusely scattered throughout the gut 1291. He proposed that substances secreted by these cells acted only in the local environment and suggested that these agents be called mediators. A large number of cells which secreted bioactive compounds were thus logically scattered throughout the gut and other endocrine organs. Pearse (1968) suggested that these cells were derived from a neuroectodermal origin and could be classified together under their common cytochemical characteristics which he designated APUD (amine content, amine precursor uptake, and decarboxylation) [7 1J. While this theory was attractive and provided a central framework to clarify the heterogenous gut endocrine system, recent work by Le Douarin using quail-chicken chimeras has suggested that not all endocrine cells are APUD and that many may be of endodermal origin, rather than ectodermal [53]. Most of the progress in the field of gastrointestinal hormones has followed recent advances in methodology. In order to understand the current status of this field of knowledge, an appreciation of the nature and scope of these techniques is necessary. Hormone

Measurement

Since hormones by definition are carried by blood from their gland of secretion to 602

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the organ of action, measurement of the circulating levels provides valuable information concerning rate of secretion, metabolism, and regulatory mechanisms. All hormones that have thus far been studied circulate in pica- and femtomolar concentrations which are lOOO-fold below the threshold of sensitivity of most bioassays [83]. In addition to being relatively insensitive, bioassays are nonspecific, have poor reproducibility, and measure endorgan activity rather than actual circulating peptide levels. The development of radioimmunoassay by Yalow and Berson, therefore, revolutionized the measurement of circulating hormones [4]. It has enabled accurate measurement of alterations in plasma levels and has thus allowed definition of the physiology of a whole new group of biologically active substances. Accurate measurement of plasma levels has allowed the correlation of abnormal findings with a number of endocrine tumors principally of the pancreas and subsequently with other disease states [3 11. Other techniques such as assays cytochemical and radioreceptor which are based on the same principles as radioimmunoassay have not as yet proved sufficiently sensitive to allow measurement of circulating peptides. Tissue Localization

Gastrointestinal hormones are produced in cells belonging to the APUD series [71]. These cells are scattered throughout the gut and nervous system. Cells which contain secretory granules some of which are characterizable by specific shape, size, and density have been identified by electron microscopy. However, the only reasonably certain method for identifying specific peptide involves the use of immunocytochemical methods and specific antisera of high avidity. These may involve either direct application of a fluorescent-labeled antibody or the indirect method using a second anti-IgG antibody carrying the fluorescent label [73]. Two further methods

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which are available involve an immunoglobulin bridge using peroxidase-antiperoxidase’ or radioimmunocytochemistry where one of the combining sites of the antibody binds the tissue antigen and the radiolabeled antigen binds to the remaining combining site [51]. Since there is incomplete knowledge as to the secretory products of some of the cells that have been labeled according to a conventional lettering system (A,B,C,D, etc.). The classification is primarily ultrastructural, although available histochemical and biochemical details are utilized. Structure, size, shape, and reactivity of secretory granules are the main criteria used in typing the cells. The system referred to in this review is the current (1977) Lausanne Classification of Gastroenteropancreatic Endocrine Cells. Purijkation

and Sequence Analysis

Most of the earliest problems in the field of gut hormone research arose because of the inability to extract and purify individual hormones. The development of sophisticated fractionation techniques such as affinity chromatography, gel filtration, ion exchange, and high-pressure liquid chromatography combined with electrophoresis and countercurrent distribution has considerably facilitated purification [66]. In order to purify hormones, they must first be extracted from tissue. Since numerous proteolytic enzymes are present in the gut, both freezing and then boiling of the material are necessary to insure complete enzyme inactivation. The hormones are then removed from the tissue homogenate and the solvent phase by use of a number of different techniques [82]. Individual hormones may exist in more than one molecular form and may require a number of procedures for separate isolation. Once a ’ The method involves the production of a histochemically detectable marker and is advantageous since nonspecific background activity is reduced and antibody-linking properties are maximally preserved allowing specific localization.

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pure peptide is obtained, the use of highly sophisticated techniques and apparatus is necessary for sequence analysis. In this respect, the development of sequencespecific radioimmunoassay has been of considerable use, especially in differentiating among similar molecular forms of one peptide [67]. This technique involves the development of an antibody which is highly specific and recognized as only a particular sequence of amino acids which may comprise only a part of the complete peptide itself. Gut Hormone

Structure

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represents the principle hormone and the other forms presumably represent either biosynthetic precusors or postsecretory degradation products [80]. The particular amino acid sequences are important since they confer a specific biological activity to the peptide and also determine the potency of action upon a particular organ (Table 1). Thus, in the gastrin-CCK family, the COOH-terminal pentapeptide amide sequence (Gly-TrpMet-Asp-Phe-NH,) is the main site of similarity and is the biologically active site of the two molecules. It is, therefore, not surprising that the two hormones share similar biological activity. The NHz-terminal part of the molecule, however, influences potency for different targets; thus CCK acts mainly on the gall bladder and pancreas, while gastrin is a more potent stimulus for parietal cells. In addition, the location of the sulfated tyrosyl residue is important not only in determining the potency of the peptide but also in influencing whether its action will be gastrinlike or CCK-like. Thus, if the sulfated residue is moved only one position, the hormone activity can be changed from gastrin-like to CCK-like, or if the sulfate radical is removed, it becomes far less potent [ 1031. The amino acid homologies are not quite as clear in the secretin family (secretin, glucagon, GIP, VIP), but when the peptides are aligned from the amino NH,-terminal there are 13 positions at which three of the four peptides have identical amino acid residues. The main functional sites in the secretin family are not yet characterized but all the members share common actions such as inhibition of acid secretion and stimulation of insulin release and hepatic glycogenolysis.

The two main families of gut hormones are the gastrin and the secretin family. Their basic structures are outlined in Table 1. The former group consists primarily of gastrin and cholecystokinin (CCK), but in addition, motilin and enkephalin share a number of structural identities. The latter group includes secretin, gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP), glucagon, and the amphibian dermal peptide, bombesin [lo]. These common structural and functional similarities presumably reflect a common ancestry and good evidence in support of peptide hormone evolution has recently been presented both for gastrin and cholecystokinin [25]. A further feature of peptide hormones, notably including gut hormones, is that they exhibit molecular heterogeneity. This may be either macroheterogeneity whereby there are a number of different hormone forms which differ considerably or microheterogeneity in which the differences involve one amino acid residue or only a side chain. Thus, gastrin has been found to circulate as four components (components: I, II, III, and IV) and each of these comBrain/Gut Axis ponents itself has various forms. This molecular heterogeneity has also been deThe original descriptions of either gut scribed for cholecystokinin and glucagon hormones or peptides occurring in the and it presumably exists in many of the gut nervous system did not deal with the conhormones. One of the molecular forms cept of bilateral distribution. In recent

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BASIC STRUCTURES Gas&in-CCK Gastrin (17) Glp GUY Pro Trp Leu GIU Glu Glu Glu Glu Ala Tyr WhW GUY Trp Met Asp Phe NH,

CCK (17-33)

Cerulein

Glp Gln Asp Tyr (SOJU Thr GUY Trp Met Asp Phe NH,

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Secretin Enkephalin

Asp Pro Ser His Arg Ile Ser Asp Arg Asp Tyr (SO,H) Met GUY Trp Met Asp Phe NH,

GUT

Motilin (14-21)

Gin Glu LYS Glu TY~ GUY GIY Phe Met

time it has become clear that a considerable number of hormones originally described in the gut are also found in the nerves and even the central nervous system, while conversely certain brain peptides have now been identified in the gut. Thus, gastrin, cholecystokinin, VIP, and motilin are found in the peripheral and central nervous system while brain peptides such as neurotensin, substance P (Sub P), enkephalin, corticotrophin, somatostatin, and thyrotrophic releasing hormone have been identified in endocrine cells of the gut [ 10, 501. A morphological explanation for this dual neuroendocrine localization has been pro-

Arg Asn LYS GUY

Secretin

Glucagon

His Ser Asp GUY Thr Phe Thr Ser Glu Leu Ser Arg Leu Arg Asp Ser Ala

His Ser Gin GIY Thr Phe Thr Ser Asp Tyr Ser LYS Tyr Leu Asp Ser Arg Arg Ala Gin Asp Phe Val Gln Tv Leu Met Asp Thr

Arg Leu Gin A% Leu Leu Glu GUY Leu Val NH,

family

VIP His Ser Asp Ala Val Phe Thr Asp Asn W Thr Arg Leu Arg LYS Gin Met Ala Val LYS LYS Tyr Leu Asn Ser Ile Leu Asn NH,

GIP (I-29)

Bombesin

Tyr Ala Glu GUY Thr Phe Ile Ser Asp W Ser Ile Ala Met Asp LYS Ile Arg Gin Gin Asp Phe Val Asn Tv Leu Leu Ala Gin

Glp Glu Arg Leu GIY Asn Gin Trp Ala Val GUY His Leu Met NH,

vided by the APUD theory which postulates that endocrine peptide-producing cells and nervous cells initially originated in common from the neuroectoderm. It has thus been suggested that the nervous system exists in three functional units: autonomic, somatic, and neuroendocrine [69]. The appreciation of the fact that gut hormones are distributed not only in endocrine cells but also in peripheral and central nerves has established the fact that these peptides can function not only as hormones, but also as neurotransmitters. The implications of the concept are far reaching and embrace the interpretation of all the

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CHARACTERISTICS OF INDIVIDUAL HORMONES AND PEPTIDES Gastrin

Gastrin is produced mainly by antral G cells and to a lesser extent by G cells of the proximal duodenum. It is released into both the blood stream and the gut lumen [52]. The significance of the latter observation has not yet been established. The principal stimulus for gastrin release is feeding. The amount of gastrin released is modified by the protein content of the food, antral distention, and vagal influence. [24]. Feedback regulation of gastrin release is mediated by antral acidification and by neural and hormonal regulatory mechanisms generally characterized by the concept of the enterogastrone axis [96].2 The administration of exogenous gastrin has been demonstrated to have a number of actions including stimulation of gastric acid secretion, sphincter tone, exocrine pancreatic secretion, gut motor activity, and a positive trophic effect on the gut [ 1051. Stimulation of gastric acid secretion is probably the most important physiological function of gastrin. The exact role of gastrin as a trophic factor is controversial [44]. Gastrin heterogeneity is complex and has previously been alluded to. Of its four main components (I,II,III, and IV), component II is gastrin 34 (big gastrin), component III is gastrin 17, and component IV is gastrin 14. Gastrin tetrapeptide probably occurs naturally. Component I has not been characterized [79]. “Big big gastrin” probably does not exist except as a radioimmunoassay artifact or in certain tumors which may produce macromolecules [78]. G17 has a half-life of 6 min while that of G34 is 40 min. Circulating concentrations 2 The concept implies the existence of a hypothetical mechanism for the control of gastric acid secretion by neural and humoral factors involving the gut and stomach.

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of G17 are five to six times more potent in all biological actions tested than equimolar concentrations of G34. Thus, while a meal releases threefold more G34 than G17, because of its greater potency, the acid secretory response to feeding is primarily due to G17 [56]. Measurement of plasma gastrin has been particularly relevant in the diagnosis of pancreatic endocrine tumors (gastrinomas) which cause peptic ulceration. The Zollinger-Ellison syndrome is characterized by high basal circulating levels of plasma gastrin. Characteristically, these levels are not significantly altered by meal stimulus, but respond with a dramatic elevation to a secretin or calcium provocation stimulus [40]. The explanation for this paradoxical response is not known. Basal plasma gastrin levels may vary depending on the gastrin standard and the laboratory in which the assay is performed. Fasting basal levels 100% above the normal for a particular institution are characterized as hypergastrinemia. Similarly, a response to secretin or calcium greater than 100% above basal represents a positive test and is almost conclusive evidence of the presence of a gastrinoma [23]. Hypergastrinemia may also occur in patients with pernicious anemia or renal failure, but these groups are usually characterized by hypochlorhydria [22]. Retained antrum is a rare cause of hypergastrinemia as is the entity of G-cell hyperfunction (hyperplasia) [ 1031. The latter condition responds to a meal stimulus with an exaggerated release of gastrin. Hyperparathyroidism, as a component of multiple endocrine neoplasia type I, may contribute to the pancreatic tumor hypergastrinemia by causing hypercalcemia [61]. There does not appear to be a consistent abnormality of gastrin secretion in patients with duodenal ulceration although an increased sensitivity to gastrin has been reported [83]. Cholecystokinin

(CCK-PZ)

In 1928, Ivy and Oldberg observed the ability of fat in the small intestinal to cause

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gallbladder contraction and coined the term cholecystokinin [42]. Fifteen years later, Harper and Raper described a peptide which caused enzyme-rich secretion by the pancreas and named it pancreozymin [373. These two substances were found to be identical 33-amino acid polypeptides now termed CCK [45]. CCK exhibits both macro- and microheterogeneity. It occurs probably in 8, 33-, and 39-amino acid residue forms and may also be sulfated or desulfated. Sulfation is of particular importance for conferring biological activity, although it is possible that unsulfated forms may have an alternative physiological role as neurotransmitters. The carboxyl-terminal octapeptide portion of CCK is at least lo-fold more potent than the intact hormone [54]. CCK is found both in the brain and the upper small intestinal mucosa. It has a number of potent biological effects which include stimulation of gallbladder contraction and pancreatic enzyme secretion. In addition, it has been reported to stimulate secretion of pepsin, Brunner’s glands, and hepatic bicarbonate and water. Other activities probably relate to its gastrin homologies and include weak stimulation of gastric acid secretion, pancreatic trophic activity, and antagonism of secretin-stimulated pancreatic bicarbonate secretion [12]. Its function in the central nervous system is not clear although it has been suggested that it plays a role in the satiety mechanism [97]. The development of a radioimmunoassay for cholecystokinin has been particularly difficult since the peptide has a low immunogenicity making antibodies not easy to raise. Radiolabeling is difficult often resulting in loss of the immunoreactivity of the ligand. Furthermore, CCK circulates in a number of molecular forms which share similarities with the gastrin-like peptides; thus exact quantification in terms of antibody recognition is problematical [80]. At this time, tissue CCK can be measured with reasonable certainty but plasma CCK

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assays have difficulty in distinguishing among gastrin components and other as yet unidentified nonspecific interference mechanisms @I]. Bioassay methods have indicated that CCK release is stimulated by fat, acid, and amino acids in the small intestine [93]. It seems evident that small quantities of CCK are released into the circulation by a mechanism which is noncholinergic in nature [60]. The development of sequence-specific antibodies against portions of the cholycystokinin molecule has greatly facilitated the accuracy of CCK measurement but these are not widely available [8 11. While obvious difficulties in the interpretation of plasma CCK values exist, it has been reported that plasma CCK levels are elevated in pancreatic exocrine insufficiency [38]. In view of its potent gallbladder contractile activity, the octapeptide has been utilized for diagnostic radiology 1341. Gastric Inhibitmy Polypeptide (GIP) -Glucose-Dependent Insulinotrophic Peptide

This 43-amino acid polypeptide with a molecular weight of 5105 daltons is predominantly found in the K cells of the jejunal mucosa and to a lesser extent in the duodenum and the ileum [16]. It was initially isolated as the acid inhibitory agent in the crude gut extract containing CCK. More recently it appears that its predominant function, however, is as a glucose-dependent insulinotrophic factor rather than as an enterogastrone [2]. Radioimmunoassay measurement of plasma GIP has been difficult since it probably circulates in multiple forms and there have been problems both with characterization of standards and purification techniques. Pharmacological studies have shown that GIP has the capacity to inhibit gastric acid and pepsin secretion as well as motility. GIP inhibits gastric acid secretion stimulated by pentagastrin, histamine, or hypo-

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glycemia to an equal extent [ 131. However, more recent studies in humans have failed to demonstrate any evidence of inhibition of gastric acid secretion when GIP is given in doses which produce plasma levels resembling those found after a meal [58]. The reason for this discrepancy is not apparent but may be related to either species differences or impurities in the original preparation tested. Furthermore, duodenal perfusion with acid does not cause GIP release [lo]. The physiological role of GIP in the control of acid secretion is thus controversial [lo]. GIP is also a potent stimulant of small intestinal fluid and electrolyte secretion. GIP is released into the plasma in substantial amounts after a meal, particularly if it is high in glucose or fat content [ 131. Since GIP releases insulin from the pancreas, it has been postulated to be the gut component of the enteroinsular axis [ 151. In the presence of moderate hyperglycemia, GIP has been shown to cause a significant increase in the amounts of insulin released into plasma. Since it appears that the acid inhibitory effects of GIP are nonphysiological and that its role is more relevant to gut glucose metabolism, GIP has been renamed the glucose-dependent insulinotrophic peptide [2]. Thus, it seems likely that the most feasible role for GIP is in the postprandial modulation of insulin release. While it is tempting to speculate that abnormalities of GIP release might be linked to the pathogenesis of diabetes, studies in this area have produced conflicting results [84]. Thus, in maturity-onset diabetes, where insulin is available but not released appropriately, GIP release has been shown to be either normal or decreased. Further investigation of GIP release in diabetes secondary to alimentary disease may be more relevant. Motilin

This 22-amino acid peptide with a molecular weight of 2700 daltons is found in the

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enterochromaffin (EC) cells of the small intestine [14]. Motilin is predominantly found in the upper small intestine, although immunohistochemical studies have suggested that it is also present in the central nervous system [75]. It is released into plasma after a meal, particularly if the fat content is high. In man and pig, duodenal acidification causes an increase in plasma motilin, while in dog alkalinization has a similar effect [9]. Since it is released by a physiological stimulus, namely a meal, it has been suggested that motilin plays a role in the modulation of gastrointestinal function. The pharmacological actions of motilin include stimulation of small intestinal and gastric motility, increase in lower esophageal sphincter pressure, and stimulation of pepsin output [47]. Recent studies have indicated that motilin accelerates gastric emptying in humans and that it is also probably responsible for the initiation of the interdigestive myoelectric complexes.” Alterations in plasma motilin levels correlate closely with patterns of bowel myoelectric activity [41]. Thus, it has been suggested that motilin is in some way responsible for emptying the small intestine between meals. No physiological role in the control of esophageal or pyloric sphincter action has yet been proven. Motilin circulates in only one molecular form and no disease has yet been recognized in which plasma motilin levels are abnormal. Vasoactive

intestinal

Peptide

(VZP)

VIP has 28 amino acids and a molecular weight of 3320 daltons. It was originally isolated from porcine intestinal mucosa and identified using bioassay measurement of its powerful vasodilatory activity [85]. It :I The pattern of electrical activity from the musclenerve areas of the gut during the initiation and progression of peristaltic activity. These are measured by implanted serosal electrodes and can be defined by activity fronts and phases.

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has subsequently been shown to be present in endocrine cells and nerves of both the gut and the central nervous system 1171. There are a number of molecular forms of VIP. In common with secretin, GIP, and glucagon, it shows a number of amino acids homologies and biological actions. VIP has a wide spectrum of activities including inhibition of gastric acid secretion, stimulation of insulin release, stimulation of pancreatic water and bicarbonate secretion, stimulation of hepatic glycogenolysis, stimulation of intestinal fluid and electrolyte secretion, stimulation of mucosal cyclic AMP, relaxation of smooth muscle, and a positive inotropic action on the heart. Its exact mode of action is uncertain but it probably functions primarily as a paracrine neurotransmitter substance rather than as a circulating hormone. [27, 861. There is no significant release of VIP into the blood after a meal. Elevated levels of plasma VIP have been found in some patients with the watery diarrhea syndrome (Verner-Morrison Syndrome) [ 111. Identification and removal of pancreatic or neural tumors secreting VIP have subsequently resulted in amelioration or cure of these patients. The clinical course has correlated well with plasma levels of VIP, and in patients with plasma VIP levels of greater than 60 pmole liter-’ Bloom and colleagues have always identified a neoplasm. False negatives have occurred, but this may reflect inadequate specimen preparation. VIP is rapidly degraded since it has two separate double basic amino-acid sequences (trypsin sensitive) and an easily oxidized methionine residue. Exogenous infusion of VIP into pigs has been demonstrated to stimulate severe watery diarrhea at plasma levels greater than 60 pmole liter’ [62]. Other agents such as prostaglandins and pancreatic polypeptide have also been implicated in the genesis of the watery diarrhea syndrome [43]. Abnormally high tissue levels of VIP have been found in Crohn’s disease, while in the aganglionic colon segment of patients with Hirsch-

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prung’s disease, VIP levels have been reported to be extremely low [lo]. The exact significance of these findings is not clear. Elevated levels of VIP have been reported in experimentally induced pancreatitis and mesenteric ischemia and it has been suggested that VIP may be involved in the pathogenesis of the severe hemodynamic sequelae found under these circumstances [63]. Pancreatic

Polypeptide

(PP)

PP is a 36-amino acid polypeptide with a molecular weight of 4200 daltons originally isolated as a contaminant of chicken insulin [19, 461. It is found almost entirely in the pancreas where it is distributed in D, cells of the islets and to a lesser extent in other parts of the gland [72]. PP is liberated into the blood in large quantities after a meal by a mechanism which appears to be vagal cholinergic dependent [.59]. Its exact physiological role has not been ascertained though it is presumed to be involved in the feedback regulation of pancreatic exocrine secretion [98]. Plasma PP levels increase with age, and although the exact mechanism for this is not clear, it has been suggested that this reflects an alteration in vagal tone [91]. The pharmacologic actions of PP include inhibition of gallbladder contraction, enhancement of choledochal tone and inhibition of pancreatic enzyme output [55]. At higher dose levels of PP, a biphasic action on pancreatic bicarbonate production has been noted with initial stimulation followed by inhibition. A similar effect occurs in the stomach since PP stimulates basal gastric secretion but inhibits pentagastrin-stimulated acid secretion [68]. Although in avian species PP appears to have a primary metabolic role (causing depletion of hepatic glycogen stores), this has not been demonstrated in mammalian species. Recent studies, however, suggest that PP may also play a role in the modulation of insulin and glucagon secretion by the islets. Regulation of pancreatic polypeptide secretion appears to involve both vagal

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and hormonal factors. Thus, insulin-induced hypoglycemia stimulates PP release as do CCK, caerulein, pentagastrin, secretin, and bombesin. Acetylcholine stimulates PP release while vagotomy and anticholinergic agents inhibit it [30]. Thus, it seems likely that PP release is modulated by an enteropancreatic reflex with both neural and hormonal components [59]. Plasma PP levels have been documented to be considerably elevated in a high percentage of patients with vipomas, insulinomas, gastrinomas, and glucagonomas [74]. While some of these tumors contain PP cells, in others only PP cell hyperplasia in the rest of the pancreas is evident. The significance of these findings is not yet clear. It has, however, been suggested that elevated plasma PP levels might serve as a biochemical marker of pancreatic endocrine tumors. A number of patients with diarrhea have been noted to have pancreatic endocrine tumors composed of only PP cells (PPomas) [92]. Although it has been suggested that PP levels might be altered in chronic pancreatitis, both basal and meal-stimulated levels do not differ appreciably from the normal range [ 1011. Pancreatic polypeptide has been implicated in the pathogenesis of diabetes mellitus, but only one study has demonstrated elevated PP levels in such patients [30]. This question still requires further exploration and is complicated by the known increase of PP antibodies in the plasma of patients treated with multicomponent insulin which contains PP. Diabetic patients treated with monocomponent insulin have normal fasting plasma PP levels. Secretin Secretin is a highly basic peptide which contains 27 amino acids and has a molecular weight of 3055 daltons [45]. It is found in S cells throughout the small intestine although its highest concentrations are in the duodenum and proximal jejunum.

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Measurement of plasma secretin has been difficult since it circulates in only very small amounts and extraction techniques are required to obviate radioimmunoassay problems of nonspecific interference [ 1031. It is probable that small amounts of secretin are liberated by food and that these plasma increments stimulate pancreatic bicarbonate secretion [88]. Secretin has numerous pharmacological actions. These include inhibition of smooth muscle contraction and of gastric acid secretion, lowering of lower esophageal sphincter pressure, and a positive trophic effect on pancreatic growth. In addition to these effects, it stimulates water and bicarbonate secretion from the liver and Brunner’s glands and augments gallbladder contraction. It has a synergistic action with CCK in the stimulation of gallbladder contraction and pancreatic enzyme secretion [77]. The primary physiological role of secretin appears to be the modulation of pancreatic bicarbonate secretion; this is accomplished by the stimulation of pancreatic ductal cyclic AMP. Duodenal acidification has been demonstrated to release secretin in amounts which are both measurable and able to stimulate pancreatic secretion [28]. Secretin is clinically useful in confirming the diagnosis of gastrinoma. For reasons which are as yet unclear, the administration of a secretin bolus (2 C.U. kg-’ iv), provokes a rapid rise in plasma gastrin in patients with gastrinomas. It has little or no effect on plasma gastrin levels when hypergastrinemia is due to any other cause [23]. Failure of adequate secretin release has been suggested as a possible pathogenetic mechanism in patients with duodenal ulcer disease, but this supposition remains to be proven. Glucagon-like

Peptides

The pancreatic (Ycells produce a 29-amino acid polypeptide with a molecular weight of 3485 daltons called pancreatic glucagon [99]. It shares numerous amino acids

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homologies with secretin and consequently, many of the biological functions are similar to those of VIP, GIP, and secretin [103]. This peptide is called pancreatic glucagon to differentiate it from other glucagon-like immunoreactivities which have been found in the gut [87]. Glucagon has a number of biological actions which include relaxation of smooth muscle, inhibition of pancreatic enzyme secretion, inhibition of gastric acid secretion, stimulation of intestinal fluid and electrolyte secretion, and stimulation of cardiac output. Pancreatic glucagon is secreted primarily in response to hypoglycemia and is important in the mobilization of hepatic glycogen stores and carbohydrate homeostasis [64]. The inhibitory action of glucagon on gastrointestinal motility has enabled it to be of clinical use to endoscopists and radiologists. Its positive effect in shock-like states has not been confirmed outside of the laboratory situation. Pancreatic endocrine tumors of a-cell origin have been described which present with a clinical syndrome of migratory skin rash, anemia, diabetes, weight loss, and glossitis. The diagnosis of glucagonoma can be confirmed by radioimmunoassay measurements of elevated plasma glucagon levels. [39]. An arginine provocation test has been reported to be of some use in the diagnosis of cases with equivocal plasma glucagon levels. Intravenous infusion of arginine causes an inappropriate elevation of plasma glucagon in patients with glucagonomas. Enteroglucagon has been accepted as the term applicable to the glucagon-like immunoreactivity found in the gut. It is found in highest concentration in the distal ileum, and appears to be produced by the L cell. Gut glucagon appears to exist in a number of different molecular forms and it is not yet clear exactly what these represent [87]. Current evidence suggests that the different gut glucagon immunoreactivities represent stages in the formation of the final active peptides. A gut glucagon-glicentin has more recently been isolated from por-

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tine intestine [65]. It contains 100 amino acid residues among which are the full sequence of pancreatic glucagon and the full sequence of the proposed proglucagon fragments. Since no pure enteroglucagon exists, it is impossible to measure it accurately in plasma or to determine its exact biological activity. Thus, radioimmunoassays for plasma enteroglucagon utilize relatively crude subtraction assay techniques. However, these assays have shown that plasma enteroglucagon levels rise after a meal and are particularly increased in patients with intestinal hurry or the dumping syndrome [lo]. A single patient who suffered from severe constipation and exhibited marked small intestinal mucosal hyperplasia was found to have a tumor of the kidney producing enteroglucagon [33]. It has thus been speculated that this group of peptides comprising the enteroglucagon family is involved in the modulation of intestinal motility and possibly mucosal growth. Somatostatin

Somatostatin is a tetradecapeptide with a molecular weight of 1640 daltons. It was originally isolated from the hypothalamus and has subsequently been found in large quantities in the gastrointestinal tract [89]. It is widely distributed in the brain, and is mainly found in the periventricular region of the anterior hypothalamus. In the gut, it is present primarily in the D cells of the pancreas and gastric antrum [76]. Somatostatin has numerous effects mainly involving the inhibition of peptide hormone secretion. Its actions are thus evident in diverse parts of the body. It inhibits the release of growth hormone, thyroid stimulating hormone, gastrin, motilin, glucagon, and insulin [ 1001. Conversely, it does not interfere with the release of adrenocorticotrophic hormone, LHRH, or follicle-stimulating hormone. The biological actions include inhibition of gastric acid and pepsin secretion, delay of gastric emptying, inhibition of gallbladder contraction and inhibition of bile and pancreatic enzyme secretion [2 11. Since somato-

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TABLE 2 statin has so many effects, it is probable that it functions as a local hormone and a HUMAN GUT HORMONES member of the paracrine system. There is evidence that somatostatin acts beyond the Classical hormones Gastrin” GIP cyclic AMP system either by interfering with Cholycystokinin” Insulin exocytosis of peptide granules or by altering Secretin GhKag0n the handling of bivalent cations [90]. Candidate hormonesA number of somatostatin producing paractine Motilin” Urogastrone Somatostatin” tumors of the pancreas have recently been Enkephalin” Neurotensin” PP described (somatostatinoma) [49]. They Enteroglucagon Substance P” Chymodenin VIP’ produce an ill-defined clinical syndrome Endorphin which includes diarrhea, gallbladder disease, Biological actions and diabetes. In patients with other panEnterogastrone Enterooxyntin Gastrone Antral chalone Villikinin Coherin creatic endocrine tumors, somatostatin cell Bulbogastrone Vagogastrone Enterocrinin hyperplasia is often present in the surrounding pancreatic tissue. The reason for this is Immunoreactivities Bombesin-like” not clear, although it has been suggested Eledoisin-like that this represents an attempt by the panThyrotrophin releasing hormone-like” creas to decrease tumor hormone production [ 1071. Since somatostatin is a potent ” Present in both brain and gut. inhibitor of peptide release in both physiological and pathological situations, its use as hormones or in terms of their described a therapeutic agent in the management of pharmacological actions [36]. Terms such tumors has been suggested. The adminisas incretin or enterogastrone have been tration of long-acting somatostatin analogs coined to designate the probable existence has resulted in inhibition of peptide secreof gut peptides which respectively share tion in a number of patients with gastrinomas, functions in either modulating insulin release glucagonomas, and VIPomas with significant or inhibiting gastric acid secretion. In many amelioration of clinical symptomatology cases, such peptides are only known in crude [S]. Somatostatin has been implicated in biological extracts, and in others the term the etiology of peptic ulceration. Recent is merely used to describe a function rather reports have suggested that the usual ratio than a known agent. of somatostatin cells to G cells in antral mucosa is altered in some ulcer patients, A. Peptides Common to the Gut and especially those with G-cell hyperplasia Nervous System [IO]. This suggestion is attractive but remains to be confirmed. A number of these peptides have already been dealt with in previous sections, and MISCELLANEOUS PEPTIDES the list at this time includes gastrin, CCK, With the advent of sophisticated methodVIP, motilin, somatostatin, neurotensin, ology and the proliferation of interest in bombesin, substance P, and enkephalin. the gut as an endocrine organ, a large numThe most recently identified peptides in this ber of substances have been postulated and group are neurotensin, substance P, and the in some cases identified (Table 2). An exact endogenous opiate group which will be disrole, if any, in the function of the gut is still cussed in more detail. largely speculative. Their presence in gut (i) Neurotensin. Neurotensin is a 13endocrine cells or nervous tissue, how- amino-acid peptide originally isolated from ever, requires explanation and they are bovine hypothalamus [IS]. In man, neurotherefore discussed either as candidate tensin is mainly localized in mucosa of the

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ileum in specific endocrine cells (N cells). A number of molecular forms of neurotensin exist in both tissue and plasma. A radioimmunoassay for plasma neurotensin has recently been described which is able to detect elevated levels of plasma neurotensin after a meal. Neurotensin appears to be released by a vagally independent mechanism but its exact physiological function is not yet apparent [6]. It has been classified as a kinin since its pharmacological actions include the contraction of smooth muscle and the production of severe hypotension. In addition, neurotensin has been shown to cause cyanosis, increase vascular permeability and release adrenal corticotrophic, luteinizand follicle-stimulating hormones. iw, Other actions include effects on insulin release, hyperglycemia, gastrin release, and the lowering of body temperature [5]. While the physiological role of neurotensin is not clear at this time, marked plasma increases have been noted in patients with the dumping syndrome. It has thus been suggested that neurotensin may play a role in the pathogenesis of this condition [6]. (ii) Substance P. This undecapeptide with molecular weight of 1348 daltons was initially identified in extracts of brain and intestine [102]. More recently it has been demonstrated in the brain, intestinal nerve plexus, and enterochromaffin cells of the gut mucosa. It has been classified as a tachykinin and shares a common biological functions with a series of related peptides isolated from amphibian skin (physalaemin, phyllomedusin and uperolein). The pharmacological actions of substance P include stimulation of intestinal motility, salivary secretion, and esophageal sphincter pressure [94]. The tachykinins characteristically produce hypotension and tachycardia. Substance P probably functions as an excitatory transmitter of sensory neurons. The large quantity and wide distribution in -I A denomination suggested by the promptness of their stimulant action on smooth muscle as opposed to the group of slow-acting kinins, the true bradykinins.

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the intestine suggests strongly that it plays a role in the function of the gut. Its physiological role in the gastrointestinal tract is not clear although it is probably involved in the modulation of motility and secretion. Elevated plasma levels of substance P have been reported in patients with the carcinoid syndrome and this peptide may be involved in the pathogenesis of the protean manifestations of this condition [94]. In Hirschprung’s disease, substance P levels are extremely low in the aganglionic segments. (iii) Endogenous opiates. This group consists of two main subgroups: the pentapeptide enkephalins and the endorphins [3]. The two enkephalins differ in structure by only 1 amino acid (carboxy-terminal methionine or leucine). They are found in high concentrations both in the brain and in the G cells and myenteric plexus of the antrum and duodenum where they probably function in a paracrine fashion [95]. It has recently been reported that both morphine and methionine enkephalin are partial agonists of gastric acid secretion in dogs and that this action can be blocked by nalaxone, atropine, or metiamide [48]. Current evidence thus suggests that this group of substances may well have a role in the regulation of gastric acid secretion and gut motility. The main member of the endorphin family is p-endorphin which is a 31-amino acid peptide found predominantly in the brain but not yet conclusively identified in the gut. B. Peptides Related to Acid Secretion (i) Urogastrone. Human urogastrone has recently been isolated and synthesized by Gregory [35]. It is a 53-amino acid polypeptide with a molecular weight of about 6000 daltons, although larger molecular forms occur [35]. Urogastrone is very similar to epidermal growth factor both in structure (37-amino acid homologies and three identical disulfide bridges) and function. Thus, both peptides are powerful inhibitors of gastric acid secretion and stimulate epithelial cell proliferation. Urogastrone is found in submaxillary glands of mice, but its source in

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humans has not yet been identified. Its inhibitory effect on gastric acid secretion is independent of gastrin and low doses (0.25 pg kg-’ hr-‘) cause 60 to 80% inhibition of stimulated gastric acid secretion from canine Heidenhein pouches [32]. The two properties of acid inhibition and promotion of cell growth have led to considerable speculation as to the role of urogastrone in the therapy of peptic ulcer disease [103]. Appropriate studies to test this hypothesis have not yet been reported. Urogastrone, like epidermal growth factors, stimulates growth of epidermal cells and fibroblasts. There is some evidence that it modulates repair and cell replication in the gut. (ii) Antral chalone. This peptide is postulated to exist in the antrum and to be responsible for the inhibition of gastric acid secretion. The recent discovery of somatostatin in this location may well resolve this physiological conundrum. (iii) Bulbogastrone. The physiological postulate of a duodenal bulb peptide distinct from secretin which would inhibit gastric acid production has obvious relevance. No such peptide has yet been isolated or sequenced although a humoral principle with this function has been identified. (iv) Gastrone. Code and colleagues partially purified a glycoprotein with a molecular weight of 12,000 to 18,000 daltons from gastric mucosa and juice which inhibited gastric acid secretion [20]. There has, however, been controversy as to whether this represented a peptide secretory product of the stomach or a contaminant bacterial endotoxin. Thus, while the existence of such an inhibitory agent is intriguing, it has not yet been clearly proven. (v) Enterogastrone. This is a substance reported to be released by fat from the intestinal mucosa and said to cause inhibition of acid secretion. It had been initially suggested that GIP might be the enterogastrone, but more recent work has rendered this suggestion unlikely. The existence of this substance thus remains only as a postulate. (vi) Vagogastrone. Vagogastrone is an

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inhibitor of Heidenhein pouch acid secretion and has been postulated to be released by vagal stimulation. No such peptide has, however, been isolated or identified. (vii) Enterooxyntin. This is the name proposed for a substance released by the presence of protein digestive products in the intestine. Its action involves augmentation of gastric acid secretory response by a gastrin-independent mechanism. The exact agent responsible has not yet been isolated. C. Peptides Related to Pancreatic-Intestinal

Function

(i) Chymodenin. This peptide has not yet been sequenced but has a molecular weight of 4900 daltons. It is present in duodenal mucosa and causes selective secretion of pancreatic chymotrypsin [ 11. The confirmation of its existence and exact action would throw serious doubt upon the current concept that pancreatic enzymes are secreted in a relatively fixed ratio corresponding to their quantities in pancreatic acinar cells. (ii) Coherin. This is a peptide isolated from the posterior pituitary which produces coordinated jejunal contraction in dogs. It has not been identified in the gastrointestinal tract of man or animal. (iii) Duocrinin. A substance which stimulates secretion of juice from Brunner’s glands, duocrinin has not yet been purified or isolated and may represent a heterogenous group of biologically active agents. (iv) Enterocrinin. A substance similar to duocrinin which stimulates fluid and electrolyte secretion by small intestinal mucosa, enterocrinin is also probably a mixture of biologically active agents. (v) Villikinin. This appears to be a small acidic peptide which has been partially purified from small intestinal extract. It stimulates villus motility, presumably therefore promoting microenvironmental mixing of substrates and facilitating nutrient absorption mechanisms. D. Amphibian Peptides In recent times, a number of peptides have been isolated from amphibian and

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marine species which have potent biological actions involving the gastrointestinal tract [26]. Although some have not as yet been isolated and sequenced from human tissue, recent immunohistochemical studies have indicated that many of these peptides probably exist in similar forms in man. Thus, four main groups of these peptides exist: bradykinin-like, eledoisin-like, caerulein-like and bombesin-like [57]. The bradykinin-like group is mainly involved in slow smooth muscle contraction activity and is thus probably related to gut motility and blood flow. The eledoisin-like group is characterized as tachykinins and include substance P, uperolein, physalaemin, and phylomedusin. It seems likely that they function in a paracrine fashion and are particularly involved in motility and possibly secretion. The caerulein-like group closely resembles cholecystokinin and appears to have much the same range of actions. The bombesin-like peptides include litorin, ranatensin, and alytensin. Bombesin-like immunoreactivity has been found throughout the human gastrointestinal tract particularly in nerve cells [104]. All members of this group are potent stimulators of gastrin, pancreatic polypeptide, gastric acid secretion and exocrine pancreatic secretion. It seems highly probable that many of these amphibian peptides will have human counterparts and be of importance in the regulation of gastrointestinal function. REFERENCES I. Adelson, J. W., and Rothman, S. S. Chymodenin, a duodenal peptide: Specific stimulation of chymotrypsinogen secretion. Amer. J. Physiol. 229: 1680, 1975. 2. Anderson, D., Elahi, D., Brown, J. C., Tobin, J. D., and Andres, R. Oral glucose augmentation of insulin secretion. J. Clin. Invest. 62(l): 152, 1978. 3. Ambinder, R. F., and Schuster, M. M. Endorphins: New gut peptides with a familiar face. Gastroenterology 77(5): 1132, 1979. 4. Berson, S. A., and Yalow, R. S. Progress in gastroenterology: Radioimmunoassay in gastroenterology. Gostroenferology 62: 1061, 1972. 5. Bisette, G., Manberg, P., Nemeroff, C. B., and

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Prange, A. J., Jr. Neurotensin, a biologically active peptide. Life Sci. 23: 2173, 1978. 6. Blackburn, A. M., and Bloom, S. R. A radioimmunoassay for neurotensin in human plasma. J. Endocrinol. 83, 175, 1979. 7. Bloom, S. R. Cur Hormones. Edinburgh: Churchill Livingstone, 1978. Pp. 1% 17. 8. Bloom, S. R. New specific long-acting somatostatin analogues in the treatment of pancreatic endocrine tumors. Gut 19: 446, 1978. 9. Bloom, S. R., Mitznegg, P., and Bryant, M. G. Measurement of human plasma motilin. Scnnd. J. Grrstvoentevol. Il(Suppl. 39): 47, 1976. 10. Bloom, S. R., and Polak, J. M. In B. Jerzy Glass (Ed.), PI-ogres5 in Gosrroenterology. New York: Grune & Stratton 1977. Pp. 109-151. I I. Bloom, S. R., Polak, J. M., and Pearse, A. G. E. Vasoactive intestinal peptide and watery diarrhea syndrome. Lancer 2: 14, 1973. 12. Brooks, A. M., and Grossman, M. I. Effect of secretin and cholecystokinin on pentagastrin stimulated gastric secretion in man. Gastroenterology 59: 114, 1970. 13. Brown, J. C. Physiology and pathophysiology of GIP. Advan. Exp. Med. Biol. 106: 169, 1978. 14. Brown, J. C., and Dryburgh, J. R. Discovery of motilin. Stand. J. Gastrornterol. ll(Supp1. 39):15, 1976. 15. Brown, J. C., and Otto, S. C. GIP and the entero-insular axis. Clin. Endocrinol. Merab. 8(2): 365, 1979. 16. Brown, J. C., Pederson, R. A., Jorpes, E., and Mutt, V. Preparation of a highly active enterogastrone. Canad. J. Physiol. Pharmacol. 47: 113, 1969. 17. Bryant, M. G., Bloom, S. R., and Polak, J. M., Abuquerque, R. H., Modlin, I. M., and Pearse, A. G. E. Possible dual role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance. Lancer 1: 991, 1976. 18. Carraway, R., and Leeman, S. The isolation of a new hypotensive peptide, neuotensin, from bovine hypothalami. J. Biol. Chem. 24(19): 6854, 1973. 19. Chance, R. E., Moon, N. E., and Johnson, M. G. Human pancreatic polypeptide (HPP) and bovine pancreatic polypeptide (BPP). In B. M. Jaffe and H. R. Behrman (Eds.), Methods of Hormone Radioimmunoassay. New York: Academic Press, 1978. P. 657. 20. Code, C. F. The recognition and assay of gastrone: gastric secretion, mechanism and control. In J. A. L. Shnitlag and R. C. Gilbert (Eds.), Proceedings of the Symposium on Gastric Secretion,

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91. Schwartz, T. W. Pancreatic polypeptide as indicator of vagal activity. In J. H. Rehfeld and E. Amdrup (Eds.), Gasrrin and the Vagus. New York: Academic Press, 1979. P. 175. 92. Schwartz, T. W. Pancreatic polypeptide and endocrine tumors of the pancreas. Scorzd. J. Gastroenrerol. 14(Suppl 53): 93, 1979. 93. Singh, M., and Webster, P. D. Neurohormonal

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