1154 Current Topics in Medicinal Chemistry, 2007, 7, 1154-1165

1568-0266/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.

The Biology of Cholecystokinin and Gastrin Peptides

Jens F. Rehfeld*, Lennart Friis-Hansen, Jens P. Goetze and Thomas V. O. Hansen

Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

Abstract: Cholecystokinin (CCK) and gastrin together constitute a family of homologous peptide hormones, which are

both physiological ligands for the gastrin/CCK-B receptor, whereas the CCK-A receptor binds only sulfated CCK-

peptides. CCK peptides are mainly produced in small intestinal endocrine I-cells and in cerebral neurons. CCK peptides

regulate pancreatic enzyme secretion and growth, gallbladder contraction, intestinal motility, satiety and inhibit gastric

acid secretion. Moreover, they are potent neurotransmitters in the brain and the periphery. CCK peptides are derived from

proCCK and have the bioactive heptasequence -Tyr(SO4)-Met-Gly-Trp-Met-Asp-Phe-NH2 as their C-terminus. The

dominant forms in plasma are CCK-58, CCK-33, CCK-22 and CCK-8, whereas CCK-8 is the major transmitterform. Due

to scarcity of specific assays, knowledge about CCK in disease is still limited. Gastrin peptides are mainly synthetized in

antroduodenal G-cells, from where they are released to blood to regulate gastric acid secretion and mucosal growth. Small

amounts are synthetized further down the intestinal tract, in the foetal pancreas, in a few cerebral and peripheral neurons,

in the pituitary gland and in spermatozoes. Gastrin peptides are derived from progastrin and all have the C-terminal

bioactive hexasequence -Tyr (SO4)-Gly-Trp-Met-Asp-Phe-NH2. The major gastrin forms in tissue and plasma are gastrin-

34 and gastrin-17, but also gastrin-71, -14 and -6 have been identified. Gastrin peptides are secreted in excessive amounts

from gastrinomas and are expressed at lower levels in bronchogenic, colorectal, gastric, ovarian and pancreatic cancers. A

carcinogenetic significance of gastrin peptides remains, however, to be proven.

INTRODUCTION

Cholecystokinin (CCK) and gastrin are members of a family of neuroendocrine peptides. Their common C-termi-nal sequence has been exceedingly well preserved during evolution [1, 2]. It is possible that their common ancestor resembles a dityrosyl-sulfated peptide, cionin, which has been isolated from the central ganglion (the “brain”) of protochordates [3]. The family includes also the frogskin peptides, caerulein and phyllocaerulein, but CCK and gastrin are the only known members of the family in mammals (Fig. 1). Phylogenetically speaking, early members of the family look more CCK- than gastrin-like; and it is first at the level of elasmobranch that separate CCK and gastrin genes are expressed [2]. Curiously, elasmobranchs are also the first organisms in evolution to produce gastric acid in the stomach.

Cholecystokinin (CCK) was discovered as a gallbladder-contracting hormone in extracts of the small intestine in 1928 [4]. The last decades, however, have shown that CCK, in addition to hormonal cholecystokinetic and pancreozymic actions, also is a growth factor for the pancreas and a potent neurotransmitter in both the central and peripheral nervous systems [for reviews, see refs. 5-7]. The long history has made the CCK literature comprehensive but confusing. Thus, early physiological studies often used impure CCK preparations with little attention paid to species differences and to physiological dosing. Also, most assays used to monitor CCK concentrations in plasma and elsewhere have until recently lacked specificity and sensitivity [8-10].

*Address correspondence to this author at the Dept. of Clinical Biochemistry, Rigshospitalet (KB 3014), DK-2100 Copenhagen, Denmark; Tel: +45 35 30 18; Fax: +45 35 46 40; E-mail: jens.f.rehfeld@rh.regionh.dk

Gastrin was discovered already in 1905 as a gastric acid-stimulating factor in extracts of the antral mucosa of the stomach [11]. Identification of gastrin and CCK peptides in the 1960’s revealed that their C-terminal sequences are identical and constitute the active site of both hormones [12-15]. During the last decades, the concept of gastrin as a simple peptide hormone from the upper digestive tract has, like that of CCK, changed considerably. Now gastrin is as CCK known to occur in multiple molecular forms. And the gastrin gene is like that of CCK expressed in a cell-specific manner in neurons, endocrine cells and other epithelial cells outside the gastrointestinal tract. Moreover, gastrin has been shown to stimulate growth on the gastric mucosa and perhaps elsewhere [16, 17].

All known biological effects of gastrin and CCK peptides reside in the conserved common C-terminal tetrapeptide amide sequence (Fig. 1). Modification of this sequence grossly reduces or abolishes receptor binding and biological effects [18]. The different N-terminal extensions of the com-mon C-terminal sequence increase the biological potency and the specificity for receptor binding. Of particular importance is the tyrosyl residue in position six of mam-malian gastrins and position seven of CCK peptides, as counted from the C-terminal phenylalanyl amide (Fig. 1). The tyrosyl residue is partly sulfated in gastrins [12, 13], and more completely sulfated in CCKs [19]. The gastrin/CCK-B receptor binds sulfated and unsulfated ligands equally well, whereas the CCK-A receptor requires sulfation of the ligand. The gastrins are consequently defined as peptides that stimulate gastric acid secretion and have the C-terminal sequence Tyr-X-Trp-Met-Asp-Phe-NH2, and CCK peptides are defined as gallbladder contracting peptides with the C-terminal sequence Tyr-Met-X-Trp-Met-Asp-Phe-NH2 (where X in most mammalian species is a glycyl residue).

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In the following, the biology of first CCK and then of gastrin will be reviewed. Receptors and signal transductions will be mentioned only briefly, since they are subject of the following articles in this volume.

CHOLECYSTOKININ

Cellular Synthesis of Cholecystokinin Peptides

The transcription unit of the CCK gene is 7 kilobases interrupted by two introns [20]. The first of the three exons is small and noncoding. Except for the homology with the gastrin gene in the coding region and in the 5’-untranslated region (5’ UTR), the structure of the CCK gene displays no unique features. Several conserved regulatory elements have been identified in first 100 bp of the human promoter, including an E-box element, a combined cAMP response element (CRE)/12-O-tetradeconoylphorbol-13-acetate res-ponse element (TRE), and a GC-rich region [21, 22]. Whereas the function of the E-box and the GC-rich region is not fully clarified [23, 24], the combined CRE/TRE sequence plays an important role in the regulation of CCK transcription. The CRE/TRE binds the transcription factor CREB, which is activated by phosphorylation by several signalling pathways, including cAMP, fibroblast growth factor (FGF), pituitary adenylate cyclase-activating polype-ptide (PACAP), calcium, hydrolysates and peptones to ultimately induce CCK transcription [25-29]. Only one CCK mRNA molecule has been found, and the CCK peptides are thus fragments of the same mRNA product. The mRNA has 750 bases, of which 345 are protein coding [30, 31]. The concentrations of CCK mRNA in cerebrocortical tissue are similar to that of the duodenal mucosa [31] and at least in the brain there is a rapid synthesis of CCK peptides [32].

The translational product, preproCCK, has 115 amino acid residues (Fig. 2). The first part is the signal peptide. The second part with considerable species variation is a spacer. The bioactive CCK peptides are derived from the subsequent 58 amino acid residues [14, 19, 33-36], and the species

variation is small in this sequence. The processing of proCCK is cell-specific, where endocrine cells contain a mixture of medium-sized CCKs. In contrast, neurons mainly release CCK-8 [34, 37]. Thus, the cell-specific synthesis is essential for the CCK functions in the different compar-tments of the body. The endoproteolysis of proCCK occur mainly at monobasic sites (Fig. 2). Tyr-77 is O-sulfated, which determines the affinity for the receptors. The specific bioactive CCKs have 83, 58, 39, 33, 22 and 8 amino acid residues (Fig. 2).

In the small intestine, CCK peptides are synthesized in endocrine I-cells [38], whose apical membrane is in direct contact with the intestinal lumen and whose basal region contains secretory granules with CCK peptides. CCK is also synthesized in pituitary corticotrophs and melanotrophs and in a few adrenal medullary cells [39, 40]. In the pituitary cells, CCK constitutes a small fraction of the hormones. Tumours originating from pituitary corticotrophs, however, produce substantial amounts of CCK [41].

It is the brain that produces most CCK [34, 39]. More-over, cerebral CCK neurons are more abundant than neurons of any other neuropeptide [34, 39, 42]. While most pepti-dergic neurons occur in subcortical regions, CCK is expressed in the highest concentrations in neocortical neurons [39, 42, 43]. The perikarya of the cortical CCK ner-ves are distributed in layers II-VI, with the highest frequency in layers II and III [39, 44]. CCK in mesencephalic dopa-mine neurons projecting to the limbic area of the forebrain [45] has aroused particular clinical interest, because these neurons are supposed to be involved in schizophrenia.

Outside the brain, the colon contains numerous CCK nerves, whereas jejunum and ileum is sparsely innervated [39]. Colonic CCK fibers occur mainly in the circular muscle layer, which they penetrate to form a neural plexus in the submucosa [39]. In accordance with these locations, CCK peptides have been shown to excite colonic smooth muscles and to release acetylcholine from neurons in both plexus myentericus and submuca [46]. Ganglionic cell bodies in the

Fig. (1). The homologous bioactive sequences of cholecystokinin, gastrin, caerulein, phyllocaerulein and cionin. The evolutionary preserved

bioactive tetrapeptide amide is boxed.

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pancreas are also surrounded by CCK nerves [47]. Moreover, in man, pig, and cat CCK nerve terminals surround pancreatic islets [48]. The origin of intestinal and pancreatic CCK nerve fibers is uncertain. Finally, afferent vagal nerve fibers also contain CCK [49, 50].

Cellular Release of Cholecystokinin

CCK in circulation originates predominantly from the intestinal endocrine cells. The release to blood was not possible to examine until specific assays were developed [8-10, 51]. These assays have confirmed most classic ideas about CCK. Thus, protein- and fat-rich food is the most important stimulus [9, 51]. Of the constituents, protein and L-amino acids as well as digested fat cause significant CCK release [51, 52]. Carbohydrates only release small amounts of CCK [51], but hydrochloric acid also stimulates release [52].

The release from neurons has been examined directly in brain slices and synaptosomes [53, 54]. Potassium-induced depolarization caused a calcium-dependent release of CCK-8. Similarly, depolarization releases CCK peptides from the

hypothalamic dopamine neurons that innervate the intermediate lobe of the pituitary [55]. It is not known to what extent neuronal CCK overflows to plasma. By analogy with other neuropeptides, CCK of neuronal origin might constitute a small part of circulating CCK.

Cholecystokinin in Plasma

By comparison with identified forms in tissue, it has been possible to deduce the molecular nature of CCK in plasma. The picture has varied [10], partly due to species differences, partly because the molecular pattern along the small intestine varies [56] so that venous blood from the duodenum contains more CCK-8 than blood from the distal gut [57]. Further-more, the distribution may vary during stimulation. In man, CCK-22 and CCK-33 predominate in plasma, but CCK-8 and CCK-58 are also present [9, 58].

In the basal state, the concentration of CCK in plasma is 1 pmol/l or less. The concentration increases within 20 min to 3-5 pmol/l during maximal stimulation, and then declines gradually to basal levels (Fig. 3). In comparison with most other pancreatic and gastrointestinal hormones [59], the

Fig. (2). Co-and posttranslational processing of procholecystokinin (proCCK). Mono- and dibasic cleavage sites are indicated.

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concentrations of CCK in plasma are low. When food-induced CCK in plasma is mimicked by infusion of exogenous CCK, the same degree of gallbladder contraction and release of enzymes as seen during meals occurs [50, 59-61]. Therefore, circulating CCK is sufficient to account for the gallbladder contraction and pancreatic enzyme secretion during meals (Fig. 3).

Because the cholecystokinetic and pancreozymic potency of CCK-33 and CCK-8 on a molar base are identical [62], it may seem less important what I-cells releases during digestion. On the other hand, CCK-58, -33 and -22 are cleared from blood at a significantly slower rate than CCK-8. It is therefore important to know the molecular pattern of CCK in plasma.

Effects of CCK

The CCK Receptors

The cellular effects of CCK peptides are mediated via two receptors [63, 64]. The “alimentary” CCK-A receptor [63] mediates gallbladder contraction, relaxation of the sphincter of Oddi, pancreatic growth and enzyme secretion, delay of gastric emptying, and inhibition of gastric acid secretion via fundic somatostatin [65]. CCK-A receptors have been found also in the anterior pituitary, the myenteric plexus and areas of the midbrain [66, 67]. The CCK-A receptor binds CCK peptides that are amidated and sulfated with high affinity, whereas the affinity for non-sulfated CCK peptides and gastrins is negligible.

The CCK-B receptor (the “brain” receptor) is the predo-minant CCK receptor in the brain [64, 68]. It is less selective than the CCK-A receptor, as it binds also non-sulfated CCK, gastrins and short C-terminal fragments with high affinity. Data on the gastrin receptor cloned from parietal cells [64] show that the gastrin and CCK-B receptor are identical [68]. The gastrin/CCK-B receptor is abundantly expressed also in the pancreas of man [69, 70].

Gallbladder Emptying, Bile Release, Pancreatic Secretion

and Growth

CCK peptides stimulate hepatic secretion mainly as bicarbonate from hepatic ductular cells [71] and act on gallbladder muscles with a potency correlated to the low plasma concentrations of sulfated CCK (Fig. 3). From the liver and gallbladder, bile is released into the duodenum via CCK-mediated rhythmic contraction and relaxation of muscles in the common bile duct and the sphincter of Oddi. CCK regulates the secretion of pancreatic enzymes so potently that it seems sufficient to account for all enzyme secretion [60-62]. CCK is also capable of releasing several small intestinal enzymes such as alkaline phosphatase [72], disaccharidase [73] and enterokinase [74]. In addition, CCK stimulates the synthesis of digestive enzymes such as pancreatic amylase, chymotrypsinogen, and trypsinogen [75-77].

While the effect of CCK on the exocrine pancreatic secretion was for many years considered restricted to enzyme secretion, it is now well established that CCK can also stimulate fluid and bicarbonate secretion. The effect on bicarbonate secretion is in itself weak, but because CCK

Fig. (3). Plasma cholecystokinin (CCK) concentrations and gallbladder volume during a meal in normal human subjects (n = 8).

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potentiates the secretin-induced bicarbonate secretion from the pancreas in the same way as secretin potentiates the CCK-induced enzyme release [78], the effect of CCK peptides on bicarbonate and fluid secretion is sufficiently potent under physiological circumstances (Fig. 4). There are species differences in the mechanism of the CCK-effect on pancreatic exocrine secretion. Hence, it is now generally assumed that CCK in man stimulates pancreatic enzyme secretion through a cholinergic pathway that is considerably less significant in rodents [79-81].

Fig. (4). Pancreatic bicarbonate output during infusion of a physio-

logical dose of cholecystokinin (CCK-8), a physiological dose of

secretin, or physiological doses of secretin plus CCK in normal

human subjects (n = 6).

In man and pig CCK-33 and -8 are weak insulin and glucagon secretagogues [48, 82], whereas CCK-5 may be more potent [48]. In contrast, CCK-8 in low concentrations release insulin and glucagon in dog and rat [83, 84]. The species differences seem to be due to innervation of pancreatic islets with terminals that release small molecular forms of CCK in man and pig [48], whereas rat and dog islets have no such innervation [47, 48]. Moreover, islet cells in man and pig also express the gastrin/CCK-B receptor [69, 70], whereas rat islet cells express mainly the CCK-A receptor [85].

Already in 1967, Rothman and Wells [77] noted that CCK increased pancreatic weight and enzyme synthesis. Also the maximal output of bicarbonate and protein from the hypertrophic pancreas was increased [86]. Although secretin in itself is without trophic effects, the combination of secretin and CCK showed additional trophic effects on ductular cells with subsequent increase of secretin-induced bicarbonate output [86].

Gastrointestinal Motility and Blood Flow

CCK peptides contribute to the control of motility of the digestive tract. The effect on gastric motility varies consi-derably between species; and it is still uncertain whether

circulating CCK affects the motility of the stomach under physiological conditions. In contrast to the stomach, the distal part of the gut is abundantly innervated with CCK neurons [39, 87]. It is therefore likely that an increase of intestinal motor activity by exogenous CCK [88] reflect control of intestinal muscles by CCK. Neuronal CCK probably acts both indirectly via acetylcholine release from postganglionic parasympathetic nerves and directly on muscle cells [30]. The observation that CCK peptides stimu-late intestinal blood flow is in harmony with the CCK nerve terminals around blood vessels in the basal lamina propria and the submucosa of all parts of the intestinal tract [39].

Satiety

In 1973, Gibbs, Young, and Smith discovered that exoge-nous CCK inhibits food intake [89]. The effect was dose dependent and specific in the sense that it mimicked the satiety induced by food and was not seen with other gut peptides known then. The effect could be demonstrated in several mammals. Vagotomy studies indicate that peripheral CCK induces satiety via gastric receptors relaying the effect into afferent vagal fibers [90]. The satiety signal then reaches the hypothalamus from the vagus via the nucleus tractus solitarius and area postrema.

Inhibition of Gastric Acid Secretion

The effect of CCK on gastric acid secretion has until recently been uncertain. On one hand, it has been suggested that CCK was an acid inhibitor released from the intestine during meals. On the other hand, the results from CCK infusions were inconsistent. Recently, the question was solved in gastrin/CCK double “knockout” mice [65], which showed that circulating CCK is a potent acid inhibitor that stimulates somatostatin release from fundic D-cells via CCK-A receptors. The local somatostatin in the fundic mucosa then inhibits acid secretion from parietal cells [65]. Thus, CCK is a potent enterogastrone.

GASTRIN

Cellular Synthesis of Gastrin Peptides

The cloning of mammalian gastrin and CCK genes shows that the genes are structurally similar, both in the overall exon-intron organisation and in certain peptide coding sequences [20, 30, 91, 92]. The gastrin gene spans 4.1 kb chromosomal DNA and contains two introns of 3041 and 130 bp, respectively. Antral G-cells generate a single mRNA of 0.7 kb, which encodes the 101 amino acid preprogastrin in man and mouse, whereas other mammalian preprogastrins have 104 amino acids due to a prolonged C-terminal flanking peptide (Fig. 5). The first exon encodes the 5'-untranslated region [for reviews, see refs. 93 and 94].

Several studies have identified important regulatory domains in the gastrin gene promoter [95-101, for review, see ref. 102]. Hence, a cell-specific regulatory element has been located in the cap-exon I region of the human gastrin gene; and a pancreatic islet cell specific regulatory domain in the gastrin promoter, containing adjacent positive and negative DNA elements have also been identified [95, 96]. This regulatory domain may be a switch controlling the transient transcription of the gastrin gene in the pancreatic

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islets during foetal and neonatal development [95, 96]. The gastrin gene transcription is stimulated by epidermal growth factor (EGF) and inhibited by somastostatin [97-101]. The EGF-responsive element is of particular relevance for the understanding of the growth promoting and oncological significance of gastrin.

So far, no tissue-specific splicing or use of alternative promoters has been described. Therefore, the tissue-specific molecular pattern of gastrin peptides is due to differences in the post-translational processing rather than alternative RNA splicing.

In the developing rat colon, gastrin mRNA concen-trations increase from birth to adult apparently without a corresponding increase in peptide synthesis [103]. Therefore, expression of the gastrin gene also appears to be regulated at the translational level. The expression of the gastrin gene is ontogenetically regulated. Consequently, expression of the gastrin gene in tumors may involve factors that normally are expressed only in foetal life.

The antral G-cells are the main site of gastrin synthesis and biosynthesis studies have so far focused on antral tissue [104-108]. After translation of gastrin mRNA in the

Fig. (5). Co- and posttranslational processing of progastrin to the predominant gastrins, -34 and -17. Dibasic sites are indicated.

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endoplasmic reticulum and removal of the N-terminal signal peptide from preprogastrin, intact progastrin is transported to the Golgi apparatus. In the trans-Golgi network, O-sulfation of the tyrosyl-66 residue neighbouring the active site, and the first endoproteolytic convertase cleavage at two monobasic processing sites occur. From the trans-Golgi network, vesicles carry the processing intermediates towards the basal part of the G-cells, where the gastrin peptides are stored in granules. The cleavage by prohormone convertases 1 and 2 (PC1/3 and PC2), the exoproteolytic carboxypeptidase E trimming as well as the subsequent glutamyl cyclization at the N-termini of gastrin-34 and gastrin-17, continue during the transport from the Golgi to the immature secretory granules (Fig. 5). The last and decisive processing steps in the synthesis of gastrin then occurs during storage in the secretory granules where the amidation enzyme removes glyoxylate from the glycine-extended intermediates, to complete the synthesis of bioactive carboxyamidated pep-tides. Notably, amidation of gastrin is a all-or-none activa-tion process, which is carefully controlled. Activation of the enzymatic amidation process requires copper, oxygen and ascorbic acid as cofactors and a pH around 5.

Cellular Release of Gastrin

As a result of the elaborate progastrin processing, the antral G-cells release a mixture of progastrin products from the secretory granules to blood. In man, a few per cent are non-amidated precursors, mainly glycine-extended gastrins, whereas more than 95% are -amidated bioactive gastrins. Of these, 85% is gastrin-17, 5-10% gastrin-34 and the rest is a mixture of gastrin-71, gastrin-14 and the short gastrin-6. Approximately half the amidated gastrins are tyrosyl-sulfated. Due to gross differences in metabolic clearance rates, the distribution pattern of gastrins in peripheral plasma changes, so that larger gastrins with their long half-lives predominate over gastrin-17 and shorter gastrins. Hence, gastrin-34 is the predominant form of gastrin in peripheral blood.

Increased gastrin synthesis changes the molecular pattern in plasma further as seen in achlorhydria, where the translational activity of gastrin mRNA in G-cells seems to be so high that the processing enzymes cannot keep up with the maturation. Consequently, G-cells release more incompletely processed non-amidated progastrin products when the synthesis is increased. Also, the carboxyamidated gastrins are less sulfated and the N-terminus of progastrin cleaved to a lesser degree.

Gastrin peptides are also released from cell types other than the antroduodenal G-cells. Quantitatively, these other cells contribute only little to circulating gastrin; partly because the secretion seems to serve local purposes, and partly because the biosynthetic processing is cell-specific. So far, expression of progastrin has been encountered also in the ileum and the colon [103, 109]; in endocrine cells in the foetal and neonatal pancreas [110, 111]; in pituitary corticotrophs and melanotrophs [112, 113]; in oxytocinergic hypothalamo-pituitary neurons [114]; in a few cerebellar and vagal neurons [115, 116]; in the bronchial mucosa [117]; in postmenopausal ovaria [118]; and in human spermatogenic cells [119]. The concentrations and presumably also the

synthesis in the extra-antral tissues are far below that of the antral “main factory”. The function of gastrin synthesized outside the antroduodenal mucosa is not yet fully known; but a possibility is paracrine or autocrine regulation of growth. It is also possible that the low expression is without significant function in the adult, but rather a relic of a more compre-hensive foetal synthesis for local stimulation of growth. A third possibility is that the low cellular and, hence, low tissue concentration is due to constitutive rather than regulated secretion.

Although the extra-antral synthesis of gastrin may be without function in the adult organism, recognition of the phenomenon has biomedical interest. Hence, tumors origi-nating from cells that express the gastrin gene at low levels in adult organisms may produce gastrin in significant amo-unts in tumors. Well-known examples are pancreatic, duode-nal and ovarian gastrinomas, which give rise to the Zollin-ger-Ellison syndrome [111, 118].

Gastrin in Plasma

The molecular nature of gastrin in plasma, and the physiological variations during meals have been well studied during the last decades [for reviews, see refs. 120, 121]. The predominant circulating forms in most mammals are gastrin-34 and gastrin-17, that both occur in sulfated and non-sulfated forms. In addition, small amounts of gastrin-71 and gastrin-14 [120] may be present. Only the cat differs, since gastrin in peripheral feline blood is all gastrin-14 [122]. During hypergastrinemia in hypo- and achlorhydria, during treatment with antacids and in gastrinoma patients, the pattern changes to be dominated by large molecular forms, gastrin-71 and gastrin-34 [107]. The shift is partly due to attenuated endoproteolytic processing of progastrin during G-cell hypersecretion [107], and partly to slower clearance of the larger forms. Thus, gastrin-34 has in man a halflife in circulation of 40 min contrasting to 4 min for gastrin-17 peptides [123].

In the basal state, the concentration of gastrin in plasma varies between 10-20 pmol/l. During stimulation with a protein-rich meal, the concentrations increase within few minutes to reach a peak around 50 pmol/l after 15-20 min. The gastrin concentrations are at the same level as most other gastrointestinal and pancreatic hormones [59]. Notably, however, they are 10-20 fold above those of CCK in plasma [9, 58]. This difference is important for receptor selection. Hence, gastrin/CCK-B type receptors in the periphery are in physiological terms the receptors only for gastrin, since the 10-20 fold lower CCK concentrations in plasma cannot compete. Thus, in normal mammals CCK effects in the periphery are elicited essentially only via CCK-A receptors, that do not bind the gastrins. The described gastrin concentrations in plasma are sufficient to account for regulation of gastric acid secretion via gastrin receptor-binding on ECL-cells, and subsequent release of histamine.

Effects of Gastrin

The Gastrin Receptor

The gastrin/CCK-B receptor is as described above the predominant receptor for gastrin and CCK peptides in the

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central nervous system [64, 69, 124]. It is expressed with particularly high density in the cerebral cortex [69] where it binds both sulfated and non-sulfated gastrin and CCK peptides as well as short C-terminal fragments of CCK (CCK-5), all with similar high affinity. The gastrin/CCK-B receptor is also abundantly expressed on ECL-cells in the stomach [125, 126] and at a lower level in the pancreas of man and pig [70, 126, 127]. In physiological terms, the CCK-B receptor expressed outside the nervous system only uses gastrins as ligands. Here, it is simply the gastrin receptor.

Gastric Acid Secretion

The main effect and purpose of gastrin is to stimulate gastric acid secretion. Gastrin was discovered and defined by its effect on gastric acid secretion [11]. Accordingly, separate gastrin peptides, distinct from CCK, first occurred during evolution in elasmobranchs in which the stomach apparently also first began to secrete acid [for review, see ref. 2]. The fundamental link between gastrin and gastric acid secretion has recently been further emphasized in genetically modified animals. For instance, the stomach in gastrin knock-out mice do not secrete acid, not even after stimulation with secretagogues such as histamine and carbachol [128, 129] (Fig. 6). Only infusion of gastrin for 1-2 weeks revives the acid producing machinery with responses to histamine and carbachol [128].

Gastric acid is essential for initiation of food digestion. In addition, however, acid constitutes a defence barrier against microbial invasion of the gut, which again appears to help prevention of cancer in the stomach. Hence, old gastrin knock-out mice develop after 1.5 years of age adenocarcino-mas in the stomach [130, 131]. Consequently, gastrin seems through its stimulation of gastric acid necessary for the maintenance of life in mammals.

Gastric acid secretion is regulated by gastrin via the gastrin/CCK-B receptor on ECL-cells, which again release histamine to stimulate parietal cells in the fundic mucosa. Parietal cells also express gastrin receptors, but at a lower level. Therefore, most of the effect of gastrin is indirect via ECL-cells and histamine [for reviews, see refs. 125, 132, 133]. Careful examination of gastrin release during meals, and subsequent infusions in physiological doses of gastrin-17 and gastrin-34 have shown that normal gastrin release to circulation during meals is sufficient to account for the gastric phase of acid secretion [for reviews, see ref. 121]. Gastrin interacts with a number of other stimulatory and inhibitory substances in the fine-tuning of gastric acid secretion [for review, see ref. 134]. But further discussion of the mechanisms is outside the scope of this review.

Mucosal Cell Growth

Four decades ago it was shown that repeated doses of pentagastrin to rats resulted in parietal cell hyperplasia [135]. Numerous studies have since then confirmed that gastrin regulates the growth of cells in the fundic mucosa [16, for reviews, see refs. 17, 136]. ECL-cells are particularly sensitive targets for the trophic effect of gastrin. Thus, prolonged hypergastrinemia of moderate degree in rats leads to carcinoid tumors of ECL-cell origin, i.e. ECLomas. Up to

Fig. (6). Gastric acid secretion in wild type (circles) and gastrin

knock-out mice (squares). Basal secretion was measured for 60

min. in one group of mice. Other groups (filled symbols) were

stimulated by subcutaneous injections of histamine (A), carbachol

(B), or gastrin (C). Each group considered of six mice (data from

ref. [116]).

25 % of female rats and a lower fraction of male rats developed ECLomas after lifelong moderate hypergastri-nemia [137, 138]. By complete lack of gastrin (knock-out mice), the stomach still contains ECL and parietal cells; but they are immature with a grossly abnormal morphology [128, 129]. Presumably, gastrin is therefore necessary also for maturation of the cells to exert normal secretion.

Gastrin has been suggested to stimulate the growth of several epithelial cells also outside the fundic mucosa [for reviews, see refs. 17, 136]. And these suggestions have in combination with low-level expression of gastrins in the bronchial, colorectal, ovarian and pancreatic cancers [93, 117, 118, 139] been used to discuss the possibility, that

1162 Current Topics in Medicinal Chemistry, 2007, Vol. 7, No. 12 Rehfeld et al.

gastrins may play important roles in the formation of major cancers [93, 140, 141]. It is obviously an important discus-sion. But a decisive carcinogenetic role of gastrin in man still remains to be proven [141, 142]. Most evidence so far is based on studies of cell cultures and experimental rodent models, and the evidence has often been controversial.

ACKNOWLEDGEMENT

The skilful secretarial assistance of Christina B. Fleischer is gratefully acknowledged. The studies from the laboratory of the authors that have contributed to the picture of CCK and gastrin described here, have been supported by grants from the Danish Medical Research Council, the Danish Cancer Union, the Danish Biotechnology Program for Pep-tide Research and the Lundbeck Foundation.

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Received: January 17, 2007 Accepted: January 17, 2007