ACE-like hydrolysis of gastrin analogs and CCK-8 by fundic mucosal cells of different species with release of the amidated C-terminal dipeptide

Various gastrin analogues and CCK-8 (Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) are hydrolyzed in vitro by angiotensin-converting enzyme (ACE), the main and initial cleavage occurring at the Met-Asp (or Leu-Asp) bond, releasing the C-terminal dipeptide amide Asp-Phe-NH2. Tetragastrin analogues (e.g., Boc-Trp-Leu-Asp-Phe-NH2) are degraded by a vesicular membrane fraction from rat gastric mucosa, yielding the C-terminal dipeptide Asp-Phe-NH2. We report here on the degradation of gastrin analogues and CCK-8 by a gastric mucosal cell preparation containing specific gastrin receptors. We have shown that gastrin analogues were specifically degraded by gastric mucosal cells from different species (e.g., rabbit and dog) at 37 degrees C (pH 7.4), releasing the C-terminal dipeptide Asp-Phe-NH2, similarly to ACE. This cleavage was found to be temperature and pH sensitive, and was inhibited by metalloproteinase inhibitors and by captopril, strongly suggesting that this enzymatic system closely resembles ACE. We have also demonstrated that a close correlation seems to exist between the apparent affinity of the gastrin analogues for gastrin receptors on gastric mucosal cells, and their ability of being hydrolyzed by this cell preparation. Moreover, all gastrin analogues which have been demonstrated to act as gastrin antagonists remained unaffected in the incubation conditions.     

The role of the Asp-32 residue of cholecystokinin in gastric acid secretion and gastrin receptor recognition

The aspartic acid residue at the penultimate position is known to be essential for the hormonal activity of CCK and gastrin on gastric acid secretion. This residue was successively replaced by beta-aspartic acid, beta-alanine, and glutamic acid in the C-terminal heptapeptide of CCK 27-33. The analogues obtained were tested on rat gastric acid secretion and for recognition by gastrin receptors. The replacement by beta-aspartic or beta-alanine decreased gastric secretion and gastrin receptor recognition. In contrast, replacement by glutamic acid affected these two parameters less. The nature of the N-blocking group (Boc or Z) also influenced these activities, Boc derivatives being more potent than Z derivatives. The results were compared to those previously obtained on pancreatic secretion and on stimulation of gall bladder contraction where the modifications were found capable of differentiating between cholecystokinin, pancreozymin and gastrin activities.     

CCKB/gastrin receptor antagonists: recent advances and potential uses in gastric secretory disorders.

Cholecystokinin (CCK) and the structurally related peptide, gastrin, have numerous effects on tissues in the central nervous system and gastrointestinal tract. Recent studies show these effect are mediated by a CCKA and CCKB receptor. Knowledge of the physiological role and role of CCKB receptors in pathologic processes has been particularly limited by the availability of selective, potent receptor antagonists. Recently, new members of five different classes of non-peptide CCKB receptor antagonists are reported and are reviewed briefly. these include compounds isolated from Streptomyces (tetronothiodin, virginiamycin analogues), ureido-acetamide analogues (RP 69758, RP 72540, RP 73870), newer benzodiazepine analogues (L-368,935, L-740,093, YM022), pyrazolidimine analogues (LY 262,691) and glutamic acid analogues (CR2194). Many of these compounds have greater than 1000-fold selectivity for the CCKB over the CCKA receptor and some have greater than 10,000-fold selectivity. The pharmacology and effects of CCKB receptor antagonists on gastric acid secretion is briefly reviewed. Furthermore, the possible clinical usefulness of CCKB receptor antagonists in treating disorders of gastric acid secretion, in inhibiting the trophic effects of gastrin and in other clinical conditions is briefly discussed.     

Phenylethylamide derivatives of the C-terminal tetrapeptide of gastrin. Potent inhibitors of gastrin-stimulated acid secretion

Peptide analogues of the C-terminal tetrapeptide of gastrin in which the phenylalanine had been replaced were synthesized and their biological activity on acid secretion evaluated. Compounds Boc-Trp-Leu-Asp phenylethylamide 6, Boc-beta-Ala-Trp-Leu-Asp phenylethylamide 9, Boc-Trp-Leu-Asp p-fluorophenylethylamide 19, Boc-Trp-psi(CH2NH)-Leu-Asp phenylethylamide 23, Boc-Trp-Leu-Asp 2,2-diphenylethylamide 15, and Boc-D Trp-Leu-Asp 2,2-diphenylethylamide 21, in which the phenylalanine had been replaced by phenylethylamine, p-fluorophenylethylamine or 2,2-diphenylethylamine were synthesized. None of these derivatives showed activity on acid secretion in the anaesthetized rat at doses as high as 5 mg/kg. However, they were potent inhibitors of gastrin-induced acid secretion, with ED50 varying from 0.1 to 0.6 mg/kg.     

CCK2 receptor antagonists: pharmacological tools to study the gastrin-ECL cell-parietal cell axis

Gastrin-recognizing CCK2 receptors are expressed in parietal cells and in so-called ECL cells in the acid-producing part of the stomach. ECL cells are endocrine/paracrine cells that produce and store histamine and chromogranin A (CGA)-derived peptides, such as pancreastatin. The ECL cells are the principal cellular transducer of the gastrin-acid signal. Activation of the CCK2 receptor results in mobilization of histamine (and pancreastatin) from the ECL cells with consequent activation of the parietal cell histamine H2 receptor. Thus, release of ECL-cell histamine is a key event in the process of gastrin-stimulated acid secretion. The oxyntic mucosal histidine decarboxylase (HDC) activity and the serum pancreastatin concentration are useful markers for the activity of the gastrin-ECL cell axis. Powerful and selective CCK2 receptor antagonits have been developed from a series of benzodiazepine compounds. These agents are useful tools to study how gastrin controls the ECL cells. Conversely, the close control of ECL cells by gastrin makes the gastrin-ECL cell axis well suited for evaluating the antagonistic potential of CCK2 receptor antagonists with the ECL-cell HDC activity as a notably sensitive and reliable parameter. The CCK2 receptor antagonists YF476, YM022, RP73870, JB93182 and AG041R were found to cause prompt inhibition of ECL-cell histamine and pancreastatin secretion and synthesis. The circulating pancreastatin concentration is raised, was lowered when the action of gastrin on the ECL cells was blocked by the CCK2 receptor antagonists. These effects were associated with inhibition of gastrin-stimulated acid secretion. In addition, sustained receptor blockade was manifested in permanently decreased oxyntic mucosal HDC activity, histamine concentration and HDC mRNA and CGA mRNA concentrations. CCK2 receptor blockade also induced hypergastrinemia, which probably reflects the impaired gastric acid secretion (no acid feedback inhibition of gastrin release). Upon withdrawal of the CCK2 receptor antagonists, their effects on the ECL cells were readily reversible. In conclusion, gastrin mobilizes histamine from the ECL cells, thereby provoking the parietal cells to secrete acid. While CCK2 receptor blockade prevents gastrin from evoking acid secretion, it is without effect on basal and vagally stimulated acid secretion. We conclude that specific and potent CCK2 receptor antagonists represent powerful tools to explore the functional significance of the ECL cells.     

Synthesis of pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin and evaluation of their biological activity on acid secretion

The syntheses of pseudo-tetrapeptides Boc-Trp-psi (CH2-NH)-Met-Asp-Phe-NH2 21 and Boc-Trp-Met-psi (CH2-NH)-Asp-Phe-NH2 20, representing the C-terminal tetrapeptide sequence of gastrin, in which amide bonds were replaced by CH2-NH bond, are described, as well as the syntheses of pseudo-peptide analogues Boc-Trp-psi (CH2-NH)-Nle-Asp-Phe-NH2 16, Boc-Trp-Nle-psi (CH2-NH)-Asp-Phe-NH2 11, and Boc-Trp-Nle-Asp-psi (CH2-NH)-Phe-NH2 5, in which the methionyl residue was replaced by a norleucyl residue. Pseudo-peptides 16 and 21, in which the amide bond between Trp and Met (or Nle) was substituted by a CH2-NH bond, stimulated gastric acid secretion in the rat in vivo. Pseudo-peptides 11 and 20, where the amide bond between Met (or Nle) and Asp was replaced by a CH2-NH bond, did not exhibit any activity on acid secretion in the rat in vivo but were potent inhibitors of pentagastrin-induced acid secretion. Peptides 11, 16, 20 and 21 all recognize the gastrin receptor on a mucosal cell preparation. Pseudo-peptide 5, in which the amide bond between Asp and Phe was replaced by a CH2-NH bond, was a less potent inhibitor of pentagastin-induced acid secretion and had a weaker affinity than the other pseudo-peptides.     

Biological effects of human gastrin I and II chemically modified at the C-terminal tetrapeptide amide.

Binding to gastrin receptors and gastric acid secretion experiments were performed with gastrin derivatives modified at the C-terminal tetrapeptide amide from HG-13 sequence. 1. When the ultimate phenylalanine amide was replaced by a phenethylester or a phenetylamide moiety, the resulting compound bound to gastrin receptors (Kd approximately 10 nM) and exhibited antagonist activity on gastrin-induced acid secretion in the anesthetized rat. 2. Changing the peptide bond between Trp and Leu residues to a -omega(CH2-NH)- bond resulted in a compound which also bound to gastrin receptors (Kd approximately 10 nM) but presented agonist activity on acid secretion in the rat. In contrast, when the peptide bond between Leu and Asp residues was replaced by a -omega(CH2-NH)- bond, the resulting compound was devoid of any affinity for gastrin receptor (Kd greater than 10(-6) M) and of any biological activity. 3. The HG-13 derivatives were synthesized in sulfated and unsulfated forms: O-sulfation of the HG-13 tyrosine residue did not change its intrinsic in vivo activity but enhanced its affinity for gastrin receptors (Kd approximately 0.3 nM). On the contrary, O-sulfation of the various chemically modified HG-13 had no significant effect in either in vitro or in vivo experiments. 4. Finally, no significant difference between binding on parietal (F3) and nonparietal (F1) cells was observed, in agreement with the presence of a gastrin-type receptor in these two cell populations.     

Isolation from chicken antrum, and primary amino acid sequence of a novel 36-residue peptide of the gastrin/CCK family

A peptide that cross-reacted with C-terminal gastrin/CCK antisera was isolated from chicken antral extracts by a combination of gel filtration and reversed-phase HPLC. The sequence was: Phe-Leu-Pro-His- Val-Phe-Ala-Glu-Leu-Ser-Asp-Arg-Lys-Gly-Phe-Val-Gln-Gly-Asn-Gly-Ala- Val-Glu-Ala-Leu-His-Asp-His-Phe-Tyr-Pro-Asp-Trp-Met-Asp-Phe(NH2). Aside from the C-terminal tetrapeptide and the Tyr residue, the molecule does not resemble other known forms of gastrin or CCK. The peptide was a potent stimulus of avian gastric acid but not pancreatic secretion. The results have important implications for the structure-activity and evolutionary relationships of the gastrin/CCK family.     

A new class of potent gastrin antagonists

The hexapeptide Z-Tyr(SO-3)-Met-Gly-Trp-Met-Asp-NH2, from the natural sequence of C-terminal cholecystokinin was found to be a competitive antagonist of cholecystokinin receptors, in vitro. In the present study, we report that this peptide inhibits gastrin-induced acid secretion in vivo, (ED50 = 1.5 mumol . kg-1), without agonist activity. Desulfation of the tyrosine residue slightly altered this effect. The tripeptide Boc-Trp-Met-Asp-NH2 showed similar effects, but had lower potency (ED50 = 12 mumol . kg-1). From these preliminary results, it can be concluded that removal of the phenylalanine residue from the C-terminal sequence of CCK or gastrin, leads to an antagonist of the natural hormones and that C-terminal phenylalanine residue is important for agonist activity. As compared with proglumide, a well known gastrin receptor antagonist, these peptides were 20-200 times more potent as inhibitors on the same model.     

Novel activity of angiotensin-converting enzyme. Hydrolysis of cholecystokinin and gastrin analogues with release of the amidated C-terminal dipeptide.

ACE (angiotensin-converting enzyme; peptidyl dipeptidase A; EC 3.4.15.1), cleaves C-terminal dipeptides from active peptides containing a free C-terminus. We investigated the hydrolysis of cholecystokinin-8 [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] and of various gastrin analogues by purified rabbit lung ACE. Although these peptides are amidated at their C-terminal end, they were metabolized by ACE to several peptide fragments. These fragments were analysed by h.p.l.c., isolated and identified by comparison with synthetic fragments, and by amino acid analysis. The initial and major site of hydrolysis was the penultimate peptide bond, which generated a major product, the C-terminal amidated dipeptide Asp-Phe-NH2. As a secondary cleavage, ACE subsequently released di- or tri-peptides from the C-terminal end of the remaining N-terminal fragments. The cleavage of CCK-8 and gastrin analogues was inhibited by ACE inhibitors (Captopril and EDTA), but not by other enzyme inhibitors (phosphoramidon, thiorphan, bestatin etc.). Hydrolysis of [Leu15]gastrin-(14-17)-peptide [Boc (t-butoxycarbonyl)-Trp-Leu-Asp-Phe-NH2] in the presence of ACE was found to be dependent on the chloride-ion concentration. Km values for the hydrolysis of CCK-8, [Leu15]gastrin-(11-17)-peptide and Boc-[Leu15]gastrin-(14-17)-peptide at an NaCl concentration of 300 mM were respectively 115, 420 and 3280 microM, and the catalytic constants were about 33, 115 and 885 min-1. The kcat/Km for the reactions at 37 degrees C was approx. 0.28 microM-1.min-1, which is approx. 35 times less than that reported for the cleavage of angiotensin I. These results suggest that ACE might be involved in the metabolism in vivo of CCK and gastrin short fragments.     

The role of luminal gastrin in the regulation of pancreatic juice secretion in preruminant calves

The effect of luminal gastrin on the secretion of pancreatic juice was studied in seven conscious preruminant calves employing luminal infusions of gastrin and cholecystokinin (CCK)-9 and pharmacological CCK1 and CCK2 receptor blocks with antagonists. The study was performed in the preprandial and prandial states. Pharmacological blocking of the CCK2 receptor, like that of the CCK1 receptor, resulted in reduction of pancreatic postprandial secretion and increased the duration of the prandial pattern of duodenal electrical activity. Exogenous luminal gastrin, like luminal CCK-9, enhanced the secretion of pancreatic juice proteins, though the overall effect of gastrin was weaker than that of CCK-9. The effect was inhibited by infusion of CCK2 but also by CCK1 receptor antagonist. In conclusion, duodenal luminal gastrin can stimulate exocrine pancreatic secretion by a mechanism that depends on CCK2 receptors in calves. Involvement of the CCK1 receptor in this mechanism needs further investigation. Prandial pancreatic secretory and duodenal motility cycles can be regulated by endogenous gastrin release.     

Characterization of [3H]pentagastrin binding in guinea pig gastric glands--an alternative convenient ligand for receptor binding assay

The binding of [3H]pentagastrin to guinea pig gastric glands was specific, saturable and of high affinity (Kd = 5 nM). The relative order of potencies for gastrin and CCK analogs in displacing [3H]pentagastrin binding correlated well with those obtained using [125I]gastrin and their reported biological potencies for stimulating acid secretion. Nonselective CCK/gastrin antagonists including carbobenzoxy-CCK (26-32), proglumide and benzotript, but not the selective peripheral CCK antagonist, asperlicin, inhibited specific [3H]pentagastrin binding. The results indicate that [3H]pentagastrin labels physiologically relevant gastrin receptors in guinea pig gastric glands.     

Inhibition of bombesin-stimulated gastrin release from isolated canine G cells by bombesin antagonists

This study investigated the effects of two putative bombesin antagonists, [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P and [Leu13-psi-CH2NH-Leu14]bombesin, on bombesin-stimulated gastrin release from isolated canine G cells following short-term culture. Canine antral tissue was dispersed by sequential collagenase and EDTA treatment, and counterflow elutriation was used to enrich for G cells. Plates were seeded with 2 x 10(6) cells/mL in each well and cultured for 2 days prior to testing. Gastrin-containing and somatostatin-containing cells were identified by immunocytochemistry using the biotin-avidin-peroxidase method and accounted for 8.5 and 1%, respectively, of adhered cells. Basal gastrin secretion was 1.91 +/- 0.48% of total cell content. After a 2-h incubation period, bombesin (0.01-100 pM) stimulated gastrin release in a concentration-dependent fashion. The substance P analog, at a concentration of 1 microM, modestly inhibited bombesin-stimulated gastrin release from canine G cells. This analog also produced weak stimulation of basal gastrin release. In contrast, the bombesin analog, at a concentration of 1 microM, did not affect basal gastrin secretion. The bombesin analog completely blocked bombesin-stimulated gastrin release from 0.01 to 1 pM and produced greater than 50% inhibition at higher doses. The ability of the bombesin analog to directly inhibit bombesin-stimulated gastrin release from cultured canine G cells underscores its usefulness in studies involving the role of bombesin and its mammalian counterpart, gastrin-releasing peptide, in the control of gastrin cell function.     

Cholecystokinin antagonists

The three classes of CCK antagonists illustrate the various factors governing affinity of the antagonists for the CCK receptor. The major influence in determining potency of the cyclic nucleotide derivatives, amino acid derivatives and C-terminal fragments of CCK, are hydrophobic forces. In contrast, structural requirements are the major influences in determining potency of the N-terminal fragments of CCK-26-33. The most potent CCK antagonist in each of the three classes is illustrated in Fig. 11. CBZ-CCK-27-32-NH2 is 30 times more potent than N-CBZ-cystine, which is, in turn, slightly more potent than Bt2 cGMP. All these CCK antagonists, however, are relatively weak. For example, CBZ-CCK-27-32-NH2 inhibits binding of 125I-CCK by 50% at a concentration of approximately 5 microM. In contrast, the agonist CCK-26-33 inhibits binding of 125I-CCK by 50% at a concentration of approximately 1 nM. The antagonists remain useful for analyzing those responses that are caused by CCK, though the relatively low potencies of the antagonists may limit their usefulness as antagonists of CCK in vivo systems. This limitation, however, may be only theoretical. For example, proglumide, which requires an in vitro concentration of 0.3 mM to cause half-maximal inhibition of binding of 125I-CCK, can inhibit the actions of CCK and gastrin in animals (Hutchison and Dockray 1980; Stubbs and Stabile 1985) and of gastrin in man (Lamers and Jansen 1983). Nevertheless, the identification of CCK antagonists with greater potency than those hitherto described will facilitate studies of the actions of CCK.     

Evidence that proglumide and benzotript antagonize secretagogue stimulation of isolated gastric parietal cells

Proglumide has been shown to be an in vivo inhibitor of secretagogue-stimulated gastric acid secretion. In the present study, we have examined the ability of proglumide and benzotript, a new tryptophan derivative, to inhibit acid output from isolated gastric fundic parietal cells from rabbit. As measured with the [14C]aminopyrine (AP) accumulation method as an index of acid secretion, the two drugs inhibited basal AP with IC-50 values of 1 X 10(-2) M for proglumide and 1 X 10(-3) M for benzotript. In the case of secretagogue stimulation (1) benzotript slightly affected histamine-induced AP (15% inhibition at 5 X 10(-3) M), proglumide did not; (2) both proglumide and benzotript inhibited in a non-competitive manner acetylcholine-induced AP; (3) these isolated cells were sensitive to gastrin and the dose-response curve for the stimulant was biphasic (maximum for 1 X 10(-9) M), suggesting a desensitization mechanism. Proglumide and benzotript competitively inhibited both [125I]gastrin binding to its receptor sites and gastrin-induced AP, suggesting they are members of a class of gastrin-receptor antagonists. But, this suggestion cannot exclude other post-receptorial mechanisms involved in the acid output from parietal cells.     

Gastrin and interleukin-1beta stimulate growth factor secretion from cultured rabbit gastric parietal cells

The hormone gastrin stimulates proliferation of the gastric mucosa. Inflammation of the stomach is also associated with increased proliferation. The proliferative response is important in the reparative response to injury but can be deleterious by predisposing to the development of cancer. Parietal cells, but not the cells in the proliferative zone of the gastric glands, express the appropriate gastrin receptor. Parietal cells may mediate the trophic effects of gastrin by secreting other growth factors. The role of parietal cells in the proliferative responses has been examined in this study. Rabbit parietal cells were cultured with gastrin or the cytokine interleukin-1beta for 18 hours. The conditioned medium from gastrin or IL-1beta stimulated parietal cells increased proliferation of HeLa cells in an epidermal growth factor-receptor dependant manner. Gastrin and IL-1beta stimulated the secretion of heparin-binding epidermal growth factor and amphiregulin but not transforming growth factor-alpha from parietal cells. Combinations of gastrin and IL-1beta on growth factor secretion were synergistic. The protein kinase C inhibitor staurosporine abolished these stimulatory effects of gastrin and IL-1beta. Divergent effects on histamine-stimulated acid secretion were observed; 18 hours pre-treatment with gastrin enhanced acid secretion by 50% but IL-1beta inhibited acid secretion in both control and gastrin pre-treated parietal cells. The acid-secreting parietal cell plays a central role in the regulation of mucosal proliferation in gastric inflammation. Secretion of paracrine growth factors by parietal cells may be an important point of integration between the endocrine and inflammatory stimuli in determining mucosal responses to injury and inflammation.     

New Chimeric Analogues of Galanin: Synthesis and Effects on the Glucose-Induced Insulin Secretion

The solid-phase synthesis and in vitro assays on the glucose-induced insulin secretion from rat pancreatic islets of Langerhans with six new chimeric peptides were performed. All the peptides were built up of the N-terminal galanin (GAL) fragment or its analogues, linked to the C-terminal portion of substance P (SP) analogues or scyliorhinin I (SCY-I) analogues. Two strong antagonists of the inhibitory effect of galanin on the glucose-induced insulin release were found: [cycloleucine⁴]GAL(1-13)-SP(5-11)-amide and GAL(1-13)-[L-norleucine¹⁰]SCY-I(3-10)-amide.     

N-isobutyryl-His-Trp-Ala-Val-D-Ala-His-Leu-NHMe (ICI 216140) a potent in vivo antaconist analogue of bombesin/gastrin releasing peptide (BN/GRP) derived from the C-terminal sequence lacking the final methionine residue

The GRP receptor mediated growth response in Swiss 3T3 cells has been used to identify BN/GRP antagonists. Analysis of bombesin antagonism by substance P analogues and by truncated GRP analogues revealed that deletion of the C-terminal methionine residue was important for antagonism. Des-Met analogues showing potent antagonist activity in the in vitro 3T3 system (IC50 approximately 2nM) were synthesized. Further structural modification of these peptides led to the identification of (CH3)2CHCO-His-Trp-Ala-Val-D-Ala-His-Leu-NHCH3 (ICI 216140) which reduced bombesin-stimulated rat pancreatic amylase secretion to basal levels when administered subcutaneously at 2.0 mg per kg.     

Regulation of gastric acid secretion.

The advent of the H2-histamine-receptor antagonists has given new life to the old hypothesis that histamine might be the final common chemical mediator of acid secretion. The available evidence, however, does not prove this hypothesis but does confer on histamine a role in the regulation of acid secretion in normal physiology. Evidence is mounting that, in addition to its stimulatory action, the vagus may play an inhibitory role in acid secretion and gastrin release. Our concepts of the gastric phase of acid secretion have been extended by the discovery of cross distension reflexes in the stomach: the pyloro-oxyntic reflex for acid secretion and the oxyntopyloric reflex for gastrin release. In addition, digested protein has been shown to stimulate directly the oxyntic gland mucosa, but the evidence is against a role for this mechanism in the intact stomach. The hormone(s) responsible for the intestinal phase have not been isolated but the physiological characteristics of entero-oxyntin (a nongastrin, enteric substance that acts on the oxyntic cell) have been defined. Gastric inhibitory polypeptide is an excellent candidate for the entero-gastrone released by fat, but whether it is the sole enterogastrone released is yet to be determined.