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Objectives: To analyse the peptidomics of mouse enteroendocrine cells (EECs) and human gastrointestinal (GI) tissue and identify novel gut derived peptides., Methods: High resolution nano-flow liquid chromatography mass spectrometry (LC-MS/MS) was performed on (i) flow-cytometry purified NeuroD1 positive cells from mouse and homogenised human intestinal biopsies, (ii) supernatants from primary murine intestinal cultures, (iii) intestinal homogenates from mice fed high fat diet. Candidate bioactive peptides were selected on the basis of species conservation, high expression/biosynthesis in EECs and evidence of regulated secretionin vitro. Candidate novel gut-derived peptides were chronically administered to mice to assess effects on food intake and glucose tolerance., Results: A large number of peptide fragments were identified from human and mouse, including known full-length gut hormones and enzymatic degradation products. EEC-specific peptides were largely from vesicular proteins, particularly prohormones, granins and processing enzymes, of which several exhibited regulated secretion in vitro. No regulated peptides were identified from previously unknown genes. High fat feeding particularly affected the distal colon, resulting in reduced peptide levels from GCG, PYY and INSL5. Of the two candidate novel peptides tested in vivo, a peptide from Chromogranin A (ChgA 435-462a) had no measurable effect, but a progastrin-derived peptide (Gast p59-79), modestly improved glucose tolerance in lean mice., Conclusion: LC-MS/MS peptidomic analysis of murine EECs and human GI tissue identified the spectrum of peptides produced by EECs, including a potential novel gut hormone, Gast p59-79, with minor effects on glucose tolerance., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: Diabetes is characterized by β-cell deficiency, and therefore restoration of β-cell function has been suggested as a potential therapy. We hypothesized that a novel glucagon-like peptide-1 (GLP-1)-gastrin dual agonist, ZP3022, improves glycaemic control via improvement of β-cell status in db/db mice., Methods: Diabetic mice were studied following short- or long-term treatment with either the GLP-1-gastrin dual agonist or the commercially available GLP-1 agonists (exendin-4 and liraglutide). The effects on glycaemic control were addressed by repeated glucose tolerance tests and/or measurements of HbA1c levels, and pancreatic islet and β-cell masses were determined by stereology., Results: ZP3022 and the pure GLP-1 agonists improved glycaemic control after both short- and long-term treatment compared with vehicle. Interestingly, the effect was sustainable only in mice treated with ZP3022. Stereology data displayed a dose-dependent increase of β-cell mass (p < 0.05) following treatment with ZP3022, whereas no significant effect of liraglutide was observed (β-cell mass: vehicle 3.7 ± 0.2 mg; liraglutide (30 nmol/kg) 3.4 ± 0.5 mg; ZP3022 (30 nmol/kg) 4.3 ± 0.4 mg and ZP3022 (100 nmol/kg) 5.2 ± 0.4 mg)., Conclusion: The novel GLP-1-gastrin dual agonist, ZP3022, improved glycaemic control in db/db mice, and pancreatic islet and β-cell mass increased significantly following treatment with ZP3022 compared with vehicle., (© 2012 Blackwell Publishing Ltd.)

Trefoil family factor 2 (TFF2) is expressed in gastrointestinal epithelial cells where it serves to maintain mucosal integrity and promote epithelial repair. The peptide hormone, gastrin, stimulates acid secretion but also induces proliferation of the acid-secreting mucosa. Because the relationship between these peptides of overlapping function is not understood, we chose to investigate the regulatory effect of gastrin on TFF2 expression. The expression of mRNA and protein of TFF2 was determined by RT-PCR and immunohistochemical staining, respectively. A series of truncated and mutant murine TFF2 promoter constructs was generated. Promoter activity was assessed using dual luciferase reporter assays. Gastrin-responsive DNA-binding sites in the TFF2 promoter were evaluated by electrophoretic mobility shift assay. Gastrin significantly increased the level of endogenous mRNA of TFF2 in the gastrin receptor-expressing AGS-E gastric cancer cell line in a time- and dose-dependent manner. TFF2 protein expression in the gastric fundus was elevated in hypergastrinemic (INS-GAS) transgenic mice and reduced in gastrin-deficient mice. Gastrin treatment increased TFF2 promoter activity through cis-acting regions, containing CCAATA- and GC-rich enhancers. Pretreatment with Y-F476, a gastrin/CCK(B) receptor antagonist, abolished gastrin-dependent promoter activity. Inhibitors of protein kinase C (PKC), mitogen/extracellular signal-regulated kinase (MEK1), and phosphatidylinositol 3-kinase (PI 3-kinase) reduced gastrin-dependent TFF2 promoter activity, whereas an epithelial growth factor receptor (EGFR) inhibitor had no effect. We found that gastrin regulates TFF2 transcription through a GC-rich DNA-binding site and a PKC-, MEK1- and PI 3-kinase-dependent but EGFR-independent pathway. Regulation of TFF2 by gastrin may play a role in the maintenance and repair of the gastrointestinal mucosa.

The elimination of progastrin-derived peptides was a first-order process, also at supraphysiological concentrations in plasma. The site of extraction was dependent on the molecular size of the peptides and not on their bioactivity. Apart from the kidneys and brain, where the extraction was nonselective, elimination was found in the gastrointestinal tract and limb (Gastrin-17, Gastrin-17 Gly and Gastrin-6). The porcine liver eliminated postprandial gastrin, while human gastrin except gastrin-6 passed unhindered. Metabolism of circulating gastrin was neither recorded in the lungs nor in the heart. In the kidneys peptides were mainly metabolized rather than excreted, since the recovery of intact peptide in urine was minimal. However, urinary clearance varied with the molecular form of the peptides under influence of glomerular filtration and handling in the renal tubules. Thus, N-terminal fragments had a higher urinary clearance than C-terminal peptides. Gastrin degrading enzymes were present in human and porcine plasma, but their contribution to whole-body metabolism was small. It is concluded that there is a differential metabolism of progastrin products in the vascular beds of which the integrated metabolism in nonorgan tissue such as limbs and trunk accounts for the major part of whole-body clearance.

Background & Aims: The cellular target for peptide YY (PYY) inhibition of gastric acid secretion is unknown. The aim of this study was to determine whether PYY inhibits histamine release from isolated enterochromaffin-like (ECL) cells by stimulation of a selective Y receptor., Methods: Isolated rat gastric ECL cells were analyzed in short-term culture for histamine release and for changes in intracellular calcium concentration using video imaging., Results: Gastrin-stimulated histamine release was inhibited with a 50% inhibiting concentration of 2.10(-9) mol/L. Inhibition of histamine release and of calcium entry by PYY and [Pro34]PYY and no effect of PYY(3-36) characterizes an inhibitory Y1 receptor subtype. Reverse-transcription polymerase chain reaction of ECL cell RNA showed that the receptor was the nontruncated Y1 isoform. The inhibitory action of PYY and related peptides on gastrin-stimulated histamine release and calcium signaling was eliminated by pretreatment with 200 ng/mL pertussis toxin. Additive but not synergistic inhibitory effects of PYY and somatostatin on gastrin-stimulated histamine release were observed., Conclusions: Activation of a Y1 inhibitory receptor subtype present on the gastric ECL cell that inhibits gastrin-induced ECL cell histamine release and Ca2+ entry by activation of a Gi or G(o) class of protein may account for inhibition of gastric acid secretion by PYY released from the small intestine.

Bombesin (BN)-like peptides injected peripherally or centrally suppress food intake in rats. The relationship between the central and peripheral actions of BN is unknown. However, experimental evidence supports a critical role for the caudal hindbrain in mediating the feeding effects of BN. To investigate this relationship further, we examined the ability of fourth ventricular infusion of a specific gastrin-releasing peptide (GRP) antagonist, [D-F5, Phe6, D-Ala11]BN-(6-13) methyl ester (BN-ME), to block suppression of glucose intake (0.5 kcal/ml) produced by intraperitoneal administration of GRP-(18-27) in 5-h food-deprived male Sprague-Dawley rats (n = 10). We found that fourth ventricular administration of 10, 32, and 100 ng BN-ME blocked the suppression of glucose intake produced by peripherally administered 10 nmol/kg GRP-(18-27). The most effective dose of BN-ME (100 ng) blocked the ability of peripheral injection of GRP-(18-27) to inhibit glucose intake but had no effect on intake when given alone. These results demonstrate that the availability of caudal hindbrain GRP receptors is necessary for peripherally administered GRP-(18-27) to reduce food intake in rats.

Stimulation of gastric acid secretion by secretagogues was measured in developing rats by in vivo and in vitro techniques. Basal acid outputs in vivo were very low in 8- and 14-day-old rats compared with those in 20- and 30-day-old rats. In 20-day-old rats, all secretagogues increased acid output in vivo, whereas only carbachol, pentagastrin, and sulfated cholecystokinin octapeptide (CCK-8S) were active in 14-day-old rats. In contrast, basal acid output in vitro and stimulation by secretagogues did not differ significantly with age. CCK-8S-stimulated acid output in vitro in 14-day-old rats was blocked by L-365,260, L-364,718, tetrodotoxin, and atropine, but not by hexamethonium, whereas gastrin-stimulated acid output was blocked only by L-365,260. Furthermore, acid output in vivo was elevated three- to fourfold by subcutaneous naloxone-methiodide or L-364,718, but not by L-365,260, in 14-day-old rats; none of these antagonists produced an effect in 20-day-old rats. These studies show that low basal gastric acid output in neonatal rats is caused by tonic inhibitory regulation by endogenous regulatory peptides.

Addition of 10 nM gastrin-releasing peptide (GRP) to Swiss 3T3 fibroblasts induced a transient (1-2 min) tyrosine phosphorylation and activation of mitogen-activated protein kinase (MAP kinase). Increased activity of 42,000 M(r) MAP kinase was detected with immunochemical methods; however, in situ kinase detection on renaturated SDS-polyacrylamide electrophoresis gel revealed activation of both 42,000 and 44,000 M(r) MAP kinase species. Furthermore, stimulation of 32P-labelled cells with 10 nM GRP for 2 min resulted in an increased phosphorylation of a protein with an approximate molecular mass of 78,000 M(r) in anti-raf kinase and anti-MAP kinase kinase immunoprecipitates of cytosolic extracts from 32P-labelled cells. The presented data demonstrated that GRP induces MAP kinase activation in Swiss 3T3 fibroblasts, and furthermore suggest a role for raf kinase in this process.

The mechanisms of action of gastrin and gastrin releasing peptide (GRP) in stimulating gastric acid secretion were examined in the anaesthetized turkey. Gastrin and GRP produced dose-dependent increases in acid secretion that were inhibited by the gastrin/CCK-B antagonist CI988. The antagonist did not affect the acid secretory responses to histamine or carbachol. A GRP antagonist (M216140) inhibited the acid response to GRP, but not gastrin. The results suggest that in birds, GRP stimulates acid secretion by release of gastrin, which acts in turn on classical gastrin/CCK-B receptors in the proventriculus.

Gastrin-releasing peptide (GRP) has previously been shown to be an autocrine growth factor for small cell lung cancer, and our objective in the study presented here was to determine whether GRP has a similar role in pancreatic cancer. Using 125I-GRP, we demonstrated binding to specific, saturable, high-affinity sites (Kd = 1 nM; Bmax = 245 fmol/mg protein) in membrane preparations from the pancreatic tumor cell line Capan. The receptors were found to be biologically active. In whole cells, a GRP analogue bound to these receptors and stimulated rapid transfer of tritium from the tritiated lipid inositol pool to inositol triphosphates. Exogenous GRP addition stimulated incorporation of [3H]thymidine into DNA 20-60%. This stimulatory effect was blocked by the addition of a monoclonal antibody that complexed specifically with the receptor-binding portion of the peptide. In addition, the monoclonal antibody inhibited the growth of Capan cells in an in vitro growth assay without exogenous peptide. Bombesin receptor-specific antagonists also inhibited growth in a similar fashion. These data suggest that paracrine production of GRP may be important in pancreatic tumor growth, or that low-levels of a GRP-like peptide may play an autocrine role in this tumor.

The proliferation of the bronchial epithelium and tumors associated with this tissue is controlled by various growth factors. The main factor is Gastrin Releasing Peptide (GRP), the human counterpart of the amphibian bombesin. These neuropeptides also act as neuromediators and gut hormones. All peptides of this family share a conserved C terminal sequence which is required for biological activity. The determination of this sequence has provided the basis for the design of specific agonist and antagonist peptides and for the generation of monoclonal antibodies (Mab). GRP interacts with a receptor coupled to a G protein and the signalling process leads to the activation of phospholipase C and kinases, and the mobilization of calcium. GRP promotes the proliferation of foetal and adult bronchial epithelium and of small cell lung cancer (SCLC) cells. GRP is also an autocrine growth factor for some SCLC cell lines. The growth of these lines is reduced in vitro and in vivo by MAb and specific antagonists. Hyperplasia of GRP producing cells has been shown in various lung diseases in adults and children. Pharmacological data on GRP suggest that its antagonists could be used in the treatment of SCLC (in addition to chemotherapy) and of interstitial lung disease. The cloning of the GRP receptor should facilitate the design of specific and potent antagonists of the peptide.

To explore the mechanisms of the effects of sucralfate on the stomach, we investigated the action of sucrose octasulfate (SOS), a constituent of sucralfate, on the function of canine gastric parietal cells and somatostatin cells and in the isolated perfused intact rat stomach. Somatostatin cells from the canine gastric fundus were isolated by EDTA-collagenase dispersion and counterflow elutriation, and somatostatin-like immunoreactivity (SLI) release in response to SOS was measured by radioimmunoassay. Similar methods were used to isolate gastric parietal cells, in which gastric acid secretion was measured by uptake of a radiolabeled weak base, [14C]aminopyrine. SLI release by the intact rat stomach was examined in an isolated vascularly perfused rat stomach model. SOS, either alone or co-administered with epinephrine or gastrin heptadecapeptide (G17), dose-dependently stimulated SLI release by isolated canine fundic D-cells. At the highest doses, SOS potentiated the effect of epinephrine but not G17. Similarly, SOS potentiated the stimulating effect of dibutyryl cyclic adenosine 3',5'-monophosphate (DBcAMP), but not 12-O-tetradecanoylphorbol 13-acetate (TPA). The effect of SOS on SLI release could be inhibited by octreotide, a somatostatin analogue. SOS did not alter acid secretion by cultured canine parietal cells either in the basal state or when coadministered with acid secretagogues. In isolated perfused rat stomach studies, SOS produced a significant (60% greater than basal) increase in SLI secretion. There was a similar effect when SOS was perfused against a background of isoproterenol. SOS stimulates SLI release from gastric somatostatin cells and from the isolated perfused stomach but has no direct effect on gastric parietal cells. These actions of SOS may mediate in part the apparent ability of sucralfate to enhance gastric mucosal defense.

This study performed on 6 conscious cats with chronic pancreatic fistulas was designed to determine the role of cholecystokinin (CCK), gastrin and gastrin-releasing peptide (GRP) in stimulation of pancreatic secretion in this species. Pancreatic response to GRP infused intravenously in graded doses appears to be mediated predominantly by CCK because a CCK receptor antagonist, L-364,718, abolished this response. Also, gastrin appears to mediate in part the secretory response to GRP because blockade of gastrin receptors by L-365,260, given at the dose that completely abolished the pancreatic response to exogenous gastrin, caused a significant reduction in the bombesin-induced pancreatic secretion. CCK and partly gastrin appear to mediate the postprandial pancreatic secretion in cats as the administration of L-364,718 and L-365,260 inhibited this secretion by over 90 and 30%, respectively. In contrast, GRP does not seem to contribute to food-induced pancreatic secretory stimulation, because the blockade of GRP receptors using novel bombesin/GRP antagonist (RC-3100) failed to affect this secretion. We conclude that CCK and partly gastrin, but not GRP, play an essential role in the postprandial pancreatic secretion.

Collagenase-isolated mouse islets were incubated with gastrin-releasing peptide (GRP). At 5.6 mmol glucose/l. 10 nmol GRP/l increased the release of insulin (by 50%) and glucagon (by twofold), decreased the release of pancreatic polypeptide (by 35%), but did not significantly affect the release of somatostatin. At 16.7 mmol glucose/l, 10 nmol GRP/l increased glucagon release (by fivefold) and decreased pancreatic polypeptide release (by 46%), without significantly altering insulin and somatostatin release. GRP (200 nmol/l) did not affect insulin release by perifused mouse islets at 2.8 mmol glucose/l, but increased both first and second phase insulin release after a square wave increase in the glucose concentration to 11.1 mmol/l. At 5.6 mmol glucose/l, GRP (100 pmol/1-100 nmol/l) increased (by 50-70%) insulin release by the RINm5F clonal cell line. GRP did not affect glucose oxidation or the cyclic adenosine monophosphate content of RINm5F cells. However, the intracellular free Ca2+ concentration of RINm5F cells was rapidly and transiently increased by GRP (maximum increase of 64% about 10 s after exposure to 1 mumol GRP/l). The rise of intracellular free Ca2+ was approximately halved in the absence of extracellular Ca2+. The results suggest that GRP may contribute to the normal regulation of the endocrine pancreas. The insulin-releasing effect of GRP is mediated via increased cytosolic free Ca2+, derived both from an increased net influx of extracellular Ca2+ and from mobilization of intracellular Ca2+ stores.

Gastrin-releasing peptide (GRP) and bombesin can stimulate pepsinogen release by both gastrin-dependent and -independent mechanisms. Using isolated guinea pig gastric chief cells, we determined that GRP can act directly on the guinea pig chief cell to cause pepsinogen release. GRP and bombesin stimulated a 2.5- to 3-fold increase in pepsinogen release above basal release. Substance P also stimulated a small but significant increase in pepsinogen release. No gastrin immunoreactivity was detected in the supernatants of cells stimulated with up to 1 microM GRP or bombesin or 1 mM carbachol. GRP-stimulated pepsinogen release was completely inhibited by GRP/bombesin receptor agonists as well as substance P receptor antagonist but not by antagonists to receptors for gastrin, the octapeptide of cholecystokinin (CCK-8), secretin, vasoactive intestinal peptide (VIP), or muscarinic agents. Substance P-stimulated pepsinogen release was completely inhibited by substance P receptor antagonist but not by GRP/bombesin receptor antagonists. An additive effect on pepsinogen release was seen when GRP was combined with maximally effective concentrations of adenosine 3',5'-cyclic monophosphate (cAMP)-mediated agents (VIP, secretin, 8-BrcAMP) but not with calcium-mediated agents (carbachol, CCK-8, gastrin). These results indicate that GRP can directly stimulate pepsinogen release from guinea pig chief cells by a specific GRP receptor that mobilizes intracellular calcium.

The absorption-promoting effect of sodium 5-methoxysalicylate was studied in the rat with respect to rectal delivery of pentagastrin and gastrin. Rectal bioavailability was quantitated by direct comparison of pharmacological effect with intravenous dose response. Coadministration of the absorption adjuvant greatly enhanced the rectal bioavailability of the model polypeptides. Sodium 5-methoxysalicylate, therefore, is representative of a new type of absorption promoter which appears to facilitate rectal absorption of polypeptide drug entities.

Epidermal growth factor (EGF) inhibits gastric acid secretion, but its effects on the growth of gut mucosa have not been examined. Fasted male rats were given six injections of EGF (10 micrograms/kg) over a 48-h period. The animals were killed and the incorporation of [3H]thymidine into various tissues was examined and compared to rats treated with NaCl, pentagastrin, and EGF plus secretin. EGF and pentagastrin significantly increased DNA synthesis of the oxyntic gland mucosa. Pentagastrin, but not EGF, stimulated DNA synthesis of duodenal and colonic mucosa as well. Neither peptide altered skin DNA synthesis. Secretin inhibited the effects of pentagastrin but not EGF. Chronic administration of EGF (5 days) caused significant increases in oxyntic gland mucosal DNA, RNA, and protein content. These results not only demonstrate that EGF is a trophic agent for the oxyntic gland mucosa but lend further support to the hypothesis that acid secretion and mitogenesis are the result of two separate mechanisms.

Cholecystokinin, bombesin or gastrin (2 microliter of 50 ng/microliter) was injected stereotaxically into the paraventricular nucleus of the hypothalamus, the arcuate/ventromedial area, the subfornical organ, the area postrema and the cerebral aqueduct of Sprague-Dawley rats and the effects of these injections on food and water intake were studied. While the injection of cholecystokinin reduced food intake when it was injected into both hypothalamic loci, food and water intake were most severely affected by the injection of this peptide into the cerebral aqueduct. Bombesin reduced food intake after its injection into all areas except the subfornical organ and reliable reductions in water intake were seen after injection of this peptide into all areas except the paraventricular nucleus. Minor reductions in food intake were seen following gastrin injection into the paraventricular nucleus while increased water consumption was observed after this peptide was injected into the paraventricular nucleus and cerebral aqueduct. In a second study 6-hydroxydopamine injections (2 microliter of 8 micrograms/microliter were made into the five areas studied 10 days before animals were injected with 100 micrograms/kg of cholecystokinin (i.p.). All 6-hydroxydopamine-injected animals reduced their food and water intake in response to the cholecystokinin challenge as did intact controls. These results indicate that while the changes in food and water intake produced by the central injection of cholecystokinin, bombesin or gastrin may involve central catecholamine systems, those occurring after its systemic administration do not. Therefore, if the release of gastrointestinal peptides during natural feeding is part of a homeostatic mechanism regulating hunger and satiety, this mechanism may operate without directly involving central catecholamine systems.

The effects of a newly synthesized peptide, human gastrin-releasing peptide (hGRP), on the pancreatic exocrine secretion and the release of pancreatic polypeptide (PP) were examined in the conscious rat. Plasma PP concentrations were determined by a recently established specific radioimmunoassay for rat PP. Amounts of 0.18, 0.35, and 3.5 nmol/kg/h hGRP significantly stimulated both pancreatic exocrine secretion and 0.35 nmol/kg/h of hGRP increased PP release. Simultaneously infused proglumide (300 mg/kg/h) did not affect either pancreatic exocrine secretion or PP release. However, simultaneous infusion of atropine (100 micrograms/kg/h) slightly inhibited PP release, but did not restrict the incremental response of pancreatic protein secretion to hGRP. These results suggest that hGRP directly stimulates pancreatic exocrine secretion and PP release.

The sulfakinins constitute a family of real and putative peptide sequences characterized from the cockroach Leucophaea maderae (leucosulfakinin subfamily) and fruitfly Drosophila melanogaster (drosulfakinin subfamily) with homology to the sulfated mammalian hormones gastrin II and cholecystokinin (CCK). The leucosulfakinin (LSK) subfamily of neuropeptides stimulate contractions of the isolated cockroach hindgut. In this paper, we have ascertained some of the primary structural requirements of the sulfakinins for myotropic (muscle-contracting) activity. The myotropic "active core" of this family has been determined to be the C-terminal hexapeptide, though the C-terminal octapeptide (Glu-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2) is required for full activity. The LSKs demonstrate considerable tolerance to Ala substitution in positions 7 and 9 within the active core without complete loss of activity. Conversely, Ala substitution in positions 8, 10 and 11 led to inactive compounds. Basicity is a critical feature of LSK position 10, while aromatic character is an important characteristic for positions 8 and 11 for myotropic activity. Only trace activity could be observed upon replacement of the Tyr(SO3H) residue in LSK-position 6 with a Ser(SO3H). One analog ([3MeHis8] LSK) proved more active as a contractile stimulant than the natural product, while another ([7-11,Tyr(SO3H)7] LSK), conversely, demonstrated inhibition of spontaneous contractions of the cockroach hindgut.

Acid secretion from the vagally innervated gastric fistula of conscious dogs was stimulated in a dose-related manner by the C-terminal penta-, tetra-, and tripetide amides of gastrin, the o-methyl esters of the tetra- and tripeptides, and the sulfone derivative of the tetrapeptide amide. The potency and efficacy of Peptavalon and the C-terminal penta- and tetrapeptide amides were about the same on a weight or molar basis. On a molar basis, the pentapeptide amide was about 4300 times as potent as the tripeptide amide. The o-methyl ester and the sulfone derivative of the tetrapeptide were about equipotent; they were, respectively, one-twenty-fifth and one-thirtieth as potent as the tetrapeptide amide. The dipetide amides did not stimulate acid secretion in a dose range of 50 to 1500 mug per kg-hr. The results indicate that (1) the C-terminal tetrapeptide amide cannot be the minimal effective "stump" for biological activity in the gastrin molecule, (2) the C-terminal amide is important but not essential for gastric stimulating power, and (3) oxidation of the sulfur atom of the methionyl residue in the tetrapeptide does not result in total loss of activity.

We examined the effects of porcine gastrin-releasing peptide (GRP) on exocrine secretion from dispersed rat pancreatic acini using a perifusion system. GRP stimulated amylase secretion, in a dose-dependent manner in the range of 10(-10)-10(-5) M. The submaximum response was observed at 10(-8) M. Stimulation with 10(-8) M GRP caused a biphasic release of amylase. Amylase secretion by GRP stimulation was not affected by the addition of carbachol, dibutyryl cyclic GMP, or atropine. W-7, a calmodulin antagonist, inhibited the amylase secretion induced by GRP. The addition of isobutylmethylxanthine or secretin increased amylase secretion caused by GRP stimulation. These observations suggest that GRP facilitates enzyme secretion by a direct action on pancreatic acini and not through muscarinic and cholecystokinin receptors. The interaction by the Ca2+-calmodulin complex was suggested.

Brain alteration of catecholaminergic, serotoninergic, dopaminergic, gabaergic and cholinergic pathways by intracisternal injection of agonists, antagonists or specific neurotoxic drugs did not significantly affect gastric acid secretion in 2 hr pylorus-ligated rats. In contrast, several neuropeptides were found to be very potent in influencing gastric secretion when administered intracisternally but not when given intravenously. Bombesin-like peptides, opioid peptides and arginine vasopressin acted within the brain to inhibit gastric acid secretion through yet unknown neurohumoral pathways, whereas TRH and some somatostatin analogs elicited brain stimulation of gastric acid output through vagal-dependent mechanisms. These observations have led to the concept that some specific neuropeptides may be important chemical messengers involved in physiologic brain processes regulating gastric acid secretion, and appear as new chemical probes to further investigate the brain-gut relationship.

A variety of stimuli can act through the central nervous system to alter gastric acid secretion. Lesioning and stimulation experiments have established roles for the lateral and ventromedial hypothalamus and the limbic system in the central regulation of gastric acid secretion. Recently a number of neuropeptides have been demonstrated to alter gastric acid secretion after central administration. Thyrotropin-releasing hormone (TRH) and gastrin both increase gastric acid secretion, whereas bombesin, calcitonin, the endogenous opioid peptides and neurotensin decrease gastric acid secretion. With the exception of bombesin, all the other neuropeptides appear to produce their effects through a vagally mediated mechanism. In addition, a number of these neuropeptides, when centrally administered, have been demonstrated to exert a potent cytoprotective effect against stress ulcer development. This review develops a peptidergic hypothesis of gastric acid secretion, suggesting that the final integration of the cephalic phase of gastric acid secretion is brought about by maintaining a delicate balance in the concentration of a number of interacting peptides and monoamines.

The effect of cholecystokinin (CCK)-gastrin family peptides (caerulein, unsulfated gastrin-17, and pentagastrin) and secretin in activating amiloride-sensitive 22Na uptake were investigated in guinea pig pancreatic acini. Secretin had no effect, but CCK-gastrin peptides stimulated the amiloride-sensitive 22Na uptake. The effect of caerulein was inhibited by dibutyryl guanosine 3',5'-cyclic monophosphate (cGMP) and asperlicin, indicating that activation of the Na+-H+ antiport caused by caerulein is mediated by CCK receptors. The effect of gastrin was dibutyryl cGMP and asperlicin insensitive, whereas the effect of pentagastrin was inhibited by the CCK antagonists but with a low affinity, indicating that the effect of gastrin and that of pentagastrin was CCK receptor independent. The calcium ionophore A23187 caused an increase in amiloride-sensitive 22Na uptake. However, the effect of caerulein, which increased internal calcium concentration, was not modified after depletion of intracellular calcium, and that of CCK-gastrin family peptides was not dependent on external calcium concentration. Activation of amiloride-sensitive 22Na uptake was also induced by 12-O-tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetyl-glycerol. Activation of protein kinase c may be involved in the mechanism of caerulein or gastrin in activating the Na+-H+ exchange.

Studies were performed to investigate the effects of neurotransmitters and neurotransmitter candidates (substance P, VIP, somatostatin, Met-enkephalin, gastrin-17, CCK-4 and -8, neurotensin and TRH) of the newly discovered peptidergic nervous system on lower oesophageal sphincter pressure in anaesthetized pigs. All neuropeptides were infused over 2 min periods in 6 different doses, separated by resting periods of at least 1 min, directly into the arterial supply of the lower oesophageal sphincter. Substance P caused a dose-dependent increase in lower oesophageal shpincter pressure; the threshold dose was 9 pmol . kg-1 . min-1 and half maximal response occurred at 72 pmol . kg-1 . min-1. None of the other polypeptides, however, influenced the resting lower oesophageal sphincter. These studies show that substance P is a potent stimulant of smooth muscle in the lower oesophageal sphincter, suggesting that this peptide may be an important regulator of lower oesophageal sphincter pressure.

The effect of intravenous administration of gastrin-releasing peptide ( GRP ) on serum gastrin and insulin levels was studied in ad libitum fed and 24-h fasted rats. Administration of GRP (55 micrograms/kg body weight) caused a significant (P less than 0.05) elevation in serum gastrin levels at 10, 30, 60, and 120 min in the rats fed ad libitum, whereas in the fasted rats, gastrin levels rose significantly only at 10 min. GRP did not cause insulin release in fasted rats, but in the fed rats, it led to a significant elevation in serum insulin levels at 10 and 30 min, in comparison to controls. GRP appears to have an insulinotropic action in addition to a gastrin-releasing effect.

In dogs with denervated fundic pouches, antrectomy, and gastrojejunostomy, feeding a meal of cooked liver and 5 percent bone dust stimualted acid secretion from the fundic pouches without increasing serum gastrin concentrations. Simultaneous administration of pentagastrin, histamine, octapeptide of cholecystokinin, or bethanecol produced potentiation of acid secretion, suggesting that the mediator of the intestinal phase is different from these secretagogues. Secretin and glucagon failed to inhibit the intestinal stimulus but both atropine and metiamide were potent inhibitors. We conclude that entero-oxyntin, the hormone responsible for the intestinal phase of gastric secretin, has a unique pattern of effects for acid secretion.

Previous studies suggested that gastrin-releasing peptide (a neuropeptide found in rat oxyntic mucosa) and oxyntomodulin (a glucagon-containing peptide of mammalian gut) could directly affect the acid secretion of the parietal cells. We therefore studied their effect on gastric acid production in vitro by measuring [14C]-aminopyrine accumulation, a reliable index of H+ generation, in isolated rat parietal cells. However, neither gastrin-releasing peptide nor oxyntomodulin influenced basal acid secretion or histamine-stimulated gastric acid secretion. Electron-microscopic studies of unstimulated and histamine-stimulated parietal cells confirmed that the cells retained the normal morphology of intracellular organelles and that the cells responded to physiological stimulation by marked expansion of the intracellular canaliculi.

Unlabelled: A humoral factor may mediate the intestinal phase of gastric acid secretion. An ex vivo perfused segment of canine jejunum maintained by an oxygenated asanguinous physiologic perfusate was used to test for release of an enterooxyntin (EO) in response to balloon distention at 30 mm Hg for 15 min. Gastric acid secretion in guinea pig fundic mucosa was determined indirectly by a quantitative cytochemical bioassay (CBA) of oxyntic cell hydroxyl ion production (HIP). An increase in the optical density (OD) caused by the cytochemical stain in the oxyntic cells reflects HIP, an index of acid secretion. Basal OD for segments with distention was 16.6 +/- 0.53 and for those without 15.5 +/- 0.68 (NS). Results are expressed as mean change of OD from basal (mean delta OD +/- SEM). (Table-see text) EO caused greater stimulation of HIP than gastrin or histamine. EO was heat stable. Trichloroacetic acid treatment decreased EO activity as did pronase digestion suggesting that EO is composed of one or more peptides., Conclusion: EO, an acid secretagogue, is a humoral agent probably composed of one or more peptides and is released by small bowel distention. Mechanical distention of the small bowel may be an important mechanism for the perpetuation of gastric acid secretion. The ex vivo perfused jejunal segment in conjunction with the CBA are ideal tools with which to study mechanisms of release of EO and the mechanism of action of EO on the oxyntic cell.

To quantitate bombesin stimulation of gastric acid and pepsin via release of gastrin, five gastric fistula dogs were given graded doses (60-1,250 pmol X kg-1 X h-1) of bombesin tetradecapeptide and 40-2,000 pmol X kg-1 X h-1 of synthetic gastrin-17 (G-17). Acid and pepsin output and serum gastrin were proportional to the dose of stimulant. The half-maximal dose of bombesin for gastrin release was 200 pmol X kg-1 X h-1. Bombesin-stimulated acid secretion related to serum gastrin concentrations was congruent with the G-17 curve, but with a maximum of only 62% of the G-17 maximum before declining by 27% despite higher serum gastrin levels. This suggested that bombesin stimulates acid secretion only via gastrin release and inhibits at higher doses by releasing another inhibitory peptide, most likely somatostatin, which is also released by bombesin. The same mechanism could apply to supramaximal inhibition of acid and pepsin seen with high doses of G-17. Because the pepsin curve related to serum gastrin was to the left of the G-17 curve, we concluded that another secretagogue released by bombesin acts synergistically with gastrin on pepsin secretion. Therefore, bombesin stimulates gastric secretion through gastrin release, but its effects are modified by peptides coreleased to a) increase pepsin output at low doses and b) limit the output of acid and pepsin to 50-60% of the G-17 maximum.

Gastrin-17 induces hypocalcemia in the rat without stimulating calcitonin release. The gastrin-induced hypocalcemia persisted after thyroparathyroidectomy or parathyroidectomy. In contrast, gastrectomy or extirpation of the acid-producing part of the stomach prevented the hypocalcemic effect, suggesting the involvement of the proximal stomach in the gastrin-evoked lowering of blood calcium. The drop in blood calcium upon injection of gastrin-17 did not reflect a loss of calcium via the gastric juice or via the urine. Extracts of the acid-producing mucosa of the rat stomach had a hypocalcemic effect. The extracts were purified by gel chromatography and reversed-phase high-performance liquid chromatography. Digestion with leucine aminopeptidase destroyed the hypocalcemic activity, while trypsin had no effect, suggesting a peptide (or peptides) with an unprotected NH2 terminus and without basic amino acid residues (or with protected basic amino acids). Both gastrin-17 and the mucosal extract stimulated the uptake of 45Ca into bone (radius and sternum). Gastrin-17 was without effect in rats that had undergone gastrectomy, while the mucosal extract was equally effective in gastrectomized and unoperated rats. We suggest that the effects of gastrin-17 on blood calcium and on calcium transfer into bone are indirect and that gastrin-17 stimulates the release of a peptide hormone, tentatively named gastrocalcin, from the acid-producing mucosa of the stomach. Gastrocalcin stimulates the uptake of 45Ca into bone, thereby causing hypocalcemia.

1. Caerulein, as expected from its amino-acid composition and sequence, has a potent stimulant action on gastric secretion in the dog, the rat and the frog.2. In the denervated fundic pouch of the dog, caerulein increases the rate of flow of gastric juice and the outputs of acid and pepsin. Acid concentration and pepsin concentration in caerulein-produced juice are generally greater than in control juice. The threshold subcutaneous dose of caerulein is 0.15-0.5 mug/kg and the threshold rate of intravenous infusion 0.25-0.5 mug/kg per hr. Rapid intravenous injection is ineffective. On a molar basis, caerulein is approximately twice as active as human gastrin I on volume and acid output of the gastric pouch and 4 times as active on pepsin output.3. Sustained acid secretion of the fundic pouch produced by histamine infusion is inhibited by caerulein, administered either intravenously or subcutaneously. In turn, acid secretion elicited by caerulein is inhibited by atropine.4. In the rat, the activity ratio of caerulein to human gastrin I is 7-30, calculated on a molar basis, and is thus considerably greater than in the dog. Further, caerulein is 3 times more active than cholecystokinin-pancreozymin. Tested on the perfused stomach preparation of the rat, the threshold dose of caerulein by rapid intravenous injection is 25 ng/kg, by intravenous infusion 0.25 mug/kg per hr, and by subcutaneous injection 0.25 to 0.5 mug/kg.5. The activity of caerulein is sharply reduced by pretreatment of the rats with the histamine liberator 48/80 and potentiated by pretreatment with the diamine oxidase inhibitor aminoguanidine. When caerulein is given by rapid intravenous injection during a priming infusion of histamine its effect is enhanced and considerably prolonged.6. The isolated mucosa of the frog stomach is extremely sensitive to caerulein which, in a concentration of a few pg/ml., stimulates active transport of chloride.7. Qualitative and quantitative differences in the action of gastrin and caerulein are pointed out, and particular emphasis is laid on the importance of esterification of the tyrosyl residue for the biological activity of caerulein.