Gastrin secretion by ovine antral mucosa in vitro

The effect on gastrin and somatostatin release in sheep of stimulatory and inhibitory peptides and pharmacological agents was investigated using an in vitro preparation of ovine antral mucosa. Carbachol stimulated gastrin release in a dose-dependent manner but had no effect on somatostatin release. As atropine blocked the effect of carbachol, cholinergic agonists appear to stimulate gastrin secretion directly through muscarinic receptors on the G-cell and not by inhibition of somatostatin secretion. Both vasoactive-intestinal peptide (VIP) and gastric-inhibitory peptide (GIP) increased somatostatin release but did not inhibit basal gastrin secretion, although VIP was effective in reducing the gastrin response to Gastrin-releasing peptide (GRP). Porcine and human GRP were stimulatory to gastrin secretion in high doses but bombesin was without effect. The relative insensitivity to GRP (not of ovine origin) previously reported from intact sheep may be caused either by a high basal release of somatostatin or by the ovine GRP receptor or peptide differing from those of other mammalian species.     

Regulation of atrial natriuretic peptide secretion by cholinergic and PACAP neurons of the gastric antrum

Atrial natriuretic peptide (ANP) released from enterochromaffin cells helps regulate antral somatostatin secretion, but the mechanisms regulating ANP secretion are not known. We superfused rat antral segments with selective neural agonists/antagonists to identify the neural pathways regulating ANP secretion. The nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) stimulated ANP secretion; the effect was abolished by hexamethonium but doubled by atropine. Atropine's effect implied that DMPP activated concomitantly cholinergic neurons that inhibit and noncholinergic neurons that stimulate ANP secretion, the latter effect predominating. Methacholine inhibited ANP secretion. Neither bombesin nor vasoactive intestinal polypeptide stimulated ANP secretion, whereas pituitary adenylate cyclase-activating polypeptide (PACAP)-27, PACAP-38, and maxadilan [PACAP type 1 (PAC1) agonist] each stimulated ANP secretion. The PAC1 antagonist M65 1) abolished PACAP-27/38-stimulated ANP secretion; 2) inhibited basal ANP secretion by 28 +/- 5%, implying that endogenous PACAP stimulates ANP secretion; and 3) converted the ANP response to DMPP from 109 +/- 21% above to 40 +/- 5% below basal, unmasking the cholinergic component and indicating that DMPP activated PACAP neurons that stimulate ANP secretion. Combined atropine and M65 restored DMPP-stimulated ANP secretion to basal levels. ANP secretion in the antrum is thus regulated by intramural cholinergic and PACAP neurons; cholinergic neurons inhibit and PACAP neurons stimulate ANP secretion.     

Dual effect of bombesin and gastrin releasing peptide on gastric emptying in conscious cats

The effect of bombesin (BBS) and gastrin releasing peptide (GRP) on gastric emptying was studied in conscious cats. This effect was measured simultaneously with antral motility. Acid and pepsin secretions as well as blood hormonal peptide release were additionally measured. A dual effect was observed. First, BBS and GRP slowed gastric emptying of liquids, while antral motility was decreased, then after 60 minutes of continuous intravenous infusion, antral motility returned to basal values and gastric emptying effect reversed. The mechanism of this peculiar action is independent of gastrin, pancreatic polypeptide, somatostatin and motilin release and most probably connected with a cholinergic stimulation induced by the peptides, the late predominance of which counterbalances the inhibitory effect of bombesin-like peptides on antral motility.     

PACAP 1-38 as neurotransmitter in the porcine antrum

The concentration of PACAP 1-38 in porcine antrum amounted to 15.4+/-7.9 and 20.3+/-8 pmol/g tissue in the mucosal and muscular layers. PACAP immunoreactive (IR) fibres innervated the muscular (co-localised with VIP) and submucosal/mucosal layers (some co-storing VIP and CGRP) including myenteric and submucosal plexus and blood vessels. Only myenteric nerve cell bodies contained PACAP-IR (co-storing VIP). In isolated perfused antrum, vagus nerve stimulation (8 Hz) and capsaicin (10(-5) M) increased PACAP 1-38 release. PACAP 1-38 (10(-9) M) increased substance P (SP), gastrin releasing peptide (GRP) and VIP release. PACAP 1-38 (10(-8) M) inhibited gastrin secretion and stimulated somatostatin secretion and motility dose-dependently. PACAP-induced motility was strongly inhibited by the antagonist PACAP 6-38 but also by atropine and substance P-antagonists (CP99994/SR48968) but PACAP 6-38 had no effect on vagus-induced secretion or motility. Conclusion: PACAP 1-38 may be involved in antral motility and secretion by interacting with cholinergic, SP-ergic, GRP-ergic and/or VIP-ergic neurones, and may also be involved in afferent reflex pathways.     

Achlorhydria induced changes in gastrin, somatostatin, H+/K(+)-ATPase and carbonic anhydrase in the sheep.

Gastrin, somatostatin, H+/K(+)-ATPase and carbonic anhydrase are principal elements of acid secretion. We investigated in the conscious sheep the effect of 24 h omeprazole (an H+/K(+)-ATPase inhibitor) infusion on these elements at the level of synthesis, storage and secretion. Omeprazole inhibited acid secretion-pH increased from 3.0 to 7.1 at 24 h. Plasma amidated and glycine extended gastrin increased 3-fold while the ratio of amidated to glycine extended gastrins (4:1) remained unchanged. Despite the increase in circulating gastrin, antral gastrin concentration and mRNA did not change significantly. Gastrin-17 (amidated and glycine extended) was the predominant form in the circulation and antrum, although there were preferential increases in larger forms following omeprazole treatment. Omeprazole had no effect on somatostatin mRNA or peptide levels in the fundus. Similarly, plasma somatostatin remained unchanged. However, antral somatostatin increased significantly (63%) following omeprazole treatment accompanied by a 4-fold increase in its mRNA. Fundic H+/K(+)-ATPase mRNA was unchanged but a significant increase (87%) in carbonic anhydrase II mRNA was observed. Omeprazole induced hypergastrinaemia occurred without a measurable reduction in storage or increased synthesis of gastrin at 24 h. Increased antral somatostatin synthesis and storage may result from stimulation by plasma gastrin on antral D cells, independent of acid. The rise in carbonic anhydrase II mRNA in the absence of any change in H+/K(+)-ATPase mRNA may reflect the differential sensitivity of the genes encoding these two enzymes to the stimulatory action of gastrin.     

Functional and anatomical relationships between antral gastrin cells and gastrin-releasing peptide neurons

The present studies were directed to examine the effect of gastrin-releasing peptide (GRP) on beta-adrenergic stimulated gastrin release by cultured rat antral mucosa and to assess the anatomical relationship between gastrin cells and GRP nerves in rat and human antrum. Peptide-containing cells were identified by application of an avidin-biotin-peroxidase immunocytochemical double staining method utilizing antibodies to GRP and gastrin prepared in rabbits. Rat antral mucosa was cultured for 60 min and gastrin released into the culture medium was measured by radioimmunoassay. Inclusion of antibodies to GRP in culture medium did not affect carbachol-stimulated gastrin release, whereas isoproterenol-stimulated gastrin release into the medium was inhibited significantly by addition of GRP antiserum to the culture medium. GRP-containing neurons and axonal fibers were stained immunocytochemically with diaminobenzidine (reddish-brown specific staining) and were located in the lamina propria adjacent to and surrounding the main lobules of antral glands. After double staining utilizing 4-Cl-1-Naphthol as substrate, blue stained gastrin-containing cells were identified in the middle and deeper regions of antral glands in close proximity to GRP neuronal elements. These studies suggest that beta-adrenergic, but not cholinergic, stimulation of gastrin release is mediated, at least in part, through GRP. They also demonstrate intimate anatomical, as well as functional, relationships between gastrin cells and GRP-containing neurons.     

Modulation of acetylcholine-induced secretion of gastric bombesin-like immunoreactivity by cholinergic and histamine H2-receptors, somatostatin and intragastric pH

Recently we have shown the release of bombesin-like immunoreactivity (BLI) from the isolated perfused rat stomach. In these experiments we have shown that BLI secretion is stimulated by acetylcholine. Gastric inhibitory peptide (GIP) exerts an inhibitory effect which is dependent on the intraluminal pH. The present study was designed to examine further the exact cholinergic mechanisms and to study the interaction between cholinergic and histaminergic mechanisms as well as the effect of the intraluminal pH. Acetylcholine elicited a dose-dependent increase in BLI and gastrin secretion (10(-6) M and 2 X 10(-6)M), whereas somatostatin release was suppressed at luminal pH 7. Blockade of muscarinic cholinergic receptors by atropine (10(-5)M) and nicotinic cholinergic receptors by hexamethonium (10(-5) M) abolished the effect of acetylcholine on all three peptides. Reduction of the intraluminal pH to 2 also abolished acetylcholine-induced stimulation of BLI and gastrin secretion and the inhibition of somatostatin secretion. Changes of intraluminal pH per se had no effect on the secretion of either peptide. Somatostatin (10(-7) M) reduced both BLI and gastrin secretion during stimulation with acetylcholine. The addition of the H2-receptor antagonist cimetidine (10(-5) M) abolished the effect of both doses of acetylcholine on BLI and somatostatin secretion and also the effect of the lower dose of acetylcholine (10(-6) M) on gastrin secretion during luminal pH 7. At luminal pH 2 cimetidine did not alter BLI and somatostatin secretion in response to acetylcholine, however, gastrin release was augmented in the presence of cimetidine. These data demonstrate that the effect of acetylcholine on BLI, gastrin, and somatostatin secretion is mediated by muscarinic and nicotinic cholinergic receptors and also by histamine H2-receptors. Somatostatin inhibits cholinergically induced BLI secretion. The cholinergic effects on BLI, somatostatin and gastrin secretion are abolished during an acidic intragastric pH. In this isolated perfused rat stomach model the inhibitory effect of intraluminal acid on gastrin secretion is, at least in part, mediated by H2-receptors. This suggests that the secretion of bombesin, a potential peptidergic neurotransmitter is modulated by neural, endocrine and local tissue factors and also by alterations of intragastric pH.     

Gastrin and somatostatin in the rat antrum. The effect of removal of acid-secreting mucosa

Female rats were subjected to operations aimed at reducing the amount of oxyntic gland mucosa draining its acid secretion to the antrum. The rats were provided either with Heidenhain or Pavlov pouches reducing the oxyntic mucosa draining its secretion to the antrum by about 50% or subjected to various degrees (75, 90 and 100%) of fundectomy. Ten weeks following surgery, plasma levels of gastrin and somatostatin were assayed. At the same time, antral mucosal content of gastrin and somatostatin was determined as well as the mucosal density of these hormone-producing cells. There was a relationship between the amount of acid-secreting mucosa removed and the ensuring plasma concentration of gastrin. Thus, a stepwise increase in plasma gastrin was found with the highest levels obtained in rats subjected to 90 or 100% fundectomy. The somatostatin concentration in plasma was reduced only in rats subjected to fundectomy with the most sustained decrease in animals in which all oxyntic gland mucosa had been removed. There was also a relationship between the amount of acid-secreting mucosa removed and the gastrin content of the antral mucosa. An inverse relationship seemed to exist between antral gastrin and somatostatin concentrations. However, a significant decrease in somatostatin concentration of the antral mucosa was seen only in rats subjected to a fundectomy. The number of gastrin cells in the antral mucosa was increased in fundectomized rats only, with the largest density seen in rats deprived of all oxyntic mucosa. A corresponding decrease in the number of somatostatin cells was noticed. Our results would suggest an apparent functional relationship between antral gastrin and somatostatin cells, where the antral acid load (or pH) appears to be the major factor of physiological significance.     

Bombesin-like immunoreactivity in human gastrointestinal tract

In the present study the distribution and molecular characteristics of bombesin-like immunoreactivity (BLI) were studied in acid extracts of human gastrointestinal tract. The highest levels were found in the fundus, antrum, pylorus and pancreas with lower levels in the duodenum, jejunum, terminal ileum and colon. BLI was also detected in both the muscle and mucosal layers of the antrum and colon. Sephadex G-50 gel chromatography under acid dissociating conditions revealed two peaks of immunoreactivity, one in the position of synthetic porcine gastrin releasing peptide (GRP) and the second eluting with synthetic amphibian bombesin. Variations in the proportions of the two molecular forms were seen in different regions of the gut. In the stomach and pancreas greater than 70% of the BLI eluted with the GRP marker while in pylorus, jejunum and terminal ileum only 20% was present in this form. Reverse-phase ODS silica HPLC of the major antral BLI peak, utilising a methanol/trifluoroacetic acid gradient indicated that this peptide was similar to porcine GRP. We have therefore (1) demonstrated the presence and heterogeneity of bombesin-like immunoreactivity throughout the human gastrointestinal tract and (2) shown for the first time that a proportion of this BLI closely resembles porcine GRP.     

Effects of porcine gastrin releasing peptide (GRP) on canine antral motility and gastrin release in vivo

The effect of GRP on the vivo canine antrum was investigated. GRP caused a dose-dependent increase in antral gastrin output which was not significantly altered by administration of tetrodotoxin. The higher doses of GRP administered also caused excitation of antral motility which was abolished by tetrodotoxin, a finding in contrast to previous in vitro results demonstrating bombesin-induced antral smooth muscle contraction to be tetrodotoxin-resistant. These data suggest that in the vivo canine model GRP causes antral gastrin release via non-reurally mediated mechanisms (probably by acting directly on the G-cell) and excites antral motility via neurally-mediated mechanisms.     

Motor effects of gastrin releasing peptide (GRP) and bombesin in the canine stomach and small intestine

Close intraarterial injections of synthetic porcine gastrin releasing peptide (GRP) or bombesin stimulated contractions in the stomach and inhibited ongoing contractile activity in the small intestine of anaesthetized dogs. Contractile activity of the circular muscle was recorded by serosal strain gauges and phasic activity when desired was elicited by local field stimulation or intraarterial motilin injections. In the stomach (corpus and antrum) following tetrodotoxin blockade of field-stimulated contractions, the contractile response to either peptide was not present, suggesting that stimulation of receptors on nerves initiated contractions in the stomach. Similarly, in the small intestine, the inhibitory response was eliminated by tetrodotoxin suggesting a neural receptor. Pre-treatment with reserpine did not alter the inhibitory response, either in the presence or absence of atropine, therefore, adrenergic inhibitory mechanisms did not appear to be involved. The concentration of bombesin producing 50% inhibition of field stimulation (ED50) was increased following treatment with the putative M1 muscarinic antagonist, pirenzipine suggesting activation of M1 cholinergic inhibitory receptors by bombesin. After blockade by atropine of field-stimulated contractions and the contractile response to intraarterial acetylcholine, the ED50 for bombesin inhibition of motilin contractions was increased. After muscarinic blockade, the residual inhibitory response of GRP/bombesin may involve activation of a neural non-cholinergic non-adrenergic inhibitory mechanism. These results suggest that GRP and bombesin act to alter motility in the dog in vivo by affecting neural activity.     

Effect of antibodies to the neuropeptide GRP on distention-induced gastric acid secretion in the rat

The present study examined and compared the effects of muscarinic blockade, beta-adrenergic blockade and immunoneutralization of the neuropeptide gastrin-releasing peptide (GRP) on distention-induced gastric acid secretion and gastrin release. In response to distention of rat stomachs with 0.9% NaCl, acid output rose from 3.5 +/- 0.5 mumol H+/30 min to 15.4 +/- 2.5 mumol H+/30 min (P less than 0.01). Intravenous administration of 4 mg/kg propranolol did not affect the acid secretory response to distention, however both 2 mg/kg atropine and 6 mg/kg pirenzepine significantly decreased gastric acid secretion by 44.8 +/- 7.8% and 40.9 +/- 5.7% (P less than 0.05), respectively. When specific antibodies to GRP were infused intravenously, the acid secretory response to distention was nearly abolished, decreasing to 5.1 +/- 0.8 mumol H+/30 min (P less than 0.01). In contrast to the effects on acid secretion, GRP antiserum did not significantly alter the gastrin release observed following distention. Results of these studies indicate that, under the conditions of these experiments, the acid secretory response to gastric distention may be independent of its effect on gastrin release. Although distention-induced gastric acid secretion may be partially governed by muscarinic pathways, the acid secretory response to distention in the rat appears to involve GRP-containing neurons.     

Gastrin-releasing peptide: neuronal distribution and spatial relation to endocrine cells in the human upper gut

By using immunocytochemical techniques, we have studied the distribution of gastrin releasing peptide (GRP)-containing neurons as well as the spatial relationship between these neurons and the endocrine cells in the human stomach and duodenum. Moderate numbers of immunoreactive fibers were distributed in the smooth muscle and submucosa of the stomach; they were more rare in the duodenal wall. Numerous GRP-containing nerve fibers were found in the oxyntic mucosa, the antral mucosa harboured only few GRP immunoreactive nerve fibers. The mucosa of the proximal duodenum was found to be virtually devoid of such fibers. Only occasionally did we observe signs of a direct contact between GRP-containing nerve fibers and gastrin and somatostatin cells in the antral mucosa. In the oxyntic mucosa GRP-containing nerve fibers sometimes seemed to contact endocrine cells, including somatostatin cells as well as individual parietal cells. In conclusion, although GRP-containing nerve fibers were quite numerous in the wall of the human upper gastro-intestinal (GI)-tract, we observed a lack of intimate spatial relationship between these fibers and endocrine cells in the antral mucosa, suggesting additive mechanisms to a direct innervation of gastrin cells and somatostatin cells by GRP nerve fibers explaining the physiological effects on hormonal release.     

The ontogeny of regulatory peptide-containing cells in the human fetal stomach: an immunocytochemical study

The antral and fundic regions of the stomachs from 24 human fetuses were examined by immunocytochemistry for the presence of three regulatory peptides (gastrin, somatostatin, and glucagon) and one amine (serotonin (5-HT)) in the epithelial endocrine cells. Gastrin- and somatostatin-containing cells were present at the earliest stage examined (8 weeks). Gastrin cells were restricted to the antrum, while somatostatin cells were found in both the antrum and the fundus. Glucagon-immunoreactive cells were detected from 10 weeks and were confined to the fundus. Serotonin-containing cells were found in both the antrum and the fundus from 11 weeks. Changes in the number of immunoreactive gastrin and somatostatin cells during gestation were quantified. The increase in the number of cells/mm length of vertically sectioned mucosal epithelium best reflects the change in cell population. The peptides and amine studied were found to be contained in separate cell types. Electron microscopic examination of the peptide-containing cells showed that the fetal cells contain granules of similar morphology to their adult counterparts.