PACAP type I receptor activation regulates ECL cells and gastric acid secretion

Pituitary adenylate cyclase activating polypeptide (PACAP) is present in gastric nerves, and PACAP receptors (PAC1) are found on gastric enterochromaffin-like (ECL) cells. Expression of PAC1 splice variants in purified ECL cells was determined by RT-PCR. PACAP effects on ECL cells were analyzed by video imaging of [Ca(2+)](i) and histamine release; its effects on gastric glands were examined by confocal microscopy of [Ca(2+)](i) in ECL and parietal cells. PACAP action on D cells was measured by [Ca(2+)](i) and radioimmunoassay. PACAP effects on acid secretion were determined in fistula rats with or without neutralizing anti-somatostatin antibodies. All splice variants of PAC1 were found, but vasoactive intestinal polypeptide (VIP) receptor (VPAC) products were absent. PACAP-27 and -38 dose-dependently raise [Ca(2+)](i) in ECL cells, and stimulated histamine release. VIP had a much lower affinity, which demonstrates the presence of PAC1 but not VPAC. PACAP elevated [Ca(2+)](i) in ECL and parietal cells of superfused gastric glands, but only the parietal cell signal was inhibited by ranitidine, showing the absence of PAC1 on parietal cells, and demonstrating functional coupling between the cell types. PACAP and VIP stimulated calcium signaling and somatostatin release from D cells with almost equal efficacy. Acid secretion was stimulated after intravenous injection of PACAP into rats treated with somatostatin antibody. PACAP is a candidate as a mediator of neural regulation of acid secretion.     

Inhibition of exocrine pancreatic secretion by opiates is mediated by suppression of cholinergic transmission: characterization of receptor subtypes

This study evaluates the hypothesis that opiates suppress pancreatic enzyme secretion by inhibiting cholinergic transmission in the pancreas. Rat pancreatic lobules were incubated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-Ringer's buffer and amylase release in response to KCl depolarization of the intrapancreatic nerve in the absence or presence of specific opiate subtype receptor agonists was studied. Depolarization by 75 mM KCl resulted in a 5-fold increase in amylase output. Pretreatment with 1 microM atropine inhibited completely the KCl-stimulated amylase release, suggesting stimulation via a cholinergic pathway. Addition of methionine enkephalin or 2-D-penicillamine-5-D-penicillamine-enkephalin [( D-Pen2, D-Pen5]enkephalin, a specific delta receptor agonist) inhibited KCl-stimulated amylase release in a dose-dependent fashion. Methionine enkephalin (1 microM) or [D-Pen2, D-Pen5]enkephalin inhibited KCl-stimulated amylase release by 32 +/- 4 and 45 +/- 4%, respectively. Addition of 1 microM ICI 174,864 (a delta opiate receptor antagonist) blocked the inhibitory effect of [D-Pen2, D-Pen5]enkephalin. Upjohn 50,488H (1 microM, a specific kappa agonist) and 1 microM Tyr-D-Ala-Gly-MePhe-Gly-ol (a specific mu agonist) had no effect. Methionine enkephalin had no effect on carbachol (1 microM)-stimulated amylase release. These data suggest that methionine enkephalin acts on a delta opiate receptor located on postganglionic cholinergic neurons. To examine the ability of methionine enkephalin to alter acetylcholine release from pancreatic tissue, pancreatic lobules were incubated with [3H]choline and the release of synthesized [3H]acetylcholine was stimulated by KCl. Depolarization of the nerves with 75 mM KCl increased [3H]acetylcholine release by 35 +/- 5% over basal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Islet amyloid polypeptide has no effect on amylase release from rat pancreatic acini stimulated by CCK-8, secretin, carbachol and VIP.

Islet amyloid polypeptide (IAPP), a novel peptide recently isolated from the amyloid substance of pancreatic islets, has been localized in normal islet B cell granules, suggesting its physiological action in exocrine pancreatic function. In the present study, the direct effect of IAPP on secretagogue-stimulated pancreatic enzyme secretion was investigated using dispersed rat pancreatic acini. Various concentrations of IAPP (10(-5) -10(-10) M) showed no significant influence on amylase release stimulated either by submaximal doses of CCK-8 (10(-11) M), secretin (0.5 x 10(-6) M) or carbachol (10(-6) M), or by a maximal dose of vasoactive intestinal peptide (10(-8) M). These results may provide negative evidence for a physiological significance of IAPP in rat pancreatic enzyme secretion.     

Influence of “calcium2+-channel blockers” on exocrine pancreatic secretion by isolated rat acini

Cytoplasmic Ca2+ is regarded as an intracellular messenger for acetylcholine- and cholecystokinin (CCK)-stimulated pancreatic enzyme secretion. We investigated in the in vitro model of isolated rat pancreatic acini whether or not Ca2+-channel blockers are able to inhibit Ca2+-mediated enzyme secretion. Isolated rat pancreatic acini were prepared via collagenase digestion. The effect of various Ca2+-channel blockers on amylase secretion stimulated by various secretagogues was monitored. Verapamil, but not nitrendipine, dose-dependently reduced CCK8- and carbachol-stimulated enzyme secretion. Higher doses of either CCK8 or carbachol could not reverse the inhibition caused by verapamil. Amylase secretion stimulated by the ionophore A23817 was not altered by verapamil. Verapamil augmented enzyme secretion stimulated by secretagogues which work through cAMP as second messenger. 3H-N-methylscopolamine- and 125I-Bolton-Hunter-CCK8-binding to pancreatic acini was dose-dependently inhibited by verapamil, but the inhibition curves did not parallel the inhibition curves with unlabeled receptor agonists. Thus, the impairment of exocrine pancreatic amylase secretion by verapamil is probably not due to its known "Ca2+-channel" blocking abilities in other tissues but is rather caused by noncompetitive effects on the level of muscarinic receptors and receptors for CCK.     

Pituitary adenylate cyclase-activating peptide as a neurotransmitter in the canine ileal circular muscle.

Pituitary adenylate cyclase-activating peptide (PACAP)1-27, PACAP1-38, and vasoactive intestinal peptide (VIP) initiated dose-dependent contractions of canine ileal circular muscle after intra-arterial injection in vivo or ex vivo. PACAP1-27- and VIP-induced contractions approached the tissue maximum; VIP was 100-fold less potent. PACAP1-38 was more potent than VIP. PACAP1-27 contractions in vivo were unaffected by hexamethonium, reduced equally by atropine or atropine plus hexamethonium, and abolished by tetrodotoxin (TTX), suggesting that PACAP released acetylcholine and another excitatory neurotransmitter from postganglionic cholinergic enteric nerves. In myenteric plexus-free circular muscle strips, PACAP1-27 at 10(-9) M and PACAP1-38 or VIP at 10(-7) M increased [3H]acetylcholine release during nerve stimulation, suggesting the locus of one postganglionic site at which PACAP1-27 acts. All agonists inhibited nerve-mediated contractions in vivo with a potency rank order similar to that for excitation. Inhibition of nitric oxide (NO) synthetase or TTX decreased the duration and amplitude of PACAP1-27- but not PACAP1-38-induced inhibition. Inhibition of NO synthetase abolished VIP-induced inhibition, but TTX did not. Submaximal contractions to acetylcholine were amplified by PACAP1-27 or VIP before TTX and inhibited after TTX. Thus, both PACAP molecules and VIP directly inhibit and indirectly excite smooth muscle contractions. PACAP1-27 and VIP also release NO. The functional potency differences between PACAP1-27 and VIP suggest PAC1 receptors mediate all responses, likely through the stimulation of adenylate cyclase.     

Gastrointestinal motor and secretory responses to cholinergic stimulation in humans. Differential modulation by muscarinic and cholecystokinin receptor blockade

Abstract. The present study investigated how a cholinergic agonist modifies interdigestive motility and secretion of the upper gastrointestinal tract and how muscarinic and cholecystokinin receptor blockade interfere with this direct cholinergic stimulation. In eight healthy volunteers, gastrointestinal motor and secretory responses to bethanechol (12.5, 25, and 50 μg kg^-1 h^-1) with and without a background of atropine (5 μg kg^-1 h^-1) or loxiglumide (10 mg kg^-1 h^-1) were studied. Stepdoses of bethanechol caused a parallel stimulation of antroduodenal motility and gastropancreatic secretion ( P < 0.01) without inducing a fed pattern. However, duration of phase I was shortened ( P < 0.05). Only high doses of bethanechol enhanced gastrin ( P < 0.05), cholecystokinin ( P < 0.05), and pancreatic polypeptide ( P < 0.01) release. Atropine completely antagonized motor and secretory responses to cholinergic stimulation. Loxiglumide left cholinergically stimulated motility and pancreatic enzyme secretion unaltered. With co-infusion of bethanechol and loxiglumide, PP release dropped by 63% ( P < 0.01); gastric acid output, gastrin and CCK release increased by 56%, 16%, and 25%, respectively ( P < 0.05). We conclude that stimulation by a cholinergic agonist preserves the interdigestive pattern. Low dose muscarinic receptor blockade abolishes cholinergic stimulation over the full dose range. Inhibition of somatostatin release would explain stimulation of gastrin release and gastric acid secretion with co-infusion of bethanechol and loxiglumide. Endogenous CCK appears to interact with direct cholinergic stimulation at the pancreatic PP cell and the gastric D-cell but not at pancreatic acinar and antroduodenal smooth muscle cells.     

VPAC2 receptors mediate vasoactive intestinal peptide-induced neuroprotection against neonatal excitotoxic brain lesions in mice

Prepro-vasoactive intestinal peptide (VIP) mRNA codes for two neuropeptides: VIP and peptide histidine isoleucine (PHI). Two VIP receptors, shared with a similar affinity by pituitary adenylate cyclase-activating polypeptide (PACAP), have been cloned: VPAC(1) and VPAC(2). PHI binds to these receptors with a lower affinity. VPAC receptors are classically associated with a cAMP-dependent pathway, although other pathways, including calcium mobilization and protein kinase C activation have been described. We previously showed that intracerebral administration of the glutamate agonist ibotenate to postnatal day 5 mice induces white matter lesions mimicking human periventricular leukomalacia. In this model, coinjection of VIP protects against white matter lesions. This neuroprotection is independent from cAMP and is mediated by protein kinase C. Using this model, this study aimed to determine the receptor involved in VIP-induced neuroprotection. VIP effects were mimicked with a similar potency by VPAC(2) agonists and PHI but not by VPAC(1) agonists, PACAP 27, or PACAP 38. VIP neuroprotective effects were lost in mice lacking VPAC(2) receptor. In situ hybridization confirmed the presence of VPAC(2) mRNA in the postnatal day 5 white matter. When analyzed between embryonic life and adulthood, VIP-specific binding site density peaked at postnatal day 5. These data suggest that, in this model, VIP-induced neuroprotection is mediated by VPAC(2) receptors. The pharmacology of this VPAC(2) receptor seems unconventional because 1) PACAP does not mimic VIP effects, 2) PHI acts with a comparable potency, and 3) PACAP 27 modestly inhibited the VIP-specific binding, whereas for PHI or VIP, inhibition was complete.     

Effects of a cholecystokinin receptor antagonist on intestinal phase of pancreatic and biliary responses in man.

The present study was designed (a) to characterize the activity of loxiglumide as a peripheral cholecystokinin (CCK) antagonist in healthy human subjects, and (b) to determine whether CCK is a physiologic regulator of the intestinal phase of meal-stimulated exocrine pancreatic and biliary secretions in man. Intravenous loxiglumide (22 mumol/kg per h) was highly potent in antagonizing CCK8-induced pancreatic enzyme and bile acid secretion as well as pancreatic polypeptide release. The potency and selectivity of loxiglumide as an antagonist of CCK provides the tool for evaluating the role of CCK as a physiological mediator of meal-induced pancreatic and biliary responses in humans. Infusion of a liquid test meal into the duodenum evoked an immediate response of pancreatic enzyme and bilirubin outputs, respectively. Intravenous loxiglumide significantly inhibited the meal-induced pancreatic amylase output by 63% (P less than 0.05), lipase output by 43% (P less than 0.05), and bilirubin output by 59% (P less than 0.05). These data suggest that CCK is a physiological mediator of the intestinal phase of meal-stimulated pancreatic and biliary responses.     

The perfused porcine pancreas as a model for testing organ protective solutions

The present study was designed to establish an in vitro perfused porcine pancreas preparation as a model for testing the effect of organ protective solutions on stimulated pancreatic endocrine and exocrine secretion. The pancreas was prepared and perfused for 10 min with Euro Collins solution, thereafter it was stored in the cold (4 degrees C) for various times. After 3-h and 6-h ischemia pancreatic insulin release in response to glucose was not significantly affected. After 12-h ischemia reduced pancreatic insulin secretin, increased perfusion pressure, and increased amylase and lipase release indicated pancreatic damage. Complete pancreatic dysfunction was seen after 24-h and 48-h ischemia with massive increase in perfusion pressure and low insulin secretion which did not follow a glucose-dependent release pattern, while amylase and lipase concentrations in the perfusion medium increased. Stimulated exocrine pancreatic secretion was significantly decreased already after 3-h ischemia and completely lost after 12 h.     

Effect of cholestyramine on plasma cholecystokinin and pancreatic polypeptide levels, and exocrine pancreatic secretion

The effect of acute and long-term administration of cholestyramine, a non-absorbable bile salt binding resin, on exocrine pancreatic secretion, plasma-cholecystokinin (CCK) and plasma-pancreatic polypeptide (PP) was investigated in 10 healthy volunteers. Oral ingestion of 12 g cholestyramine augmented the stimulatory effect of a liquid test meal on plasma-CCK (3.5-fold) and plasma-PP (2-fold). During prolonged treatment with 3 x 12 g cholestyramine daily for 4 weeks, the most pronounced increase in basal hormone levels was observed after 1 day, but progressively decreased during treatment and had normalized after 4 weeks. However, the stimulated plasma-CCK output was still significantly elevated after cessation of treatment, compared with pretreatment values. After acute and chronic cholestyramine administration only stimulated lipase secretion was elevated, whereas trypsin and amylase remained unchanged. It is suggested that removal of bile salts enhances CCK and thereby PP release and pancreatic lipase secretion.     

Cutaneous nociception and neurogenic inflammation evoked by PACAP38 and VIP

Pituitary adenylate cyclase-activating peptide-38 (PACAP38) and vasoactive intestinal peptide (VIP) belong to the same secretin–glucagon superfamily and are present in nerve fibers in dura and skin. Using a model of acute cutaneous pain we explored differences in pain perception and vasomotor responses between PACAP38 and VIP in 16 healthy volunteers in a double-blind, placebo-controlled, crossover study. All participants received intradermal injections of 200 pmol PACAP38, 200 pmol VIP and placebo into the volar forearm. Measurements included pain intensity on a visual analog scale (VAS), blood flow by laser Doppler flowmetry, visual flare and wheal. Pain intensities after PACAP38 and VIP were mild and limited to a short time of about 100 s after injection. The area under the VAS-time curve was larger following PACAP38 (P = 0.004) and VIP (P = 0.01) compared to placebo. We found no statistical difference in pain perception between PACAP38 and VIP. Skin blood flow increase, flare and wheal were larger after both PACAP38 (P = 0.011) and VIP (P = 0.001) compared to placebo. VIP induced a considerably larger increase in skin blood flow, flare and wheal than PACAP38 (P = 0.002). In conclusion, we found that peripheral nociceptive cutaneous responses elicited by PACAP38 and VIP are similar in healthy volunteers. This suggests that acute pain and vasomotor responses following intradermal injections of PACAP38 and VIP are primarily mediated by VPAC receptors.     

Role of bile acids in the control of pancreatic secretion and CCK release

Abstract. In most species stimulated pancreatic enzyme secretion and CCK release are increased in the absence and inhibited in the presence of luminal bile acids. Changes in CCK release are almost unequivocal in all investigated species. With respect to enzyme secretion, physiological bile acid concentrations seem to be necessary to exert an inhibitory effect on stimulated enzyme output in humans. Bile acids administered in higher concentrations may enhance basal and stimulated pancreatic secretion. Furthermore, the chemical properties of different bile acids (i.e., hydroxylation, conjugation) seem to contribute to their stimulating effect on enzyme secretion as was observed in several species. The rank order of bile acids inhibiting stimulated enzyme secretion in humans is taurocholate taurodeoxycholate > taurocheno-deoxycholate. On the other hand, chenodeoxycholic acid exerts the strongest stimulating effect on secretin release, which may account for the stimulating effect of this bile acid on exocrine pancreatic secretion.
The strongest candidate for the mediator role in bile-acid-induced changes of exocrine pancreatic secretion is CCK (at least in dogs and rats).
The CCK cell may be influenced either directly or indirectly.
In conclusion, bile acids modulate pancreatic enzyme secretion and CCK release. CCK is a major candidate for this regulatory role under physiological conditions.     

Cyclic AMP and bicarbonate responses of the dog pancreas to vasoactive intestinal peptide (VIP) and secretion.

The effects of secretin (3 CU per kilogram) and vasoactive intestinal peptide (VIP; 8 mug per kilogram) on bicarbonate and cyclic AMP secretions in pancreatic juice (with pancreatic duct perfusion) and on pancreatic tissue cyclic AMP were investigated as a function of time in 13 anesthetized dogs. The peptides were given by rapid intravenous injection. Even 30 sec. after peptide administration, tissue cyclic AMP levels were elevated, reaching peak values within the first minute and a second peak at about 3 min. Bicarbonate and cyclic AMP secretions in pancreatic juice started with a lag of 1 min. after peptide injection. Following the injection of VIP, peak pancreatic response developed within the first 5 min. and the pancreatic response actually disappeared after 15 min., whereas secretion evoked by secretin was sustained for at least 30 min. The mean +/- S.D. observed maximal bicarbonate response to VIP (100 +/- 49 muEq./5 min.) was about one sixth of the maximum output following secretin (592 +/- 181 muEq./5 min.). Increases in pancreatic tissue and juice cyclic AMP caused by VIP were significant (p less than 0.05) at 1 and 4 min.; however, they were but moderate if compared with the rise achieved by secretin. The results presented confirm previous reports that VIP is a secretin-like partial agonist of pancreatic bicarbonate secretion and are compatible with the hypothesis that both secretin and VIP elicit canine pancreatic bicarbonate secretion via the second messenger system of cyclic AMP.     

Effect of Atropine on Vagal Release of Gastrin and Pancreatic Polypeptide

We studied the effect of several doses of atropine on the serum gastrin and pancreatic polypeptide responses to vagal stimulation in healthy human subjects. Vagal stimulation was induced by sham feeding. To eliminate the effect of gastric acidity on gastrin release, gastric pH was held constant (pH 5) and acid secretion was measured by intragastric titration. Although a small dose of atropine (2.3 mug/kg) significantly inhibited the acid secretory response and completely abolished the pancreatic polypeptide response to sham feeding, this dose of atropine significantly enhanced the gastrin response. Higher atropine doses (7.0 and 21.0 mug/kg) had effects on gastrin and pancreatic polypeptide release which were similar to the 2.3-mug/kg dose. Atropine (0.78 and 2.3 mug/kg) without sham feeding significantly inhibited basal acid secretion and also led to significant increases in serum gastrin above basal levels. The gastrin response to sham feeding with 2.3 mug/kg atropine was significantly greater than the sum of the gastrin responses to sham feeding alone and to 2.3 mug/kg atropine alone, indicating potentiation of vagal gastrin release by atropine. We conclude: (a) Unlike vagally mediated gastric acid secretion and pancreatic polypeptide release which can be blocked by atropine, vagal gastrin release is potentiated by atropine. This observation suggests the existence of a vagal-cholinergic pathway which normally (i.e., in the absence of atropine) inhibits gastrin release. (b) Because atropine (without sham feeding) increased basal gastrin levels, it is likely that the cholinergic pathway which inhibits gastrin release is active even when the vagus nerve is not stimulated by sham feeding.     

PAC1 receptor-deficient mice display impaired insulinotropic response to glucose and reduced glucose tolerance

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a ubiquitous neuropeptide of the vasoactive intestinal peptide (VIP) family that potentiates glucose-stimulated insulin secretion. Pancreatic beta cells express two PACAP receptor subtypes, a PACAP-preferring (PAC1) and a VIP-shared (VPAC2) receptor. We have applied a gene targeting approach to create a mouse lacking the PAC1 receptor (PAC1(-/-)). These mice were viable and normoglycemic, but exhibited a slight feeding hyperinsulinemia. In vitro, in the isolated perfused pancreas, the insulin secretory response to PACAP was reduced by 50% in PAC1(-/-) mice, whereas the response to VIP was unaffected. In vivo, the insulinotropic action of PACAP was also acutely reduced, and the peptide induced impairment of glucose tolerance after an intravenous glucose injection. This demonstrates that PAC1 receptor is involved in the insulinotropic action of the peptide. Moreover, PAC1(-/-) mice exhibited reduced glucose-stimulated insulin secretion in vitro and in vivo, showing that the PAC1 receptor is required to maintain normal insulin secretory responsiveness to glucose. The defective insulinotropic action of glucose was associated with marked glucose intolerance after both intravenous and gastric glucose administration. Thus, these results are consistent with a physiological role for the PAC1 receptor in glucose homeostasis, notably during food intake.     

Vasoactive intestinal polypeptide stimulates cholecystokinin secretion in perfused rat duodenum

We examined the effect of vasoactive intestinal polypeptide (VIP) on cholecystokinin (CCK) secretion from the isolated perfused rat duodenum. VIP stimulated CCK secretion mono-phasically in a concentration-dependent manner in concentrations ranging from 10(-9) to 10(-7) M, and 10(-7) M of VIP led to an increment of 82 +/- 25.8 fmole/3 min. The stimulatory effect of VIP on CCK was not inhibited by 10(-5) M atropine. These results suggest that VIP may directly stimulate CCK secretion from the duodenum and work as a non-cholinergic, peptidergic neurotransmitter.     

Feedback regulation of stimulated pancreatic enzyme secretion during intraduodenal perfusion of trypsin in man

The pancreatic enzyme secretion in several species is controlled by a negative feedback mechanism induced by the presence of active proteases in the duodenum. The existence of this mechanism in man is controversial. The purpose of the present study was to evaluate the effect of tryptic activity in the duodenum on phenylalanine-stimulated pancreatic enzyme secretion in healthy volunteers. A double-balloon, multi-lumen tube was used for the collection of duodenal juice containing pancreatic enzymes. The continuous infusion of phenylalanine (100 mM) into the duodenum evoked an almost constant secretion of trypsin, amylase and lipase for 160 min. The infusion of trypsin (150 mg h-1; 1.25 g l-1) caused a reduction of phenylalanine-stimulated amylase and lipase output by 25%. The subsequent infusion of aprotinin at a dose of 1.5 X 10(6) KIU for 30 min led to an almost complete inhibition of trypsin. Simultaneously, the amylase and lipase output returned to the values seen before trypsin perfusion. Infusion of a higher dose of trypsin (300 mg h-1; 2.5 g l-1) caused a more pronounced decrease in phenylalanine-stimulated amylase and lipase output by 45%. These data indicate that active trypsin in the duodenum is responsible for the inhibition of phenylalanine-stimulated pancreatic enzyme secretion in man in a dose-dependent fashion, thus confirming the existence of a feedback control of pancreatic secretion regulated by the amount of proteases in the gut.