Stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system. Determination of GTP/peptide bond ratios during codon-specific elongation and misreading

Pancreatic secretory factor (PSF), a 17.5-kDa protein purified from the venom of Gila monster (Heloderma suspectum), stimulated amylase secretion from dispersed rat pancreatic acini more efficiently than CCK-8, bombesin, carbachol and secretin, and without increasing 45Ca2+ efflux and cyclic AMP levels. The secretory action was dependent on the presence of extracellular calcium and was additive to the secretion induced by agents acting via cyclic AMP or via Ca2+ efflux.     

Interaction of Gila monster venom with secretin receptors in rat pancreatic membranes

The stimulatory effect of Gila monster venom on adenylate cyclase activity in rat pancreatic membranes was compared to that of porcine secretin and porcine VIP. The maximal effect exerted by the venom was identical to that of VIP but significantly lower than that of secretin. The effect of Gila monster venom could, however, be attributed to its interaction with secretin receptors rather than with VIP receptors, at variance with its previously described action on guinea pig pancreatic acini. Adenylate cyclase activation by both Gila monster venom and secretin in rat pancreatic membranes was, indeed: (1) dose-dependently inhibited by two secretin fragments secretin-(4-27) and secretin-(7-27), and (2) more severely depressed than VIP stimulation, after pretreating pancreatic membranes with dithiothreitol (DTT).     

Evidence that helodermin, a newly extracted peptide from Gila monster venom, is a member of the secretin/VIP/PHI family of peptides with an original pattern of biological properties

Helodermin, a newly isolated peptide from the venom of Gila monster (Heloderma suspectum) was shown to stimulate the adenylate cyclase activity of rat pancreatic membranes as efficiently as secretin and VIP. It also increased cyclic AMP levels and inhibited [125I]VIP binding in rat pancreatic acini. Finally, helodermin activated adenylate cyclase in membranes from rat heart, rat brain, and human heart, showing properties analogous yet distinct from those of secretin, VIP and PHI.     

Interaction of Gila monster venom with VIP receptors in intestinal epithelium of human. A comparison with rat

Gila monster venom (1-300 micrograms/ml) is shown to inhibit completely the binding of [125I]VIP to human and rat intestinal epithelial cell membranes. In both models, the venom inhibits [125I]VIP binding and stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP and a potency that is 10000-50000 times lower than that of the peptide, on a weight basis. At maximal doses, VIP and Gila monster venom do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the venom occurs through VIP receptors. As is the case for VIP, adenylate cyclase activation by Gila monster venom requires the presence of GTP in the incubation medium. Finally, no VIP-like immunoreactivity was detected in the venom using an antiserum raised against mammalian VIP. All these data suggest the presence in the venom of the Gila monster, of a new substance which behaves as a VIP agonist in human as well as rat intestine.     

Regulation of protein phosphorylation in pancreatic acini by cyclic AMP-mediated secretagogues: interaction with carbamylcholine

The effects on protein phosphorylation in mouse pancreatic acini of cyclic AMP-mediated secretagogues and the Ca2+-mediated agonist carbamylcholine were compared. Under the conditions adopted for the study of protein phosphorylation, carbamylcholine (3 microM) stimulated amylase release from pancreatic acini 6-fold, whereas vasoactive intestinal polypeptide (VIP) (100 nM) and the cyclic AMP analogue 8-bromo-cyclic AMP (1 mM) caused little or no increase in secretion. However, VIP and 8-bromo-cyclic AMP, when added in combination with carbamylcholine, potentiated the stimulation of amylase release to 170-180% of that caused by carbamylcholine alone. As assessed by two-dimensional gel electrophoresis, VIP reproduced four of the ten changes in protein phosphorylation elicited by carbamylcholine, these changes being the increased phosphorylation of one soluble protein and the decreased phosphorylation of three soluble proteins. VIP enhanced the carbamylcholine-induced changes in phosphorylation for three proteins. In addition, VIP increased the phosphorylation of a unique protein of Mr 52,000 and pI 5.66 which was not affected by carbamylcholine. All of the effects on protein phosphorylation exerted by VIP in the presence or absence of carbamylcholine were mimicked by 8-bromo-cyclic AMP. Secretin also reproduced most of the changes in protein phosphorylation caused by VIP, although concentrations of secretin of at least 100-fold higher were required to elicit a maximal response. It is concluded that cyclic AMP-mediated secretagogues alter the phosphorylation of a unique protein as well as of several pancreatic proteins affected by carbamylcholine. Moreover, these effects appear to be mediated primarily by VIP-preferring receptors and may be involved in the synergistic action of VIP to promote carbamylcholine-induced amylase release.     

Forskolin potentiates calcium-dependent amylase secretion from rat pancreatic acinar cells

The cellular and molecular effects of forskolin, a direct, nonhormonal activator of adenylate cyclase, were assessed on the enzyme secretory process in dispersed rat pancreatic acinar cells. Forskolin stimulated adenylate cyclase activity in the absence of guanyl nucleotide. It promoted a rapid and marked increase in cellular accumulation of cyclic AMP alone or in combination with vasoactive intestinal peptide (VIP) but was itself a weak pancreatic agonist and did not increase the secretory response to VIP or other cyclic AMP dependent agonists. Somatostatin was a partial antagonist of forskolin stimulated cyclic AMP synthesis and forskolin plus cholecystokinin-octapeptide (CCK-OP) induced amylase release. Forskolin potentiated amylase secretion in response to calcium-dependent agonists such as CCK-OP, carbachol and A-23187, but did not affect the ability of CCK-OP and (or) carbachol to mobilize 45Ca from isotope preloaded cells; forskolin alone did not stimulate 45Ca release. In calcium-poor media, the secretory response to forskolin and CCK-OP was reduced in a both absolute and relative manner. The data suggests that calcium plays the primary role as intracellular mediator of enzyme secretion and that the role of cyclic AMP may be to modulate the efficiency of calcium utilization.     

Evidence against cyclic GMP as a mediator of the actions of secretagogues on amylase release from guinea-pig pancreas

In dispersed acini from guinea-pig pancrease several pancreatic secretagogues increased calcium outflux, cyclic GMP and amylase secretion, whereas nitroprusside and hydroxylamide increased cyclic GMP but did not increase calcium outflux or amylase secretion and did not alter the action of secretagogues on calcium outflux or amylase secretion. Secretin and vasoactive intestinal peptide increased cyclic AMP and increased secretion but did not alter cyclic GMP. Nitroprusside and hydroxylamine did not alter cyclic AMP or the action of secretin or vasoactive intestinal peptide on cyclic AMP and enzyme secretion. Agents that increased cyclic GMP also caused release of the nucleotide into the extracellular medium; however, this release did not correlate with secretion of amylase into the extracellular medium. 8-Bromo cyclic AMP as well as 8-bromo cyclic GMP increased enzyme secretion and potentiated the increase in enzyme secretion caused by cholecystokinin or carbachol. The increase in amylase secretion caused by vasoactive intestinal peptide or secretin plus either of the cyclic nucleotide derivatives was the same as that caused by the peptide alone. These results indicate that cyclic GMP does not mediate the action of secretagogues on pancreatic enzyme secretion, that the release of cyclic GMP into the extracellular medium does not occur by exocytosis and that the increase in enzyme secretion caused by 8-bromo cyclic GMP results from its stability to mimic the action of endogenous cyclic AMP.     

Amylase release from rat parotid glands. I. General characteristics.

The involvement of calcium, ATP, and cyclic AMP-dependent protein kinase activity in the release of amylase from rat parotid glands was examined. Pretreatment of the glandular tissue in 11.25 mM Ca2+ medium potentiated the secretory responses to: dibutyryl cyclic AMP, elevation of the extracellular K+ concentration, reduction of the H+ concentration, La3+, and caffeine. Uncoupling of oxidative phosphorylation blocked release induced by dibutyryl cyclic AMP, K+, and reduction of H+, but had no effect on La3+, caffeine or tolbutamide-stimulated release. Inhibition of cyclic AMP-dependent protein kinase activity blocked only dibutyryl cyclic AMP-induced release and did not inhibit the responses to K+, reduction of H+ or caffeine. The loss of lactate dehydrogenase was used to access the integrity of the tissue during amylase release. No significant increase in the release of lactate dehydrogenase was observed during the secretory responses to: dibutyryl cyclic AMP, La3+, caffeine, or tolbutamide. Triton X-100 and ethanol increased the efflux of both amylase and lactate dehydrogenase. The differential involvement of Ca2+, ATP, and cyclic AMP-dependent protein kinase activity in amylase release induced by the various secretagogues suggests that three types of reactions are involved in the release of amylase.     

Receptors for vasoactive intestinal peptide and secretin on guinea pig pancreatic acini

We investigated the abilities of VIP and secretin to occupy receptors and to increase cellular cyclic AMP using dispersed acini from guinea pig pancreas. The dose-inhibition curve for inhibition of binding of 125I-VIP by VIP was broad with detectable inhibition at 0.1 nM VIP, half-maximal inhibition at 2 nM VIP and complete inhibition at 10 microM VIP. Secretin also inhibited binding of 125I-VIP was compatible with two VIP-preferring receptors with one class having a high affinity for VIP (Kd 1.1 nM) and a low affinity for secretin (Kd 5 microM) and the other class having an intermediate affinity for VIP (Kd 470 nM). The dose inhibition curve for inhibition of binding of 125I-secretin by secretin was not broad. Half-maximal inhibition occurred with 7 nM secretin or with 10 microM VIP. Computer analysis was compatible with a single secretin-preferring receptor with a high affinity for secretin (Kd 7 nM) and a low affinity for VIP (Kd 5.9 microM). Comparison of the ability of VIP to increase cyclic AMP with or without the secretin-receptor antagonist, secretin-5-27, demonstrated only occupation of the high affinity VIP-preferring or high affinity secretin-preferring receptors increase cyclic AMP. Our results demonstrate that, in contrast to previous reports, guinea pig pancreatic acini possess 3 classes of receptors that interact with VIP and secretin. The low affinity receptor seen with 125I-VIP is not the same as the secretin-preferring receptor and does not increase cellular cyclic AMP.     

Evidence for a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum

The effects of secretin and vasointestinal peptide (VIP) on the production of cyclic AMP have been studied in gastric glands isolated by means of EDTA from rat fundic and antral mucosa. (1) In gastric fundus, secretin and VIP caused a time- and temperature-dependent stimulation of cyclic AMP production that was maximal when the test agents were incubated for 60 min at 20 degrees C in the presence of 0.5 mM 3-isobutyl-1-methylxanthine as a phosphodiesterase inhibitor. The dose-response curve was monophasic for both peptides, the production of cyclic AMP being sensitive to 10(-10) M secretin and to 5 . 10(-8) M VIP. Half-maximal stimulation was obtained with 2.9 10(-9) M secretin or 2 . 10(-7) M VIP and the maximal stimulation represented a 21-fold and a 19-fold increase above control for secretin and VIP, respectively. Histamine also stimulated cyclic AMP production, with a Km of about 5 . 10(-4) M. No additive effect on cyclic AMP production was oberved when secretin and VIP were simultaneously added at maximally active concentrations, while an additive effect was observed when secretin and histamine were added together. (2) In gastric antrum, the characteristics of the secretin- and VIP-stimulated cyclic AMP production were similar to those observed in gastric fundus. Histamine nevertheless failed to stimulate the formation of cyclic AMP in antral mucosa. (3) These data demonstrate the existence of a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum and suggest that VIP operates through this system. (4) It is proposed that the pepsinogen- and/or mucous-secreting cells are implicated in the regulation of cyclic AMP production by secretin in gastric glands of the rat.     

Interaction of porcine vasoactive intestinal peptide with dispersed pancreatic acinar cells from the guinea pig. Structural requirements for effects of vasoactive intestinal peptide and secretin on cellular adenosine 3':5'-monophosphate.

Secretin and vasoactive intestinal peptide (VIP), but not glucagon, stimulate accumulation of cyclic AMP in dispersed guinea pig pancreatic acinar cells. Secretin stimulated cellular accumulation of cyclic AMP by interacting with a single class of high affinity receptors. On the other hand, the dose-response curve for VIP-stimulated cellular cyclic AMP was biphasic and reflected interaction of this peptide with two classes of receptors. Results obtained with synthetic fragments of VIP and secretin indicate that the receptor having a high affinity for VIP has a low affinity for secretin, interacts with, but does not distinguish among, secretin, secretin 5-27 and [6-tyrosine] secretin or among secretin 14-27, VIP 14-28, VIP 15-28, and increases cellular cyclic AMP when occupied by VIP, but not when occupied by secretin, [6-tyrosine] secretin, or secretin 1-14. The receptor having a low affinity for VIP has a high affinity for secretin, interacts with and distinguishes among secretin, secretin 5-27, and [6-tyrosine] secretin, interacts with secretin 14-27 but not with VIP 14-28 or VIP 15-28, and increases cellular cyclic AMP when occupied by VIP, secretin, [6-tyrosine] secretin, or secretin 1-14.     

PACAP-38, a novel peptide from ovine hypothalamus, is a potent modulator of amylase release from dispersed acini from rat pancreas.

Despite studies indicating the presence of specific pancreatic acinar receptors for PACAP-38, a peptide that was recently isolated from ovine hypothalamus, the actions of the new peptide on pancreatic enzyme secretion have not been examined. The present study demonstrates that in terms of cAMP production and amylase release from dispersed acini from rat pancreatic acini, PACAP-38 and an N-terminal fragment, PACAP-27, have the same potency and efficacy as vasoactive intestinal peptide (VIP). As with VIP, these actions are potentiated by adding an inhibitor of cyclic nucleotide phosphodiesterase, and combination of PACAP-38 with bombesin, CCK-8, carbachol or the calcium ionophore A23187 results in 2-fold augmentation of the secretory actions of these agents. Inhibition of PACAP-38-induced cAMP production and amylase release by two VIP-receptor antagonists indicates that the secretory effects of PACAP-38 are mediated by interaction with VIP receptors. PACAP-38, a new brain-gut peptide, may be a physiological modulator of pancreatic enzyme secretion.     

Amylase release from rat parotid glands I. General characteristics

The involvement of calcium, ATP, and cyclic AMP-dependent protein kinase activity in the release of amylase from rat parotid glands was examined. Pretreatment of the glandular tissue in 11.25 mM Ca²⁺ medium potentiated the secretory responses to: dibutyryl cyclic AMP, elevation of the extracellular K⁺ concentration, reduction of the H⁺ concentration, La³⁺, and caffeine. Uncoupling of oxidative phosphorylation blocked release induced by dibutyryl cyclic AMP, K⁺, and reduction of H⁺, but had no effect on La³⁺, caffeine or tolbutamide-stimulated release. Inhibition of cyclic AMP-dependent protein kinase activity blocked only dibutyryl cyclic AMP-induced release and did not inhibit the responses to K⁺, reduction of H⁺ or caffeine.The loss of lactate dehydrogenase was used to access the integrity of the tissue during amylase release. No significant increase in the release of lactate dehydrogenase was observed during the secretory responses to: dibutyryl cyclic AMP, La³⁺, caffeine, or tolbutamide. Triton X-100 and ethanol increased the efflux of both amylase and lactate dehydrogenase.The differential involvement of Ca²⁺, ATP, and cyclic AMP-dependent protein kinase activity in amylase release induced by the various secretagogues suggests that three types of reactions are involved in the release of amylase.     

Exendin-3, a novel peptide from Heloderma horridum venom, interacts with vasoactive intestinal peptide receptors and a newly described receptor on dispersed acini from guinea pig pancreas. Description of exendin-3(9-39) amide, a specific exendin receptor antagonist.

Exendin-3 increased cellular cAMP levels and amylase release from dispersed acini from guinea pig pancreas. Low concentrations (0.1-3 nM) caused a 12-fold increase in cAMP, whereas higher concentrations (0.3-3 microM) caused an additional 24-fold increase in cAMP. Maximal cAMP with the highest concentration tested was the same as the maximal response with secretin, vasoactive intestinal peptide (VIP), peptide histidine isoleucine, helodermin, or helospectin-I. In terms of amylase release, exendin-3 had the same efficacy but was the least potent of these peptides. Exendin-3-induced increases in amylase release were inhibited by VIP receptor antagonists and the new peptide (greater than 0.1 microM) competed with radiolabeled VIP for binding sites on dispersed acini. Increasing concentrations of an exendin-3 fragment, exendin-3(9-39) amide, did not increase cAMP or amylase release but inhibited the increase in cAMP observed with 0.1-3 nM exendin-3. The fragment did not alter the effects of other peptides that are known to increase acinar cAMP. We conclude that exendin-3 interacts with at least two receptors on guinea pig pancreatic acini; at high concentrations (greater than 100 nM) the peptide interacts with VIP receptors, thereby causing a large increase in cAMP and stimulating amylase release; at lower concentrations (0.1-3 nM) the peptide interacts with a putative exendin receptor, thereby causing a smaller increase in cAMP of undetermined function. Exendin-3(9-39) amide is a specific exendin receptor antagonist.     

Action of cholera toxin on dispersed acini from guinea pig pancreas.

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of binding of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretin or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Stimulatory effect of vasoactive intestinal peptide (VIP) on cyclic AMP production in rat peritoneal macrophages.

Vasoactive intestinal peptide (VIP) stimulated cyclic AMP production in rat peritoneal macrophages. The stimulatory effect of VIP was dependent on time, temperature and cell concentration, and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). At 15 degrees C, the response occurred in the 0.1-1000 nM range of VIP concentrations. Half maximal stimulation of cellular cyclic AMP (ED50) was obtained at 1.2 +/- 0.5 nM VIP, and maximal stimulation (about 3-fold basal level) was obtained between 100-1000 nM. The cyclic AMP system of rat peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, pancreastatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 microM. The beta-adrenergic agonist isoproterenol increased the cyclic AMP production and show additive effect with VIP. Somatostatin inhibits the accumulation of cyclic AMP in the presence of both vasoactive intestinal peptide and isoproterenol. The finding of a VIP-stimulated cyclic AMP system in rat peritoneal macrophages, together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, strongly suggest that VIP may be involved in the regulation of macrophage function.     

Secretin Stimulates Cyclic AMP Formation in the Rat Brain

The effects of secretin on cyclic AMP levels in the rat brain were determined. Incubation of rat brain frontal cortex slices with secretin or the structurally related peptides peptide histidine leucine (PHI) or vasoactive intestinal polypeptide (VIP) in the presence of 10 mM theophylline resulted in a dose-dependent increase in the cyclic AMP levels. The half-maximal increase in cyclic AMP occurred using a 1 microM dose of secretin or a 2 microM dose of PHI or VIP. Preincubation of slices with secretin-(5-27) produced a dose-dependent inhibition of the secretin but not VIP- or PHI-stimulated increase in the cyclic AMP content. Also, in receptor binding studies, secretin-(5-27) produced a dose-dependent inhibition (Ki = 400 nM) of 125I-secretin but not of 125I-VIP binding to rat brain membranes. Guanyl-5'-yl imidodiphosphate decreased the affinity of radiolabelled secretin binding as a result of an increased rate of dissociation of bound 125I-secretin. These data suggest that secretin receptors in the rat brain may be coupled to adenylate cyclase in a stimulatory manner and that secretin-(5-27) may function as a central secretin receptor antagonist.     

Stimulatory effects of human pancreatic polypeptide on rat pancreatic acini

The effect of human pancreatic polypeptide (HPP) on rat pancreatic acini has been studied. It was found that HPP stimulated amylase and lipase release from the acini. The secretory response of acini to HPP was dose-dependent in a sigmoidal fashion. Between 10(-9) M and 10(-8) M concentration of HPP there was a slow increase of enzyme release to about 40-60% over basal release. At concentrations of HPP above 10(-8) M there was a rapid increase of enzyme release, amounting to 4-6 times over basal release at 10(-6) M concentration of HPP. The potency of HPP compared to other secretagogues at 10(-7) M concentration was 45% of CCK, 60% of carbachol and 75% of secretin. HPP did not inhibit the effect of CCK, secretin and carbachol on amylase release. The amylase release stimulated by HPP was accompanied by an increase in 45Ca2+ efflux. Atropine or dibutyryl cyclic GMP did not influence the effect of HPP. It is concluded that HPP stimulates the release of enzymes from rat pancreatic acini and that Ca2+ may be a mediator for this secretion.     

Effect of substance P and eledoisin on K+ efflux, amylase release and cyclic nucleotide levels in slices of rat parotid gland.

The undecapeptides, substance P and eledoisin, caused a rapid, concentration-dependent increase in K+ efflux and amylase release from parotid tissue slices. The effects were not blocked by beta-adrenergic, alpha-adrenergic, or cholinergic antagonists. Incubation buffer calcium was required for stimulation of K efflux and amylase release. The action of the undecapepides was independent of any effects on parotid cyclic AMP or cyclic GMP levels. Since the actions of the undecapeptides were Ca2+ dependent and no effects on cyclic nucleotide levels were discerned it was concluded that Ca2+ plays a primary role in agonist regulation of K+ efflux from the parotid.     

Secretin induces rapid increases in inositol trisphosphate, cytosolic Ca2+ and diacylglycerol as well as cyclic AMP in rat pancreatic acini.

Previous studies have shown that the dose-response relationship for secretin-stimulated cyclic AMP accumulation is different from that for secretin-stimulated enzyme secretion in the rat exocrine pancreas. Here we show that secretin concentrations of 10(-10) M and higher stimulated a rise in cyclic AMP levels, with maximum effect on cyclic AMP accumulation being achieved already with 10(-8) M-secretin. However, at this concentration of secretin, enzyme secretion rates were approximately half-maximal. Unexpectedly, at concentrations of secretin greater than 10(-8) M there was evidence suggestive of phosphatidylinositol bisphosphate hydrolysis with rapid increases in inositol trisphosphate, cytosolic free calcium and diacylglycerol content of rat pancreatic acini. Furthermore, there was a dose-response relationship among secretin concentration (in the range 10(-8) M-2 X 10(-6) M), increases in inositol trisphosphate and increases in cytosolic free calcium ([Ca2+]i). Contrary to what has been previously believed, these results clearly indicate that in rat pancreatic acini secretin not only stimulates cyclic AMP accumulation but also raises inositol trisphosphate, [Ca2+]i and diacylglycerol. Thus, two second messenger systems may play a role in the regulation of secretin-induced amylase release.