Effects of calcium and chelating agents on the ability of various agonists to increase cyclic GMP in pancreatic acinar cells.

In dispersed acinar cells from guinea pig pancreas we found that chelating extracellular calcium with EDTA did not alter cellular cyclic GMP but caused a 50% reduction in the increase in cyclic GMP caused by the synthetic C-terminal octapeptide of porcine cholecystokinin (cholecystokinin octapeptide). This effect was maximal within 2 min and preincubating the cells with EDTA for as long as 30 min caused no further reduction in the action of cholecystokinin octapeptide. In acinar cells preincubated without calcium, adding calcium caused a time dependent increase in the action of cholecystokinin octapeptide and this increase was maximal after 10 min of incubation. An effect of extracellular calcium on the action of cholecystokinin octapeptide could be detected with 0.5 mM calcium and was maximal with 2.0 mM calcium. Magnesium alone or with calcium did not alter the action of cholecystokinin octapeptide. Extracellular calcium did not alter the time course or the configuration of the dose vs. response curve for the action of cholecystokinin octapeptide on cellular cyclic GMP. Low concentrations of EGTA (0.1 mM) decreased the effect of cholecystokinin octapeptide on cellular cyclic GMP to the same extent as did EDTA or preincubating acinar cells without calcium. Increasing EGTA above 0.1 mM caused progressive augmentation of the action of cholecystokinin octapeptide on cellular cyclic GMP and this augmentation did not require extracellular calcium or magnesium. Results similar to those obtained with cholecystokinin octapeptide were also obtained with bombesin, carbamylcholine, litorin and eledoisin. In contrast, the action of sodium nitroprusside on cyclic GMP in pancreatic acinar cells was not altered by adding EDTA or EGTA. These results indicate that the ability of extracellular calcium to influence the action of cholecystokinin octapeptide and other agents on cyclic GMP results from changes in cellular calcium and not from effects of extracellular calcium per se. The action of low concentrations of EGTA on the increase in cyclic GMP caused by various agents reflects the ability of EGTA to chelate extracellular calcium. The actions of high concentrations of EGTA were independent of extracellular calcium or magnesium and appear to reflect a direct action of EGTA on pancreatic acinar cells.     

Effects of calcium and chelating agents on the ability of various agonists to increase cyclic GMP in pancreatic acinar cells

In dispersed acinar cells from guinea pig pancreas we found that chelating extracellular calcium with EDTA did not alter cellular cyclic GMP but caused a 50% reduction in the increase in cyclic GMP caused by the synthetic C-terminal octapeptide of porcine cholecystokinin (cholecystokinin octapeptide). This effect was maximal within 2 min and preincubating the cells with EDTA for as long as 30 min caused no further reduction in the action of cholecystokinin octapeptide. In acinar cells preincubated without calcium, adding calcium caused a time dependent increase in the action of cholecystokinin octapeptide and this increase was maximal after 10 min of incubation. An effect of extracellular calcium on the action of cholecystokinin octapeptide could be detected with 0.5 mM calcium and was maximal with 2.0 mM calcium. Magnesium alone or with calcium did not alter the action of cholecystokinin octapeptide. Extracellular calcium did not alter the time course or the configuration of the dose vs. response curve for the action of cholecystokinin octapeptide on cellular cyclic GMP. Low concentrations of EGTA (0.1 mM) decreased the effect of cholecystokinin octapeptide on cellular cyclic GMP to the same extent as did EDTA or preincubating acinar cells without calcium. Increasing EGTA above 0.1 mM caused progressive augmentation of the action of cholecystokinin octapeptide on cellular cyclic GMP and this augmentation did not require extracellular calcium or magnesium. Results similar to those obtained with cholecystokinin octapeptide were also obtained with bombesin, carbamylcholine, litorin and eledoisin. In contrast, the action of sodium nitroprusside on cyclic GMP in pancreatic acinar cells was not altered by adding EDTA or EGTA.These results indicate that the ability of extracellular calcium to influence the action of cholecystokinin octapeptide and other agents on cyclic GMP results from changes in cellular calcium and not from effects of extracellular calcium per se. The action of low concentrations of EGTA on the increase in cyclic GMP caused by various agents reflects the ability of EGTA to chelate extracellular calcium. The actions of high concentrations of EGTA were independent of extracellular calcium or magnesium and appear to reflect a direct action of EGTA on pancreatic acinar cells.     

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.     

Effect of AA861, a 5-lipoxygenase inhibitor,on amylase secretion from rat pancreatic acini

We have examined the effects of 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), a selective inhibitor of 5-lipoxygenase, on the action of cholecystokinin (CCK) and other secretagogues in the stimulation of amylase secretion from dispersed rat pancreatic acini. AA861 inhibited amylase secretion caused by CCK, carbamylcholine (carbachol), bombesin or calcium ionophore A23187 but failed to affect amylase secretion by vasoactive intestinal peptide or 12-O-tetradecanoyl-phorbol 13-acetate. Inhibition by AA861 of CCK or carbachol-induced amylase secretion was confined to the relatively lower concentrations of these secretagogues. AA861 did not inhibit receptor binding of CCK or alter the cellular calcium mobilization induced by CCK. In kinetic studies, AA861 was effective only on amylase secretion from pancreatic acini incubated with CCK for more than 5 min. Indomethacin, a known inhibitor of cyclooxygenase, did not affect the amylase secretion caused by all secretagogues used. These results indicate that the 5-lipoxygenase pathway of arachidonate metabolism may be involved in the actions of calcium-dependent secretagogues of amylase secretion in rat dispersed pancreatic acini, especially for sustaining stimulation of amylase secretion by CCK.     

Action of cholecystokinin, cholinergic agents, and A-23187 on accumulation of guanosine 3':5'-monophosphate in dispersed guinea pig pancreatic acinar cells.

The COOH-terminal octapeptide of cholecystokinin (CCK-OP) and carbamylcholine each increased calcium outflux, cellular cyclic GMP and amylase secretion in dispersed guinea pig pancreatic acinar cells. Following addition of CCK-OP or carbamylcholine, cellular cyclic GMP increased as early as 15 s, became maximal after 1 to 2 min, and then decreased steadily during the subsequent incubation. For both CCK-OP and carbamylcholine there was close agreement between the dose-response curve for stimulation of calcium outflux and that for increase of cellular cyclic GMP. With CCK-OP an effect on both functions could be detected at 10(-10) M and maximal stimulation occurred at 3 X 10(-8) M. With carbamylcholine an effect on both functions could be detected at 10(-5) M and maximal stimulation occurred at 3 X 10(-3) M. Atropine inhibited stimulation of both cyclic GMP and calcium outflux by carbamylcholine but not by CCK-OP. Stimulation of calcium outflux or cellular cyclic GMP by CCK-OP or carbamylcholine did not require extracellular calcium since stimulation occurred in a calcium-free, ethylene glycol bis(beta, beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA)-containing solution. The divalent cation ionophore A-23187 increased bidirectional fluxes of calcium, cellular cyclic GMP and secretion of amylase from dispersed pancreatic acinar cells. Like CCK-OP and carbamylcholine, the ionophore stimulated calcium outflux and cellular cyclic GMP in a calcium-free, EGTA-containing solution. These results suggest that in pancreatic acinar cells the initial step in the sequence of events mediating the action of ionophore as well as that of CCK-OP and carbamylcholine is stimulation of calcium outflux, and that this stimulation then increases cellular cyclic GMP.     

Morphological changes in rat pancreatic slices associated with inhibition of enzyme secretion by high concentrations of secretagogues

Stimulation of enzyme secretion in rat pancreatic slices by cholinergic agonists or by cholecystokinin-pancreozymin (CCK-PZ) and its peptide analogs showed a biphasic dose response curve. The optimal concentrations eliciting an efficient rate of enzyme secretion were 1 microM for carbamylcholine or acetylcholine, and 5 nM and 20 nM for CCK-PZ octapeptide and CCK-PZ, respectively. At higher concentrations of secretagogues, however, the rate of secretion progressively declined, and almost complete inhibition was achieved at 1 mM of carbamylcholine or acetylcholine and at 0.1 microM of CCK-PZ or its octapeptide analog. Atropine displaced the dose-response curve for carbamylcholine to the right so that in the presence of 7 microM atropine a concentration of 1 mM carbamylcholine now gave an optimal rate of enzyme secretion. The ionophore A-23187 which bypasses the receptor and elicits enzyme secretion did not relieve the inhibition caused by supraoptimal concentrations of secretagogues, indicating that the inhibition occurs at the cellular rather than at the receptor level. Secretin had no effect on the inhibition of enzyme secretion by a high concentration of carbamylcholine, indicating that the inhibition was not caused by lack of water and electrolyte secretion. The energy-producing metabolism was not affected since the ATP level in the pancreatic slices was the same in the presence of either inhibitory or optimal concentrations of secretagogues. The inhibition of enzyme secretion was reversible since restoration of efficient enzyme secretion occurred after removal of carbamylcholine (1 mM) by washing, followed by addition of an optimal concentration of CCK-PZ octapeptide. Morphological studies revealed that the presence of inhibitory concentrations of secretagogues caused severe distortion of the lumen structure: disruption of the filamentous system surrounding the lumen, disappearance of microvilli, and production of distended evaginations of the luminal membrane containing cellular material. These changes eventually caused a reduction in the size of the lumen which becomes plugged with secretory material. It is suggested that these changes in the microtubular microfilamentous system could account for the inhibition of enzyme secretion.     

The actions of tetraethylammonium on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas, replacing extracellular sodium by tetraethylammonium (1) abolished carbamylcholine-stimulated amylase secretion but did not alter the increase in amylase secretion caused by the C-terminal octapeptide of cholecystokinin, bombesin, ionophore A23187, vasoactive intestinal peptide or 8-bromoadenosine 3':5' monophosphate, (2) caused a parallel rightward shift in the dose-response curve for carbamylcholine-stimulated amylase secretion and (3) inhibited binding of N-[3H]methyl scopolamine to muscarinic cholinergic receptors. Detectable inhibition of carbamylcholine-stimulated amylase secretion and binding of N-[3H]methyl scopolamine occurred with 300 microM tetraethylammonium, and half-maximal inhibition of these functions occurred with 1-2 mM tetraethylammonium. Replacing extracellular sodium by Tris did not alter the stimulation of enzyme secretion caused by any secretagogue tested. These results indicate that the tetraethylammonium is a muscarinic cholinergic receptor antagonist and that enzyme secretion from pancreatic acini does not depend on extracellular sodium.     

An analogue of substance P with broad receptor antagonist activity

[DPro4,DTrp7,9,10]Substance P-4-11 functions as a substance P receptor antagonist in several different systems. Because some analogues of substance P can function as receptor antagonists for bombesin as well as substance P, we tested [DPro4,DTrp7,9,10]substance P-4-11 for its ability to modify the interaction of various pancreatic secretagogues with their receptors in dispersed acini from guinea pig pancreas. [DPro4,DTrp7,9,19]Substance P-4-11 did not stimulate amylase secretion and did not alter the stimulation of amylase secretion caused by secretin, vasoactive intestinal peptide, calcitonin gene-related peptide or carbachol, but did inhibit the stimulation of amylase secretion caused by substance P, bombesin or cholecystokinin. With substance P, bombesin and cholecystokinin, [DPro4,DTrp7,9,10]substance P-4-11 caused a parallel rightward shift in the dose-response curve for stimulation of amylase secretion with no change in the maximal response. Schild plots of these results gave straight lines with slopes that were not significantly different from unity. [DPro4,DTrp7,9,10]Substance P-4-11 inhibited binding of 125I-labeled substance P, 125I-[Tyr4]bombesin and 125I-cholecystokinin octapeptide over the same range of concentrations as that in which it inhibited biologic activity of each of these peptides. Half-maximal inhibition of binding of 125I-substance P occurred with 4 microM, of 125I-[Tyr4]bombesin with 17 microM and of 125I-cholecystokinin octapeptide with 5 microM. With each radiolabeled peptide the value of Ki for inhibition of binding by [DPro4,DTrp7,9,10]substance P-4-11 was not significantly different from the corresponding value of Ki calculated from the appropriate Schild plot. The present results indicate that [DPro4,DTrp7,9,10]substance P-4-11 is a competitive antagonist at receptors for substance P, for bombesin and for cholecystokinin. Thus, these receptors must share a common peptide recognition mechanism even though they interact with agonists that have no obvious structural similarity.     

Action of cholera toxin on dispersed acini from rat pancreas. Post-receptor modulation involving cyclic AMP and calcium

In dispersed acini from rat pancreas, cholera toxin caused a significant increase in cellular cyclic AMP but little or no change in amylase secretion. The presence of a secretagogue that causes mobilization of cellular calcium (e.g., cholecystokinin, carbamylcholine, bombesin or ionophore A23187) caused a substantial increase in the effect of cholera toxin on enzyme secretion. Cholera toxin did not alter calcium transport or the changes in calcium transport caused by other secretagogues, and secretagogues that mobilize cellular calcium did not alter cellular cyclic AMP or the increase in cyclic AMP caused by cholera toxin. These results indicate that in dispersed acini from rat pancreas there is post-receptor modulation of the action of cholera toxin by secretagogues that mobilize cellular calcium and that this modulation is a major determinant of the effect of the toxin on enzyme secretion.     

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.     

Action of cholera toxin on dispersed acini from rat pancreas: Post-receptor modulation involving cyclic AMP and calcium

In dispersed acini from rat pancreas, cholera toxin caused a significant increase in cellular cyclic AMP but little or no change in amylase secretion. The presence of a secretagogue that causes mobilization of cellular calcium (e.g., cholecystokinin, carbamylcholine, bombesin or ionophore A23187) caused a substantial increase in the effect of cholera toxin on enzyme secretion. Cholera toxin did not alter calcium transport or the changes in calcium transport caused by other secretagogues, and secretagogues that mobilize cellular calcium did not alter cellular cyclic AMP or the increase in cyclic AMP caused by cholera toxin. These results indicate that in dispersed acini from rat pancreas there is post-receptor modulation of the action of cholera toxin by secretagogues that mobilize cellular calcium and that this modulation is a major determinant of the effect of the toxin on enzyme secretion.     

Calcium ion uptake in isolated pancreas cells induced by secretagogues.

1. Secretagogues of pancreatic enzyme secretion: pancreozymin, carbamylcholine, gastrin I, the octapeptide of pancreozymin, caerulein and the Ca2+ ionophore A 23187 stimulate 45Ca uptake into isolated rat pancreatic cells, whereas adrenaline, isoproterenol, secretin, dibutyrylic cyclic adenosine 3',5'-monophosphate and dibutyrylic cyclic guanosine 3',5'-monophosphate have no effect on 45Ca uptake. 2. A graphical analysis of the Ca2+ uptake curves reveals at least two phases: a fast phase, probably due to binding of Ca2+ to the membrane and a slow phase representing Ca2+ transport into cells. Both phases are stimulated by pancreozymin and carbamylcholine. 3. The 45Ca-exchangeable pool size is increased by both carbamylcholine and pancreozymin, whereas a significant increase of total content of cell calcium was too small to be detected. 4. Atropine blocks the stimulatory effect of carbamylcholine completely but not that of pancreozymin. The Ca2+ antagonist D600 blocks the stimulatory effects of both carbamylcholine and pancreozymin only partially. 5. The data suggest that secretagogues of pancreatic enzyme secretion act by increasing the rate of Ca2+ transfer into the cell most probably through an increase of the cell membrane permeability for Ca2+.     

Phorbol ester attenuates cholecystokinin-stimulated amylase release in pancreatic acini of rats

12-O-tetradecanoylphorbol 13-acetate (TPA) and cholecystokinin octapeptide stimulate amylase secretion in dispersed pancreatic acini, presumably acting via the activation of protein kinase C. In this study, we examined TPA pretreatment on the subsequent response of rat pancreatic acini to secretagogues. Acini exposed to TPA (3 X 10(-7) M) at 37 degrees C reduced the subsequent amylase secretion as stimulated by cholecystokinin octapeptide and carbachol, but not by A23187 or VIP. The optimal effect was obtained after 5 min of preincubation with TPA. Longer incubation did not result in greater attenuation. The degree of attenuation was dependent on the concentration of TPA used in the pretreatment. Maximal effect was seen at TPA concentrations of 10(-7) M and higher. Preincubation with TPA resulted in alterations of the dose response of pancreatic acini to cholecystokinin octapeptide. A decrease in amylase secretion was obtained at optimal and suboptimal but not at supraoptimal concentrations of cholecystokinin octapeptide. The peak response to cholecystokinin octapeptide, furthermore, was shifted almost 1 log unit to the right, suggesting a decrease in cholecystokinin binding of the acini following TPA treatment. Binding studies demonstrated a reduction in the specific binding of 125I-labelled cholecystokinin octapeptide to acini following TPA treatment. Analysis of binding data revealed a decrease in affinity and binding capacity of the high-affinity component. No significant change in the binding capacity was detected with the low-affinity component, but a great increase in its affinity was observed. This suggests that the attenuation effect by TPA on the cholecystokinin octapeptide response in rat pancreatic acini in vitro is at the receptor level.     

The influence of localized chemical modifications of the basic pancreatic trypsin inhibitor on static and dynamic aspects of the molecular conformation in solution.

Porcine vasoactive intestinal peptide stimulated adenosine 3':5'-monophosphate (cyclic AMP) production in rat intestinal epithelial cells. The stimulation was dependent on time and temperature and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Under optimal conditions (at 15 degrees C, with 0.2 mM 3-isobutyl-1-methylaxanthine, at a cell concentration up to 18 microgram DNA/ml), the cyclic AMP production produced by vasoactive intestinal peptide was constant for 10 min and stopped after 15 min incubation, at either low (1 nM) or high (30 nM) concentration of the peptide. This plateau effect was demonstrated not to be due to an inactivation of vasoactive intestinal peptide in the medium nor to an alteration of receptors for the peptide. Cyclic AMP production was sensitive to a concentration as low as 0.1 nM vasoactive intestinal peptide. Maximal stimulation of cyclic AMP levels by vasoactive intestinal peptide was observed with 30 nM vasoactive intestinal peptide and represented an 11-fold increased above basal. The dorse-response curve was monophasic with a Km of 2.3 x 10(-9) M. No cooperative effects were detected by Hill analysis. The positive non-linear relationship observed between stimulation of cyclic AMP production and occupancy of binding site was not time-dependent as indicated by experiments performed after 15, 45 and 120 min incubation. Maximal and half-maximal responses were obtained at about 70% and 7% occupation of binding sites, respectively. Chicken vasoactive intestinal peptide and porcine secretin were agonists of porcine vasoactive intestinal peptide with a 6-times and a 120-times lower potency, respectively. Among secretin analogs that were found to have low affinity for vasoactive intestinal peptide binding sites, [4-alanine, 5-valine]secretin, that resembles vasoactive intestinal peptide at the first seven amino acids at the N-terminal end, was a partial agonist of vasoactive peptide at the first seven amino acids at the N-terminal end, was a partial agonist of vasoactive intestinal peptide and others failed to stimulate cyclic AMP production. Glucagon (10microM), gastric inhibitory peptide (0.1 microM), substance, P, neurotensin, octapeptide of cholecystokinin, bovine pancreatic polypeptide, human gastrin I with leucine at residue 15, Leu-enkephalinand somatostatin (1 microM) did not alter cyclicAMP levels. Non-peptide mediators such as dopamine, serotonin, acetylcholine and histamine, tested at 10 microM, were also ineffective. Prostaglandins E2, E1 and isoproterenol, tested at 10 microM, induced an increase of cyclic AMP levels above basal but were 9.5, 13.7 and 17.5 times less efficient than vasoactive intestinal peptide, respectively. Thus vasoactive intestinal peptide is a unique stimulus of cyclic AMP production in rat intestinal epithelial cells.     

Guinea pig pancreatic acini prepared with purified collagenase

Dispersed guinea pig pancreatic acinar cells have been used to investigate several aspects of stimulus-secretion coupling but possess the disadvantage that they are less sensitive and less responsive to secretagogues than in vitro preparations of intact pancreatic tissue (lobules). To overcome the poor responsiveness of isolated acinar cells, we have developed a new procedure for preparing dispersed, intact pancreatic acini whose sensitivity to secretagogues and morphological characteristics are similar to those of pancreatic lobules. Dispersed acini can be manipulated as suspensions of cells and full access of macromolecular probes to apical and basolateral plasmalemmal domains is obtained. Acini were prepared in good yields (~70% on a DNA basis) using only purified collagenase and mild mechanical shear in medium containing 2.0 mM Ca²⁺. Morphologically, acinar cells in the preparations retained intact junctional complexes, asymmetrical distribution of intramembranous particles between apical and basolateral plasmalemmal domains, and polarized distribution of intracellular organelles as found in intact pancreas. Dose-response curves of acini and mechanically prepared lobules to caerulein, carbachol, and bombesin were similar though acini were more sensitive to the C-terminal octapeptide of cholecystokinin. Net stimulated secretory protein discharge was ~36% over 2 h. Crude collagenase was purified for use in preparation of acini by Sephadex G-75 column chromatography which resolved collagenase from clostripain and a non-sulfhydryl-requiring protease. The purified collagenase contained at least four proteins with molecular weights between 85 000 and 110 000. Collagenase with <0.14 units of protease per unit of collagenase produced highly responsive acini; collagenase with >0.9 units of protease per unit of collagenase yielded unresponsive acini. Acini incubated with crude collagenase, chymotrypsin, or the non-sulfhydryl-requiring protease showed depressed secretory response to caerulein. Freeze-fracture electron microscopy of protease-treated acini indicated that the intramembranous particles aggregated and that many of the tight junctions had undergone a proliferation of non-cross-linked sealing strands which extended far down the basolateral plasma membrane and encircled gap junctions. Acini incubated with purified collagenase or with a clostripain-containing fraction from the Sephadex G-75 column appeared unaltered. This procedure produces acini which are morphologically and biochemically similar to the in situ pancreas and overcomes the poor response to secretagogues by isolated pancreatic acinar cells.     

Potassium transport in dispersed mucosal cells from guinea pig stomach

Dispersed mucosal cells (approx. 70% parietal cells) prepared from guinea pig stomach maintained their cellular concentration of potassium (65--80 nmol potassium/10(6) cells) for at least 5 h in vitro. Uptake of 42K by dispersed gastric mucosal cells depended on temperature, H+ concentration and oxidative metabolism. Carbachol and, in some instances, gastrin caused a 40--50% increase in cellular uptake of 42K as a consequence of the ability of these agents to increase 42K influx. Ouabain reduced uptake of 42K by 70% but did not alter the effect of carbachol. Cellular uptake of 42K was not altered by histamine, prostaglandin, E1, glucagon, secretin, vasoactive intestinal peptide or C-terminal octapeptide of cholecystokinin. Uptake of 42K was also increased by dibutyryl cyclic AMP or dibutyryl cyclic GMP but not by cyclic AMP, cyclic GMP or their 8-bromo derivatives. Theophylline caused a small (10--15%) increase in 42K uptake and potentiated the increase caused by submaximal concentrations of carbachol. The increase in 42K uptake caused by either dibutyryl cyclic nucleotide and carbachol was additive.     

A possible role for guanosine 3′,5′-monophosphate in the stimulus-secretion coupling in exocrine pancreas

Carbamylcholine, caerulein and cholecystokinin octapeptide rapidly increased the cyclic GMP concentration and amylase secretion in isolated guinea pig pancreatic slices. The cyclic GMP concentration was increased eight-fold over the basal concentration in 30 s, with concomitant increase in the rate of amylase secretion. The tissue concentration of cyclic GMP then rapidly declined to a plateau value of approx. 16% of the peak level within 10 min and was maintained at that concentration for the duration of the experiment. We have shown earlier (Kapoor, C.L. and Krishna, G. (1977) Science 196, 1003–1005) that the decrease of tissue cyclic GMP was due mainly to the secretion of cyclic GMP into the medium. The cyclic AMP concentration in the tissue was not changed, nor was it secreted into the medium.There was a correlation between the concentration response to various agents for the increase in cyclic GMP concentration and amylase secretion in pancreatic slices. Carbamylcholine increased both the cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 1.5 μM concentration. Caerulein and cholecystokinin octapeptide were 5000 times more potent than carbamylcholine in increasing cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 0.3 nM concentration. Atropine, which completely inhibited the increase in cyclic GMP and amylase secretion induced by carbamylcholine, did not block the effects of caerulein or cholecystokinin octapeptide. These results suggest that various secretagogues induced amylase secretion by increasing the cyclic GMP concentration, but the mechanism by which cyclic GMP caused amylase secretion remains to be elucidated.     

A possible role for guanosine 3',5'-monophosphate in the stimulus-secretion coupling in exocrine pancreas.

Carbamylcholine, caerulein and cholecystokinin octapeptide rapidly increased the cyclic GMP concentration and amylase secretion in isolated guinea pig pancreatic slices. The cyclic GMP concentration was increased eight-fold over the basal concentration in 30 s, with concomitant increase in the rate of amylase secretion. The tissue concentration of cyclic GMP then rapidly declined to a plateau value of approx. 16% of the peak level within 10 min and was maintained at that concentration for the duration of the experiment. We have shown earlier (Kapoor, CL. and Krishna, G. (1977) Science 196, 1003--1005) that the decrease of tissue cyclic GMP was due mainly to the secretion of cyclic GMP into the medium. The cyclic AMP concentration in the tissue was not changed, nor was it secreted into the medium. There was a correlation between the concentration response to various agents for the increase in cyclic GMP concentration and amylase secretion in pancreatic slices. Carbamylcholine increased both the cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 1.5 micrometer concentration. Caerulein and cholecystokinin octapeptide were 5000 times more potent than carbamylcholine in increasing cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 0.3 nM concentration. Atropine, which completely inhibited the increase in cyclic GMP and amylase secretion induced by carbamylcholine, did not block the effects of caerulein or cholecystokinin octapeptide. These results suggest that various secretagogues induced amylase secretion by increasing the cyclic GMP concentration, but the mechanism by which cyclic GMP caused amylase secretion remains to be elucidated.     

COOH-terminal fragments of cholecystokinin. A new class of cholecystokinin receptor antagonists

COOH-terminal fragments of cholecystokinin varying in length from 1 to 3 amino acids and their NH2-terminal butyloxycarbonyl derivatives were investigated for their ability to interact with the cholecystokinin receptor on dispersed acini from guinea pig pancreas. No fragment stimulated amylase secretion when present alone, but each of the butyloxycarbonyl derivatives and the COOH-terminal tripeptide amide inhibited the stimulation of enzyme secretion by cholecystokinin. In each case the inhibition was surmounted by increasing the concentration of cholecystokinin. Each fragment also inhibited binding of 125I-labeled cholecystokinin, with significant inhibition occurring with 30 microM butyloxycarbonyl tripeptide amide, 0.3 mM butyloxycarbonyl dipeptide amide, 10 mM butyloxycarbonyl phenylalanine amide and 3 mM tripeptide amide of cholecystokinin. In each case, there was a close correlation between the ability of the fragment to inhibit binding of 125I-labeled cholecystokinin and its ability to inhibit cholecystokinin-stimulated amylase release, cholecystokinin-stimulated 45Ca outflux and cholecystokinin-stimulated residual stimulation of amylase secretion. The inhibition of amylase secretion caused by the butyloxycarbonyl tripeptide of cholecystokinin was reversible and specific for those peptides which interact with the cholecystokinin receptor (i.e., cholecystokinin, caerulein, gastrin); it did not inhibit the actions of bombesin, carbachol, physalaemin, vasoactive intestinal peptide, secretin, PHI, ionophore A23187 or 8-bromo cyclic AMP. These results demonstrate that COOH-terminal fragments of cholecystokinin comprise a new class of cholecystokinin receptor antagonists.     

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.