Inhibition by mepacrine and amylase secretion from intact and permeabilized rat pancreatic acini.

In intact rat pancreatic acini, the phospholipase A2 inhibitor mepacrine did not affect basal amylase release but dose-dependently inhibited the carbachol (IC50 65 microM) and CCK-8 (IC50 210 microM)-stimulated amylase release. In permeabilized acini, mepacrine shifted the dose-response curve for calcium to the right by a factor 2 and inhibited the release of amylase stimulated by GTPrS. From these results we conclude that carbachol, CCK-8 and GTPrS probably activate a phospholipase A2 closely coupled to exocytosis.     

Inositol trisphosphate independent increase of intracellular free calcium and amylase secretion in pancreatic acini

It is generally believed that the activation of various cell surface receptors results in the phospholipase C-catalyzed production of inositol trisphosphate which, in turn, increases the intracellular concentration of free Ca2+ by stimulating its release from nonmitochondrial sources. We have investigated both the production of inositol trisphosphate and changes in intracellular Ca2+ concentration in rat pancreatic acini in response to caerulein and CCK-JMV-180, two analogs of cholecystokinin. Both of these analogs cause comparable increases in the rate of amylase secretion and in intracellular Ca2+ concentration but their effects on inositol phosphate generation are dramatically different; caerulein stimulates significant production of inositol phosphates within 1 min of its addition, whereas no detectable levels of inositol phosphates were generated within the same time after addition of CCK-JMV-180. These results suggest that the CCK-JMV-180 stimulated release of intracellular Ca2+ is not mediated by inositol trisphosphate but some other as yet unidentified messenger.     

Inositol trisphosphate production and amylase secretion in mouse pancreatic acini

Dispersed mouse pancreatic acini prelabelled with (3H)-myoinositol generated (3H)-inositol trisphosphate (3H-IP3), (3H)-IP2 and (3H)-IP1 in response to both cholinergic and cholecystokinin analogues. The generation of (3H)-IP3 was very rapid, reaching a maximal value within 5 seconds following hormone stimulation. Stimulation with 10(-3)M carbachol increased (3H)-IP3 to a value which was 13 times that found in unstimulated acini. These results indicate that the mechanism of stimulus-secretion coupling in mouse pancreatic acini may proceed by a mechanism similar to many other systems, including rat pancreatic acini. This sequence includes hormone-stimulated phosphatidylinositol turnover and Ca2+ mobilization, i.e. secretagogue-stimulated generation of IP3 which induces the subsequent release of intracellular Ca2+. These observations differ from those recently reported by Hokin-Neaverson and Sadeghian (J. Biol. Chem. 259: 1346, 1984), in which no hormone stimulated IP3 generation was detected in mouse pancreatic acini.     

Factors regulating amylase secretion from chicken pancreatic acini in vitro

In mammals, cholecystokinin regulates pancreatic exocrine secretion under physiological conditions. We have shown, however, that cholecystokinin at physiological concentrations does not induce pancreatic amylase secretion in birds. Therefore, we investigated the effects of various neurotransmitters and gut hormones on the pancreatic amylase secretory response in isolated chicken pancreatic acini. Acetylcholine (half-maximal stimulation at 800 nM) and vasoactive intestinal polypeptide (half-maximal stimulation at 40 pM) produced a concentration-dependent increase in amylase secretion at physiological concentrations. The combination of acetylcholine and vasoactive intestinal polypeptide produced an additive response in amylase secretion. Sodium nitroprusside, a spontaneous nitric oxide releaser, and bombesin, induced amylase secretion at concentrations greater than 10 nM and 100 nM, respectively. Gastrin and secretin increased amylase secretion at pharmacological concentrations (10 to 100 nM). Our findings suggest that neural regulation is important for pancreatic enzyme secretion in birds and the contribution of gut hormones seems to be physiologically unimportant.     

Protein tyrosine phosphatase stimulates Ca(2+)-dependent amylase secretion from pancreatic acini.

Eukaryotic cells respond to various stimuli by an increase or decrease in levels of phosphoproteins. Phosphotyrosine levels on eukaryotic cellular proteins are tightly regulated by the opposing actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPases, EC 3.1.3.48). Studies on permeabilized mast cells suggest that the enabling reaction for exocytosis might involve protein dephosphorylation. In the present studies, a recombinant form of rat brain PTPase (rrbPTP-1) has been used to examine the potential role of PTPases in Ca(2+)-dependent amylase secretion from permeabilized rat pancreatic acini. Additionally, the concentrations and subcellular distributions of endogenous PTPase activity in rat pancreas were determined. The results from these experiments indicate that addition of exogenous PTPase stimulated Ca(2+)-dependent amylase secretion from pancreatic acinar cells and that endogenous PTPase activity was associated with the postgranule supernatant, zymogen granules, and in particular zymogen granule membranes. Our data suggest that protein tyrosine dephosphorylation is potentially involved in regulated secretion at a site(s) distal to receptor-mediated elevation of intracellular second messengers.     

Calcium dependence of proteinase-activated receptor 2 and cholecystokinin-mediated amylase secretion from pancreatic acini

Pancreatic acini secrete digestive enzymes in response to a variety of secretagogues including CCK and agonists acting via proteinase-activated receptor-2 (PAR2). We employed the CCK analog caerulein and the PAR2-activating peptide SLIGRL-NH(2) to compare and contrast Ca(2+) changes and amylase secretion triggered by CCK receptor and PAR2 stimulation. We found that secretion stimulated by both agonists is dependent on a rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and that this rise in [Ca(2+)](i) reflects both the release of Ca(2+) from intracellular stores and accelerated Ca(2+) influx. Both agonists, at low concentrations, elicit oscillatory [Ca(2+)](i) changes, and both trigger a peak plateau [Ca(2+)](i) change at high concentrations. Although the two agonists elicit similar rates of amylase secretion, the rise in [Ca(2+)](i) elicited by caerulein is greater than that elicited by SLIGRL-NH(2). In Ca(2+)-free medium, the rise in [Ca(2+)](i) elicited by SLIGRL-NH(2) is prevented by the prior addition of a supramaximally stimulating concentration of caerulein, but the reverse is not true; the rise elicited by caerulein is neither prevented nor reduced by prior addition of SLIGRL-NH(2). Both the oscillatory and the peak plateau [Ca(2+)](i) changes that follow PAR2 stimulation are prevented by the phospholipase C (PLC) inhibitor U73122, but U73122 prevents only the oscillatory [Ca(2+)](i) changes triggered by caerulein. We conclude that 1) both PAR2 and CCK stimulation trigger amylase secretion that is dependent on a rise in [Ca(2+)](i) and that [Ca(2+)](i) rise reflects release of calcium from intracellular stores as well as accelerated influx of extracellular calcium; 2) PLC mediates both the oscillatory and the peak plateau rise in [Ca(2+)](i) elicited by PAR2 but only the oscillatory rise in [Ca(2+)](i) elicited by CCK stimulation; and 3) the rate of amylase secretion elicited by agonists acting via different types of receptors may not correlate with the magnitude of the [Ca(2+)](i) rise triggered by those different types of secretagogue.     

Reversible acetaldehyde inhibition of A23187-stimulated amylase secretion from isolated rat pancreatic acini

The Ca2+ ionophore, A23187, stimulated amylase secretion from isolated rat pancreatic acini in a dose-dependent manner with a maximal effect at 6 microM. Acetaldehyde, a metabolite of ethanol, caused a reduction in the magnitude of ionophore-stimulated secretion with no evidence of competitive inhibition. Furthermore, 6 microM ionophore-stimulated amylase secretion was dose-dependently inhibited by acetaldehyde. This inhibitory effect of acetaldehyde, however, was reversible on washing and reincubating acetaldehyde-treated acini. These results suggest that acetaldehyde reversibly inhibits intracellular components mediating stimulated secretion and this inhibition requires a continuous chemical interaction between acetaldehyde and intracellular component(s) regulating stimulated enzyme secretion.     

Phorbol ester induces intracellular translocation of phospholipid/Ca2+-dependent protein kinase and stimulates amylase secretion in isolated pancreatic acini

Treatment of intact rat pancreatic acini with phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate, TPA) resulted in a time- and concentration-dependent translocation of phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK) from the soluble fraction. Redistribution of PL/Ca-PK was concurrent with stimulation of amylase secretion by TPA-treated acini. Polymyxin B, a potent and selective inhibitor of PL/Ca-PK completely inhibited TPA-induced amylase secretion. These findings are consistent with a role for PL/Ca-PK in the regulation of pancreatic exocrine secretion.     

Influence of thyroid status on Ca2+ mobilization and amylase secretion in rat pancreatic acini

Thyroid hormones influence Ca2+ homeostasis in both skeletal and cardiac muscle. Since secretory cells, like muscle cells, store and use Ca2+ in stimulus-response coupling, we have studied the effects of thyroid status on Ca2+ mobilization and secretion in a model secretory tissue, the pancreatic acinar cell. Hyperthyroidism was induced by rats by daily, subcutaneous injections of triiodothyronine for 8 days and hypothyroidism by adding 6-n-propyl-2-thiouracil to the drinking water for 14 days. Pancreatic acini were prepared by collagenase digestion of pancreatic tissue from hyper- and hypo-thyroid animals and from euthyroid controls. Ca2(+)-mobilization was assessed using Quin-2 fluorescence and secretion by assaying amylase release. The data indicate that the amount of Ca2+ mobilized by the muscarinic agonist carbachol or by cholecystokinin octapeptide increases with increasing thyroid hormone concentrations. Only in hypothyroidism was this change in Ca2+ homeostasis reflected by a parallel change in amylase secretion. This implies the existence of some compensatory mechanism which stabilizes secretory rate in the face of stimulus-evoked increases in intracellular Ca2+ concentration.     

The effects of γ-hexachlorocyclohexane on amylase secretion and inositol phospholipid metabolism in mouse pancreatic acini

Dispersed mouse and guinea-pig pancreatic acini were used to examine the effects of the inositol analogue, γ-hexachlorocyclohexane (lindane) on agonist-stimulated amylase secretion. Secretion from mouse acini in response to carbachol and cholecystokinin octapeptide (CCK-8) was reduced by lindane. Similarly, amylase release from guinea-pig acini stimulated by carbachol was abolished by lindane. These acini, however, still remained responsive to dibutyryl-cAMP with only a slightly diminished secretion to this agent. Inositol phospholipid synthesis and hydrolysis was stimulated in mouse acini by both carbachol and CCK-8. Although hydrolysis of these lipids in response to CCK-8 was reduced by only 18%, stimulation of inositol phospholipid synthesis by either agonist was abolished by lindane. Dose-response curves for inositol phospholipid synthesis stimulated by carbachol and CCK-8 in mouse acini were biphasic and superimposable with those of amylase secretion. In contrast, the dose-response curve for phosphoinositide hydrolysis was sigmoid and clearly separable from that of synthesis. Reducing the external Ca²⁺ concentration caused the dose-response curves for carbachol- and CCK-8-induced inositol phospholipid synthesis to be displaced to the right, as has been observed for amylase secretion. A23187 was also found to induce amylase secretion and inositol phospholipid synthesis, and both of these responses were inhibited by lindane. Amylase secretion and inositol phospholipid synthesis may, therefore, be closely related events in the exocrine pancreas. Lindane may provide a valuable tool with which to determine the role of inositol phospholipid metabolism in stimulus-response coupling.     

Effects of an inhibitor of myosin light chain kinase on amylase secretion from rat pancreatic acini

Ca(2+)/calmodulin-dependent protein (CaM) kinases play an important role in Ca(2+)-mediated secretory mechanisms. Previously, we demonstrated that a CaM kinase II inhibitor KN-62 had a small inhibitory effect on amylase secretion stimulated by CCK. In the present study, we investigated the effects of a myosin light chain kinase (MLCK) inhibitor on amylase secretion and Ca(2+) signaling in rat pancreatic acini. A specific inhibitor of MLCK, wortmannin, inhibited amylase secretion stimulated by CCK-8 (30 pM) in a concentration-dependent manner. Wortmannin (10 microM) had no effects on basal secretion but reduced amylase secretion stimulated by CCK-8 (30 pM) by 67 +/- 3%. Wortmannin inhibited amylase secretion stimulated by calcium ionophore (A23187) and phorbol ester (TPA). Wortmannin also inhibited amylase response to thapsigargin by 76 +/- 8% and to both thapsigargin and TPA by 52 +/- 10%. Ca(2+) oscillations evoked by CCK-8 (10 pM) were inhibited by wortmannin (10 microM). Wortmannin had a little inhibitory effect on an initial rise in [Ca(2+)](i), and abolished a subsequent sustained elevation of [Ca(2+)](i) evoked by 1 nM CCK-8. In conclusion, MLCK plays a crucial role in amylase secretion from pancreatic acini and regulates Ca(2+) entry from the extracellular space.     

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.     

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.     

Galanin inhibits pancreatic amylase secretion in the pentobarbital-anesthetized rat.

The potent inhibitory effect of galanin on basal and pentagastrin-stimulated gastric acid secretion in vivo, and the presence of galanin-containing nerves in gastrointestinal tract and pancreas, suggested that this peptide may regulate the exocrine secretion of the GI system. Male rats were anesthetized with pentobarbital and the dose-dependent inhibitory effects of galanin on basal and stimulated pancreatic protein and amylase secretions were investigated in separate experiments. Galanin was administered intravenously in the following doses: 3, 6, 10, 15 and 20 micrograms/kg/h (0.93, 1.86, 3.1, 4.65 and 6.2 nmol/kg/h), and pancreatic secretions measured. The maximal effective dose of galanin (3.1 nmol/kg/h) on basal pancreatic secretions was found, and was used for evaluating the inhibitory effect of galanin on pancreatic protein and amylase secretions stimulated by bombesin, secretin and cholecystokinin. Galanin potently inhibited basal, bombesin-, secretin- and cholecystokinin-stimulated pancreatic protein and amylase secretion. Inhibitory effect of galanin was dose-dependent and biphasic.     

Phenylarsine oxide evokes intracellular calcium increases and amylase secretion in isolated rat pancreatic acinar cells

The effects of the thiol reagent, phenylarsine oxide (PAO, 10(-5)-10(-3) M ), a membrane-permeable trivalent arsenical compound that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures, were studied by monitoring intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. PAO increased [Ca2+]i by mobilizing calcium from intracellular stores, since this increase was observed in the absence of extracellular calcium. PAO also prevented the CCK-8-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF-4). In addition to the effects of PAO on calcium mobilization, it caused a significant increase in amylase secretion and reduced the secretory response to either CCK-8 or AlF-4. The effects of PAO on both [Ca2+]i and amylase release were reversed by the sulfhydryl reducing agent, dithiothreitol (2 mM). Pretreatment of acinar cells with high concentration of ryanodine (50 microM) reduced the PAO-evoked calcium release. However, PAO was still able to release a small fraction of Ca2+ from acinar cells in which agonist-releasable Ca2+ pools had been previously depleted by thapsigargin (0.5 microM) and ryanodine receptors were blocked by 50 microM ryanodine. We conclude that, in pancreatic acinar cells, PAO mainly releases Ca2+ from the ryanodine-sensitive calcium pool and consequently induces amylase secretion. These effects are likely to be due to the oxidizing effects of this compound.     

Effect of an intracellular calcium antagonist (TMB-8) on carbamylcholine-induced amylase release from dispersed rat pancreatic acini

During 10-min incubation with increasing concentrations of carbamylcholine (carbachol), amylase release from dispersed rat pancreatic acini increased, became maximal at 2 X 10(-6)M and then decreased. In the concentration range of 10(-7)M to 10(-4)M, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) caused a dose-dependent inhibition of amylase release induced by a submaximal concentration of carbachol. No inhibitory effect was observed on basal and secretin-stimulated amylase release. TMB-8 showed a significantly greater ability of blocking the action of carbachol than verapamil and diltiazem. TMB-8 could reverse the submaximal stimulation of amylase release caused by supramaximal concentrations of carbachol to a maximal stimulation, while verapamil and diltiazem could not. These results confirm the hypothesis that mobilization of intracellular calcium is the primary step in the action of carbachol on pancreatin acinar cells and contributes to the submaximal secretory response of acinar cells induced by high concentrations of carbachol.     

Cholecystokinin-induced secretion and synthesis of amylase and cationic trypsinogen by pancreatic acini isolated from rats given an ethanol diet

Effects of chronic alcohol intake on secretion and synthesis of amylase and cationic trypsinogen (CT) were studied with pancreatic acini isolated from male Sprague-Dawley rats fed a Lieber-DeCarli ethanol or control diet for 30 days. Pancreatic acini were incubated in a media containing increasing concentrations of cholecystokinin octapeptide (CCK-8: 0-1000pM) followed by addition of [3H]leucine. Amylase and CT secreted in the media and those labeled in acini were quantitated. Basal and CCK-stimulated secretion of CT was not different in alcoholic and control groups. On the other hand, a dose-response curve of CCK-stimulated amylase secretion from alcoholic acini was markedly reduced with both basal and maximal secretion decreased to only 40% of controls. Basal incorporation of [3H]leucine into amylase was reduced by 70% in alcoholic acini compared to controls while that into CT was not different in the two groups. CCK-8 exhibited a biphasic effect on [3H]leucine incorporation into both enzymes in alcoholic acini: low concentrations of CCK-8 (less than 100pM) increased the incorporation whereas high concentrations (greater than 100pM) decreased it. However, in control acini, CCK-8 induced progressive suppression of the incorporation into these enzymes, the pattern of which was similar to that previously observed in fasted rats (Am. J. Physiol. 241:G116-G112, 1981). This difference in the response pattern resulted in significantly higher rates of CCK-8 induced incorporation into CT in alcoholic acini. These results suggest that the differences observed may possibly be attributable not only to alcohol intake but also to the differences in carbohydrate intake and in temporal patterns of diet consumption.     

Effect of proton pump inhibitor on amylase release from isolated pancreatic acini

Proton pump inhibitors (PPIs) could inhibit the secretion of gastric acid. Meanwhile, it could also decrease the secretion of other digestive glands besides gastric parietal cell. As we know, PPIs have been widely used to treat acute pancreatitis, and it is effective in clinical practice. However, research showed the side effect of PPIs on acute pancreatitis. The direct effect of PPI on pancreatic secretion is still unknown. Our experiment investigated the direct effect of PPIs on pancreatic exocrine by isolated pancreatic acini. In our study, isolated pancreatic acini were prepared as previously described by Williams, and cerulein was added to stimulate its secretion. The amylase release in the suspension was determined after the administration of different concentrations of omeprazole and Sandostatin; and its activity was also observed in different time phases. In our in vitro study, all results suggest that omeprazole has no direct repression on amylase release from isolated pancreatic acini.