Dynamic analysis of secretagogue-induced amylase secretion from rat pancreatic acini studied by perifusion system

A perifusion system was applied for the study on stimulus-enzyme secretion coupling in dispersed pancreatic acini. The system is highly simple, preserves the acini up to more than 3 hr, and makes feasible clear-cut examination on the time course of enzyme secretion caused by secretagogues. Caerulein (10(-9) M) and carbamylcholine (10(-5) M) caused a biphasic amylase secretory pattern consisting of an initial burst secretion and a sustained one. Caerulein induced a persistent amylase release even after cessation of the stimulation, while carbamylcholine-stimulated amylase release returned to basal levels. Atropine inhibited completely carbamylcholine-stimulated amylase release and the successive stimulation by caerulein evoked the amylase secretion with a decreased initial burst secretion. In calcium free medium, caerulein and carbamylcholine induced only a slight secretion, particularly in the sustained secretion phase and a gradual increase occurred with the addition of calcium.     

The inhibitory effect of CR-1409 on amylase secretion and intracellular Ca2+ mobilization in rat pancreatic acini in vitro

The inhibitory effects of CR-1409, a new glutaramic acid derivative developed as a cholecystokinin (CCK) receptor antagonist, on caerulein-stimulated amylase secretion and on intracellular Ca2+ ([Ca2+]i) mobilization were studied in isolated rat pancreatic acini. Pancreatic acini were prepared by collagenase digestion method and loaded with 1 microM fura-2/AM for measurement of the intracellular free Ca2+ concentration. Amylase release was examined by a perifusion method. Stimulation with 10(-10) M caerulein, 10(-5) M carbachol, or 10(-8) M gastrin-releasing peptide (GRP) led to biphasic amylase release and increase in [Ca2+]i. CR-1409 at 1 and 5 microM inhibited, by 50 and 84%, respectively, the amylase secretion and increase in [Ca2+]i induced by 10(-10) M caerulein, and 25 microM CR-1409 completely inhibited both amylase secretion and increase in [Ca2+]i induced by caerulein. However, 25 microM CR-1409 did not inhibit unstimulated secretion of amylase or the secretions induced by carbachol and GRP, which are also mediated by changes in intracellular Ca2+. We conclude that CR-1409 acts as a specific inhibitor of the CCK receptor in the pancreas, and is useful in studies on the involvement of the release and action of CCK in vitro.     

The relationship between free cytosolic calcium and amylase release in rat pancreatic acini

We have studied the effects of various pancreatic secretagogues on free cytosolic calcium ([Ca2+]i) and amylase release in dispersed rat pancreatic acini, to determine the role of [Ca2+]i in stimulated enzyme secretion from the exocrine pancreas. Dispersed rat pancreatic acini were loaded with the new Ca2+-sensitive fluorescent indicator, fura-2. Resting [Ca2+]i was 110 +/- 2 nM (a mean +/- S.E.). Carbachol, caerulein, bombesin, and neuromedin B and C each caused a rapid increase in [Ca2+]i; maximal increases of 100 to 400-500 nM were reached within 20s following the secretagogue addition, and this was followed by a return to a lower sustained level within 2 min. When enzyme secretion from the acini was monitored as a function of time using a perifusion system, secretagogue-induced amylase release took a biphasic pattern consisting of an initial burst phase for a several minutes and a second sustained phase during stimulation. Although sustained amylase secretion occurred at near resting [Ca2+]i, the peak [Ca2+]i correlated with the amount of stimulated amylase release as well as with the initial release, during submaximal and maximal stimulation by these agents. At supramaximal concentrations of carbachol and caerulein, amylase release, but not the increase in [Ca2+]i, was attenuated. On the other hand, in response to supramaximal concentrations of bombesin, and neuromedin B and C, both the amount of amylase released and the peak [Ca2+]i were similar to those obtained in response to maximal concentrations. From a standpoint of time course analysis of enzyme secretion, both the first burst phase and the second sustained phase were inhibited during stimulation by 10(-3) M carbachol, compared with 10(-5) M carbachol, while supramaximal stimulation by neuromedin C caused a pattern of amylase release similar to that produced by maximal stimulation. These data suggest that in pancreatic acinar cells an increase in [Ca2+]i plays an important role in stimulus-secretion coupling; however, other factors may be indispensable in regulating enzyme secretion. Furthermore, it is suggested that there is a difference in the intracellular messenger system between carbachol and caerulein, and neurotransmitters belonging to the bombesin family, especially during supramaximal stimulations.     

Decreased pancreatic CCK receptor binding and CCK-stimulated amylase release in Zucker obese rats

In Zucker obese rats the response to the effects of CCK on food intake and pancreatic exocrine function are decreased. However, it is unknown whether the decreased responsiveness is due to decreased receptor number and/or sensitivity or abnormal circulating concentrations of CCK. In these experiments percent total binding of 125I-CCK-33 to pancreatic acini from obese rats was one-half that in lean rats when data was expressed on a per microgram DNA basis (19.6 +/- 5.1 vs. 47.4 +/- 11.4, p less than 0.01). In a second experiment while the maximally effective dose of CCK for stimulating amylase secretion from dispersed pancreatic acini was similar in obese and lean rats (10(-10) M), less amylase was secreted in obese rats across the dose range tested (p less than 0.001). In contrast, carbachol had similar potency and efficacy in stimulating amylase release from obese and lean pancreatic acini. The increase of pancreas size by use of a trypsin inhibitor was greater in lean than obese rats (p less than 0.03). In addition, stimulation of amylase release by CCK from obese trypsin inhibitor-treated compared with control obese rats was greater than that from lean trypsin inhibitor-treated compared with control lean rats (p less than 0.002). However, overall, stimulation of amylase secretion by CCK was only 36% of control (p less than 0.001) and by carbachol was only 20% of control (p less than 0.001). Thus, increased size by increased cell number was associated with decreased response per cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Participation of Ca2+ and calmodulin in rat pancreatic enzyme secretion induced by secretin, forskolin, and dibutyryl cyclic AMP

The role of Ca2+ and calmodulin in stimulation of the rat pancreatic acini induced by secretin, forskolin, and dibutyryl cyclic AMP (dbcAMP) was studied using W-7, a calmodulin antagonist, and a low Ca2+ medium. The time course of amylase secretion was studied in a perfusion system using dispersed rat pancreatic acini. The amylase release patterns of each secretagogue were as follows: a biphasic amylase release pattern under the stimulation of secretin, a one peak pattern during the stimulation of forskolin and a rapid response after cessation of the stimulation, and a gradual increased pattern during the stimulation of dbcAMP followed by a rapid response. The amylase release under the stimulation by forskolin and dbcAMP was slightly weaker as compared with that of secretin stimulation. The amylase secretion stimulated by secretin (5 X 10(-7) M), forskolin (50 microM), and dbcAMP (2 mM) was inhibited by W-7 (50 microM). In a low Ca2+ medium (4.7-5.1 X 10(-6) M), the secretory rate did not increase during the stimulation by secretin, forskolin, and dbcAMP, and a rapid amylase response remained after cessation of the stimulation of forskolin and dbcAMP. The pretreatment with EDTA (1 mM) suppressed both the gradual amylase release and the rapid response induced by dbcAMP in a low Ca2+ medium. These results suggested that each secretagogue, via cyclic AMP (cAMP), induced a different amylase secretory pattern dependent on an intracellular Ca2+ content, and was mediated by the Ca2+-calmodulin complex.     

Calcitonin gene-related peptide inhibits exocrine secretion from the rat pancreas by a neurally mediated mechanism

The mechanism by which calcitonin gene-related peptide (CGRP) inhibits exocrine secretion from the rat pancreas was examined in the isolated, vascularly perfused pancreas and in vitro using freshly isolated pancreatic acini. CGRP (10(-10)-10(-7) M) inhibited the volume and protein output from the perfused pancreas, stimulated by a mixture of the cholecystokinin octapeptide CCK8 (10(-10) M) and secretin (10(-8) M). The inhibition by CGRP was dose related and maximal at 10(-8) M (P less than 0.05). CGRP (10(-8) M) failed to inhibit amylase secretion from isolated pancreatic acini, stimulated by graded concentrations of CCK8 (10(-13)-10(-8) M). This implies an indirect mechanism of inhibition. The mechanism of inhibition was investigated in the isolated, vascularly perfused pancreas using tetrodotoxin, atropine and hexamethonium (all 10(-7) M). Tetrodotoxin and atropine but not hexamethonium prevented the inhibition of volume and protein secretion by CGRP (10(-8) M) (P less than 0.05). Tetrodotoxin, atropine and hexamethonium were without effect on exocrine secretion stimulated by CCK8 and secretin (controls). These results indicate that CGRP inhibits pancreatic exocrine secretion by an indirect, neurally mediated mechanism involving cholinergic-muscarinic transmission.     

Bombesin-induced desensitization of enzyme secretion in dispersed acini from guinea pig pancreas

Incubating dispersed acini from guinea pig pancreas with bombesin and then washing the cells to remove bombesin reduced the subsequent stimulation of amylase secretion caused by bombesin, litorin, or ranatensin by as much as 80%, but did not alter the stimulation of amylase secretion caused by cholecystokinin, carbamylcholine, A23187 or vasoactive intestinal peptide. This bombesin-induced desensitization was reversible, and the onset of the process, as well as its reversal, were time and temperature dependent. Neither desensitization or resensitization were inhibited by abolishing protein synthesis. The concentrations of bombesin required to cause desensitization were in the same range as those required to stimulate amylase secretion. Incubating pancreatic acini with vasoactive intestinal peptide did not reduce the subsequent stimulation of amylase secretion caused by vasoactive intestinal peptide, bombesin, or cholecystokinin. These results indicate that bombesin-induced desensitization of pancreatic acini reflects changes that occur at or close to the bombesin receptor.     

Secretion of fluid and amylase in the perfused rat pancreas.

1. The isolated rat pancreas was perfused with physiological salt solutions of varying composition. Flow of pancreatic juice and output of amylase during rest and after stimulation with pure secretin, pure cholecystokinin-pancreozymin (CCK-PZ), caerulein or acetylcholine (ACh) were measured. 2. Basal fluid secretion was abolished replacing perfusion fluid NA+ or Cl- by Tris+ or SO42- respectively. Readmission of Na+ or Cl- caused a transient increase above the normal control level of both fluid and amylase output. Exposure to K+-free solution severely reduced fluid output and K+ readmission resulted in a transient increase in secretory rate. 3. Maximal stimulation with ACh (10(-7) M), CCK-PZ (1-5 X 10(-10) M) or caerulein (10(-10) M) caused marked sustained fluid and amylase secretion. Maximal secretin stimulation (5-7 X 10(-9) M) caused marked sustained fluid but only a small sustained amylase secretion following an initial transient. 4. Under continuous secretin stimulation, replacement of the CO2/HCO3-buffered control fluid by a CO2/HCO3-free Tris buffered solution caused a sharp decrease in pancreatic juice flow. In the absence of extracellular CO2/HCO3-secretin did not evoke fluid or enzyme secretion. In contrast the effects of ACh, CCK-PZ or caerulein were independent on CO2/HCO3-. Monobutyryl cyclic AMP (10(-3) M) caused marked sustained fluid secretion and transient enzyme secretion. The effect was entirely dependent on the presence of CO2/HCO3-in the perfusion fluid. 5. Ouabain (10(-4)-10(-3) M) markedly inhibited both secretin- and caerulein-evoked fluid secretion while caerulein-evoked amylase secretion was hardly affected. Similar findings were made with K+-free solution. 6. The effect of maximal secretin stimulation on amylase secretion was greatly augmented in the presence of a maximally stimulating concentration of caerulein. The effects on fluid secretion of secretin and caerulein were simply additive. The effects of secretin on both amylase and fluid secretion, in the presence of caerulein, were entirely dependent on the presence of CO2/HCO3- in the perfusion fluid. 7. We conclude that two different fluid secretion processes occur in the rat exocrine pancreas. One stimulated by ACh and CCK-PZ, that is independent of extracellular CO2/HCO3- and another stimulated by secretin involving H+ or HCO3-transport. Only the effects of secretin seem to be mediated by intracellular cyclic AMP.     

Action of theophylline on secretagogue-stimulated amylase release from dispersed pancreatic acini

In dispersed acini from guinea pig pancreas, theophylline did not alter basal amylase release, but had three functionally distinct modes of action on the stimulation of amylase release caused by various secretagogues. 1) At relatively low concentrations (0.1-1.0 mM), theophylline augmented the increase in enzyme secretion caused by vasoactive intestinal peptide, secretin, or 8-bromoadenosine 3',5'-monophosphate, but did not alter the increase in amylase release caused by other secretagogues. 2) At intermediate concentrations (1-10 mM), theophylline selectively altered the increase in enzyme secretion caused by carbamylcholine, but did not alter the effects of cholecystokinin or bombesin, secretagogues whose modes of action are similar to that of cabamylcholine. 3) At high concentrations (greater than 10 mM), theophylline inhibited the increase in enzyme secretion caused by all secretagogues tested.     

Effect of extracellular calcium on amylase release from dispersed pancreatic acini.

In dispersed acini prepared from guinea pig pancreas, removing extracellular calcium did not alter the basal rate of amylase release but reduced the stimulation of enzyme release caused by cholecystokinin, carbachol, secretin, and vasoactive intestinal peptide as well as that caused by derivatives of cyclic nucleotides. In acini incubated in a calcium-free, EGTA-containing medium the increase in amylase release caused by each secretagogue tested did not change during the initial 10 min of incubation, decreased by approximately 65% during the subsequent 40 min, and remained constant thereafter. Removing extracellular calcium did not alter the maximally effective concentrations of cholecystokinin or vasoactive intestinal peptide but abolished the decrease in stimulated enzyme secretion seen with supramaximal concentrations of cholecystokinin. Incubating pancreatic acini with cholecystokinin or carbachol plus secretin or vasoactive intestinal peptide caused potentiation of amylase release, and removing extracellular calcium reduced the stimulation of enzyme release caused by the two secretagogues in combination but did not alter their potentiating interactions.     

Effect of uncoupling NO/cGMP pathways on carbachol- and CCK-stimulated Ca2+ entry and amylase secretion from the rat pancreas

 Nitric oxide (NO) production reportedly regulates guanosine 3′,5′-cyclic monophosphate (cGMP) formation and Ca²⁺ influx in pancreatic acini. We have investigated the functional roles of the NO/cGMP messenger system in rat pancreatic acini. In dispersed acini, the levels of amylase secretion, cytosolic [Ca²⁺]([Ca²⁺]_i), NO synthase, and cGMP were measured. The NO synthase inhibitor N ^G-nitro-L-arginine methyl ester (L-NAME, 0.01–100 μM) had no effect on amylase secretion induced by various concentrations of carbachol, cholecystokinin octapeptide (CCK-8) or the high affinity CCK agonist, JMV-180. Similarly, L-NAME up to 100 μM did not affect the changes in Ca²⁺ spiking evoked by these secretagogues; nor was Ca²⁺ entry, refilling or oscillation altered by L-NAME. Sub- and supramaximal concentrations of these secretagogues did not change NO synthase activities compared with basal levels. While sodium nitroprusside (SNP), a NO donor, caused a 9.4-fold increase in cGMP levels compared with basal levels, carbachol, CCK-8 and JMV-180 had no effect. In addition, the guanylate cyclase inhibitor LY 83583 (10 nM to 10 μM) altered neither amylase secretion nor Ca²⁺ signaling induced by these secretagogues. These findings indicate that the stimulatory action of carbachol or CCK-8 is not mediated by NO or cGMP. To investigate whether cGMP stimulates pancreatic secretion we showed that both SNP and a cell-permeant cGMP analog at 0.1–1 mM stimulated amylase secretion and Ca²⁺ transients to a level equal to 10–15% and 13–24%, respectively, of those observed with maximal concentrations of secretagogues. The guanylate cyclase activator guanylin (1–10 μM), which increased cGMP levels 2.4-fold compared with basal levels, elicited a small amount of amylase secretion and a small Ca²⁺ transient. In conclusion, exogenous NO is capable of increasing endogenous cGMP, which results in a modest increase in the [Ca²⁺]_i transient and pancreatic amylase secretion. However, the NO/cGMP system does not appear to be involved significantly in the mediation of Ca²⁺ signaling and amylase secretion stimulated by carbachol and CCK-8. Received: 30 October 1996 / Received after revision and accepted: 13 January 1997     

Metabolism of arachidonic acid by pancreatic acini: relation to amylase secretion

Isolated guinea pig pancreatic acini were incubated with exogenous [14C]arachidonic acid (10 microM) at 37 degrees C for 3 min. The lipids were extracted and separated by thin-layer chromatography. Radiolabeled metabolites were identified by comigration with standards: 0.024% of the recovered radioactivity comigrated with prostaglandin E2 (PGE2), 0.016% comigrated with PGF2 alpha, 4.9% was incorporated into triglycerides, 1.8% was incorporated into phospholipids, and 93.2% remained as arachidonic acid. The synthesis of PGE2 and PGF2 alpha was inhibited by indomethacin (ID50, 30 nM). Simultaneous addition of carbachol or caerulein with the [14C]arachidonic acid did not alter the metabolism of the arachidonate. Further studies were done on the role of arachidonate metabolites in the secretion of amylase. Exogenously added PGE2 and PGF2 alpha (0.3-100 nM) did not induce amylase secretion from isolated acini. Incubation of isolated acini with indomethacin (0.1-28 microM) did not inhibit the release of amylase induced by carbachol or caerulein. From these data, we conclude that isolated guinea pig pancreatic acini are capable of converting a small percentage of exogenous arachidonate to PGE2 and PGF2 alpha. However, there is no evidence for a role of these compounds in stimulus-secretion coupling.     

Interaction between secretin and cholecystokinin-octapeptide in the exocrine rat pancreas in vivo and in vitro.

This study investigates the interaction between physiological doses of the synthetic gut hormones, cholecystokinin-octapeptide (CCK8) and secretin on pancreatic juice secretion in the anaesthetized rat and on amylase secretion and Ca2+ and Mg2+ mobilization in isolated pancreatic segments and acinar cells. CCK8 (150 pmol kg-1 h-1) and secretin (100 pmol kg-1 h-1) evoked marked time course increases in pancreatic juice flow, total protein output and amylase secretion in the anaesthetized rat when administered separately compared to saline controls. Simultaneous intravenous infusion of CCK8 and secretin did not yield either an additive response or a potentiation but instead it caused a decrease in secretory responses. Administration of either polymyxin B (10(-8) mol kg-1 h-1) or staurosporine (10(-8) mol kg-1 h-1), two protein kinase C inhibitors, simultaneously with both CCK8 and secretin caused a further decrease in all secretory parameters. Superfusing pancreatic segments with either CCK8 (10(-11) M) or secretin (10(-11) M) elevated amylase output compared to the smaller response with a combination of CCK8 and secretin. Combining staurosporine (10(-6) M) with CCK8 and secretin resulted in a further decrease in amylase output. CCK8 (10(-11) M) evoked a large increase in radiolabelled Ca2+ influx into pancreatic segments and elevated cytosolic free Ca2+ concentration ([Ca2+]i) in acinar cells loaded with the fluorescent dye, Fura-2. Secretin (10(-11) M) alone had no significant effect on Ca2+ mobilization but it markedly attenuated the increases in radiolabelled Ca2+ influx and [Ca2+]i elicited by CCK8. In superfused pancreatic segments CCK8 (10(-11) M) evoked a net efflux of Mg2+ whereas secretin (10(-11) M) induced a net uptake of Mg2+. Combining secretin with CCK8 also resulted in a net uptake of Mg2+. The results indicate that both Ca2+ and Mg2+ mobilization may be associated with the interaction between CCK8 and secretin in the rat pancreas.     

Gastrin releasing peptide stimulates the secretion of insulin, but not that of glucagon or somatostatin, from the isolated perfused dog pancreas

Gastrin releasing peptide (GRP) is an intrapancreatic peptide, but its physiological function is unknown. Previously, the peptide has been shown to increase plasma levels of insulin and glucagon in vivo in dogs, but no studies on the possible direct actions on islet hormone secretion from the dog pancreas have been undertaken. Therefore, we examined the effects of a 10-min perfusion of synthetic porcine GRP at four different dose rates over a wide range (0.1-50 nmol l-1) on the islet hormone release from the isolated dog pancreas (n = 5-6 in each group) at 5.5 mM glucose. We found that, at all four concentrations tested, GRP rapidly and markedly stimulated insulin secretion. The stimulation was, however, transient: the increased insulin secretion returned to basal levels within 7-8 min despite the ongoing GRP perfusion for 10 min. In contrast, GRP did not affect the pancreatic secretion of glucagon or somatostatin. We conclude that GRP stimulates insulin secretion by a direct pancreatic action without affecting the secretion of glucagon or somatostatin.     

Postreceptor modulation of action of VIP and secretin on pancreatic enzyme secretion by secretagogues that mobilize cellular calcium

In dispersed acini from rat pancreas, secretagogues that act through or mimic the action of AMP [vasoactive intestinal peptide (VIP), secretin, or 8-bromo-AMP] caused a twofold increase in amylase secretion. Secretagogues that mobilize cellular calcium (carbamylcholine, C-terminal octapeptide of cholecystokinin, bombesin, or A23187) caused a sevenfold augmentation of the actions of VIP, secretin, or 8-bromo-cAMP on enzyme secretion. Carbamylcholine and the C-terminal octapeptide of cholecystokinin also augmented the action of VIP on amylase secretion from mouse pancreatic acini. Secretagogues that mobilize cellular calcium did not alter binding of 125I-VIP, cellular cAMP, or the increase in cellular cAMP caused by VIP or secretin. Similarly, secretagogues that increase cellular cAMP did not alter 45Ca outflux or the increase in 45Ca outflux caused by carbamylcholine, C-terminal octapeptide of cholecystokinin, bombesin, or A23187. These results indicate that in dispersed acini from rat pancreas there is postreceptor modulation of the actions of VIP and secretin on enzyme secretion by secretagogues that mobilize cellular calcium and that this modulation is a major determinant of the magnitude of the effect of VIP and secretin on enzyme secretion. This modulation, in turn, reflects the ability of cellular calcium, mobilized from intracellular stores, to amplify the action of cellular cAMP on the enzyme secretory process.     

Non-parallel enzyme secretion from rat pancreas in vitro studies.

1. The relative variations of rat pancreatic amylase versus lipase and chymotrypsinogen secretions have been studied in vitro with the help of two different techniques: in situ organ perfusion and incubation of pancreatic lobules. 2. In experiments on the perfused pancreas, with 8 C u.kg-1 hr-1 secretin added to the perfusion fluid, cholecystokinin-pancreozymin (8 ID u.kg-1) or pilocarpine (15 mg kg-1) both resulted in a significant change in the enzyme proportions in the juice. 3. In experiments on pancreatic lobules, the addition to the incubation medium of secretin (10(-7) M), alone or associated with cholecystokinin-pancreozymin (8 x 10(-7) M) or pilocarpine (10(-4) M) did not induce any change in the enzyme proportions in secretion. 4. It was concluded that the non-parallelism between enzyme secretions can occur in the rat upon pancreozyminic or cholinergic stimulation in vitro as well as in vivo (Dagorn, 1978) provided basal protein output is low enough. This is not the case when the tissular integrity of the pancreas is lost, such as in experiments on lobules. 5. This work confirms that pancreatic secretion derives from two intrapancreatic pools of different enzymatic composition, and gives a possible explanation for some discrepancies from the literature on the existence of non-parallel secretion.     

Cholecystokinin-induced desensitization of enzyme secretion in dispersed acini from guinea pig pancreas

Incubating dispersed acini from guinea pig pancreas with cholecystokinin and then washing the cells to remove cholecystokinin reduced the subsequent stimulation of amylase secretion caused by pancreatic secretagogues, whose actions are mediated by release of cellular calcium (i.e., cholecystokinin, carbamylcholine, bombesin, litorin, physalaemin, and A23187), but did not alter the stimulation caused by secretagogues whose actions are mediated by cAMP (i.e., vasoactive intestinal peptide and secretin). This cholecystokinin-induced desensitization was reversible, and the onset of the process as well as its reversal were time- and temperature-dependent changes. The concentrations of cholecystokinin required to cause desensitization were greater than those required to cause maximal stimulation of amylase secretion, and this finding suggests that the submaximal stimulation of enzyme secretion seen with supramaximal concentrations of cholecystokinin may be caused by cholecystokinin-induced desensitization.     

Selective modification of the ability of cholecystokinin to cause residual stimulation of pancreatic enzyme secretion

In the C-terminal heptapeptide of cholecystokinin (-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2), replacing the aspartic acid residue by beta-aspartic acid did not alter the ability of the peptide to cause stimulation, desensitization, or residual stimulation of enzyme secretion from dispersed pancreatic acini. Replacing the tyrosine sulfate residue by hydroxynorleucine sulfate did not alter the ability of the heptapeptide to cause stimulation or desensitization, but caused a 50-fold decrease in the potency with which the peptide caused residual stimulation of enzyme secretion. These findings suggest that a modification of the N-terminal region of cholecystokinin heptapeptide, which does not alter the ability of the peptide to bind to its receptor on pancreatic acini and by so doing cause stimulation and desensitization of enzyme secretion, can increase the rate at which the bound peptide dissociates when the acini are washed and reincubated. This increased dissociation is reflected by a reduction in the potency with which the peptide causes residual stimulation of enzyme secretion.     

Adenovirus-mediated gene expression in isolated rat pancreatic acini and individual pancreatic acinar cells

 In this study we have examined the feasibility of using replication-deficient recombinant adenoviral vectors to transfer and express genes in pancreatic acinar cellsin vitro. We infected primary cultures of both isolated pancreatic acini and individual acinar cells with a recombinant adenovirus containing the coding sequence for β-galactosidase. Our data demonstrate that recombinant adenoviruses readily infect pancreatic acinar cellsin vitro. Close to 100% infection and maximal β-galactosidase expression were obtained, when acini or acinar cells were infected with 5×10⁶ or 10⁶ plaque-forming units (pfu) of virus per millitre of acini or acinar cell suspension, respectively. Examination of the time-course of β-galactosidase expression showed that there was a lag of approximately 6 h before β-galactosidase levels increased. Thereafter β-galactosidase expression increased rapidly. By 20 h post-infection β-galactosidase activity had increased from undetectable levels to 2.5–3.0 units/mg of cellular protein. Acini/acinar cells maintained a robust secretory response after adenoviral infection. The cholecystokinin-octapeptide (CCK8) dose/response curves for amylase secretion for acini and acinar cells infected with 5×10⁵ and 1×10⁵ pfu/ml of virus, respectively, were biphasic, with maximal amylase secretion being stimulated by 1 nM CCK8. In addition, the dose/response curves were identical to those obtained from control, sham-infected, acini/acinar cells. Our findings indicate that replication-deficient recombinant adenoviral vectors will be excellent tools to transfer and express genes in isolated pancreatic acini or acinar cells. Received: 27 March 1998 / Received after remission: 6 May 1998 / Accepted: 7 May 1998     

Influence of stepwise uncoupling and temporary anoxia on pancreatic enzyme secretion by isolated rat acini.

For special studies on pancreatic diseases a parameter is needed to record alterations of the cellular energy metabolism. In the in vitro model of isolated pancreatic acini, we investigated whether or not at standardized cholecystokinin stimulation the energy-consuming process of enzyme secretion can be used to monitor changes of the energy-supplying capacity. Rat pancreatic acini were isolated via collagenase digestion and characterized by basal and stimulated release of amylase and trypsin, oxygen uptake under resting and maximally uncoupled conditions and by their ability to accumulate actively rhodamine-6G, as a measure of the mitochondrial membrane potential. The stimulation of enzyme release did not find a measurable reflection in rhodamine-6G accumulation and in the respiratory rat. Stepwise uncoupling of oxidative phosphorylation by 2,4-dinitrophenol (DNP) and temporary anoxia were used to simulate disturbances of the pancreatic energy metabolism in vitro. With increasing DNP concentration the enzyme release was significantly reduced. While after 30 min anoxia the enzyme release still exceeded that of unstimulated control, after 60 min anoxia there was no further response to hormonal stimulation. At standardized stimulation and after suitable calibration the enzyme release by acini may be used to monitor alterations of the pancreatic energy metabolism in vitro.