Cyclic GMP does not inhibit protein kinase C-mediated enzyme secretion in rat pancreatic acini

In pancreatic acini, cGMP can be increased by secretagogues such as cholecystokinin (CCK), cholinergic agents, and bombesin, whose actions on enzyme secretion are believed to be mediated by protein kinase C. However, the role of cGMP in acinar cell function has been unclear. A recent paper by Rogers et al. (Rogers, J., Hughes, R.G., and Matthews, E. K. (1988) J. Biol. Chem. 263, 3713-3719) reported that two analogues of cGMP, N2,O2-dibutyl guanosine 3':5'-monophosphate (Bt2cGMP) and 8-bromoguanosine 3':5'-monophosphate (8Br-cGMP), at concentrations in the nanomolar range, inhibited the stimulation of amylase secretion caused by CCK-8, bethanechol, bombesin, and 12-O-tetradecanoylphorbol-13-acetate (TPA). Rogers et al. also reported that sodium nitroprusside inhibited the stimulation of enzyme secretion caused by CCK-8 or TPA. These authors concluded that cGMP inhibits protein kinase C-mediated secretion in pancreatic acini. In the present study we attempted to confirm the findings of Rogers et al., We found, however, that Bt2cGMP inhibited CCK-8-stimulated amylase release only at concentrations of the nucleotide above 10 microM. Moreover, there was a close correlation between the ability of Bt2cGMP to inhibit CCK-8-stimulated amylase release and its ability to inhibit binding of 125I-CCK-8. Bt2cGMP, at concentrations as high as 3 mM, did not alter the stimulation of amylase release caused by carbachol, bombesin, TPA, or A23187. 8Br-cGMP, at concentrations up to 1 mM, did not inhibit the stimulation of amylase release caused by CCK-8 or TPA. At concentrations above 0.1 mM, 8Br-cGMP augmented the stimulation of amylase release caused by CCK-8, carbachol, bombesin, or TPA. Sodium nitroprusside, at a concentration that causes a 60-fold increase in cGMP, did not inhibit the stimulation of amylase release caused by CCK-8, carbachol, bombesin, or TPA. Our results do not confirm the findings of Rogers et al. and indicate that cGMP does not inhibit protein kinase C-mediated secretion in pancreatic acini.     

Stimulatory effect of PG-KII, an NK3 tachykinin receptor agonist, on isolated pancreatic acini: species-related differences

More information is needed on the physiological role of the tachykinins (TKs), especially neurokinin3-receptor (NK3) agonists, in the pancreas. In this paper we investigated and compared the effect of PG-KII (10(-9) to 10(-6) M), a natural NK3-receptor agonist, with that of the known secretagogues substance P (10(-9) to 10(-6)M), caerulein (10(-11) to 10(-8) M) and carbachol (10(-8) to 10(-5) M), on amylase secretion from dispersed pancreatic acini of the guinea pig and rat. PG-KII (10(-7) M) significantly increased basal amylase release from guinea pig pancreatic acini (from 5.4+/-0.9% to 11.3+/-0.5%, P < 0.05) but left basal release in the rat unchanged (6.5+/-0.5%). The stimulant effect of PG-KII on guinea pig acini was significantly reduced by the NK3-receptor antagonist, SR 142801 (5 x 10(-7) M), and left unchanged by the NK1-receptor antagonist, SR 140333 (5 x 10(-7) M). Conversely, substance P (10(-7) M) significantly stimulated amylase secretion from rat and guinea pig acini (12.6+/-0.6% and 12.1+/-0.7%, P < 0.05). This stimulated effect of substance P was antagonized by the NK1--receptor antagonist (5 x 10(-7) M), but not by the NK3-receptor antagonist (5 x 10(-7) M). The PG-KII- and substance P-evoked maximal responses were lower than those evoked by caerulein (10(-9) M) (guinea pig, 19.1+/-1.3%; rat, 1802+/-0.9%, P < 0.01) and carbachol (10(-5) M) (guinea pig, 23.3+/-1.2%; rat, 24.0+/-1.1%, P < 0.01). The inhibitors of phospholipase C U-73122 (10(-5) M), phospholipase A2 quinacrine (10(-5)M), and protein tyrosine kinase genistein (10(-4) M), partly but significantly inhibited PG-KII, as well as carbachol-stimulated amylase release. Coincubation of PG-KII 10(-7) M with submaximal doses of caerulein (10(-11) to 10(-10) M) and carbachol (10(-7) to 10(-6) M) had an additive effect on amylase release. Pre-incubation with PG-KII (10(-7) M) for 30 min significantly reduced the subsequent amylase response to PG-KII, whereas pre-incubation with caerulein 10(-10) M or carbachol 10(-6) M did not. These findings suggest that PG-KII directly contributes to pancreatic exocrine secretion by interacting with acinar NK3 receptors of the guinea pig but not of the rat. PG-KII signal transduction involves the intracellular phospholipase C, phospholipase A2 and protein tyrosine kinase pathways. The NK3 receptor system cooperates with the other known secretagogues in regulating guinea pig exocrine pancreatic secretion and undergoes rapid homologous desensitization.     

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.     

Effects of 1-oleoyl-2-acetyl glycerol are distinct from those of phorbol ester in rat pancreatic acini

1-oleoyl-2-acetyl glycerol (OAG), a potent activator of protein kinase C, inhibited the binding of 125I-labelled epidermal growth factor (EGF) in isolated rat pancreatic acini. Unlike cholecystokinin-octapeptide (CCK8) and the C-kinase activator 12-O-tetradecanoyl phorbol-13-acetate (TPA), two inhibitors of 125I-EGF endocytosis in the pancreas, OAG had no effect on the distribution of bound ligand between the cell surface and intracellular compartments. Unlike TPA, OAG failed to potentiate the inhibitory effects of the calcium ionophore A23187 on 125I-EGF cell-associated radioactivity and had no effect on either basal or carbachol-stimulated amylase release in acini. These data suggest that the actions of the synthetic diacyl-glycerol OAG are not fully equivalent with the action of other known activators of protein kinase C in the pancreatic acinar cell.     

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.     

Stimulatory effects of human pancreatic polypeptide on rat pancreatic acini

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

Two functionally distinct cholecystokinin receptors show different modes of action on Ca2+ mobilization and phospholipid hydrolysis in isolated rat pancreatic acini. Studies using a new cholecystokinin analog, JMV-180.

A new hepatapeptide cholecystokinin (CCK) analog, JMV-180 (Boc-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester), acts as an agonist at high affinity CCK receptors on rat pancreatic acini to stimulate amylase release but unlike cholecystokinin octapeptide (CCK8) does not act on low affinity CCK receptors to inhibit amylase release (Galas, M. D., Lignon, M. F., Rodriguez, M., Mendre, C., Fulcrand, P., Laur, J., and Martinez, J. (1988) Am. J. Physiol. 254, G176-G188). To investigate the biochemical mechanisms initiated by CCK acting on each class of CCK receptor, the effects of JMV-180 and CCK8 on amylase release, Ca2+ mobilization, and phospholipid hydrolysis were studied in isolated rat pancreatic acini. When acini were loaded with the intracellular Ca2+ chelator BAPTA, amylase release stimulated by both JMV-180 and CCK8 was reduced. Measurement of 45Ca2+ efflux and cytosolic free calcium concentration ([Ca2+]i) by the fluorescence of fura-2-loaded acini in a stirred cuvette showed that JMV-180 induced a concentration-dependent increase but with a maximal response only two-thirds that induced by CCK8. When [Ca2+]i of individual fura-2-loaded acinar cells was measured by microspectrofluorometry, all concentrations of JMV-180 (1 nM-10 microM) induced repetitive transient [Ca2+]i spikes (Ca2+ oscillations). By contrast, stimulation with a high concentration of CCK8 (1 nM) caused a large increase in [CA2+]i followed by a small sustained elevation of [Ca2+]i. The measurement of inositol trisphosphate (IP3) production by both [3H]inositol labeling and 1,4,5-IP3 radioreceptor assay showed that JMV-180 had only minimal effects at 10 microM in contrast to the large increase induced by high concentrations of CCK8 (more than 1 nM). JMV-180 blocked the effect of a high concentration of CCK8 on both [Ca2+]i and 1,4,5-IP3 productions but did not affect the response to carbamylcholine. JMV-180 caused a delayed monophasic stimulation of 1,2-diacylglycerol (DAG) sustained to 60 min without the early increase in DAG observed in response to CCK8. Furthermore, JMV-180 stimulated the release of [3H]choline metabolites, primarily phosphorylated choline, from [3H]choline-labeled acini at low concentrations and to the same extent as CCK8. Since JMV-180 interacts not only with high affinity CCK receptors as an agonist but also with low affinity CCK receptors as a functional antagonist, the present results indicate that the occupancy of high affinity state receptors by CCK induces Ca2+ oscillations, DAG formation from phosphatidylcholine hydrolysis, and amylase release with minimal phosphatidylinositol 4,5-bisphosphate hydrolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mutual dependence of VIP/PACAP and CCK receptor signaling for a physiological role in duck exocrine pancreatic secretion

Unlike in rodents, CCK has not been established as a physiological regulator in avian exocrine pancreatic secretion. In the isolated duck pancreatic acini, 1 nM CCK was required for stimulation of amylase secretion, maximal effect being achieved at 10 nM; picomolar CCK was without effect. Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 (10(-12)-10(-7) M) alone had no effect, but made picomolar CCK effective. VPAC agonist VIP 10(-10)-10(-7) M stimulated amylase secretion marginally, but made CCK 10(-12)-10(-10) M effective also. PACAP-27 and VIP both shifted the maximal CCK concentration from 10(-8) to 10(-9) M. This sensitizing effect was mimicked by forskolin. CCK dose dependently induced intracellular Ca2+ concentration ([Ca2+]i) oscillations. PACAP-38 (1 nM), PACAP-27 (1 nM), VIP (10 nM), or forskolin (10 microM) alone did not stimulate [Ca2+]i increase, neither did they modulate CCK (1 nM)-induced oscillations; but when they were added to cells simultaneously exposed to subthreshold CCK (10 pM), calcium spikes emerged. Amylase secretion induced by the simultaneous presence of 10 pM CCK and VPAC agonists was completely blocked by removing extracellular calcium, but the protein kinase C inhibitor staurosporine (1 microM) was without effect. CCK (10 nM)-induced secretion was inhibited by CCK1 receptor antagonist FK480 (1 microM). Gastrin from 10(-12) to 10(-6) M did not stimulate amylase secretion nor did it (100 nM) induce [Ca2+]i increase. The above data suggest that duck pancreatic acini possess both CCK1 and VPAC receptors; simultaneous activation of both is required for each to play a physiological role.     

Multiple sources of 1,2-diacylglycerol in isolated rat pancreatic acini stimulated by cholecystokinin. Involvement of phosphatidylinositol bisphosphate and phosphatidylcholine hydrolysis

Changes in the cellular content of 1,2-diacylglycerol (DAG) in isolated rat pancreatic acini in response to agonist stimulation were studied using a sensitive mass assay. When acini were stimulated by 10 nM COOH-terminal cholecystokinin-octapeptide (CCK8), the increase in DAG was biphasic, consisting of an early peak at 5 s and a second, larger, gradual increase that was maximal by 15 min. The basal level of DAG in acini was 1.04 nmol/mg of protein, which was increased to 1.24 nmol/mg of protein at 5 s and 2.76 nmol/mg of protein at 30 min. In comparison, the increase in DAG stimulated by 30 pM CCK8, a submaximal concentration for amylase release, was monophasic, increasing without an early peak but sustained to 60 min. Other Ca2+-mobilizing secretagogues such as carbamylcholine and bombesin increased DAG in acini, whereas vasoactive intestinal peptide, which acts to increase cAMP, had no effect. Phorbol ester and Ca2+ ionophore also stimulated DAG production. Analysis of the mass level of inositol 1,4,5-trisphosphate (1,4,5-IP3) showed that the generation of 1,4,5-IP3 stimulated by 10 nM CCK8 peaked at 5 s, a finding consistent with the early peak of DAG. The basal level was 4.7 pmol/mg of protein, which was increased to 144.6 pmol/mg of protein at 5 s by 10 nM CCK8. The levels of 1,4,5-IP3 then returned toward basal in contrast to the gradual and sustained increase of DAG. The dose dependencies of 1,4,5-IP3 and DAG formation at 5 s with respect to CCK8 were almost identical. This suggests that phosphatidylinositol 4,5-bisphosphate hydrolysis is a major source of the early increase in DAG but not of the sustained increase in DAG. Therefore, a possible contribution of phosphatidylcholine hydrolysis to DAG formation was examined utilizing acini prelabeled with [3H]choline. CCK8 (1 nM) maximally increased [3H]choline metabolite release by 133% of control at 30 min. Separation of these metabolites by thin layer chromatography showed that the products of CCK8-stimulated release were almost entirely phosphorylcholine, indicating the activation of a phospholipase C specific for phosphatidylcholine. By comparison, 1 nM CCK8 stimulated [3H]ethanolamine metabolite release from [3H]ethanolamine-labeled acini by only 22% of control. These data suggest that CCK stimulates both phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine hydrolysis; the latter may contribute to the sustained generation of DAG and hence the maintained activation of protein kinase C.     

Relationship between cytosolic Ca2+ concentration and amylase release in rat parotid acinar cells following muscarinic stimulation.

Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cytosolic free calcium concentration ([Ca2+]i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), or the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA), strongly attenuated the increases in [Ca2+]i evoked by CCh, but amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02-0.5 microM) of ionomycin, a Ca2+ ionophore, caused increases in [Ca2+]i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change in [Ca2+]i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca2+]i elicited by CCh does not play an essential role for inducing amylase release in rat parotid acini. Amylase release by muscarinic stimulation may be mediated mainly by activation of protein kinase C.     

Thyroidal and neural control of myosin transitions during development of rat fast and slow muscles

The uptake of 125I-labeled epidermal growth factor (125I-EGF) by mouse pancreatic acini was inhibited (40-50%) by the secretagogue cholecystokinin octapeptide (CCK8). Analysis of competitive binding data showed that the apparent Kd of EGF binding increased 135% while the binding capacity was only slightly altered (30% increase). That the effect of CCK8 on acini was mediated by intracellular Ca2+ was indicated by the following: (i) Inhibition of 125I-EGF binding to acini was dose-dependent and paralleled the known abilities of CCK8, its analogs, and the cholinergic secretagogue carbachol to induce Ca2+ efflux from acini; and (ii) addition of the Ca2+ ionophore A23187 also inhibited 125I-EGF binding. In addition, EGF association with A431 cells was also inhibited by A23187 in the presence but not the absence of Ca2+.     

A synthetic peptide derivative that is a cholecystokinin receptor antagonist

So far, there are no known peptidic effective receptor antagonists of both peripheral and central effects of cholecystokinin (CCK). Here, we describe a synthetic peptide derivative of CCK, t-butyloxycarbonyl-Tyr(SO3-)-Met-Gly-D-Trp-Nle-Asp 2-phenylethyl ester 1 (where Nle is norleucine), which is a potent CCK receptor antagonist. In rat and guinea pig dispersed pancreatic acini, this peptide derivative did not alter amylase secretion, but was able to antagonize the stimulation caused by cholecystokinin-related agonists. It caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion with half-maximal inhibition of CCK-8-stimulated amylase release at a concentration of about 0.1 microM. Compound 1 was able to inhibit the binding of labeled CCK-9 (the C-terminal nonapeptide of CCK) to rat and guinea pig pancreatic acini (IC50 = 5 X 10(-8) M) as well as to guinea pig cerebral cortical membranes (IC50 = 5 X 10(-7) M). These results indicate that Compound 1 is a potent competitive CCK receptor antagonist.     

Nonparallel discharge of digestive enzymes from isolated pancreatic acini

The secretion of amylase, trypsinogen, chymotrypsinogen and proelastase from isolated rat dispersed pancreatic acini was investigated in the absence (basal) and presence of two concentrations of CCK8 (50 and 500 pM), carbachol (2.5 and 7.5 microM) and secretin (10 nM and 1 microM). The unstimulated (basal) rate of release of each of the digestive enzymes was essentially the same. However, whereas both doses of CCK8 and carbachol caused a preferential release of chymotrypsinogen over that of amylase and trypsinogen, the magnitude of stimulated release of amylase, trypsinogen and chymotrypsinogen by 1 microM secretin was found to be similar for each of the enzymes. Furthermore, none of the secretagogues caused a significant enhancement in proelastase release. The present data demonstrate that whereas CCK8 and carbachol induce a greater release of chymotrypsinogen over that of amylase or trypsinogen, release of all three enzymes was equally stimulated by secretin from isolated pancreatic acini.     

Different sensitivity to phorbol esters and pertussis toxin of bombesin- and platelet-derived growth factor-induced, phospholipase C-mediated hydrolysis of phosphoinositides in NIH/3T3 cells

Incubation of the serum-deprived cultures of NIH/3T3 cells with bombesin or platelet-derived growth factor (PDGF) induced the phospholipase C-mediated hydrolysis of phosphoinositides. Protein kinase C-activating 12-O-tetradecanoylphorbol 13-acetate (TPA) and pertussis toxin inhibited the bombesin-induced phospholipase C reactions. AlF4-, a direct activator of GTP-binding proteins (G proteins), also induced the phospholipase C reactions and TPA inhibited the AlF4- -induced reactions. These results suggest that a pertussis toxin-sensitive G protein is involved in the coupling of the bombesin receptor to the phospholipase C and that the coupling of the G protein to the phospholipase C is inhibited by protein kinase C. In contrast, neither TPA nor pertussis toxin inhibited the PDGF-induced phospholipase C reactions, indicating that a pertussis toxin-sensitive G protein is not involved in the coupling of the PDGF receptor to the phospholipase C and that this coupling is insensitive to protein kinase C. These results suggest that the regulatory mechanism of the PDGF receptor for the phospholipase C activation is different from that of the bombesin receptor.     

Cholecystokinin receptor occupation and cholecystokinin-induced calcium mobilization in the early phase in rat pancreatic acini.

We examined receptor occupation, calcium mobilization and amylase release for cholecystokinin octapeptide (CCK-8) within a 3-min incubation period at 37 degrees C using dispersed acini from rat pancreas. Analysis of competitive binding inhibition data obtained after a 3-min incubation revealed the presence of only a single class of CCK receptors, while two classes of CCK receptor, i.e., high-affinity and low-affinity CCK receptors, were detected when binding reached a steady-state after a 60-min incubation. The IC50 of CCK receptors calculated from the 3-min binding data was 19.0 +/- 0.5 nM (mean +/- S.D.), close to the Kd of the low-affinity CCK receptors determined by equilibrium binding studies. Exposure of fura-2-loaded acini to 10-1000 pM CCK-8 caused an immediate and dose-dependent increase in [Ca2+]i followed by a gradual decrease in [Ca2+]i. The CCK-stimulated amylase release after 3 min of incubation was biphasic; amylase release increased over the dose range of 3-300 pM CCK-8, peaked at 300 pM CCK-8 and decreased with supramaximal concentrations of CCK-8. Our data suggest that occupation of the low-affinity, but not the high-affinity, CCK receptors is more directly associated with calcium mobilization and subsequent stimulation of amylase release in rat pancreatic acini.     

Rap1 activation plays a regulatory role in pancreatic amylase secretion

Rap1 is a member of the Ras superfamily of small GTP-binding proteins and is localized on pancreatic zymogen granules. The current study was designed to determine whether GTP-Rap1 is involved in the regulation of amylase secretion. Rap1A/B and the two Rap1 guanine nucleotide exchange factors, Epac1 and CalDAG-GEF III, were identified in mouse pancreatic acini. A fraction of both Rap1 and Epac1 colocalized with amylase in zymogen granules, but only Rap1 was integral to the zymogen granule membranes. Stimulation with cholecystokinin (CCK), carbachol, and vasoactive intestinal peptide all induced Rap1 activation, as did calcium ionophore A23187, phorbol ester, forskolin, 8-bromo-cyclic AMP, and the Epac-specific cAMP analog 8-pCPT-2'-O-Me-cAMP. The phospholipase C inhibitor U-73122 abolished carbachol- but not forskolin-induced Rap1 activation. Co-stimulation with carbachol and 8-pCPT-2'-O-Me-cAMP led to an additive effect on Rap1 activation, whereas a synergistic effect was seen on amylase release. Although the protein kinase A inhibitor H-89 abolished forskolin-stimulated CREB phosphorylation, it did not modify forskolin-induced GTP-Rap1 levels, excluding PKA participation. Overexpression of Rap1 GTPase-activating protein, which blocked Rap1 activation, reduced the effect of 8-bromo-cyclic AMP, 8-pCPT-2'-O-Me-cAMP, and vasoactive intestinal peptide on amylase release by 60% and reduced CCK- as well as carbachol-stimulated pancreatic amylase release by 40%. These findings indicate that GTP-Rap1 is required for pancreatic amylase release. Rap1 activation not only mediates the cAMP-evoked response via Epac1 but is also involved in CCK- and carbachol-induced amylase release, with their action most likely mediated by CalDAG-GEF III.     

Potential mediation of prostaglandin E2 release from isolated human parietal cells by protein kinase C

Parietal cells are a major source of gastric mucosal prostaglandins in various species. We examined cholinergic stimulation of prostaglandin E2 (PGE2) release from human parietal cells; using activators of the protein kinase C we attempted to get an indirect insight into cellular mechanisms which control PGE2 release. Gastric mucosal specimens were obtained at surgery and the cells were dispersed by collagenase and pronase E. Parietal cells were enriched to 65-80% by a Percoll gradient, and were incubated for 30 min. PGE2 release into the medium (radioimmunoassay) was 74-126 pg/10(6) cells/30 min under basal conditions and was 2.6-fold increased by carbachol (10(-5) and 10(-4) M). Similarly, PGE2 release was stimulated by phospholipase C (20-200 mU/ml, 364% above basal), 1-oleoyl-2-acetyl-sn-glycerol (10(-9)-10(-5) M, 229%), 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-9)-10(-5) M, 283%) and calcium ionophore A23187 (10(-7)-10(-5) M, 219%). Simultaneous presence of A23187 and TPA synergistically induced stimulation which was slightly higher than the sum of the individual responses. N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide W-7, a putative calmodulin antagonist, inhibited TPA-induced PGE2 release at concentrations regarded specific for blocking calmodulin (IC50 = 1.5 X 0(-6) M). We conclude that in human parietal cells PGE2 is released upon cholinergic stimulation and that phospholipase C and protein kinase C are involved in the control of PGE2 release. We speculate that calmodulin might interact with a protein phosphorylated by protein kinase C to cause PGE2 release.     

Augmentation of cholinergic-mediated amylase release by forskolin in mouse parotid gland

Cholinergic-mediated amylase release in mouse parotid acini was augmented by forskolin; the potency but not the maximal response to carbachol was altered. Amylase released by carbachol plus forskolin was dependent on extracellular calcium and was mimicked by the calcium ionophore, A23187 plus forskolin. Forskolin was also shown to enhance carbachol-stimulated 45Ca2+ uptake into isolated acini. Hydroxylamine, nitroprusside, and 8-bromo-c-GMP each in combination with forskolin mimicked the effects of carbachol plus forskolin on amylase release. In the presence of carbachol (10(-8)M) forskolin did not augment c-AMP levels. However, in the presence of carbachol (5 X 10(-7) M) or hydroxylamine (50 microM) forskolin did significantly augment c-AMP accumulation. These results suggest that calcium and c-GMP may mediate the augmentation of cholinergic-mediated amylase release by effects on c-AMP metabolism.     

Cyclic GMP inhibits protein kinase C-mediated secretion in rat pancreatic acini

Dibutyryl cyclic GMP (Bu2cGMP) inhibited agonist-induced secretion of amylase from isolated rat pancreatic acini. In contrast to previous studies, this inhibitory action was not confined to butyryl derivatives of cyclic GMP, since the membrane-permeant cyclic GMP analogues Bu2cGMP and cyclic 8-bromo-GMP (8-Br-cGMP) were equipotent (IC50 2 nM) in their inhibition of amylase secretion stimulated by cholecystokinin-(26-33)-octapeptide (CCK8): at extracellular concentrations up to 1 mM, cyclic GMP itself was devoid of inhibitory activity. Both Bu2cGMP and 8-Br-cGMP also potently inhibited secretion stimulated by 4 beta-phorbol 12-myristate 13-acetate (PMA) (IC50 6 nM), but only partially inhibited responses elicited by bethanechol or bombesin and were without effect on A23187-evoked secretion. Furthermore, agents that are known to raise intracellular cyclic GMP levels (MB22948 (2-o-propoxyphenyl-8-azapurin-6-one) or nitroprusside) or antagonize the actions of protein kinase C (4 alpha-PMA or staurosporine), also inhibited CCK8- or PMA-stimulated secretion but not secretion elicited by bombesin, bethanechol, or A23187. It is concluded from these and other observations reported here that protein kinase C is the major intracellular mediator of amylase secretion stimulated by CCK8 and that this pathway may be regulated by cyclic GMP at a step that follows protein kinase C activation.     

Estrogens influence cholecystokinin stimulated pancreatic amylase release and acinar cell membrane cholecystokinin receptors in rat.

This study examines the influence of ovariectomy and administration of a pharmacologic dose of estradiol on amylase release from isolated-dispersed rat pancreatic acini and cholecystokinin receptors on rat acinar cell membranes. Rats were sham ovariectomized (intact) or ovariectomized (Ovx) and 21 day timed release pellets containing either estradiol (2.5 mg) or vehicle, were implanted subcutaneously. Eighteen days later, pancreatic acini were isolated from rats by collagenase digestion and differential centrifugation. Total cellular amylase, basal and cholecystokinin octapeptide (CCK8) stimulated amylase release and CCK membrane receptors were measured. Acini isolated from estradiol treated Ovx rats had significantly greater total cellular amylase, compared to acini isolated from either intact or Ovx rats. The amplitude of both total stimulated amylase release and percent total stimulated amylase release were significantly greater for acini isolated from vehicle treated Ovx rats, than acini isolated from either intact or estradiol treated Ovx rats. The magnitude of percent total amylase release of acini isolated from estradiol treated Ovx rats was significantly lower than that of acini isolated from intact rats. Cholecystokinin receptor concentration was significantly greater on membranes prepared from vehicle treated Ovx rats, compared to membranes prepared from either intact or estradiol treated Ovx rats. These data indicate that ovariectomy is associated with increased responsiveness of pancreatic acini to CCK stimulation, while chronic estradiol treatment of ovariectomized rats is associated with increased total cellular amylase and decreased acinar cell responsiveness to CCK8. Estrogen mediated alterations in acinar cell amylase content and amylase release may play a role in estrogen related pancreatitis.