The diacylglycerol kinase inhibitor, R59022, potentiates cholecystokinin-induced enzyme secretion from rabbit pancreatic acini.

The putative inhibitor of diacylglycerol kinase activity, 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)-ethyl-7-meth yl-5H- thiazolo[3,2-a]pyrimidin-5-one (R59022), markedly potentiated cholecystokinin-C-terminal-octapeptide(CCK-8-)stimulated enzyme secretion from isolated rabbit pancreatic acini. Maximal potentiation occurred when acini were stimulated in the presence of 5-10 microM R59022. Potentiation depended both on the concentration of R59022 and CCK-8. No potentiation was observed when acini were half-maximally stimulated, whereas the secretory response to maximal and supramaximal concentrations of secretagogue was increased by 50-60%. R59022 alone had no effect on basal enzyme secretion and the drug did not potentiate the secretory response to the Ca2+ ionophore A23187 or to the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Moreover, no increase in basal secretion was observed when acini were incubated in the presence of both R59022 and forskolin. These observations strongly suggest that receptor-mediated activation of the inositol phospholipid pathway is required for R59022-induced potentiation. R59022 inhibited the CCK-8-stimulated incorporation of 32Pi into phosphatidic acid dose dependently, without affecting the CCK-8-stimulated hydrolysis of 32P-labelled phosphatidylinositol 4,5-bisphosphate. This is consistent with an inhibitory effect of R59022 on acinar cell diacylglycerol kinase activity. The potentiating effect of R59022 was mimicked by 12-O-tetradecanoylphorbol 13-acetate added simultaneously with CCK-8. Therefore, it is concluded that in the presence of 5-10 microM R59022 the receptor-mediated increase in acinar cell diacylglycerol content is enhanced leading to enhanced activation of protein kinase C and to potentiation of the secretory response. The fact that the secretory response to maximal and supramaximal concentrations of CCK-8 is potentiated by R59022 suggests that at these concentrations of secretagogue the diacylglycerol/protein kinase C branch of the signal-transduction route is rate-limiting.     

Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas.

The recent identification in Heloderma horridum venom of exendin-3, a new member of the glucagon superfamily that acts as a pancreatic secretagogue, prompted a search for a similar peptide in Heloderma suspectum venom. An amino acid sequencing assay for peptides containing an amino-terminal histidine residue (His1) was used to isolate a 39-amino acid peptide, exendin-4, from H. suspectum venom. Exendin-4 differs from exendin-3 by two amino acid substitutions, Gly2-Glu3 in place of Ser2-Asp3, but is otherwise identical. The structural differences make exendin-4 distinct from exendin-3 in its bioactivity. In dispersed acini from guinea pig pancreas, natural and synthetic exendin-4 stimulate a monophasic increase in cAMP beginning at 100 pM that plateaus at 10 nM. The exendin-4-induced increase in cAMP is inhibited progressively by increasing concentrations of the exendin receptor antagonist, exendin-(9-39) amide. Unlike exendin-3, exendin-4 does not stimulate a second rise in acinar cAMP at concentrations greater than 100 nM, does not stimulate amylase release, and does not inhibit the binding of radiolabeled vasoactive intestinal peptide to acini. This indicates that in dispersed pancreatic acini, exendin-4 interacts only with the recently described exendin receptor.     

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.     

Purification and structure of exendin-3, a new pancreatic secretagogue isolated from Heloderma horridum venom.

An amino-terminal histidyl structure (His1) is characteristic of most peptides in the glucagon superfamily. An assay for His1 peptides performed by amino-terminal amino acid sequencing was used to screen venom from the Gila monster lizard, Heloderma horridum. Two His1 peptides were identified: helospectin and a new His1 peptide that has been named exendin-3 to indicate that it is the third peptide to be found in an exocrine secretion of Heloderma lizards which has endocrine activity, the first two being helospectin (exendin-1) and helodermin (exendin-2). In the lot of H. horridum venom tested, exendin-3 was 5-10-fold more abundant in molar concentration than helospectin. The structure of exendin-3 was analyzed by amino acid sequencing and mass spectrometry. Exendin-3 is a 39-amino acid peptide with a mass of 4200. It contains a carboxyl-terminal amide and has a strong homology with secretin at its amino-terminal 12 amino acids. The complete structure of exendin-3 is His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala- Val-Arg - Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser- amide. It is 32 and 26% homologous with helospectin and helodermin, respectively. It has greatest homology with glucagon (48%) and human glucagon-like peptide-1 (50%). Exendin-3 (3 microM) stimulated increases in cellular cAMP and amylase release from dispersed guinea pig pancreatic acini.     

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.     

Dominant negative Rab3D inhibits amylase release from mouse pancreatic acini

Rab3 proteins are believed to play an important role in regulated exocytosis and previous work has demonstrated the presence of Rab3D on pancreatic zymogen granules. To further understand the function of Rab3D in acinar cell exocytosis, adenoviral constructs were prepared encoding hemagglutinin-tagged wild type Rab3D and three mutant forms, N135I and T36N (both deficient in guanine nucleotide binding) and Q81L (deficient in GTP hydrolysis), which also expressed enhanced green fluorescent protein driven by a separate promoter. When isolated mouse pancreatic acini were cultured with 5 x 10(6) pfu/ml adenovirus, nearly 100% of acini were infected as visualized by expression of green fluorescent protein. Cultured acini showed a biphasic dose-response to cholecystokinin (CCK); basal amylase secretion was 1.8 +/- 0.3%/30 min, peak release was 7.3 +/- 0.2%/30 min at 30 pm CCK and reduced secretion was observed at higher CCK concentrations. Control beta-galactosidase virus infection had no effect on either basal or CCK-induced secretion in the titer range from 0.5 to 10 x 10(6) pfu/ml. While the expression of Rab3D and Rab3D Q81L had no effect on amylase secretion, Rab3D N135I and T36N functioned as dominant negative mutants and inhibited CCK-induced amylase release by 40-50% at all points on the CCK dose-response curve from 3 to 300 pm. Inhibition was stronger during the first 5 min (71 +/- 5%) than over 30 min (36%+/-5%). Similar inhibition was found using other agonists including bombesin, carbachol, and cAMP. Localization of adenoviral expressed Rab protein showed wild type Rab3D localized to zymogen granules. The two dominant negative mutants did not localize to granules and were primarily in the basolateral region of the cell. Since both dominant negative Rab3D mutants had no effect on intracellular calcium increase induced by CCK, it is unlikely that they acted at receptors or transmembrane signaling. These results suggest that Rab3D plays an important role in regulating the terminal steps of acinar exocytosis and that this effect is greatest on the early phase of amylase release.     

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

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

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.     

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.     

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

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

Photodynamic action of rose bengal on isolated rat pancreatic acini: stimulation of amylase release

The halogenated fluorescein derivative, rose bengal, upon photon activation, elicits amylase secretion from isolated, perifused pancreatic acini. This effect is due to production of highly reactive singlet delta oxygen which can permeabilize the cell membrane and may also react chemically with secretagogue receptors, or other functional components of the membrane such as the G-proteins. The profile of photodynamically induced amylase secretion is anion-dependent: it becomes biphasic when the chloride ion is substituted by the glutamate ion, an effect attributed to the action of glutamate on the ionic transport systems of the zymogen granule membrane.     

Introduction of calcium chelators into isolated rat pancreatic acini inhibits amylase release in response to carbamylcholine

The Ca2+ chelators, EGTA and BAPTA, have been introduced into intact, isolated rat pancreatic acini using a hypotonic swelling method. This resulted in complete inhibition of amylase release, stimulated by carbamylcholine at a submaximal concentration and 82 - 85% inhibition at maximal concentrations. Acini swollen in the absence of Ca2+ chelators showed similar secretory responses to those of unswollen acini. Treatment of unswollen acini with chelators inhibited the maximum response to carbamylcholine by only 23%. The inhibitory effect of intracellular chelators was not due to ATP depletion or a lowering of the total cell Ca2+ content. Thus, these results provide the first direct demonstration that an increase in intracellular Ca2+ concentration is necessary for the stimulation of enzyme release from pancreatic acinar cells.     

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 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.     

Chemical, immunological and biological properties of peptides like vasoactive-intestinal-peptide and peptide-histidine-isoleucinamide extracted from the venom of two lizards (Heloderma horridum and Heloderma suspectum)

Having previously isolated helodermin, the major peptide like vasoactive-intestinal-peptide and peptide-histidine-isoleucinamide, from the venom of the lizard Heloderma suspectum, we decided on a systematic exploration of all (VIP-PHI)-like peptides present in the venom of another lizard of the Helodermatidae family: Heloderma horridum. Six (VIP-PHI)-like peptides (PHH1 to 6) were purified to homogeneity from the venom of the lizard H. horridum with PHH3 and PHH4 representing two minor forms. All peptides cross-reacted in radioimmunoassays for helodermin and PHI but not for VIP. They yielded four fragments (T1 to T4) after trypsin digestion. T1, T2 and T3 showed the same retention time by reverse-phase HPLC and the same amino acid composition; the differences were confined to T4, the C-terminal sequence. PHH5 and PHH6 were found to be identical to synthetic helospectins I and II respectively. PHH1 and PHH3 probably resulted from a secondary modification of PHH5, while PHH2 and PHH4 derived from PHH6. Thus, the VIP-like peptides, previously called helospectins, are in fact typical of H. horridum venom. We confirmed that helodermin is the major (VIP-PHI)-like peptide of the venom of H. suspectum and observed its absence in H. horridum venom. Also, we found that positions 8 and 9 of helodermin are occupied by two Glu residues instead of two Gln as previously published. Helospectin-like material was also present in H. suspectum venom but in very small amount. In both venoms all VIP-like peptides were equally potent and efficient when tested for (a) their ability to occupy VIP as well as secretin receptors in rat pancreatic membranes and VIP receptors in rat liver membranes, and (b) the ensuing activation of adenylate cyclase in both membrane preparations.     

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.     

Galanin inhibits rat pancreatic amylase release via cholinergic suppression.

The effects of galanin on pancreatic exocrine function were examined using rat pancreatic tissues. In anesthetized rats, galanin (40 micrograms/kg/h) decreased amylase secretion stimulated by 2-deoxy glucose (5.8 +/- 0.1 vs. 3.1 +/- 0.1 times basal) and cholecystokinin octapeptide (21.5 +/- 0.6 vs. 16.8 +/- 0.5), while not inhibiting bethanechol-stimulated secretion. In dispersed acini, there was no effect of galanin alone (10(-8) to 10(-13) M) on amylase release, nor did galanin (10(-6) or 10(-8) M) coincubation affect amylase release stimulated by bethanechol (10(-3) to 10(-7) M) or CCK-8 (10(-8) to 10(-13) M). Using pancreatic lobules, coincubation with galanin (10(-6) M) suppressed 75 mM KCl-stimulated amylase secretion and ACh release (10.1 +/- 0.6% vs. 7.3 +/- 0.4%). Veratridine-stimulated (10(-4) M) amylase secretion and ACh release (12.4 +/- 1.7% vs. 8.5 +/- 0.7%) were similarly diminished.     

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.     

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.