Pretreatment Of Acini Research Articles

Src-mediated Cortactin Phosphorylation Regulates Actin Localization and Injurious Blebbing in Acinar Cells

Suprastimulation of pancreatic acini is a well-known model for pancreatitis, and it is characterized by actin reorganization and cell blebbing. Currently, however, the mechanisms underlying regulation of these aberrant cytoskeletal and membrane dynamics and how they contribute to cell injury are unclear. We observed that suprastimulation results in a rapid activation of Src and relocalization of the actin-binding protein cortactin from the apical to the basolateral domain at the necks of membrane blebs. Furthermore, Src-mediated cortactin tyrosine phosphorylation was markedly increased after suprastimulation. Pretreatment of acini with Src inhibitors or expression of a cortactin tyrosine phospho-inhibitory mutant reduced actin redistribution and bleb formation induced by suprastimulation in vitro. Importantly, inhibition of Src activity in rat models of suprastimulation-induced pancreatitis substantially reduced disease severity, as indicated by a reduction in serum amylase and pancreatic edema and a striking improvement in tissue histology. These findings indicate a novel, disease-relevant role for Src-mediated cortactin phosphorylation in aberrant reorganization of the actin cytoskeleton, a mechanism that is likely to have implications in other types of cell injury. In addition, they suggest a potential use for Src inhibitors as an approach to reduce cell injury.

LOW-DOSE ALCOHOL REDIRECTS POSTPRANDIAL CHOLINERGIC-STIMULATED APICAL EXOCYTOSIS TO THE BASOLATERAL MEMBRANE IN PANCREATIC ACINAR CELLS, THE SUSCEPTIBILITY MECHANISM OF ALCOHOLIC PANCREATITIS

Background: We previously demonstrated that alcohol (EtOH) pretreatment of pancreatic acini caused the basal plasma membrane (BPM) to become receptive to aberrant BPM exocytosis upon stimulation with submaximal CCK-8. These actions of alcohol on the acinar cell were due to induction of PM-bound Munc18c to dissociate into the cytosol, rendering the PM Syntaxin4 to interact with PM-SNAP-23 and granule-VAMP to form a complex capable of mediating basolateral exocytosis into the interstitial space, which we had postulated to be a mechanism of alcoholic pancreatitis. Rat pancreas however lacks CCK receptors and instead responds to cholinergic stimulation. Aim: We therefore examined whether 1- low EtOH concentration (20 mM) is able to alter submaximal cholinergic stimulation of rat pancreatic acinar cell secretion and exocytosis; 2- alcohol diet-fed rats are susceptible to develop pancreatitis upon low postprandial cholinergic stimulation. Methods: 1- Dispersed rat pancreatic acini were pretreated with non-stimulatory EtOH (20 mM-1 h) followed by stimulation with postprandial carbachol (3 uM-1 h), a dose stimulating only half maximal amylase secretion; 2- rats were fed with either a control or ethanol (36% of total calories intake replacement) liquid diet for 6 weeks and then challenged with low carbachol (5 ip injections of 0.5 uM/kg/h) stimulation.We then examined: a) PM levels of Munc18c and SNARE proteins, and Munc18c displacement into the cytosol by confocal microscopy and cell subfractionation; b) amylase release; c) real-time epifluorescent imaging of exocytosis using FM1-43; d) electron microscopy to visualize the distribution of granules within the cell; e) tissue hematoxylin and eosin (H&E) staining to examine morphologic changes of pancreatitis; f) serum amylase, lipase and ethanol concentrations as clinical parameters of pancreatitis. Results: 1- a) Whereas 20 mM EtOH has no effect on amylase secretion, pretreatment with 20 mM EtOH inhibited 3 uM carbachol stimulated secretion. b) Whereas 3 uM carbachol evokes apical exocytosis, pretreatment with 20 mM EtOH redirected exocytosis to the basal (by FM1-43 imaging) and lateral plasma membrane (by E.M.). c) While neither 20 mM EtOH nor 3 uM carbachol affected PM-Munc18c (confocal microscopy and subcellular fractionation), pretreatment of acini with 20 mM ETOH induced 3 uM carbachol to displace Munc18c from the BPM into the cytosol. 2-Alcohol diet-fed rats (serum ETOH levels of 20-30 mM) were normal and did not exhibit major morphologic defects in the pancreas. However, subsequent exposure to low-dose carbachol induced mild pancreatitis, observed as altered morphology (E.M and H&E) showing vacuolization in acinar cells, leukocyte infiltrate, hemorrhage and necrotic spots; along with clinical parameters of elevated serum levels of amylase and lipase. These morphologic changes correlated to Munc18c displacement from the BPM and its degradation in the cytosol. Conclusion: Postprandial EtOH renders acini PM-Munc18c conducive for postprandial cholinergic stimulation to displace into the cytosol, relieving Syntaxin4 to bind SNAP-23 and ZG VAMP proteins to promote basolateral exocytosis. We propose this to be a 'susceptibility' mechanism by which alcohol predisposes to human pancreatitis. (Funded by: Alcohol Beverage Medical Research Foundation and NIH -R21 AA015579-01).

Activated Polyamine Catabolism in Acute Pancreatitis: α-Methylated Polyamine Analogues Prevent Trypsinogen Activation and Pancreatitis-Associated Mortality

Polyamines are essential for normal cellular growth and function. Activation of polyamine catabolism in transgenic rats overexpressing spermidine/spermine N(1)-acetyltransferase, the key enzyme in polyamine catabolism, results in severe acute pancreatitis. Here, we investigated the role of polyamine catabolism in pancreatitis and studied the effect of polyamine analogues on the outcome of the disease. Polyamine depletion was associated with arginine- and cerulein-induced pancreatitis as well as with human acute necrotizing and chronic secondary pancreatitis. Substitution of depleted polyamine pools with methylspermidine partially prevented arginine-induced necrotizing pancreatitis whereas cerulein-induced edematous pancreatitis remained unaffected. Transgenic rats receiving methylated polyamine analogues after the induction of pancreatitis showed less pancreatic damage than the untreated rats. Most importantly, polyamine analogues dramatically rescued the animals from pancreatitis-associated mortality. Induction of spermidine/spermine N(1)-acetyltransferase in acinar cells isolated from transgenic rats resulted in increased trypsinogen activation. Pretreatment of acini with bismethylspermine prevented trypsinogen activation, indicating that premature proteolytic activation is one of the effects triggered by polyamine depletion. Our data suggest that activation of polyamine catabolism is a general pathway in the pathogenesis of acute pancreatitis and that experimental disease can be ameliorated with stable polyamine analogues.

Reactive oxygen species activate mitogen-activated protein kinases in pancreatic acinar cells.

It has been recently reported that kinases that belong to the mitogen-activated protein kinase (MAPK) family are rapidly activated by cholecystokinin (CCK) in rat pancreas both in vitro and in vivo. It is known that reactive oxygen species (ROS) play an important role in the pathogenesis of acute pancreatitis induced by supraphysiologic stimulation with CCK analogue, cerulein. The aim of our study was to evaluate whether MAPKs are activated by ROS in pancreatic acini. The activity of MAPK, c-Jun amino-terminal kinase (JNK), and p38 MAPK was determined in isolated rat pancreatic acinar cells by means of Western blotting, with the use of specific antibody that recognizes active, dually phosphorylated kinases. Incubation of acini with ROS donors, hydrogen peroxide (H2O2) and/or menadione (MND), strongly activated all three kinases. Activation of these kinases by ROS, but not by CCK, was substantially inhibited by pretreatment of acini with antioxidant N-acetylo-L-cysteine (NAC). Whereas CCK-induced activation of MAPK or JNK was totally or partially blocked by protein kinase C (PKC) inhibitor GF-109203X, ROS-induced activation of MAPK, JNK, and p38 MAPK was PKC independent. In conclusion, ROS strongly activate MAPK, JNK, and p38 MAPK in pancreatic acinar cells. It may be of importance in acute pancreatitis, because ROS are involved in the pathogenesis of this disease.

Involvement of Rab4 in regulated exocytosis of rat pancreatic acini

Background & Aims: Rab4, a Ras-related small guanosine triphosphate (GTP)-binding protein, has been suggested to participate in exocytosis. The function of Rab4 in regulated exocytosis of pancreatic acini was examined in this study. Methods: Subcellular localization of Rab4 was determined by Western blotting and immunohistochemistry. The Rab4 function in regulated exocytosis was examined by introducing Rab4 hypervariable carboxy-terminal domain peptide (Rab4 peptide) and anti-Rab4 antibody into streptolysin O–permeabilized acini. The regulation of Rab4 by cholecystokinin (CCK) and 12- O-tetradecanoyl-phorbol 13-acetate (TPA) was investigated by examining their effects on [ 32P]GTP binding rate into the Rab4 immunoprecipitates. The participation of protein kinase C in the Rab4 regulation by CCK was confirmed by calphostin C pretreatment of acini. Results: Rab4 was localized on zymogen granule membranes. Both Rab4 peptide and anti-Rab4 antibody enhanced calcium-stimulated amylase release from streptolysin O–permeabilized acini, suggesting the inhibitory role of Rab4 in exocytosis. CCK and TPA increased GTP binding to Rab4. Calphostin C attenuated the stimulatory effect of CCK on GTP binding to Rab4. Conclusions: Rab4 negatively modulates regulated exocytosis of pancreatic acini and is controlled by CCK through a protein kinase C pathway. GASTROENTEROLOGY 1999;116:943-952

Purification and Characterization of a Novel Physiological Substrate for Calcineurin in Mammalian Cells

Although the calcium/calmodulin-regulated protein phosphatase calcineurin has been shown to play a role in a number of intracellular processes, relatively few of the downstream phosphoproteins that are dephosphorylated by this enzyme in cells have been described. Calcineurin was previously shown to play a role in amylase secretion by rat pancreatic acinar cells and to specifically dephosphorylate a 24-kDa cytosolic protein. The present study describes the purification and characterization of this novel phosphoprotein, termed CRHSP-24 (calcium-regulated heat-stable protein with a molecular mass of 24 kDa). Microgram quantities of CRHSP-24 were purified from a large-scale rat pancreas preparation in a procedure involving heat and acid precipitation, anion-exchange chromatography, preparative electrophoresis, electroelution, and two-dimensional electrophoresis. Internal amino acid sequence was obtained from two peptides following trypsin digestion and high pressure liquid chromatography. Both sequences matched with 100% identity nucleotide sequences of expressed sequence tags from human placenta and rat PC-12 cells. Two CRHSP-24 transcripts of 0.7 and 2. 9 kilobases were detected in multiple rat tissues by Northern analysis, whereas a single 24-kDa protein was observed by Western blotting. The CRHSP-24 protein is 147 amino acids in length, is composed of nearly 14% proline, and is phosphorylated entirely on serine residues. Western analysis and 32P metabolic labeling of acini revealed CRHSP-24 to be maximally phosphorylated in control cells and to undergo a rapid sustained dephosphorylation on at least 3 serine residues in response to calcium-mobilizing stimuli. Dephosphorylation of CRHSP-24 was completely inhibited by pretreatment of acini with cyclosporin A or FK506. Furthermore, the inhibitory effects of FK506 were blocked by excess rapamycin. The ubiquitous expression of CRHSP-24 in rat tissues suggests that this novel calcineurin substrate plays a common role in calcium-mediated signal transduction.

Stimulatory effects of vanadate on amylase release from isolated rat pancreatic acini

The effects of vanadate on exocrine pancreatic function were examined in isolated rat pancreatic acini. Vanadate caused a concentration-dependent stimulation of amylase release above a concentration of 1 mM. Co-incubation of vanadate with vasoactive intestinal polypeptide, 8-bromoadenosine 3′:5′-cyclic monophosphate, and the Ca 2+ ionophore A23187 produced a synergistic pattern of amylase release, whereas co-incubation with cholecystokinin octapeptide (CCK-8), carbamylcholine, and 12-O-tetradecanoylphorbol 13-acetate produced an additive effect. Vanadate alone had no influence on acinar cyclic AMP content, Ca 2+ efflux, or intracellular Ca 2+ concentration. However, preincubation with vanadate prevented the plateau phase of CCK-8-induced Ca 2+ transient increase from returning to baseline. Moreover, depletion of the intracellular Ca 2+ pool by pretreatment of acini with CCK-8 in Ca 2+-free medium (plus ethyleneglycol bis[β-aminoethylether]-N ,N′-tetraacetic acid) had no effect on subsequent stimulation by vanadate, although it abolished the response to both CCK-8 and carbamylcholine stimulation. The protein kinase C (PKC) inhibitors staurosporine and calphostin C significantly inhibited vanadate-stimulated amylase release, whereas the protein tyrosine kinase inhibitor genistein had no inhibitory effect. Moreover, vanadate caused a significant translocation of PKC from cytosol to membrane fraction in pancreatic acinar cells. This translocation was inhibited significantly by staurosporine and calphostin C but not by genistein. These results suggest that vanadate acts directly on pancreatic acini and stimulates amylase release by activating PKC without an effect on Ca 2+ mobilization, cyclic AMP, or protein tyrosine kinase.

Pertussis toxin-sensitive G-proteins inhibit fibroblast growth factor-induced signaling in pancreatic acini.

Signal transduction of fibroblast growth factor (FGF) receptors is known to involve tyrosine phosphorylation of several substrates, including Grb2, phospholipase C-gamma, and phosphatidylinositol 3-kinase, whereas the role of G-proteins in FGF receptor signaling is controversial. In the present study we investigated the role of G-proteins in FGF receptor signaling in rat pancreatic acini. Immunological analysis revealed the presence of FGF receptor and phospholipase C-gamma1 in rat pancreatic acini. Both basic fibroblast growth factor (FGF-2) and guanosine 5'-(gamma-O-thio)triphosphate (GTPgammaS) caused an increase in inositol 1,4,5-trisphosphate (1,4,5-IP3) production and amylase release. Combined stimulation of the acini with GTPgammaS and FGF-2 led to a decrease of these responses as compared to the effect of the single substances. When pancreatic acini were preincubated with FGF-2 (1 nM) or vehicle (water) ADP-ribosylation of the alpha-subunit of Gi-type G-proteins by pertussis toxin was reduced in membranes prepared from FGF-2 pretreated acini as compared to control acini, suggesting functional interaction of FGF receptors with Gi-proteins. Pretreatment of acini with pertussis toxin which inhibits Gi-type G-proteins abolished the inhibitory effect of GTPgammaS on FGF-induced 1,4,5-IP3 production and amylase release, whereas the stimulatory effects of FGF-2 and GTPgammaS on these parameters remained unchanged. In conclusion, these results show communication of FGF receptors and Gi-type G-proteins and that Gi-type G-proteins exert an inhibitory influence on FGF-induced activation of phosphoinositide-specific phospholipase C in pancreatic acinar cells.

Pertussis toxin‐sensitive G‐proteins inhibit fibroblast growth factor‐induced signaling in pancreatic acini

Signal transduction of fibroblast growth factor (FGF) receptors is known to involve tyrosine phosphorylation of several substrates, including Grb2, phospholipase C-γ, and phosphatidylinositol 3-kinase, whereas the role of G-proteins in FGF receptor signaling is controversial. In the present study we investigated the role of G-proteins in FGF receptor signaling in rat pancreatic acini. Immunological analysis revealed the presence of FGF receptor and phospholipase C-γ1 in rat pancreatic acini. Both basic fibroblast growth factor (FGF-2) and guanosine 5′-(γ-O-thio)triphosphate (GTPγS) caused an increase in inositol 1,4,5-trisphosphate (1,4,5-IP3) production and amylase release. Combined stimulation of the acini with GTPγS and FGF-2 led to a decrease of these responses as compared to the effect of the single substances. When pancreatic acini were preincubated with FGF-2 (1 nM) or vehicle (water) ADP-ribosylation of the α-subunit of Gi-type G-proteins by pertussis toxin was reduced in membranes prepared from FGF-2 pretreated acini as compared to control acini, suggesting functional interaction of FGF receptors with Gi-proteins. Pretreatment of acini with pertussis toxin which inhibits Gi-type G-proteins abolished the inhibitory effect of GTPγS on FGF-induced 1,4,5-IP3 production and amylase release, whereas the stimulatory effects of FGF-2 and GTPγS on these parameters remained unchanged. In conclusion, these results show communication of FGF receptors and Gi-type G-proteins and that Gi-type G-proteins exert an inhibitory influence on FGF-induced activation of phosphoinositide-specific phospholipase C in pancreatic acinar cells. © 1996 Wiley-Liss, Inc.

Inhibition by somatostatin of amylase secretion induced by calcium and cyclic AMP in rat pancreatic acini.

It has recently been shown that somatostatin inhibits amylase secretion from isolated pancreatic acini by reducing cyclic AMP (cAMP) production [Matsushita, Okabayashi, Hasegawa, Koide, Kido, Okutani, Sugimoto and Kasuga (1993) Gastroenterology 104, 1146-1152]. To date, however, little is known as to the other mechanism(s) by which somatostatin inhibits amylase secretion in exocrine pancreas. To investigate the action of somatostatin independent of cAMP generation, we examined the effect of somatostatin in isolated rat pancreatic acini stimulated by 1 microM calcium ionophore A23187 and 1 mM 8-bromo-cyclic AMP (8Br-cAMP). Somatostatin inhibited amylase secretion evoked by a combination of A23187 and 8Br-cAMP in a dose-dependent manner. The maximum inhibition was obtained by 10(-7) M somatostatin, and at this concentration somatostatin inhibited the effect of A23187 and 8Br-cAMP by approximately 30%. In electrically permeabilized acini, an elevation of free calcium concentration resulted in an increase in amylase secretion and cAMP enhanced the secretion evoked by calcium. cAMP shifted the dose-response curve for calcium-induced secretion leftwards and elevated the peak value of secretion. Somatostatin inhibited the effect of cAMP on calcium-induced amylase secretion by shifting the dose-response curve to the right. To determine the involvement of a G-protein(s), we examined the effect of somatostatin in acini pretreated with pertussis toxin. Pretreatment of acini with pertussis toxin completely blocked somatostatin-inhibition of amylase-secretion evoked by A23187 and 8Br-cAMP. These results indicate that somatostatin decreases amylase secretion induced by cAMP and calcium by reducing the calcium sensitivity of exocytosis. A pertussis toxin-sensitive G-protein is also involved in this step.

Biphasic effects of carbachol on stimulated cAMP accumulation in mouse parotid acini.

Carbachol (0.1-10 microM) augmented the isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by approximately 50% in mouse parotid acini; at carbachol concentrations > 10 microM the stimulatory trend was reduced. These effects were time dependent. In the presence of 3-isobutyl-1-methylxanthine (IBMX), the overall response to carbachol was an inhibition of the isoproterenol response. Pretreatment of acini with pertussis toxin failed to reverse this inhibition, suggesting that the effects of carbachol were not related to effects on the GTP binding protein, Gi. A-23187 mimicked the effects of carbachol on isoproterenol-stimulated cAMP accumulation in the presence and absence of IBMX. In the presence of IBMX, carbachol failed to inhibit isoproterenol-stimulated cAMP accumulation when calcium was absent from the extracellular media and depleted from intracellular stores by thapsigargin. By contrast, in the absence of IBMX, removal of calcium abolished augmentation of isoproterenol responses by low concentrations of carbachol, whereas at higher carbachol concentrations isoproterenol responses were significantly inhibited; the time to maximal cAMP accumulation was decreased approximately eightfold. The results show that the mechanisms underlying the effects of carbachol on cAMP metabolism involve both the enzymes that synthesize and degrade cAMP.

PGE 2 regulates cholecystokinin-octapeptide (CCK-8)-stimulated Cl − conductance in isolated zymogen granules from rat pancreas

In this study we have examined the effects of prostaglandin E 2 (PGE 2), the cyclooxygenase inhibitor, indomethacin, and a protein kinase A inhibitor (PKA-I) on the Cl − conductance in isolated zymogen granules (ZG) from cholecystokinin octapeptide (CCK-8) pre-stimulated pancreatic acini. The Cl − conductance in isolated ZG from CCK-8 pre-stimulated rat pancreatic acini increases with increasing CCK-8 concentrations and decreases at supramaximal CCK-8 concentrations. The basal and CCK-8-stimulated Cl − conductance in ZG is inhibited by pretreatment of acini with PGE 2 (10 −6 M). This PGE 2-induced inhibition is abolished in the presence of PKA-I (20 U/ml). Furthermore, pretreatment of acini with indomethacin (10 −5 M) or PKA-I (20 U/ml) abolishes the decrease in the Cl − conductance at supramaximal CCK-8 concentrations (10 −9 M). We conclude that the inhibition of the Cl − conductance in isolated ZG at high CCK-8 concentrations is mediated by an enhanced production of PGE 2, and that PGE 2 operates by stimulating adenylate cyclase (AC) with a consequent rise in cAMP and activation of PKA.

Carbachol acts through protein kinase C to modulate cholecystokinin receptors on pancreatic acini

Cholecystokinin (CCK) and cholinergic agonists are both major stimulants of pancreatic enzyme secretion and both utilize a common calcium-phosphoinositide-mediated receptor coupling system. In this study we investigated the modulation of pancreatic acinar CCK receptors by the muscarinic agonist carbachol (CCh) and investigated the intracellular mechanisms involved in the modulation. Acini were isolated from rat pancreas and dispersed in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-Ringer solution. Preincubation with 0.1 mM carbachol for 60 min reduced the CCK octapeptide (CCK-8; 100 pM)-stimulated amylase release by 43 +/- 5%. Binding of 125I-Bolton-Hunter-labeled CCK-8 (125I-BH-CCK-8) revealed two classes of CCK receptors, a high affinity with a dissociation constant (Kd) of 20 pM and a low affinity with a Kd of 2.3 nM. Pretreatment with 100 microM CCh decreased total binding by 35 +/- 6%, affecting the binding capacity of the high-affinity site, without change in the maximal binding capacity of the low-affinity site and no change in the Kd of either site. Preincubation of acini with 12-O-tetradecanoylphorbol 12,13-acetate (TPA, 1 microM), an activator of protein kinase C (PKC), decreased subsequent CCK-8-stimulated amylase release, and total binding of 125I-BH-CCK-8 to a similar extent as with pretreatment with CCh. The inhibitory effect of TPA or CCh on CCK-8-stimulated amylase release was reversed by simultaneous preincubation with H-7, an inhibitor of PKC. Pretreatment of acini with the calcium ionophore A23187, vasoactive intestinal peptide, or 8-bromoadenosine 3',5'-cyclic monophosphate had no effect on 125I-BH-CCK-8 binding. After CCh or TPA preincubation, CCK-8-stimulated production of [3H]inositol phosphates was inhibited by at least 49%.(ABSTRACT TRUNCATED AT 250 WORDS)

Basic fibroblast growth factor is a calcium-mobilizing secretagogue in rat pancreatic acini

Basic fibroblast growth factor (bFGF) induced a marked increase in the levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and a rapid rise in cytosolic free calcium [Ca 2+] i levels in rat pancreatic acini. The bFGF-mediated calcium transient was not dependent on the presence of extracellular calcium, and was abolished by pretreatment of acini with carbachol. bFGF stimulated amylase release in pancreatic acini in a monophasic, dose-dependent manner, and this effect was blocked by neutralizing anti-bFGF antibodies. At much higher concentrations, epidermal growth factor (EGF), but not insulin-like growth factor-I (IGF-I), partially mimicked some of the actions of bFGF. These findings suggest that bFGF is a previously unrecognized calcium-mobilizing pancreatic secretagogue that may participate in the regulation of pancreatic exocrine function.

Phorbol Ester Potentiates VIP-Stimulated Amylase Release in Rat Pancreatic Acini

Vasoactive intestinal peptide (VIP) or 12-O-tetradecanoylphorbol-13-acetate (TPA) individually stimulated amylase release in dispersed rat pancreatic acini. Pretreatment of acini with TPA (10(-6) M) for 5 min at 37 degrees C potentiated their subsequent response to stimulation by VIP at a dose range of 10(-8)-10(-6) M in that the treated pancreatic acini released more amylase than could be accounted for by the additive effects of VIP or TPA acting individually. This potentiation effect of TPA was still evident when isobutyl methylxanthine was given together with VIP. Further, the maximal' dose-response curve to VIP shifted 2 log units to the left (3 x 10(-9) versus 3 x 10(-7) M). The TPA preincubation was found also to potentiate VIP-stimulated net increases in intracellular cyclic AMP (cAMP) levels. A close correlation existed between the net releases of amylase and the net increases in intracellular cAMP levels (r = 0.97). This suggested that TPA potentiated the response of rat pancreatic acini to VIP by modulating the cAMP system. The TPA as a potent activator of protein kinase C may act as a modulator of the adenylate cylase-cAMP system in rat pancreatic acini.

Pancreatic secretagogues regulate somatostatin binding to acinar cell membranes via two-functionally distinct pathways.

Pretreatment of pancreatic acini with vasoactive intestinal peptide (VIP) or secretin for 120 min reduced subsequent [125I-Tyr1]somatostatin binding to membranes prepared from these acini, with a maximally reduced binding being 79.2% or 77.4% of control, respectively. In addition, exogenously added cyclic AMP derivatives or a phosphodiesterase inhibitor mimicked the effect of VIP or secretin. Scatchard analysis of [125I-Tyr1]somatostatin binding demonstrated that the decrease in the labeled somatostatin binding induced by VIP or dibutyryl cyclic AMP (dbcAMP) pretreatment was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The effect of simultaneous pretreatment of acini with VIP and carbamylcholine (carbachol) on subsequent labeled somatostatin binding appeared to be almost equal to the calculated additive value for each peptide. Results obtained, therefore, indicate that the binding of somatostatin to its receptors in the pancreas may be regulated via two functionally distinct pathways.

Pancreatic secretagogues regulate somatostatin binding to its receptors on rat pancreatic acinar membranes.

Labeled somatostatin binding to pancreatic acinar membranes was examined to investigate how pancreatic secretagogues regulate somatostatin receptors in the pancreas. Pretreatment of pancreatic acini at 37 degrees C for 120 min with not only pancreatic secretagogues, such as carbachol and bombesin, but also vasoactive intestinal peptide (VIP) and secretin reduced subsequent labeled somatostatin binding to the acinar membranes in a dose-dependent manner. The inhibitory effects of these secretagogues on labeled somatostatin binding were also time dependent. Both types of secretagogues maximally reduced subsequent somatostatin binding when acini were incubated with them for more than 120 min. However, the degree of the inhibition was greater with carbachol or bombesin than VIP or secretin; the former secretagogues reduced the binding to 40-45%, and the latter to 75-80% of a control, respectively. These pancreatic secretagogues had no inhibitory effect on somatostatin binding when added directly to the binding media. Furthermore, the inhibitory effect of carbachol was attenuated by the presence of 1 mM EDTA in media for pretreatment, suggesting that intracellular pathways activated by pancreatic secretagogues may be responsible for somatostatin receptor modulation. Interestingly, when combined with VIP, pretreatment of acini with carbachol produced an additive inhibition of labeled somatostatin binding to the membranes. Results, therefore, suggest that somatostatin binding to its receptors in the pancreas may be regulated via two functionally distinct intracellular pathways.

Downregulation of protein kinase C in guinea pig pancreatic acini: effects on secretion

Pretreatment of guinea pig pancreatic acini with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a time- and concentration-dependent down-regulation of protein kinase C. In control acini almost all of the protein kinase C activity was present in a cytosolic fraction. Incubation with TPA initially shifted protein kinase C activity to a particular fraction which then disappeared over the following 24-h incubation with TPA. To study the role of protein kinase C in stimulus-secretion coupling, acini were pretreated with TPA and then amylase release was studied in response to various secretagogues. Preincubation of acini with TPA led to a time- and concentration-dependent decrease in TPA-stimulated amylase release that correlated with protein kinase C downregulation. Preincubation of acini with 1 microM TPA for 24 h, resulting in complete loss of protein kinase C activity, abolished the secretory effect of subsequently added TPA. By contrast, the secretory effects of cholecystokinin octapeptide (CCK-8) and carbamylcholine chloride (CCh) were only inhibited by 44 and 34%, respectively, and amylase release stimulated by the Ca2+ ionophore A23187 and an adenosine 3',5'-cyclic monophosphate-mediated agonist, vasoactive intestinal peptide, was unaffected. Dose-response curves for CCK-8- or CCh-stimulated amylase release in TPA-pretreated acini revealed attenuation of both maximal efficacy and sensitivity. However, the CCh-stimulated intracellular Ca2+ increase as determined by use of the fluorescent probe fura-2 was not affected by the long-term TPA pretreatment of acini. This study strongly suggests that both protein kinase C and intracellular Ca2+ play a significant role in CCK-8- and CCh-stimulated amylase release.

Secretagogue-induced changes in intracellular pH and amylase release in mouse pancreatic acini

The response of the intracellular pH (pHi) to stimulation of enzyme secretion in pancreatic acini was measured using the fluorescent dye 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Acini suspended in pH 7.40 buffer demonstrated cytoplasmic alkalinization of 0.17, 0.14, and 0.15 pH units 2 min after addition of the secretagogues carbachol (10(-5) M), caerulein (10(-10) M), and bromo-A23187 (10(-6) M). Corresponding net stimulated amylase secretion over 30 min was 9.2, 10.3, and 5.6% of total content, respectively. Pretreatment of acini with atropine blocked the pHi rise induced by carbachol; addition of atropine 2 min after the carbachol did not reverse the alkalinization. Acini suspended in Ca2+ free buffer containing 0.1 or 0.2 mM ethylene glycol tetraacetic acid showed 0.21 and 0.10 pH unit alkalinization in response to caerulein (10(-10) M) and carbachol (10(-5) M) but no change in pHi after addition of bromo-A23187. Amylase release in response to increasing concentrations of caerulein was maximal at 10(-10) M, with decreasing rates of amylase release at higher drug concentrations (10(-8), 10(-7) M). Alkalinization in response to stimulation of secretion was maximal at 10(-8) M caerulein (0.30 pH units at 2 min) but was of lesser magnitude at 10(-7) M. Pancreatic acini demonstrated autoregulation of pHi over a range of external pH from 7.4 to 7.1. Net amylase release over 30 min in response to 10(-5) M carbachol was sustained at normal levels in buffers of pH varying between 7.7 and 6.5. In contrast, cytoplasmic alkalinization in response to carbachol occurred only in buffers with pH values between 7.40 and 7.10. These results indicate that amylase release occurs over a wide range of pHi and is not invariably associated with secretagogue-induced alkalinization.

Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on rat pancreatic acinar membranes

To investigate whether somatostatin receptors couple to guanine nucleotide inhibitory protein, Ni, on rat pancreatic acinar membranes, the effects of guanine nucleotide analogs or pretreatment of acini with islet activating protein (IAP), pertussis toxin on labeled somatostatin binding were examined. Guanine nucleotides reduced labeled somatostatin binding to acinar membranes up to 80%, with rank order of potency being guanyl-5'-yl imidodiphosphate (Gpp(NH)p) greater than GTP greater than GDP greater than GMP. Scatchard analysis of the labeled somatostatin binding revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg2+ and Na+ also reduced labeled somatostatin binding. Furthermore, inhibitory effects of 100mM Na+ and Gpp(NH)p were additive in reducing labeled somatostatin binding. A half maximal inhibitory concentration of Gpp(NH)p was decreased to 10(-7)M in the presence of 100mM Na+ and 5mM Mg2+ as compared to 10(-6)M in the presence of 5mM Mg2+ alone. Results therefore suggest that Gpp(NH)p requires Mg2+ for Ni activation and Na+ increases sensitivity of Ni to guanine nucleotide analogs. When pancreatic acini were treated for 4 hours with varying concentrations of IAP, which has been shown to uncouple Ni-mediated communication between inhibitory receptors and adenylate cyclase catalytic unit, subsequent labeled somatostatin binding to the acinar membranes was decreased in a dose dependent manner. These results indicate that somatostatin receptors on pancreatic acinar membranes couple to guanine nucleotide inhibitory protein, Ni and thus somatostatin probably functions in the pancreas to regulate intracellular signal transduction via Ni.