Pancreatic Acinar Membranes Research Articles

Alcoholic chronic pancreatitis involves displacement of Munc18c from the pancreatic acinar basal membrane surface.

The minimal machinery for fusion of secretory vesicles with the cell membrane is a cognate set of v- and t-SNAREs on opposing membranes. Spontaneous SNARE complex assembly leading to unregulated membrane fusion is prevented by Munc18 proteins that bind membrane SNAREs syntaxins. Munc18 blocks syntaxin interactions with cognate SNARE proteins and thereby act as an inhibitor of exocytosis. The pancreatic acinar cell contains several sets of cognate SNAREs and Munc18 proteins that mediate the distinct exocytic events. We had reported that in the rat pancreas, Munc18c co-localizes with t-SNAREs syntaxin4 and SNAP23 on the acinar cell basolateral plasma membrane. Under conditions that induce pancreatitis in vivo, displacement of Munc18c from the basolateral plasma membrane relieved its blockade of SNARE-mediated membrane fusion in this region and thereby redirected apical exocytosis to the basal membrane surface. Here we show in a case of human mild alcoholic chronic pancreatitis that Munc18c is also displaced from the plasma membrane of intact acinar cells, which would render these cells receptive to pathologic basolateral exocytosis and further episodes of pancreatitis.

Bombesin induces a reduction of somatostatin inhibition of adenylyl cyclase activity, G i function, and somatostatin receptors in rat exocrine pancreas

To analyze the effect of bombesin on the somatostatin (SS) mechanism of action in the exocrine pancreas, male Wistar rats (250–270 g) were injected intraperitoneally with bombesin (10 μg/kg) three times daily at 8-h intervals for 7 or 14 days. Bombesin attenuated the ability of SS to inhibit forskolin-stimulated adenylyl cyclase activity in pancreatic acinar membranes. However, it did not decrease the ability of forskolin to stimulate the adenylyl cyclase catalytic subunit. The ability of 5′-guanylylimidodiphosphate [Gpp(NH)p] (a nonhydrolyzable GTP analog) to inhibit forskolin-stimulated adenylyl cyclase activity was diminished in pancreatic acinar cell membranes from bombesin-treated rats. Bombesin administration did not affect the ADP-ribosylation of a 41-kDa G protein catalyzed by pertussis toxin. The maximal SS binding capacity of pancreatic acinar membranes from bombesin-treated rats was decreased when compared with controls at the two time periods studied. The bombesin/gastrin-releasing peptide antagonist [ d-Tpi 6,Leu 13ψ(CH 2NH)Leu 14]bombesin (6–14) (RC-3095) (10 μg/kg ip), injected three times daily at 8-h intervals for 7 or 14 days, had a similar effect to that of bombesin on the SS mechanism of action. The combined administration of bombesin and its antagonist RC-3095 had a greater effect on the SS receptor–effector system than when administered separately. The present study indicates that the pancreatic SS receptor–effector system may be regulated by bombesin in vivo.

Effect of nitric oxide on the somatostatinergic system in the rat exocrine pancreas

Nitric oxide (NO) and somatostatin (SS) are two important mediators of the exocrine and endocrine pancreas, exerting opposite effects on this organ. There is strong evidence suggesting an interaction between pancreatic NO and SS. The aim of this study was to determine whether L-arginine ( L-Arg), the substrate for NO synthase (NOS), and N ω-nitro- L-arginine methyl ester ( L-NAME), a NOS inhibitor, regulate pancreatic somatostatin-like immunoreactivity (SSLI) content and the SS mechanism of action in pancreatic acinar cell membranes. L-Arg (150 mg/kg, intraperitoneally (i.p.)), L-NAME (50 mg/kg, i.p.) or L-NAME plus L-Arg were injected twice daily at 8 h intervals for 8 days. L-Arg decreased pancreatic SSLI content as well as the number of SS receptors in pancreatic acinar cell membranes whereas L-NAME increased both parameters. The stable SS analogue SMS 201-995 induced a significantly lower inhibition of forskolin-stimulated adenylyl cyclase activity in pancreatic acinar cell membranes from L-Arg-treated rats whereas an increased inhibition was observed in pancreatic acinar membranes from L-NAME-treated rats. These results indicate that the NO system may contribute to the regulation of the pancreatic somatostatinergic system.

Epidermal growth factor inhibits bombesin-induced activation of phospholipase C-beta1 in rat pancreatic acinar cells

BACKGROUND & AIMS: Epidermal growth factor (EGF) inhibits bombesin- induced activation of phosphoinositide-specific phospholipase C (PLC) in pancreatic acini. The aim of this study was to investigate the mechanism by which EGF inhibits bombesin-induced activation of PLC.METHODS: Intact pancreatic acini were pretreated with pertussis toxin to study the role of Gi/o-type heterotrimeric guanosine triphosphate- binding regulatory proteins (G proteins) in EGF-induced modulation of PLC activity. To identify the PLC isoenzyme(s) and Gi/o protein subtype(s) involved in EGF-induced signaling, PLC activity was measured in isolated pancreatic acinar membranes that had been preincubated with immunoneutralizing antibodies raised against various PLC-beta isoenzymes or G protein alpha-subunits. The association of PLC-beta1 and Gi/o-type G proteins was studied by pertussis toxin-catalyzed [32P]adenosine diphosphate ribosylation of PLC-beta1 immunoprecipitates.RESULTS: Pertussis toxin pretreatment of pancreatic acini abolished the inhibitory effect of EGF on bombesin-induced PLC activation and amylase release. Anti-PLC-beta1, -beta3, and Gq/11alpha antibodies inhibited bombesin-induced PLC activity by 50%, 35%, and 65%, respectively. Anti-Gi1-2alpha, but not a Gi3alpha-specific antibody, abolished the inhibitory effect of EGF on bombesin-induced PLC activity. Pertussis toxin-sensitive G proteins coimmunoprecipitated with PLC-beta1 in an EGF-dependent fashion.CONCLUSIONS: EGF inhibits bombesin-induced activation of PLC-beta1 by a mechanism involving activation of Gi1-2 proteins in pancreatic acinar membranes.(Gastroenterology 1997 Nov;113(5):1747-55)

The somatostatin receptor-adenylate cyclase system in rat pancreatic acinar membranes after temporary pancreaticobiliary duct ligation

The mechanism whereby somatostatin (SS) produces beneficial effects in established pancreatitis induced by pancreaticobiliary duct ligation (PBDL) is still not clear. The aim of the work was to evaluate the possibility of a direct action of SS on pancreatic acinar cells from rats with acute pancreatitis. For this purpose, we studied the SS-receptor-adenylate cyclase system in pancreatic acinar membranes from both, control rats and rats with experimentally induced acute pancreatitis. On the other hand, it has been reported that cholecystokinin (CCK) diminishes the number of SS receptors in pancreatic acinar cells. Proglumide, a CCK receptor antagonist reduces the severity of acute pancreatitis in the rat. Therefore, we have also examined the effect of proglumide on the somatostatinergic system in controls and rats with acute pancreatitis. Fourteen hours after PBDL, the SS receptors, the capacity of the SS analogue SMS 201–995 to inhibit forskolin-stimulated adenylate cyclase activity and PTX-catalyzed [ 32P] ADP-ribosylation of the α 1 subunits of Gi proteins could not be detected in pancreatic acinar membranes. One month after reopening the closed pancreaticobiliary duct (PBD), the pancreas showed regeneration of acinar cells, and the above-mentioned parameters were significantly lower than in the control group. Two months after reopening the closed PBD, all these parameters had returned to control values. The administration of proglumide (20 mg kg i.p.), a cholecystokinin receptor antagonist, accelerated pancreatic regeneration and approached all these parameters to control values one month after reopening the closed PBD. The present study suggests that the beneficial effects of SS on established pancreatitis induced by PBDL may not be due to a direct action of the peptide on pancreatic acinar cells at least at 14 hours after PBDL. In addition, these findings suggest that in established pancreatitis the effect of proglumide on the SS receptor-adenylate cyclase system could be due to its action on pancreatic regeneration.

Synthetic peptides containing a BXBXXXB(B) motif activate phospholipase C-beta1.

We have recently shown that synthetic peptides of the effector domain of the low-molecular-mass GTP-binding protein Rab3 stimulate inositol 1,4,5-trisphosphate production in various permeabilized cells. To investigate the mechanism of the peptide-induced activation of phospholipase C (PLC) and to identify the PLC isoenzyme(s) targeted by these peptides, isolated pancreatic acinar membranes and cytosol were preincubated with anti-PLC antibodies before examination of PLC activity in response to the Rab3B/D effector-domain peptide (VSTVGIDFKVKTVYRH, peptide P1). Western blot analysis revealed the presence of PLC-beta1, -beta3, -gamma1 and -delta1 in membrane and cytosolic fractions. P1 stimulated PLC activity in both membrane and cytosolic fractions. Anti-(PLC-beta1) antibody inhibited P1-induced PLC activity in both subcellular fractions almost completely. Moreover, P1-induced amylase release in digitonin-permeabilized pancreatic acini was also inhibited. Other immunoneutralizing anti-PLC antibodies had no effect, suggesting that P1 activates PLC-beta1 but not PLC-beta3, -gamma1 or -delta1. P1 also activated recombinant PLC-beta1, indicating direct activation of PLC-beta1 by Rab3 effector-domain peptides. To investigate further the structure-function relationship of the peptides, truncated peptides of P1 were tested for their ability to activate PLC in isolated pancreatic acinar membranes and to stimulate amylase release from digitonin-permeabilized pancreatic acini. Peptides containing a BXBXXXB(B) motif (where B represents a basic residue and X any residue)[KVKTVYRH (EC50 of 1 nM to stimulate amylase release) approximately TVGIDFKVKTVYRH > TVGIDFKVKTVYR] were potent stimulators of amylase release and PLC activity, whereas deletion of the C-terminus (VSTVGIDF), of the two basic C-terminal amino acid residues (VSTVGIDFKVKTVY and KVKTVY), or destruction of the BXB motif (VKTVYR) resulted in inactive peptides. In conclusion, the results of the present study show that short peptides containing a BXBXXXB motif represent promising pharmacological agents to activate the PLC-beta1 isoenzyme.

Epidermal growth factor activates phospholipase C-gamma 1 via G(i)1-2 proteins in isolated pancreatic acinar membranes

In the present study, isolated pancreatic acinar membranes were used to investigate the mechanism of epidermal growth factor (EGF)-induced activation of phospholipase C (PLC). The data show that EGF caused a rapid and strong increase in tyrosine phosphorylation of the EGF receptor, with a maximum 5-15 s after the beginning of the incubation followed by a decline. With use of [3H]phosphatidylinositol 4,5-bisphosphate as an exogenous substrate, PLC activity increased fourfold on exposure of the membranes to EGF (85 nM). In contrast, EGF-induced tyrosine phosphorylation of PLC-gamma 1 was rather small, indicating that tyrosine phosphorylation of PLC-gamma 1 is not proportional to changes in PLC activity. EGF-induced activation of PLC was strongly inhibited by pretreatment of the membranes with pertussis toxin, by an antibody raised against a COOH-terminal sequence shared by alpha-subunits of the inhibitory G proteins G(i)1 and G(i)2, and by an anti-PLC-gamma 1 antibody, whereas anti-G(i) alpha 3, anti-Gq/11 alpha, and anti-PLC-beta 1 antibodies had no effect. In contrast, pertussis toxin or the anti-G(i) alpha 1-2 antibody had no effect on EGF-induced tyrosine phosphorylation of PLC-gamma 1. EGF promoted association of G(i) proteins with both the EGF receptor and PLC-gamma 1 with similar kinetics as EGF-receptor autophosphorylation. All EGF-induced responses were abolished by the specific tyrosine kinase inhibitor pp60v-arc (137-157), suggesting that EGF-receptor tyrosine kinase activity is essential for G(i)1-2-mediated activation of PLC-gamma 1. However, there was no evidence of tyrosine phosphorylation of G(i) alpha 1-2. Taken together, these data show that EGF causes activation of PLC-gamma 1 by a mechanism requiring activation of G(i)1-2 and only a small increase in tyrosine phosphorylation of PLC-gamma 1.

5-Hydroxytryptamine decreases somatostatin receptors and somatostatin-responsive adenylyl cyclase in rat pancreatic acinar membranes

Pretreatment of pancreatic acini with 5-hydroxytryptamine (5-HT) reduced the binding of the labeled somatostatin (SS) analogue 125I-Tyr 3-SMS to pancreatic acinar membranes. This effect was dependent of the dose of 5-HT used and length of pretreatment. This inhibitory effect of 5-HT was abolished when pancreatic acini were pretreated with 5-HT in the presence of the 5-HT 1P receptor-antagonist 5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP). Pretreatment of pancreatic acini with 5-HT reduced the inhibition by the stable SS analogue SMS 201-995 of basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity in pancreatic acinar membranes. There was no statistical difference established between IC 50 values for the stable GTP analogue 5'-guanylylimidodiphosphate (Gpp(NH)p) which inhibits ligand binding to SMS receptors in controls and in 5-HT treated pancreatic cells, respectively. In addition, no significant differences were seen in the level of Gi proteins in the control and 5-HT treated panceatic acini. These data suggest that the decrease of the number of 125I-Tyr 3-SMS receptors, would explain the decreased sensitivity of AC to SMS 201-995 in membranes from 5-HT-pretreated acini.

CCK, carbachol, and bombesin activate distinct PLC-beta isoenzymes via Gq/11 in rat pancreatic acinar membranes.

Four different isoforms of phospholipase C-beta (PLC-beta 1-4) have been discovered, raising the important question of whether a distinct receptor activates a single PLC-beta isoenzyme or a subset of PLC-beta isoenzymes. The present study was designed to investigate activation of PLC-beta isoenzymes by three different PLC-activating agonists that bind to different receptor entities, i.e., cholecystokinin octapeptide (CCK-8), bombesin, and carbachol in rat pancreatic acinar membranes. PLC activity was measured using exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate. Western blot analysis of pancreatic acinar membranes revealed the presence of PLC-beta 1, -beta 3, -gamma 1, and -delta 1, but not of PLC-beta 2, -beta 4, -gamma 2, and -delta 2. Preincubation of the membranes with anti-PLC-beta 1 or -beta 3 antibody reduced agonist-induced activation of PLC. The order of sensitivity toward inhibition by anti-PLC-beta 1 antibody was CCK-8 > bombesin > carbachol. An opposite order of sensitivity was found for inhibition of PLC activity by anti-PLC-beta 3 antibody (carbachol > bombesin > CCK-8). Anti-PLC-beta 2, -beta 4, -gamma 1, -gamma 2, -delta 1, and -delta 2 antibodies had no effect. Preincubation of the membranes with an antibody raised against the COOH terminus of the alpha-subunit of Gq/11 proteins inhibited PLC activity in response to all three different receptor agonists to a similar extent, whereas anti-Gi alpha 1-2 and anti-Gi alpha 3 antibodies had no effect. In conclusion, the data of the present study indicate that CCK-8 and carbachol activate PLC-beta 1 and PLC-beta 3, respectively, whereas bombesin activates both PLC-beta 1 and PLC-beta 3. Activation of PLC-beta by these receptor agonists is mediated by Gq/11.

Octapeptide somatostatin analog SMS 201-995 induces translocation of intracellular PTP1C to membranes in MCF-7 human breast adenocarcinoma cells.

Somatostatin (SST) analogs exert direct antiproliferative actions in pancreatic, pituitary, and mammary tumor cells in vitro. SST receptor (SSTR)-mediated induction of membrane-associated protein tyrosine phosphatase (PTP) activity has been implicated in its anti-proliferative signaling by virtue of its ability to dephosphorylate and inactivate growth factor receptor kinases. Recently, a PTP-containing Src homology 2 domain, identified as PTP1C/SHPTP1/SHP/HCP, was found to be associated with SSTR in rat pancreatic acinar cell membranes. In the present study we investigated the antiproliferative action of the octapeptide SST analog SMS 201-995 (OCT) and its effect on PTP activity in MCF-7 human breast adenocarcinoma cells. We report here that OCT does not directly stimulate membrane-associated PTP activity, but induces translocation of intracellular PTP to the membrane in MCF-7 cells preincubated with the peptide in a time- and concentration-dependent manner. We demonstrate that this is due at least in part to OCT-induced recruitment of cytosolic PTP1C. OCT-induced recruitment of PTP1C to the cell surface as well as its ability to inhibit the growth of MCF-7 cells was G protein dependent and inhibited by orthovanadate. These findings suggest that translocation of cytosolic PTP1C by SST analogs to the cell surface is an early event in its antiproliferative signaling in tumor cells.

Lactational changes in the rat exocrine pancreas somatostatin receptors and modulation of guanylate cyclase

Hyperplasia of the pancreatic tissue during late lactation (third week) and lasting for at least the first two weeks after weaning has been observed by several authors. Since the tetradecapeptide somatostatin (SS) inhibits pancreatic growth and its plasma levels are elevated during these periods, the aim of the present study was to determine the possible implication of the somatostatinergic system in the pancreatic changes cited above. Thus, the present study investigated 125I-Tyr 11-somatostatin ( 125I-Tyr 11-SS) binding and the effects of SS on guanylate cyclase activity as well as pancreatic somatostatin-like immunoreactivity (SSLI) levels in pancreatic acinar membranes from control, lactating and weaning rats. SS receptors were identified using 125I-Tyr 11-SS and isolated pancreatic acinar membranes in vitro. There was an increase in the number of SS receptors after the third week of lactation (244 ± 6 vs. 155 ± 12 mol/mg protein, P < 0.01) and the first two weeks after weaning (327 ± 8 vs. 155 ± 10 mol/mg protein, P < 0.001). No change in the affinity of the receptor site was detected at either study time. In addition, SS-stimulated guanylate cyclase activity was markedly increased at the third week of lactation (119%) and at the second week after weaning (158%) when compared with the control group. In contrast, basal guanylate cyclase activity was not modified at either study period. Thus, SS-stimulated guanylate cyclase activity is increased in pancreatic acinar membranes at late lactation and at the second week after beginning weaning probably due to an increase in the number of SS receptors. Significant decreases in SSLI content were observed at the third week of lactation (69%) and the second week after weaning (37%) when compared with the respective controls. The present results suggest that pancreatic acinar cell growth observed at the third week of lactation and the second week after weaning is associated with up-regulation of SS receptors which would represent a mechanism promoted by the cell that would negatively regulate the mitogenic activity of the increased number of pancreatic growth factors observed during both periods.

Epidermal growth factor regulates adenylate cyclase activity via Gs and Gi1-2 proteins in pancreatic acinar membranes

In the present study, Western-blot and radioreceptor analyses have revealed the presence of the epidermal growth factor (EGF) receptor in pancreatic acinar membranes. Isolated pancreatic acinar membranes, which allow access of functional antibodies to individual components of the signal transduction cascade, were used to examine EGF-induced regulation of adenylate cyclase activity. Forskolin, vasoactive intestinal peptide (VIP) and to a smaller extent EGF increased cAMP production in pancreatic acinar membranes. Preincubation of the membranes with anti-GS alpha antibody abolished EGF- and VIP-induced cAMP production, but had no effect on forskolin-induced cAMP accumulation. In the presence of either VIP or forskolin, EGF inhibited the VIP- and forskolin-induced cAMP production with an IC50 of 5 nM. Anti-G alpha i1-2 protein antibody, but not anti-G alpha i3 antibody, increased basal cAMP production, indicating that Gi proteins exert an inhibitory influence on basal adenylate cyclase activity. Anti-G alpha i1-2 antibody, but not anti-G alpha i3 antibody, abolished the inhibitory effect of EGF on the forskolin- and VIP-induced cAMP accumulation. A peptide corresponding to the juxtamembrane region in the cytosolic domain of the rat EGF receptor increased cAMP production in pancreatic acinar membranes in an anti-G alpha s antibody-sensitive fashion, whereas the EGF receptor peptide did not mimic the inhibitory effect of the native EGF receptor. The tyrosine kinase inhibitors genistein and pp60v-src (137-157) inhibited both the stimulatory and the inhibitory effects of EGF on cAMP production. Thus the data of the present study show that EGF regulates adenylate cyclase via activation of Gs and Gi proteins by a tyrosine phosphorylation-dependent mechanism in pancreatic acinar membranes. This leads to stimulation of basal and inhibition of forskolin- and VIP-induced adenylate cyclase activity respectively.

Adenylate cyclase activity during exocrine pancreatic proliferation in the rat

Adenylate cyclase activity in pancreatic acinar cell membranes was determined in rats that had undergone a treatment with pentagastrin (250 μg/kg, intraperitoneal three times daily) for 1 week or that had undergone small bowel resection (90%) and were sacrified at 2 weeks, 1 month and 6 months after intervention. Both treatments are potent stimulators of pancreatic acinar cell proliferation. Adenylate cyclase activity was similar under basal conditions and after the diterpene forskolin stimulation in pancreatic acinar membranes from all groups studied. The ability of low concentrations of the stable GTP analogue, 5′-guanylylimidodiphosphate (Gpp[NH]p) to inhibit forskolin- stimulated adenylate cyclase activity was decreased in pancreatic acinar membranes from enterectomized rats at 2 weeks and 1 month after the operation and returned to control values at 6 months after enterectomy. Stimulation of adenylate cyclase by high concentration of Gpp[NH]p or by secretin (10 −8 M) was higher in both pancreatic hyperplasia conditions as compared with control animals. These findings suggest that the coupling efficiency of the G s protein to adenylate cyclase from pancreatic acinar membranes is enhanced without any alterations in the catalytic activity of the enzyme during pancreatic proliferation. In addition, it is possible that the highly regulated pancreatic acinar adenylate cyclase activity may be necessary to regulate pancreatic acinar cell proliferation.

Stimulation of inositol 1,4,5-trisphosphate production by peptides corresponding to the effector domain of different Rab3 isoforms and cross-linking of an effector domain peptide target.

Rab3 proteins are localized on secretory vesicles and appear to be involved in regulated exocytosis. We have previously shown that a modified peptide corresponding to the effector domain of the small molecular mass GTP-binding protein Rab3A, Rab3AAL, stimulates inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production and amylase release in digitonin-permeabilized pancreatic acini. Experiments using monoclonal antibodies reveal that the Rab3-like protein present in pancreatic acini is not the Rab3A isoform. However, since the putative effector domains of the four as yet known Rab3 proteins (A, B, C and D) differ only in the C-terminal four amino acid residues, Rab3A effector domain peptide could mimic the action of the pancreas-specific Rab3 isoform. In the present study we report that peptides corresponding to the different Rab3 isoforms stimulate both Ins(1,4,5)P3 production and amylase secretion with an order of potency Rab3B/D > Rab3AAL > Rab3A = Rab3C. For Rab3A, B/D and C effector domain peptides the concentrations causing half-maximal response (EC50) were 3, 0.2 and 3 nM for Ins(1,4,5)P3 accumulation and 0.3, 0.02 and 0.3 nM for amylase release, respectively. A Rab1A effector domain peptide, Rab1AAL, and a scrambled peptide of Rab3AAL were less potent by several orders of magnitude in eliciting these responses compared with native Rab3 effector domain peptides. None of the peptides influenced Ins(1,4,5)P3 production and amylase release in intact acini. Cross-linking of 125I-Rab3B/D peptide to pancreatic acinar membranes showed a band at 70 to 75 kDa with maximum intensity at 75 kDa. Radiolabelling of the substrates could be displaced by unlabelled Rab3B/D peptide, and to a lesser extend by Rab3A peptide, whereas the scrambled peptide of Rab3AAL had no effect. These data suggest that phospholipase C and exocytosis might be regulated by Rab3B-or Rab3D-like proteins in pancreatic acinar cells. A 75 kDa protein that preferentially cross-linked to 125I-Rab3B/D effector domain peptide is a potential candidate as an effector protein of Rab3 effector domain peptides.

Ethanol-induced modification of somatostatin-responsive adenylyl cyclase in rat exocrine pancreas

Male rats were given 10% (w/v) ethanol in drinking fluid during the first week, 15% (w/v) during the second week, 20% (w/v) during the third, and 25% (w/v) during the fourth week, at the end of which they were kept on 25% (w/v) ethanol drinking water for 3 weeks. Some animals were then allowed the withdrawal of ethanol for a period of 2 weeks or 7 weeks. No significant differences were seen for the basal and forskolin (FK_-stimulated adenylate cyclase (AC) enzyme activities in the pancreatic acinar membranes of ethanol-treated and ethanol withdrawal rats as compared to the control group. Chronic ethanol ingestion resulted in an attenuation of somatostatin(SS)-inhibited FK-stimulated AC in rat pancreatic acinar membranes. The ability of the stable GTP analogue 5′-guanylylimidodiphosphate (Gpp[NH]p) to inhibit FK-stimulated AC activity was also decreased in pancreatic acinar membranes from ethanol-treated rats. Gpp[NH]p was a much less potent inhibitor of SS binding in the pancreatic acinar membranes from chronic ethanol-treated animals than in those from controls, suggesting a change of G 1. A significant reduction in the number of 125I-Tyr 11-SS receptors was observed after ethanol ingestion, when compared with control values. Two weeks after the replacement of the ethanol solution by water, the ethanol effect on the parameters cited above persisted. At week 7 of withdrawal, these parameters reached the level of water controls. Ethanol administration did not affect either the number or the affinity of secretin receptors as compared to control values which suggests that the change in SS binding is not a non-specific effect. Neither chronic ethanol consumption nor withdrawal affected somatostatin-like immunoreactivity (SSLI). These results suggest that the attenuated inhibition of AC by SS in pancreatic acinar membranes from ethanol-treated rats and ethanol withdrawal (2 weeks) rats may be caused by decreases in both G i activity and in the number of SS receptors. Alternatively, an uncoupling of SS receptors from G i and/or a decrease in the level of functional G i may result in both a decrease in apparent B max for SS binding and in SS-mediated inhibition of AC. Since SS has been suggested to be an inhibitor of basal and cholecystokinin (CCK)- and/or secretin-stimulated exocrine pancreatic secretion, it is tempting to speculate that the impairment of the SS receptor/effector system seen in the present study can participate in the increase of basal pancreatic exocrine secretion described after chronic ethanol consumption.

Somatostatin receptor-effector system in rat pancreatic acinar membranes after subtotal enterectomy

In the present study we found that exocrine pancreatic hyperplasia observed after proximal small bowel resection is accompanied by an increase in pancreatic somatostatin (SS) content at 1 mo and an increase in the number of SS receptors at 2 wk and 1 mo after intestinal surgery. At 6 mo after small bowel resection SS content and SS receptors had returned to control values. However, the original increase in SS receptor number was accompanied by a decrease in the ability of SS to inhibit forskolin-stimulated adenylyl cyclase (AC) activity. In addition, the ability of 5'-guanylylimidodiphosphate (a nonhydrolyzable GTP analogue) to inhibit SS receptor binding was decreased in pancreatic acinar membranes from enterectomized rats at 2 wk and 1 mo after jejunoileal resection. These data suggest that there is an abnormality in the integrity of SS receptor binding site-G protein interactions and would explain the decreased inactivation of AC by SS at 2 wk and 1 mo after proximal small bowel resection.

Somatostatin binding capacity, guanylate cyclase and tyrosine phosphatase activities during pancreatic proliferation in the rat induced by gastrectomy

Gastrectomy increased pancreatic growth and this effect was associated with an increase in the number of somatostatin-14 (SS) receptors (146% of control) without altering their affinity. SS increased guanylate cyclase activity twofold in pancreatic acinar membranes from gastrectomized rats. The gastrectomy decreased pancreatic SS-like immunoreactivity (SS-LI) content (55% of control levels) and tyrosine phosphatase activity (74% of control levels). Administration of proglumide (20 mg/kg, IP), a gastrin/cholecystokinin (CCK) receptor antagonist, suppressed the inhibitory effect of gastrectomy on basal tyrosine phosphatase activity and SS-LI content, which returned to control levels. Furthermore, proglumide suppressed the increase of the number of SS receptors and of SS-stimulated guanylate cyclase activity induced by gastrectomy. All this suggests that pancreatic acinar cell growth is associated with upregulation of SS receptors, which could represent a mechanism promoted by the cell to negatively regulate the mitogenic activity of pancreatic growth factors such as CCK. In addition, the results also suggest that the negative regulation of tyrosine phosphatase activity may be important in the events involved in the pancreatic hyperplasia observed after gastrectomy.

The effect of pentagastrin on the somatostatin receptor/effector system in rat pancreatic acinar membranes

An intraperitoneal (i.p.) injection of pentagastrin (250 μg/kg, three times daily) for 1 week increased somatostatin like-immunoreactivity (SSLI) content in the pancreas and the number of somatostatin (SS) receptors in pancreatic acinar membranes without influencing their apparent affinity as compared with control animals. No significant differences were seen in basal or forskolin (FK)-stimulated adenylate cyclase (AC) enzyme activities in the control and pentagastrin treated rats. In spite of the increase in the number of SS receptors, SS caused a significantly lower inhibition in AC activity in these membranes. This finding is related to the fact that the stable GTP analogue, 5′-guanylylimidodiphosphate (Gpp[NH]p) was a much less potent inhibitor of binding in the pancreatic acinar cell membranes from pentagastrin-treated animals than in those from controls. In addition the ability of Gpp(NH)p to inibit FK-stimulated AC activity was also decreased in pancreatic acinar cell membranes from pentagastrin-treated rats. Pretreatment with proglumide, (20 mg/kg i.p.) a gastrin/cholecystokinin (CCK) receptor antagonist, prevented the pentagastrin-induced changes in SS level and binding as well as the inhibitory effect of SS on AC activity in pancreatic acinar cell membranse. Proglumide alone had no observable effect on the somatostatinergic system. These data suggest a SS receptor/G protein uncoupling as a result of binding of pentagastrin to gastrin receptors present in pancreatic acinar cell membranes.

Co-purification of a protein tyrosine phosphatase with activated somatostatin receptors from rat pancreatic acinar membranes.

We have previously shown that somatostatin promotes the stimulation of a membrane tyrosine phosphatase activity in pancreatic cells. To gain insight into the mechanism of somatostatin action, we purified somatostatin-receptor complexes from somatostatin 28-prelabelled rat pancreatic plasma membranes by immunoaffinity chromatography using immobilized antibodies raised against the N-terminal part of somatostatin 28, somatostatin 28 (1-14), which is not involved in receptor-binding-site recognition. After SDS gel electrophoresis a band with a molecular mass of 87 kDa was identified in the affinity-purified material as the somatostatin receptor. The 87 kDa protein was not observed when the membrane receptors were solubilized in a free unoccupied or somatostatin 14-occupied form, or when nonimmune serum replaced the anti-[somatostatin 28 (1-14)] anti-serum. Somatostatin 14 inhibited the appearance of the 87 kDa protein in the same range of concentrations that inhibit radioligand binding on pancreatic membranes. After somatostatin 28 treatment of membranes, purified somatostatin receptor preparations exhibited an elevated tyrosine phosphatase activity that dephosphorylated phosphorylated epidermal growth factor receptor and poly(Glu,Tyr). This activity was related to the presence of somatostatin receptors in purified material. It was increased by dithiothreitol and inhibited by orthovanadate. In purified material containing somatostatin receptors, anti-[Src homology 2 domains (SH2)]-containing tyrosine phosphatase SHPTP1 polyclonal antibodies identified a protein of 66 kDa which was not detected in the absence of somatostatin receptor. Furthermore, the anti-SHPTP1 antibodies immunoprecipitated specific somatostatin receptors from somatostatin-prelabelled pancreatic membranes and from untreated membranes. These results indicate that a 66 kDa tyrosine phosphatase related to SHPTP1 co-purifies with the pancreatic somatostatin receptors, and suggest that this protein is associated with somatostatin receptors at the membrane level.

Tyrphostins inhibit secretagogue-induced 1,4,5-IP3 production and amylase release in pancreatic acini.

We examined the role of protein tyrosine kinase inhibitors (tyrphostins) in secretagogue-induced inositol 1,4,5-trisphosphate (1,4,5-IP3) production and amylase secretion in rat pancreatic acinar cells. The data show that various specific cell-permeant tyrphostins (methyl 2,5-dihydroxycinnamate, tyrphostin 25, and genistein) inhibited the cholecystokinin octapeptide-, carbachol-, and bombesin-induced 1,4,5-IP3 production and amylase release. In digitonin-permeabilized cells, tyrphostins decreased 1,4,5-IP3 accumulation and amylase release generated by directly stimulating G proteins with the weakly hydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate). Tyrphostins had no effect on vasoactive intestinal peptide-induced amylase secretion. In isolated pancreatic acinar membranes, cholecystokinin octapeptide caused a rapid increase in tyrosine phosphorylation of a synthetic peptide containing the 12-amino acid sequence around a tyrosine phosphorylation site in pp6osrc. These results provide evidence that tyrosine kinases are involved in the activation of phospholipase C by G protein-coupled receptors in pancreatic acinar cells.