Bombesin induces a reduction of somatostatin inhibition of adenylyl cyclase activity, Gi 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 microg/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-Tpi6,Leu13psi(CH2NH)Leu14]bombesin (6-14) (RC-3095) (10 microg/kg i.p.), 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.

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 alpha1 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.

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 Gi. A significant reduction in the number of 125I-Tyr11-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 Gi activity and in the number of SS receptors. Alternatively, an uncoupling of SS receptors from Gi and/or a decrease in the level of functional Gi may result in both a decrease in apparent Bmax 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.

Pancreatic changes in somatostatin content and receptor/effector system after intrapancreatic injection of 5,7-dihydroxytryptamine.

To date, it is unknown whether intrapancreatic serotonergic nerves can influence pancreatic somatostatin (SS) content and the SS receptor/effector system in the exocrine pancreas. In this study, the intrapancreatic serotonergic nerves were chemically ablated by injecting a specific serotonin (5-HT) neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), into the substance of the gland. Three days after the injection, the 5-HT-like immunoreactive levels in the pancreas were reduced by more than 85% whereas somatostatin-like immunoreactive levels had increased (86%). The number of SS receptors in the pancreatic acinar cell membranes of the 5,7-DHT-treated rats was also increased (72%). No significant differences were seen in basal or forskolin-stimulated adenylate cyclase (AC) enzyme activities in the control and the 5,7-DHT-treated groups. In spite of the increase in the number of SS receptors in the pancreatic acinar cell membranes of 5,7-DHT-treated rats, SS caused a significantly lower inhibition of AC activity in these membranes. This finding is related to the observed decrease of a 41 kD pertussis toxin-sensitive substrate, presumably the alpha i subunit of the guanine nucleotide inhibitory protein, in pancreatic acinar cell membranes 3 days after intrapancreatic 5,7-DHT administration when compared with the corresponding controls. The functions of pancreatic serotonergic nerves seem to be associated with enteropancreatic communication. These data together with the present results suggest that pancreatic SS content and the SS receptor/effector system in the exocrine pancreas may be regulated by enteropancreatic serotonergic nerve fibers and may participate in enteropancreatic reflexes.

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.

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 micrograms/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 Gs 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.

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

An intraperitoneal (i.p.) injection of pentagastrin (250 micrograms/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 inhibit 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 membranes. 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.