Inhibitory effect of somatostatin on cholecystokinin release is independent of luminal cholecystokinin-releasing factor content in conscious rats.

INTRODUCTION: Exclusion of bile-pancreatic juice from the intestine increases pancreatic secretion via cholecystokinin (CCK) release in conscious rats. Luminal CCK-releasing factor (LCRF), purified from rat intestinal secretions, is an intraluminal regulator of CCK secretion during bile-pancreatic juice diversion. AIMS: Because somatostatin is a potent inhibitor of CCK release and pancreatic secretion, the inhibitory effect of somatostatin on LCRF was examined. METHODOLOGY: Rats were prepared with bile and pancreatic cannulae and two duodenal cannulae and with an external jugular vein cannula. The experiments were conducted without anesthesia. After 1.5-hour basal collection of pancreatic juice with bile-pancreatic juice return, bile-pancreatic juice was diverted for 2 hours, during which time somatostatin (2, 10 nmol/kg/h) was infused intravenously. The rats were killed before and 1 and 2 hours after bile-pancreatic juice diversion. To examine the effect of luminal somatostatin, 50 or 200 nmol/kg/h of somatostatin was infused into the duodenum. The plasma CCK and luminal content of LCRF were measured by specific radioimmunoassays. RESULTS: Bile-pancreatic juice diversion significantly increased pancreatic secretion, plasma CCK, and LCRF levels. Intravenous infusion of somatostatin inhibited CCK release and pancreatic secretion, but not LCRF content. Luminal administration of somatostatin did not show any effect. CONCLUSION: Inhibitory effect of circulating somatostatin on CCK release and pancreatic secretion is independent of LCRF content.     

Role of cholecystokinin in the negative feedback control of pancreatic enzyme secretion in conscious rats

Using a specific radioimmunoassay for cholecystokinin (CCK) we have studied the relation between circulating CCK concentrations and the feedback regulation of pancreatic enzyme secretion in conscious rats. Recirculation of diverted bile-pancreatic juice into the duodenum or intraduodenal perfusion of trypsin during biliary-pancreatic juice diversion produced basal output of amylase and trypsin and low portal CCK levels (less than 10 pmol/L). Biliary-pancreatic juice diversion or inactivation of trypsin caused increased CCK concentrations (peak values 50-100 pmol/L) and enzyme outputs. During biliary-pancreatic juice diversion, infusion of the CCK receptor antagonist proglumide suppressed the enzyme response without altering the increase in CCK. Measurement of portal and peripheral CCK during biliary-pancreatic juice diversion yielded values of 131 +/- 37 and 32 +/- 5 pmol/L, respectively. The peripheral CCK levels corresponded to concentrations achieved during exogenous CCK-8 infusion which resulted in similar enzyme outputs. Gel chromatography of portal plasma during diversion of biliary-pancreatic juice revealed one peak of CCK corresponding to CCK-8, and a larger peak eluted between CCK-33 and CCK-8, probably representing CCK-22. Similar CCK components were found in water extracts of jejunal mucosa, whereas the acetic acid extracts mainly contained CCK-33/39. We conclude that the negative feedback regulation of pancreatic enzyme secretion in rats is mediated by the release of CCK from the intestine and that the major molecular form of CCK in plasma is probably CCK-22.     

Stimulatory effect of monitor peptide and human pancreatic secretory trypsin inhibitor on pancreatic secretion and cholecystokinin release in conscious rats

The stimulatory effects of monitor peptide (MP) that was recently purified from rat bile-pancreatic juice on cholecystokinin (CCK) release and pancreatic exocrine secretions were examined in the conscious rat. As the sequence of MP has some homology with human pancreatic secretory trypsin inhibitor (hPSTI), the effects of these two materials were compared with each other. Rats were prepared with external bile and pancreatic fistulae. Pancreatic juice diversion significantly increased pancreatic secretions, but the intraduodenal injection of MP (0.9 micrograms per rat) could further increase pancreatic secretions. The MP injection produced significantly higher plasma CCK concentrations than the injection of isotonic saline solution did. Trasylol was infused simultaneously with pancreatic juice diversion to completely eliminate residual luminal protease activities. The MP (0.9 micrograms per rat) still showed the stimulatory effect, but hPSTI did not show any stimulatory effect on pancreatic secretion. Plasma CCK concentrations produced by MP were significantly higher than those produced by hPSTI. It was concluded that MP has a strong species specificity and that MP could stimulate CCK release and pancreatic exocrine secretions, not only via inhibiting luminal protease activities but also probably with a direct effect.     

Direct,concentration-dependent inhibition by taurocholate of pancreatic exocrine secretion and CCK release in conscious rats

In conscious rats, bile inhibits pancreatic secretion. The role of luminal taurocholate (TC), a major component of rat bile, in the regulation of pancreatic secretion was studied in conscious rats with external bile and pancreatic fistulae. On the fourth postoperative day, after the basal collection of bile and pancreatic juice (PJ) returned to the duodenum, graded doses of TC (0, 0.4, 4, 40 mM) containing 10 mM CaCl₂ were infused into the duodenum instead of bile and PJ for 2 hr (1 ml/hr), with or without 1 mg/ml of porcine trypsin. Luminal trypsin activities were not affected by any dose of TC. The increases in pancreatic secretion in response to diversion of bile and PJ were progressively inhibited with increasing doses of infused TC from 0 mM to 4 mM both with and without trypsin infusion. The effects with 4 and 40 mM TC were not significantly different. Changes in plasma cholecystokinin concentrations roughly correlated with changes in protein output in rats without trypsin infusion. We concluded that TC directly inhibited pancreatic secretion independent of the luminal trypsin activity and that its inhibitory action was concentration dependent.     

Inhibitory effects of octreotide on luminal cholecystokinin-releasing factor, plasma cholecystokinin, and pancreatic secretion in conscious rats

INTRODUCTION: Luminal cholecystokinin-releasing factor (LCRF), purified from rat intestinal secretions, is an intraluminal regulator of cholecystokinin (CCK) secretion during bile and pancreatic juice diversion. AIM: Because the LCRF content was not influenced by intravenous administration of atropine or somatostatin, the inhibitory effect of a potent somatostatin analog octreotide on LCRF content, the plasma CCK level, and pancreatic secretion was examined. METHODOLOGY: Rats were prepared with bile and pancreatic cannulae and two duodenal cannulae and with an external jugular vein cannula. After 1.5-hour basal collection, bile and pancreatic juice was diverted for 2 hours, during which octreotide was infused intravenously or into the duodenal lumen. The changes in pancreatic secretion were recorded for 2 hours, and the plasma CCK level and LCRF content 2 hours after the treatment were measured. RESULTS: Bile and pancreatic juice diversion significantly increased pancreatic secretion and plasma CCK and LCRF levels. Intravenous infusion of octreotide (0.2 and 0.5 nmol/kg/hour) inhibited all parameters. Intraduodenal infusion of a lower dose of octreotide (33 nmol/kg/hour) inhibited pancreatic secretion, but did not inhibit CCK release or LCRF content. The higher doses (100 and 300 nmol/kg/hour) inhibited all parameters. CONCLUSION: Intravenous and intraduodenal administrations of octreotide inhibited CCK release and LCRF content and pancreatic secretion induced by bile and pancreatic juice diversion.     

Effect of taurocholate on CCK release and pancreatic secretion produced by two CCK-releasing peptides in conscious rats.

The role of luminal bile salts (taurocholate) in regulation of rat pancreatic secretion was examined by studies on the effects of luminal stimulants on the pancreas during infusion of various concentrations of taurocholate into the duodenum of conscious rats. Rats with external bile and pancreatic fistulae were used. For 24 h before the experiment, pancreatic juice was excluded from the intestine but bile was continuously returned to the duodenum. From the beginning of the experiment, 8-200 mM of taurocholate was infused at a rate of 1 ml/h instead of returning the bile. Pancreatic juice was collected for a 2-h period and then 2 micrograms of pancreatic secretory trypsin inhibitor-61 (PSTI-61) (= monitor peptide) or partially purified putative CCK-releasing peptide from rat intestine (intestinal CCK-RP) was injected into the duodenum (1 ml/min). Continuous infusion of taurocholate maintained a constant rate of pancreatic secretion, except at a concentration of 8 mM, which resulted in a slight increase in pancreatic secretion. Both PSTI-61 and intestinal CCK-RP significantly increased pancreatic secretions during infusion of 20 or 40 mM taurocholate, but had no significant effect during infusion of 80 or 200 mM taurocholate. Therefore, higher concentrations of taurocholate in the intestine prevented the stimulatory effects of luminal stimulants, probably by preventing the latter from reaching CCK cells.     

CCK-releasing activity of rat intestinal secretion: effect of atropine and comparison with monitor peptide

A bioassay for studying the cholecystokinin (CCK)-releasing activity of intraluminal protease-sensitive bioactive peptides was developed. In conscious rats, bile and pancreatic juice were chronically diverted from the proximal intestine to the ileum to cause chronic stimulation of CCK release and pancreatic protein secretion. CCK-releasing activity of test substances was assayed during transient inhibition of CCK release by intraduodenal sodium taurocholate (78 mumols/h). Intestinal secretion as a source of the putative trypsin-sensitive intestinal CCK-releasing peptide was obtained by rapid intestinal perfusion of isolated Thiry-Vella fistulae of jejunum in conscious rats, collected with or without atropine pretreatment. Partially purified rat pancreatic secretory trypsin inhibitor (PSTI, or "monitor peptide") was compared with ovomucoid trypsin inhibitor (OMTI) and with concentrated jejunal secretions for CCK-releasing activity and trypsin inhibitor activity. Concentrated, heat-treated jejunal secretions were the strongest stimulants of CCK release and pancreatic protein secretion in this model. OMTI had no CCK-releasing activity in this model, whereas a larger amount (approximately 5x, based on trypsin inhibitor activity) of PSTI weakly but significantly stimulated CCK release. CCK-releasing activity manifested by pancreatic protein secretion was equivalent in intestinal washes from atropine-treated and control Thiry-Vella fistula donor rats. Concentrated jejunal secretions had no trypsin inhibitory activity, indicating that the putative intestinal CCK-releasing peptide and "monitor peptide" are different substances.     

Effect of intestinal hormones on pentagastrin- and histamine-evoked gastric acid secretion and duodenal ulcerations in cats

In conscious cats with gastric fistulae and the diversion of bile and pancreatic juice to jejunum, secretin (1 unit/kg-hr), cholecystokinin (CCK) (4 units/kg-hr), and duodenal acidification (1 mEq HCl/hr) caused a marked inhibition of half maximal gastric acid response to pentagastrin (8 μg/kg-hr) but did not alter the response to histamine (160 μg/kg-hr). Secretin, CCK, and jejunal acidification prevented the formation of duodenal ulcerations in conscious cats infused with pentagastrin or histamine for 36 hours. The antiulcer action of the intestinal hormones can be attributed to their inhibitory effect on gastric acid secretion and to the stimulation of pancreatic juice and bile secretion resulting in the increased neutralization of gastric acid in the duodenum.     

Involvement of pancreatic polypeptide (PP) in luminal feedback regulation in the conscious rat

The possibility of the involvement of pancreatic polypeptide (PP) release in luminal feedback regulation in the conscious rat was examined. Pancreatic secretion in the intestinal phase in the rat is regulated by negative feedback control so that a decrease in luminal protease activities produced by a diversion of bile-pancreatic juice (BPJ) from the intestine stimulates pancreatic secretion. Plasma concentration of rat PP and the effect of exogenous infusion of rat PP on pancreatic secretions during BPJ diversion were determined. Plasma PP concentration significantly increased with BPJ diversion and peaked at 90 min after BPJ diversion began, almost paralleling changes in protein output. Exogenous PP infusion (1, 2, and 10 g/kg/hr) inhibited pancreatic protein and fluid outputs but not the bicarbonate output during BPJ diversion. PP was shown to be physiologically released in the intestinal phase of pancreatic secretion; however, the physiological role of endogenous PP remains unknown.This study was supported in part by the Uehara Memorial Foundation and a grant in aid from the Japanese Ministry of Education.     

Release of pancreatic polypeptide and its mechanism in luminal feedback regulation in the conscious rat

Exclusion of bile and pancreatic juice (BPJ) from the proximal intestine increases the release of pancreatic polypeptide (PP) from 4.4 to 14.3 pM and its increase was diminished by the intravenous infusion of atropine (100 micrograms/kg/h) in conscious rats. Neither intravenous bolus injection nor continuous infusion of cerulein did increase plasma PP concentration. It is suggested that the increase in plasma PP concentration produced by BPJ diversion is regulated by cholinergic mechanism, but not by cholecystokinin (CCK) released despite the known fact that BPJ diversion increases plasma CCK concentration.     

Isolation and bioactivity of putative cholecystokinin-releasing peptide from rat small intestinal mucosa

Pancreatic exocrine secretion in the conscious rat is regulated by proteases in the intestine secreted by the pancreas, and cholecystokinin (CCK) is known to be involved in the mechanism. The authors proposed that the release of CCK was regulated by a CCK-releasing factor secreted into the intestinal lumen from the proximal intestine. We isolated and partially purified a CCK-releasing factor from rat small intestine by gel filtration and high performance liquid chromatography. The partially purified CCK-releasing factor increased pancreatic exocrine secretions and plasma CCK concentrations in conscious rats and this activity was abolished after the incubation with trypsin. The bioactivity of the partially purified CCK-releasing factor was confirmed.     

Luminal bile regulates cholecystokinin release in conscious rats

The effects of intraluminal bile on cholecystokinin release and pancreatic exocrine secretion were studied in conscious rats. Since it has been suggested that bile acid may influence pancreatic secretion indirectly by interacting with luminal protease activities, intraduodenal protease activities were eliminated by pancreatic juice diversion accompanied with simultaneous intraduodenal infusion of aprotinin. This treatment resulted in gradual increases in pancreatic juice flow, bicarbonate and protein outputs, and an increase in plasma cholecystokinin levels, reaching plateau levels 2 hr after the start of the treatment. When endogenous bile was excluded from the intestine, the pancreatic secretion and plasma cholecystokinin concentrations further increased. The intraduodenal infusion of sodium taurocholate during bile pancreatic juice diversion inhibited cholecystokinin release, while pancreatic protein output was only transiently decreased. The results indicate that bile in the duodenum directly regulates cholecystokinin release, probably through its major components, bile salts.     

Isolation and bioactivity of putative cholecystokinin-releasing peptide from rat small intestinal mucosa.

Pancreatic exocrine secretion in the conscious rat is regulated by proteases in the intestine secreted by the pancreas, and cholecystokinin (CCK) is known to be involved in the mechanism. The authors proposed that the release of CCK was regulated by a CCK-releasing factor secreted into the intestinal lumen from the proximal intestine. We isolated and partially purified a CCK-releasing factor from rat small intestine by gel filtration and high performance liquid chromatography. The partially purified CCK-releasing factor increased pancreatic exocrine secretions and plasma CCK concentrations in conscious rats and this activity was abolished after the incubation with trypsin. The bioactivity of the partially purified CCK-releasing factor was confirmed.     

Effect of intraluminal bile on the feedback regulatory mechanism of pancreatic enzyme secretion in conscious rats

The effect of intraluminal bile on the well-known feedback regulatory mechanism of exocrine pancreatic secretion exerted by intraluminal trypsin was investigated in conscious rats with pancreatic, biliary and duodenal fistulae. The stimulated pancreatic enzyme secretion caused by diversion of bile-pancreatic juice from the intestine was apparently suppressed by intraduodenal reintroduction of pancreatic juice or bile-pancreatic juice, while it was slightly suppressed by intraduodenal reintroduction of bile. Although additional reintroduction of bile did not alter the already suppressed pancreatic enzyme secretion by the presence of pancreatic juice in the intestine, diversion of bile stimulated the suppressed pancreatic enzyme secretion by intraluminal bile-pancreatic juice. Infusion of sodium taurocholate into the duodenum with diversion of bile-pancreatic juice effectively inhibited pancreatic enzyme secretion. The inhibitory effect seemed to be dependent on the concentration of taurocholate infused into the duodenum. The results suggest that bile and bile acid have an important role in the feedback regulatory mechanism of pancreatic enzyme secretion, at least partly directly inhibiting the secretion.     

Role of gastric juice in feedback regulation of rat pancreatic secretion by luminal proteases

The role of gastric juice in the intestine on the pancreatic secretory response to intraduodenal infusion of trypsin inhibitors or to diversion of bile and pancreatic juice from the intestine was studied in conscious rats with pylorus ligation and gastric juice drainage. In absence of gastric juice in the intestine, diversion of bile and pancreatic juice from the intestine stimulated pancreatic secretion, but the incremental protein and fluid secretory responses to diversion of bile and pancreatic juice were increased approximately 2.9-fold and 2.5-fold, respectively, by intraduodenal infusion of HCl (60 microEq/h). Intraduodenal infusion of HCl (240 microEq/h) had no effect on the pancreatic secretory response to infusion of lima bean trypsin inhibitor (20 mg). These results support the hypothesis that the inhibitory effect of atropine on the pancreatic secretory response to diversion of pancreatic juice or bile and pancreatic juice is secondary to inhibition of gastric acid secretion. The lack of effect of HCl on the pancreatic response to trypsin inhibitor contradicts the hypothesis that acid in the intestine is important or necessary for the feedback response to loss of intraluminal protease activity. It is proposed that acid in the intestine augments the pancreatic response to diversion of pancreatic juice or bile and pancreatic juice by reducing intraluminal pH and thereby inactivating residual pancreatic proteases.     

Importance of bile in regulation of intraluminal proteolytic enzyme activities in the rat

The effects of biliary diversion on pancreatic enzyme activities of intestinal contents was studied in conscious rats prepared with biliary and pancreatic fistulae. Diversion of bile from the intestine for 1 day caused on 80% decrease in trypsin and chymotrypsin activities of intestinal contents, in spite of increased (230%) pancreatic trypsin and chymotrypsin secretion. Bile diversion in fed rats caused a smaller decrease (58%) in trypsin and chymotrypsin activities of intestinal contents. Sodium taurocholate (100 mumol/hr intraduodenally) partially reversed the changes in pancreatic secretion and intestinal contents' activities of trypsin and chymotrypsin caused by bile diversion. The results indicated that bile was important in controlling the rate of disappearance of trypsin and chymotrypsin activities from the small intestine. The mechanism for this was studied by comparing the rate of disappearance of trypsin activity in vivo and in vitro. Bovine trypsin, with or without sodium taurocholate, was infused intraduodenally into conscious rats deprived of bile-pancreatic juice and the recovery of trypsin activity from the small intestine determined. Taurocholate increased recovery of trypsin from the small intestine more than threefold, but inactivation of bovine trypsin in vitro was not retarded by sodium taurocholate. The results indicate that bile in the small intestine controls the rate of disappearance of intraluminal trypsin and chymotrypsin activities, probably by inhibiting their autodigestion in vivo. We previously reported that bile duct ligation in rats caused decreased trypsin and chymotrypsin activities in the small intestine, but increased pancreatic enzyme secretion. We concluded that trypsin and chymotrypsin underwent accelerated inactivation in the small intestine in the absence of bile. The present study was designed to explore the mechanism for the effects of bile deprivation on intraluminal proteolytic enzyme activities in the rat.     

Effect of bile duct obstruction on pancreatic enzyme secretion and intestinal proteolytic enzyme activity in the rat

The role of bile in regulation of intestinal proteolytic activity in rats was investigated by studying the effects of bile diversion and bile duct obstruction on pancreatic protease secretion and on recovery of protease from the intestine. Diversion of bile and pancreatic juice from the intestine caused a large increase in pancreatic enzyme secretion; replacement of bile partially suppressed this response. Bile duct obstruction resulted (3–4 days postobstruction) in a threefold increase in pancreatic juice chymotrypsin but caused large decreases inintestinal trypsin and chymotrypsin activities and total proteolytic activity. Recovery of pancreatic juice protein (labeled with¹⁴C) from intestinal contents was markedly decreased in bile duct obstruction, indicating a more rapid rate of degradation and absorption of pancreatic juice protein. The evidence suggests that interruption of bile flow results in an accelerated rate of degradation of pancreatic proteolytic enzymes, and that the increase in pancreatic enzyme secretion is an adaptation to decreased intestinal proteolytic activity.     

Effects of secretin and caerulein on luminal feedback regulation of pancreatic enzyme secretion in rats

The stimulatory pancreatic response to exclusion of pancreatic proteases from the intestine was compared with the response to stepwise increasing doses of secretin and caerulein in conscious rats. Secretin stimulated pancreatic fluid secretion in a dose-related manner with or without intraduodenal return of pancreatic juice, while it could not significantly affect enzyme secretion. The dose response curve for enzyme secretion to caerulein was smooth during return of the juice. However, the already increased enzyme secretion by pancreatic juice diversion was only stimulated with the smallest dose of caerulein. The maximal dose of caerulein for enzyme secretion during return had been supramaximal dose during diversion. Intraduodenal trypsin inhibitor failed to stimulate enzyme secretion during diversion but induced the same stimulatory effect as the submaximal dose of caerulein during return. Different doses of intraduodenal trypsin caused an almost dose-related inhibition. It is concluded that a submaximal level of endogenous CCK might participate in the feedback regulation of pancreatic enzyme secretion in rats.     

Regulation of plasma cholecystokinin levels by bile and bile acids in the rat

To determine whether intraduodenal bile acids inhibit pancreatic secretion and cholecystokinin (CCK) release independent of pancreatic proteases, experiments were conducted in rats with bile and pancreatic juice chronically diverted to the ileum. Diversion of bile and pancreatic juice increased plasma CCK concentration to 19.1 +/- 4.0 pmol/L. Intraduodenal sodium taurocholate (78 mumol/h) reduced plasma CCK concentration to 6.6 +/- 1.5 pmol/L after 1 hour, but values increased to 17.3 +/- 2.3 pmol/L after 13.5 hours despite continued taurocholate infusion. Pancreatic protein secretion was also significantly but transiently inhibited by taurocholate. However, neither acute nor chronic intraduodenal bile infusion significantly reduced plasma CCK concentration compared with sodium bicarbonate infusion (13.4 +/- 1.9 pmol/L vs. 15.0 +/- 1.7 pmol/L, respectively). Chronic (13.5 hours) intraduodenal infusion of taurocholate plus pancreatic juice caused a sustained reduction of plasma CCK level to 3.1 +/- 0.5 pmol/L, which significantly increased to 9.4 +/- 1.1 pmol/L after cessation of taurocholate but with continued infusion of pancreatic juice. The results indicate that bile does not inhibit CCK release and that bile acids do not physiologically inhibit pancreatic secretion or CCK release independent of the presence of pancreatic proteases.     

A difference in stimulatory effects on pancreatic exocrine secretion between ursodeoxycholate and trypsin inhibitor in the rat

We previously reported that intraduodenally infused ursodeoxycholate produced hypersecretion of pancreas in bicarbonate and fluid secretion in the rabbit (Digestive Diseases and Sciences, 28942, 1983). Since trypsin inhibitor stimulates pancreatic secretion in the rat whose pancreatic exocrine secretion is regulated by a luminal feedback mechanism, in the present study wer examined the stimulatory effect of ursodeoxycholate in comparison to Trasylol in unanesthetized rats with both the presence and the absence of returning bile-pancreatic juice. Under the condition in which bile-pancreatic juice were continuously returned to the intestine, the intraduodenally infused ursodeoxycholate produced significant increases in juice flow and bicarbonate and protein outputs, while Trasylol significantly increased protein output only. After an 8-to 10-hr period of bile-pancreatic juice diversion, Trasylol no longer affected pancreatic secretion, whereas ursodeoxycholate still stimulated the bicarbonate output significantly. Trypsin activities in the proximal half of the small intestine were not decreased by the infusion of UDCA. The mechanism of stimulatory effect of ursodeoxycholate on pancreatic secretion is independent of luminal feedback regulation and appears to differ from that of trypsin inhibitor.A part of this study was supported by Grants in Aid from the Agency of Science and Technology of Japan.