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.     

Regulation of intestinal concentration of cholecystokinin by bile and/or pancreatic juice

Pancreatic exocrine secretion in conscious rats is regulated by intraluminal bile and/or pancreatic juice. Exclusion of bile and/or pancreatic juice from the intestinal lumen caused cholecystokinin (CCK) release and stimulated pancreatic secretion. CCK in the plasma is mainly derived from endocrine cells in the proximal small intestinal mucosa. We examined the changes in CCK concentrations in the intestinal mucosa and compared them to those of plasma CCK concentrations and the changes of luminal trypsin activities after bile and/or pancreatic juice diversion in conscious rats. Rats with bile and pancreatic fistulae were used. Each treatment of bile, pancreatic juice, and bile-pancreatic juice diversion decreased luminal trypsin activity and increased plasma and intestinal CCK concentrations. The potency of the stimulatory effect on plasma and intestinal CCK concentrations was bilepancreatic juice diversion>pancreatic juice diversionbile diversion. Neither plasma CCK concentration nor intestinal CCK concentration was in inverse proportion to trypsin activity. The plasma CCK concentration did not parallel intestinal CCK concentration. Intravenous infusion of CCK-8 (300 pmol/kg/hr) did not increase CCK concentration in the intestinal mucosa. It was proposed that bile and/or pancreatic juice in the intestinal lumen regulated CCK concentrations not only in the plasma but also in the intestinal mucosa.     

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.     

Immunoglobulin A Secretion into Pancreatic Juice as a Novel Marker of Local Immune Defense and Exocrine Pancreatic Function

The importance of secretory immunoglobulin A (IgA) of local immune defense in the gastrointestinal tract has gained increasing acceptance. Bacterial contamination is a major factor related to mortality in acute pancreatitis. However, very little is known about IgA in pancreatic juice. Pure pancreatic juice was collected from 40 patients undergoing pancreatoduodenectomy. The patients were divided into three groups according to the degree of preoperative pancreatic duct obstruction, as follows: normal, narrowed, and obstructed. IgA concentration, amylase activity, and daily volume of pancreatic juice were measured. Daily IgA secretion into pancreatic juice was constant during the early period after the operation. The concentration of IgA in the control group was 5 ± 0.8 g/ml, and IgA daily secretion was 1.2 ± 0.2 mg/day. Pancreatic duct obstruction resulted in a marked decrease in both amylase and pancreatic juice secretion. The concentration of IgA, however, was markedly increased in the narrowed group (11.1 ± 2.4 g/ml) and the obstructed group (32.5 ± 5.4 g/ml). The concentration of amylase increased with the increase in pancreatic juice. Conversely, the concentration of IgA increased with the decrease in volume of pancreatic juice. Similarly, the increased in IgA concentrations positively correlated with the decrease in amylase activity. In conclusion, the mechanisms that modulate IgA secretion in the human pancreas are essentially different from those that modulate digestive enzyme and fluid secretion. IgA in pancreatic juice may play an important role in pathological conditions such as pancreatic duct obstruction. As such, the measurement of IgA in pancreatic juice may potentially be used as a new marker of local immune defense and exocrine pancreatic function.     

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.     

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.     

Exocrine pancreatic secretion and immunoreactive secretin release after repeated intraduodenal infusions of bile in man

The exocrine pancreatic secretion of water, bicarbonate, amylase, trypsin, chymotrypsin, and lipase and the plasma concentration of immunoreactive secretin (IRS) were studied before and after repeated intraduodenal infusions of cattle bile in man. After endoscopic cannulation of the main pancreatic duct, juice was collected in 5-min samples for 20 min. A solution of 6 g dried cattle bile in 60 ml water was then infused into the duodenum through a separate catheter attached to the outside of the duodenoscope. Juice was collected for another 20 min. After this period a solution of 6 g dried cattle bile in 40 ml water was infused into the duodenum, and juice again collected for 20 min. Blood was frequently drawn from an arm vein for estimation of plasma concentration of secretin by radioimmunoassay. Both bile infusions caused significant rises in flow rate, bicarbonate concentration and output, and IRS (p less than 0.05). Enzyme concentrations decreased significantly after intraduodenal bile infusions (p less than 0.05). Outputs of enzymes rose significantly after the first bile infusion; however, a rise after the second bile infusion was found only for amylase. Further, a significant decrease in amylase and lipase concentration was found after the second bile infusion. The findings indicate that the increase in proteolytic enzyme and lipase secretion was due to a washout phenomenon. The increase in the plasma concentration of secretin after repeated bile infusions, with a corresponding effect on flow rate and bicarbonate secretion, indicates that secretin may be the main factor responsible for the exocrine pancreatic secretion caused by intraduodenal bile infusions.     

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.     

Electrophoretic separation of proteolytic enzymes in pancreatic juice collected with the pouch or catheter method

Summary Conclusion The results of this study demonstrated that proteolytic enzymes in pancreatic juice from pigs prepared with the pouch method (PM) were nearly fully active or were fully active. When activation with enterokinase was carried out further inactivation and/or breakdown occurred for chymotrypsin C and cathodal trypsin. In addition, some inactivation and/or breakdown of proteolytic enzymes in pancreatic juice occurred during collection of pancreatic juice from PM pigs. Methods Samples of pancreatic juice were collected from growing pigs using either the PM or the catheter method (CM). An isolated pouch was prepared where the pancreatic duct enters the duodenum, and three pigs were fitted with a pancreatic pouch re-entrant cannula. Three different pigs had a catheter surgically inserted into the pancreatic duct. Pooled 8-h samples of pancreatic juice were analyzed before and after activation with enterokinase. Chymotrypsin, trypsin, and elastase activities were identified in pancreatic juice after separation by electrophoresis in 1% agarose gels at pH 8.6 usingN-acetyl-dl-phenylalanine-β-naphthyl ester (Ac-Phe?ne) as a substrate. Results This qualitative enzyme assay indicated that a considerable amount of chymotrypsin C, anodal trypsin, chymotrypsins A and B, elastase II, and cathodal trypsin were present in samples of nonactivated pancreatic juice from PM pigs. In contrast, the only active enzymes identified in pancreatic juice from CM pigs were very small amounts of chymotrypsin A and elastase II. The amounts of chymotrypsin C and cathodal trypsin were lower in activated than in nonactivated pancreatic juice from PM pigs. However, there were increases in the amounts of the other enzymes when pancreatic juice from PM pigs was activated. As expected, the activation of pancreatic juice from CM pigs resulted in the measurement of very high amounts, of all the proteolytic enzymes. The amounts of anodal trypsin, chymotrypsins A and B, and elastase II were higher in activated pancreatic juice from CM pigs than from PM pigs.     

Biliary immunoreactive trypsin in man

Small, but significant amounts of immunoreactive trypsin/trypsinogen (IRT) are excreted in bile, obtained via percutaneous transhepatic catheter in patients with complete distal bile duct obstruction, and thus uncontaminated with pancreatic juice. The major serum proteolytic enzyme inhibitors alpha 2-macroglobulin and alpha 1-antitrypsin are also present in very small amounts in bile. The bile-to-serum ratios of these inhibitors are much lower (approximately two orders of magnitude) than that for IRT. The role of these inhibitors and their relative importance in biliary proteolytic enzyme inhibition is unknown.     

Intestinal disaccharidase activity following pancreatic duct occlusion in the rat

The influence of pancreatic secretions on growth and brush-border enzyme activity, throughout the entire small intestine, was examined in the rat. Pancreatic secretions were excluded from the gut lumen by stapling the pancreatic ducts, without interruption of bile flow. The entire small intestine was studied as four segments; the duodenum and three distal segments of equal length. Weight of intestine and mucosa, and mucosal sucrase, isomaltase, lactase, and alkaline phosphatase activity were measured 10-15 days following pancreatic duct occlusion, or sham-operation. The duodenum of pancreatic duct-occluded animals exhibited significant hypertrophy. In general, specific and total disaccharidase activities were greater in duct-occluded animals than in controls throughout the intestine. The increase was more pronounced in distal than in proximal segments. The sucrase/isomaltase ratio was significantly greater in pancreatic duct-occluded animals than in controls in the two distal segments. Alkaline phosphatase activity was not affected by pancreatic duct occlusion. The greater relative increase of disaccharidase activities and sucrase/isomaltase activity ratios in the distal segments of duct-occluded animals, indicates a more important regulatory role of pancreatic enzymes in the distal small intestine. It is concluded that regulation of intestinal brush-border enzyme activity by pancreatic secretion is selective for enzyme and site as follows: disaccharidases, but not alkaline phosphatase, are regulated; the sucrase subunit of the sucrase/isomaltase complex is most sensitive to regulation, while lactase is least sensitive; and the regulatory effect on disaccharidases is greater in distal than in proximal intestine.     

Inhibition of rat basal pancreatic secretion by intraduodenal bile

The effect on basal exocrine pancreatic secretion of diversion from and reintroduction into the duodenum of bile has been studied on conscious rats provided with pancreatic, biliary and duodenal fistulae. Diversion of bile from the intestine augmented protein output by 30%. After an eight-hour diversion recirculation of bile into the duodenum reduced pancreatic protein output by 30%; volume being not significantly modified. When either bile was diverted or the main bile duct was ligated, a similar inhibition of protein secretion was observed after intraduodenal injections of 20 mM solutions of taurocholate, taurochenodeoxycholate, chenodeoxycholate, and cholate, and of synthetic mixed micelles (bile salts, lecithin). Inhibitory action of bile salts on pancreatic secretion was seen equally whether or not the bile salts were in free or conjugated form or pancreatic juice returned to the intestine. We conclude that unlike man and the dog, bile as well as pancreatic juice inhibits the basal pancreatic exocrine secretion of the rat.     

How to protect human pancreatic enzyme activities in frozen duodenal juice

We determined whether activity of pancreatic enzymes could be maintained in frozen duodenal juice by diluting the specimens or by adding nutrients or a chymotrypsin inhibitor. Human duodenal juice was obtained during cholecystokinin octapeptide IV administration. Trypsin, chymotrypsin, lipolytic, lipase, and colipase activities were measured in fresh undiluted or diluted (1:4 and 1:16 with saline and T-tube bile) duodenal juice as well as after adding CaCl2, casein, triolein, or a chymotrypsin inhibitor. Subsequently, the samples were frozen at -20 degrees C, and enzyme activities were measured at 1, 2, 3, 7, 14, 28, and 56 days. Activities of chymotrypsin and colipase did not change during freezer storage. Trypsin survival was variable in juice from different subjects. By contrast, in duodenal juice to which no nutrient or only CaCl2 had been added, 90%, 65%, and 40% (P = 0.05 vs. undiluted) of lipolytic activity was lost by 56 days in undiluted and 1:4 or 1:16 diluted duodenal juice samples, respectively. The loss of lipolytic activity was prevented (P less than 0.05) by adding casein or casein and triolein to undiluted and 1:4 diluted samples and turkey egg white to undiluted samples. The loss of lipolytic activity was strongly associated with loss of lipase activity (r = 0.97) but only weakly associated with loss of colipase activity (r = 0.49). In summary, chymotrypsin and colipase are well preserved in frozen duodenal juice and can be used to accurately assess concentrations of pancreatic enzymes after thawing frozen duodenal samples. If it is necessary to measure lipolytic activity after freezing samples, lipase can be maintained by adding casein or a chymotrypsin inhibitor to juice before freezing.     

Influence of bile flow obstruction vs bile diversion on pancreatic secretion in the conscious rat

Changes in pancreatic exocrine functions were compared between conscious rats with bile duct ligation and bile diversion from the duodenum on the first, third, fifth, and seventh postoperative days. Body weight was significantly decreased with time in both groups. Basal secretions of fluid, bicarbonate, and protein remained unchanged throughout the experimental period in bile duct ligated rats, whereas in bile diverted rats, the basal bicarbonate secretion with returning of pancreatic juice to the duodenum increased on the third postoperative day, and the basal protein output significantly increased with time. Basal secretions with returning of pancreatic juice to the duodenum in both groups were higher than that in control (bile and pancreatic juice returned to the intestine) rats. Stepwise increases in fluid and bicarbonate outputs responding to the graded doses of secretin were observed in bile duct ligated rats on the first and third postoperative days, as has been observed in bile diverted rats. However, on the fifth and seventh postoperative days, stepwise responses to graded doses of secretin were no longer observed in bile duct ligated rats. The pancreatic response to cerulein was greater in bile diverted rats than in bile duct ligated rats. Plasma CCK concentration in 7-d bile duct ligated rats (4.7 pM) was significantly higher than that in 7-d bile diverted rats (1.6 pM), although the pancreatic wet weight, protein concentration, and total content were comparable for the two groups. It was suggested that the presence of bile in the duodenum is required to maintain normal pancreatic secretion, and that the removal of bile from the intestine has quite different effects, depending on whether the bile flow is obstructed or diverted.     

Influence of bile flow obstruction vs bile diversion on pancreatic secretion in the conscious rat

Changes in pancreatic exocrine functions were compared between conscious rats with bile duct ligation and bile diversion from the duodenum on the first, third, fifth, and seventh postoperative days. Body weight was significantly decreased with time in both groups. Basal secretions of fluid, bicarbonate, and protein remained unchanged throughout the experimental period in bile duct ligated rats, whereas in bile diverted rats, the basal bicarbonate secretion with returning of pancreatic juice to the duodenum increased on the third postoperative day, and the basal protein output significantly increased with time. Basal secretions with returning of pancreatic juice to the duodenum in both groups were higher than that in control (bile and pancreatic juice returned to the intestine) rats. Stepwise increases in fluid and bicarbonate outputs responding to the graded doses of secretin were observed in bile duct ligated rats on the first and third postoperative days, as has been observed in bile diverted rats. However, on the fifth and seventh postoperative days, stepwise responses to graded doses of secretin were no longer observed in bile duct ligated rats. The pancreatic response to cerulein was greater in bile diverted rats than in bile duct ligated rats. Plasma CCK concentration in 7-d bile duct ligated rats (4.7 pM) was significantly higher than that in 7-d bile diverted rats (1.6 pM), although the pancreatic wet weight, protein concentration, and total content were comparable for the two groups. It was suggested that the presence of bile in the duodenum is required to maintain normal pancreatic secretion, and that the removal of bile from the intestine has quite different effects, depending on whether the bile flow is obstructed or diverted.     

Exocrine pancreatic function in the early period after pancreatoduodenectomy and effects of preoperative pancreatic duct obstruction

Exocrine pancreatic function in the early period after pancreatoduodenectomy was investigated. The effects of preoperative pancreatic duct obstruction on exocrine pancreatic function were also investigated. The volume of pancreatic juice and its amylase activity were investigated in 39 patients who underwent pancreatoduodenectomy (including pylorus-preserving pancreatoduodenectomy). TheN-benzoyl-l-tyrosyl-p-aminobenzoic acid (BT-PABA) test was performed on 23 of 39 patients about 40 days after pancreatoduodenectomy. The exocrine pancreatic function was inhibited three to eight days after pancreatoduodenectomy (amylase activity: 23,700±4300 IU/day), and recovered on days 9–15 (48,000±8400 IU/day) in patients with a normal main pancreatic duct. In patients with pancreatic duct obstruction, the exocrine pancreatic function was almost eliminated (amylase activity: 440±260 IU/day) and BT-PABA test results were low (45±17%). In patients with narrowed pancreatic duct, amylase secretion was significantly inhibited even in patients with a normal number of acinar cells. There was a good positive correlation (Spearman's rank correlation coefficient,rs=0.715,P<0.01) between amylase secretion and BT-PABA test. Amylase secretion more than 10,000 IU/day is essential for a normal BT-PABA test and normal digestive function. The inhibited digestive function in patients with pancreatic duct obstruction may be due to the decreased number of acinar cells and the inhibition of exocrine pancreatic function.     

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.     

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.     

Feedback regulation of basal pancreatic secretion in humans

The effect of intrajejunal infusion of pancreatic juice on basal pancreatic secretion was studied in patients who had received pancreatoduodenectomy for pancreatic, biliary, or duodenal malignancy. Pure pancreatic juice was obtained through a drainage tube inserted into the main pancreatic duct. There was little fibrosis in the pancreatic remnant and daily pancreatic juice output was more than 200 ml. After intraluminal infusion of pancreatic juice, water, protein, bicarbonate, and enzyme outputs were decreased significantly by about 30%. Intraluminal trypsin also reduced pancreatic secretion. Trypsin inhibitor (aprotinin) suppressed the significant decrease caused by autopancreatic juice or trypsin solution. We conclude that basal pancreatic secretion in humans is under negative feedback control by intestinal pancreatic juice or tryptic activity.     

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.