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Information concerning the cellular localization of cholecystokinin (CCK)-1 receptors has been discrepant and remained scanty at ultrastructural levels. The present immunohistochemical study at light and electron microscopic levels revealed the distinct localization of CCK1 receptors in visceral organs. Immunohistochemistry by use of a purified antibody against mouse CCK1 receptor was applied to fixed tissue sections of the pancreas, gallbladder, stomach, and intestine of mice. A silver-intensified immunogold method revealed the subcellular localization under electron microscope. The immunoreactivity for CCK1 receptors was selectively found in the basolateral membrane of pancreatic acinar cells and gastric chief cells but was absent in pancreatic islets and gastric D cells. Another intense expression in the gut was seen in the myenteric nerve plexus of the antro-duodenal region and some populations of c-Kit-expressing pacemaker cells in the duodenal musculature. The gallbladder contained smooth muscle fibers with an intense immunoreactivity of CCK1 receptors on cell surfaces. The restricted localization of CCK1 receptors on the basolateral membrane of pancreatic acinar cells and gastric chief cells, along with their absence in the islets of Langerhans and gastric D cells, provides definitive information concerning the regulatory mechanism by circulating CCK. Especially, the subcellular localization in the acinar cells completes the investigation for the detection of circulating CCK by the basolateral membrane.

Objectives: Pancreatic functions were determined in a Ki-ras-induced actin-interacting protein (KRAP)-deficient (-/-) mouse mutant., Methods: Pancreatic enzyme, protein, and DNA contents were measured, and histological examinations were conducted. The mixture of bile-pancreatic juice was collected, and amylase and bile acid outputs were determined. Oral glucose tolerance test was determined. Moreover, the gene expression of KRAP was determined in cholecystokinin (CCK)-A(1) receptor (-/-) mice., Results: The body weight was smaller, and the ratio of pancreatic wet weight/body weight was higher in KRAP(-/-) mice compared with wild-type mice. The enzyme contents, but not DNA content, in the pancreas of KRAP(-/-) mice were higher than those of wild-type mice. Histological examination revealed the increase in the number of zymogen granules in the pancreatic acinar cells of KRAP(-/-) mice. Amylase secretions in response to CCK-octapeptide sulfate were significantly higher in KRAP(-/-) than wild-type mice, whereas the basal secretion did not differ between the 2 genotypes. A normal glucose tolerance was observed in KRAP(-/-) mice. The gene expression of KRAP in CCK-A(1) receptor (-/-) mice was significantly lower than in wild-type mice., Conclusions: The lack and/or decrease in KRAP level in the pancreas may promote the pancreatic growth and hypertrophy.

Background: Alcoholic beverages stimulate gastric acid secretion and increase the appetite. Although ingested ethanol stimulates pancreatic secretion, alcoholic beverages contain several congeners. N-methyltyramine (NMT) was isolated from beer as a factor in stimulating gastric acid secretion. In this study, we examined NMT to determine whether the congener stimulated pancreatic secretion in conscious rats., Methods: Cannulae were inserted into male Wistar rats to separately drain bile and pancreatic secretions: 2 duodenal cannulae, a gastric cannula, and an external jugular vein cannula. The rats were placed in modified Bollman-type restraint cages. After a 4-day recovery period, experiments were conducted on unanesthetized rats. Different concentrations of NMT (5, 25, and 50 microg/kg) solutions were infused into the stomach. To examine the mechanism, the effects of the proton pump inhibitor, cholecystokinin (CCK-BR) antagonist (YM022), CCK-AR antagonist (CR1505), and atropine were administered prior to the NMT (25 microg/kg) infusion. The effect of intravenous infusion of NMT (7.5 microg/kg) was then determined. Moreover, dispersed acini were prepared, and the effect of different concentrations of NMT on amylase release was determined., Results: Intragastric administration of NMT significantly increased pancreatic exocrine secretion in a dose-dependent manner. Atropine eliminated the stimulatory effect of NMT, but the infusion of the proton pump inhibitor, YM022, and CR1505 did not. Intravenous infusion of NMT did not affect pancreatic secretion, and NMT did not stimulate amylase release in vitro., Conclusions: N-methyltyramine stimulates pancreatic secretion via the cholinergic gastro-pancreatic reflex. The NMT content in beer was 2 mg/l, so that if a person weighing 60 kg consumes a 750 ml of beer, 25 microg/kg NMT will be ingested. Therefore, the stimulatory effect of beer on pancreatic secretion was produced not only by ethanol but also by the congener, NMT.

Objectives: Although many studies have investigated the acute effect of ethanol on the rat pancreas, the effect of intravenous injection of ethanol on pancreatic secretion has been controversial. The purposes of this study were to examine whether intraduodenal and intravenous administration of ethanol stimulates pancreatic secretion in Wistar rats. To elucidate the mechanism, we examined whether ethanol influences pancreatic secretion in CCK-A receptor-deficient rats., Methods: Rats were prepared with cannulae for draining bile and pancreatic juice separately. The experiments were conducted in unanesthetized rats. Different concentrations of ethanol solutions were infused at the rate of 1.5 mL/h into the duodenum, and half concentrations of the respective ethanol solutions were prepared and infused into the duodenum at a rate of 3 mL/h. Then we determined the pancreatic responses to simultaneous administration of a liquid meal with ethanol. Moreover, the effect of intravenous infusion of ethanol on pancreatic secretion was examined. Finally, the effect of intraduodenal and intravenous infusion of ethanol was determined in CCK-A receptor-deficient rats., Results: Intraduodenal infusion of ethanol significantly increased pancreatic secretion, and these responses depended on the amount of ethanol instilled into the lumen, except for 3% ethanol solution, which did not affect pancreatic secretion. Simultaneous administration of a liquid meal with ethanol augmented pancreatic response in rats treated with the higher 2 ethanol concentrations (33% and 66%). Intravenous administration of ethanol slightly but significantly increased pancreatic secretion. However, no stimulation was observed in CCK-A receptor-deficient rats regardless of ethanol administration route., Conclusions: Both intraduodenal and intravenous administration of ethanol stimulated pancreatic secretion, and this stimulation required CCK-A receptor.

Ghrelin, a novel growth-hormone releasing peptide, was originally isolated from rat and human stomach. Immunohistochemical analyses revealed that ghrelin-immunoreactive neurons were localized in the hypothalamic arcuate nucleus. The function of the digestive organs is controlled from the central nervous system, and the vagus nerve plays an important role. Intracerebroventricular and intravenous administration of ghrelin significantly increased gastric acid secretion, and its effect was abolished by vagotomy. In the present study, the effect of centrally injected ghrelin on pancreatic exocrine secretion was examined in conscious rats. Moreover, an electrophysiologic study was conducted in anesthetized rats to examine whether the excitation of vagal efferent nerve could be induced by ghrelin. To determine the pancreatic exocrine secretion, rats were prepared with cannulae draining bile and pancreatic juice separately. The experiments were conducted in conscious rats on day 4 or 5 after the operation. To exclude the involvement of gastric acid, a proton pump inhibitor omeprazole (5 micromol/kg) was administered into the duodenum 1 h before ghrelin injection. An intracerebroventricular administration of ghrelin (12, 60, and 300 pmol/10 microl) significantly increased pancreatic fluid and protein output in a dose-dependent manner. Pretreatment with the ganglion blocker hexamethonium and with atropine completely abolished the stimulatory effect of central ghrelin. In contrast, an intravenous injection of ghrelin (300 pmol/10 microl) had no effect. Centrally administered ghrelin stimulated the vagal efferent nerve in anesthetized rats. In conclusion, centrally administered ghrelin stimulates pancreatic exocrine secretion through the vagal efferent nerve, and the stimulatory action is independent of gastric acid secretion.

Introduction: Digestive organs are controlled from the central nervous system, and the vagus nerve plays an important role. Orexins are recently purified neuropeptides localized in neurons within the lateral hypothalamus., Aim: To examine the effects of centrally injected Orexin-A and B on pancreatic exocrine secretion in conscious rats., Methodology: Rats were prepared with cannulae draining bile and pancreatic juice separately. The experiments were conducted without anesthesia on day 4 or 5 after the operation., Results: Intracerebroventricular administration of Orexin-A (0.25, 0.5, and 1.0 nmol) significantly increased pancreatic fluid and protein output in a dose-dependent manner. A significant stimulatory effect of Orexin-B was not observed. Pretreatment with the ganglion blocker hexamethonium and with atropine completely abolished the stimulatory effect of central Orexin-A. Central Orexin-A significantly increased pancreatic secretion after pretreatment with omeprazole. Intravenous injection of Orexin-A had no effect. Centrally administered Orexin-A stimulated the vagal efferent nerve in anesthetized rats., Conclusions: Centrally administered Orexin-A stimulates pancreatic exocrine secretion through the vagal efferent nerve, and the stimulatory action is independent of gastric acid secretion.

Introduction: In humans, cholecystokinin (CCK) stimulates pancreatic secretion, and CCK-A receptor antagonists prevent it in vivo. However, the human pancreas has been reported to express mainly CCK-B receptors., Aim: To elucidate this discrepancy., Methodology: We prepared dispersed acini from human pancreas and examined whether various doses of CCK stimulated the release of amylase, in comparison with the effects of neuromedin C, carbamylcholine, and secretin., Results: Human pancreatic acini did not respond to any dose of CCK or secretin. Amylase release was stimulated by carbamylcholine and neuromedin C dose-dependently and was inhibited by respective antagonists. The localizations of CCK receptors in the human duodenum were determined. High concentrations of CCK-A receptors were detected in the mucosa as well as in smooth muscle of the duodenum by microautoradiography., Conclusion: In conclusion, human pancreatic acinar cells are responsible for carbamylcholine and neuromedin C but not for secretin. Neither CCK-A nor CCK-B receptor mediates amylase release from human pancreatic acini in vitro. Pancreatic secretion in humans in vivo may be regulated indirectly by CCK (via CCK-A receptors).

The synthetic trypsin inhibitor camostat has been used for the treatment of acute and chronic pancreatitis in Japan based on the evidences obtained from a rat experimental model. However, rats differ from other rodents and from humans in terms of lacking a gallbladder and no response of pancreatic bicarbonate secretion to cholecystokinin (CCK). In the present study, we determined whether oral administration of camostat showed a trophic effect in mice as observed in rats and whether the trophic effect, if substantial, was mediated via the CCK-A receptor, using CCK-A receptor gene targeting mice. The chow containing 0.1% camostat was fed to 8-month-old mice. Three- and seven-day treatments with camostat did not affect pancreatic wet weight in CCK-A receptor (+/-) mice. After 14-day treatment, the ratio of pancreatic wet weight/body weight was significantly lower in CCK-A receptor (-/-) than (+/+) mice. The protein and chymotrypsin contents were lower and amylase content was higher in CCK-A receptor (-/-) mice, compared to (+/+) mice. No pathological findings were observed by histological examination. Camostat has a trophic effect on the pancreas in mice and this effect is mediated via the CCK-A receptor, but is less potent than in 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.

Introduction: The cholecystokinin (CCK) family of peptides and receptors is present throughout the brain and gastrointestinal tract. The CCK receptors can be pharmacologically subdivided into two subtypes: CCK-A and CCK-B. CCK-A receptor is enriched in the pancreas of mice., Aims: To determine pancreatic functions in a CCK-A receptor deficient mouse mutant generated by gene targeting in embryonic stem cells. The targeting vector contained lacZ and neo insertions in exon 2., Methodology: To examine exocrine functions, amylase release from the dispersed acini in vitro was examined. In the in vivo study, the mixture of bile-pancreatic juice was collected, and amylase, bicarbonate, and bile acid outputs were determined after the administration of various stimulants. The cystic duct of the gallbladder and the pylorus were ligated to exclude the involvement of gallbladder contraction and gastric acid. Pancreatic enzyme content was measured, and histologic examinations by HE and lacZ staining were conducted. To examine endocrine functions, oral glucose tolerance test (2 g/kg) was determined., Results: The body weight, pancreatic wet weight, and enzyme content in the pancreas were similar among the three genotypes. Amylase release in vivo and in vitro and bicarbonate secretion in vivo were not stimulated by CCK-8 in CCK-AR (-/-) mice, whereas the responses to other stimulants were substantial in (-/-) mice. Administration of secretin did not increase bicarbonate secretion regardless of genotype. A normal glucose tolerance was observed in (-/-) mice. Acinar cells, islets, and duct cells were stained by lacZ, and HE staining revealed no pathologic findings., Conclusion: The CCK-A receptor is important for pancreatic exocrine secretion, but not essential for maintaining glucose concentration and pancreatic growth in mice.

Expressions of the cholecystokinin (CCK)-A and -B receptor genes in human duodenum, pancreas and gallbladder were examined by Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) followed by Southern blot hybridization. The autoradiographic study of CCK-A and -B receptors in the human duodenum and pancreas was examined in vitro. To determine the subtypes to CCK receptors in the pancreas or duodenum, we studied the abilities of CCK-A and -B receptor agonists (CCK-8 and gastrin) and antagonists (loxiglumide, L-364,718 and L-365,260) to inhibit binding of [sup 125] I-CCK-8. CCK-A receptor mRNA was not expressed in the human pancreas, but was expressed in the gallbladder and duodenum, although it was expressed in the pancreas by RT-PCR. CCK-B receptor mRNA was expressed in the pancreas, but not in gallbladder and duodenum. Using autoradiography, high concentrations of CCK-A receptors were detected in the duodenal mucosa, although in the pancreas only CCK-B receptors were detected by this method. These results suggest that localization of CCK-A receptor in human duodenum provides a biochemical and morphological basis for some physiological functions of CCK. [ABSTRACT FROM AUTHOR]