Hirotsugu Shinozaki

Role of CCK-A Receptor for Pancreatic Function in Mice : A Study in CCK-A Receptor Knockout Mice

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.

Elevated plasma levels of pancreastatin (PST) in patients with non-insulin-dependent diabetes mellitus (NIDDM)

Pancreastatin (PST) is known as the peptide which inhibits first phase of glucose-stimulated insulin secretion. Fasting plasma PST levels and responses of PST after oral glucose ingestion in patients with non-insulin-dependent diabetes mellitus (NIDDM) were studied with human PST-specific radioimmunoassay. Fasting plasma PST in NIDDM patients was not different from healthy controls, although a slightly higher level of PST was observed in patients treated with sulfonylurea among NIDDM patients. No significant increase in plasma PST was observed after a glucose ingestion in healthy controls. In contrast, plasma PST levels in NIDDM patients rose significantly after glucose ingestion. These results suggest a possible pathophysiological role for PST in NIDDM.

Amylase Secretion from Dispersed Human Pancreatic Acini : Neither Cholecystokinin A Nor Cholecystokinin B Receptors Mediate Amylase Secretion In Vitro

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).

Disruption of cholecystokinin (CCK)-B receptor gene did not modify bile or pancreatic secretion or pancreatic growth : A study in CCK-B receptor gene knockout mice

Pancreatic exocrine function and bile secretion were examined in cholecystokinin (CCK)-B receptor gene-targeted mice and compared among different genotypes [i.e., CCK-B receptor gene: (+/+), wild-type; (+/-), heterozygous; and (-/-), homozygous deficient]. The histology and protein concentrations in the pancreas also were examined. Amylase release from the dispersed acini was examined in vitro by using the various doses of CCK-8, carbachol, and secretin. In vivo, the bile and pancreatic juice were collected, and the concentrations of amylase and bile acid were measured in anesthetized mice. The responses to CCK (100 pmol/kg) or acetyl-beta-methylcholine (500 nmol/kg) were examined. In vitro studies showed that the maximal effective concentrations of CCK-8 (10(-l0) M), carbachol (10(-5) M), and secretin (5 x 10(-7) M) were comparable for all genotypes. Fluid, amylase, and bile acid outputs in vivo also were comparable for all genotypes. Pancreatic wet weight and protein concentrations were not significantly different, and no abnormal findings were observed on histologic examination in any genotype. These results indicated that the CCK-B receptor has no role in pancreatic growth, exocrine secretion, or bile secretion in adult mice.

High plasma cholecystokinin response following ingestion of test meal by patients with non-insulin dependent diabetes mellitus.

Postprandial responses of plasma cholecystokinin (CCK) in patients with non-insulin dependent diabetes mellitus (NIDDM) were studied with a CCK specific radioimmunoassay. After the ingestion of a liquid test meal, plasma CCK levels increased from the basal level of 9.8 +/- 1.1 pg/ml to a peak of 19.4 +/- 1.8 pg/ml at 20 min in healthy subjects (n = 10). The ingestion of a test meal in patients with NIDDM (n = 10) resulted in a significantly greater increase of plasma CCK than in healthy subjects and a significant increase of plasma CCK from a basal level of 14.2 +/- 4.4 pg/ml to a peak of 47.4 +/- 12.4 pg/ml at 10 min.

Clinical usefulness of serum phospholipase A2 determination in patients with pancreatic diseases.

A new kit for radioimmunoassay of serum phospholipase A2 (PLA2) with monoclonal antibody (S-0932, Shionogi, Osaka, Japan) was used to examine PLA2 levels in patients with various diseases. Patients with acute pancreatitis showed significantly increased serum PLA2 levels. In patients with chronic pancreatitis, significant correlations were observed between the levels of factors evaluated by the secretin test and serum PLA2 levels. In patients with pancreatic cancer, serum PLA2 levels varied with disease severity. Serum PLA2 concentrations were within the normal range in patients with other malignant tumors, diabetes mellitus, and chronic liver diseases but were increased in patients with chronic renal failure. S-Sepharose column analysis of sera showed a small peak of pro-PLA2 and a large peak of PLA2 in sera from patients with severe acute pancreatitis, but a large peak of pro-PLA2 in healthy controls and patients with other diseases. On G-100 gel filtration, high-molecular-weight PLA2 immunoreactivity was detected in sera of patients with chronic renal failure, whereas a single peak of PLA2 immunoreactivity coinciding with that of standard PLA2 was detected in sera of patients with acute pancreatitis. These results suggest that (a) measurement of serum PLA2 is clinically useful for diagnosis and monitoring of pancreatitis, (b) active PLA2 in the circulation is dominant in severe acute pancreatitis, and (c) the kidney may be the main site of PLA2 degradation or excretion.

Plasma pancreastatin responses after intrajejunal infusion of liquid meal in patients with chronic pancreatitis

The plasma concentrations of pancreastatin and cholescystokinin (CCK), exocrine pancreatic responses, and gallbladder contraction following intrajejunal ingestion of 100 kcallhr semidigested liquid meal (Clinimeal) were simultaneously studied in six controls and six patients with chronic pancreatitis. An intrajejunal infusion of Clinimeal resulted in significant rises of pancreastatin and CCK, which paralleled the pancreatic secretion and gallbladder contraction. On the other hand, an intrajejunal infusion of Clinimeal resulted in a delayed rise of pancreastatin and no rise of CCK in chronic pancreatitis. Pancreatic secretion did not increase, and gallbladder contraction was not induced in these patients. It is suggested that pancreastatin may play an important role in the regulation of intestinal phase of exocrine pancreas. The impaired pancreastatin and CCK release in chronic pancreatitis may be due to the inappropriate stimuli in the lumen, which is attributed to pancreatic exocrine dysfunction, or to disturbed physiological regulation between the pancreas and gastrointestinal tract.

Effect of somatostatin on pancreatic enzyme secretion

SummaryThe effect of somatostatin (SS) on the pancreatic enzyme secretion was studied in a perfusion system using dispersed pancreatic rat acini in vitro. In addition the effect of SS on pancreatic secretion in vivo was also studied in conscious rats for comparison. In an in vitro study, 6×10-7M SS-14 caused no significant change in amylase release when added 20 min before stimulation by 10-5M carbamylcholine (Cch), 10-6M A23187, 5×10-7M secretin and 2mM dibutyryl cyclic AMP. The addition of 6×10-7M SS-28 also caused no significant change in amylase release stimulated by 10-5M Cch. High performance liquid chromatographic examination indicated that no degradation of either SS-14 or SS-28 occurred after reaction with dispersed acini. In an in vivo study SS-14 caused marked inhibition of basal pancreatic secretion and stimulated pancreatic secretion by bile-pancreatic juice diversion. These results indicate that SS has no direct inhibitory action on rat pancreatic secretion, and that SS may inhibit the pancreatic secretion by indirect mechanisms.

Bioactivity of synthetic human cholecystokinin (CCK)-33 in vitro and in vivo

SummaryThe relative potencies of synthetic human cholecystokinin (h-CCK)-33, porcine CCK-33 (p-CCK-33) and CCK-8 were examined by measuring pancreatic secretion in the conscious rat (in vivo) and amylase release from rat pancreatic acini using a perifusion study (in vitro). The increments of protein output during an 1-hr infusion of 100 pmol/kg/hr of h-CCK-33, p-CCK-33 and CCK-8 were 27.0±2.9 mg/hr (M±SE), 19.3±2.8 and 14.0± 1.8 mg/hr, respectively. H-CCK-33 and p-CCK-33 showed significantly higher responses of protein output than CCK-8 in a same molar ratio, in vivo. In vitro, the stimulation with 10-10M h-CCK-33, p-CCK-33 and CCK-8 led to a similar biphasic amylase release in a perifusion study. Twenty-fiveμ M CR-1409, an antagonist for CCK receptor, completely inhibited the 10-10M h-CCK-33-stimulated amylase release. Although it was found that h-CCK-33 and p-CCK-33 were more potent than CCK-8 in vivo, 10-10M CCK-8, h-CCK-33 and p-CCK-33 were equipotent on rat pancreatic acini in vitro. It is suggested that the discrepancy in potencies of the large molecular form and small molecular form of CCK in vivo and in vitro may be attributed to the delay of degradation of the large molecular form of CCK in vivo.

Cholinergic independent dopaminergic regulation of motilin release in man

SummaryThis study investigated the dopaminergic control of motilin secretion in normal volunteers. Dopamine infusion caused a significant decline of plasma motilin levels, but had no effect on human pancreatic polypeptide (hpp) secretion. Dopamine antagonism with domperidone resulted in a significant elevation of motilin 10 and 20 min after the drug administration, and a significant elevation of hpp at 20 and 30 min. Atropine suppressed the basal levels of motilin and hpp but did not alter the increment of motilin levels after domperidone administration, while atropine suppressed domperidone induced secretory response of hpp.These results suggest that dopaminergic mechanisms exert a tonic inhibitory effect on motilin secretion in normal subjects.