Makoto Koide

New model of acute necrotizing pancreatitis induced by excessive doses of arginine in rats

We examined the biological and histologic characteristics of a new experimental model of acute necrotizing pancreatitis induced by excessive doses of arginine in rats. Rats were given a single intraperitoneal injection of 500 mg/100 g body weight ofl-arginine. At 12–24 hr after the arginine injection, serum levels of amylase, lipase, and anionic trypsin(ogen) reached respective peak values 2, 5, and 20 times those of control rats without arginine and returned to control levels after 24–48 hr. The contents of pancreatic protein, DNA, and digestive enzymes were markedly reduced after the arginine injection and reached their nadirs at 72 hr. After 14 days these levels were almost normal. Histologic examination revealed a number of small vesicles within acinar cells at 6 hr, which were identified as markedly swollen mitochondria by the electron microscope. Other intracellular organelles and nuclei also showed degenerative changes. At 12 hr interstitial edema appeared, and acinar cell necrosis was seen after 24 hr. The extent and severity of necrotic changes of pancreatic exocrine tissue with inflammatory cell infiltration were maximal at 72 hr. At seven days, pancreatic acinar cells began to regenerate, and pancreatic architecture appeared almost normal after 14 days. The present study has demonstrated that the administration of excessive doses of arginine induces a new, noninvasive experimental model of acute necrotizing pancreatitis.

Effect of Gymnema sylvestre, R.Br. on glucose homeostasis in rats

Effect of Gymnema sylvestre, R.Br. (G. sylvestre; GS4) on glucose homeostasis was studied in rats. In the first set of experiments, the acute effect of GS4 was examined in both non-diabetic and streptozocin (30 mg/kg)-induced mildly diabetic rats. Administration of 1 g/kg body weight of GS4 to 18-h fasted non-diabetic rats significantly attenuated the serum glucose response to oral administration of 1 g/kg glucose. The immunoreactive insulin (IRI) response in GS4-administered rats was lower, but not significantly, than that in control rats. In mildly diabetic rats, a 60 min increment in serum glucose concentrations was significantly reduced by GS4 administration. No IRI response was observed in these diabetic rats irrespective of GS4 administration. In the second set of experiments, the chronic effect of GS4 was examined in mildly diabetic rats. Two weeks after the induction of diabetes, the rats were divided into two groups that had similar impairment of glucose tolerance assessed by an oral glucose loading test. The rats were fed for 32-35 days with either a control diet or a diet supplemented with GS4. After 4 weeks, GS4 showed a tendency to reduce the serum glucose concentrations in the fed state and to improve the glucose tolerance. Gain in body weight, food intake, pancreas weight and the pancreatic contents of IRI, protein, amylase and trypsinogen were unaltered in the GS4-treated group compared with the control. These results suggest the usefulness of G. sylvestre in the treatment of certain classes of non-insulin-dependent diabetes mellitus.

Role of endogenous bile on basal and postprandial CCK release in humans

The role of intraduodenal bile in regulation of plasma cholecystokinin (CCK) levels were investigated in patients with obstructive jaundice under external bile diversion and under physiological bile flow into the duodenum by internal bile drainage. Basal plasma CCK levels determined by a specific and sensitive bioassay in patients under external bile drainage (2.2±0.2 pmol/liter; mean±se) were significantly higher than those in control subjects (1.0±0.3 pmol/liter). In control subjects, the peak CCK response (6.2±0.7 pmol/liter) to a test meal was seen at 45 min, whereas that in patients under external bile drainage, it was seen at 20 min after a test meal (17.6±3.2 pmol/liter;P<0.01 vs controls). After peak response, plasma CCK levels in controls gradually decreased, but remained significantly elevated during a 3-hr observation period. In patients under bile diversion, the test meal caused a prompt plasma CCK peak, with a transient fall followed by a continuous rise until 180 min postprandially. In six patients, external bile diversion was changed to internal biliary drainage with a stent tube within two weeks to maintain physiological bile flow into the duodenum. Internal bile drainage normalized basal (0.9±0.2 pmol/liter) as well as meal-stimulated CCK release (peak value: 5.0±0.8 pmol/liter). These results demonstrate that endogenous bile exerts tonic inhibition on basal and postprandial plasma CCK levels in humans.

Involvement of endogenous cholecystokinin in the development of acute pancreatitis induced by closed duodenal loop

Involvement of endogenous cholecystokinin (CCK) in the development of acute pancreatitis induced in rats by closed duodenal loop (CDL) was examined, and the effects of the potent and specific CCK receptor antagonist loxiglumide on this model of acute pancreatitis were evaluated. Plasma CCK bioactivity was markedly elevated 3 and 6 h after onset of acute pancreatitis. A single subcutaneous injection of 50 mgkg body wt of loxiglumide 30 min before the induction of acute pancreatitis completely eliminated the hypercholecystokinemia. Loxiglumide given 3 h after the induction of acute pancreatitis suppressed plasma CCK bioactivity, which had risen up to 30-fold over basal value (0 h) at 3 h, to nearly the basal level. Loxiglumide pretreatment, in addition, significantly prevented the rise in serum amylase and lipase activity, as well as the increase in ascitic volume. It also ameliorated histological alterations of hemorrhagic and necrotizing pancreatitis. Reduction of plasma CCK bioactivity by loxiglumide after the onset of pancreatitis slowed the rate of progression of pancreatitis. However, pancreatic wet weight and cellular infiltration were not significantly influenced by loxiglumide treatment. These observations suggest that endogenous CCK is not involved in the initiation of acute hemorrhagic and necrotizing pancreatitis induced by CDL, but is involved in the development of pancreatitis in this model.

Loxiglumide. A new proglumide analog with potent cholecystokinin antagonistic activity in the rat pancreas.

D,L-4-(3,4-dichlorobenzoylamino)-5-(N-3-methoxypropyl-pentylami no)-5- oxopentanoic acid (CR 1505; loxiglumide) is a newly developed analog of proglumide. We examined the inhibitory effects of loxiglumide on pancreatic exocrine function in the isolated pancreatic acini and the isolated perfused pancreata of rats. Loxiglumide inhibited cholecystokinin octapeptide (CCK-8)-stimulated amylase release and, similarly, binding of [125I]CCK-8 to isolated rat pancreatic acini. Loxiglumide was about 3000 times more potent than the reference substance proglumide, but was about 1000 times less potent than L-364,718, another new CCK antagonist having benzodiazepine ring, in inhibiting CCK-8-stimulated amylase release. The inhibitory effect of loxiglumide displayed competitive kinetics and was specific for CCK in that the effects of other receptor secretagogues or agents bypassing receptors were not altered. The inhibitory effect of loxiglumide was fully reversible in isolated acini. However, the pancreata perfused with 10 microM loxiglumide for 20 min did not respond to CCK-8 for more than 20 min even after the removal of loxiglumide infusion. In contrast, an immediate increase in pancreatic exocrine secretion was observed after proglumide removal. Loxiglumide appeared to be bound to the receptors on acinar cells in a slowly dissociating state. These results indicate that loxiglumide acts as a potent, competitive, and specific CCK antagonist on the exocrine pancreas and, because of its prolonged inhibitory action, may be useful as a therapeutic agent in pancreatic disease.

Oral glucose ingestion stimulates cholecystokinin release in normal subjects and patients with non-insulin-dependent diabetes mellitus

The role of glucose in the regulation of plasma cholecystokinin (CCK) level was investigated in healthy control subjects and patients with non-insulin-dependent diabetes mellitus (NIDDM). Plasma CCK concentration was determined by a specific and sensitive bioassay and by a highly sensitive and reliable double-antibody radioimmunoassay using OAL-656 as an antiserum. In control subjects, ingestion of Trelan G-75 (1,200 mOsm/L,225 mL), which is equivalent to 75 g glucose as metabolic products, caused a rapid and significant increase in plasma CCK bioactivity from 1.3 +/- 0.2 to a peak of 5.8 +/- 0.6 pmol/L and immunoreactive CCK concentration from 1.2 +/- 0.1 to 4.6 +/- 0.6 pmol/L. Ingestion of 75 g glucose in 225 mL water (33.3% solution) increased plasma CCK bioactivity to a similar degree to that observed following Trelan G-75 (peak response, 4.5 +/- 0.4 pmol/L). The same volume of 0.9% NaCl solution or water failed to increase plasma CCK concentration. A smaller dose of glucose (50 b/150 mL water) increased plasma CCK concentration, although the peak level (3.0 +/- 0.5 pmol/L) was less than that observed following 75 g glucose. In patients with NIDDM, Trelan G-75 ingestion increased CCK concentration, but the peak level was lower, albeit insignificantly, than that of normal subjects. When the maximal increment of plasma CCK above the basal value was compared between control and NIDDM subjects, the differences were statistically significant (NIDDM, 3.6 +/- 0.1 pmol/L; control, 5.0 +/- 0.4; P < .01). However, integrated CCK responses to Trelan G-75 in NIDDM (165.8 +/- 15.5 pmol/120 min) were not significantly different from those in control subjects (189.8 +/- 15.9 pmol/120 min). Peak CCK bioactivity occurred within 10 to 30 minutes of ingestion, preceding the increase in glucose and insulin. These results suggest a possible effect of CCK on insulin release in humans, and that the CCK secretory response to glucose in well-controlled diabetic patients is not significantly altered.

Potentiating effect of insulin on exocrine secretory function in isolated rat pancreatic acini

BACKGROUND/AIMS Insulin is shown to exert various regulatory effects on the exocrine pancreatic function. We investigated the direct effect of insulin on exocrine pancreatic secretion. METHODS The effects of insulin on amylase release, 125I-secretin binding and Na(+)- and K(+)-activated adenosine triphosphate phosphohydrolase (Na+,K(+)-ATPase) activity were measured using the isolated rat pancreatic acini. RESULTS Insulin potentiated the amylase release elicited by secretin plus cholecystokinin (CCK), but not by either secretin or CCK alone. The potentiating effect of insulin was dependent on the concentration and preincubation time. Insulin had no effect on 125I-secretin binding. Ouabain, a specific Na+,K(+)-ATPase inhibitor, caused a concentration-dependent inhibition of the potentiated secretion by insulin without affecting the secretory response to secretin plus CCK. In membranes prepared from acini treated with insulin, Na+,K(+)-ATPase activity was significantly increased. Similar results were obtained when acini were treated with insulin in combination with secretin plus CCK. CONCLUSIONS Insulin exerts a direct effect on pancreatic acinar cells and potentiates exocrine secretion elicited by secretin in combination with CCK, in part, by increasing Na+,K(+)-ATPase activity.

Effect of islet hormones on secretin-stimulated exocrine secretion in isolated perfused rat pancreas

To clarify the effect of islet hormones on pancreatic ductular cell function, we measured the exocrine secretion elicited by 10 pM secretin in the presence or absence of islet hormones using an isolated perfused rat pancreas model. Insulin significantly increased secretin-stimulated pancreatic juice secretion, but not protein secretion. The potentiating effect of insulin on pancreatic juice secretion was concentration-dependent, and the maximal effect was observed with 1 μM insulin. Ouabain, a specific Na+,K+-ATPase inhibitor, caused concentration-dependent inhibition of the potentiating effect of insulin without affecting secretin action. Glucagon (100 nM) significantly inhibited secretin-stimulated pancreatic juice secretion and also tended to inhibit protein secretion. A somatostatin analog, SMS 201-995 (10 nM) significantly inhibited both the pancreatic juice and protein secretion stimulated by secretin. The inhibitory effect of SMS 201-995 was concentration-dependent and was maximal at 1–10 nM. These results demonstrate that insulin potentiates the secretory response to secretin, at least partly by increasing Na+,K+-ATPase activity, whereas glucagon and somatostatin inhibit this response. Thus, pancreatic islet hormones regulate the secretory function of pancreatic ductular and centroacinar cells.

Bioassay of plasma cholecystokinin in rat and human: inhibition of protein synthesis prevents the decrease in the sensitivity and responsiveness of isolated rat pancreatic acini to CCK-8

Isolated rat pancreatic acini were most sensitive and responsive when stimulated directly with cholecystokinin octapeptide (CCK-8) without preincubation. Both the responsiveness and sensitivity of acini to CCKd decreased time dependently with prolonged preincubation. When acini were stimulated with CCK-8 following pulse labeling with radioactive leucine, old protein was discharged together with newly synthesized (labeled) protein. However, the sensitivity and responsiveness of these acini for release of labeled protein were primarily reduced with preincubation, indicating that newly synthesized protein was contributing to the time-dependent loss of sensitivity and responsiveness. Then isolated acini were treated with 300 μM cycloheximide for 2 h. This treatment prevented the decreases in the sensitivity and responsiveness of amylase release to CCK-8 stimulation and made these alterations in one series of experiments negligible. Using these acini, a sensitive and specific bioassay for the measurement of CCK in human and rat plasma was developed.

Proglumide analogues CR 1409 and CR 1392 inhibit cholecystokinin-stimulated insulin release more potently than exocrine secretion from the isolated perfused rat pancreas

The effects of proglumide-related cholecystokinin (CCK) receptor antagonists CR 1409 and CR 1392 on CCK-octapeptide (CCK-8)-stimulated immunoreactive insulin (IRI) release and exocrine secretion were examined simultaneously in the isolated perfused rat pancreas. The CR 1409, at concentrations of 10–100 nM, significantly inhibited CCK-8 (100 pM) stimulation on IRI release but failed to inhibit the stimulatory effect of CCK-8 on both pancreatic juice flow and protein secretion. Increasing concentrations of CR 1409 inhibited both CCK-%stimulated IRI release and exocrine secretion. Halfmaximal inhibition was observed with approximately 2 nM for IRI release and 1 pM for protein secretion. When a higher dose (1 nM) of CCK-8 was used, the inhibitory effect of 10 μM CR 1409 on CCK-stimulated IRI release was abolished, whereas 10 pM CR 1409 retained significant inhibitory effect. Furthermore, l μM carbachol-induced IRI release was not altered by the addition of 10 μM CR 1409. The CR 1392 also had an inhibitory effect on both CCK-stimulated IRI release and exocrine secretion. The concentration of CR 1392 that caused half-maximal inhibition of CCK-%stimulated IRI release was 300 times lower than that of exocrine secretion. In addition, 1 μM carbacholstimulated IRI release was not altered by 100 pM CR 1392. Thus, the inhibitory effects of CR 1409 and CR 1392 on IRI release were mediated through the interaction at the CCK receptor and were more potent than those on juice and protein secretion. This study suggests, therefore, that CCK receptors on B cells might be different from those on acinar cells in terms of their relative affinities for antagonists.