Atsushi Ohki

Effect of synthetic protease inhibitor camostate on pancreatic exocrine function in rats

Pancreatic exocrine function in rats given synthetic protease inhibitor camostate (200 mg/kg body weight) perorally once daily for 10 days was investigated, Pancreatic wet weight was significantly increased in the camostate-treated rats. The increase in pancreatic weight was associated with pronounced hypertrophy and moderate hyperplasia. Total amylase, trypsin, and lipase contents in the pancreas were also increased in the camostate-treated group compared with the control rats. Secretory patterns of pancreatic juice and amylase in response to caerulein were similar in both groups, whereas the dose-response curve for pancreatic juice secretion in the camostate-treated rats was shifted tenfold toward higher concentrations of caerulein. Basal and caerulein-stimulated flow rates of pancreatic juice were significantly greater in the camostate-treated rats than the control rats, although both groups showed a threefold increase over basal secretion in response to maximal stimulation. Amylase outputs in basal state and in response to submaximal doses of caerulein were significantly lower, whereas those to maximal and supramaximal doses were significantly greater in the camostate-treated animals than that in the control rats. These results indicate that treatment with camostate induces pancreatic hypertrophy and hyperplasia, and that the secretory function of the hypertrophied pancreas is quantitatively but not qualitatively altered.

Intrahepatic cholangiocarcinoma with increased serum CYFRA 21-1 level.

Abstract: CYFRA 21-1 is a fragment of cytokeratin 19 (CK 19). Four patients with large intrahepatic (or peripheral) cholangiocarcinoma (CC) and high serum levels of CYFRA 21-1 (normal, ≤2 ng/ml) are reported. CYFRA 21-1 levels exceeded 9 ng/ml in all 4 patients. Carcinoembryonic antigen (CEA), was high in 1 (CEA; normal range, ≤5.0 ng/ml) and carbohydrate antigen 19-9 (CA 19-9) was high in 3 (CA19-9; normal range, ≤36 U/ml). We also measured serum levels of CYFRA 21-1 in 13 patients with hepatocellular carcinoma (HCC) more than 5 cm in diameter. Levels of CYFRA 21-1 exceeded 2 ng/ml in 9 of the HCC patients and were higher than 9 ng/ml in 2 of the HCC patients. Levels of alpha fetoprotein (AFP) and/or protein induced by vitamin K absence or antagonist II (PIVKA II) were elevated in all HCC patients (AFP, PIVKA II, respectively; normal range, ≤10.0 ng/ml and ≤0.1 AU/ml) CYFRA 21-1 levels were measured twice or three times during the clinical course in 2 CC patients and in 6 HCC patients, and increased gradually with tumor growth in the 2 CC patients and in 3 of the 6 HCC patients. Marked increases in serum CYFRA 21-1 levels in patients with large liver cancers, particularly in those with normal levels of AFP and PIVKA II, would suggest the existence of intrahepatic CC rather than HCC.

Relationship between the severity of diabetes mellitus and pancreatic exocrine dysfunction in rats

The relationship between the severity of diabetes mellitus and pancreatic exocrine function was investigated in rats made diabetic by injecting 3 different doses of streptozotocin (30, 45 or 60 mg/kg body weight). The expected correlation was obtained between the dose of streptozotocin and degree of elevation of blood glucose and decrease in pancreatic insulin content. Pancreatic amylase content of the diabetic rats was less than that of control rats and was in parallel with less values in pancreatic insulin content. On the other hand, trypsinogen content of diabetic rats was greater than that of control. Basal and caerulein-stimulated flow rates of pancreatic juice and protein output were similar in the control and in all 3 groups of diabetic rats. In contrast, there was a graded response of amylase and trypsinogen, depending upon the content of each enzyme in the pancreas. Both basal and caerulein-stimulated amylase outputs from diabetic rat pancreas were significantly reduced in parallel with the severity of diabetic state, but were similar to those from the control rats when related to the total pancreatic content. The present findings indicate that pancreatic exocrine dysfunction in diabetes mellitus is closely related to the severity of the disease, but the secretory dynamics in the perfused pancreas are not altered.

Effect of diabetes mellitus on pancreatic exocrine secretion from isolated perfused pancreas in rats

We studied pancreatic exocrine function in response to cerulein and carbamylcholine in isolated perfused pancreas obtained from control, streptozotocin-induced diabetic, and insulin-treated diabetic rats. The time course of pancreatic juice, protein, amylase, and trypsinogen secretion in response to cerulein or carbamylcholine in diabetic rats was similar to that in control rats. Basal as well as cerulein- or carbamylcholine-stimulated output of amylase from diabetic rat pancreas was significantly reduced, whereas that of trypsinogen was similar to the control. Amylase and trypsinogen outputs in response to 620 pM (1.0 ng/ml) cerulein from insulin-treated diabetic rat pancreas were significantly lower than those from control rat pancreas, although the pancreatic contents of these enzymes were similar to or greater than those in control rats. The dose-response curves of pancreatic juice, protein, amylase, and trypsinogen for cerulein and carbamylcholine were biphasic in both control and diabetic rats. The minimal and the maximal release in response to cerulein occurred with higher concentrations in diabetic rats compared with control rats. In contrast, the maximal responses were obtained with 1 μM carbamyl-choline in control rats and with 0.1–1 μM carbamylcholine in diabetic rats. The present study demonstrates that the concentration of cerulein required to elicit maximal response was increased, whereas that to carbamylcholine was reduced in diabetic rat pancreas, and that the protein and enzyme outputs in response to cerulein were significantly reduced in insulin-treated diabetic rat pancreas despite restoration of the pancreatic enzyme contents to control levels.

Increased β-Cell Secretory Responsiveness to Ceruletide and TPA in Streptozocin-Induced Mildly Diabetic Rats

We examined the effects of various stimuli on immunoreactive insulin (IRI) and glucagon (IRG) release from perfused pancreases isolated from control and streptozocin-induced diabetic (STZ-D) rats. Diabetes was induced by injecting 30 mg/kg STZ into rats fasted for 16–18 h 12–17 days before our experiments. Glucose (11.1 mM) caused a distinct biphasic pattern of IRI release from the control pancreas, whereas the first phase was marginal and the second phase was absent in the diabetic pancreas. Arginine (20 mM)-induced IRI release was similar in both groups, whereas IRG release was greater in the control rats than in the diabetic rats. Thus, this model of STZ-D simulates a certain class of non-insulin-dependent diabetes mellitus (NIDDM). In these diabetic animals, the cholecystokinin (CCK) analogue ceruletide (620 pM) caused a significantly greater increase in IRI release in the presence of 5.6 mM glucose than in the control rats, but ceruletide caused a similar IRG release in both groups. Because CCK and ceruletide stimulate phosphoinositide turnover in pancreatic islets, we examined the effects of carbachol and phorbol ester TPA on IRI release in the presence of 5.6 mM glucose. Carbachol (10 μM), which is thought to generate similar second messengers as ceruletide, induced greater IRI release in diabetic than in control rats. TPA (100 nM) caused a significantly greater increase in IRI release from the diabetic than the control pancreas. Our results demonstrate that the insulin-releasing mechanism involved in protein kinase C activation is enhanced in this model of NIDDM. Considering the diminished glucose sensitivity of islets from STZ-D rats, the augmented insulinotropic effect of CCK may facilitate the maintenance of glucose homeostasis in these animals.

Secretin-induced exocrine secretion in perfused pancreas isolated from diabetic rats

Exocrine secretory function in response to 10 pM to 10 nM synthetic secretin was evaluated in perfused pancreas isolated from control, streptozocin-induced diabetic (STZ-D), alloxan-induced diabetic (ALX-D), and insulin-treated STZ-D rats. In STZ-D rats, the basal rate of pancreatic juice flow was significantly increased (10.3 ± 1.0 μl/20 min) compared with control rats (4.4 ± 0.2 μl/20 min). The basal rate of amylase output as well as pancreatic amylase content were significantly decreased to <5% of control values. The basal rates of protein and trypsinogen outputs were similar in both groups. In both control and diabetic rats, secretin caused a dose-dependent increase in exocrine secretion. Secretin (10 pM to 10 nM) induced 1.1- to 11.7-fold increases in exocrine secretion in STZ-D rats. These increases were significantly lower than the 2.1- to 20.8-fold increases in control rats. Furthermore, there was no significant increase in exocrine secretion from STZ-D rats in response to 10 pM secretin, although this concentration of secretin caused a significant increase in control rats. Secretin-induced exocrine secretion in ALX-D rats was similar to that in STZ-D rats. In insulin-treated STZ-D rats, the basal rates of pancreatic secretion were not significantly different from those of control rats. Secretin, at a concentration of 10 pM, caused a significant increase in juice flow (from 8.6 ± 1.6 to 30.7 ± 2.0 μl/20 min), protein output (from 227.9 ± 47.3 to 661.0 ± 127.5 μg/20 min), and trypsinogen output (from 37.4 ± 5.3 to 125.8 ± 27.6 μg/20 min), but not in amylase output (from 247.9 ± 57.2 to 327.4 ± 80.6 Somogyi units/20 min). Pancreatic juice flow from insulin-treated STZ-D rats in response to 10 nM secretin (40.3 ± 2.0 μl/20 min) was similar to that of control rats (46.5 ± 5.5 μl/20 min), whereas protein and enzyme outputs in response to 10 nM secretin were significantly reduced compared with those of control rats. This study indicates, therefore, that the sensitivity to secretin is reduced in the diabetic rat pancreas. In addition, this change is, in part, reversible by insulin treatment.

Dual effects of hydrocortisone on exocrine rat pancreas

The acute and chronic effects of hydrocortisone on exocrine pancreatic function were examined in the isolated perfused rat pancreas. In the first part of this study, rats were given subcutaneous injections of hydrocortisone at doses of 1.25, 2.5, 5, and 10 mg/kg body wt once daily for 7 days. Trypsin and lipase secretion in response to 100 pM cholecystokinin-octapeptide was significantly increased in rats with the two highest doses of hydrocortisone compared with controls, irrespective of whether calculated as the total amount of stimulated output of enzymes or related to the secretion of enzyme to the pancreas content. On the other hand, the secretory responsiveness of amylase to 100 pM cholecystokinin-octapeptide was maximal at the 5-mg dose, and decreased with higher doses. In the second part, 100 microM hydrocortisone was superimposed for 20 min on 100 pM cholecystokinin-octapeptide stimulation to examine the acute effects of hydrocortisone on exocrine pancreatic function in the isolated perfused rat pancreas. Addition of hydrocortisone caused a significant inhibition of the secretion of pancreatic juice and amylase. The present study has clearly demonstrated the dual effects of glucocorticoids on the pancreas: inhibition and potentiation. There is a possibility that chronic treatment with large doses of glucocorticoid may sensitize the acinar cells an induce hypersecretion of trypsin and lipase, whereas acute treatment inhibits secretory function of exocrine pancreas.

Exocrine pancreatic function in rats after acute trypsin inhibitor treatment.

A single oral dose of synthetic trypsin inhibitor (TI, 20 mg/100 g) was given to rats by orogastric tube 6, 12, 18, or 24 hr before the removal of the pancreas and the preparation of isolated perfused pancreas. TI treatment induced no significant changes in body weight and total amount of DNA content in the pancreas, but pancreatic wet weight, total pancreatic protein and amylase, and the concentration of total protein and amylase relative to DNA were significantly decreased at 6 or 12 hr posttreatment, with a partial return toward control values at 18–24 hr after TI treatment. In isolated perfused pancreas, basal amylase output was similar in the control and in all 4 groups of TI-pretreated rats, while basal rate of flow of pancreatic juice was significantly increased at 12–24 hr posttreatment. Caerulein (0.1 ng/ml; 64 pM) stimulated pancreatic juice flow was greatly increased in rats pretreated with TI 12–24 hr earlier. In contrast, caerulein-stimulated amylase output was significantly lower in TI-pretreated groups compared with the control. However, when amylase output was related to the total content in the pancreas, the secretory responsiveness for amylase release was significantly higher in rats at 6–18 hr posttreatment compared with the control. The present study indicates that a single oral administration of TI modulates biological response to caerulein in the isolated perfused pancreas. The enhanced responsiveness of amylase release to subsequent stimulation is seen in early periods, while that of pancreatic juice flow is observed in late periods.

Serum pancreatic secretory trypsin inhibitor in pancreatic disease

The clinical usefulness of serum pancreatic secretory trypsin inhibitor (PSTI) in pancreatic diseases was evaluated. The mean serum PSTI level of 41 healthy normal persons was 9.4 ng/ml (ranging from 5.2 to 16.7 ng/ml). Serum PSTI levels were abnormally raised in all patients with acute pancreatitis ranging from 35.0 to 4500 ng/ml, but were almost within normal range in patients with chronic pancreatitis, pancreatic cyst, acute abdominal emergencies such as perforated ulcer and intestinal obstruction, and macroamylasemia. There was no correlation between serum PSTI levels and total or pancreatic-type isoamylase activity. Patients with acute pancreatitis in whom the elevation of serum PSTI was transient and occurred after that of serum amylase activity had relatively mild symptoms and recovered along with normalization of serum PSTI levels. On the other hand, patients whose serum PSTI values became increased coincidentally with serum amylase activity and remained elevated, had severe clinical symptoms and unfavorable clinical outcome. Of 2 patients who underwent partial pancreatectomy, the serum PSTI level increased markedly in one who developed postoperative pancreatitis but not in the other without pancreatitis. In contrast to patients with acute pancreatitis, the serum response to the secretin stimulation in patients with chronic pancreatitis, was only small and transient, reaching the maximum at 10 min after administration of secretin. These results suggest that measurement of serum PSTI concentration may be useful in the diagnosis of acute pancreatitis and that the degree of rise and the duration of the elevated levels of serum PSTI are closely related to the severity of acute pancreatitis.

Pancreatic exocrine secretion and immunoreactive secretin release after intraduodenal instillation of 1-phenyl-1-hydroxy-n-pentane and HCl in rats.

Portal plasma immunoreactive secretin (IRS) concentrations, pancreatic juice flow, and amylase output were simultaneously measured in response to intraduodenal infusion of 1-phenyl-1-hydroxy-n-pentane (PHP), as well as infusion of hydrochloric acid (HCl). These data were compared with those obtained from intravenous bolus injections of synthetic porcine secretin in anesthetized rats. The intraduodenal infusion of PHP or HCl at a rate of 2 ml/min for 2 min produced a dose-related increase in portal plasma secretin concentrations, pancreatic juice flow, and amylase output. However, the mechanism of secretin release by PHP seems to differ from that of HCl. The secretin response to 0.1 N HCl infused at a rate of 0.1 ml/min for 30 min was complete after 10 min, despite continued infusion, while PHP stimulated a secetin release which persisted for 10 min after cessation of infusion. The pH in the second portion of the duodenum, following PHP infusion, remained consisitently greater than 6.3. PHP-stimulated pancreatic exocrine secretions were only partially suppressed by somatostation, while secretin release was almost completely inhibited. However, intraduodenal PHP may stimulate the release of secretin along with other gastrointestinal hormones, and the endogenous release of these hormones may not be inhibited by somatostatin.