佐田 尚宏

BACKGROUND/AIMS: A large, sustained increase in acinar [Ca2+](i) may play a key role in the pathogenesis of acute pancreatitis. Many mechanisms which lead to cell damage in vitro and pancreatitis in, vivo, such as free radicals or supraphysiological cerulein concentrations, cause a rapid increase in [Ca2+](i) in pancreatic acinar cells. Little is known about why [Ca2+](i) increases in some instances stimulate secretion and in other instances initiate cell death. So far, [Ca2+](i) increases were thought to represent physiological signals when they occurred as oscillations at the single cell level. METHODOLOGY: This paper reviews recent literature and our own original research about the role of calcium in the function of pancreatic acinar cells and the development of pancreatitis. RESULTS: Recent studies showed that exposure of acinar cells' to free radicals not only caused a bulk increase in [Ca2+](i) but also resulted in calcium oscillations which had a lower frequency than, but similar amplitude to oscillations occurring after physiological stimuli. The absolute increase in [Ca2+](i) did not definitely determine the cellular response. Instead, the duration of [Ca2+](i) increase may have been more important. In contrast to previous belief of a direct relationship between [Ca2+](i) oscillations and exocytosis, recent results show that radicals can induce [Ca2+](i) oscillations which do not exert exocytosis but inhibit the secretory response to physiological stimuli. Further experiments showed that the [Ca2+](i) release caused by radicals originates from thapsigargin-insensitive, ryanodine-sensitive stores. CONCLUSIONS: The origin and duration of [Ca2+](i) increases rather than their extent or oscillatory nature, determine whether the cell will secrete or die. An abnormal [Ca2+](i) increase can trigger trypsin activation, acinar cell damage and acute pancreatitis. This hypothesis is supported by studies which show that calcium chelators inhibit radical-induced trypsin activation as well as cell necrosis and apoptosis. Thus, an inhibition of pathological [Ca2+](i) release may have a therapeutic potential.

Activation of trypsinogen is thought to trigger the autodigestive process in acute pancreatitis. The lysosomal enzyme cathepsin B was suggested to cause the activation of trypsinogen because it is known that cathepsin B is able to activate trypsinogen in special circumstances and that lysosomal and digestive enzymes are colocalized within intracellular vacuoles in the early stage of pancreatitis, As yet this hypothesis has been difficult to prove because activated trypsin is difficult to quantify in pancreatitis by conventional enzymatic measurements. We therefore employed an ELISA for trypsin activating peptide (TAP), which is a small peptide cleaved during the activation of trypsinogen and can be determined reliably. Supraphysiological concentrations of cerulein (1 nM-1 mu M) resulted in a marked increase in TAP in freshly isolated pancreatic acinar cells, indicating activation of trypsinogen. This activation as determined by the TAP increase was significantly reduced by the serine protease inhibitor Fut-175 but not by the cathepsin B inhibitors E-64 and NCO-700. The concentrations of NCO-700 and E-64 abolished the cathepsin B activity of pancreatic acinar cells but did not significantly reduce the trypsin activity (after enterokinase preincubation); correspondingly the concentrations of Fut-175 used abolished the trypsin activity but did not reduce the cathepsin B activity. The results indicate that an autoactivation of trypsin rather than an activation of trypsinogen by cathepsin B triggers trypsin activation by supramaximal cerulein concentrations.

Peroxynitrite (0.5-50 mu M) induced does-dependent cytotoxic effects in rat pancreatic acinar AR4-2J cells. Glutathione (2 mM) and ebselen (10 mu M) partially reduced the cytotoxicity caused by 1-10 mu M concentrations of peroxynitrite. Higher concentrations (10-50 mu M) of peroxynitrite induced DNA smear suggestive of necrosis, while lower concentrations (2-5 mu M) induced DNA fragmentations suggestive of apoptosis. The effects of peroxynitrite on [Ca2+](i) showed a similar dose dependency. Peroxynitrite concentrations >10 mu M rapidly increased [Ca2+](i) in a dose-dependent manner, while concentrations <5 mu M did not affect [Ca2+](i). In contrast, the presentation of wild-type P53 was accelerated at lower concentrations of peroxynitrite (less than or equal to 10 mu M) but not at higher concentrations (50 mu M). The present study suggests that peroxynitrite at lower concentrations (2-5 mu M) induces wildtype P53 and apoptosis, which is potentially a protective response toward the DNA damage caused by peroxynitrite. On the other hand, higher concentrations of peroxynitrite (10-50 mu M) rapidly increase [Ca2+](i) and eventually induce necrosis.

This study evaluated the action of menadione on cell proliferation and integrity of the rot pancreatic acinar cell line, AR4-2J. Menadione at 1-20 mu M dose-and time-dependently inhibited cell proliferation of AR4-2J cells. In contrast, a high concentration of menadione (100 mu M) caused rapid cell death (> 90% of cells took up trypan blue within 4-h). While the high concentration of menadione (100 mu M) induced DNA smear in electrophoresis indicative of necrosis, lower concentrations (10-20 mu M) induced a DNA ladder indicative of apoptosis, Similar results were obtained using a DNA fragmentation ELISA. Glutathione (1 mM), the calcium chelator EGTA (500 mu M), and the cystein protease inhibitor NCO-700 (5 mM) partly inhibited the effect of 1-10 mu M menadione on cell proliferation and DNA fragmentation. Menadione at 1-20 mu M induced wild-type P53, whereas the 100 mu M menadione had a minor effect on wild-type P53. It is concluded that menadione induced necrosis at high concentrations and apoptosis at low concentrations in AR4-2J cells. Apoptosis induced by lower concentrations of menadione may be mediated by wild-type P53, intracellular calcium, and mechanisms which decrease the intracellular concentration of reduced glutathione. (C) 1997 Elsevier Science Inc.

A 55-year-old man with gallbladder cancer was surgically treated in our hospital in July 1988. The tumor was about 8 cm in diameter, replaced the entire gallbladder, and invaded the liver and the hepatoduodenal ligament. In addition, extensive tumor metastasis to lymph nodes, including those of the para-aortic area was noted (Stage IV). Extended cholecystectomy with resection of the liver and lymph node dissection were performed. Although all of the macroscopic tumors were removed surgically, toe believed that the tumor would recur in. the near future, since all of the excised para-aortic lymph nodes were involved by carcinoma histologically. After surgery, the patient received 5'-deoxy-5-fluorouridine (5'-DFUR) orally at a dose of 600mg per day. In December 1993, more than 5 years after the primary operation, cancer recurrence in para-aortic lymph nodes was demonstrated by computed tomography (CT). In June 1994, the patient underwent a second operation for treatment of recurrent tumor. The lymph nodes firmly adhered to both the aorta and left renal vein, and could not be removed. Since August 1994, he has received external radiation therapy, and there has been no further enlargement of the nodes. This is the first reported case of gallbladder cancer with para-aortic lymph node metastasis who survived more than, seven years after the primary extended radical operation with cholecystectomy, resection of the liver, and extended lymph node dissection.

Exocrine function was studied in anesthetized rats that had received two specific doses of caerulein (maximal stimulation and supramaximal stimulation). Male Wistar rats (body weight, 200-250 g) were divided into three groups: the control group (4-h saline infusion), the maximal stimulation group (0.25 mu g/kg per h caerulein for 4h), and the caerulein pancreatitis group (10 mu g/kg per h for 4h). Histologically, interstitial edema and cytoplasmic vacuolization were observed only in the caerulein pancreatitis group, with no abnormal findings in the other groups. The volume of pancreatic juice was significantly increased in both the maximal stimulation group and the caerulein pancreatitis group. The protein ouput and the amylase output in the 1st h of caerulein infusion were also significantly increased, to 459% and 338% in the maximal stimulation group, and to 925% and 1430% respectively, in the caerulein pancreatitis compared to the baseline values. We also found that the pancreatic juice of the caerulein pancreatitis group contained precipitated protein, and high trypsin activity, and protein degradation was confirmed by electrophoresis. These findings were not observed in the other groups. These results strongly suggest that hypersecretion and the appearance of trypsin activity in pancreatic juice plays an important role in the induction of histological changes in this pancreatitis model in anesthetized rats.

We report herein a rare case of malignant insulinoma which recurred as multiple liver metastasis 8 years after the initial resection, The patient was a 51-year-old Japanese man who originally presented in 1985 at the age of 43 years suffering from general malaise and syncope, The initial surgery in 1985 involved complete enucleation of a 15 X 13mm insulinoma located in the uncus of the pancreas, Histopathologically, the tumor was diagnosed as a benign adenoma (insulinoma) which was immunohistochemically stained with only the anti-insulin monoclonal antibody, Macroscopically, there were no signs of either invasion or metastasis. During the subsequent 7 years, he did not show any symptoms or significant abnormality in laboratory data, However, in 1993, the patient again experienced syncope with hypoglycemia and hyperinsulinemia. Ultrasonography revealed multiple echogenic lesions in the liver and a second laparotomy confirmed multiple hepatic metastases from insulinoma, the histopathological findings of which were similar to those of the primary tumor from 8 gears before, The patient is currently being treated with streptozotocin and 5-fluorouracil via a catheter in the hepatic artery.

Two cases of pancreatic cancer accompanied by pseudocyst are reported. Case 1 was a 60-year-old man who was admitted to our hospital complaining of left lower abdominal discomfort. A cystic lesion, about 3 cm in diameter, was found in the pancreatic tail by ultrasonography (US) and computed tomography (CT). No signs of chronic pancreatitis were found. At operation, an elastic, hard, white tumor, about 1 cm in diameter, was felt adjacent to the cystic lesion on the duodenal side. Histologically, this tumor was a duct cell carcinoma with an adjacent pseudocyst upstream of the pancreas. Case 2 was a 57-year-old man who complained of back pain and loss of body weight. US and CT examination revealed a cystic lesion, 11×7 cm in size, in the tail of the pancreas. Histological examination of the resected speciment revealed both a duct cell carcinoma, 3 cm in size, in the body of the pancreas and a pseudocyst, 9 cm in size. Pseudocysts accompanying carcinoma are thought to develop from obstruction of the pancreatic duct by the carcinoma, followed by intraductal high pressure and disruption of ductules upstream of the pancreas. Thus, we should pay careful attention to pseudocyst of the pancreas, especially when signs of diffuse chronic inflammation cannot be found, to help identify duct cell carcinoma in the early stage. Further detailed examinations of the cyst fluid or pancreatic juice, such as cytology, tumor marker determinations, or establishment of K-ras codon 12 mutation, are needed. © 1994 Springer-Verlag.

The intracellular distribution and action of a new synthetic protease inhibitor, E3123, were studied in cerulein-induced acute pancreatitis in rats. Acute pancreatitis was induced by a 4-h iv infusion of a supramaximal dose of cerulein, and was treated by prophylactic (pretreatment) or therapeutic (posttreatment) continuous administration of E3123. Pancreatic edema and hyperamylasemia were ameriolated only by prophylactic treatment. A subcellular fractionation study showed that the activities of cathepsin-B and trypsin in the zymogen granule-enriched fraction of the cerulein-pancreatitis group were remarkably increased. Both prophylactic and therapeutic treatment significantly prevented the elevation of these enzyme activities. These effects were accompanied by amelioration of pancreatic histopathological features, including intracellular vacuolization and fat necrosis. A microscopic autoradiographic study using2H-labeled E3123 showed diffuse intracellular distribution of E3123, and the radioactivity of2H-E3123 in the posttreatment group was three times greater than that in the pretreatment group. This study provides the first experimental evidence that, even when administered therapeutically, exogenous protease inhibitors are transported into pancreatic acinar cells, thereby reducing the seventy of early intracellular alterations in cerulein-induced acute pancreatitis. © 1994 Humana Press Inc.

The intracellular distribution and action of a new synthetic protease inhibitor, E3123, were studied in cerulein-induced acute pancreatitis in rats. Acute pancreatitis was induced by a 4-h iv infusion of a supramaximal dose of cerulein, and was treated by prophylactic (pretreatment) or therapeutic (posttreatment) continuous administration of E3123. Pancreatic edema and hyperamylasemia were ameriolated only by prophylactic treatment. A subcellular fractionation study showed that the activities of cathepsin-B and trypsin in the zymogen granule-enriched fraction of the cerulein-pancreatitis group were remarkably increased. Both prophylactic and therapeutic treatment significantly prevented the elevation of these enzyme activities. These effects were accompanied by amelioration of pancreatic histopathological features, including intracellular vacuolization and fat necrosis. A microscopic autoradiographic study using H-3-labeled E3 123 showed diffuse intracellular distribution of E3 123, and the radioactivity of H-3-E3 123 in the posttreatment group was three times greater than that in the pretreatment group. This study provides the first experimental evidence that, even when administered therapeutically, exogenous protease inhibitors are transported into pancreatic acinar cells, thereby reducing the severity of early intracellular alterations in cerulein-induced acute pancreatitis.