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Local tissue pressure is higher in chronic pancreatitis than in the normal pancreas. We reported recently that pressure application induces synthesis of extracellular matrix (ECM) and cytokines in pancreatic stellate cells (PSCs) and that epigallocatechin gallate (EGCG), a potent antioxidant, inhibits the transformation of PSCs from quiescent to activated phenotype and ethanol-induced synthesis of ECM and cytokines in PSCs. These results suggest that oxidative stress and reactive oxygen species (ROS) are important in PSC activation. The aim of this study was to clarify the effects of ROS on activation and functions of pressure-stimulated PSCs. We used freshly isolated rat PSCs and culture-activated PSCs. Pressure was applied on rat cultured PSCs by adding compressed helium gas into a pressure-loading apparatus. PSCs were cultured with or without antioxidants (EGCG and N-acetyl cysteine) under normal or elevated pressure. Externally applied high pressure (80 mmHg) resulted in a gradual decrease of superoxide dismutase activity in PSCs and increased intracellular ROS generation as early as 30 s, reaching a peak level at 1 h. Antioxidants significantly inhibited ROS generation. Pressure increased the expression levels of a-smooth muscle actin, [[alpha].sub.1](I)-procollagen, and TGF-[beta]1 in PSCs. EGCG suppressed these alterations, abolished pressure-induced phosphorylation of p38 MAPK, and suppressed pressure-induced PSC transformation to activated phenotype. Our results indicated that ROS is a key player in pressure-induced PSC activation and ECM synthesis. Antioxidants could be potentially effective against the development of pancreatic fibrosis in patients with chronic pancreatitis. antioxidants; pancreatic fibrosis

This study was designed to examine whether continuous pancreatic ductal hypertension (PDH) plays an important role in the onset and development of chronic pancreatitis (CP). Pancreatic, biliary, and duodenal cannulas were implanted in male Wistar rats. PDH was induced by vertically raising the free end of the pancreatic duct cannula to exert a hydrostatic pressure and maintained for 2 wk. PDH was gradually increased, but when the pancreatic juice (PJ) flow was interrupted, PDH was decreased to restore PJ flow. The induction of PDH resulted in a marked reduction of amylase activity in PJ and an increase in serum amylase activity. At 2 wk after persistent PDH, pancreatic exocrine function was markedly decreased in response to a bolus injection of secretin (100 pmol/kg) compared with the control group. Histological examination revealed interlobular as well as intralobular fibrosis in the form of nodular pancreatitis at 2 wk after the induction of PDH. Immunohistochemistry revealed the expression of fibronectin and collagen types I and III. Quantitative real-time RT-PCR showed an increase in transforming growth factor-[[beta].sub.1] mRNA expression in the pancreas during PDH. The present results suggest that PDH plays an important role in the onset and development of CP. Furthermore, our animal model seems useful for investigating the mechanisms of CP in rats. pancreatic fibrosis; pancreatic ductal hypertension

Pancreatic stellate cells (PSCs) play a central role in development of pancreatic fibrosis. In chronic pancreatitis, pancreatic tissue pressure is higher than that of the normal pancreas. We here evaluate the effects of pressure on the activation of rat PSCs. PSCs were isolated from the pancreas of Wistar rat using collagenase digestion and centrifugation with Nycodenz gradient. Pressure was applied to cultured rat PSCs by adding compressed helium gas into the pressure-loading apparatus to raise the internal pressure. Cell proliferation rate was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation. MAPK protein levels and [alpha]-smooth muscle actin ([alpha]-SMA) expression were evaluated by Western blot analysis. Concentration of activated transforming growth factor-[beta]1 (TGF-[beta]1) secreted from PSCs into culture medium was determined by ELISA. Collagen type I mRNA expression and collagen secretion were assessed by quantitative PCR and Sirius red dye binding assay, respectively. Application of pressure significantly increased BrdU incorporation and [alpha]-SMA expression. In addition, pressure rapidly increased the phosphorylation of p44/42 and p38 MAPK. Treatment of PSCs with an MEK inhibitor and p38 MAPK inhibitor suppressed pressure-induced cell proliferation and [alpha]-SMA expression, respectively. Moreover, pressure significantly promoted activated TGF-[beta]1 secretion, collagen type 1 mRNA expression, and collagen secretion. Our results demonstrate that pressure itself activates rat PSCs and suggest that increased pancreatic tissue pressure may accelerate the development of pancreatic fibrosis in chronic pancreatitis. activation; fibrosis; chronic pancreatitis: mitogen-activated protein kinase; transforming growth factor-[beta]

The reninangiotensin system (RAS) plays important roles in various pathophysiological processes. However, the role of the RAS in pancreatic fibrosis has not been established. We investigated the role of angiotensin II (ANG II)-ANG II type 1 (A[T.sub.1]) receptor pathway in the development of pancreatic fibrosis with A[T.sub.1a] receptor-deficient [A[T.sub.1a](-/-)] mice. To induce pancreatic fibrosis, A[T.sub.1a](-/-) and wild-type (WT) mice were submitted to three episodes of acute pancreatitis induced by six intraperitoneal injections of 50 [micro]g/kg body wt cerulein at hourly intervals, per week, for four consecutive weeks. Pancreatic fibrosis was assessed by histology and hydroxyproline content. Pancreatic stellate cell (PSC) activation and the localization of A[T.sub.1] receptors were assessed by Western blot analysis for [alpha]-smooth muscle actin and immunostaining. Transforming growth factor-[[beta].sub.1] (TGF-[[beta].sub.1]) mRNA expression in the pancreas was assessed by RTPCR. Six intraperitoneal injections of cerulein induced acute pancreatitis in both A[T.sub.1a](-/-) and WT mice. There were no significant differences between two groups with regard to serum amylase and histological changes. Pancreatic fibrosis induced by repeated episodes of acute pancreatitis was significantly attenuated in A[T.sub.1a](-/-) mice compared with that in WT mice. This finding was accompanied by a reduction of activated PSCs. Dual-immunofluorescence staining in WT mice revealed that activated PSCs express A[T.sub.1] receptors. The level of TGF-[[beta].sub.1] mRNA was lower in A[T.sub.1a](-/-) mice than in WT mice. Our results demonstrate that the ANG II-A[T.sub.1] receptor pathway is not essential for the local pancreatic injury in acute pancreatitis but plays an important role in the development of pancreatic fibrosis through PSC activation and proliferation. pancreatic stellate cells; renin-angiotensin system; transforming growth factor-[[beta].sub.1]

Recent studies demonstrated that cholecystokinin (CCK) at physiological levels stimulates pancreatic enzyme secretion via a capsaicin-sensitive afferent vagal pathway. This study examined whether chemical ablation of afferent vagal fibers influences pancreatic growth and secretion in rats. Bilateral subdiaphragmatic vagal trunks were exposed, and capsaicin solution was applied. Pancreatic wet weight and pancreatic secretion and growth in response to endogenous and exogenous CCK were examined 7 days after capsaicin treatment. Perivagal application of capsaicin increased plasma CCK levels and significantly increased pancreatic wet weight compared with those in the control rats. Oral administration of CCK-1 receptor antagonist loxiglumide prevented the increase in pancreatic wet weight after capsaicin treatment. In addition, continuous intraduodenal infusion of trypsin prevented the increase in plasma CCK levels and pancreatic wet weight after capsaicin treatment. There were no significant differences in the expression levels of CCK-1 receptor mRNA and protein in the pancreas in capsaicin-treated and control rats. Intraduodenal administration of camostat or intravenous infusion of CCK-8 stimulated pancreatic secretion in control rats but not in capsaicin-treated rats. In contrast, repeated oral administrations of camostat or intraperitoneal injections of CCK-8 significantly increased pancreatic wet weight in both capsaicin-treated and control rats. Present results suggest that perivagal application of capsaicin stimulates pancreatic growth via an increase in endogenous CCK and that exogenous and endogenous CCK stimulate pancreatic growth not via vagal afferent fibers but directly in rats. cholecystokinin; pancreatic growth; capsaicin

Recently established Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of naturally occurring obesity diabetes, exhibit progressive accumulation of connective tissue in the pancreas. The present study was designed to determine the pathogenic role of transforming growth factor-[beta]1 (TGF-[beta]1) in the development of pancreatic fibrosis in OLETF rats by investigating the serial changes in the expression of TGF-[beta]1 and extracellular matrix (ECM) in the pancreas. Progressive proliferation of connective tissue arose from the interstitial region surrounding islets at 20 wk of age and extended to the exocrine pancreas adjacent to the islets. TGF-[beta]1 mRNA levels in the pancreas increased at 20 wk of age and reached a peak value at 30 wk of age. Fibronectin (FN) and procollagen types I and III mRNAs peaked at 20 wk of age and remained at higher levels than those in the nondiabetic counterparts Long-Evans Tokushima Otsuka rats until 50 wk of age. Immunoreactivities for TGF-[beta]1 and FN were found in islets of OLETF rats at 20 wk of age and were seen in acinar and interstitial cells at 50 wk of age. Moreover, [alpha]-smooth muscle actin was located at interstitial region surrounding the islets. Proliferation of the connective tissue in the pancreas of OLETF rats closely correlated with expression of TGF-[beta]1 and ECM. Out results suggest that the development of pancreatic fibrosis in OLETF rats extends from endocrine to exocrine pancreas and that TGF-[beta]1 is involved in pancreatic fibrosis of OLETF rats. extracellular matrix; myofibroblast; Type 2 diabetes; pancreatic stellate cell; transforming growth factor-[beta]1

Stimulatory effects of bilirubin on amylase release from isolated rat pancreatic acini. Am J Physiol Gastrointest Liver Physiol 282: G249-G256, 2002; 10.1152/ajpgi.00429.2002.--Considered to be an etiologic factor of acute pancreatitis, hypersecretion of pancreatic juice and digestive enzymes is often associated with hyperbilirubinemia. We explored the intracellular mechanisms through which bilirubin affects pancreatic exocrine secretory function by examining the effect of bilirubin on isolated rat pancreatic acini. Bilirubin stimulated amylase release in a concentration- and time-dependent manner, significantly increasing amylase release at concentrations >5 mg/100 ml and after 15 min of incubation. Coincubation of bilirubin with vasoactive intestinal polypeptide, 8-bromo-cAMP, or A-23187 had a synergistic effect on amylase release, whereas coincubation with CCK-8, carbamylcholine, or 12-O-tetradecanoylphorbol 13-acetate had an additive effect. Bilirubin did not affect acinar cAMP content or [Ca.sup.2+] efflux. Intracellular [Ca.sup.2+] pool depletion had no influence on bilirubin-evoked amylase release. The protein kinase C (PKC) inhibitors staurosporine and calphostin C partially but significantly inhibited bilirubin-stimulated amylase release, whereas the PKA inhibitor H-89 did not. The tyrosine kinase (TK) inhibitor genistein, phospholipase [A.sup.2] (PL[A.sub.2]) inhibitor indoxam, and PLC inhibitor U-73122 also inhibited amylase release. Bilirubin significantly translocated PKC activity from the cytosol to the membrane fraction and activated TK in cytosol and membrane fractions. These results indicate that bilirubin stimulates amylase release by activating PKC and TK in rat pancreatic acini and that PLC and PL[A.sub.2] partly mediate this process. signal transduction; protein kinase C; tyrosine kinase; phospholipase C; phospholipase [A.sub.2]