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Background: Smad6 is implicated in the inhibition of bone morphogenetic protein signalling. However, the function of Smad6 in the pancreas remains obscure. Methods: To elucidate the unknown function of Smad6, we developed transgenic mice selectively expressing Smad6 in pancreatic acinar cells using a plasmid construct coding rat elastase 1 enhancer/promoter. Results: Smad6 transgenic mice had no specific distinguishing phenotype such as body weight, pancreatic wet weight and concentrations of pancreatic protein. However, Smad6 transgenic mice reacted to hyperstimulation by caerulein injection or a diet containing 0.5% ethionine. Maximal amylase release stimulated by CCK-8 was significantly decreased in Smad6 transgenic mice acini, and trypsin activities in transgenic mice acini were significantly increased after stimulation of CCK-8. There was no difference in effect of CCK-8 stimulation on the subsequent increase in intracellular free Ca 2+ concentration ([Ca 2+ ] i ) between wild-type and transgenic mice acini. These findings suggest that reduced pancreatic enzyme secretion was caused by the disorder of its downstream signal transduction pathways in acinar cells. The amino acid sequence at the N-terminus of Smad6 was similar to that of synaptosome-associated protein (SNAP) 25 interacting protein, which plays an important role in regulating exocytosis of pancreatic enzymes in acinar cells. Pancreatic SNAP25 protein levels in transgenic mice were decreased after caerulein-induced pancreatitis. Conclusions: These results suggest that elevated expression of Smad6 inhibits normal function of SNAP25-interacting protein and SNAP25, reduces amylase secretion in acinar cells, and increases the susceptibility of acinar cells to the onset of pancreatitis.

Objectives To elucidate the role of transforming growth factor (TGF) beta1 and extracellular matrix (ECM) after acute necrotizing pancreatitis, we studied the regulation of TGF-beta1 and ECM after induction of pancreatitis. Methods We examined the serial changes of levels of plasma TGF-beta1 by enzyme-linked immunoassay and expression of TGF-beta1 and ECM by Northern and Western blot analyses, respectively, in the pancreas after induction of sodium taurocholate-induced acute pancreatitis. Results Plasma total (active and inactive) TGF-beta1 levels at 3 hours after induction of pancreatitis were significantly increased compared with baseline values. The levels of TGF-beta1 messenger RNA (mRNA) were unaltered by day 2. Levels of fibronectin mRNA at 3 hours after induction of pancreatitis were already higher than the baseline values. Latency-associated peptide-TGF-beta1 showed a peak on day 7. Thus, the expression of ECM mRNA increased earlier than that of TGF-beta1 mRNA. Conclusions These results suggest that the increase in plasma TGF-beta1 concentration probably results from the elevated secretion of TGF-beta1 from several cells and/or the redistribution of TGF-beta1 protein and that the increase in expression of ECM probably is associated with the activation of TGF-beta1. It is conceivable that both increased plasma concentration and focal activation of TGF-beta1 play an important role in ECM production during the early stage of acute pancreatitis.

We developed a catheter drive with two rotors that pinch an aspiration catheter between them and incline along the perpendicular of the catheter axis at mutually opposite angles. Rotors are driven with the same torque in opposite directions to minimize residual torque devolving on the patient being treated. The catheter drive enables a flexible 4 mm diameter plastic catheter to be inserted into and withdrawn from a respiratory cannula over 200 mm long at a constant speed of 30 mm/s accompanying catheter rotation. The catheter drive is about 30 g mass, including a joint to a ventilator and a joint to a vacuum resource for aspiration. Aspiration tests of 5 types of dummy sputum confirmed the feasibility of aspiration by the catheter drive we developed.

PURPOSE: Premedication with glucagon or hyoscyamine is reported to be effective in reducing colonic spasm. However, these drugs can be associated with unfavorable events. This prospective study was designed to compare the effects of premedication with glucagon with those of scopolamine butylbromide on cardiopulmonary parameters, intubation time, and patient discomfort in unsedated patients undergoing diagnostic colonoscopy. METHODS: One hundred consecutive adult patients (65 males) undergoing colonoscopy without sedation were randomized to receive 1 mg of glucagon (n = 50) or 20 mg of scopolamine butylbromide (n = 50), intramuscularly. Physiologic changes, including systolic blood pressure, heart rate, and oxygen saturation, were monitored before colonoscope insertion and at three-minute intervals during colonoscopy. The percentages of completed procedure and time to cecal intubation were recorded. Patients were asked to rate pain by using a five-point pain score (0 = no pain; 4 = severe pain). RESULTS: The percentages of completed procedure (96 vs. 98 percent), time to cecal intubation (16.3 vs. 14.5 minutes), and pain score (1.7 vs. 1.5) did not differ significantly between two groups. An increase in heart rate of more than ten beats per minute from baseline during colonoscopy occurred significantly more often in scopolamine group (44 percent of 50 patients) than in the glucagon group (12 percent of 50 patients; P = 0.0004). There were no significant differences between the two study groups with regard to changes in systolic blood pressure and decrease in oxygen saturation during colonoscopy. CONCLUSIONS: Premedication with 1 mg of glucagon facilitates favorable examination with respect to physiologic changes compared with 20 mg of scopolamine. These features favor glucagon as the preferred premedication for patients undergoing colonoscopy.

Background and aims The imbalance between synthesis and degradation of extracellular matrix (ECM) proteins is a characteristic feature in chronic pancreatitis. We evaluated the relationship between type IV collagen structure in the basement membrane (BM) and the development of acinar atrophy or the regeneration from acinar injury. Methods Three different models of pancreatitis were induced in rats by repetitive intraperitoneal injections of 500 mg/100 g body weight of arginine (Arg) or 20 microg/kg body weight of caerulein (Cn) or a single retrograde intraductal infusion of 40 microL/100 g body weight of 3% sodium taurocholate (NaTc). We examined the changes in type IV collagen structure by immunostaining, and the serial changes in the gelatinolytic activity of pro- and active matrix metalloproteinase-2 by zymography in these models of pancreatitis. Results The pancreas appeared to be histologically normal on day 35 after the first intraperitoneal Cn injection and on day 42 after intraductal infusion of NaTc, whereas 85% to 90% of acinar tissue was replaced by fatty tissue and dilated pancreatic ducts on day 54 after the first intraperitoneal Arg injection. Immunoreactivity for type IV collagen appeared as a discontinuous line along the BM of ducts, vessels, tubular complexes, and acinar cells on day 40 in Arg-induced pancreatitis, whereas it was detected as a continuous line along the BM on day 35 in Cn-induced pancreatitis and on day 42 in NaTc-induced pancreatitits. Gelatinolytic activity of active MMP-2 increased significantly from day 13 to day 40 after the first intraperitoneal Arg injection, whereas it decreased to the baseline level on day 35 after the first intraperitoneal Cn injection and on day 42 after intraductal infusion of NaTc. Conclusions Our findings indicate that a long-term increase in gelatinolytic activity of active MMP-2 in Arg-induced pancreatitis causes continuous disorganization of type IV collagen in the BM and progressive acinar atrophy, whereas a transient increase in gelatinolytic activity of active MMP-2 is involved in the regeneration of type IV collagen structure in the BM and recovery from acinar injury.

CCK acts on pancreatic acinar cells to increase intracellular Ca2+leading to secretion of digestive enzymes and, in the long term, pancreatic growth. Calcineurin (CN) is a serine/threonine-specific protein phosphatase activated by Ca2+and calmodulin that recently has been shown to participate in the growth regulation of cardiac and skeletal myocytes. We therefore tested the effect of two different CN inhibitors, cyclosporine A (CsA) and FK506, on mouse pancreatic growth induced by oral administration of the synthetic protease inhibitor camostat, a known stimulator of endogenous CCK release. Mice were fed a powdered diet with or without 0.1% camostat. Pancreatic wet weight, protein, and DNA were increased in response to camostat in a time-dependent manner over 10 days in ICR mice but not in CCK-deficient mice. Both CsA (15 mg/kg) and FK506 (3 mg/kg) given twice daily blocked the increase in pancreatic wet weight and protein and DNA content induced by camostat. The increase in plasma CCK induced by camostat was not blocked by CsA or FK506. Camostat feeding also increased the relative amount of CN protein, whereas levels of MAPKs, ERKs, and p38 were not altered. In summary, 1) CCK released by chronic camostat feeding induces pancreatic growth in mice; 2) this growth is blocked by treatment with both CsA and FK506, indicating a role for CN; 3) CCK stimulation also increases CN protein. In conclusion, activation and possibly upregulation of CN may participate in regulation of pancreatic growth by CCK in mice.

Introduction: Survivin is one of the inhibitors of the apoptosis family and has dual effects: antiapoptotic effect and regulation of the cell cycle. Aim: To show involvement of survivin in acute pancreatitis. Methodology: Acute necrohemorrhagic pancreatitis was induced in male Wistar rats by intraductal infusion of 4% sodium taurocholate. Results: By northern blotting, the survivin mRNA level was significantly increased at 36 hours and peaked at 48 hours after induction of acute pancreatitis. Survivin protein was found in cytoplasm of ductal cells by immunohistochemical analysis at 48-72 hours. It was also observed in nuclei of both acinar and ductal cells as well as infiltrating cells. Apoptotic cells were observed in pancreatic acinar cells. Survivin protein partially colocalized with 5-bromo-2'-deoxyuridine in some nuclei of ductal cells. Conclusions: We showed involvement of survivin in acute pancreatitis in rats. Survivin may have some roles in the regulation of pancreatic regeneration and proliferation as well as an antiapoptotic effect after acute pancreatitis.

Cholecystokinin (CCK) acting through its G protein-coupled receptor is now known to activate a variety of intracellular signaling mechanisms and thereby regulate a complex array of cellular functions in pancreatic acinar cells. The best studied mechanism is the coupling through heterotrimeric G proteins of the Gq family to activate a phospholipase C leading to an increase in inositol trisphosphate and release of intracellular Ca2+. This pathway along with protein kinase C activation in response to the increase in diacylglycerol stimulates the secretion of digestive enzymes by the process of exocytosis. CCK also activates signaling pathways in acini more related to other processes. The three mitogen activated protein kinase cascades leading to ERKs, JNKs and p38 MAPK are all activated by CCK. CCK activates the ERK cascade by PKC activation of Raf which in turn activates MEK and ERKs. JNKs are activated by a distinct mechanism which requires higher concentrations of CCK. Both ERKs and JNKs are presumed to regulate gene expression. CCK activation of p38 MAPK also plays a role in regulating the actin cytoskeleton through phosphorylation of the small heat shock protein HSP27. The PI3K-PKB-mTOR pathway is activated by CCK and plays a major role in regulating protein synthesis at the translational level. This includes both activation of p70 S6K leading to phosphorylation of ribosomal protein S6 and the phosphorylation of the binding protein for initiation factor 4E leading to formation of the mRNA cap binding complex. Other signaling pathways activated by CCK receptors include NF-kappaB and a variety of tyrosine kinases. Further work is needed to understand how CCK receptors activate most of the above pathways and to better understand the biological events regulated by these diverse signaling pathways.

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 Ca2+efflux. Intracellular Ca2+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 A2(PLA2) 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 PLA2partly mediate this process.

Heat shock proteins (HSPs) which are induced by stress can provide protection against subsequent cellular damage. Whole body hyperthermia in rats leading to induction of HSP70 has been shown to protect against subsequent caerulein-induced acute pancreatitis. We studied the effect of hyperthermia on pancreatic HSP expression and found a significant increase in HSP70 (26.0-fold) and HPS27 (6.0-fold) but no change in HSP60, HSP90 or GRP78. Hyperthermia conferred significant protection against subsequent arginine-induced acute pancreatitis. More specifically, the degradation and disorganization of the actin cytoskeleton, an important early component of acute pancreatitis, was prevented. These results generalize previous work on caerulein-induced pancreatitis to another model of experimental pancreatitis, arginine-induced pancreatitis, and suggest that multiple HSPs may be involved in the cytoprotective effect in rat pancreas.

INTRODUCTION Pancreatic tissues are almost completely restored to normal after an attack of acute pancreatitis, once the cause of the disease is removed. Transforming growth factor (TGF)-beta and extracellular matrix (ECM) are known to play an important role in the process of wound healing in pathologic diseases. Tissue repair is a process regulated by a balance between synthesis and degradation of ECM. AIMS To elucidate the role of TGF-beta, ECM, and matrix metalloproteinase (MMP) in the process of regeneration occurring after acute necrotizing pancreatitis. METHODOLOGY Acute necrotizing pancreatitis was induced by intraperitoneal injection of 500 mg/100 g body weight of L-arginine in male Wistar rats. Expression of TGF-beta1 and ECM messenger RNA (mRNA) was determined by Northern blot analysis, and that of MMP-1 and MMP-2 mRNA was examined by the reverse transcription polymerase chain reaction (RT-PCR). Immunoreactivity for ECM components, TGF-beta1, and MMP-2 in the pancreas was assessed by using a monoclonal antibody. RESULTS TGF-beta1 mRNA expression reached a peak value on day 2.5, with a decrease on day 3, and reached the control level on day 7. Procollagen types III and IV and fibronectin mRNA reached a peak value on day 2.5, whereas the expression level of procollagen type I mRNA was maximal on day 3, and gradually decreased to control levels by day 7. MMP-2 mRNA was significantly elevated on day 3, and peaked on day 5, whereas MMP-1 mRNA levels did not change throughout the observation period. Immunoreactivity for MMP-2 was observed around disrupted acinar cells and interstitial spaces on day 3, and maximally on day 7. Immunoreactivity for fibronectin was detected around disrupted acinar cells and interstitial spaces. On day 7, it was less than on day 5 around disrupted acinar cells and interstitial spaces, whereas in the regenerated acinar cells, it was undetected. CONCLUSION Our results show that TGF-beta1 mRNA expression peaked earlier than that of ECM mRNA. Furthermore, increased level of the MMP-2 transcript was followed by disappearance of fibronectin. Our findings suggest that TGF-beta1 plays an important role in ECM production in the early phase of acute pancreatitis, and that MMP-2 is involved in the subsequent healing process.

A 73-year-old man, who was diagnosed as having advanced anorectal malignant melanoma (Stage IV), was treated with combination chemotherapy using dacarbazine, nimustine, cisplatin, and tamoxifen plus interferon-beta. After the first course of chemotherapy, rectal tumor was decreased in size with less anal pain and liver tumor was disappeared. Twenty-four months after the first treatment, the patient is survived. DAC-Tam IFN-beta therapy may improve the management of patients who have advanced MM of the anorectum.

A 62-year-old Japanese woman with ulcerative colitis (UC) had episcleritis and erythema nodosum. Oral administration of corticosteroid with granulocytapheresis improved all these diseases. Extraintestinal manifestations such as ocular and skin complications are infrequent, especially in Japan, in patients with UC. Although concurrent onset of episcleritis and erythema nodosum associated with UC is also extremely rare, clinical course in this case suggests a possible association among ulcerative colitis, episcleritis and erythema nodosum.

A 30-years-old Japanese woman with a liver tumor was found to have congenital absence of the portal vein (CAPV). Both three-dimensional CT and angiography revealed that the superior mesenteric vein and splenic vein flowed into inferior vena cava and there was us portal vein, CAPV is an extremely rare congenital anomaly and liver tumor. Most cases on diagnosed in childhood, although this case was found in on adult. We reviewed the literature on reported CAPV cases.

Background Recent investigations suggest that activation of coagulation and fibrinolysis occurs in patients with ulcerative colitis (UC). However, the role of the hypercoagulable state in UC has not been determined. On the other hand, there are no reports dealing with coagulation in ischemic colitis (IC), in which acute bowel inflammation and reversible vascular occlusion affect the colon. Thus, our aim was to evaluate the hyper states of coagulation and fibrinolysis in UC by comparing activations of coagulation and fibrinolysis in patients with active UC and in those with IC. Methods Twenty-four patients with active UC and 12 patients with IC were studied, with 18 patients with inactive UC serving as controls. We investigated the activation of the coagulation system, including platelet counts, activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), serum concentrations of von Willebrand factor (vWF), activated factors XII, XI, X, IX, VIII, VII, V, II, fibrinogen, prothrombin fragments 1+2 (F1+2), thrombin-antithrombin complexes (TAT), protein S, protein C, plasminogen, α-2 plasminogen inhibitor (α-2PI) and D-dimer (D-D). Results Median serum vWF concentrations, F1+2, TAT, fibrinogen, activated factor XI, IX, VIII and V were significantly elevated in patients with active UC and IC compared to those in patients with inactive UC. There was no significant difference between active UC and IC patients in the mean values of any of the factors that were measured. Conclusion The results of the present study indicate that the coagulation-fibrinolysis system is activated in patients with active bowel inflammation such as active UC and IC, and that the hyper states of coagulation and fibrinolysis in patients with active UC are secondary to bowel inflammation.

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 α-smooth muscle actin, α1(I)-procollagen, and TGF-β1 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.

Although many workers suffer from chronic hepatitis, the influence of labor on its clinical course is not clear. We prospectively followed 89 workers with chronic hepatitis for 3 years, and examined the relationship between job-related factors, such as job class, job type, working hours and work effort, and the liver function test. There were no job-related factors that had any influence on the activity of hepatitis. Moreover, no significant relationship was found between job-related factors, including tiredness, and the acute exacerbation of hepatitis. No significant changes of aminotransferase levels and of platelet counts divided by each job-related factor were found during the observation period, but the platelet counts decreased in workers with acute exacerbation, but without clinical significance. These results suggest that job-related factors have little influence on the clinical course of chronic hepatitis during a relatively short observation period.

OBJECTIVE—To determine the association between nonalcoholic fatty liver disease and the risk for development of diabetes. RESEARCH DESIGN AND METHODS—We conducted an observational cohort study in male workers ≥40 years old in a Japanese company from 1997 to 2005. We excluded workers with alcohol intake ≥20 g/day and those with impaired glucose tolerance by a 75-g oral glucose tolerance test. The remaining 3,189 workers were classified into fatty liver (FL) and non-FL group based on the findings of abdominal ultrasonography. Both groups were followed for the development of diabetes. Hazard ratio (HR) was determined in Cox proportional hazard analysis. A nested case-control study was conducted to determine the odds ratio (OR). RESULTS—The average age of participants was 48.0 years at the entry, and the average follow-up period was 4.0 years. The incidence of diabetes in the FL group was 2,073 per 100,000 person-years (65 cases), whereas 452 per 100,000 person-years (44 cases) in the non-FL group. The age- and BMI-adjusted HR of diabetes associated with FL was 5.5 (95% CI 3.6–8.5, P < 0.001). In the nested case-control analysis, the OR adjusted for age and BMI was 4.6 (3.0–6.9, P < 0.001). CONCLUSIONS—Nonalcoholic fatty liver disease significantly increases the risk of diabetes in middle-aged Japanese men.

Objective Hyperglycemia is implicated in fibrosis in many organs. Exocrine and endocrine pancreas are closely linked both anatomically and physiologically, and pathological conditions in the exocrine gland can cause impairment of endocrine function and vice versa. Chronic pancreatitis causes pancreatic fibrosis and sometimes results in diabetes mellitus. Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrogenesis. However, the effects of high glucose concentrations on PSC activation have not been fully elucidated. Methods Cultured PSCs were incubated in the presence of various concentrations of glucose. Pancreatic stellate cell proliferation, alpha-smooth muscle actin (alpha-SMA) expression, and collagen production were determined by colorimetric conversion assay, Western blot analysis, and Sirius red dye binding assay, respectively. Results High glucose concentrations significantly increased PSC proliferation, alpha-SMA expression, and collagen type I production in PSCs. High glucose concentrations activated protein kinase C (PKC) in PSCs, and PKC inhibitor GF109203X inhibited glucose-stimulated PSC proliferation, alpha-SMA expression, and collagen secretion. High glucose also activated p38 mitogen-activated protein kinase (MAPK) in PSCs, and p38 MAPK inhibitor SB203580 inhibited glucose-stimulated collagen secretion. Conclusions Our results indicate that high glucose concentrations stimulate PSC activation via PKC-p38 MAP kinase pathway and suggest that high glucose may aggravate pancreatic fibrosis.

In Japan, the number of patients with both chronic pancreatitis (CP) and pancreatic cancer (PC) is increasing. A nationwide survey on CP revealed that the total number of patients treated for CP in Japan in 2002 was estimated as 45,200 (95% confidence interval, 35,600-54,700), and 20,137 patients died of PC in 2002. Alcoholic pancreatitis was the most common type of pancreatitis (67.5 %). Cigarette smoking was an independent and significant risk factor for CP. The risks of pancreatic and nonpancreatic cancers increased in the course of CP. While alcohol consumption may increase the risk of PC via CP, smoking was important as a risk factor for both CP and PC. The increasing incidence of PC was closely related to the increasing intake of animal fat. Lifestyle in patients with CP appeared to be the same as that in patients with PC. Environmental factors such as lifestyle in combination with genetic factors may increase the risk for both CP and PC. Therefore, changing and improving lifestyle habits such as drinking, smoking and nutrition may reduce the risks for both CP and PC.

Feeding stimulates pancreatic digestive enzyme synthesis at the translational level, and this is thought to be mediated by hormones and neurotransmitters. However, BCAAs, particularly leucine, stimulate protein synthesis in several tissues. We investigated whether BCAA stimulated the translational machinery in murine pancreas and whether their effects were independent of hormones. Rats and mice were administered (i.g. gavage) individual BCAA at 1.35 mg/g (body weight) and rat isolated pancreatic acini were incubated with BCAA under different conditions. Activation of translation initiation factors and total protein synthesis were analyzed. BCAA gavage stimulated the phosphorylation of the initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and the ribosomal protein S6 kinase (S6K), with leucine being the most effective. Leucine also increased the association of the initiation factors eIF4E and eIF4G, but did not affect the activity of the guanine nucleotide exchange factor eIF2B, nor total protein synthesis. BCAA acted independently of insulin signaling on isolated pancreatic acini from diabetic rats. The ability of leucine to promote phosphorylation of 4E-BP1 and S6K as well as enhance the assembly of the eIF4F complex was unimpaired in CCK-deficient mice. Finally, rapamycin (0.75 mg/kg) administered to rats 2 h before leucine gavage inhibited the phosphorylation of S6 and 4E-BP1 induced by leucine. We conclude that leucine may participate, as a signal as well as a substrate, in activating the translational machinery in pancreatic acinar cells independently of hormonal effects and that this action is through the mTOR pathway.

OBJECTIVE We have recently reported that pancreatic growth driven by cholecystokinin released endogenously by feeding the synthetic trypsin inhibitor camostat requires the Ca-activated phosphatase calcineurin. In the present study, we evaluated a number of signal transduction pathways for their activation as part of the growth response and whether their activation was dependent on calcineurin. METHODS Male ICR mice were fed with either chow or chow plus 1 mg/g of camostat. FK506 was administered at 3 mg/kg. After various times from 12 hours to 10 days, pancreatic samples were prepared and assayed for activity of various signal transduction pathway components. RESULTS Camostat feeding increased the activation of extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and phosphorylation of the translation factor eukaryotic initiation factor 4E and activated the mammalian target of rapamycin pathway that leads to phosphorylation of the ribosomal protein S6 and of the eukaryotic initiation factor 4E binding protein but with different time courses. Treatment of mice with the calcineurin inhibitor FK506 totally blocked c-Jun NH2-terminal kinase activation, partially blocked the mammalian target of rapamycin pathway, and had no effect on extracellular signal-regulated kinase activation or the phosphorylation of eukaryotic initiation factor 4E. CONCLUSIONS The pancreatic growth response is accompanied by activation of a number of signaling pathways regulating transcription and translation, some of which are dependent on and some independent of calcineurin.

Background Activated pancreatic stellate cells (PSCs) play a central role in the pathogenesis of pancreatic fibrogenesis and inflammation. Ethanol, a major cause of chronic pancreatitis, directly induces PSC activation and oxidative stress. Inhibition of PSC activation or stimulation to PSC might be an effective therapeutic strategy for the prevention of pancreatic fibrosis, and (–)-epigallocatechin-3-gallate (EGCG), a major component of green tea extracts, is a potent antioxidant of polyphenols. Therefore, we examined the mechanisms through which ethanol induces oxidative stress on PSCs and evaluated the effect of EGCG on activation and cell functions of ethanol-stimulated PSCs. Materials and methods The PSCs were isolated from the pancreas of male Wister rats with Nycodenz gradient methods and cells between passages one and four were used. Isolated PSCs were cultured with ethanol (50 mM) in the absence or presence of EGCG (5 µM or 25 µM). Results The EGCG pre-treatment abolished ethanol-induced lipid peroxidation of the cell membrane, loss of total superoxide dismutase (SOD) activity and suppressed ethanol-induced gene expressions of Mn- and Cu/Zn-SOD. EGCG also suppressed ethanol-induced p38 mitogen-activated protein (MAP) kinase phosphorylation, α-smooth muscle actin production in PSCs and activated transforming growth factor-β1 secretion into the medium. Furthermore, EGCG inhibited ethanol-induced type-I procollagen production and collagen secretion. In addition, EGCG inhibited transformation of freshly isolated cells to activated myofibroblast-like phenotype. Conclusions Our results suggest that green tea and polyphenols could prevent pancreatic fibrosis by inhibiting PSC activation through the antioxidative effect.

Transforming growth factor-beta (TGF-beta) is an important cytokine in the fibrogenesis in many organs, including the pancreas. Using an adenoviral vector expressing the entire extracellular domain of type II human TGF-beta receptor (AdTbeta-ExR), we investigated whether inhibition of TGF-beta action is effective against persistent pancreatic fibrosis, and whether it exerts a beneficial effect on the pancreas in the process of chronic injury. To induce chronic pancreatic injury and pancreatic fibrosis, mice were subjected to three episodes of acute pancreatitis induced by six intraperitoneal injections of 50 microg/kg body weight cerulein at hourly intervals, per week for 3 consecutive weeks. Mice were infected once with AdTbeta-ExR, or with a control adenoviral vector expressing bacterial beta-galactosidase (AdLacZ). Pancreatic fibrosis was evaluated by histology and hydroxyproline content. Activation of pancreatic stellate cells (PSCs) was assessed by immunostaining for alpha-smooth muscle actin. Apoptosis and proliferation of acinar cells were assessed by immunostaining of ssDNA and Ki-67, respectively. Three-week cerulein injection induced pancreatic fibrosis and pancreatic atrophy with proliferation of activated PSCs. In AdTbeta-ExR-injected mice, but not AdLacZ-injected mice, pancreatic fibrosis was significantly attenuated. This finding was accompanied by a reduction of activated PSCs. AdTbeta-ExR, but not AdLacZ, significantly increased pancreas weight after chronic pancreatic injury. AdTbeta-ExR did not change the proportion of proliferating acinar cells, whereas it reduced the number of apoptotic acinar cells. Our results demonstrate that inhibition of TGF-beta action not only decreases pancreatic fibrosis but also protects the pancreas against chronic injury by preventing acinar cell apoptosis.

Background Cholecystokinin (CCK) plays an important role in regeneration after acute pancreatitis in rats. The present study was aimed to elucidate the role of CCK-1 receptor (CCK-1R) in acute pancreatitis. We investigated the serial changes in CCK-1R mRNA and protein levels and their immunolocalization after acute haemorrhagic pancreatitis induced in male Wistar rats by retrograde intraductal infusion of 4% sodium taurocholate (100 µL 100 g−1 body weight). Methods Histological changes were evaluated by haematoxylin and eosin staining. Pancreatic CCK-1R mRNA was determined by Northern blot analysis. Pancreatic CCK-1R protein was evaluated by immunoblot analysis and immunohistochemistry with a polyclonal antibody against rat CCK-1R protein. Results Histological findings revealed that newly formed acinar cells were detected at the periphery of tubular complexes on day 14, and normal architecture of lobules was observed focally on day 21. Pancreatic CCK-1R mRNA peaked on day 3 and thereafter gradually decreased. Cholecystokinin-1R protein rapidly increased after induction of pancreatitits, reaching a maximal level on day 3. On day 3, intense immunoreactivity for CCK-1R protein was observed in both the cytoplasm of vacuolized acinar cells and the tubular complexes. In the regenerative process after acute haemorrhagic pancreatitis in rats, the expression of pancreatic CCK-1R mRNA and protein increased, and intense immunoreactivity for CCK-1R protein was observed in tubular complexes in the cytoplasm of regenerated acinar cells. Conclusion These results suggest that CCK-1R contributes to pancreatic regeneration after acute haemorrhagic pancreatitis and that tubular complexes are involved in the process of acinar cell regeneration following pancreatic injury.

The renin-angiotensin 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 (AT1) receptor pathway in the development of pancreatic fibrosis with AT1areceptor-deficient [AT1a(−/−)] mice. To induce pancreatic fibrosis, AT1a(−/−) and wild-type (WT) mice were submitted to three episodes of acute pancreatitis induced by six intraperitoneal injections of 50 μg/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 AT1receptors were assessed by Western blot analysis for α-smooth muscle actin and immunostaining. Transforming growth factor-β1(TGF-β1) mRNA expression in the pancreas was assessed by RT-PCR. Six intraperitoneal injections of cerulein induced acute pancreatitis in both AT1a(−/−) 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 AT1a(−/−) 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 AT1receptors. The level of TGF-β1mRNA was lower in AT1a(−/−) mice than in WT mice. Our results demonstrate that the ANG II-AT1receptor 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.

The aim of the present study was to determine the underlying cellular mechanisms in the pancreas after acute pancreatitis and to study the pathogenesis of pancreatitis-associated lung injury. We applied a differential display analysis to normal pancreas and to the pancreas with acute pancreatitis in rats, and we examined the expression of the identified gene in the lung as well as the pancreas after acute pancreatitis.Controlled animal study.Research laboratory of an academic institution.Ninety male Wistar rats.Pancreatitis was induced by retrograde intraductal infusion of 4% sodium taurocholate (100 microL/100 g of body weight). Data were compared with data from controls (sham).We cloned some expressed sequence tags and identified one complementary DNA fragment. The deduced protein was a polypeptide of 218 amino acids, which was almost identical to human 19-kD interacting protein-3-like (NIP3L) protein. The expression of rat NIP3L identified in this study increased slightly in the pancreas after induction of acute pancreatitis but showed a marked increase in the lung by both Northern and Western blot analysis. NIP3L immunoreactivity was noted in alveolar and epithelial cells of the control (sham) lung, and the immunoreactivity in these cells was elevated after induction of acute pancreatitis. Moreover, acute pancreatitis increased terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive alveolar and bronchiolar cells in the lung.NIP3L may be involved in lung injury, which is one of the major causes of death in cases of severe acute pancreatitis.

Cholecystokinin (CCK) acting through its G protein-coupled receptor is now known to activate a variety of intracellular signaling mechanisms and thereby regulate a complex array of cellular functions in pancreatic acinar cells. The best studied mechanism is the coupling through heterotrimeric G proteins of the Gq family to activate a phospholipase C leading to an increase in inositol trisphosphate and release of intracellular Ca2+. This pathway along with protein kinase C activation in response to the increase in diacylglycerol stimulates the secretion of digestive enzymes by the process of exocytosis. CCK also activates signaling pathways in acini more related to other processes. The three mitogen activated protein kinase cascades leading to ERKs, JNKs and p38 MAPK are all activated by CCK. CCK activates the ERK cascade by PKC activation of Raf which in turn activates MEK and ERKs. JNKs are activated by a distinct mechanism which requires higher concentrations of CCK. Both ERKs and JNKs are presumed to regulate gene expression. CCK activation of p38 MAPK also plays a role in regulating the actin cytoskeleton through phosphorylation of the small heat shock protein HSP27. The PI3K-PKB-mTOR pathway is activated by CCK and plays a major role in regulating protein synthesis at the translational level. This includes both activation of p70 S6K leading to phosphorylation of ribosomal protein S6 and the phosphorylation of the binding protein for initiation factor 4E leading to formation of the mRNA cap binding complex. Other signaling pathways activated by CCK receptors include NF-kappaB and a variety of tyrosine kinases. Further work is needed to understand how CCK receptors activate most of the above pathways and to better understand the biological events regulated by these diverse signaling pathways.

INTRODUCTION AND AIMS Extracellular matrix (ECM) components participate in the process of tissue repair and development of fibrosis in the pancreas. We studied the production kinetics of ECM components and transforming growth factor (TGF)-beta1 and identified their production sites in the pancreas following pancreatitis. METHODOLOGY Pancreatitis was induced in rats by a single intraductal infusion of oleic acid. Gene expression of TGF-betas and ECM components was studied by northern blotting. Pancreatic stellate cell activation was assessed by immunostaining for alpha-smooth muscle actin (alphaSMA) and desmin. RESULTS Gene expression of TGF-betas and ECM components was increased in association with pancreatic fibrosis after 1-2 weeks and remained higher than the control levels for the ensuing 12 weeks. Both alphaSMA and desmin were strongly immunostained around small vessels and faintly stained in mesenchymal cells and tubular complexes at 1 week. The combination of staining for alphaSMA plus in situ hybridization for procollagen type III mRNA revealed that procollagen type III mRNA was expressed in both alphaSMA-positive and alphaSMA-negative cells in the mesenchyma. CONCLUSIONS Our findings demonstrate that expression of genes for both TGF-betas and ECM components was increased and that both alphaSMA-positive myofibroblasts and mesenchymal cells are the major sources of ECM components after pancreatitis.

Arginine induced acute pancreatitis was evaluated as a novel and distinct form of experimental pancreatitis with particular attention to the actin cytoskeleton and expression of heat shock or stress proteins. Arginine induced a dose related necrotising pancreatitis in rats, as shown by histological evaluation, and an increase in serum amylase. Severe pancreatitis induced by 4.5 g/kg arginine was accompanied by dramatic changes in the actin cytoskeleton, as visualised with rhodamine phallodin. Intermediate filaments were also disrupted, as visualised by cytokeratin 8/18 immunocytochemistry. Arginine pancreatitis was accompanied by a stress response with a large increase in the small heat shock protein HSP27, as well as HSP70, peaking at 24 hours and localised to acinar cells. There was a lower increase in HSP60 and HSP90 and no effect on GRP78. HSP27 was also shifted to phosphorylated forms during pancreatitis. A lower dose of arginine (3.0 g/kg) induced less pancreatitis but a larger increase in HSP70 and HSP27 expression and phosphorylation of HSP27. Thus HSP expression can be overwhelmed by severe damage. The present work in conjunction with earlier work on caerulein induced pancreatitis indicates that changes in the actin cytoskeleton are an early component in experimental pancreatitis. Keywords: experimental pancreatitis; pancreas; actin cytoskeleton; cytokeratins; heat shock proteins; HSP27; rat

Background & Aims: Cholecystokinin (CCK) is known to stimulate the synthesis of digestive enzymes in the pancreas at the translational level. We investigated in vivo the biochemical regulation of initiation factors important for the stimulation of translation of digestive enzyme protein in rat pancreas by CCK. Methods: Intraperitoneal injection of CCK or intragastric administration of a trypsin inhibitor to elicit endogenous CCK release was followed by removal and preparation of pancreas for protein evaluation. Isoelectric focusing was used to evaluate the phosphorylation of the initiation factor eIF4E, and Western blotting and immunoprecipitation followed by Western blotting were used to study the phosphorylation state and amount of other interacting factors. Results: CCK treatment induced a time- and dose-dependent phosphorylation of pancreatic eIF4E and its binding protein (PHAS-I). Because the release of eIF4E from its binding protein as a result of phosphorylation is followed by formation of a messenger RNA cap–binding complex that includes the initiation factor eIF4G, we evaluated the association of eIF4G with released eIF4E and showed that it was increased by CCK. These events occurred over a range of CCK doses from 0.2 to 5 μg/kg. We also evaluated the effect of endogenous CCK by administering a synthetic trypsin inhibitor, camostat (100 mg/kg). Camostat treatment markedly increased the phosphorylation of both PHAS-I and eIF4E and the formation of eIF4E-eIF4G complex. Thus, both exogenous and endogenous CCK activate translational initiation factors in vivo. Conclusions: Activation of translational machinery necessary for initiation of protein synthesis likely contributes to the normal postprandial synthesis of pancreatic digestive enzymes. GASTROENTEROLOGY 2000;119:1731-1739

We investigated the pharmacologic characteristics of a newly developed benzodiazepine derivative (S)-(-)-N-[2,3-dihydro-2-oxo-5-phenyl-1-[(1H-tetrazol-5-yl)methyl] -1H-1,4-benzodiazepine-3-yl]-2-indolecarboxamide (TS-941), a cholecystokinin type A (CCK-A)-receptor antagonist, in the isolated rat pancreatic acini and compared with those of well-known CCK-A-receptor antagonists, devazepide and loxiglumide. TS-941 inhibited CCK-8-stimulated amylase release concentration dependently, as did devazepide and loxiglumide, with a half-maximal inhibition (IC50) at 78.6 +/- 10.3 nM. TS-941 was approximately 23 times less potent than devazepide (IC50, 3.4 +/- 0.3 nM), but was 50 times more potent than loxiglumide (IC50, 3,966 +/- 544 nM) in inhibiting 100 pM CCK-8-stimulated amylase release from rat pancreatic acini. TS-941 had a fivefold lower selectivity than devazepide for pancreatic CCK (CCK-A) over brain CCK (CCK-B) receptors but fourfold greater than loxiglumide when IC50 values for inhibition of [125I]CCK-8 binding in isolated acini and cerebral cortex were compared. The antagonism produced by TS-941 was specific for CCK in that the effects of other receptor secretagogues or agents bypassing receptors were not altered. TS-941 caused a parallel rightward shift of the entire dose-response curve for CCK-8-stimulated amylase release without altering the maximal increase, as did devazepide and loxiglumide. TS-941, whether added at the beginning or 20 min after the CCK-8 stimulation, inhibited amylase release. TS-941 caused a concentration-dependent residual inhibition of the action of CCK-8. The acini, once incubated with a high concentration of TS-941 (10 microM; 127 times IC50) for 30 min, was 10-fold less sensitive to CCK-8 than the acini preincubated without TS-941, whereas the sensitivity and the responsiveness to CCK-8 stimulation of those incubated with a low concentration of TS-941 (1.0 microM) were similar to the control acini. These results indicate that TS-941 is a potent, competitive, and selective CCK-A receptor antagonist for the pancreas.

Recent studies in genetically obese and diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats suggest defects of cholecystokinin (CCK)-A receptor gene expression and CCK-A receptor-mediated biological functions such as pancreatic juice, protein, and gastric acid secretion. The present studies were undertaken to further examine CCK-A receptor gene expression and CCK-A receptor-mediated biological functions in the pancreas, stomach, and brain of OLETF rats. Expression of the CCK-A receptor gene could not be detected in the stomach, pancreas and brain by the reverse-transcription polymerase chain reaction (RT-PCR) method and Southern blotting of the PCR products. Southern blot analysis of genomic DNA from OLETF and control Long-Evans Tokushima Otsuka (LETO) rats with CCK-A receptor fragment as a probe revealed different restriction bands. Expression of the CCK-B receptor gene was observed in the stomach, pancreas, and brain in both OLETF and LETO rats by the RT-PCR method, with expression of the CCK-B receptor gene markedly enhanced in OLETF rats compared with that in LETO rats. Consistent with the defect of CCK-A receptor gene expression, CCK-A receptor-mediated biological functions were not observed in these organs. Perfused exocrine and endocrine pancreas of OLETF rats were insensitive to CCK stimulation but not to carbamylcholine stimulation. Basal gastric acid and pepsinogen secretions in OLETF rats were higher than in LETO rats. OLETF rats showed a significantly higher average daily food intake, gained body weight faster, and were heavier than LETO rats. The present study confirmed that OLETF rats have CCK-A receptor gene anomalies and demonstrated deficient CCK-A receptor-mediated biological function in the pancreas, stomach, and brain.

A 62-year-old male was referred to our hospital because of liver dysfunction, diffuse pancreatic swelling, and trachelophyma. At admission, the patient was free of pain. Physical examination showed enlarged and palpable bilateral submandibular masses, but no palpable mass or organomegaly in the abdomen. Laboratory findings were as follows: total protein 90 g/L with gamma-globulin of 37.3% (33 g/L), total bilirubin 4 mg/L, aspartate aminotransferase 39 IU/L, alanine aminotransferase 67 IU/L, gamma-glutamyl transpeptidase 1 647 IU/L, and amylase 135 IU/L. Autoantibodies were negative, and tumor markers were within the normal range. Serum IgG4 level was markedly elevated (18 900 mg/L). Computed tomography (CT) showed diffuse swelling of the pancreas and dilatation of both common and intra-hepatic bile ducts. Endoscopic retrograde pancreatography (ERP) revealed diffuse irregular and narrow main pancreatic duct and stenosis of the lower common bile duct. Biopsy specimens from the pancreas, salivary gland and liver showed marked periductal IgG4-positive plasma cell infiltration with fibrosis. We considered this patient to be autoimmune pancreatitis (AIP) with fibrosclerosis of the salivary gland and biliary tract, prescribed prednisolone at an initial dose of 40 mg/d. Three months later, the laboratory data improved almost to normal. Abdominal CT reflected prominent improvement in the pancreatic lesion. Swelling of the salivary gland also improved. At present, the patient is on 10 mg/d of prednisolone without recurrence of the pancreatitis. We present here a case of AIP with fibrosclerosis of salivary gland and biliary tract.