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Novel Expression of Protein C in Pancreatic Acinar Cells and Its Suppression During Acute Pancreatitis
exposed to 250 μM H2O2 with or without the Ca2+ chelator BAPTA-AM, the calpain inhibitors PD150606, calpeptin or z-VF methyl ester, and the pan-caspase inhibitor z-VADfmk. Calpain activation was detected by a fluorometric assay, autolysis of the catalytic subunit of m-calpain and calpain-specific cleavage of αII-spectrin. Apoptosis was evaluated by electron-microscopic analysis, detection of caspase-3-specific αII-spectrin breakdown and activation of caspase-12, -8 and -3 studied by specific fluorometric assays. Cellular viability was assessed by the trypan blue exclusion test and measuring the mitochondrial dehydrogenase activity (WST-1 assay). Results: H2O2 induced a 2-fold increase in the calpain activity compared with the vehicle-treated cultures (P<0.05) immediately after starting the experiments, and after 1 and 8 h. Western blot analysis showed that the ubiquitous mcalpain isoform was responsible for this. Whereas no activities of caspase-12, -8 and -3 were measured within 0.5 h, a 5.7-fold (P<0.01), 4.7-fold (P<0.001) and 5.2-fold (P<0.001) elevation was observed within 8 h, respectively, compared with the control. At the same time, cells showed first ultrastructural hallmarks of apoptosis and a decrease in the viability. In addition, a fragmentation of the actin cytoskeleton-associated protein αII-spectrin was found by immunoblotting that could be attributed to both calpain and caspase-3. Inhibition of the calpain activity by BAPTA-AM or different calpain inhibitors decreased the activities of caspase-12, -8 and -3 that was accompanied by a decrease in the ultrastructural alterations. Similarly, inhibition of caspases by z-VAD-fmk exerted protection against H2O2-induced apoptosis as well. Blocking calpain and caspases was associated with an increase in the cellular survival rate but failed to increase the mitochondrial dehydrogenase activity. Conclusions: In AR42J cells, H2O2-induced apoptosis requires an activation of calpain that consequently leads to activation of the endoplasmic reticulum stress-induced caspase-12 pathway and the extrinsic caspase-8 pathway in a mitochondria-independent manner. The findings suggest that calpain may be involved in both the signalling process and the execution phase of apoptosis. Elucidating the specific apoptotic pathways induced by oxidative stress may have therapeutic implications for oxidant-induced diseases including acute pancreatitis.
Sa2092 Tobacco Carcinogen NNK (4-[Methylnitrosamino]-1-[3-Pyridyl]-1-Butanone) Initiates and Sensitizes Acute Pancreatitis Responses Martine Alexandre, Samantha N. Minervini, Vikhil Patel, Abdul-Kareem Uduman, Christine Shugrue, Fred S. Gorelick, Edwin C. Thrower Clinical studies suggest that cigarette smoking increases the risk for developing acute pancreatitis. A major constituent of tobacco is the compound NNK (4-[methylnitrosamino]-1-[3pyridyl]-1-butanone) which is a potent agonist of several signaling pathways that could mediate the effects of smoking-related pancreatitis. We hypothesized that NNK could directly mediate or sensitize to pancreatitis responses. The effects of NNK were first examined in isolated rat pancreatic acinar cells. NNK caused a concentration-dependent (0.1 nM-100 nM) increase of up to 3-fold in trypsinogen activation over control. Further, NNK pretreatment sensitized acinar cells to cerulein (100 nM)-induced trypsinogen activation. NNK (100 nM) increased amylase secretion when given alone but did not enhance ceruleininduced amylase secretion. Similar effects of NNK on zymogen activation were seen in acinar cells treated with hyperstimulatory concentrations of the muscarinic agonist carbachol (1 mM). In-Vivo studies showed that NNK given to rats by IP injection (100mg/kg body weight) caused a 6-fold increase in trypsinogen activation within 4 hours. NNK alone did not cause edema or inflammation, but pre-treatment of animals with NNK, followed by cerulein (40 μg/kg) tended to increase levels of edema, inflammation and necrosis compared to cerulein alone. Potential targets of NNK include non-neuronal nicotinic acetylcholine receptors and β adrenergic receptors; both types of receptors were detected in the acinar cell using PCR. NNK mediated acinar cell responses were unaffected by the β-blocker propranolol, but reduced by the nicotinic receptor antagonist mecamylamine, suggesting NNK works through a nicotinic receptor. These studies indicate that NNK may act directly on the pancreatic acinar cell to initiate and/or sensitize pancreatitis responses.
Sa2095 Ethanol/CCK Treatment Impaired Breakdown of Actin Cytoskeleton and CellCell Adhesion via Gα13-Vav-2-RHOA Pathway in Rat Pancreatic Acini Tomoyuki Iwata, Fumihiko Nozu, Michio Imawari Background: Although ethanol is one of common risk factors for acute and chronic pancreatitis, the effect of ethanol for RhoA signaling pathway has not been investigated in pancreatic acini. Aim: We attempted to evaluate interactions of ethanol with RhoA signaling pathway, actin cytoskeleton and cell-cell adherens protein in CCK-stimulated pancreatic acini. Methods: Isolated intact acini were prepared from male Sprague-Dawley rat and preincubated with or without ethanol (EtOH) and stimulated by CCK. Western immunoblotting and immunoprecipitation were performed to detect expressions and associations of Gα13, Vav2, RhoA, catenin p120 (p120), E-cadherin (EC). Translocation of RhoA and Vav-2 were examined by cell fractionation assay. Amylase secretion was also measured. Immunolocalization of Gα13, Vav-2, RhoA, p120, EC and F-actin were analyzed by conforcal laser microscopy. Results: CCK (10 pM, 10 nM) enhanced expressions and associations of Gα13, Vav2, RhoA, p120 and EC. Pretreatment of 20 mM EtOH inhibited their expressions and associations. Furthermore, cell fractionation assay revealed that not only RhoA, but also Vav-2 was mainly located in the cytosol fraction in resting state. After CCK stimulation, RhoA and Vav-2 translocated from cytosol fraction to membrane fraction. Pretreatment of EtOH inhibited CCK-induced translocations of RhoA and Vav-2. Amylase secretion was also inhibited by these treatments without altering basal secretion. Conforcal microscopy images showed that RhoA and Vav-2 were diffusely distributed throughout of acinar cell and Factin located around apical site and lateral lumen. After 10 pM CCK stimulation, RhoA, Vav-2 and F-actin were mainly located to apical site. The distributions of these proteins were diminished after 10 nM CCK stimulation. Pretreatment of EtOH and following 10pM CCK stimulation resulted similar distribution of which 10 nM CCK induced. Although immunolocalization of p120 and EC were well-defined to the lateral lumen in resting state, CCK (10 pM, 10 nM) stimulation and pretreatment of EtOH and following 10pM CCK stimulation, pretreatment of EtOH and following 10 nM CCK stimulation diminished these well-defined localizations. Conclusion: We conclude that ethanol/CCK impaired the assembly and disassembly of actin cytoskeleton and cell-cell adhesion via Gα13, Vav-2, RhoA, p120 and EC in CCK-stimulated pancreatic acini.
Sa2093 Novel Expression of Protein C in Pancreatic Acinar Cells and Its Suppression During Acute Pancreatitis Jun Ienaga, Hitoshi Takahashi, Daiki Okamura, Marlene E. Starr, B. M. Evers, Hiroshi Saito Protein C (PC) is a zymogen of the serine protease activated protein C (aPC) which has multiple functions including anti-coagulation, anti-inflammation, and cell protection. While PC is abundantly present in plasma, aPC is generated from PC cleavage by thrombin upon thrombotic stimuli or tissue injury. The liver is thought to be the major source of PC, and pancreatic production of PC has not previously been reported. The objectives of this study were to compare PC expression in the pancreas and other tissues including liver, and to examine pancreatic PC expression during acute pancreatitis. METHODS: (1) The distribution of PC expression in 14 different organs of male C57BL/6 mice was determined by Western blot analysis. (2) Pancreatic expression of PC was confirmed by immunohistochemical staining. (3) Pancreatic PC expression was further examined in mouse pancreatic acini and islets that were isolated and cultured separately In Vitro. (4) Acute pancreatitis was induced in mice by sequential intraperitoneal injections of the CCK analogue, caerulein (50mg/kg, 9 times, at 1hour intervals), and changes in the level of PC in the pancreas, liver, and plasma were monitored. RESULTS: (1) The pancreas showed the highest PC expression level among all the tissues examined. The pancreatic PC level was 8 times greater than the hepatic level (total protein weight per tissue). By measuring total protein weight of each organ, the pancreas was estimated to produce approximately 64% of total PC in the body, whereas the liver produced only 30%. (2) Immunohistochemical staining revealed that pancreatic expression of PC was localized mainly to acinar cells, rather than islet or duct cells, indicating that acinar cells are the single major source of pancreatic PC. (3) PC production by pancreatic acinar cells, but not islet cells, was also confirmed In Vitro. (4) During acute pancreatitis, pancreatic PC levels diminished dramatically; the levels after caerulein injection decreased to below 5% of the control level (p<0.05). Plasma PC levels also decreased during acute pancreatitis; the levels dropped to below 29% of the control (p<0.05). Time course analysis showed a reduction in plasma PC levels following a decrease in pancreatic PC levels. In contrast, hepatic PC levels remained constant during acute pancreatitis. CONCLUSIONS: This is the first report that the pancreas expresses PC more abundantly than the liver. The time course analysis suggests that pancreas-derived PC has a significant impact on plasma PC levels during acute pancreatitis. Since PC is an important anti-coagulant and antiinflammatory factor, the decrease in pancreatic and plasma PC levels during acute pancreatitis may be causally linked to systemic inflammation and disseminated intravascular coagulation (DIC), a late and fatal complication of severe acute pancreatitis.
Su1029 Effects of Acid-Suppressive Medications Use on Bone Metabolism Daisuke Asaoka, Akihito Nagahara, Taro Osada, Masako Oguro, Mariko Hojo, Takashi Yoshizawa, Michiro Otaka, Sumio Watanabe Background: Acid-suppressive medications is wildly used in treatment for GERD or peptic ulcer. However it has been indicated a relationship between acid-suppressive medications and fracture risk in Europe and the United States. In Japan, osteoporosis patients are increasing as the population grows older. On the other hand it has been indicated a relationship between reflux esophagitis and osteoporosis. In this study, our aim is to demonstrate the relationship between use of acid-suppressive medications and findings from bone metabolism and upper GI endoscopy. Methods: Patients who visited our clinic from Apr. 2008 to Sep. 2010 and being taken all measurements by lumber spine DXA (Bone Mineral Density(BMD), T-score, YAM% and Z-score), by bone metabolism marker (serum NTX and serum BAP) and by upper GI endoscopy were enrolled. These factors were compared between patient who take proton pump inhibitor(PPI) or H2 receptor antagosist(H2RA) as acidsuppressive medications(the use group) and patient who do not take either medications(the non-use group). Patients who previously received steroid drug and/or bisphosphonate and/ or vitamin D, and have disease of thyroid or parathyroid were excluded. Results: The use group was assigned 68 patients (female 32, male 36, mean age 63.9±9.6 years) and the non-use group was assigned 39 patients (female 17, male 22, mean age 62.5±9.0 years). There was no significant difference in age and gender. Compared with the non-use group, the use group was significantly low value on BMD, T-score, YAM % and Z-score (p<0.05). Date are shown in the table. Between both groups, no siginificant differences in bone
Sa2094 Calpain Activation Contributes to Apoptosis in Pancreatic Acinar Cells AR42J Induced by Oxidative Stress Heike Weber, Leon Müller, Peter Schuff-Werner Several studies have shown that oxidative stress induces apoptosis in a variety of cellular systems including pancreatic acinar cells but the exact mechanisms remain incompletely understood. The aim of the present study was to investigate the role of the cytosolic cysteine protease calpain in oxidant-induced apoptosis. Methods: Pancreatic acinar cells, AR42J, were
S-387
AGA Abstracts
AGA Abstracts
of NF-κB is mainly at the transcriptional level but not upstream of it (consistent with the cytosolic location of the NF-κB/IκB complex and the nuclear location of AP-1 proteins). In summary, our In Vitro findings suggest that improved survival following ERK inhibition in murine pancreatitis could result from reduced crosstalk between NF-κB and AP-1 (in addition to inhibiting ERK-mediated activation of NF-κB and AP-1). We conclude that — in exocrine pancreatic cells — NF-κB modulates AP-1 and that AP-1 regulates NF-κB, indicating that crosstalk between these two key nuclear transcription factor pathways may augment proinflammatory mediator production and thus exacerbate acute pancreatitis.
Sa2092 Tobacco Carcinogen NNK (4-[Methylnitrosamino]-1-[3-Pyridyl]-1-Butanone) Initiates and Sensitizes Acute Pancreatitis Responses Martine Alexandre, Samantha N. Minervini, Vikhil Patel, Abdul-Kareem Uduman, Christine Shugrue, Fred S. Gorelick, Edwin C. Thrower Clinical studies suggest that cigarette smoking increases the risk for developing acute pancreatitis. A major constituent of tobacco is the compound NNK (4-[methylnitrosamino]-1-[3pyridyl]-1-butanone) which is a potent agonist of several signaling pathways that could mediate the effects of smoking-related pancreatitis. We hypothesized that NNK could directly mediate or sensitize to pancreatitis responses. The effects of NNK were first examined in isolated rat pancreatic acinar cells. NNK caused a concentration-dependent (0.1 nM-100 nM) increase of up to 3-fold in trypsinogen activation over control. Further, NNK pretreatment sensitized acinar cells to cerulein (100 nM)-induced trypsinogen activation. NNK (100 nM) increased amylase secretion when given alone but did not enhance ceruleininduced amylase secretion. Similar effects of NNK on zymogen activation were seen in acinar cells treated with hyperstimulatory concentrations of the muscarinic agonist carbachol (1 mM). In-Vivo studies showed that NNK given to rats by IP injection (100mg/kg body weight) caused a 6-fold increase in trypsinogen activation within 4 hours. NNK alone did not cause edema or inflammation, but pre-treatment of animals with NNK, followed by cerulein (40 μg/kg) tended to increase levels of edema, inflammation and necrosis compared to cerulein alone. Potential targets of NNK include non-neuronal nicotinic acetylcholine receptors and β adrenergic receptors; both types of receptors were detected in the acinar cell using PCR. NNK mediated acinar cell responses were unaffected by the β-blocker propranolol, but reduced by the nicotinic receptor antagonist mecamylamine, suggesting NNK works through a nicotinic receptor. These studies indicate that NNK may act directly on the pancreatic acinar cell to initiate and/or sensitize pancreatitis responses.
Sa2095 Ethanol/CCK Treatment Impaired Breakdown of Actin Cytoskeleton and CellCell Adhesion via Gα13-Vav-2-RHOA Pathway in Rat Pancreatic Acini Tomoyuki Iwata, Fumihiko Nozu, Michio Imawari Background: Although ethanol is one of common risk factors for acute and chronic pancreatitis, the effect of ethanol for RhoA signaling pathway has not been investigated in pancreatic acini. Aim: We attempted to evaluate interactions of ethanol with RhoA signaling pathway, actin cytoskeleton and cell-cell adherens protein in CCK-stimulated pancreatic acini. Methods: Isolated intact acini were prepared from male Sprague-Dawley rat and preincubated with or without ethanol (EtOH) and stimulated by CCK. Western immunoblotting and immunoprecipitation were performed to detect expressions and associations of Gα13, Vav2, RhoA, catenin p120 (p120), E-cadherin (EC). Translocation of RhoA and Vav-2 were examined by cell fractionation assay. Amylase secretion was also measured. Immunolocalization of Gα13, Vav-2, RhoA, p120, EC and F-actin were analyzed by conforcal laser microscopy. Results: CCK (10 pM, 10 nM) enhanced expressions and associations of Gα13, Vav2, RhoA, p120 and EC. Pretreatment of 20 mM EtOH inhibited their expressions and associations. Furthermore, cell fractionation assay revealed that not only RhoA, but also Vav-2 was mainly located in the cytosol fraction in resting state. After CCK stimulation, RhoA and Vav-2 translocated from cytosol fraction to membrane fraction. Pretreatment of EtOH inhibited CCK-induced translocations of RhoA and Vav-2. Amylase secretion was also inhibited by these treatments without altering basal secretion. Conforcal microscopy images showed that RhoA and Vav-2 were diffusely distributed throughout of acinar cell and Factin located around apical site and lateral lumen. After 10 pM CCK stimulation, RhoA, Vav-2 and F-actin were mainly located to apical site. The distributions of these proteins were diminished after 10 nM CCK stimulation. Pretreatment of EtOH and following 10pM CCK stimulation resulted similar distribution of which 10 nM CCK induced. Although immunolocalization of p120 and EC were well-defined to the lateral lumen in resting state, CCK (10 pM, 10 nM) stimulation and pretreatment of EtOH and following 10pM CCK stimulation, pretreatment of EtOH and following 10 nM CCK stimulation diminished these well-defined localizations. Conclusion: We conclude that ethanol/CCK impaired the assembly and disassembly of actin cytoskeleton and cell-cell adhesion via Gα13, Vav-2, RhoA, p120 and EC in CCK-stimulated pancreatic acini.
Sa2093 Novel Expression of Protein C in Pancreatic Acinar Cells and Its Suppression During Acute Pancreatitis Jun Ienaga, Hitoshi Takahashi, Daiki Okamura, Marlene E. Starr, B. M. Evers, Hiroshi Saito Protein C (PC) is a zymogen of the serine protease activated protein C (aPC) which has multiple functions including anti-coagulation, anti-inflammation, and cell protection. While PC is abundantly present in plasma, aPC is generated from PC cleavage by thrombin upon thrombotic stimuli or tissue injury. The liver is thought to be the major source of PC, and pancreatic production of PC has not previously been reported. The objectives of this study were to compare PC expression in the pancreas and other tissues including liver, and to examine pancreatic PC expression during acute pancreatitis. METHODS: (1) The distribution of PC expression in 14 different organs of male C57BL/6 mice was determined by Western blot analysis. (2) Pancreatic expression of PC was confirmed by immunohistochemical staining. (3) Pancreatic PC expression was further examined in mouse pancreatic acini and islets that were isolated and cultured separately In Vitro. (4) Acute pancreatitis was induced in mice by sequential intraperitoneal injections of the CCK analogue, caerulein (50mg/kg, 9 times, at 1hour intervals), and changes in the level of PC in the pancreas, liver, and plasma were monitored. RESULTS: (1) The pancreas showed the highest PC expression level among all the tissues examined. The pancreatic PC level was 8 times greater than the hepatic level (total protein weight per tissue). By measuring total protein weight of each organ, the pancreas was estimated to produce approximately 64% of total PC in the body, whereas the liver produced only 30%. (2) Immunohistochemical staining revealed that pancreatic expression of PC was localized mainly to acinar cells, rather than islet or duct cells, indicating that acinar cells are the single major source of pancreatic PC. (3) PC production by pancreatic acinar cells, but not islet cells, was also confirmed In Vitro. (4) During acute pancreatitis, pancreatic PC levels diminished dramatically; the levels after caerulein injection decreased to below 5% of the control level (p<0.05). Plasma PC levels also decreased during acute pancreatitis; the levels dropped to below 29% of the control (p<0.05). Time course analysis showed a reduction in plasma PC levels following a decrease in pancreatic PC levels. In contrast, hepatic PC levels remained constant during acute pancreatitis. CONCLUSIONS: This is the first report that the pancreas expresses PC more abundantly than the liver. The time course analysis suggests that pancreas-derived PC has a significant impact on plasma PC levels during acute pancreatitis. Since PC is an important anti-coagulant and antiinflammatory factor, the decrease in pancreatic and plasma PC levels during acute pancreatitis may be causally linked to systemic inflammation and disseminated intravascular coagulation (DIC), a late and fatal complication of severe acute pancreatitis.
Su1029 Effects of Acid-Suppressive Medications Use on Bone Metabolism Daisuke Asaoka, Akihito Nagahara, Taro Osada, Masako Oguro, Mariko Hojo, Takashi Yoshizawa, Michiro Otaka, Sumio Watanabe Background: Acid-suppressive medications is wildly used in treatment for GERD or peptic ulcer. However it has been indicated a relationship between acid-suppressive medications and fracture risk in Europe and the United States. In Japan, osteoporosis patients are increasing as the population grows older. On the other hand it has been indicated a relationship between reflux esophagitis and osteoporosis. In this study, our aim is to demonstrate the relationship between use of acid-suppressive medications and findings from bone metabolism and upper GI endoscopy. Methods: Patients who visited our clinic from Apr. 2008 to Sep. 2010 and being taken all measurements by lumber spine DXA (Bone Mineral Density(BMD), T-score, YAM% and Z-score), by bone metabolism marker (serum NTX and serum BAP) and by upper GI endoscopy were enrolled. These factors were compared between patient who take proton pump inhibitor(PPI) or H2 receptor antagosist(H2RA) as acidsuppressive medications(the use group) and patient who do not take either medications(the non-use group). Patients who previously received steroid drug and/or bisphosphonate and/ or vitamin D, and have disease of thyroid or parathyroid were excluded. Results: The use group was assigned 68 patients (female 32, male 36, mean age 63.9±9.6 years) and the non-use group was assigned 39 patients (female 17, male 22, mean age 62.5±9.0 years). There was no significant difference in age and gender. Compared with the non-use group, the use group was significantly low value on BMD, T-score, YAM % and Z-score (p<0.05). Date are shown in the table. Between both groups, no siginificant differences in bone
Sa2094 Calpain Activation Contributes to Apoptosis in Pancreatic Acinar Cells AR42J Induced by Oxidative Stress Heike Weber, Leon Müller, Peter Schuff-Werner Several studies have shown that oxidative stress induces apoptosis in a variety of cellular systems including pancreatic acinar cells but the exact mechanisms remain incompletely understood. The aim of the present study was to investigate the role of the cytosolic cysteine protease calpain in oxidant-induced apoptosis. Methods: Pancreatic acinar cells, AR42J, were
S-387
AGA Abstracts
AGA Abstracts
of NF-κB is mainly at the transcriptional level but not upstream of it (consistent with the cytosolic location of the NF-κB/IκB complex and the nuclear location of AP-1 proteins). In summary, our In Vitro findings suggest that improved survival following ERK inhibition in murine pancreatitis could result from reduced crosstalk between NF-κB and AP-1 (in addition to inhibiting ERK-mediated activation of NF-κB and AP-1). We conclude that — in exocrine pancreatic cells — NF-κB modulates AP-1 and that AP-1 regulates NF-κB, indicating that crosstalk between these two key nuclear transcription factor pathways may augment proinflammatory mediator production and thus exacerbate acute pancreatitis.