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Pancreatic duct ligated rat under bombesin stimulation causes TAP generation and cytoskeleton disassembly
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blinded pathologist scored pancreatic tissue samples for edema, inflammatory cell infiltrate, vacuolization and necrosis. RESULTS:KO mice showed milder pancreatitis based on biochemical parameters. Interestingly. trypsin did not appear to increase significantly in response to cerulein in the KO mice. Pancreatic trypsin Increased from 0.095 + / - 0.009 to 0.296 +/ - 0.132 rig/rag protein in WT mice and from 0 105 +0.029 to 0.118 +0.13 ng/mg protein in KO mice. Serum amylase increased 4.4 + / - 0.4 fold in wr mice and 3.2 + / - 0.3 fold in KO mice. Pancreatic edema was elevated but not selectively. Pancreatic histologic changes in the KO and WT mice were indistinguishable but characterized by diffuse expansion of interlohutar septa, marked perilobular cellular debri without inflammation or vacuolization. CONCLUSION: Cerulein-induced pancreatitis in nNOS KO mice is less severe at five hours compared with controls with the primary effect being a decrease in active trypsin. Endogenous NO may contribute to early pancreatitis, most likely being produced by nNOS.
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The Role of Nuclear Factor K B and Phosphutidylinositol 3-1dnau ie TrypllNBn Activation In Cerulein-Stimutated Rat Pancreatic Acini Yoon Jun Kim, Seoul National Univ Coil of Medicine, Seoul South Korea; Jin Kim, YongTae Kim, Yong Bum Yoon, Chung Yong Kim Background & Aims: Intra-acinar cell activation of trypsmogen is a critical event in caruleininduced pancreatitis. However little is known about the identities of the intracellular signal transduction systems that are involved in trypsinogen activation. This study focused on the role of NF-KB and PI3-K as intracellular signal transduction systems to activate trypsinogen. Methods: Pancreatic acinar cells were prepared from male Wistar rats and NF-KB inhibitor, pyrolidine dithiocarbamate and N-acetylcysteine,or PI3-K inhibitor, woMmanninand LY294002 were applied to each of a series of acinar cell cultures. Then a suprapbysiological (lOOnM) concentration of cerulein was applied to the pretreated cells and the untreated control cells. Trypsinogen activation was detected by measuring trypsin activity from acinar cell homogee nates and expressed as arbitrary units/time/DNA. The level of NF-KB activity was checked from the nuclear extracts using EMSA. Results: NF-KB activation in the aciuar calls after cerulein hyperstimulation displayed a biphasic process over time. Both NF-KB inhibitors partially suppressed trypsinogen activation while the PI3-K inhibitors inhibited trypsinogen activation almost completely. NF-KB activity was blocked by pyrolidine dithiocarbomete or Nacetytcysteine but not by wortmannin or LY294002. Conclusion: These results suggest that trypsinogen activation in cerulein hyperstimuleted acinar cells requires both PI3-K and NF-K8 pathways independently.
2745 Pancreatic Duct Ligated Rat Under Bombesin Stimulation Causes TAP Generation And Cyloskeleton Disassembly Taiichi Otani, Yasuhito Shimizu, Takaaki Sugiki, Fumiaki Ozawa, Akira Matsukura, Takeshi Takamoto, Hidehiro Shinozaki, Atsuko Takeuchi, Hiroshi Usui, Masatoshi Makuuchi, Univ of Tokyo, Tokyo Japan; Suresh Karne, Fred S. Gorelick, Yale UniversdyNAMC, West Haven, CT Ceruleincausesenzymes activation within the acinar cell, inhibited secretion of active enz~es, and pancreatitis. Bombesin causes enzyme activation, but the enzymes are secreted from the cell and there is no pancreatitis. Duct obstruction may cause inhibition of enzyme secretion from the acinar cell. In preliminary studies, we have found that bombesin treatment in the pancreatic duct ligated rat results in pancreatitis (Pancreas 21:460,2000). To examine this issue further, rats were subject to sham operation or pancreatic duet ligation (ligate). A continuous IV infusion of saline (sham and ligate), bombesin (5 pg/kg/h - sham or ligate) or cerulein hyperstimulation (5 p,0/kg/h - sham) were administered. After 3hrs in vivo pertusion fixation was done and frozen sections of the pancreas were labeled using antibodies to trypsinogen activation peptide (TAP) or phalloidin (f-actin). In sham pancreas, virtually no TAP immunotluorescence was observed; f-actin fluorescence was normally distributed at luminal and lateral cellular membranes. After cerulein-hyperstimuiation, TAP immunoreactivity was diffusely distributed in the acin cell (AJP 275:G999,1998); actin labeling was also diffuse. After bombesin stimulation, TAP was present in some vacuoles; actin did not differ from sham. After duct-ligatation, there was virtually no TAP; actin did not differ from sham. Finally, in bombesin stimulated duct-ligated, TAP was frequently localized to vacuoles in acinar cells and actin was diffuse. These studies suggest that duct obstruction may lead to inhibited secretion of active enzyme and the formation of cytoplasmic vacuoles. Distruption of the secretory mechanisms and the apical actin cytoskeleton may cause the active enzymes to be retained in the acinar cell and be required for the initiation of pancreatitis.
Inhibition Of PIIolpholnositide-3-Klnasa (PI-3K) Protects Against Both CaeruleinImluced Aid Taurockolute-lndusad Acute Experimental Pancreatitis. Vijay P. Singh, Ashok K. Saluja, Gijs Jd Van Acker, Lakshmi Bhagat, Albert M. Song, Andreas Mykoniatis, Michael L. Steer, Beth Israel Deaconess Medical Ctr and Harvard Medical Sch, Boston, MA BACKGROUND:The PI-3Ks are a widely expressed group of enzymes, which play important roles in signal traneduction. The role of PI-3Ks in pancreatitis, however, has not been previously noted. We have used the fungal metabolite, wortmannin, to investigate this issue since, at low concentrabons, wortmannin, is supposed to act solely as a PI-3K inhibitor. METHODS: Two experimental models of pancreatitis were employed. Secretagogue induced pancreatitis was elicited by giving mice hourly (x6) intraperitoneal injections of a supramaximally-stimulating dose (50/~l/kg) of caerutein. Duct injection-induced pancreatitis was elicited by retrograde infusion of the pancreatic duct of rats (200-250 grams) with 5% Na-taurocholate at a rate of 0.1ml/minete for 2 minutes. When given, wortmannin (1.5 mg/kg/i.p.) was administered 4 hours before the induction of pancreatitis. In the duct injection model an additional injection of wortmannin was given 12 hours after the pancreatic duct infusion. Animals were sacrificed either 1 hour after the last caerulein injection or 20 hours after the Na-taurocholate injection. The severity of paocreatitis was evaluated by measuring pancreatic myeloperoxidase (MPO) activity (an index of neetrophil sequestration into the pancreas), pancreatic trypsin activity (spactrofluommefrically), and the extent of pancreatic acinar cell necrosis and pancreatic inflammebon (by morphometry). RESULTS:Administration of wortmannin resulted in a reduction in the rise in pancreatic MPO activity, a reduction in the rise in trypsin activity, and a reduction in the extent of pancreatic acinar cell necrosis and pancreatic inflammation. CONCLUSION:PI-3Ks play an important role in regulating the severity of these two dissimilar models of experimental acute pancreatitis. It is likely that PI-3Ks also play an important role in regulating the severity of clinical acute pancreatitis
Lysssamat Hydrolase / Oige~iw Zymogen Co-Localization And Intra-Acinar Cell Activation Of Tqtllninogen: Which Is The Horse And Which Is The Cad? Lakshmi Bhagat, Ashok K. Saluja, Gijs Jd Van Acker, Vijay P. Singh, Albert M. Song, Andruas Mykoniatis, Michael L. Steer, Beth Israel Deaconess Medical Ctr and Harvard Medical Sch, Boston, MA During the very early stages of secretagogue (caerulein)-induced pancreatitis and after in v/tro supramaximal stimulation of acini with caerulein, two phenomena are observed: (a) the co-localization of lysosomal hydmlases with digestive enzyme zymogens in cytoplasmic vacuoles and (b) intra-acinar cell cathepsin B-mediated activation of trypsinogen. The relationship between these two phenomena is controversial. We have argued that co-localization leads to cathepsin B-mediated trypsinogen activation and cell injury while others (Luthen et al., Gastroeeterology 1995, 109:573-581) have suggested that enzyme activation and cell injury lead to the co-localization phenomenon. In the current studies, the cathepsin B inhibitors E64d and CA-O74me were used to prevent in vitro caerulein-induced trypsinogen activation in acini and the effects of that treatment on the co-localization phenomenon (monitored in subcellular fractionation experiments tracking the distribution of the lysosomal hydrolase aryl suitatase) were evaluated. METHODS:Rat pancreatic acini, prepared by collagenase digestion were preiocubeted with or without E64d (1 mM) or CA-074 (0.5 mM) for 15 minutes and then supramaximally stimulated with caerulein (0.1 p.M) for 30 minutes. They were then homogenized, subcellularly fractionated, and the distribution of aryl sulfatase in the various fractions determined spectrofluorometrically. Trypsin activity and trypsinogen activation peptide (TAP) levels were determined by spectrofluommutric assay and by ELISA respectively. RESULTS:Supramaximal stimulation of acini with caerulein resulted in the shift of aryf suffatase activity from the lysosome-enriched to the zymogen granule-enrichedfraction and trypsinogen activation. Preincubation of acini with either E64d or CA-O74meprevented trypsinogen activation bet did not alter the redistribution of aryl sulfatase. CONCLUSIONS:These results indicate that trypsinogen activation does not lead to subcellular redistribution of lysosomal hydrolases such as aryl sulfatase (and by extension, cathepsin B). Rather, these findings indicate that redistribution (and presumably, co-localization) leads to trypsinogen activation and that this activation is dependent upon the presence of active cathepsin B.
2746 Reduced Active Trypsin in Experimental Pancreutitis in Mice Lacking nNOS Matthew J. DiMagno, John A. Williams, Chung Owyang, Univ of Michigan Health System, Ann Arbor, MI BACKGROUND: Pharmacologic inhibition of nitric oxide synthase (NOS) or enhancement of nitric oxide (NO) in acute experimental pancreatitis has yielded mixed results. NOS inhibitors have not proven sufficiently specific to clarity the role of endogenous (NOS-derived) NO in acute pancreatitis, in part becausethree NOS isoforms exist: neuronal- (nNOS), endothelial(eNOS) and inducible- (iNOS) NOS. NNOS expression has correlated with disease severity in models of reactive airway disease, stroke and inflammatory bowel disease. We therefore investigated a possible protective effect of nNOS deletion in pancreatitis. METHODS:66,129S knockout (KO) mice with targeted deletion of the nNOS gene were compared to B6,129S controls (WT). Mice received variable hourly intraperitoneal injections of either cerulein (50 ug/kg) or saline and were sacrificed one hour after the final injection. A time course experiment with WT mice showed that biochemical parameters of pancreatitis were established but not maxima/at five hours. Evidence of pancreatic edema, trypsin activation and actin cytoskeleton disruption occurred within one-half hour of the first cerulein injection and serum amylase elevation occurred between one and three hours. An end point of five hours was chosen in order to discern a biochemical difference in outcome. Parameters of pancreatitis measured in this experiment included pancreatic trypsin, pancreatic wet weight and serum amylase. A
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blinded pathologist scored pancreatic tissue samples for edema, inflammatory cell infiltrate, vacuolization and necrosis. RESULTS:KO mice showed milder pancreatitis based on biochemical parameters. Interestingly. trypsin did not appear to increase significantly in response to cerulein in the KO mice. Pancreatic trypsin Increased from 0.095 + / - 0.009 to 0.296 +/ - 0.132 rig/rag protein in WT mice and from 0 105 +0.029 to 0.118 +0.13 ng/mg protein in KO mice. Serum amylase increased 4.4 + / - 0.4 fold in wr mice and 3.2 + / - 0.3 fold in KO mice. Pancreatic edema was elevated but not selectively. Pancreatic histologic changes in the KO and WT mice were indistinguishable but characterized by diffuse expansion of interlohutar septa, marked perilobular cellular debri without inflammation or vacuolization. CONCLUSION: Cerulein-induced pancreatitis in nNOS KO mice is less severe at five hours compared with controls with the primary effect being a decrease in active trypsin. Endogenous NO may contribute to early pancreatitis, most likely being produced by nNOS.
2744 2747
The Role of Nuclear Factor K B and Phosphutidylinositol 3-1dnau ie TrypllNBn Activation In Cerulein-Stimutated Rat Pancreatic Acini Yoon Jun Kim, Seoul National Univ Coil of Medicine, Seoul South Korea; Jin Kim, YongTae Kim, Yong Bum Yoon, Chung Yong Kim Background & Aims: Intra-acinar cell activation of trypsmogen is a critical event in caruleininduced pancreatitis. However little is known about the identities of the intracellular signal transduction systems that are involved in trypsinogen activation. This study focused on the role of NF-KB and PI3-K as intracellular signal transduction systems to activate trypsinogen. Methods: Pancreatic acinar cells were prepared from male Wistar rats and NF-KB inhibitor, pyrolidine dithiocarbamate and N-acetylcysteine,or PI3-K inhibitor, woMmanninand LY294002 were applied to each of a series of acinar cell cultures. Then a suprapbysiological (lOOnM) concentration of cerulein was applied to the pretreated cells and the untreated control cells. Trypsinogen activation was detected by measuring trypsin activity from acinar cell homogee nates and expressed as arbitrary units/time/DNA. The level of NF-KB activity was checked from the nuclear extracts using EMSA. Results: NF-KB activation in the aciuar calls after cerulein hyperstimulation displayed a biphasic process over time. Both NF-KB inhibitors partially suppressed trypsinogen activation while the PI3-K inhibitors inhibited trypsinogen activation almost completely. NF-KB activity was blocked by pyrolidine dithiocarbomete or Nacetytcysteine but not by wortmannin or LY294002. Conclusion: These results suggest that trypsinogen activation in cerulein hyperstimuleted acinar cells requires both PI3-K and NF-K8 pathways independently.
2745 Pancreatic Duct Ligated Rat Under Bombesin Stimulation Causes TAP Generation And Cyloskeleton Disassembly Taiichi Otani, Yasuhito Shimizu, Takaaki Sugiki, Fumiaki Ozawa, Akira Matsukura, Takeshi Takamoto, Hidehiro Shinozaki, Atsuko Takeuchi, Hiroshi Usui, Masatoshi Makuuchi, Univ of Tokyo, Tokyo Japan; Suresh Karne, Fred S. Gorelick, Yale UniversdyNAMC, West Haven, CT Ceruleincausesenzymes activation within the acinar cell, inhibited secretion of active enz~es, and pancreatitis. Bombesin causes enzyme activation, but the enzymes are secreted from the cell and there is no pancreatitis. Duct obstruction may cause inhibition of enzyme secretion from the acinar cell. In preliminary studies, we have found that bombesin treatment in the pancreatic duct ligated rat results in pancreatitis (Pancreas 21:460,2000). To examine this issue further, rats were subject to sham operation or pancreatic duet ligation (ligate). A continuous IV infusion of saline (sham and ligate), bombesin (5 pg/kg/h - sham or ligate) or cerulein hyperstimulation (5 p,0/kg/h - sham) were administered. After 3hrs in vivo pertusion fixation was done and frozen sections of the pancreas were labeled using antibodies to trypsinogen activation peptide (TAP) or phalloidin (f-actin). In sham pancreas, virtually no TAP immunotluorescence was observed; f-actin fluorescence was normally distributed at luminal and lateral cellular membranes. After cerulein-hyperstimuiation, TAP immunoreactivity was diffusely distributed in the acin cell (AJP 275:G999,1998); actin labeling was also diffuse. After bombesin stimulation, TAP was present in some vacuoles; actin did not differ from sham. After duct-ligatation, there was virtually no TAP; actin did not differ from sham. Finally, in bombesin stimulated duct-ligated, TAP was frequently localized to vacuoles in acinar cells and actin was diffuse. These studies suggest that duct obstruction may lead to inhibited secretion of active enzyme and the formation of cytoplasmic vacuoles. Distruption of the secretory mechanisms and the apical actin cytoskeleton may cause the active enzymes to be retained in the acinar cell and be required for the initiation of pancreatitis.
Inhibition Of PIIolpholnositide-3-Klnasa (PI-3K) Protects Against Both CaeruleinImluced Aid Taurockolute-lndusad Acute Experimental Pancreatitis. Vijay P. Singh, Ashok K. Saluja, Gijs Jd Van Acker, Lakshmi Bhagat, Albert M. Song, Andreas Mykoniatis, Michael L. Steer, Beth Israel Deaconess Medical Ctr and Harvard Medical Sch, Boston, MA BACKGROUND:The PI-3Ks are a widely expressed group of enzymes, which play important roles in signal traneduction. The role of PI-3Ks in pancreatitis, however, has not been previously noted. We have used the fungal metabolite, wortmannin, to investigate this issue since, at low concentrabons, wortmannin, is supposed to act solely as a PI-3K inhibitor. METHODS: Two experimental models of pancreatitis were employed. Secretagogue induced pancreatitis was elicited by giving mice hourly (x6) intraperitoneal injections of a supramaximally-stimulating dose (50/~l/kg) of caerutein. Duct injection-induced pancreatitis was elicited by retrograde infusion of the pancreatic duct of rats (200-250 grams) with 5% Na-taurocholate at a rate of 0.1ml/minete for 2 minutes. When given, wortmannin (1.5 mg/kg/i.p.) was administered 4 hours before the induction of pancreatitis. In the duct injection model an additional injection of wortmannin was given 12 hours after the pancreatic duct infusion. Animals were sacrificed either 1 hour after the last caerulein injection or 20 hours after the Na-taurocholate injection. The severity of paocreatitis was evaluated by measuring pancreatic myeloperoxidase (MPO) activity (an index of neetrophil sequestration into the pancreas), pancreatic trypsin activity (spactrofluommefrically), and the extent of pancreatic acinar cell necrosis and pancreatic inflammebon (by morphometry). RESULTS:Administration of wortmannin resulted in a reduction in the rise in pancreatic MPO activity, a reduction in the rise in trypsin activity, and a reduction in the extent of pancreatic acinar cell necrosis and pancreatic inflammation. CONCLUSION:PI-3Ks play an important role in regulating the severity of these two dissimilar models of experimental acute pancreatitis. It is likely that PI-3Ks also play an important role in regulating the severity of clinical acute pancreatitis
Lysssamat Hydrolase / Oige~iw Zymogen Co-Localization And Intra-Acinar Cell Activation Of Tqtllninogen: Which Is The Horse And Which Is The Cad? Lakshmi Bhagat, Ashok K. Saluja, Gijs Jd Van Acker, Vijay P. Singh, Albert M. Song, Andruas Mykoniatis, Michael L. Steer, Beth Israel Deaconess Medical Ctr and Harvard Medical Sch, Boston, MA During the very early stages of secretagogue (caerulein)-induced pancreatitis and after in v/tro supramaximal stimulation of acini with caerulein, two phenomena are observed: (a) the co-localization of lysosomal hydmlases with digestive enzyme zymogens in cytoplasmic vacuoles and (b) intra-acinar cell cathepsin B-mediated activation of trypsinogen. The relationship between these two phenomena is controversial. We have argued that co-localization leads to cathepsin B-mediated trypsinogen activation and cell injury while others (Luthen et al., Gastroeeterology 1995, 109:573-581) have suggested that enzyme activation and cell injury lead to the co-localization phenomenon. In the current studies, the cathepsin B inhibitors E64d and CA-O74me were used to prevent in vitro caerulein-induced trypsinogen activation in acini and the effects of that treatment on the co-localization phenomenon (monitored in subcellular fractionation experiments tracking the distribution of the lysosomal hydrolase aryl suitatase) were evaluated. METHODS:Rat pancreatic acini, prepared by collagenase digestion were preiocubeted with or without E64d (1 mM) or CA-074 (0.5 mM) for 15 minutes and then supramaximally stimulated with caerulein (0.1 p.M) for 30 minutes. They were then homogenized, subcellularly fractionated, and the distribution of aryl sulfatase in the various fractions determined spectrofluorometrically. Trypsin activity and trypsinogen activation peptide (TAP) levels were determined by spectrofluommutric assay and by ELISA respectively. RESULTS:Supramaximal stimulation of acini with caerulein resulted in the shift of aryf suffatase activity from the lysosome-enriched to the zymogen granule-enrichedfraction and trypsinogen activation. Preincubation of acini with either E64d or CA-O74meprevented trypsinogen activation bet did not alter the redistribution of aryl sulfatase. CONCLUSIONS:These results indicate that trypsinogen activation does not lead to subcellular redistribution of lysosomal hydrolases such as aryl sulfatase (and by extension, cathepsin B). Rather, these findings indicate that redistribution (and presumably, co-localization) leads to trypsinogen activation and that this activation is dependent upon the presence of active cathepsin B.
2746 Reduced Active Trypsin in Experimental Pancreutitis in Mice Lacking nNOS Matthew J. DiMagno, John A. Williams, Chung Owyang, Univ of Michigan Health System, Ann Arbor, MI BACKGROUND: Pharmacologic inhibition of nitric oxide synthase (NOS) or enhancement of nitric oxide (NO) in acute experimental pancreatitis has yielded mixed results. NOS inhibitors have not proven sufficiently specific to clarity the role of endogenous (NOS-derived) NO in acute pancreatitis, in part becausethree NOS isoforms exist: neuronal- (nNOS), endothelial(eNOS) and inducible- (iNOS) NOS. NNOS expression has correlated with disease severity in models of reactive airway disease, stroke and inflammatory bowel disease. We therefore investigated a possible protective effect of nNOS deletion in pancreatitis. METHODS:66,129S knockout (KO) mice with targeted deletion of the nNOS gene were compared to B6,129S controls (WT). Mice received variable hourly intraperitoneal injections of either cerulein (50 ug/kg) or saline and were sacrificed one hour after the final injection. A time course experiment with WT mice showed that biochemical parameters of pancreatitis were established but not maxima/at five hours. Evidence of pancreatic edema, trypsin activation and actin cytoskeleton disruption occurred within one-half hour of the first cerulein injection and serum amylase elevation occurred between one and three hours. An end point of five hours was chosen in order to discern a biochemical difference in outcome. Parameters of pancreatitis measured in this experiment included pancreatic trypsin, pancreatic wet weight and serum amylase. A
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