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Pancreatitis-associated protein-I is expressed in rat pancreas in pseudomonas pneumonia induced Sepsis
concenuntions of caerulin strongly induced CRItSP-28 phosphorylation within 15 m m Similarly, CRHSP-28 was markedly phosphorylated following cousumption of a semi-purified AIN-93 chet, Dual immunoilnorescence microscopy and snbcelMar t~'actionation demonstrated that CRHSP-28 co-localized on vesicular structures in tile apical cytoplasm with early endosomal antigen 1 (EEAI). Association of CRHSP-28 with apical endosomes was Ieversibly disrupted by incubaliou of lobules with the ADP ribosylation tactor inhibitor breMdin A ( 10 Ixg/ml, 20 min). Immurmelectron microscopy revealed a peripheral association of CRttSP 28 with early endusome-like structures in the apical cytoplasm. Comparably smaller amounta of immunoreactivity were associated with the limiting membrane of zymogen granules. Consistent wi(h irnmunofluorescence data, CRHSP-28 was found to accunmlate in clusters on endos<~mes and between juxtaposinoned zymogen granules below the cell apex upon CCK stimulation These data suppm~ that CRHSP-28 phosphorylation is a nntnent-driven event, which correlates with activation of 1he protein within the apical secretory pathway of acina~ cells,
normotensive sepsis model with intratracbeal instillation of Pseudomonas aernginosa in Sprague Dawley rats. Twentydour hours after induction of Pseudomonas pneumonia associated sepsis, the pancreas showed upregularion of Pancreatitis-associated protein (PAP)-I mRNA (2/3 pneumonia vs 0/3 in shams) and of amylase mRNA (3L3 in pneumonia vs 1/ 3 in shams) mRNA samples were normalized for L32. A concomitant increase at tissuePAP-I was confirmed by Western Blot in 717 pneumonia animals (22-270% increase compat~ to the positive control) and 1/5 sham animals (7% increase compare to the positive control). Sepsis was accompanied by a significant decrease in WBC, but not by marked alterations of serum amylase and lipase. Histological examination of the pancreas did not show significant changes in cellular infiltration, edema, and necrosis or apoptosis of acinar cells. Myeloperoxidase content of pancreatic tissue- as marker of leukocyte aggregation- was not statistically different between groups. These findings indicate remote organ injup/in the pancreas in a model of Pseudomonas pneumonia induced sepsis with transcriptional and posttranscriptional modifications. In addition, data suggest that not only the liver but also the pancreas is involved into the acute phase reaction in sepsis.
M2207 M2210 Maintenance and Proliferation of Purified Pancreatic [~-Cells In Vitro Bmr~o Schmied, Karin Blacuer, Michael Kremer, Alexis Ulrich, Huberrus SchmitzWirmenthal, Beate Kuttler, Kaspar Zgraggen, Markns W Buechler
Endogenous NO Production Reduces the CCK Induced Cell-Damage in Pancreatic Exocrine Cells Juergen Schnekenburger, Iris Keiner, igor Buchwalow, Markus M. Lerch
Introduction: Diabetes mellitus is one of the leading metabolic diseases in humans Transplantation of the whole pancreas or Langerhans islets is restricted only to a limited number o} patients. Lately, ettorts have been made in isolating and expanding embD'omc or adult endocrine stem cells in vit~z~,to generate a pool of transplantable cells. However, up to now it has been thought that terminally differentiated endocrine cells do not have the capacity to prohti:mte and theretore an in vitro culture of aduh, terminally-dift~erentiated [:?,-cellshas never been considered. Mateml and Methods: Langerhans Islets of adult hamsters were isolated, purified and hand picked under a light microscope. Islets were dispersed into single cells by dispase digestion and ~-cells extracted by ~sitive immunomagnetic selection with a specific antibody tar hamster {g-cells lmmunohistochemistry, immunofluorescence microscopy and RT-PCR were performed at defined time points during culture. Basal and glucosestimulated secretion at insulin was measured by RIA The proliferation index was measured. Results: At day" zero, imnnmohistochemistry and RT-PCR show that the purified cells are ~
The role of nitric oxide (NO) in the pathogenesis of experimental pancrearitis remains controversial. NO may either act as a reactive oxygen species (ROSY scavenger or potentiate the initial cell damage induced by an oxidative burst. In a pancreatic cell culture system we studied CCK-induced radical production, the role of NO in this process, and its eftect on cell damage. Methods: Exocrine pancreatic AR42J cells were seeded at 50 000 cells per well and preincubated with the cell pem~eant tluorogenic ROS substrate dichlorodihydrofluorescein diacetate (DCF) or the NO specific indicator DAF FM DA. Cells were coincubated with inhibitors of known CCK-activated signalling pathways and stimulated with physiology cal and supramaximal concentrations of CCK The fluorescence emission of the oxidized indicators was quantitated and cell-damage was detected by immunohistochemistry for oxidized DNA with 8-hydroxy guanidine specific antibodies. Results: Pancreatic AR42j cells expressed the constitutive NO synthases l and Ill. The inhibition of NO synthases increased CCK induced radical production and DNA oxidation within 5 rain. Stimulation with CCK in the presence of a NO donor increased the amount of radicals measured with the bispecific indicator DCF but inhibited DNA oxidation completely. The NO specific indicator DAF FM DA showed that CCK did not directly increase NO s}mthesis but reduced NO levels after incubation with a NO donor. Conclusions: These experiments indicate that CCK does not increase endogenous NO production in AR42J cells, that NO and ROS are endogenous antagonists, and that the available NO concentration regulates the degree of CCK-dependent, ROS-induced DNA damage. NO production in exocrine AR42J cells has therelbre a protective ['unction against the CCK mediated ceil-damage by reactive oxygen species. M2211
M2208 Protein Kinase C lsoforms Mediate Tyrosine Phosphorylation of Paxilfin Induced by Physiologically Relevant Ethanol Concentrations in Rat Pancreatic AR4-2J Cells Peter Feick, Eric Henn, Manfred V. Singer
Ettects of CGRP and Leptin on Gene Expression for TNF A and Ob Receptor in AR42J Cells Subjected to Caerulein Overstimulation Joanna Bonior, Jolanta jaworek, Piotr Pierzchalski, Stanislaw Konturek, Wieslaw Pawlik Background: Leptin is capable of protecting the pancreas from the acute damage produced by caerulein overstimulatlon. However the eitects of leptin, on gene expression of tumor necrosis tactv*r alpha (TNF alpha), or of leptin receptor subtyTaes in the pancreatic acmar cells have not been examined. Aims: 1. to investigate the Sane expression of TNF alpha in the pancreatic AR42J cells subjecled to caernlein and leptin stimulation. 2. to assess the effect of leptin, calcitonin gene-related peptide (CGRP) and caerulein overstinmlation on mRNA signal for leptin receptor dfi in pancreatic AR42J cells Methods: 1.Gene expression of I-NF alpha or that of db receptor was measured in AR42J ceils using RT-PCR The mRNA for bactin was determined in all samples as a housekeeping gene. 2 AR42j cells were incubated m standard medium at 37o C for: 0, 1, 3, 5, 12 and 24 h, under basal conditions, or in the prepuce of increasing concentration of caerulein (10 ~0_ 10 " r MY, leptin (10 -s. 10 My, CGRP (10 ~ - 10 ~ M) or combination of above Results: 1. The signals for leptin receptors subb]~es dbl-3 and signal t)_~rTNF alpha have been observed in unstimulated ceils All these signals were more pronounced after 3 h of incubation with caerulein (10" My, leptin (10 MY, CGRP (10 ~ - 10 ~ M) or the combination of above 2. The incubation at these ceils in tile presence of caemlein, leptin or combination of above resulted in the significant increase at gene expression tar all db receptor subtypes. 3.The signal for leptin receptor was significantly and dose-dependently decreased following the exposure of these cells to the combination at caerulein and CGRP. 4. The sigual [or TNF alpha was markedly increased in AR~2J cells subjected to caerulein stimulation and this eftect was reversed by the addition of leptin to incubation medium. Conclusions: 1Gene expression for leptin receptor and for TNF alpha was detected in pancreatic AR42] cells under basal conditions and this was |urther enMnced by caerulein overstimnahion of these ceils 2. Addition of CGRP sigmflcantly decreased fhe mRNA expression ti)r db receptor and TNF alpha mRNA was decreased by leptin in AR42J cells stimulated by caemlein
Acute and chronic administration of ethanol affects protein secretion in pancreatic acinar cells. PKC are key enzymes m stimulus-secretion coupling m rat pancreatic acinar cells with the delta, epsilon and zeta isolorms being the most abundant PKCs m these cells. Experiments in neuronal cell lines and cardiomyocytes have shown that the novel isoforms delta and epsilon are target proteins of physiologically relevant ethanol concentrations (10-25 mM). The aim of our stud), is to determine the effects of physiologically relevant concentrations of ethanol on protein secretion and signal transdnction in the rat pancreatic acinar ceil line AR4-2J. Incubation oI AR4-2J ceils with ethanol (10-400 mM) alone had no effect on basal protein secretion as measured by amylase release into the medium. Bombesin-stimulated amylase secretion was dose-dependently inhibited at ethanol concentrations higher than 100 mM. In contrast, Western blot analyses showed that ethanol alone was able to reduce tyrosine phosphorylation of proteins *Mth apparent molecular masses of 120-130 and 70 kDa both time- and dose-dependently. A dose as low as 20 mM ethanol for 60 rain already increased tyrosine phosphorylation of 120-130 kDa and 70 kDa protein band about 4 and 3dbld, respectively,, as compared to basal phosphorylation in control cells Immunoprecipltation and immunoblotting experiments using specific antibodies showed that the protein band with an apparent molecular mass of' 70 kDa corresponds to the focal adhesion protein paxilfin. Further, protein tyrosine phosphoryIation induced by ethanol could be inhibited with tyrosine kinase inhibitor genistein and PKC-inhibitor Ro 31-8220 (specific for conventional and novel PKC iso{brms), but not with the PKC-inhibitor GO 6976 (specific for conventional PKC isoforms only) Ethanol concentrations as low as 20 mM were able to induce changes in the amount of the novel PKC isoforms delta and epsilon in the menlbrane fraction as well as in the nucleus of AR4-2J cells indicating activation and translocation of these proteins. These data suggest that physiologically' relevant doses of ethanol induce activation of the novel PKC isoforms delta and epsilon leading to tyrosine phosphorylation of paxillin in AR4-2J cells. Since paxilfin is a component of the cell cytoskeleton our data support the notion that ethanol can induce changes in the organization of the cyloskeleton and can thus subsequently affect different cell functions.
M2209 Pancreatitis-Associated Protein-I Is Expressed in Rat Pancreas in Pseudomonas Pneumonia Induced Sepsis Barbara Ttibl, Hans P.<~deker, Dominik Fihpp, Pei Yu, Colin McKerhe, Volker Keim, William J. Sibbald Sepsis is often complicated by development of multiple organ dysflmctinn. However, pancreatic dysfunction in sepsis is not well characterized. Pancreatic exocrine dystnnction has been demonstrated in a torroer clinical study in sepsis and septic shock patients. The purpose of our study was to harlber chal~cterize sepsis associated alterations in the pancreas using a
A-437
AGA
Abstracts
normotensive sepsis model with intratracbeal instillation of Pseudomonas aernginosa in Sprague Dawley rats. Twentydour hours after induction of Pseudomonas pneumonia associated sepsis, the pancreas showed upregularion of Pancreatitis-associated protein (PAP)-I mRNA (2/3 pneumonia vs 0/3 in shams) and of amylase mRNA (3L3 in pneumonia vs 1/ 3 in shams) mRNA samples were normalized for L32. A concomitant increase at tissuePAP-I was confirmed by Western Blot in 717 pneumonia animals (22-270% increase compat~ to the positive control) and 1/5 sham animals (7% increase compare to the positive control). Sepsis was accompanied by a significant decrease in WBC, but not by marked alterations of serum amylase and lipase. Histological examination of the pancreas did not show significant changes in cellular infiltration, edema, and necrosis or apoptosis of acinar cells. Myeloperoxidase content of pancreatic tissue- as marker of leukocyte aggregation- was not statistically different between groups. These findings indicate remote organ injup/in the pancreas in a model of Pseudomonas pneumonia induced sepsis with transcriptional and posttranscriptional modifications. In addition, data suggest that not only the liver but also the pancreas is involved into the acute phase reaction in sepsis.
M2207 M2210 Maintenance and Proliferation of Purified Pancreatic [~-Cells In Vitro Bmr~o Schmied, Karin Blacuer, Michael Kremer, Alexis Ulrich, Huberrus SchmitzWirmenthal, Beate Kuttler, Kaspar Zgraggen, Markns W Buechler
Endogenous NO Production Reduces the CCK Induced Cell-Damage in Pancreatic Exocrine Cells Juergen Schnekenburger, Iris Keiner, igor Buchwalow, Markus M. Lerch
Introduction: Diabetes mellitus is one of the leading metabolic diseases in humans Transplantation of the whole pancreas or Langerhans islets is restricted only to a limited number o} patients. Lately, ettorts have been made in isolating and expanding embD'omc or adult endocrine stem cells in vit~z~,to generate a pool of transplantable cells. However, up to now it has been thought that terminally differentiated endocrine cells do not have the capacity to prohti:mte and theretore an in vitro culture of aduh, terminally-dift~erentiated [:?,-cellshas never been considered. Mateml and Methods: Langerhans Islets of adult hamsters were isolated, purified and hand picked under a light microscope. Islets were dispersed into single cells by dispase digestion and ~-cells extracted by ~sitive immunomagnetic selection with a specific antibody tar hamster {g-cells lmmunohistochemistry, immunofluorescence microscopy and RT-PCR were performed at defined time points during culture. Basal and glucosestimulated secretion at insulin was measured by RIA The proliferation index was measured. Results: At day" zero, imnnmohistochemistry and RT-PCR show that the purified cells are ~
The role of nitric oxide (NO) in the pathogenesis of experimental pancrearitis remains controversial. NO may either act as a reactive oxygen species (ROSY scavenger or potentiate the initial cell damage induced by an oxidative burst. In a pancreatic cell culture system we studied CCK-induced radical production, the role of NO in this process, and its eftect on cell damage. Methods: Exocrine pancreatic AR42J cells were seeded at 50 000 cells per well and preincubated with the cell pem~eant tluorogenic ROS substrate dichlorodihydrofluorescein diacetate (DCF) or the NO specific indicator DAF FM DA. Cells were coincubated with inhibitors of known CCK-activated signalling pathways and stimulated with physiology cal and supramaximal concentrations of CCK The fluorescence emission of the oxidized indicators was quantitated and cell-damage was detected by immunohistochemistry for oxidized DNA with 8-hydroxy guanidine specific antibodies. Results: Pancreatic AR42j cells expressed the constitutive NO synthases l and Ill. The inhibition of NO synthases increased CCK induced radical production and DNA oxidation within 5 rain. Stimulation with CCK in the presence of a NO donor increased the amount of radicals measured with the bispecific indicator DCF but inhibited DNA oxidation completely. The NO specific indicator DAF FM DA showed that CCK did not directly increase NO s}mthesis but reduced NO levels after incubation with a NO donor. Conclusions: These experiments indicate that CCK does not increase endogenous NO production in AR42J cells, that NO and ROS are endogenous antagonists, and that the available NO concentration regulates the degree of CCK-dependent, ROS-induced DNA damage. NO production in exocrine AR42J cells has therelbre a protective ['unction against the CCK mediated ceil-damage by reactive oxygen species. M2211
M2208 Protein Kinase C lsoforms Mediate Tyrosine Phosphorylation of Paxilfin Induced by Physiologically Relevant Ethanol Concentrations in Rat Pancreatic AR4-2J Cells Peter Feick, Eric Henn, Manfred V. Singer
Ettects of CGRP and Leptin on Gene Expression for TNF A and Ob Receptor in AR42J Cells Subjected to Caerulein Overstimulation Joanna Bonior, Jolanta jaworek, Piotr Pierzchalski, Stanislaw Konturek, Wieslaw Pawlik Background: Leptin is capable of protecting the pancreas from the acute damage produced by caerulein overstimulatlon. However the eitects of leptin, on gene expression of tumor necrosis tactv*r alpha (TNF alpha), or of leptin receptor subtyTaes in the pancreatic acmar cells have not been examined. Aims: 1. to investigate the Sane expression of TNF alpha in the pancreatic AR42J cells subjecled to caernlein and leptin stimulation. 2. to assess the effect of leptin, calcitonin gene-related peptide (CGRP) and caerulein overstinmlation on mRNA signal for leptin receptor dfi in pancreatic AR42J cells Methods: 1.Gene expression of I-NF alpha or that of db receptor was measured in AR42J ceils using RT-PCR The mRNA for bactin was determined in all samples as a housekeeping gene. 2 AR42j cells were incubated m standard medium at 37o C for: 0, 1, 3, 5, 12 and 24 h, under basal conditions, or in the prepuce of increasing concentration of caerulein (10 ~0_ 10 " r MY, leptin (10 -s. 10 My, CGRP (10 ~ - 10 ~ M) or combination of above Results: 1. The signals for leptin receptors subb]~es dbl-3 and signal t)_~rTNF alpha have been observed in unstimulated ceils All these signals were more pronounced after 3 h of incubation with caerulein (10" My, leptin (10 MY, CGRP (10 ~ - 10 ~ M) or the combination of above 2. The incubation at these ceils in tile presence of caemlein, leptin or combination of above resulted in the significant increase at gene expression tar all db receptor subtypes. 3.The signal for leptin receptor was significantly and dose-dependently decreased following the exposure of these cells to the combination at caerulein and CGRP. 4. The sigual [or TNF alpha was markedly increased in AR~2J cells subjected to caerulein stimulation and this eftect was reversed by the addition of leptin to incubation medium. Conclusions: 1Gene expression for leptin receptor and for TNF alpha was detected in pancreatic AR42] cells under basal conditions and this was |urther enMnced by caerulein overstimnahion of these ceils 2. Addition of CGRP sigmflcantly decreased fhe mRNA expression ti)r db receptor and TNF alpha mRNA was decreased by leptin in AR42J cells stimulated by caemlein
Acute and chronic administration of ethanol affects protein secretion in pancreatic acinar cells. PKC are key enzymes m stimulus-secretion coupling m rat pancreatic acinar cells with the delta, epsilon and zeta isolorms being the most abundant PKCs m these cells. Experiments in neuronal cell lines and cardiomyocytes have shown that the novel isoforms delta and epsilon are target proteins of physiologically relevant ethanol concentrations (10-25 mM). The aim of our stud), is to determine the effects of physiologically relevant concentrations of ethanol on protein secretion and signal transdnction in the rat pancreatic acinar ceil line AR4-2J. Incubation oI AR4-2J ceils with ethanol (10-400 mM) alone had no effect on basal protein secretion as measured by amylase release into the medium. Bombesin-stimulated amylase secretion was dose-dependently inhibited at ethanol concentrations higher than 100 mM. In contrast, Western blot analyses showed that ethanol alone was able to reduce tyrosine phosphorylation of proteins *Mth apparent molecular masses of 120-130 and 70 kDa both time- and dose-dependently. A dose as low as 20 mM ethanol for 60 rain already increased tyrosine phosphorylation of 120-130 kDa and 70 kDa protein band about 4 and 3dbld, respectively,, as compared to basal phosphorylation in control cells Immunoprecipltation and immunoblotting experiments using specific antibodies showed that the protein band with an apparent molecular mass of' 70 kDa corresponds to the focal adhesion protein paxilfin. Further, protein tyrosine phosphoryIation induced by ethanol could be inhibited with tyrosine kinase inhibitor genistein and PKC-inhibitor Ro 31-8220 (specific for conventional and novel PKC iso{brms), but not with the PKC-inhibitor GO 6976 (specific for conventional PKC isoforms only) Ethanol concentrations as low as 20 mM were able to induce changes in the amount of the novel PKC isoforms delta and epsilon in the menlbrane fraction as well as in the nucleus of AR4-2J cells indicating activation and translocation of these proteins. These data suggest that physiologically' relevant doses of ethanol induce activation of the novel PKC isoforms delta and epsilon leading to tyrosine phosphorylation of paxillin in AR4-2J cells. Since paxilfin is a component of the cell cytoskeleton our data support the notion that ethanol can induce changes in the organization of the cyloskeleton and can thus subsequently affect different cell functions.
M2209 Pancreatitis-Associated Protein-I Is Expressed in Rat Pancreas in Pseudomonas Pneumonia Induced Sepsis Barbara Ttibl, Hans P.<~deker, Dominik Fihpp, Pei Yu, Colin McKerhe, Volker Keim, William J. Sibbald Sepsis is often complicated by development of multiple organ dysflmctinn. However, pancreatic dysfunction in sepsis is not well characterized. Pancreatic exocrine dystnnction has been demonstrated in a torroer clinical study in sepsis and septic shock patients. The purpose of our study was to harlber chal~cterize sepsis associated alterations in the pancreas using a
A-437
AGA
Abstracts