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Lack of mild irritant-induced adaptive cytoprotection in IP receptor knockout mice. —The involvement of endogenous calcitonin gene-related peptide—
A232 AGA ABSTRACTS
1383 MODULATION OF INTESTINAL EPITHELIAL TIGHT JUNCTION PERMEABILITY IS ASSOCIATED WITH BIOCHEMICAL AND MORPHOLOGIC ALTERATION OF OCCLUDIN PROTEIN. Babu Bassa, Neil T. Hoa, Daniel Tran, Margaret Merryfield, Nathan Nguyen, Thomas Y. Ma, Long Beach VA Med Ctr, Long Beach, CA; Univ of CA, Irvine, Irvine, CA. The apico-Iaterally located tight junctions (TJ) form a paracellular seal between the lateral membranes of the adjacent cells. This seal acts as a structural barrier against the paracellular penetration of luminal substances. Disruption of the TJ barrier leads to rapid influx of toxic luminal substances resulting in mucosal injury. Occludin is a transmembrane TJ protein which appears to be involved in forming the intercellular seal (J Cell BioI 146;683-693,1999). In this study, we investigated the sequential alteration of occludin proteins during Cytochalasin-B (Cyto-B) induced modulation of intestinal monolayer TJ permeability, using an in-vitro intestinal epithelial model (consisting of Caco-Z intestinal epithelial cells grown on permeable inserts). Methods: Caco-2 intestinal epithelial TJ permeability was assessed by measuring epithelial resistance and paracellular permeability. Cellular localization of occludin was assessed by immunofluorescent antibody labeling. Distribution and phosphorylation state of occludin was determined by detergent extraction and quantitation by Western blot analysis. Results: Cyto-B (5 mg/ml), an actin disrupting agent, produced a progressive increase in Caco-Z TJ permeability over the 60 min experimental period. Cyto-B induced increase in TJ permeability was associated with progressive disassembly and separation of occludin protein from the junctional regions, and formation of large openings in the paracellular spaces. Double antibody labeling of actin micro filaments and occludin revealed co-localization of occludin protein and peri-junctional actin filaments during the opening of the TJ barrier. Additionally, TJ opening was accompanied by a shift in distribution of occludin from membrane (detergent soluble) to cytoskeletal (detergent insoluble) fraction and decrease in phosphorylated-occludin in the membrane fraction. Conclusion: Our results suggest that Cyto-B induced opening of the intestinal monolayer TJ barrier was related to the alteration of occludin protein as demonstrated by I) disassembly and separation from the cellular junction, 2) shift in distribution from membrane to cytoskeletal fraction, and 3) dephosphorylation of the protein in the membrane fraction.
1384 GENERATION AND CHARACTERIZATION OF HUMAN ESOPHAGEAL MICROVASCULAR ENDOTHELIAL CELLS (HEMEC): REGIONAL MICROVASCULAR SPECIALIZATION IN THE GI TRACT. David G. Binion, Muhammad Aslam, Pamela J. Fisher, Shannon 1. Graewin, Nicole M. Collins, Subra Kugathasan, Parvaneh Rafiee, Christopher P. Johnson, Michael Bousamra, Reza Shaker, Med Coli of Wisconsin, Milwaukee, WI. Background and aims: Microvascular endothelial cells play an essential regulatory role in inflammation through their ability to selectively bind and recruit circulating leukocytes into tissues. There is evidence that vascular inflammatory mechanisms are involved in esophageal inflammation, as there is selective recruitment of eosinophils in gastroesphageal reflux disease (GERD), and severe esophagitis is characterized by hemorrhage and neutrophil infiltration. The role of microvascular endothelial cells in human esophageal inflammation is unexplored. The aim of this study was to isolate and characterize HEMEC from resected esophageal tissue in order to define the ability of these endothelial cells to interact with leukocytes. Methods and results: Five HEMEC lines were isolated using a modification of our human intestinal microvascular endothelial cell (HIMEC) isolation protocol (GE 112: 1895-1907, 1997). HEMEC survived in culture for up to 18 passages, and displayed classic endothelial cell scavenger receptor function, with avid uptake of DiI-ac-LDL. Cell adhesion molecule ewression was assessed using biotinylated monoclonal antibodies and 12 I streptavidin. In marked contrast to HlMEC, HEMEC displayed significantly higher (p
GASTROENTEROLOGY Vol. 118, No.4
above levels seen with either TNFalLPS or IL-I{3lTNFaactivation alone. TNFalLPS activation of HEMEC resulted in tyrosine phosphorylation of several proteins, including p38 MAPK and JNK, as detected by Western blotting. VCAM-l expression in activated HEMEC was completely blocked with the antioxidant curcumin. HEMEC secreted the chemokines IL-8 and MCP-l in response to TNFaILPS, but did not secrete eotaxin as measured by ELISA. Conclusion: Our data demonstrate that HEMEC are a unique population of microvascular endothelial cells, and further investigation of HEMEC leukocyte interaction will shed new insight into cellular and molecular mechanisms underlying esophageal inflammation in GERD.
1385 LACK OF MILD IRRITANT-INDUCED ADAPTIVE CYTOPROTECTION IN IP RECEPTOR KNOCKOUT MICE. -THE INVOLVEMENT OF ENDOGENOUS CALCITONIN GENE-RELA TED PEPTIDE-. Katsuharu Boku, Takeo Saeki, Takashi Ohno, Kazuhisa Kamata, Katsunori Saigenji, Izumi Hayashi, Makoto Katori, Takahiko Murata, Shuh Narumiya, Masataka Majima, Dept of Internal Medicine, Kitasato Univ Sch of Med, Sagamihara, Japan; Dept of Pharmacology, Kitasato Univ Sch of Med, Sagarnihara, Japan; Dept of Pharmacology, Kyoto Univ Sch of Med, Kyoto, Japan. Background: Pretreatment with hyperosmotic saline (1M NaCI) into gastric lumen increased release of endogenous prostaglandins (PGs) from stomach, and inhibited ethanol (EtOH)-induced gastric mucosal injury. During this protective action, the release of CGRP, which has a protective action, was increased. Administration of 50% EtOH after PGE2 did not released CGRP, but administration of 50% EtOH after beraprost sodium, a PGI analogue released CGRP. In the present study, we tested whether or not PGI2 analogue generated by 1M NaCI has a role in prevention of gastric mucosal injury through the release of CGRP using IP knock out mice. Methods: Stomach of C57IBL6 mice and IP,f,) anesthetized with urethane (1.225 g!kg, IP) was doubly cannulated from the esophageal and duodenal sides, and the gastric mucosa was perfused (l mllmin) with physiological saline. Perfusate was changed to 1M NaCI or 50% EtOH. Injured area was estimated at the end of each perfusion experiment. In some animals, CGRP(8-37) (0.3 mglkg, IV), a CGRP antagonist or indomethacin (IDM, 1 mglkg, IV) was injected before hyperosmotic saline perfusion. Results: I) Wild type mice (JP+f+): 50% EtOH; The injured area was 19.8%. 1M NaCI -?50% EtOH; The injured area was reduced to 12.2%. Pretreatment with CGRP(8-37) restored the injured area (19.0%). Pretreatment with IDM restored the injured area (21.1 %). 2) IP knockout mice (fp-f,): 50% EtOH; The injured area was 20.3%. 1M NaCI -?50% EtOH; The injured area was not reduced (18.8%). Conclusions: The present results suggested that the endogenous PGI2 generated by 1M NaCl may prevent EtOH-induced mucosal injury through the enhancement of CGRP release.
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1386 INVOLVEMENT OF PROSTAGLANDINS (PG), NITRIC OXIDE (NO) AND ADENOSINE (ADE) IN THE MECHANISM OF GASTRIC PRECONDITIONING INDUCED BY SHORT ISCHAEMIA. Tomasz Brzozowski, Peter Ch Konturek, Zbigniew Sliwowski, Jerzy Stachura, Stanislaw J. Konturek, Eckhart G. Hahn, Dept Physiol , Univ Med Sch, Cracow, Poland; Dept Med I, Univ Med, Erlangen, Germany. Gastric mucosa subjected to repeated brief episodes of ischaemia exhibits an increased resistance to damage caused by a subsequent more prolonged ischaemic insult and this is called gastric preconditioning, but the mechanism of this phenomenon remains unknown. In this study, the mucosal expression of COX-I and COX-2 mRNA was examined by RT-PCR and Western blot and COX-I and COX-2, NO and ADE inhibitors were used to determine the involvement of PG, NO and ADE in ischaemia preconditioning. This preconditioning was induced by short ischaemia (occlusion of celiac artery twice for 5 min) applied 30 min before subsequent exposure to 0.5 h of ischaemia induced by clamping of celiac artery followed by 3 h
1383 MODULATION OF INTESTINAL EPITHELIAL TIGHT JUNCTION PERMEABILITY IS ASSOCIATED WITH BIOCHEMICAL AND MORPHOLOGIC ALTERATION OF OCCLUDIN PROTEIN. Babu Bassa, Neil T. Hoa, Daniel Tran, Margaret Merryfield, Nathan Nguyen, Thomas Y. Ma, Long Beach VA Med Ctr, Long Beach, CA; Univ of CA, Irvine, Irvine, CA. The apico-Iaterally located tight junctions (TJ) form a paracellular seal between the lateral membranes of the adjacent cells. This seal acts as a structural barrier against the paracellular penetration of luminal substances. Disruption of the TJ barrier leads to rapid influx of toxic luminal substances resulting in mucosal injury. Occludin is a transmembrane TJ protein which appears to be involved in forming the intercellular seal (J Cell BioI 146;683-693,1999). In this study, we investigated the sequential alteration of occludin proteins during Cytochalasin-B (Cyto-B) induced modulation of intestinal monolayer TJ permeability, using an in-vitro intestinal epithelial model (consisting of Caco-Z intestinal epithelial cells grown on permeable inserts). Methods: Caco-2 intestinal epithelial TJ permeability was assessed by measuring epithelial resistance and paracellular permeability. Cellular localization of occludin was assessed by immunofluorescent antibody labeling. Distribution and phosphorylation state of occludin was determined by detergent extraction and quantitation by Western blot analysis. Results: Cyto-B (5 mg/ml), an actin disrupting agent, produced a progressive increase in Caco-Z TJ permeability over the 60 min experimental period. Cyto-B induced increase in TJ permeability was associated with progressive disassembly and separation of occludin protein from the junctional regions, and formation of large openings in the paracellular spaces. Double antibody labeling of actin micro filaments and occludin revealed co-localization of occludin protein and peri-junctional actin filaments during the opening of the TJ barrier. Additionally, TJ opening was accompanied by a shift in distribution of occludin from membrane (detergent soluble) to cytoskeletal (detergent insoluble) fraction and decrease in phosphorylated-occludin in the membrane fraction. Conclusion: Our results suggest that Cyto-B induced opening of the intestinal monolayer TJ barrier was related to the alteration of occludin protein as demonstrated by I) disassembly and separation from the cellular junction, 2) shift in distribution from membrane to cytoskeletal fraction, and 3) dephosphorylation of the protein in the membrane fraction.
1384 GENERATION AND CHARACTERIZATION OF HUMAN ESOPHAGEAL MICROVASCULAR ENDOTHELIAL CELLS (HEMEC): REGIONAL MICROVASCULAR SPECIALIZATION IN THE GI TRACT. David G. Binion, Muhammad Aslam, Pamela J. Fisher, Shannon 1. Graewin, Nicole M. Collins, Subra Kugathasan, Parvaneh Rafiee, Christopher P. Johnson, Michael Bousamra, Reza Shaker, Med Coli of Wisconsin, Milwaukee, WI. Background and aims: Microvascular endothelial cells play an essential regulatory role in inflammation through their ability to selectively bind and recruit circulating leukocytes into tissues. There is evidence that vascular inflammatory mechanisms are involved in esophageal inflammation, as there is selective recruitment of eosinophils in gastroesphageal reflux disease (GERD), and severe esophagitis is characterized by hemorrhage and neutrophil infiltration. The role of microvascular endothelial cells in human esophageal inflammation is unexplored. The aim of this study was to isolate and characterize HEMEC from resected esophageal tissue in order to define the ability of these endothelial cells to interact with leukocytes. Methods and results: Five HEMEC lines were isolated using a modification of our human intestinal microvascular endothelial cell (HIMEC) isolation protocol (GE 112: 1895-1907, 1997). HEMEC survived in culture for up to 18 passages, and displayed classic endothelial cell scavenger receptor function, with avid uptake of DiI-ac-LDL. Cell adhesion molecule ewression was assessed using biotinylated monoclonal antibodies and 12 I streptavidin. In marked contrast to HlMEC, HEMEC displayed significantly higher (p
GASTROENTEROLOGY Vol. 118, No.4
above levels seen with either TNFalLPS or IL-I{3lTNFaactivation alone. TNFalLPS activation of HEMEC resulted in tyrosine phosphorylation of several proteins, including p38 MAPK and JNK, as detected by Western blotting. VCAM-l expression in activated HEMEC was completely blocked with the antioxidant curcumin. HEMEC secreted the chemokines IL-8 and MCP-l in response to TNFaILPS, but did not secrete eotaxin as measured by ELISA. Conclusion: Our data demonstrate that HEMEC are a unique population of microvascular endothelial cells, and further investigation of HEMEC leukocyte interaction will shed new insight into cellular and molecular mechanisms underlying esophageal inflammation in GERD.
1385 LACK OF MILD IRRITANT-INDUCED ADAPTIVE CYTOPROTECTION IN IP RECEPTOR KNOCKOUT MICE. -THE INVOLVEMENT OF ENDOGENOUS CALCITONIN GENE-RELA TED PEPTIDE-. Katsuharu Boku, Takeo Saeki, Takashi Ohno, Kazuhisa Kamata, Katsunori Saigenji, Izumi Hayashi, Makoto Katori, Takahiko Murata, Shuh Narumiya, Masataka Majima, Dept of Internal Medicine, Kitasato Univ Sch of Med, Sagamihara, Japan; Dept of Pharmacology, Kitasato Univ Sch of Med, Sagarnihara, Japan; Dept of Pharmacology, Kyoto Univ Sch of Med, Kyoto, Japan. Background: Pretreatment with hyperosmotic saline (1M NaCI) into gastric lumen increased release of endogenous prostaglandins (PGs) from stomach, and inhibited ethanol (EtOH)-induced gastric mucosal injury. During this protective action, the release of CGRP, which has a protective action, was increased. Administration of 50% EtOH after PGE2 did not released CGRP, but administration of 50% EtOH after beraprost sodium, a PGI analogue released CGRP. In the present study, we tested whether or not PGI2 analogue generated by 1M NaCI has a role in prevention of gastric mucosal injury through the release of CGRP using IP knock out mice. Methods: Stomach of C57IBL6 mice and IP,f,) anesthetized with urethane (1.225 g!kg, IP) was doubly cannulated from the esophageal and duodenal sides, and the gastric mucosa was perfused (l mllmin) with physiological saline. Perfusate was changed to 1M NaCI or 50% EtOH. Injured area was estimated at the end of each perfusion experiment. In some animals, CGRP(8-37) (0.3 mglkg, IV), a CGRP antagonist or indomethacin (IDM, 1 mglkg, IV) was injected before hyperosmotic saline perfusion. Results: I) Wild type mice (JP+f+): 50% EtOH; The injured area was 19.8%. 1M NaCI -?50% EtOH; The injured area was reduced to 12.2%. Pretreatment with CGRP(8-37) restored the injured area (19.0%). Pretreatment with IDM restored the injured area (21.1 %). 2) IP knockout mice (fp-f,): 50% EtOH; The injured area was 20.3%. 1M NaCI -?50% EtOH; The injured area was not reduced (18.8%). Conclusions: The present results suggested that the endogenous PGI2 generated by 1M NaCl may prevent EtOH-induced mucosal injury through the enhancement of CGRP release.
ur":
1386 INVOLVEMENT OF PROSTAGLANDINS (PG), NITRIC OXIDE (NO) AND ADENOSINE (ADE) IN THE MECHANISM OF GASTRIC PRECONDITIONING INDUCED BY SHORT ISCHAEMIA. Tomasz Brzozowski, Peter Ch Konturek, Zbigniew Sliwowski, Jerzy Stachura, Stanislaw J. Konturek, Eckhart G. Hahn, Dept Physiol , Univ Med Sch, Cracow, Poland; Dept Med I, Univ Med, Erlangen, Germany. Gastric mucosa subjected to repeated brief episodes of ischaemia exhibits an increased resistance to damage caused by a subsequent more prolonged ischaemic insult and this is called gastric preconditioning, but the mechanism of this phenomenon remains unknown. In this study, the mucosal expression of COX-I and COX-2 mRNA was examined by RT-PCR and Western blot and COX-I and COX-2, NO and ADE inhibitors were used to determine the involvement of PG, NO and ADE in ischaemia preconditioning. This preconditioning was induced by short ischaemia (occlusion of celiac artery twice for 5 min) applied 30 min before subsequent exposure to 0.5 h of ischaemia induced by clamping of celiac artery followed by 3 h