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Mechanism of PGI2-mediated adaptive cytoprotection by hypertonic saline against Ethanol-induced gastric mucosal injury. An intravital microscopic study
A1134 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
5225
5227
EXPRESSION OF TRANSFORMING GROWTH FACTOR·B (TGF·B) BY HUMAN INTESTINAL LAMINA PROPRIA MONO· NUCLEAR CELLS. Felicity R. Rose, Elaine Y. Lee, Brian C. McKaig, Patrick J. Tighe, Yashwant R. Mahida, Div of Gastroenterology, Univ of Nottingham, Nottingham, United Kingdom; Div of Immunology, Univ of Nottingham, Nottingham, United Kingdom. Introduction. TGF-,B, of which three isoforms are known in humans, mediates a wide range of biological functions involved in mucosal immunological responses and repair and remodelling. The synthesized latent form of TGF-,B requires processing to the active form following secretion. The aim of this study was to investigate the expression of TGF-,B by isolated normal human colonic lamina propria cells. Methods. Lamina propria cells (lymphocytes and macrophages) were isolated from histologically normal colonic mucosal samples (n=8) and cultured for 24 h at concentration of 106/ml in serum-deprived medium. Following culture, adherent and non-adherent cells were pooled and expression of TGF-,B-I, -2 & -3 mRNA was studied by reverse transcriptase-polymerase chain reaction (RT-PCR). TGF-,B bioactivity was investigated in untreated and acid-treated (to activate latent TGF-,B) supernatant samples using the specific MvlLu bioassay. Data are expressed as mean(:!::SEM) and analysed by paired t test. Results. Viability of isolated lamina propria cells following 24 h culture: 93.8(:!::0.65). All the lamina propria cell preparations expressed transcripts for TGF-,B-I, -2 and 3. TGF-,B bioactivity in supernatant samples was 359.0(:!:: 112.2) pg/ml in untreated samples and 605.0(:!:: 151.0) pg/ml in acid-treated samples (p=0.008). Conclusions. Isolated and cultured normal human colonic lamina propria mononuclear cells (lymphocytes and macrophages) express transcripts for all three isoforms of TGF-,B. Both latent and biologically active (processed) forms of TGF-,B are secreted by these cells, which may playa major role in the regulation of mucosal immunological responses and also repair and remodelling following injury and inflammation. This study was supported by the National Association for Colitis and Crohn's disease (UK).
REGULATION OF COLONIC ION TRANSPORT BY VASOCON· STRICTIVE PEPTIDES. Yoshihiko Sato, Hiroyuki Hanai, Yoshisuke Hosoda, Takayuki Iida, Koki Hirasawa, Liu Wei Xin, Atsuhiro Nogaki, Hisayoshi Hayashi, Yuichi Suzuki, Hamamatsu Univ Sch of Medicine, Hamamatsu, Japan; Univ of Shizuoka, Shizuoka, Japan. Background/Aim: Vasoconstrictive peptides, including vasopressin (VP), angiotensin II (ATII) and endothelin-I (ET-I), play important roles in the maintenance of fluid and electrolyte homeostasis mainly through the effects on kidney and circulation. However, in pathophysiological condition such as inflammatory bowel disease and renal dysfunction, their effects on the regulation of intestinal fluid and electrolyte transport could have greater significance. In this study, we examined and compared the effects of VP, ATII and ET-I on colonic electrogenic ion transport. Method: We measured the effects of these peptides on the short circuit current (lsc) and conductance (Gt) in stripped distal colonic mucosa of Guinea pig using Ussing chamber. We also studied the effects on cytosolic calcium of colonic crypt using fura-2, and the receptor SUbtypes included in their action. Results: All three peptides stimulated electrogenic K secretion (Table). VP and ATII, but not ET-I, inhibited electrogenic Na absorption. VP and ET-I, but not ATII, stimulated electrogenic CI secretion. These peptides caused changes in Isc in a concentration dependent manner. These effects were through VI, AT!, ETA receptor subtype, respectively. Mobilization of cytosolic calcium in colonic crypt was induced by VP and ATII, but not by ET-1. The time course of chages in cytosolic calcium induced by VP and ATII were quite different. Conclusion: The stimulation of K secretion, which was stimulated by all three peptides, could be most important function. The stimulation of CI secretion was induced by VP and ET-l, and the inhibition of Na absorption was induced by VP and ATII. The mechanism of these effects on electrolyte transport may be different because the effects on crypt cytosolic calciurn were different among these three peptides. EFFECTS OF VASOCONSTRICTIVE PEPTIDES
5226 MECHANISM OF PGI2·MEDIATED ADAPTIVE CYTOPROTEC· TION BY HYPERTONIC SALINE AGAINST ETHANOL·IN· DUCED GASTRIC MUCOSAL INJURY. AN INTRAVITAL MI· CROSCOPIC STUDY. Takeo Saeki, Takashi Ohno, Katsuharu Boku, Kazuhisa Kamata, Katsunori Saigenji, Makoto Katori, Masataka Majima, Sagamihara, Dept of Internal Medicine, Kitasato Univ Sch of Med, Sagamihara, Japan; Dept of Internal Medicine, Isehara Kyodo Hosp, Isehara, Japan; Dept of Pharmacology, Kitasato Univ Sch of Med, Sagamihara, Japan. Background: Our previous intravital microscopic study revieled that exposure of 50% EtOH to the mucosa developed gastric mucosal injury, which was induced by mucosal congestion as a result of the constrictions of collecting venules and venules. Pretreatment of gastric mucosa with 1M NaCI prevented EtOH-induced injury. This prevention was completely abolished by indomethacin (10M), suggesting the involvement of endogenous prostaglandins (PGs). We investigated the preventive mechanism of 1M NaCI against EtOH-induced mucosal injury using intravital microscopy. Methods: The greater curvature of the anesthetized (urethane 0.875 g/kg, i.p.) SO strain male rat stomach was cut. The posterior wall of the glandular stomach was fixed in a chamber and perfused with Tyrode's solution, and the gastric microcirculation of the mucosal base was observed through a window with transilluminations. The diameter of the arterioles, collecting venules and venules were measured by analyzer. EtOH (50%; I m!) or NaCI (1M; I ml) was placed between the chamber and the gastric mucosa. In some animals, 10M (10 mg/kg, IV) was infused before application of 1M NaCI. CGRP 8-37, a CGRP receptor antagonist (lIJ-M; 20 IJ-l) and Beraprost sodium, a PGI2 analogue (I nM; 20 IJ-l) were applied in the window. Results: 1) Pretreatment of Beraprost sodium (I nM) to the microvasculature, which did not influence the original size of gastric mucosal vessels including arterioles, completely inhibited constrictions of collecting venules and venules induced by 50% EtOH. This effect was abolished by CGRP 8-37 pretreated. 2) CGRP pretreatment blocked the venular constriction induced by 50% EtOH. 3) The exposure of 1M NaCI into the gastric lumen significantly increased the release of 6-keto-PGFI a, a P0I2 metabolite to the gastric perfusates. 4) Pretreatment with 1M NaCI inhibited EtOH-induced constrictive changes in collecting venu1es and venules, and the administration of CGRP 8-37 abolished the cancellation of constrictions of collecting venules and venules by 1M NaCI. Conclusions: The present results suggested that the preventive effect of 1M NaCI against the EtOH-induced injury is mediated by CGRP which is released by P0I2 generated.
Vasopressin Inhibition of Na absorption Stimulaton of K secretion Stimulation ofCIsecretion Mobilization of crypt[Cali Receptor subtype
Angiotensin II
Endothelin·1
+
V1
An
ETA
5228 ROLE OF APOPTOSIS IN THE T CELL·DEPENDENT ENTEROP· ATHY INDUCED BY ANTI·Cm ANTIBODY. Jrgen Schlmerich, Klaus Schlottmann, Univ of Regensburg, Regensburg, Germany. Background: The small intestinal, T cell-dependent enteropathy (aCD3-E) induced by systemic administration of anti-CD3 antibody is characterized by the appearance of numerous apoptotic bodies in the epithelium. Therefore, we investigated the role of apoptosis in the aCD3-E. Methods: aCD3-E was induced by intraperitoneal injection of Balb/c mice with 50 IJ-g of anti-CD3 antibody 2Cll-145. DNA fragmentation was shown by TUNEL assay of small intestinal tissue sections. Cleavage of Caspase 3 was assessed by a colorimetric activity assay of epithelial cell protein, and by immunhistochemistry on in toto isolated epithelial crypts using an antibody recognizing only activated caspase 3. Apoptosis inhibitors (ZVAD-fmk, DEVD-fmk) and inducers (anti-Fas antibody, staurosporine) were injected i.p. at various time points after anti-CD3 administration. Results: Positive staining for apoptosis by both caspase 3 antibody and TUNEL was largely restricted to the lower crypt regions. Increased caspase 3 activity was readily detected 4h after anti-CD3 injection. Treatment of mice with apoptosis inhibitors did not reduce intestinal tissue damage but rather worsened disease. Additional treatment of anti-CD3 treated mice with usually sub-lethal doses of anti-Fas antibody resulted in premature death of some animals but no discernible change of the aCD3-E. However, concomitant treatment of mice with staurosporine led to a distinct reduction of tissue damage. Conclusion: Epithelial cell damage following nonspecific T cell activation involves apoptotic mechanisms. However, broad inhibition of apoptosis does not necessarily ameliorate tissue damage, and apoptosis of immune effector cells may contribute to limiting tissue damage in intestinal inflammatory diseases.
GASTROENTEROLOGY Vol. 118, No.4
5225
5227
EXPRESSION OF TRANSFORMING GROWTH FACTOR·B (TGF·B) BY HUMAN INTESTINAL LAMINA PROPRIA MONO· NUCLEAR CELLS. Felicity R. Rose, Elaine Y. Lee, Brian C. McKaig, Patrick J. Tighe, Yashwant R. Mahida, Div of Gastroenterology, Univ of Nottingham, Nottingham, United Kingdom; Div of Immunology, Univ of Nottingham, Nottingham, United Kingdom. Introduction. TGF-,B, of which three isoforms are known in humans, mediates a wide range of biological functions involved in mucosal immunological responses and repair and remodelling. The synthesized latent form of TGF-,B requires processing to the active form following secretion. The aim of this study was to investigate the expression of TGF-,B by isolated normal human colonic lamina propria cells. Methods. Lamina propria cells (lymphocytes and macrophages) were isolated from histologically normal colonic mucosal samples (n=8) and cultured for 24 h at concentration of 106/ml in serum-deprived medium. Following culture, adherent and non-adherent cells were pooled and expression of TGF-,B-I, -2 & -3 mRNA was studied by reverse transcriptase-polymerase chain reaction (RT-PCR). TGF-,B bioactivity was investigated in untreated and acid-treated (to activate latent TGF-,B) supernatant samples using the specific MvlLu bioassay. Data are expressed as mean(:!::SEM) and analysed by paired t test. Results. Viability of isolated lamina propria cells following 24 h culture: 93.8(:!::0.65). All the lamina propria cell preparations expressed transcripts for TGF-,B-I, -2 and 3. TGF-,B bioactivity in supernatant samples was 359.0(:!:: 112.2) pg/ml in untreated samples and 605.0(:!:: 151.0) pg/ml in acid-treated samples (p=0.008). Conclusions. Isolated and cultured normal human colonic lamina propria mononuclear cells (lymphocytes and macrophages) express transcripts for all three isoforms of TGF-,B. Both latent and biologically active (processed) forms of TGF-,B are secreted by these cells, which may playa major role in the regulation of mucosal immunological responses and also repair and remodelling following injury and inflammation. This study was supported by the National Association for Colitis and Crohn's disease (UK).
REGULATION OF COLONIC ION TRANSPORT BY VASOCON· STRICTIVE PEPTIDES. Yoshihiko Sato, Hiroyuki Hanai, Yoshisuke Hosoda, Takayuki Iida, Koki Hirasawa, Liu Wei Xin, Atsuhiro Nogaki, Hisayoshi Hayashi, Yuichi Suzuki, Hamamatsu Univ Sch of Medicine, Hamamatsu, Japan; Univ of Shizuoka, Shizuoka, Japan. Background/Aim: Vasoconstrictive peptides, including vasopressin (VP), angiotensin II (ATII) and endothelin-I (ET-I), play important roles in the maintenance of fluid and electrolyte homeostasis mainly through the effects on kidney and circulation. However, in pathophysiological condition such as inflammatory bowel disease and renal dysfunction, their effects on the regulation of intestinal fluid and electrolyte transport could have greater significance. In this study, we examined and compared the effects of VP, ATII and ET-I on colonic electrogenic ion transport. Method: We measured the effects of these peptides on the short circuit current (lsc) and conductance (Gt) in stripped distal colonic mucosa of Guinea pig using Ussing chamber. We also studied the effects on cytosolic calcium of colonic crypt using fura-2, and the receptor SUbtypes included in their action. Results: All three peptides stimulated electrogenic K secretion (Table). VP and ATII, but not ET-I, inhibited electrogenic Na absorption. VP and ET-I, but not ATII, stimulated electrogenic CI secretion. These peptides caused changes in Isc in a concentration dependent manner. These effects were through VI, AT!, ETA receptor subtype, respectively. Mobilization of cytosolic calcium in colonic crypt was induced by VP and ATII, but not by ET-1. The time course of chages in cytosolic calcium induced by VP and ATII were quite different. Conclusion: The stimulation of K secretion, which was stimulated by all three peptides, could be most important function. The stimulation of CI secretion was induced by VP and ET-l, and the inhibition of Na absorption was induced by VP and ATII. The mechanism of these effects on electrolyte transport may be different because the effects on crypt cytosolic calciurn were different among these three peptides. EFFECTS OF VASOCONSTRICTIVE PEPTIDES
5226 MECHANISM OF PGI2·MEDIATED ADAPTIVE CYTOPROTEC· TION BY HYPERTONIC SALINE AGAINST ETHANOL·IN· DUCED GASTRIC MUCOSAL INJURY. AN INTRAVITAL MI· CROSCOPIC STUDY. Takeo Saeki, Takashi Ohno, Katsuharu Boku, Kazuhisa Kamata, Katsunori Saigenji, Makoto Katori, Masataka Majima, Sagamihara, Dept of Internal Medicine, Kitasato Univ Sch of Med, Sagamihara, Japan; Dept of Internal Medicine, Isehara Kyodo Hosp, Isehara, Japan; Dept of Pharmacology, Kitasato Univ Sch of Med, Sagamihara, Japan. Background: Our previous intravital microscopic study revieled that exposure of 50% EtOH to the mucosa developed gastric mucosal injury, which was induced by mucosal congestion as a result of the constrictions of collecting venules and venules. Pretreatment of gastric mucosa with 1M NaCI prevented EtOH-induced injury. This prevention was completely abolished by indomethacin (10M), suggesting the involvement of endogenous prostaglandins (PGs). We investigated the preventive mechanism of 1M NaCI against EtOH-induced mucosal injury using intravital microscopy. Methods: The greater curvature of the anesthetized (urethane 0.875 g/kg, i.p.) SO strain male rat stomach was cut. The posterior wall of the glandular stomach was fixed in a chamber and perfused with Tyrode's solution, and the gastric microcirculation of the mucosal base was observed through a window with transilluminations. The diameter of the arterioles, collecting venules and venules were measured by analyzer. EtOH (50%; I m!) or NaCI (1M; I ml) was placed between the chamber and the gastric mucosa. In some animals, 10M (10 mg/kg, IV) was infused before application of 1M NaCI. CGRP 8-37, a CGRP receptor antagonist (lIJ-M; 20 IJ-l) and Beraprost sodium, a PGI2 analogue (I nM; 20 IJ-l) were applied in the window. Results: 1) Pretreatment of Beraprost sodium (I nM) to the microvasculature, which did not influence the original size of gastric mucosal vessels including arterioles, completely inhibited constrictions of collecting venules and venules induced by 50% EtOH. This effect was abolished by CGRP 8-37 pretreated. 2) CGRP pretreatment blocked the venular constriction induced by 50% EtOH. 3) The exposure of 1M NaCI into the gastric lumen significantly increased the release of 6-keto-PGFI a, a P0I2 metabolite to the gastric perfusates. 4) Pretreatment with 1M NaCI inhibited EtOH-induced constrictive changes in collecting venu1es and venules, and the administration of CGRP 8-37 abolished the cancellation of constrictions of collecting venules and venules by 1M NaCI. Conclusions: The present results suggested that the preventive effect of 1M NaCI against the EtOH-induced injury is mediated by CGRP which is released by P0I2 generated.
Vasopressin Inhibition of Na absorption Stimulaton of K secretion Stimulation ofCIsecretion Mobilization of crypt[Cali Receptor subtype
Angiotensin II
Endothelin·1
+
V1
An
ETA
5228 ROLE OF APOPTOSIS IN THE T CELL·DEPENDENT ENTEROP· ATHY INDUCED BY ANTI·Cm ANTIBODY. Jrgen Schlmerich, Klaus Schlottmann, Univ of Regensburg, Regensburg, Germany. Background: The small intestinal, T cell-dependent enteropathy (aCD3-E) induced by systemic administration of anti-CD3 antibody is characterized by the appearance of numerous apoptotic bodies in the epithelium. Therefore, we investigated the role of apoptosis in the aCD3-E. Methods: aCD3-E was induced by intraperitoneal injection of Balb/c mice with 50 IJ-g of anti-CD3 antibody 2Cll-145. DNA fragmentation was shown by TUNEL assay of small intestinal tissue sections. Cleavage of Caspase 3 was assessed by a colorimetric activity assay of epithelial cell protein, and by immunhistochemistry on in toto isolated epithelial crypts using an antibody recognizing only activated caspase 3. Apoptosis inhibitors (ZVAD-fmk, DEVD-fmk) and inducers (anti-Fas antibody, staurosporine) were injected i.p. at various time points after anti-CD3 administration. Results: Positive staining for apoptosis by both caspase 3 antibody and TUNEL was largely restricted to the lower crypt regions. Increased caspase 3 activity was readily detected 4h after anti-CD3 injection. Treatment of mice with apoptosis inhibitors did not reduce intestinal tissue damage but rather worsened disease. Additional treatment of anti-CD3 treated mice with usually sub-lethal doses of anti-Fas antibody resulted in premature death of some animals but no discernible change of the aCD3-E. However, concomitant treatment of mice with staurosporine led to a distinct reduction of tissue damage. Conclusion: Epithelial cell damage following nonspecific T cell activation involves apoptotic mechanisms. However, broad inhibition of apoptosis does not necessarily ameliorate tissue damage, and apoptosis of immune effector cells may contribute to limiting tissue damage in intestinal inflammatory diseases.