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Interferon-γ regulates epithelial chloride secretion: Role of EGF receptor-dependent pathways
A426 AGA ABSTRACTS
• G1735 5-HT 3 ANTAGONIST, GRANISETRON, DIRECTLY INHIBITS CHOLERA TOXIN (CT)-INDUCED 5-HT RELEASE FROM ENTEROCHROMAFFIN CELLS. Turvill J[_,, Connor P, Farthing MJG. Digestive Diseases Research Centre, St Bartholomew's & The Royal London School of Medicine, London, UK. Background CT-induced secretion is accompanied by 5-HT release from enterochromaffin (EC) cells. 5-FIT~ receptor antagonists are known to inhibit CT-induced secretion. Recently, 5-HT~ receptors have been identified on the basolateral membrane of EC cells. We postulated that these basolateral 5-HT3 autoreceptors and additional neuronal inputs may modulate CT-induced 5-HT release and so modify secretion. Methods Anesthetised adult male Wistar rats were pre-treated with granisetron (5-HT~ antagonist), 75lag/kg ip, 6mg/kg lidocaine or saline. 20cm jejunal segments were isolated between cannulae and 25lag CT in 2ml saline or saline alone was instilled for 30rain. The residual luminal contents of the segments were then gently collected for gravimetric assessment of net fluid movement and determination of 5-HT levels by HPLC. A further 2ml isotonic saline was then instilled into the segments. This was repeated six times over 180min so that sequential 30min collections of luminal contents were obtained. Results CT induced a net fluid secretion (median 7.3~tl/mg dry weight [IQR 5.0 to 8.5]; n=8) compared to control (-7.5 [-11,3 to -6.1]; n=7, p < 0.0001). This was reversed by granisetron (-4.4 [-5.0 to -3.2]; n=8, p < 0.0001). The CT induced increase in 5-HT release (1.7 pmol/mg dw [1.6 to 2.3]) over basal levels (0.6 [0.5 to 0.7]; p < 0.0001) was inhibited by granisetron (1.3 [1.1 to 1.5]; p <0.001). Linear regression confirmed a correlation between net fluid movement and 5-HT release (r=0.7 [95% CI 0.4 to 0.9]; p <0.0007). By contrast, lidocaine inhibited secretion (-8.0 [-11.2 to -5.6]; n=9, p < 0.001) without affecting 5-HT release (2.8 [2.4 to 4.0], ns). Summary and conclusion CT-induced 5-HT release is inhibited by the 5-HT3 antagonist, granisetron, but not by lidocaine. This observation suggests that granisetron has a direct effect on EC cells possibly acting via 5-HT antoreceptors. The degree by which granisetron inhibits 5-HT release is closely correlated to the degree by which it reverses secretion. • G1736 ACTION OF NON-PEPTIDE SUBSTANCE P ANTAGONIST, CP 96 345, ON CHOLERA TOXIN AND E . C O L I ENTEROTOXIN-INDUCED SECRETION. Turvill JL, P Connor, Farthing MJG. Digestive Diseases Research Centre, St Bartholomew's & The Royal London School of Medicine, UK. Introduction, The role of substance P (SP) in non-inflammatory secretory states remains unclear. We recently found that cholera toxin (CT)-induced secretion was inhibited by the SP antagonist, D Pro 2 D Trp 7,9 SP. This peptide has a poor selectivity for SP, however, and so it is uncertain whether the effect was due directly to SP blockade. We assessed whether the novel, highly selective, non-peptide SP antagonist, CP 96,345 and its inactive enantiomer CP 96,344 could modify the non-inflammatory secretory states induced by CT and E. coli heat labile (LT) and heat stable enterotoxins (STa) in vivo. Methods. CT and LT experiments: Anesthetised male Wistar rats (180-220g) were pre-treated with CP 96,345 0.3, 1, 3 or 10mg/kg, CP 96,344 10mg/kg or saline. 20era small intestinal segments were isolated between cannulae into which 25 lag LT or saline was instilled and incubated. After 2h, in situ perfusion was performed with plasma electrolyte solution (Na 140, K 4, C1 104, HCO 3 40 mmoYl) containing [14C]-polyethylene glycol as a nonabsorbable marker. After equilibration, 3xl0min collections were made and net fluid and electrolyte movements were determined. STa experiments: After pre-treatment with CP 96,345 3mg/kg, CP 96,344 3mg/kg or saline, the jejunal segment was perfused with PES containing []4C]-PEG to which 200lag/l STa had been added. Results. Neither CP 96,345 nor CP 96,344 affected basal transport. CT-induced net water secretion (median -100lal/min/g dw [IQR -119 to -85]; n=8) was dose-dependently reduced by CP 96,345. CP 96,345, 3 and 10mg/kg, significantly inhibited secretion (-58 [-68 to -49]; n=8 and -65 [-79 to -53]; n=8, respectively, p < 0.03 KW), but not CP 96,444, 10mg/kg (-128 [-163 to -91]; n=8, ns). By contrast LT- and STa-induced secretion (-73 [-97 to -64]; n=8 and -83 [-102 to -81]; n=6 respectively) were not inhibited by CP 96,345, 3mg/kg (-76 [-140 to -38]; n=7 and -98 [-129 to -82]; n=6 respectively, ns) or by CP 96,344. Net electrolyte movement paralleled water. Conclusion. These findings support our earlier observation that SP mediates CT-induced secretion. The failure to inhibit LT- and STa-induced secretion is consistent with differences in the way these three enterotoxins activate a secretory reflex arc in the host. • G1737 GLUCOCORTICOID INDUCTION OF INTESTINAL EPITHELIAL HEAT SHOCK PROTEIN 72 INVOLVES TRANSCRIPTIONAL GENE ACTIVATION. S Urayama, MW Musch, D. Strans, EB Chang. IBD Center; Univ. Chicago; Chicago, IL. Back,,round/Aims. Mucosal injury and dysfunction are cardinal features of inflammatory bowel diseases, arising when injurious immune and inflammatory elements overwhelm cellular defense mechanisms. Inducible heat shock proteins (hsp), particularly hsp72, appear to have a major role in
GASTROENTEROLOGY Vol. 114, No. 4
conferring cellular protection to intestinal epithelial cells against inflammation-associated injury and stress. We previously reported that glucocorticoids (GC), commonly used for the treatment of inflammatory bowel diseases, protect intestinal epithelial cells against oxidant exposure by induction of hsp72. In this study, potential mechanisms of the GC-induced hsp 72 response were studied. Methods. Confluent, normal, diploid, rat intestinal IEC-18 epithelial cells were studied because of their low endogenous hsp72 expression. Cells were stimulated with the GC, dexamethasone (DEX), and the hsp72 protein response determined by Western blotting. As a measure of transcriptional hsp72 gene activation, IECI8 cells were transfected with a CAT/hsp72 promoter construct containing the 2.5 kB upstream region of the human hsp72 gene (a construct beating the heat shock element and previously shown to be activated appropriately in rat intestinal epithelial ceils). Transfected cells were exposed to DEX (10"7 M) for 48 h and electroporated at capacitance of 1000~tF and voltage of 330V to transfect the CAT/hsp72 promoter construct. CAT assays were performed with appropriate controls as previously described. Re..su!ts. In contrast to the immediate hsp72 response to thermal stress of IEC18 cells, DEX stimulates a dose-dependent hsp72 response that first appears after 24 h and peaks at 72-96 h. The response is specific, as no changes in the constitutively expressed homolog, hsc73, were observed. After 24 h exposure, radiolabeled (14C-chloramphenicol) TLC templates (n=3)) showed a significant acetylation of chloramphenicol in IEC-18 samples with DEX exposure as well as in the thermal stress controls. In contrast, no reporter activity was observed in any of the untreated control groups. Conclusion. Glucocorticoid induction of intestinal epithelial hsp 72 involves transcriptional activation of the gene. This may involve an activation process distinct from thermal stress (which requires the heat shock element), as the time course and magnitude of hsp72 induction differ significantly. This process confers cellular protection to intestinal epithelial cells subjected to inflammation-associated stress. • G1738 INTERFERON-T REGULATES EPITHELIAL CHLORIDE SECRETION: ROLE OF E G F R E C E P T O R - D E P E N D E N T P A T H W A Y S . J.M. Uribe and K.E. Barrett, Dept. of Med., UCSD School of Medicine, San Diego, CA. Interferon-'/ (IFN-V) is known to alter epithelial permeability and CIsecretion. A long-term inhibitory effect of IFN-'y on CI" secretion has been attributed to down-regulation of CFTR expression. However, signaling events leading to this response have yet to be elucidated. We have shown that epidermal growth factor (EGF) family members also inhibit calcium-activated CI- secretion, such as that stimulated by carbachol (CCh). Likewise, effects of IFN-T in other systems are mediated via upregulated expression of EGF family members, including transforming growth factor-or (TGF-c0. This could lead to activation of the 170 kDa EGF receptor (EGF-R). Our goal was to determine if IFN-'/ inhibits calcium-activated C1- secretion via EGF-R activation. Studies utilized the intestinal epithelial cell line, T~4. CI- secretion was measured as changes in short circuit current (I,o, in laA/cm2) in Ussing chambers. Levels of cellular TGF-ct and tyrosine-phosphorylated proteins were determined by Western blotting with antibodies (Abs) to TGF-ct or phosphotyrosine. EGF-R activation was assessed in anti-EGF-R immunoprecipitates by Western blotting with Abs to phosphotyrosine. Secreted TGF-et was measured by ELISA. Data are means ± SEM and were analyzed by ANOVA. Stimulation of T~4 cells with IFN-y (100ng/ml) led to a progressive increase in tyrosine-phosphorylation of a 170 kDa protein, peaking between 12-24 hrs. IFN-;, also significantly inhibited CCh-indueed C1- secretion at 1 hr ( A I CCh: 27.2 -+ 2.4 vs. IFN-T+CCh: 16.7 ± 1.7, n=3, p < 0.05) or 24 hrs ( A I CCh: 37.5 ± 0.3 vs. IFN-y+CCh: 9.0--. 2.0, n=3-4, p<0.001) of preincubation~ 24 hr IFN-~/ treatment increased tyrosinephosphorylation of EGF-R. Furthermore, 1 and 24 hr IFN-'/ treatment increased levels of the 20 kDa TGF-ct precursor. However, no free 6 kDa TGF-Gt was found in the medium of IFN-'¢ treated cells. Moreover, an inhibitor of EGF-R kinase activity, tyrphostin AG1478 (Tyr, llaM), did not reverse inhibition of CCh-induced CI- secretion induced by 24h pretreatment with IFN-~/ ( A I CCh: 37.5 ±0.3 vs. IFN-'/+CCh: 9.0-+ 2.0 vs. Tyr+IFN")'+CCh: 10.3 ± 1.6, n=3-4, p <0.001), nor did it inhibit IFN-y-induced phosphorylation of EGF-R. These data suggest that while IFN-'/ increases expression of transmembrane TGF-ct and activates EGF-R in Tg~ cells, receptor activation likely occurs via mechanisms other than ligand-binding. These novel mechanisms for EGF-R transactivation may contribute to the ability of IFN-'/to inhibit CI- secretion. Overall, this may have implications for our understanding of epithelial dysfunction in inflammation, where IFN-'/ levels are reportedly elevated. • G1739 MUC2 IS THE PREDOMINANT SECRETORY MUCIN IN THE MOUSE COLON. B,J.W. Van Klinken, L.A. Duits, M. Verburg, M.K. Makkink, K.M.A.L Tytgat, I.B. Renes, H.A. BUllet#, A.W.C. Einerhand, J Dekker. Pediatric Gastroenterology and Nutrition, Acad. Med. Ctr, Amsterdam, and #Sophia's Children Hospital, Rotterdam, The Netherlands. We have shown previously that the predominant human colonic mucin (MUC2), which is responsible for the mucus-layer in the colon [Tytgat et al., 1994, Gastroenterology 107, 1352], is deficient in active ulcerative colitis [Tytgat et aL, 1996, Biochem Biophys Res Comm 224, 397]. We aim to investigate the role of MUC2 in vivo in the protection of the colon during
• G1735 5-HT 3 ANTAGONIST, GRANISETRON, DIRECTLY INHIBITS CHOLERA TOXIN (CT)-INDUCED 5-HT RELEASE FROM ENTEROCHROMAFFIN CELLS. Turvill J[_,, Connor P, Farthing MJG. Digestive Diseases Research Centre, St Bartholomew's & The Royal London School of Medicine, London, UK. Background CT-induced secretion is accompanied by 5-HT release from enterochromaffin (EC) cells. 5-FIT~ receptor antagonists are known to inhibit CT-induced secretion. Recently, 5-HT~ receptors have been identified on the basolateral membrane of EC cells. We postulated that these basolateral 5-HT3 autoreceptors and additional neuronal inputs may modulate CT-induced 5-HT release and so modify secretion. Methods Anesthetised adult male Wistar rats were pre-treated with granisetron (5-HT~ antagonist), 75lag/kg ip, 6mg/kg lidocaine or saline. 20cm jejunal segments were isolated between cannulae and 25lag CT in 2ml saline or saline alone was instilled for 30rain. The residual luminal contents of the segments were then gently collected for gravimetric assessment of net fluid movement and determination of 5-HT levels by HPLC. A further 2ml isotonic saline was then instilled into the segments. This was repeated six times over 180min so that sequential 30min collections of luminal contents were obtained. Results CT induced a net fluid secretion (median 7.3~tl/mg dry weight [IQR 5.0 to 8.5]; n=8) compared to control (-7.5 [-11,3 to -6.1]; n=7, p < 0.0001). This was reversed by granisetron (-4.4 [-5.0 to -3.2]; n=8, p < 0.0001). The CT induced increase in 5-HT release (1.7 pmol/mg dw [1.6 to 2.3]) over basal levels (0.6 [0.5 to 0.7]; p < 0.0001) was inhibited by granisetron (1.3 [1.1 to 1.5]; p <0.001). Linear regression confirmed a correlation between net fluid movement and 5-HT release (r=0.7 [95% CI 0.4 to 0.9]; p <0.0007). By contrast, lidocaine inhibited secretion (-8.0 [-11.2 to -5.6]; n=9, p < 0.001) without affecting 5-HT release (2.8 [2.4 to 4.0], ns). Summary and conclusion CT-induced 5-HT release is inhibited by the 5-HT3 antagonist, granisetron, but not by lidocaine. This observation suggests that granisetron has a direct effect on EC cells possibly acting via 5-HT antoreceptors. The degree by which granisetron inhibits 5-HT release is closely correlated to the degree by which it reverses secretion. • G1736 ACTION OF NON-PEPTIDE SUBSTANCE P ANTAGONIST, CP 96 345, ON CHOLERA TOXIN AND E . C O L I ENTEROTOXIN-INDUCED SECRETION. Turvill JL, P Connor, Farthing MJG. Digestive Diseases Research Centre, St Bartholomew's & The Royal London School of Medicine, UK. Introduction, The role of substance P (SP) in non-inflammatory secretory states remains unclear. We recently found that cholera toxin (CT)-induced secretion was inhibited by the SP antagonist, D Pro 2 D Trp 7,9 SP. This peptide has a poor selectivity for SP, however, and so it is uncertain whether the effect was due directly to SP blockade. We assessed whether the novel, highly selective, non-peptide SP antagonist, CP 96,345 and its inactive enantiomer CP 96,344 could modify the non-inflammatory secretory states induced by CT and E. coli heat labile (LT) and heat stable enterotoxins (STa) in vivo. Methods. CT and LT experiments: Anesthetised male Wistar rats (180-220g) were pre-treated with CP 96,345 0.3, 1, 3 or 10mg/kg, CP 96,344 10mg/kg or saline. 20era small intestinal segments were isolated between cannulae into which 25 lag LT or saline was instilled and incubated. After 2h, in situ perfusion was performed with plasma electrolyte solution (Na 140, K 4, C1 104, HCO 3 40 mmoYl) containing [14C]-polyethylene glycol as a nonabsorbable marker. After equilibration, 3xl0min collections were made and net fluid and electrolyte movements were determined. STa experiments: After pre-treatment with CP 96,345 3mg/kg, CP 96,344 3mg/kg or saline, the jejunal segment was perfused with PES containing []4C]-PEG to which 200lag/l STa had been added. Results. Neither CP 96,345 nor CP 96,344 affected basal transport. CT-induced net water secretion (median -100lal/min/g dw [IQR -119 to -85]; n=8) was dose-dependently reduced by CP 96,345. CP 96,345, 3 and 10mg/kg, significantly inhibited secretion (-58 [-68 to -49]; n=8 and -65 [-79 to -53]; n=8, respectively, p < 0.03 KW), but not CP 96,444, 10mg/kg (-128 [-163 to -91]; n=8, ns). By contrast LT- and STa-induced secretion (-73 [-97 to -64]; n=8 and -83 [-102 to -81]; n=6 respectively) were not inhibited by CP 96,345, 3mg/kg (-76 [-140 to -38]; n=7 and -98 [-129 to -82]; n=6 respectively, ns) or by CP 96,344. Net electrolyte movement paralleled water. Conclusion. These findings support our earlier observation that SP mediates CT-induced secretion. The failure to inhibit LT- and STa-induced secretion is consistent with differences in the way these three enterotoxins activate a secretory reflex arc in the host. • G1737 GLUCOCORTICOID INDUCTION OF INTESTINAL EPITHELIAL HEAT SHOCK PROTEIN 72 INVOLVES TRANSCRIPTIONAL GENE ACTIVATION. S Urayama, MW Musch, D. Strans, EB Chang. IBD Center; Univ. Chicago; Chicago, IL. Back,,round/Aims. Mucosal injury and dysfunction are cardinal features of inflammatory bowel diseases, arising when injurious immune and inflammatory elements overwhelm cellular defense mechanisms. Inducible heat shock proteins (hsp), particularly hsp72, appear to have a major role in
GASTROENTEROLOGY Vol. 114, No. 4
conferring cellular protection to intestinal epithelial cells against inflammation-associated injury and stress. We previously reported that glucocorticoids (GC), commonly used for the treatment of inflammatory bowel diseases, protect intestinal epithelial cells against oxidant exposure by induction of hsp72. In this study, potential mechanisms of the GC-induced hsp 72 response were studied. Methods. Confluent, normal, diploid, rat intestinal IEC-18 epithelial cells were studied because of their low endogenous hsp72 expression. Cells were stimulated with the GC, dexamethasone (DEX), and the hsp72 protein response determined by Western blotting. As a measure of transcriptional hsp72 gene activation, IECI8 cells were transfected with a CAT/hsp72 promoter construct containing the 2.5 kB upstream region of the human hsp72 gene (a construct beating the heat shock element and previously shown to be activated appropriately in rat intestinal epithelial ceils). Transfected cells were exposed to DEX (10"7 M) for 48 h and electroporated at capacitance of 1000~tF and voltage of 330V to transfect the CAT/hsp72 promoter construct. CAT assays were performed with appropriate controls as previously described. Re..su!ts. In contrast to the immediate hsp72 response to thermal stress of IEC18 cells, DEX stimulates a dose-dependent hsp72 response that first appears after 24 h and peaks at 72-96 h. The response is specific, as no changes in the constitutively expressed homolog, hsc73, were observed. After 24 h exposure, radiolabeled (14C-chloramphenicol) TLC templates (n=3)) showed a significant acetylation of chloramphenicol in IEC-18 samples with DEX exposure as well as in the thermal stress controls. In contrast, no reporter activity was observed in any of the untreated control groups. Conclusion. Glucocorticoid induction of intestinal epithelial hsp 72 involves transcriptional activation of the gene. This may involve an activation process distinct from thermal stress (which requires the heat shock element), as the time course and magnitude of hsp72 induction differ significantly. This process confers cellular protection to intestinal epithelial cells subjected to inflammation-associated stress. • G1738 INTERFERON-T REGULATES EPITHELIAL CHLORIDE SECRETION: ROLE OF E G F R E C E P T O R - D E P E N D E N T P A T H W A Y S . J.M. Uribe and K.E. Barrett, Dept. of Med., UCSD School of Medicine, San Diego, CA. Interferon-'/ (IFN-V) is known to alter epithelial permeability and CIsecretion. A long-term inhibitory effect of IFN-'y on CI" secretion has been attributed to down-regulation of CFTR expression. However, signaling events leading to this response have yet to be elucidated. We have shown that epidermal growth factor (EGF) family members also inhibit calcium-activated CI- secretion, such as that stimulated by carbachol (CCh). Likewise, effects of IFN-T in other systems are mediated via upregulated expression of EGF family members, including transforming growth factor-or (TGF-c0. This could lead to activation of the 170 kDa EGF receptor (EGF-R). Our goal was to determine if IFN-'/ inhibits calcium-activated C1- secretion via EGF-R activation. Studies utilized the intestinal epithelial cell line, T~4. CI- secretion was measured as changes in short circuit current (I,o, in laA/cm2) in Ussing chambers. Levels of cellular TGF-ct and tyrosine-phosphorylated proteins were determined by Western blotting with antibodies (Abs) to TGF-ct or phosphotyrosine. EGF-R activation was assessed in anti-EGF-R immunoprecipitates by Western blotting with Abs to phosphotyrosine. Secreted TGF-et was measured by ELISA. Data are means ± SEM and were analyzed by ANOVA. Stimulation of T~4 cells with IFN-y (100ng/ml) led to a progressive increase in tyrosine-phosphorylation of a 170 kDa protein, peaking between 12-24 hrs. IFN-;, also significantly inhibited CCh-indueed C1- secretion at 1 hr ( A I CCh: 27.2 -+ 2.4 vs. IFN-T+CCh: 16.7 ± 1.7, n=3, p < 0.05) or 24 hrs ( A I CCh: 37.5 ± 0.3 vs. IFN-y+CCh: 9.0--. 2.0, n=3-4, p<0.001) of preincubation~ 24 hr IFN-~/ treatment increased tyrosinephosphorylation of EGF-R. Furthermore, 1 and 24 hr IFN-'/ treatment increased levels of the 20 kDa TGF-ct precursor. However, no free 6 kDa TGF-Gt was found in the medium of IFN-'¢ treated cells. Moreover, an inhibitor of EGF-R kinase activity, tyrphostin AG1478 (Tyr, llaM), did not reverse inhibition of CCh-induced CI- secretion induced by 24h pretreatment with IFN-~/ ( A I CCh: 37.5 ±0.3 vs. IFN-'/+CCh: 9.0-+ 2.0 vs. Tyr+IFN")'+CCh: 10.3 ± 1.6, n=3-4, p <0.001), nor did it inhibit IFN-y-induced phosphorylation of EGF-R. These data suggest that while IFN-'/ increases expression of transmembrane TGF-ct and activates EGF-R in Tg~ cells, receptor activation likely occurs via mechanisms other than ligand-binding. These novel mechanisms for EGF-R transactivation may contribute to the ability of IFN-'/to inhibit CI- secretion. Overall, this may have implications for our understanding of epithelial dysfunction in inflammation, where IFN-'/ levels are reportedly elevated. • G1739 MUC2 IS THE PREDOMINANT SECRETORY MUCIN IN THE MOUSE COLON. B,J.W. Van Klinken, L.A. Duits, M. Verburg, M.K. Makkink, K.M.A.L Tytgat, I.B. Renes, H.A. BUllet#, A.W.C. Einerhand, J Dekker. Pediatric Gastroenterology and Nutrition, Acad. Med. Ctr, Amsterdam, and #Sophia's Children Hospital, Rotterdam, The Netherlands. We have shown previously that the predominant human colonic mucin (MUC2), which is responsible for the mucus-layer in the colon [Tytgat et al., 1994, Gastroenterology 107, 1352], is deficient in active ulcerative colitis [Tytgat et aL, 1996, Biochem Biophys Res Comm 224, 397]. We aim to investigate the role of MUC2 in vivo in the protection of the colon during