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Effects of gaba on stress-induced gastroduodenal ulceration
GASTROENTEROLOGYVol. 114, No. 4
A1174 AGA ABSTRACTS • G4801
COMPARISON OF SYNTHETIC R A T CCK-58 A N D CCK-8 REVEALS DISSOCIATION OF PANCREATIC FLUID AND ENZYME SECRETION AND GREATER POTENCY OF CCK-58 I N VIVO. J.R. Reeve, Jr., Peixian Gong, Tamer Coskun, Yumei Zong, FIJ. Ho, T.E. Solomon. CURE Dig. Dis. Res. Center; Dig. Dis. Div., UCLA Sch. Of Med.; Res. Serv., West Los Angeles VAMC; Los Angeles, CA. Purpose: Physiological actions of CCK-58 in vivo have not been studied in detail because of limited availability of peptide. We synthesized rat CCK-58 and compared its effects on pancreatic secretion to those of CCK-8. Methods: Rats were anesthetized with Inactin and prepared with separate bile and pancreatic catheters. Bile and pancreatic juice were collected in 30 min portions. After collecting basal secretion for 1 h, a single dose of either CCK-58 or CCK-8 (3, 10, 30, 100, 200, 300, 1000, 1500, or 3000 prnol/kg-h) was infused IV in 1% BSA for 2 h (n=3 to 18 per dose and peptide). Sample volume was determined gravimetrical!y, bicarbonate by back-titration, and amylase by measurement of maltose production from starch. Results: CCK-58 was 3-fold more potent than CCK-8 for stimulation of both volume and amylase secretion. Significant increases in volume and amylase occurred at 10 pmol/kg-h CCK-58, 30 and 10 pmol/kg-h CCK-8. Neither peptide increased bicarbonate concentration of pancreatic juice. Maximal volume and amylase responses to CCK-58 and CCK-8 were not significantly different. However, maximal volume response to CCK-58 was seen at a 3-fold lower dose than amylase, while no dissociation was seen with CCK-8. Conclusions: Although less potent than CCK-8 for CCK-A receptor binding in vitro, CCK-58 is substantially more potent for stimulation of pancreatic secretion in vivo (likely because of lower catabolic rates for CCK-58). As the major product of preprocholecystokinin processing in gut mucosa, CCK-58 is probably the dominant mediator of the physiological actions of circulating CCK. Dissociation of maximal fluid and amylase responses to CCK-58 suggest different mechanisms of action, an effect not seen with CCK-8. ~X:Q-
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• G4802
NOVEL HUMAN IL-17 RECEPTORS EXPRESSED IN INTESTINAL EPITHELIAL CELLS. H.C. Reinecker, D.K. Podolsky, D.J. Li. Gastrointestinal Unit, Department of Medicine and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital & Harvard Medical School Boston, MA. Background: IL-17 is a recently identified cytokine which is secreted by activated lymphocytes. IL-17 is able to activate NF-KB and the subsequent stimulation of proinflammatory cytokine expression. Efforts were undertaken to clone the human receptor for IL-17 expressed in intestinal epithelial ceils. Methods: Degenerate primers recognizing motifs in the N-terminus and distal of the transmembrane domain of the mouse IL-17 receptor were used to generate a segment of the human IL-17 receptor from reverse transcribed mRNA isolated from the intestinal epithelial cell line Caco-2. ~. eDNA library screens and 5' as well as 3' RACE using a poly A+ primed adaptor linked small intestinal library yielded the remaining extracellular and cytoplasmic regions of the human IL-17 receptors. Tissue and intestinal epithelial cell line specific expression of human IL-17 receptor variant mRNA was determined by Northern blot analysis and RT-PCR. Results: The obtained cDNAs predict four novel variants of the human IL-17 receptor with openreading frames for proteins of 677, 553, 364 and 597 aminoacids. The human IL-17 receptor variants share a common extracellular domain which has a homology of 79% with the mouse IL-17 receptor on the nucleotide level. The cytoplasmic parts of three of these receptors are derived from exons which share 69%-79% homology with the mouse IL-17 receptor. A eDNA probe corresponding to the extracellular domain of the IL-17 receptor hybridized to single digestion products of human genomic DNA, indicating that the human IL-17 receptor variants are derived by splice events from a single gene. Northern blot analysis revealed the differential expression of human IL-17 receptor transcript of 2.6 kb, 4.4 kb, and 6.6 kb in multiple human tissues and tumor derived intestinal epithelial cell lines. Splice variantspecific RT-PCRs confirmed the expression of IL-17 receptor variants in human intestinal epithelial cell lines. One of the variants lacks the transmembrane domain and thus may encode for a soluble receptor. Conclusion: In contrast to the mouse IL-17 receptor which was described as a single receptor, the human IL-17 receptor is expressed as different splice variants which may mediate distinct functions regulating separate cellular
responses activated by IL-17 in intestinal epithelial cells. These novel IL-17 receptor variants are differentially expressed and may regulate and determine targets of IL-17 during inflammatory responses. G4803
EFFECTS OF GABA ON STRESS-INDUCED GASTRODUODENAL ULCERATION. I Ren, Y Xia, L Qian, P Chotiprasidhi and R Harty Department of Medicine, University of Oklahoma Health Sciences Center and Oklahoma City VA Medical Center, Oklahoma City, OK. CGRP-containing sensory afferent nerves in the stomach participate in gastric defense. Furthermore, peripheral GABAergic mechanism has been shown to protect gastric mucosa against ethanol injury. This protective effect is mediated, in part, by activation of sensory afferent nerves. The effects of GABA on stress-induced gastric ulceration have not been examined. Objectives of this study were to evaluate the effects of GABA on water immersion restraint-induced gastric injury in rats. In addition, participation of capsaicin-sensitive sensory nerves in GABA effects on stress-induced ulceration was determined. Method: Male Sprague-Dawley rats were denied food and allowed free access to water for 24 h prior to experimentation. Experimental protocols included the following treatment prior to stress: 1. Control: saline 1 ml, intragastric (ig), 2.GABA, 400mg/kg (ig), 3. Sensory denervation by chronic capsaicin treatment (total 125 mg subcutaneously 2 weeks prior to stress experiment) and 4. GABA administration to capsaicin treated rats. Stress experiments were performed by placing rats in rigid plastic tubes which were immersed in a water bath at 24°C for four hours. After the stress interval rats were euthanized and gastroduodenal mucosa was inspected and the area of ulceration quantitated (mm2). CGRP content was measured by RIA after tissue extraction. Data represent the result of 6-8 experiments. Statistical analysis was determined by ANOVA. Results: Water immersion stress caused both severe gastric and duodenal ulceration: areas of gastric injury measured 47.8 ± 5.6 mm2 and duodenal injury measured 5.7 -+0.6 mm2 respectively. Administration of GABA significantly reduced stress-induced gastric lesion by 81% to 8.7 +-.0.5 mm2 and duodenal lesion by 90% to 0.6 ± 0.2 mm2. Capsaicin-induced sensory denervation enhanced significantly stress ulceration in the stomach (61.8 +_7.2 mm2; p<0.01 vs stress alone) and duodenum (7.8 ± 0.8 mm2; p<0.01 vs stress alone). GABA protection against stress ulceration was prevented by capsaicin-inudced sensory denervation. Gastric and duodenal CGRP contents (pmol/g tissue) were significantly reduced by stress when compared to values from non-stressed animals: 22.8 + 3.3 vs 48.7 ± 5.4 (p<0.001) in corpus and 25.5 + 3.1 vs 37.9 ± 4.8 (P<0.05) in duodenum. GABA pretreatment prevented stress-induced reduction in CGRP content in both stomach and duodenum. Condusion: orally administrated GABA prevented stress-induced ulceration in the stomach and duodenum. The data suggest that the protective effects of GABA in this stress model is associated with activation of sensory afferent nerves. • G4804
NEUROTENSIN (NT) INDUCES CHLORIDE SECRETION IN HUMAN COLONIC M U C O S A T H R O U G H A N ADENOSINE-DEPENDENT P A T H W A Y . ].2Riegler M, 1I Castagliuolo, 3JB Matthews, 2M Mlk, 2T
Sogukoglu, 2E Wenzl, 1C Pothoulakis. Division of Gastroenterology I and 3Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School; Boston MA, and 2University Clinic of Surgery, Vienna, Austria. Animal studies showed that NT modulates fluid secretion in the gastrointestinal tract possibly through a neural reflex in the enteric nervous system. Our goal was to investigate whether NT stimulates chloride secretion in normal human colon and study the cellular pathway(s) involved in this response. Methods: Human colonic mucosal strips were mounted in Ussing chambers and after 30 rain equilibration NT (10 .6 to 10.9 M) was added. Changes (A) in short circuit current (Isc) were recorded every 2 rain for 120 min. Atropine, lodoxamide, indomethacin, tetrodototoxin (TTX), and the NT receptor antagonist SR-48,942 (Sanofi Reserche, Toulouse France) (all inhibitors at 10.6 M) were added to the sernsal side 30 rain before NT (10-6 M) exposure. Results: Serosal, but not luminal, addition of NT induced a dosedependent, C1--dependent and transient rise of colonic Isc, which peaked at 10 min and was completely abolished by the specific NTR antagonist, SR 48,692 (p<0.01). Addition of NT at 10-6 M caused a 20-fold rise in Alsc (p<0.001), whereas at 10-7 and 10.8 M induced a 13-fold and 6.5-fold increase (p<0.01), respectively. NT at 10.9 M had no significant secretory effect. The nerve blocker "I~X almost completely inhibited NT-induced changes on Alsc (p<0.001), while the mast cell stabilizer lodoxamide had no inhibitory effect. Since the effects of NT appear to involve intestinal nerves, we studied the role of acetylcholine, adenosine and vasoactive intestinal peptide (VIP) on NT-induced secretion. The muscatinic receptor antagonist atropine had no effect, whereas the adenosine receptor antagonists 8-phenyltheophylline and 3,7-dimethyl-1-propargylxanthine), and the VIP antagonist [Dcpa 6, Leul7]VIP significantly inhibited NT-induced changes in AIsc (by 75%, 65% and 58% respectively). Conclusion: NT-stimulated chloride secretion in human colon is selectively sensitive to inhibitors of adenosine and VIP receptors and thus may involve purinergic and/or VIPergic neural pathways.
A1174 AGA ABSTRACTS • G4801
COMPARISON OF SYNTHETIC R A T CCK-58 A N D CCK-8 REVEALS DISSOCIATION OF PANCREATIC FLUID AND ENZYME SECRETION AND GREATER POTENCY OF CCK-58 I N VIVO. J.R. Reeve, Jr., Peixian Gong, Tamer Coskun, Yumei Zong, FIJ. Ho, T.E. Solomon. CURE Dig. Dis. Res. Center; Dig. Dis. Div., UCLA Sch. Of Med.; Res. Serv., West Los Angeles VAMC; Los Angeles, CA. Purpose: Physiological actions of CCK-58 in vivo have not been studied in detail because of limited availability of peptide. We synthesized rat CCK-58 and compared its effects on pancreatic secretion to those of CCK-8. Methods: Rats were anesthetized with Inactin and prepared with separate bile and pancreatic catheters. Bile and pancreatic juice were collected in 30 min portions. After collecting basal secretion for 1 h, a single dose of either CCK-58 or CCK-8 (3, 10, 30, 100, 200, 300, 1000, 1500, or 3000 prnol/kg-h) was infused IV in 1% BSA for 2 h (n=3 to 18 per dose and peptide). Sample volume was determined gravimetrical!y, bicarbonate by back-titration, and amylase by measurement of maltose production from starch. Results: CCK-58 was 3-fold more potent than CCK-8 for stimulation of both volume and amylase secretion. Significant increases in volume and amylase occurred at 10 pmol/kg-h CCK-58, 30 and 10 pmol/kg-h CCK-8. Neither peptide increased bicarbonate concentration of pancreatic juice. Maximal volume and amylase responses to CCK-58 and CCK-8 were not significantly different. However, maximal volume response to CCK-58 was seen at a 3-fold lower dose than amylase, while no dissociation was seen with CCK-8. Conclusions: Although less potent than CCK-8 for CCK-A receptor binding in vitro, CCK-58 is substantially more potent for stimulation of pancreatic secretion in vivo (likely because of lower catabolic rates for CCK-58). As the major product of preprocholecystokinin processing in gut mucosa, CCK-58 is probably the dominant mediator of the physiological actions of circulating CCK. Dissociation of maximal fluid and amylase responses to CCK-58 suggest different mechanisms of action, an effect not seen with CCK-8. ~X:Q-
1at 16tl140,
| too~
100.
| ~. .i
Q.
o
• G4802
NOVEL HUMAN IL-17 RECEPTORS EXPRESSED IN INTESTINAL EPITHELIAL CELLS. H.C. Reinecker, D.K. Podolsky, D.J. Li. Gastrointestinal Unit, Department of Medicine and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital & Harvard Medical School Boston, MA. Background: IL-17 is a recently identified cytokine which is secreted by activated lymphocytes. IL-17 is able to activate NF-KB and the subsequent stimulation of proinflammatory cytokine expression. Efforts were undertaken to clone the human receptor for IL-17 expressed in intestinal epithelial ceils. Methods: Degenerate primers recognizing motifs in the N-terminus and distal of the transmembrane domain of the mouse IL-17 receptor were used to generate a segment of the human IL-17 receptor from reverse transcribed mRNA isolated from the intestinal epithelial cell line Caco-2. ~. eDNA library screens and 5' as well as 3' RACE using a poly A+ primed adaptor linked small intestinal library yielded the remaining extracellular and cytoplasmic regions of the human IL-17 receptors. Tissue and intestinal epithelial cell line specific expression of human IL-17 receptor variant mRNA was determined by Northern blot analysis and RT-PCR. Results: The obtained cDNAs predict four novel variants of the human IL-17 receptor with openreading frames for proteins of 677, 553, 364 and 597 aminoacids. The human IL-17 receptor variants share a common extracellular domain which has a homology of 79% with the mouse IL-17 receptor on the nucleotide level. The cytoplasmic parts of three of these receptors are derived from exons which share 69%-79% homology with the mouse IL-17 receptor. A eDNA probe corresponding to the extracellular domain of the IL-17 receptor hybridized to single digestion products of human genomic DNA, indicating that the human IL-17 receptor variants are derived by splice events from a single gene. Northern blot analysis revealed the differential expression of human IL-17 receptor transcript of 2.6 kb, 4.4 kb, and 6.6 kb in multiple human tissues and tumor derived intestinal epithelial cell lines. Splice variantspecific RT-PCRs confirmed the expression of IL-17 receptor variants in human intestinal epithelial cell lines. One of the variants lacks the transmembrane domain and thus may encode for a soluble receptor. Conclusion: In contrast to the mouse IL-17 receptor which was described as a single receptor, the human IL-17 receptor is expressed as different splice variants which may mediate distinct functions regulating separate cellular
responses activated by IL-17 in intestinal epithelial cells. These novel IL-17 receptor variants are differentially expressed and may regulate and determine targets of IL-17 during inflammatory responses. G4803
EFFECTS OF GABA ON STRESS-INDUCED GASTRODUODENAL ULCERATION. I Ren, Y Xia, L Qian, P Chotiprasidhi and R Harty Department of Medicine, University of Oklahoma Health Sciences Center and Oklahoma City VA Medical Center, Oklahoma City, OK. CGRP-containing sensory afferent nerves in the stomach participate in gastric defense. Furthermore, peripheral GABAergic mechanism has been shown to protect gastric mucosa against ethanol injury. This protective effect is mediated, in part, by activation of sensory afferent nerves. The effects of GABA on stress-induced gastric ulceration have not been examined. Objectives of this study were to evaluate the effects of GABA on water immersion restraint-induced gastric injury in rats. In addition, participation of capsaicin-sensitive sensory nerves in GABA effects on stress-induced ulceration was determined. Method: Male Sprague-Dawley rats were denied food and allowed free access to water for 24 h prior to experimentation. Experimental protocols included the following treatment prior to stress: 1. Control: saline 1 ml, intragastric (ig), 2.GABA, 400mg/kg (ig), 3. Sensory denervation by chronic capsaicin treatment (total 125 mg subcutaneously 2 weeks prior to stress experiment) and 4. GABA administration to capsaicin treated rats. Stress experiments were performed by placing rats in rigid plastic tubes which were immersed in a water bath at 24°C for four hours. After the stress interval rats were euthanized and gastroduodenal mucosa was inspected and the area of ulceration quantitated (mm2). CGRP content was measured by RIA after tissue extraction. Data represent the result of 6-8 experiments. Statistical analysis was determined by ANOVA. Results: Water immersion stress caused both severe gastric and duodenal ulceration: areas of gastric injury measured 47.8 ± 5.6 mm2 and duodenal injury measured 5.7 -+0.6 mm2 respectively. Administration of GABA significantly reduced stress-induced gastric lesion by 81% to 8.7 +-.0.5 mm2 and duodenal lesion by 90% to 0.6 ± 0.2 mm2. Capsaicin-induced sensory denervation enhanced significantly stress ulceration in the stomach (61.8 +_7.2 mm2; p<0.01 vs stress alone) and duodenum (7.8 ± 0.8 mm2; p<0.01 vs stress alone). GABA protection against stress ulceration was prevented by capsaicin-inudced sensory denervation. Gastric and duodenal CGRP contents (pmol/g tissue) were significantly reduced by stress when compared to values from non-stressed animals: 22.8 + 3.3 vs 48.7 ± 5.4 (p<0.001) in corpus and 25.5 + 3.1 vs 37.9 ± 4.8 (P<0.05) in duodenum. GABA pretreatment prevented stress-induced reduction in CGRP content in both stomach and duodenum. Condusion: orally administrated GABA prevented stress-induced ulceration in the stomach and duodenum. The data suggest that the protective effects of GABA in this stress model is associated with activation of sensory afferent nerves. • G4804
NEUROTENSIN (NT) INDUCES CHLORIDE SECRETION IN HUMAN COLONIC M U C O S A T H R O U G H A N ADENOSINE-DEPENDENT P A T H W A Y . ].2Riegler M, 1I Castagliuolo, 3JB Matthews, 2M Mlk, 2T
Sogukoglu, 2E Wenzl, 1C Pothoulakis. Division of Gastroenterology I and 3Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School; Boston MA, and 2University Clinic of Surgery, Vienna, Austria. Animal studies showed that NT modulates fluid secretion in the gastrointestinal tract possibly through a neural reflex in the enteric nervous system. Our goal was to investigate whether NT stimulates chloride secretion in normal human colon and study the cellular pathway(s) involved in this response. Methods: Human colonic mucosal strips were mounted in Ussing chambers and after 30 rain equilibration NT (10 .6 to 10.9 M) was added. Changes (A) in short circuit current (Isc) were recorded every 2 rain for 120 min. Atropine, lodoxamide, indomethacin, tetrodototoxin (TTX), and the NT receptor antagonist SR-48,942 (Sanofi Reserche, Toulouse France) (all inhibitors at 10.6 M) were added to the sernsal side 30 rain before NT (10-6 M) exposure. Results: Serosal, but not luminal, addition of NT induced a dosedependent, C1--dependent and transient rise of colonic Isc, which peaked at 10 min and was completely abolished by the specific NTR antagonist, SR 48,692 (p<0.01). Addition of NT at 10-6 M caused a 20-fold rise in Alsc (p<0.001), whereas at 10-7 and 10.8 M induced a 13-fold and 6.5-fold increase (p<0.01), respectively. NT at 10.9 M had no significant secretory effect. The nerve blocker "I~X almost completely inhibited NT-induced changes on Alsc (p<0.001), while the mast cell stabilizer lodoxamide had no inhibitory effect. Since the effects of NT appear to involve intestinal nerves, we studied the role of acetylcholine, adenosine and vasoactive intestinal peptide (VIP) on NT-induced secretion. The muscatinic receptor antagonist atropine had no effect, whereas the adenosine receptor antagonists 8-phenyltheophylline and 3,7-dimethyl-1-propargylxanthine), and the VIP antagonist [Dcpa 6, Leul7]VIP significantly inhibited NT-induced changes in AIsc (by 75%, 65% and 58% respectively). Conclusion: NT-stimulated chloride secretion in human colon is selectively sensitive to inhibitors of adenosine and VIP receptors and thus may involve purinergic and/or VIPergic neural pathways.