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Calmodulin antagonism by zaldaride maleate significantly ameliorates cholera toxin induced secretion
April 1995
CALMODULIN ANTAGONISM BY ZALDARIDE MALEATE SIGNIFICANTLY AMELIORATES CHOLERA TOXIN INDUCED SECRETION. JL Turvill, FH Mourad, MJG Farthing. Digestive Diseases Research Centre, Medical College of St. Bartholomew's Hospital, London, UK. Introduction: The majority of known intestinal secretagogues act via intracellular calcium and calmodulin which open enterocyte chloride channels resulting in water and electrolyte secretion. Theoretically, blocking this pathway will inhibit secretion. Aim: The aim of this study was to investigate the effect of a new, specific ealmodulin antagonist, zaldaride maleate, on cholera toxin (CT)-induced water and electrolyte secretion in rat small intestine in vivo. Methods: Under phenobarbitone anesthesia, the entire small intestine of fasted adult male Wistar rats (180-220g) was isolated between two carmulae. Zaldaride maleate (30mg/kg dissolved in dimethyl-sulfoxide (DMSO) and then diluted in lml distilled water) or DMSO in distilled water alone was instilled into the isolated segment for 30 minutes prior to the instillation of 751.tg CT. After 2h incubation with CT, in situ small intestinal perfusion was performed with plasma dectrolyte solution (PES= Na 140, K 4, C1 104, HCO3 40mmoFL) containing [14C]polyethylene glycol 4000 as a non-absorbable volume marker to assess net water and electrolyte movement. Control animals were perfused without exposure to CT. Results: CT induced net water secretion (median: -50.1pl/min/g dry intestinal weight [interquartile range: -29.8 to -59.5], n=13). Zaldaride maleate markedly reduced the water secretion (-1.5 [-9.5 to 9.7], n=6; p<0.01) and reversed secretion to absorption in 50% of the rats. However, water absorption was less than in control rats without CT (50 [41 to 61], n=8; p<0.01 compared to CT and zaldaride maleate). Sodium and chloride movement paralleled that of water. Summary and conclusion: Our findings indicate that calmodulin antagonism by zaldaride maleate can significantly decrease CT-induced water secretion and thus may be of benefit in the treatment of secretory diarrheas where calmodulin plays an important role.
• TISSUE AND LUMINAL 5-HT LEVELS IN CHOLERA TOXIN AND E.COLI HEAT LABILE TOXIN INDUCED SECRETION. JL Turvill, FH Mournd, D Perrett, MJG Farthing. Digestive Diseases Research Centre, Medical College of St Bartholomew's Hospital, London, UK. 5-HT is a potent intestinal secretagogue. We have previously demonstrated that 5-HT3 receptor antagonism reverses cholera toxin (CT) induced secretory state but fails to inhibit E.coli heat labile toxin (LT) induced secretion. Both LT and CT act by binding to the GMt receptor, causing the induction of cyclic AMP. The aim of this study was to measure tissue and luminal 5-HT levels after exposing rat intestine to CT and LT. 75lag CT, 50ttg LT or saline was instilled into isolated whole small intestine of adult male Wistar rats (180-220g). After 2h incubation, in situ small intestinal perfusion was performed with plasma electrolyte solution (Na 140, K 4, CI 104, HCO3 40mmol/L) containing a non-absorbable volume marker to assess net water movement. 5-HT levels were then determined in feeze-clamped, full thickness small intestinal segments by high performance liquid chromatography with fluorimetric detection. In parallel experiments, 25~tg CT, 17.5~tg LT or saline was incubated for 2h in a 20cm loop of isolated jejunum. ThereaRer, the 5-HT accumulating in the lumen during a 30rain period was determined. CT and LT induced a comparable net water secretion (CT: median -50.11al/mirdg [interquartile range -29.8 to -59.5], n=13; LT: -49 [-30 to -58], n=10) compared to control (51 [40 to 60], n=8: p<0.01). CT decreased tissue 5-HT levels (32pmol/mg dry weight [25.8 to 38], n=12) and increased luminal 5-HT (0.31pmol/mg/h [0.17 to 0.32], n=10.) compared with control (tissue 5-HT: 73 [58.5 to 80], n=ll; p<0.01; luminal 5-HT: 0.11 [0.08 to 0.18], n=10: p<0.01). No changes were detected, however, after LT, compared to control (tissue 5-HT: 64 [53 to70], n=8; luminal 5-HT: 0.08 [0.07 to 0.16], n=10: NS ). Our findings quantitatively confirm the release of 5-HT occuring after CT but not LT exposure and support our observation that 5-HT3 receptor antagonism fails to reverse LT-induced secretion. Thus, while both LT and CT induce cyclic AMP their mechanisms of action differ with respect to involvement of 5-HT.
Intestinal Disorders
A333
• EFFECT OF 5-HT DEPLETION ON BASAL INTESTINAL WATER MOVEMENT AND ON CHOLERA TOXIN INDUCED SECRETION. JL TurviU, FH Mourad, D Perrett, MIG Farthing. Digestive Diseases Research Centre, Medical College of St Bartholomew's Hospital, London, UK. 5-Hydroxytryptamine (5-HT) is a potent intestinal secretagogue. It is released from enterochromaffin cells and is implicated in cholera toxin (CT) induced secretion, para-Chlorophenylalanine (PCPA) Selectively inhibits tryptophan hydroxylase, the enzyme acting at the rate limiting step of 5-HT synthesis. We sought to assess whether depletion of small intestinal 5-HT by pretreatment with PCPA would effect basal water movement and CT induced secretion. Adult male Wistar rats (180-220g) were gavaged daily for 3 days with either 500mg/kg PCPA in lmi water or water alone (control). 75p.g CT or saline was then instilled into isolated whole small intestine After 2h, in situ perfusion was performed with plasma electrolyte solution (Na'140, K 4, CI 104, HCO3 40mmol/L) to assess net water and electrolyte movement. At the end of the experiment 5-HT levels were determined in freeze-clamped, full thickness segments of small intestine by high performance liquid chromatography with fluorimetric detection. Tissue 5-HT levels in controls (median 73pmol/mg dry weight [interquartile range 58.5 to 80], n=ll) were significantly decreased by PCPA pretreatment (6 [4 to 7], n=13; p<0.01). CT reduced tissue 5-HT levels in the controls (32 [25.5 to 38.5], n=12; p<0.01) but not in PCPA pretreated rats (4 [6 to 3.5], n=6). Net water absorption in controls (50 [41 to 61], n=8) was enhanced by PCPA (811~l/min/g [74.5 to 85], n=8; p<0.05). In addition, CT-induced net water secretion (-75.2 [-58 to -89], n=l 1) was reduced by pretreatment with PCPA (-38.3 [-34.3 to =43], n=6; p<0.05). Electrolyte movement paralleled water. These findings support a role for 5-HT in mediating CT induced secretion. The failure of PCPA pretreatment to reverse the secretion points towards the efficacy of sub-maximal levels of 5-HT in the secretory state. That 5HT depletion enhances water absorption in normal intestine indicates a role for 5-HT in basal water transport.
MECHANISM(S) OF SODIUM TRANSPORT ACROSS THE HUMAN COLONIC BASOLATERAL MEMBRANE. S.Tvapi, V. Joshi, K. E. Kim, K. Ramaswamy and P.K. Dudeja. Dept. of Medicine, University of Illinois at Chicago and Westside VA Med Center, Chicago, IL Transcellular transport of Na+ across the polarized epithelial cells involves contribution of transporters localized on both the apical and basolateral membrane domains. Recent studies from our laboratory have suggested that Na+ uptake by apical membrane vesicles isolated from both the proximal and distal human colon is via an electroneutral Na+-H+ exchange and Na + channels. Mechanism(s) of Na + transport across the basolateral membranes in the human colon, however, are not well understood. Current studies were, therefore, undertaken to elucidate Na+ transport mechanisms in isolated basolateral membrane vesicles (BLMV) utilizing a rapid filtration 22Na uptake technique. BLMV were purified from mucosal scrapings of organ donor proximal colons utilizing a Percoll continuous density gradient centrifugation technique developed in our laboratory, with a 7-11 fold enrichment in Na+-K+ ATPase activity compared to crude homogenate. Our results are summarized as follows: i) an outwardly directed H+ gradient (pHin5.5/pHout7.5) stimulated 22Na uptake into these vesicles; ii) 22Na uptake was markedly inhibited (80-100%) by Na+-H + exchange inhibitors EIPA (Ki : 8.0 + 1.6 I.tM) and amiloride (Ki; 120 + 50 btM); iii) H+ gradient-stimulated 22Na uptake was not inhibited by other transport inhibitors e.g. bumetanide, SITS, DIDS, acetazolamide, phenamil and benzamil; iv) voltage clamping the membrane vesicles by K+/valinomycin failed to influence the H+ gradient-stimulated 22Na uptake; v) 22Na uptake was saturable with a Km for Na+ of 16.9 + 5.9 mM and a Vmax of 1.47 ± 0.42 nmol/mg prot./15 sec; vi) lmM 22Na uptake was markedly inhibited by 10 mM concentrations of non radioactive Na+ and Li+ but was unaffected by 10 mM N-Methyl Glucamine+, choline + and NH4+ and vii) an inwardly directed HCO3gradient (100 mM) significantly stimulated 22Na uptake (5.6 + 1.0 fold) compared to uptake in the presence of an outwardly directed H + gradient (pHin5.5/pHout7.5) alone. Conclusion: Our results indicate that in addition to a Na+-K+ ATPase, Na+ transport pathways in the human colonic basolateral domains involve an electroneutral Na+-H + exchange process and a possible Na+-HCO3- cotransport mechanism. (Supported by BRSG, NIDDK and the Dept. of Veterans Affairs).
CALMODULIN ANTAGONISM BY ZALDARIDE MALEATE SIGNIFICANTLY AMELIORATES CHOLERA TOXIN INDUCED SECRETION. JL Turvill, FH Mourad, MJG Farthing. Digestive Diseases Research Centre, Medical College of St. Bartholomew's Hospital, London, UK. Introduction: The majority of known intestinal secretagogues act via intracellular calcium and calmodulin which open enterocyte chloride channels resulting in water and electrolyte secretion. Theoretically, blocking this pathway will inhibit secretion. Aim: The aim of this study was to investigate the effect of a new, specific ealmodulin antagonist, zaldaride maleate, on cholera toxin (CT)-induced water and electrolyte secretion in rat small intestine in vivo. Methods: Under phenobarbitone anesthesia, the entire small intestine of fasted adult male Wistar rats (180-220g) was isolated between two carmulae. Zaldaride maleate (30mg/kg dissolved in dimethyl-sulfoxide (DMSO) and then diluted in lml distilled water) or DMSO in distilled water alone was instilled into the isolated segment for 30 minutes prior to the instillation of 751.tg CT. After 2h incubation with CT, in situ small intestinal perfusion was performed with plasma dectrolyte solution (PES= Na 140, K 4, C1 104, HCO3 40mmoFL) containing [14C]polyethylene glycol 4000 as a non-absorbable volume marker to assess net water and electrolyte movement. Control animals were perfused without exposure to CT. Results: CT induced net water secretion (median: -50.1pl/min/g dry intestinal weight [interquartile range: -29.8 to -59.5], n=13). Zaldaride maleate markedly reduced the water secretion (-1.5 [-9.5 to 9.7], n=6; p<0.01) and reversed secretion to absorption in 50% of the rats. However, water absorption was less than in control rats without CT (50 [41 to 61], n=8; p<0.01 compared to CT and zaldaride maleate). Sodium and chloride movement paralleled that of water. Summary and conclusion: Our findings indicate that calmodulin antagonism by zaldaride maleate can significantly decrease CT-induced water secretion and thus may be of benefit in the treatment of secretory diarrheas where calmodulin plays an important role.
• TISSUE AND LUMINAL 5-HT LEVELS IN CHOLERA TOXIN AND E.COLI HEAT LABILE TOXIN INDUCED SECRETION. JL Turvill, FH Mournd, D Perrett, MJG Farthing. Digestive Diseases Research Centre, Medical College of St Bartholomew's Hospital, London, UK. 5-HT is a potent intestinal secretagogue. We have previously demonstrated that 5-HT3 receptor antagonism reverses cholera toxin (CT) induced secretory state but fails to inhibit E.coli heat labile toxin (LT) induced secretion. Both LT and CT act by binding to the GMt receptor, causing the induction of cyclic AMP. The aim of this study was to measure tissue and luminal 5-HT levels after exposing rat intestine to CT and LT. 75lag CT, 50ttg LT or saline was instilled into isolated whole small intestine of adult male Wistar rats (180-220g). After 2h incubation, in situ small intestinal perfusion was performed with plasma electrolyte solution (Na 140, K 4, CI 104, HCO3 40mmol/L) containing a non-absorbable volume marker to assess net water movement. 5-HT levels were then determined in feeze-clamped, full thickness small intestinal segments by high performance liquid chromatography with fluorimetric detection. In parallel experiments, 25~tg CT, 17.5~tg LT or saline was incubated for 2h in a 20cm loop of isolated jejunum. ThereaRer, the 5-HT accumulating in the lumen during a 30rain period was determined. CT and LT induced a comparable net water secretion (CT: median -50.11al/mirdg [interquartile range -29.8 to -59.5], n=13; LT: -49 [-30 to -58], n=10) compared to control (51 [40 to 60], n=8: p<0.01). CT decreased tissue 5-HT levels (32pmol/mg dry weight [25.8 to 38], n=12) and increased luminal 5-HT (0.31pmol/mg/h [0.17 to 0.32], n=10.) compared with control (tissue 5-HT: 73 [58.5 to 80], n=ll; p<0.01; luminal 5-HT: 0.11 [0.08 to 0.18], n=10: p<0.01). No changes were detected, however, after LT, compared to control (tissue 5-HT: 64 [53 to70], n=8; luminal 5-HT: 0.08 [0.07 to 0.16], n=10: NS ). Our findings quantitatively confirm the release of 5-HT occuring after CT but not LT exposure and support our observation that 5-HT3 receptor antagonism fails to reverse LT-induced secretion. Thus, while both LT and CT induce cyclic AMP their mechanisms of action differ with respect to involvement of 5-HT.
Intestinal Disorders
A333
• EFFECT OF 5-HT DEPLETION ON BASAL INTESTINAL WATER MOVEMENT AND ON CHOLERA TOXIN INDUCED SECRETION. JL TurviU, FH Mourad, D Perrett, MIG Farthing. Digestive Diseases Research Centre, Medical College of St Bartholomew's Hospital, London, UK. 5-Hydroxytryptamine (5-HT) is a potent intestinal secretagogue. It is released from enterochromaffin cells and is implicated in cholera toxin (CT) induced secretion, para-Chlorophenylalanine (PCPA) Selectively inhibits tryptophan hydroxylase, the enzyme acting at the rate limiting step of 5-HT synthesis. We sought to assess whether depletion of small intestinal 5-HT by pretreatment with PCPA would effect basal water movement and CT induced secretion. Adult male Wistar rats (180-220g) were gavaged daily for 3 days with either 500mg/kg PCPA in lmi water or water alone (control). 75p.g CT or saline was then instilled into isolated whole small intestine After 2h, in situ perfusion was performed with plasma electrolyte solution (Na'140, K 4, CI 104, HCO3 40mmol/L) to assess net water and electrolyte movement. At the end of the experiment 5-HT levels were determined in freeze-clamped, full thickness segments of small intestine by high performance liquid chromatography with fluorimetric detection. Tissue 5-HT levels in controls (median 73pmol/mg dry weight [interquartile range 58.5 to 80], n=ll) were significantly decreased by PCPA pretreatment (6 [4 to 7], n=13; p<0.01). CT reduced tissue 5-HT levels in the controls (32 [25.5 to 38.5], n=12; p<0.01) but not in PCPA pretreated rats (4 [6 to 3.5], n=6). Net water absorption in controls (50 [41 to 61], n=8) was enhanced by PCPA (811~l/min/g [74.5 to 85], n=8; p<0.05). In addition, CT-induced net water secretion (-75.2 [-58 to -89], n=l 1) was reduced by pretreatment with PCPA (-38.3 [-34.3 to =43], n=6; p<0.05). Electrolyte movement paralleled water. These findings support a role for 5-HT in mediating CT induced secretion. The failure of PCPA pretreatment to reverse the secretion points towards the efficacy of sub-maximal levels of 5-HT in the secretory state. That 5HT depletion enhances water absorption in normal intestine indicates a role for 5-HT in basal water transport.
MECHANISM(S) OF SODIUM TRANSPORT ACROSS THE HUMAN COLONIC BASOLATERAL MEMBRANE. S.Tvapi, V. Joshi, K. E. Kim, K. Ramaswamy and P.K. Dudeja. Dept. of Medicine, University of Illinois at Chicago and Westside VA Med Center, Chicago, IL Transcellular transport of Na+ across the polarized epithelial cells involves contribution of transporters localized on both the apical and basolateral membrane domains. Recent studies from our laboratory have suggested that Na+ uptake by apical membrane vesicles isolated from both the proximal and distal human colon is via an electroneutral Na+-H+ exchange and Na + channels. Mechanism(s) of Na + transport across the basolateral membranes in the human colon, however, are not well understood. Current studies were, therefore, undertaken to elucidate Na+ transport mechanisms in isolated basolateral membrane vesicles (BLMV) utilizing a rapid filtration 22Na uptake technique. BLMV were purified from mucosal scrapings of organ donor proximal colons utilizing a Percoll continuous density gradient centrifugation technique developed in our laboratory, with a 7-11 fold enrichment in Na+-K+ ATPase activity compared to crude homogenate. Our results are summarized as follows: i) an outwardly directed H+ gradient (pHin5.5/pHout7.5) stimulated 22Na uptake into these vesicles; ii) 22Na uptake was markedly inhibited (80-100%) by Na+-H + exchange inhibitors EIPA (Ki : 8.0 + 1.6 I.tM) and amiloride (Ki; 120 + 50 btM); iii) H+ gradient-stimulated 22Na uptake was not inhibited by other transport inhibitors e.g. bumetanide, SITS, DIDS, acetazolamide, phenamil and benzamil; iv) voltage clamping the membrane vesicles by K+/valinomycin failed to influence the H+ gradient-stimulated 22Na uptake; v) 22Na uptake was saturable with a Km for Na+ of 16.9 + 5.9 mM and a Vmax of 1.47 ± 0.42 nmol/mg prot./15 sec; vi) lmM 22Na uptake was markedly inhibited by 10 mM concentrations of non radioactive Na+ and Li+ but was unaffected by 10 mM N-Methyl Glucamine+, choline + and NH4+ and vii) an inwardly directed HCO3gradient (100 mM) significantly stimulated 22Na uptake (5.6 + 1.0 fold) compared to uptake in the presence of an outwardly directed H + gradient (pHin5.5/pHout7.5) alone. Conclusion: Our results indicate that in addition to a Na+-K+ ATPase, Na+ transport pathways in the human colonic basolateral domains involve an electroneutral Na+-H + exchange process and a possible Na+-HCO3- cotransport mechanism. (Supported by BRSG, NIDDK and the Dept. of Veterans Affairs).