title: Inhibition of cAMP-activated intestinal chloride secretion by diclofenac: cellular mechanism and potential application in cholera.
abstract: Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84) cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl- current showed that diclofenac reversibly inhibited CFTR Cl- channel activity (IC50 ∼ 10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na(+)-K(+) ATPases and Na(+)-K(+)-Cl- cotransporters, but inhibited cAMP-activated basolateral K(+) channels with IC50 of ∼ 3 µM. In addition, diclofenac suppressed Ca(2+)-activated Cl- channels, inwardly rectifying Cl- channels, and Ca(2+)-activated basolateral K(+) channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl- secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼ 70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca(2+)-activated Cl- secretion by inhibiting both apical Cl- channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal hypersecretion of Cl-.
authors.affiliation: Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand; Research Center of Transport Protein for Medical Innovation, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Pawin
authors.initials: P
authors.lastname: Pongkorpsakol
authors.affiliation: Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand; Research Center of Transport Protein for Medical Innovation, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Nutthapoom
authors.initials: N
authors.lastname: Pathomthongtaweechai
authors.affiliation: Graduate Study, Faculty of Allied Health Sciences, Thammasat University, Pathumtanee, Thailand.
authors.forename: Potjanee
authors.initials: P
authors.lastname: Srimanote
authors.affiliation: Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand; Research Center of Transport Protein for Medical Innovation, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Sunhapas
authors.initials: S
authors.lastname: Soodvilai
authors.affiliation: Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand; Research Center of Transport Protein for Medical Innovation, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Varanuj
authors.initials: V
authors.lastname: Chatsudthipong
authors.affiliation: Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand; Research Center of Transport Protein for Medical Innovation, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Chatchai
authors.initials: C
authors.lastname: Muanprasat
mesh_terms: D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D002712:Chlorides / Q000378:metabolism*; D002771:Cholera / Q000378:metabolism* / Q000473:pathology; D002772:Cholera Toxin; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004008:Diclofenac / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects; D008297:Male; D051379:Mice; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism
chemical_list: C505032:CFTR protein, human; D002712:Chlorides; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004008:Diclofenac; D002772:Cholera Toxin; D000242:Cyclic AMP; D002118:Calcium