Mechanisms of chloride secretion induced by thermostable direct haemolysinof Vibrio parahaemolyticus in human colonic tissue and a human intestinal epithelial cell line

Thermostable direct haemolysin (TDH) produced by Vibrio parahaemolyticus is thought to play an important role in the severe diarrhoea caused by this o rganism. This study investigated the enterotoxicity of TDH for human intest inal cells. Addition of TDH to the mucosal side of human colonic tissue in Ussing chambers caused increased short circuit currents (Isc), a process th at was inhibited by 4,4 '-diisothiocyanatostilbene-2,2'-disulphonic acid (D IDS), an inhibitor of Ca2+-activated chloride (Cl-) channels. With human co lonic epithelial (Caco-2) cells, high Ise and intracellular Ca2+ concentrat ions ([Ca2+](in)) were detected after the addition of TDH to the apical sid e of the cell monolayer, The Ise decreased with the addition of DIDS, but n ot with glybenclamide, 5-nitro-2-(3-phenylpropylamino) benzoic acid, or gad olinium chloride, No Ise increase with TDH was observed when the Cl- in the medium was replaced by gluconate or when Ca2+ was depleted, Similarly, TDH did not raise [Ca2+](in) after depletion of extracellular Ca2+, R7, a muta nt form of TDH, reduced the effects of TDH on Isc and [Ca2+](in) as did pro tein kinase C (PKC) inhibitors. Thus, TDH increases Cl- secretion in human colonic epithelial cells, apparently through mechanisms involving cell bind ing and Ca2+ influx, followed by elevation of [Ca2+](in) associated with PK C phosphorylation.