Mechanisms of chloride secretion induced by thermostable direct haemolysin of 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 organism. This study investigated the enterotoxicity of TDH for human intestinal cells. Addition of TDH to the mucosal side of human colonic tissue in Ussing chambers caused increased short circuit currents (Isc), a process that was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of Ca2+ -activated chloride (Cl-) channels. With human colonic epithelial (Caco-2) cells, high Isc and intracellular Ca2+ concentrations ([Ca2+]in) were detected after the addition of TDH to the apical side of the cell monolayer. The Isc decreased with the addition of DIDS, but not with glybenclamide, 5-nitro-2-(3-phenylpropylamino) benzoic acid, or gadolinium chloride. No Isc 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 mutant form of TDH, reduced the effects of TDH on Isc and [Ca2+]in, as did protein kinase C (PKC) inhibitors. Thus, TDH increases Cl- secretion in human colonic epithelial cells, apparently through mechanisms involving cell binding and Ca2+ influx, followed by elevation of [Ca2+]in associated with PKC phosphorylation.
Takahashi
A
A
Sato
Y
Y
Shiomi
Y
Y
Cantarelli
V V
VV
Iida
T
T
Lee
M
M
Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamadaoka, Suita, Osaka 565-0871 and *Department of Urology, School of Medicine, University of Kobe, 7-5-1 Kusunoki, Tyuoku, Kobe, Hyogo 650-0017, Japan.
Honda
T
T
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D001427:Bacterial Toxins; D001692:Biological Transport; D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018118:Chloride Channels / Q000037:antagonists & inhibitors; D002712:Chlorides / Q000378:metabolism*; D003106:Colon / Q000166:cytology / Q000187:drug effects; D015194:Diffusion Chambers, Culture; D004553:Electric Conductivity; D004768:Enterotoxins / Q000235:genetics / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects; D007422:Intestines / Q000166:cytology / Q000187:drug effects*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000494:pharmacology; D014736:Vibrio parahaemolyticus / Q000472:pathogenicity*
D001427:Bacterial Toxins; D018118:Chloride Channels; D002712:Chlorides; D004768:Enterotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C068489:thermostable direct hemolysin; D011493:Protein Kinase C; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium