title: Haemolysin produced by Vibrio mimicus activates two Cl- secretory pathways in cultured intestinal-like Caco-2 cells.
abstract: Haemolysin (VMH) is a virulent factor produced by Vibrio mimicus, a human pathogen that causes diarrhoea. As intestinal epithelial cells are the primary targets of haemolysin, we investigated its effects on ion transport in human colonic epithelial Caco-2 cells. VMH increased the cellular short circuit current (Isc), used to estimated ion fluxes, and 125I efflux of the cells. The VMH-induced increases in Isc and 125I efflux were suppressed by depleting Ca2+ from the medium or by pretreating the cells with BAPTA-AM or by Rp-adenosin 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS). The Cl- channel inhibitors 4,4'-disothiocyanatostibene-2,2'-disulfonic acid (DIDS), glybenclamide, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) suppressed the VMH-induced increases in Isc and 125I efflux. Moreover, VMH increased the intracellular concentrations of Ca2+ and cAMP. Thus, VMH stimulates Caco-2 cells to secrete Cl- by activating both Ca2+ -dependent and cAMP-dependent Cl- secretion mechanisms. VMH forms ion-permeable pores in the lipid bilayer that are non-selectively permeable to small ions. However, the ion permeability of these pores was not inhibited by glybenclamide and DIDS, and VMH did not change the cell membrane potential. These observations indicate that the pores formed on the cell membrane by VMH are unlikely to be involved in VMH-induced Cl- secretion. Notably, VMH stimulated fluid accumulation in the iliac loop test that was fully suppressed by a combination of DIDS and glybenclamide. Thus, Ca2+-dependent and cAMP-dependent Cl- secretion may be important therapeutic targets with regard to the diarrhoea that is induced by Vibrio mimicus.
authors.affiliation: Department of Nutrition, School of Medicine, Tokushima University, 3-18-5 Kuramoto-cho, Tokushima City, Tokushima 770-8503, Japan. akiratak@nutr.med.tokushima-u.ac.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Takahashi
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Miyoshi
authors.forename: Noriko
authors.initials: N
authors.lastname: Takata
authors.forename: Masayuki
authors.initials: M
authors.lastname: Nakano
authors.forename: Akiko
authors.initials: A
authors.lastname: Hamamoto
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Mawatari
authors.forename: Nagakatsu
authors.initials: N
authors.lastname: Harada
authors.forename: Sumio
authors.initials: S
authors.lastname: Shinoda
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Nakaya
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005905:Glyburide / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000187:drug effects / Q000473:pathology; D007457:Iodine Radioisotopes / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D044164:Vibrio mimicus / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002712:Chlorides; D006460:Hemolysin Proteins; D007457:Iodine Radioisotopes; D004533:Egtazic Acid; D000242:Cyclic AMP; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D005905:Glyburide; D002118:Calcium