Background & aims: Vibrio parahaemolyticus is a major agent of seafood gastroenteritis that induces intestinal secretion in the rabbit through its thermostable direct hemolysin. The aim of this study was to characterize the enterotoxicity of purified hemolysin in vitro.
Methods: Rabbit ileum was mounted in Ussing chambers, and changes in potential difference and short-circuit current were monitored after addition of hemolysin. Intracellular calcium concentrations in the nontumoral rat crypt-derived cell line IEC-6 were measured using microspectrofluorometry.
Results: In Ussing chamber experiments, mucosal toxin addition up to 50 hemolytic units per milliliter induced a proportional increase of the electrical parameters in normal but not Cl(-)-free Ringer's solution. The response to the toxin was not additive to that of calcium ionophore A23187 and was eliminated by preloading the tissue with 1-2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA), a calcium buffer. In IEC-6 cells, a 10-fold increase in intracellular calcium level was found after addition of hemolysin. Such an increase was totally quenched by BAPTA. Finally, preincubation with trisialoganglioside GT1b, but not monosialoganglioside GM1, eliminated toxin-induced increases in potential difference and short-circuit current.
Conclusions: These data support the hypothesis that the thermostable direct hemolysin induces intestinal chloride secretion using GT1b as a putative receptor and Ca2+ as a second messenger.