Endogenous nitric oxide in MDCK-I cells modulates the Vibrio cholerae haemagglutinin/protease (HA/P)-mediated cytotoxicity.

Previously, we have shown that the Vibrio cholerae haemagglutinin/protease (HA/P) accounts for significant remaining toxicity of CVD110, an attenuated V. cholerae 01 El Tor live oral vaccine-strain. The present report demonstrates that endogenous nitric oxide (NO) production modulates HA/P-mediated cytotoxicity in Madin-Darby canine kidney cell strain I (MDCK-I) epithelial cells. The basal levels of endogenous NO suppressed the cytotoxicity of HA/P, whereas inhibition of NO production with nitro-L-arginine methyl-ester (L-NAME) made the MDCK-I cells susceptible even to low concentrations of the cytotoxin. The inhibition of NO production caused a reinforcement of the HA/P- mediated distortion of a tight junction-associated protein ZO-1 and increment of filamentous actin at the apical and the lateral membrane domains. The mechanism by which NO exerts its modulatory action is not likely to be from its direct interaction with the zinc-containing catalytic domain of HA/P, since two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetyl-D, L-penicillamine (SNAP), did not affect the proteolytic activity of HA/P. In conclusion, the endogenous NO in the MDCK-I cells has a modulating effect on the cytotoxicity of HA/P and thus protects the cells against the cytotoxin.