Induction of mitochondrial nitric oxide synthase during endotoxemia

LISDERO C; BOCZKOWSKI J; BOVERIS A; PODEROSO JJ

Florianópolis, Brasil

Congreso; First Meeting of the South American Group for Free Radical Research; 1999

South American Group for Free Radical Research

Recently, we and others have reported that liver mitochondria express a NOS isoform in the inner membrane (mtNOS). This mtNOS has epitopes which react with iNOS antibodies but its complete structure remains to be elucidated. In addition, it was recently demonstrated by our group that mitochondrial proteins are spontaneously nitrated during endotoxemia in connection with LPS induced iNOS expression and the formation of superoxide anion by the respiratory chain in the rat diaphragm. The aim of this study was to determine whether iNOS targeted to the mitochondrial membrane promoting in situ formation of ONOO- and, whether the recently described mitochondrial NOS can be induced during endotoxemia. The study was done with Sprague-Dawley rats inoculated i.p. with E. Coli LPS (2mg/kg). Expression of iNOS protein, detected by Western Blot, enzymatic activity and cofactor requirements, measured by the conversion of [3H] L-arginine to [3H] L-citrulline were studied in diaphragm, heart and liver mitochondria 24 h after inoculation. The results showed that LPS induced the expression of liver and diaphragm mitochondrial NOS protein and activity but not in heart mitochondria; increase in NOS activity was essentially Ca2+ independent (diaphragm mitochondria: 13±3 in septic animals vs 3.4±1.5 pmoles/min/mg prot in control animals, p<0.05; liver mitochondria: 12±2 in septic animals vs 4.4±0.9 pmoles/min/mg prot in control animals, p<0.05) and was independent of the presence of cofactors in the reaction media. It is concluded that iNOS is targeted to mitochondrial membranes in diaphragm and liver, and that, the expression of liver mitochondrial NOS is subjected to regulation by LPS and cytokines. The independence of cofactors, is probably dew to the localization of the enzyme to the inner mitochondrial membrane. These results determine that mtNOS can, in pathological situations, contribute to protein nitration and consequently, to the impairment of mitochondrial function.