Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia

It has been shown that nitric oxide (NO), synthesized by the inducible NO s ynthase (iNOS) expressed in the diaphragm during endotoxemia, participates in the development of muscular contractile failure. The aim of the present study was to investigate whether this deleterious action of NO was related to its effects on cellular oxidative pathways. Rats were inoculated with E. coli lipopolysaccharide (LPS) or sterile saline solution (controls) and st udied at 3 and 6 h after inoculation. iNOS protein and activity could be de tected in the rat diaphragm as early as 3 h after LPS, with a sustained ste ady-state concentration of 0.5 mu M NO in the muscle associated with increa sed detection of hydrogen peroxide (H2O2). In vitro, the same NO concentrat ion produced a marked increase in H2O2 production by isolated control diaph ragm mitochondria, thus reflecting a higher intramitochondrial concentratio n of nondiffusible superoxide anion (O-2(-.)). In a similar way, whole diap hragmatic muscle and diaphragm mitochondria from endotoxemic rats showed a progressive increase in H2O2 production associated with uncoupling and decr eased phosphorylating capacity. Simultaneous with the maximal impairment in respiration (6 h after LPS), nitration of mitochondrial proteins (a peroxy nitrite footprint) was detected and diaphragmatic force was reduced. Functi onal mitochondrial abnormalities, nitration of mitochondrial proteins, and the decrease in force were significantly attenuated by administration of th e NOS inhibitor L-NMMA. These results show that increased and sustained NO levels lead to a consecutive formation of O-2(-.) that reacts with NO to fo rm peroxynitrite, which in turn impairs mitochondrial function, which proba bly contributes to the impairment of muscle contractility.