Bioenergetic regulation of heart mitochondrial nitric oxide synthase (mtNOS)

VALDEZ LB; ZAOBORNYJ T; BOMBICINO SS; IGLESIAS DE; BOVERIS A

Santiago, Chile

Congreso; Free Radicals and Antioxidants in Chile 2009: VI Meeting of the Society for Free Radical Biology and Medicine, South American Group.; 2009

VI Meeting of the Society for Free Radical Biology and Medicine, South American Group

The recognition of a mtNOS activity in a series of organs, the dependence of mtNOS activity on metabolic state and membrane potential, the physical interaction among mtNOS, cytochrome oxidase and complex I proteins, support the hypothesis that mtNOS is an oxygenase that uses the electron transport chain as an electron source instead of its own reductase domain. The aim of this work was to characterise mtNOS activity regulation by the redox state of the respiratory chain and membrane potential, using rat heart intact mitochondria and phosphorylating electron transfer particles (ETPH(Mg2+)). Not only heart mitochondrial membranes (2.2±0.1 nmol/min.mg protein), but also heart coupled mitochondria, exhibit an enzymatic production of NO. MtNOS activity is 40% lower in state 3 than in state 4, and shows an exponential dependence on membrane potential. In the presence of succinate and ATP, ETPH(Mg2+) particles showed a high NAD+ reductase activity, which was inhibited by the addition of rotenone (93%), oligomycin (96%) and m-CCCP (90%). The generation of NO by heart ETPH(Mg2+) particles resulted 0.53±0.03 nmol/min.mg protein, and this value was enhanced up to 0.73±0.03 nmol/min.mg protein when mtNOS activity was assessed in the presence of succinate and ATP. The addition of rotenone, oligomycin and m-CCCP inhibited by 30%, 29% and 49% this reversed electron transfer supported-mtNOS activity. These data suggest the key role of mitochondrial membrane potential on the mtNOS activity and that the respiratory flow modulates NO production, in agreement with the reported physical interaction of mtNOS and respiratory chain components.