The mitochondrial metabolic state regulates the rate of NO release from coupled mitochondria: NO release by heart, liver and kidney mitochondria was about 40-45% lower in state 3 (1.2, 0.7 and 0.4 nmol/min mg protein) than in state 4 (2.2, 1.3 and 0.7 nmol/min mg protein). The activity of mtNOS, responsible for NO release, appears driven by the membrane potential component and not by intramitochondrial pH of the proton motive force. The intramitochondrial concentrations of the NOS substrates, L-arginine (about 310 microM) and NADPH (1.04-1.78 mM) are 60-1000 times higher than their KM values. Moreover, the changes in their concentrations in the state 4-state 3 transition are not enough to explain the changes in NO release. Nitric oxide release was exponentially dependent on membrane potential as reported for mitochondrial H2O2 production [S.S. Korshunov, V.P. Skulachev, A.A. Satarkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15-18.]. Agents that decrease or abolish membrane potential minimize NO release while the addition of oligomycin that produces mitochondrial hyperpolarization generates the maximal NO release. The regulation of mtNOS activity, an apparently voltage-dependent enzyme, by membrane potential is marked at the physiological range of membrane potentials.

Mitochondria isolated from rat heart, liver, kidney and brain (respiratory control 4.0-6.5) release NO and H2O2 at rates that depend on the mitochondrial metabolic state: releases are higher in state 4, about 1.7-2.0 times for NO and 4-16 times for H2O2, than in state 3. NO release in rat liver mitochondria showed an exponential dependence on membrane potential in the range 55 to 180 mV, as determined by Rh-123 fluorescence. A similar behavior was reported for mitochondrial H2O2 production by [S.S. Korshunov, V.P. Skulachev, A.A. Starkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15_18.]. Transition from state 4 to state 3 of brain cortex mitochondria was associated to a decrease in NO release (50%) and in membrane potential (24-53%), this latter determined by flow cytometry and DiOC6 and JC-1 fluorescence. The fraction of cytosolic NO provided by diffusion from mitochondria was 61% in heart, 47% in liver, 30% in kidney, and 18% in brain. The data supports the speculation that NO and H2O2 report a high mitochondrial energy charge to the cytosol. Regulation of mtNOS activity by membrane potential makes mtNOS a regulable enzyme that in turn regulates mitochondrial O2 uptake and H2O2 production.

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Mitochondrial nitric oxide (NO) production was assayed in rats submitted to hypobaric hypoxia and in normoxic controls (53.8 and 101.3 kPa air pressure, respectively). Heart mitochondria from young normoxic animals produced 0.62 and 0.37 nmol NO.min(-1).mg protein(-1) in metabolic states 4 and 3, respectively. This production accounts for a release to the cytosol of 29 nmol NO.min(-1).g heart(-1) and for 55% of the NO generation. The mitochondrial NO synthase (mtNOS) activity measured in submitochondrial membranes at pH 7.4 was 0.69 nmol NO.min(-1).mg protein(-1). Rats exposed to hypobaric hypoxia for 2-18 mo showed 20-60% increased left ventricle mtNOS activity compared with their normoxic siblings. Left ventricle NADH-cytochrome-c reductase and cytochrome oxidase activities decreased by 36 and 12%, respectively, from 2 to 18 mo of age, but they were not affected by hypoxia. mtNOS upregulation in hypoxia was associated with a retardation of the decline in the mechanical activity of papillary muscle upon aging and an improved recovery after anoxia-reoxygenation. The correlation of left ventricle mtNOS activity with papillary muscle contractility (determined as developed tension, maximal rates of contraction and relaxation) showed an optimal mtNOS activity (0.69 nmol.min(-1).mg protein(-1)). Heart mtNOS activity is regulated by O(2) in the inspired air and seems to play a role in NO-mediated signaling and myocardial contractility.