Heart mitochondrial nitric oxide synthase. Effects of hypoxia and aging

The production of NO by heart mitochondria was 0.7-1.1 nmol NO/min.mg protein, an activity similar to the ones observed in mitochondrial membranes from other organs. Heart mtNOS seems to contribute with about 56% of the total cellular NO production. The immunological nature of the mtNOS isoform of cardiac tissue remains unclear; in our laboratory, heart mtNOS reacted with an anti-iNOS anti-body. Heart mtNOS expression and activity are regulated by physiological and pharmacological effectors. The state 4/state 3 transition regulates heart mtNOS activity and NO release in intact respiring mitochondria: NO production rates in state 3 were 40% lower than in state 4. Heart mtNOS expression was selectively regulated by O(2) availability in hypobaric conditions and the activity was 20-60% higher in hypoxic rats than in control animals, depending on age. In contrast, NADH-cytochrome c reductase and cytochrome oxidase activities were not affected by hypoxia. The activity of rat heart mtNOS decreased 20% on aging from 12 to 72 weeks of age. On the pharmacological side, mitochondrial NO production was increased after enalapril treatment (the inhibitor of the angiotensin converting enzyme) with modification of heart mtNOS functional activity in the regulation of mitochondrial O(2) uptake and H(2)O(2) production. Thus, heart mtNOS is a highly regulated mitochondrial enzyme, which in turn, plays a regulatory role through mitochondrial NO steady state levels that modulate O(2) uptake and O(2)(-) and H(2)O(2) production rates. Nitric oxide and H(2)O(2) constitute signals for metabolic control that are involved in the regulation of cellular processes, such as proliferation and apoptosis.     

Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria. Evidence for intramitochondrial peroxynitrite formation.

Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca(2+) dependence, and its relevance for mitochondrial bioenergetics was reported (Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291-296; Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038-11043). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca(2+) by mitochondria triggers mtNOS activity and causes the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner. mtNOS-induced cytochrome c release was paralleled by increased lipid peroxidation. The release of cytochrome c as well as increase in lipid peroxidation were prevented by NOS inhibitors, a superoxide dismutase mimic, and a peroxynitrite scavenger. We show that mtNOS-induced cytochrome c release is not mediated via the mitochondrial permeability transition pore because the release was aggravated by cyclosporin A and abolished by blockade of mitochondrial calcium uptake by ruthenium red. We conclude that, upon Ca(2+)-induced mtNOS activation, peroxynitrite is formed within mitochondria, which causes the release of cytochrome c from isolated mitochondria, and we propose a mechanism by which elevated Ca(2+) levels induce apoptosis.     

Mitochondrial metabolic states regulate nitric oxide and hydrogen peroxide diffusion to the cytosol

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.     

Mitochondrial metabolic states and membrane potential modulate mtNOS activity

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.     

Effect of sustained hypobaric hypoxia during maturation and aging on rat myocardium. II. mtNOS activity.

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.     

Brain mitochondrial nitric oxide synthase: in vitro and in vivo inhibition by chlorpromazine

Mouse brain mitochondria have a nitric oxide synthase (mtNOS) of 147 kDa that reacts with anti-nNOS antibodies and that shows an enzymatic activity of 0.31-0.48 nmol NO/min mg protein. Addition of chlorpromazine to brain submitochondrial membranes inhibited mtNOS activity (IC50 = 2.0 +/- 0.1 microM). Brain mitochondria isolated from chlorpromazine-treated mice (10 mg/kg, i.p.) show a marked (48%) inhibition of mtNOS activity and a markedly increased state 3 respiration (40 and 29% with malate-glutamate and succinate as substrates, respectively). Respiration of mitochondria isolated from control mice was 16% decreased by arginine and 56% increased by NNA (Nomega-nitro-L-arginine) indicating a regulatory activity of mtNOS and NO on mitochondrial respiration. Similarly, mitochondrial H2O2 production was 55% decreased by NNA. The effect of NNA on mitochondrial respiration and H2O2 production was significantly lower in chlorpromazine-added mitochondria and absent in mitochondria isolated from chlorpromazine-treated mice. Results indicate that chlorpromazine inhibits brain mtNOS activity in vitro and can exert the same action in vivo.     

Mitochondrial metabolic states and membrane potential modulate mtNOS activity

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 μM) and NADPH (1.04-1.78 mM) are 60-1000 times higher than their K_M 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.     

Differing roles of mitochondrial nitric oxide synthase in cardiomyocytes and urothelial cells

The existence of mitochondrial nitric oxide (NO) synthase (mtNOS) has been controversial since it was first reported in 1995. We have addressed this issue by making direct microsensor measurements of NO production in the mitochondria isolated from mouse hearts. Mitochondrial NO production was stimulated by Ca2+ and inhibited by blocking electrogenic Ca2+ uptake or by using NOS antagonists. Cardiac mtNOS was identified as the neuronal isoform by the absence of NO production in the mitochondria of mice lacking the neuronal but not the endothelial or inducible isoforms. In cardiomyocytes from dystrophin-deficient (mdx) mice, elevated intracellular Ca2+, increased mitochondrial NO production, slower oxidative phosphorylation, and decreased ATP production were detected. Inhibition of mtNOS increased contractility in mdx but not in wild-type cardiomyocytes, indicating that mtNOS may protect the cells from overcontracting. mtNOS was also implicated in radiation-induced cell damage. In irradiated rat/mouse urinary bladders, we have evidence that mitochondrially produced NO damages the urothelial "umbrella" cells that line the bladder lumen. This damage disrupts the permeability barrier thereby creating the potential to develop radiation cystitis. RT-PCR and Southern blot analyses indicate that mtNOS is restricted to the umbrella cells, which scanning electron micrographs show are selectively damaged by radiation. Simultaneous microsensor measurements demonstrate that radiation increases NO and peroxynitrite (ONOO-) production in these cells, which can be prevented by transfection with manganese superoxide dismutase (MnSOD) or instillation of NOS antagonists during irradiation or irradiation of bladders devoid of mtNOS. These studies demonstrate that mtNOS is in the cardiomyocytes and urothelial cells, that it is derived from the neuronal isoform, and that it can be either protective or detrimental.     

Mitochondrial nitric oxide synthase regulates mitochondrial matrix pH.

Nitric oxide (nitrogen monoxide, NO) exerts a wide profile of its biological activities via regulation of respiration and respiration-dependent functions. The presence of nitric oxide synthase (NOS) in mitochondria (mtNOS) was recently reported by us (Ghafourifar and Richter, FEBS Lett. 418, 291-296, 1997) and others (Giulivi et al., J. Biol. Chem. 273, 11038-11043, 1998). Here we report that NO, provided by an NO donor as well as by mtNOS stimulation, regulates mitochondrial matrix pH, transmembrane potential and Ca2+ buffering capacity. Exogenously-added NO causes a dose-dependent matrix acidification. Also mtNOS stimulation, induced by loading mitochondria with Ca2+, causes mitochondrial matrix acidification and a drop in mitochondrial transmembrane potential. Inhibition of mtNOS's basal activity causes mitochondrial matrix alkalinization and provides a resistance to the sudden drop of mitochondrial transmembrane potential induced by mitochondrial Ca2+ uptake. We conclude that mtNOS plays a critical role in regulating mitochondrial delta(pH).     

Free radical chemistry in biological systems

Mitochondria are an active source of the free radical superoxide (O2-) and nitric oxide (NO), whose production accounts for about 2% and 0.5% respectively, of mitochondrial O2 uptake under physiological conditions. Superoxide is produced by the auto-oxidation of the semiquinones of ubiquinol and the NADH dehydrogenase flavin and NO by the enzymatic action of the nitric oxide synthase of the inner mitochondrial membrane (mtNOS). Nitric oxide reversibly inhibits cytochrome oxidase activity in competition with O2. The balance between NO production and its utilization results in a NO intramitochondrial steady-state concentration of 20-50 nM, which regulates mitochondrial O2 uptake and energy supply. The regulation of cellular respiration and energy production by NO and its ability to switch the pathway of cell death from apoptosis to necrosis in physiological and pathological conditions could take place primarily through the inhibition of mitochondrial ATP production. Nitric oxide reacts with O2- in a termination reaction in the mitochondrial matrix, yielding peroxynitrite (ONOO-), which is a strong oxidizing and nitrating species. This reaction accounts for approximately 85% of the rate of mitochondrial NO utilization in aerobic conditions. Mitochondrial aging by oxyradical- and peroxynitrite-induced damage would occur through selective mtDNA damage and protein inactivation, leading to dysfunctional mitochondria unable to keep membrane potential and ATP synthesis.     

Mitochondrial nitric oxide metabolism in rat muscle during endotoxemia

In this study, heart and diaphragm mitochondria produced 0.69 and 0.77 nmol nitric oxide (NO)/min mg protein, rates that account for 67 and 24% of maximal cellular NO production, respectively. Endotoxemia and septic shock occur with an exacerbated inflammatory response that damages tissue mitochondria. Skeletal muscle seems to be one of the main target organs in septic shock, showing an increased NO production and early oxidative stress. The kinetic properties of mitochondrial nitric oxide synthase (mtNOS) of heart and diaphragm were determined. For diaphragm, the KM values for O2 and L-Arg were 4.6 and 37 microM and for heart were 3.3 and 36 microM. The optimal pH for mtNOS activity was 6.5 for diaphragm and 7.0 for heart. A marked increase in mtNOS activity was observed in endotoxemic rats, 90% in diaphragm and 30% in heart. Diaphragm and heart mitochondrial O2*- and H2O2 production were 2- to 3-fold increased during endotoxemia and Mn-SOD activity showed a 2-fold increase in treated animals, whereas catalase activity was unchanged. One of the current hypotheses for the molecular mechanisms underlying the complex condition of septic shock is that the enhanced NO production by mtNOS leads to excessive peroxynitrite production and protein nitration in the mitochondrial matrix, causing mitochondrial dysfunction and contractile failure.     

12(S)-hydroperoxyeicosatetraenoic acid (12-HETE) increases mitochondrial nitric oxide by increasing intramitochondrial calcium

12(S)-Hydroxyeicosatetraenoic acid (12-HETE) is one of the metabolites of arachidonic acid involved in pathological conditions associated with mitochondria and oxidative stress. The present study tested effects of 12-HETE on mitochondrial functions. In isolated rat heart mitochondria, 12-HETE increases intramitochondrial ionized calcium concentration that stimulates mitochondrial nitric oxide (NO) synthase (mtNOS) activity. mtNOS-derived NO causes mitochondrial dysfunctions by decreasing mitochondrial respiration and transmembrane potential. mtNOS-derived NO also produces peroxynitrite that induces release of cytochrome c and stimulates aggregation of mitochondria. Similarly, in HL-1 cardiac myocytes, 12-HETE increases intramitochondrial calcium and mitochondrial NO, and induces apoptosis. The present study suggests a novel mechanism for 12-HETE toxicity.     

Mitochondrial calcium uptake stimulates nitric oxide production in mitochondria of bovine vascular endothelial cells

Although nitric oxide (NO) is a known modulator of cell respiration in vascular endothelium, the presence of a mitochondria-specific nitric oxide synthase (mtNOS) in these cells is still a controversial issue. We have used laser scanning confocal microscopy in combination with the NO-sensitive fluorescent dye DAF-2 to monitor changes in NO production by mitochondria of calf vascular endothelial (CPAE) cells. Cells were loaded with the membrane-permeant NO-sensitive dye 4,5-diaminofluorescein (DAF-2) diacetate and subsequently permeabilized with digitonin to remove cytosolic DAF-2 to allow measurements of NO production in mitochondria ([NO]_mt). Stimulation of mitochondrial Ca²⁺ uptake by exposure to different cytoplasmic Ca²⁺ concentrations (1, 2, and 5 µM) resulted in a dose-dependent increase of NO production by mitochondria. This increase of [NO]_mt was sensitive to the NOS antagonist L-N⁵-(1-iminoethyl)ornithine and the calmodulin antagonist calmidazolium (R-24571), demonstrating the endogenous origin of NO synthesis and its calmodulin dependence. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca²⁺ uniporter with ruthenium red, as well as blocking the respiratory chain with antimycin A in combination with oligomycin, inhibited mitochondrial NO production. Addition of the NO donor spermine NONOate caused a profound increase in DAF-2 fluorescence that was not affected by either of these treatments. The mitochondrial origin of the DAF-2 signals was confirmed by colocalization with the mitochondrial marker MitoTracker Red and by the observation that disruption of caveolae (where cytoplasmic NOS is localized) formation with methyl--cyclodextrin did not prevent the increase of DAF-2 fluorescence. The activation of mitochondrial calcium uptake stimulates mtNOS phosphorylation (at Ser-1177) which was prevented by FCCP. The data demonstrate that stimulation of mitochondrial Ca²⁺ uptake activates NO production in mitochondria of CPAE cells. This indicates the presence of a mitochondria-specific NOS that can provide a fast local modulatory effect of NO on cell respiration, membrane potential, and apoptosis. nitric oxide; nitric oxide synthase; calcium; endothelium; mitochondria     

Functional argument for the existence of an avian nitric oxide synthase in muscle mitochondria: effect of cold acclimation

We report the first evidence of a mitochondrial NO synthase (mtNOS) in bird skeletal muscle. In vitro, mtNOS activity stimulated by l-arginine reduced intermyofibrillar mitochondrial oxygen uptake and ATP synthesis rates, stimulated endogenous H₂O₂ generation, but had no effect on oxidative phosphorylation efficiency. Arginine-induced effects were fully reversed by l-NAME, a known NOS inhibitor. When ducklings were cold exposed for 4 weeks, muscle mitochondria displayed an increased state 3 respiration, a reduced H₂O₂ generation but no significant alteration in mtNOS activity. We conclude that mtNOS is expressed in avian skeletal muscle.     

High doses of vitamin E improve mitochondrial dysfunction in rat hippocampus and frontal cortex upon aging

Rat aging from 4 to 12 mo was accompanied by hippocampus and frontal cortex mitochondrial dysfunction, with decreases of 23 to 53% in tissue and mitochondrial respiration and in the activities of complexes I and IV and of mitochondrial nitric oxide synthase (mtNOS) (P < 0.02). In aged rats, the two brain areas showed mitochondria with higher content (35-78%) of oxidation products of phospholipids and proteins and with higher (59-95%) rates of O(2)(-) and H(2)O(2) production (P < 0.02). Dietary supplementation with vitamin E (2.0 or 5.0 g/kg of food) from 9 to 12 mo of rat age, restored in a dose-dependent manner, the decreases in tissue and mitochondrial respiration (to 90-96%) and complexes I and IV and mtNOS activities (to 86-88%) of the values of 4-mo-old rats (P < 0.02). Vitamin E prevented, by 73-80%, the increases in oxidation products, and by 62-68%, the increases in O(2)(-) and H(2)O(2) production (P < 0.05). High resolution histochemistry of cytochrome oxidase in the hippocampal CA1 region showed higher staining in vitamin E-treated rats than in control animals. Aging decreased (19%) hippocampus mitochondrial mass, an effect that was restored by vitamin E. High doses of vitamin E seem to sustain mitochondrial biogenesis in synaptic areas.     

Oxygen dependence of mitochondrial nitric oxide synthase activity

The effect of O(2) concentration on mitochondrial nitric oxide synthase (mtNOS) activity and on O(2)(-) production was determined in rat liver, brain, and kidney submitochondrial membranes. The K(mO(2)) for mtNOS were 40, 73, and 37 microM O(2) and the V(max) were 0.51, 0.49, and 0.42 nmol NO/minmg protein for liver, brain, and kidney mitochondria, respectively. The rates of O(2)(-) production, 0.5-12.8 nmol O(2)(-)/minmg protein, depended on O(2) concentration up to 1.1mM O(2). Intramitochondrial NO, O(2)(-), and ONOO(-) steady-state concentrations were calculated for the physiological level of 20 microM O(2); they were 20-39 nM NO, 0.17-0.33 pM O(2)(-), and 0.6-2.2 nM ONOO(-) for the three organs. These levels establish O(2)/NO ratios of 513-1000 that correspond to physiological inhibitions of cytochrome oxidase by intramitochondrial NO of 16-25%. The production of NO by mtNOS appears as a regulatory process that modulates mitochondrial oxygen uptake and cellular energy production.     

Modulation of mitochondrial nitric oxide synthase and energy expenditure in rats during cold acclimation

To preserve thermoneutrality, cold exposure is followed by changes in energy expenditure and basal metabolic rate (BMR). Because nitric oxide (NO) modulates mitochondrial O(2) uptake and energy levels, we analyzed cold effects (30 days at 4 degrees C) on rat liver and skeletal muscle mitochondrial NO synthases (mtNOS) and their putative impact on BMR. Cold exposure delimited two periods: A (days 1-10), with high systemic O(2) uptake and weight loss, and B (days 10-30), with lower O(2) uptake and fat deposition. mtNOS activity and expression decreased in period A and then increased in period B by 60-100% in liver and skeletal muscle (P < 0.05). Conversely, mitochondrial O(2) uptake remained initially high in the presence of l-arginine and later fell by 30-50% (P < 0.05). On this basis, the estimated fractional contribution of liver plus muscle to total BMR varied from 40% in period A to 25% in period B. The transitional modulation of mtNOS in rat cold acclimation could participate in adaptive responses that favor calorigenesis or conservative energy-saving mechanisms.     

Hyperglycemic switch from mitochondrial nitric oxide to superoxide production in endothelial cells

The accumulated ultrastructural and biochemical evidence is highly suggestive of the existence of mitochondrial nitric oxide (NO) synthase (mtNOS), where local production of NO regulates the electron transport along the respiratory chain. Here, the functional competence of mtNOS in situ in a living cell was examined using an intravital fluorescent NO indicator, 4,5-diaminofluorescein, employing a new procedure for loading it into the mitochondria to demonstrate local NO generation in undisrupted endothelial cells and in isolated mitochondria as well as in human embryonic kidney cells stably expressing endothelial NOS. With the use of this approach, we showed that endothelial cells incubated in the presence of high concentration of D-glucose (but not L-glucose) are characterized by the reduced NO synthetic function of mitochondria despite the unaltered abundance of the enzyme. In parallel, mitochondrial generation of superoxide was augmented in endothelial cells incubated in the presence of a high concentration of D-glucose. Both the NO generation and superoxide production in hyperglycemic environment could be restored to control levels by treating cells with a cell-permeable superoxide dismutase mimetic. In addition, enhanced mitochondrial superoxide production could be suppressed with an inhibitor of NOS in stimulated endothelial cells. In conclusion, the data 1) provide direct evidence of mitochondrial NO production in endothelial cells, 2) demonstrate its suppression and enhanced superoxide generation in hyperglycemic environment, and 3) provide evidence that "uncoupled" mtNOS represents an important source of superoxide anions in endothelial cells incubated in high glucose-containing medium.     

Heart mitochondrial nitric oxide synthase is upregulated in male rats exposed to high altitude (4,340 m)

Male rats exposed for 21 days to high altitude (4,340 m) responded with arrest of weight gain and increased hematocrit and testosterone levels. High altitude significantly (58%) increased heart mitochondrial nitric oxide (NO) synthase (mtNOS) activity, whereas heart cytosolic endothelial NOS (eNOS) and liver mtNOS were not affected. Western blot analysis found heart mitochondria reacting only with anti-inducible NOS (iNOS) antibodies, whereas the postmitochondrial fraction reacted with anti-iNOS and anti-eNOS antibodies. In vitro-measured NOS activities allowed the estimation of cardiomyocyte capacity for NO production, a value that increased from 57% (sea level) to 79 nmol NO.min(-1).g heart(-1) (4,340 m). The contribution of mtNOS to total cell NO production increased from 62% (sea level) to 71% (4340 m). Heart mtNOS activity showed a linear relationship with hematocrit and a biphasic quadratic association with estradiol and testosterone. Multivariate analysis showed that exposure to high altitude linearly associates with hematocrit and heart mtNOS activity, and that testosterone-to-estradiol ratio and heart weight were not linearly associated with mtNOS activity. We conclude that high altitude triggers a physiological adaptive response that upregulates heart mtNOS activity and is associated in an opposed manner with the serum levels of testosterone and estradiol.