Reprint of: Production of Superoxide Radicals and Hydrogen Peroxide by NADH- Ubiquinone Reductase and Ubiquinol-Cytochrome c Reductase from Beef-Heart Mitochondria.

Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O2-at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H2O2 at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H2O2 was essentially produced by disproportionation of O2-, which constitutes the precursor of H2O2. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O2- and 0.63 mol of H2O2/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O2 to O2-, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H202 generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H2O2 generation. The efficiency of added quinones as peroxide generators decreased in the order Q1 > Q0 > Q2 > Q6 = Q10, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymati- cally by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O2- and H2O2 during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q0H2), benzoquinol, duroquinol and menadiol generated O2-with k3 values of 0.1 to 1.4 M-1 s-1 and H2O2 with k4 values of 0.009 to 4.3 m-1·s-1.

Loss of dystrophin is associated with increased myocardial stiffness in a model of left ventricular hypertrophy.

Transition from compensated to decompensated left ventricular hypertrophy (LVH) is accompanied by functional and structural changes. Here, the aim was to evaluate dystrophin expression in murine models and human subjects with LVH by transverse aortic constriction (TAC) and aortic stenosis (AS), respectively. We determined whether doxycycline (Doxy) prevented dystrophin expression and myocardial stiffness in mice. Additionally, ventricular function recovery was evaluated in patients 1 year after surgery. Mice were subjected to TAC and monitored for 3 weeks. A second group received Doxy treatment after TAC. Patients with AS were stratified by normal left ventricular end-diastolic wall stress (LVEDWS) and high LVEDWS, and groups were compared. In mice, LVH decreased inotropism and increased myocardial stiffness associated with a dystrophin breakdown and a decreased mitochondrial O2 uptake (MitoMVO2). These alterations were attenuated by Doxy. Patients with high LVEDWS showed similar results to those observed in mice. A correlation between dystrophin and myocardial stiffness was observed in both mice and humans. Systolic function at 1 year post-surgery was only recovered in the normal-LVEDWS group. In summary, mice and humans present diastolic dysfunction associated with dystrophin degradation. The recovery of ventricular function was observed only in patients with normal LVEDWS and without dystrophin degradation. In mice, Doxy improved MitoMVO2. Based on our results it is concluded that the LVH with high LVEDWS is associated to a degradation of dystrophin and increase of myocardial stiffness. At least in a murine model these alterations were attenuated after the administration of a matrix metalloprotease inhibitor.

Mitochondrial nitric oxide production supported by reverse electron transfer.

Heart phosphorylating electron transfer particles (ETPH) produced NO at 1.2 ± 0.1 nmol NO. min(-1) mg protein(-1) by the mtNOS catalyzed reaction. These particles showed a NAD(+) reductase activity of 64 ± 3 nmol min(-1) mg protein(-1) sustained by reverse electron transfer (RET) at expenses of ATP and succinate. The same particles, without NADPH and in conditions of RET produced 0.97 ± 0.07 nmol NO. min(-1) mg protein(-1). Rotenone inhibited NO production supported by RET measured in ETPH and in coupled mitochondria, but did not reduce the activity of recombinant nNOS, indicating that the inhibitory effect of rotenone on NO production is due to an electron flow inhibition and not to a direct action on mtNOS structure. NO production sustained by RET corresponds to 20% of the total amount of NO released from heart coupled mitochondria. A mitochondrial fraction enriched in complex I produced 1.7 ± 0.2 nmol NO. min(-1) mg protein(-1) and reacted with anti-75 kDa complex I subunit and anti-nNOS antibodies, suggesting that complex I and mtNOS are located contiguously. These data show that mitochondrial NO production can be supported by RET, and suggest that mtNOS is next to complex I, reaffirming the idea of a functional association between these proteins.

Role of thioredoxin-1 in ischemic preconditioning, postconditioning and aged ischemic hearts.

Thioredoxin is one of the most important cellular antioxidant systems known to date, and is responsible of maintaining the reduced state of the intracellular space. Trx-1 is a small cytosolic protein whose transcription is induced by stress. Therefore it is possible that this antioxidant plays a protective role against the oxidative stress caused by an increase of reactive oxygen species concentration, as occurs during the reperfusion after an ischemic episode. However, in addition to its antioxidant properties, it is able to activate other cytoplasmic and nuclear mediators that confer cardioprotection. It is remarkable that Trx-1 also participates in myocardial protection mechanisms such as ischemic preconditioning and postconditioning, activating proteins related to cellular survival. In this sense, it has been shown that Trx-1 inhibition abolished the preconditioning cardioprotective effect, evidenced through apoptosis and infarct size. Furthermore, ischemic postconditioning preserves Trx-1 content at reperfusion, after ischemia. However, comorbidities such as aging can modify this powerful cellular defense leading to decrease cardioprotection. Even ischemic preconditioning and postconditioning protocols performed in aged animal models failed to decrease infarct size. Therefore, the lack of success of antioxidants therapies to treat ischemic heart disease could be solved, at least in part, avoiding the damage of Trx system.

Endotoxemia impairs heart mitochondrial function by decreasing electron transfer, ATP synthesis and ATP content without affecting membrane potential.

Acute endotoxemia (LPS, 10 mg/kg ip, Sprague Dawley rats, 45 days old, 180 g) decreased the O2 consumption of rat heart (1 mm³ tissue cubes) by 33% (from 4.69 to 3.11 µmol O2/min. g tissue). Mitochondrial O2 consumption and complex I activity were also decreased by 27% and 29%, respectively. Impaired respiration was associated to decreased ATP synthesis (from 417 to 168 nmol/min. mg protein) and ATP content (from 5.40 to 4.18 nmol ATP/mg protein), without affecting mitochondrial membrane potential. This scenario is accompanied by an increased production of O2·â» and H2O2 due to complex I inhibition. The increased NO production, as shown by 38% increased mtNOS biochemical activity and 31% increased mtNOS functional activity, is expected to fuel an increased ONOO⁻ generation that is considered relevant in terms of the biochemical mechanism. Heart mitochondrial bioenergetic dysfunction with decreased O2 uptake, ATP production and contents may indicate that preservation of mitochondrial function will prevent heart failure in endotoxemia.

Diastolic function during hemorrhagic shock in rabbits.

Hemorrhage (H) is associated with a left ventricular (LV) dysfunction. However, the diastolic function has not been studied in detail. The main goal was to assess the diastolic function both during and 120 min after bleeding, in the absence and in the presence of L-NAME. Also, the changes in mRNA and protein expression of nitric oxide synthase (NOS) isoforms were determined. New Zealand rabbits were divided into three groups: Sham group, H group (hemorrhage 20% blood volume), and H L-NAME group (hemorrhage treated with L-NAME). We evaluated systolic and diastolic ventricular functions in vivo and in vitro (Langendorff technique). Hemodynamic parameters and LV function were measured before, during, and at 120 min after bleeding. We analyzed the isovolumic relaxation using t ½ in vivo (closed chest). After that, hearts were excised and perfused in vitro to measure myocardial stiffness. Samples were frozen to measure NOS mRNA and protein expression. The t½ increased during bleeding and returned to basal values 120 min after bleeding. L-NAME blunted this effect. Data from the H group revealed a shift to the left in the LV end diastolic pressure-volume curve at 120 min after bleeding, which was blocked by L-NAME. iNOS and nNOS protein expression and mRNA levels increased at 120 min after the hemorrhage. Acute hemorrhage induces early and transient isovolumic relaxation impairment and an increase in myocardial stiffness 120 min after bleeding. L-NAME blunted the LV dysfunction, suggesting that NO modulates ventricular function through iNOS and nNOS isoforms.

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.

Effects of rotenone and pyridaben on complex I electron transfer and on mitochondrial nitric oxide synthase functional activity.

Rotenone and pyridaben were tested on activities and properties of rat brain mitochondria determining Ki (inhibitor concentration at half maximal inhibition) and Imax (% of inhibition at maximal inhibitor concentration). The assayed activities were complexes I, II and IV, respiration in states 3, 3u (uncoupled) and 4, biochemical and functional activities of mitochondrial nitric oxide synthase (mtNOS), and inner membrane potential. Selective inhibitions of complex I activity, mitochondrial respiration and membrane potential with malate-glutamate as substrate were observed, with a Ki of 0.28-0.36 nmol inhibitor/mg of mitochondrial protein. Functional mtNOS activity was half-inhibited at 0.70-0.74 nmol inhibitor/mg protein in state 3 mitochondria and at 2.52-2.98 nmol inhibitor/mg protein in state 3u mitochondria. This fact is interpreted as an indication of mtNOS being structurally adjacent to complex I with an intermolecular mtNOS-complex I hydrophobic bonding that is stronger at high Δψ and weaker at low Δψ.

Role of matrix metalloproteinase-2 in the cardioprotective effect of ischaemic postconditioning.

The activation of matrix metalloproteinases (MMPs) contributes to myocardial injury at the onset of reperfusion; however, their role in ischaemic postconditioning is unknown. The aim of the present study was to examine the effects of ischaemic postconditioning on MMP activity in isolated rabbit hearts. The isolated rabbit hearts were subjected to 30 min of global ischaemia followed by 180 min of reperfusion (I/R group; n = 8). In the ischaemic postconditioning group (n = 8), a postconditioning protocol was performed (2 cycles of 30 s reperfusion-ischaemia). In other experiments, we added doxycycline, an MMP inhibitor, at 25 (n = 7) or 50 micromol l(1) (n = 8) during the first 2 min of reperfusion. Coronary effluent and left ventricular tissue were collected during pre-ischaemic conditions and at different times during the reperfusion period to measure MMP-2 activity and cardiac protein nitration. We evaluated ventricular function and infarct size. In the I/R group, infarct size was 32.1 +/- 5.2%; Postcon reduced infarct size to 9.5 +/- 3.8% (P < 0.05) and inhibited MMP-2 activity during reperfusion. The administration of doxycycline at 50 micromol l(1) inhibited MMP-2 activity and cardiac protein nitration and reduced the infarct size to 9.7 +/- 2.8% (P < 0.05). A lower dose of doxycycline (25 micromol l(1)) failed to inhibit MMP-2 activity and did not modify the infarct size. Our results strongly suggest that ischaemic postconditioning may exert part of its cardioprotective effects through the inhibition of MMP-2 activity.

Oxidative damage: the biochemical mechanism of cellular injury and necrosis in choline deficiency.

Oxidative stress and damage are characterized by decreased tissue antioxidant levels, consumption of tissue alpha-tocopherol, and increased lipid peroxidation. These processes occur earlier than necrosis in the liver, heart, kidney, and brain of weanling rats fed a choline deficient (CD) diet. In tissues, water-soluble antioxidants were analyzed as total reactive antioxidant potential (TRAP), alpha-tocopherol content was estimated from homogenate chemiluminescence (homogenate-CL), and lipid peroxidation was evaluated by thiobarbituric acid reactive substances (TBARS). Histopathology showed hepatic steatosis at days 1-7, tubular and glomerular necrosis in kidney at days 6 and 7, and inflammation and necrosis in heart at days 6 and 7. TRAP levels decreased by 18%, 48%, 56%, and 66% at day 7, with t(1/2) (times for half maximal change) of 2.0, 1.8, 2.5, and 3.0 days in liver, kidney, heart, and brain, respectively. Homogenate-CL increased by 97%, 113%, 18%, and 297% at day 7, with t(1/2) of 2.5, 2.6, 2.8, and 3.2 days in the four organs, respectively. TBARS contents increased by 98%, 157%, 104%, and 347% at day 7, with t(1/2) of 2.6, 2.8, 3.0, and 5.0 days in the four organs, respectively. Plasma showed a 33% decrease in TRAP and a 5-fold increase in TBARS at day 5. Oxidative stress and damage are processes occurring earlier than necrosis in the kidney and heart. In case of steatosis prior to antioxidant consumption and increased lipid peroxidation, no necrosis is observed in the liver.

The involvement of transition metal ions on iron-dependent lipid peroxidation.

The metals iron (Fe) and copper (Cu) are considered trace elements, and the metals cobalt (Co) and nickel (Ni) are known as ultra-trace elements, considering their presence in low to very low quantity in humans. The biologic activity of these transition metals is associated with the presence of unpaired electrons that favor their participation in redox reactions. They are part of important enzymes involved in vital biologic processes. However, these transition metals become toxic to cells when they reach elevated tissue concentrations and produce cellular oxidative damage. Phospholipid liposomes (0.5 mg/ml, phosphatidylcholine (PC)/phosphatidylserine (PS), 60/40) were incubated for 60 min at 37 degrees C with 25 microM of Fe2+ in the absence and in the presence of Cu2+, Co2+, and Ni2+ (0-100 microM) with and without the addition of hydrogen peroxide (H2O2, 5-50 microM). Iron-dependent lipid peroxidation in PC/PS liposomes was assessed by thiobarbituric acid-reactive substances (TBARS) production. Metal transition ions promoted lipid peroxidation by H2O2 decomposition and direct homolysis of endogenous hydroperoxides. The Fe2+-H2O2-mediated lipid peroxidation takes place by a pseudo-second order process, and the Cu2+-mediated process by a pseudo-first order reaction. Co2+ and Ni2+ alone do not induce lipid peroxidation. Nevertheless, when they are combined with Fe2+, Fe2+-H2O2-mediated lipid peroxidation was stimulated in the presence of Ni2+ and was inhibited in the presence of Co2+. The understanding of the effects of transition metal ions on phospholipids is relevant to the prevention of oxidative damage in biologic systems.

Brain mitochondrial dysfunction and oxidative damage in Parkinson's disease.

Complex factors contribute to the appearance of Parkinson's disease (PD), but with a constant mitochondrial involvement. There are two interdependent conditions in PD: brain mitochondrial dysfunction and brain mitochondrial oxidative damage. Mitochondrial dysfunction and reduced complex I activity are recognized in substantia nigra and in frontal cortex in PD patients. The molecular mechanism involved in the inactivation of complex I is likely accounted by the sum of ONOO(-) mediated reactions, reactions with free radical intermediates of the lipid peroxidation process and amine-aldehyde adduction reactions. The inhibitory effects on complex I lead synergistically to denaturation of the protein structure and to further increases of O(2)(-) and ONOO(-) production at the vicinity of complex I. An adaptive response in PD patients has been described with increases in mtNOS activity, mitochondrial mass and mitochondrial biogenesis. Mitochondrial dysfunction in the human frontal cortex is to be considered a factor contributing to impaired cognition in PD.

Systemic oxidative stress associated with the neurological diseases of aging.

Markers of oxidative stress were measured in blood samples of 338 subjects (965 observations): Alzheimer's, vascular dementia, diabetes (type II) superimposed to dementias, Parkinson's disease and controls. Patients showed increased thiobarbituric acid reactive substances (+21%; P < 0.05), copper-zinc superoxide dismutase (+64%; P < 0.001) and decreased antioxidant capacity (-28%; P < 0.001); pairs of variables resulted linearly related across groups (P < 0.001). Catalase and glutathione peroxidase, involved in discrimination between diseases, resulted non-significant. When diabetes is superimposed with dementias, changes resulted less marked but significant. Also, superoxide dismutase resulted not linearly correlated with any other variable or age-related (pure Alzheimer's peaks at 70 years, P < 0.001). Systemic oxidative stress was significantly associated (P << 0.001) with all diseases indicating a disbalance in peripheral/adaptive responses to oxidative disorders through different free radical metabolic pathways. While other changes - methionine cycle, insulin correlation - are also associated with dementias, the responses presented here show a simple linear relation between prooxidants and antioxidant defenses.

(+)-Catechin inhibits heart mitochondrial complex I and nitric oxide synthase: functional consequences on membrane potential and hydrogen peroxide production.

In order to study the in vitro effect of flavan-3-ol (+)-catechin on the enzymatic activities of mitochondrial complex I and nitric oxide synthase (mtNOS), as well as the consequences on the membrane potential and H2O2 production rate, isolated mitochondria from rat heart were exposed to 3 nM to 100 µM (+)-catechin. NADH-Q1 reductase (complex I) and mtNOS activities were inhibited 25% and 50%, respectively, by the addition of 10 nM (+)-catechin to the reaction medium. Moreover, in the nM range, (+)-catechin decreased state 4 mitochondrial membrane potential by about 10 mV, but failed to change the membrane potential measured in the presence of ADP. (+)-Catechin (10 nM) inhibited not only complex I activity, but also the H2O2 production rate (35%) sustained by malate-glutamate, in accordance with the decrease observed in mitochondrial membrane potential. Considering (+)-catechin concentrations lower than 10 nM, linear and positive correlations were obtained between mitochondrial complex I activity and either NO (r2 = 0.973) or H2O2 production rates (r2 = 0.958), suggesting a functional association among these parameters. Altogether, the results indicate that (+)-catechin, at nM concentrations, inhibits mitochondrial complex I activity, leading to membrane potential decline and consequently to reduction in H2O2 and NO production rates. The decrease in mtNOS activity could also be a consequence of the direct action of (+)-catechin on the NOS structure, this effect being in accordance with the functional interaction between complex I and mtNOS, as previously reported.

Complex I syndrome in striatum and frontal cortex in a rat model of Parkinson disease.

Mitochondrial dysfunction named complex I syndrome was observed in striatum mitochondria of rotenone treated rats (2 mg rotenone/kg, i. p., for 30 or 60 days) in an animal model of Parkinson disease. After 60 days of rotenone treatment, the animals showed: (a) 6-fold increased bradykinesia and 60% decreased locomotor activity; (b) 35-34% decreases in striatum O2 uptake and in state 3 mitochondrial respiration with malate-glutamate as substrate; (c) 43-57% diminished striatum complex I activity with 60-71% decreased striatum mitochondrial NOS activity, determined both as biochemical activity and as functional activity (by the NO inhibition of active respiration); (d) 34-40% increased rates of mitochondrial O2•- and H2O2 productions and 36-46% increased contents of the products of phospholipid peroxidation and of protein oxidation; and (e) 24% decreased striatum mitochondrial content, likely associated to decreased NO-dependent mitochondrial biogenesis. Intermediate values were observed after 30 days of rotenone treatment. Frontal cortex tissue and mitochondria showed similar but less marked changes. Rotenone-treated rats showed mitochondrial complex I syndrome associated with cellular oxidative stress in the dopaminergic brain areas of striatum and frontal cortex, a fact that describes the high sensitivity of mitochondrial complex I to inactivation by oxidative reactions.

Systemic and mitochondrial adaptive responses to moderate exercise in rodents.

The systemic and nonmuscular adaptive response to moderate exercise is reviewed and compared with muscle responses to moderate and exhaustive exercise. Rats participating in voluntary wheel running and mice subjected to treadmill exercise on a lifelong basis showed 10-19% increased median life span. Mice also showed improved neurological functions, such as better (35-216%) neuromuscular coordination (tightrope test) and better (11-27%) exploratory activity (T maze). These effects are consistent with the systemic effects of moderate exercise lowering hyperglycemia, hypercholesterolemia, and hypertension. Mitochondria isolated from brain, liver, heart, and kidney of exercised mice show a 12-32% selectively increased complex IV activity, with a significant correlation between complex IV activity and performance in the tightrope test. Chronic exercise decreases (10-20%) the mitochondrial content of TBARS and protein carbonyls in the four organs after 24 weeks of training. Protein carbonyls were linearly and negatively related to complex IV activity. Exercise increased the levels of nNOSmu in human muscle and of nNOS in mouse brain. It is concluded that chronic moderate exercise exerts a whole-body beneficial effect that exceeds muscle adaptation, likely through mechanosensitive afferent nerves and beta-endorphin release to brain and plasma that promote mitochondrial biogenesis in distant organs.

Cardiac mitochondrial nitric oxide: a regulator of heart rate?

Alterations in autonomic control and myocardial nitric-oxide (NO) production are likely linked to the development and progression of heart dysfunction. By focusing on heart rate, the complexity of the actions of NO at distinct levels throughout the autonomic nervous system and its relationship with other regulators can be demonstrated. Given the multiple and opposing actions of NO on cardiac control, it is difficult to interpret a response after a global intervention in the NO system. The diversity of intracellular pathways activated by NO, and their differing sensitivities to different levels of NO, might account for some aspects of reported specific but opposite effects. We discuss factors that might contribute to this diversity of actions. A proper elucidation of the effects of NO on metabolic pathways and on energy generation could lead to novel therapeutic strategies aimed at the early treatment of heart dysfunction.

Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells.

Activation of ERK1/2 implies the phosphorylation of tyrosine (pTyr) and threonine (pThr) by MEK1/2; both reactions were thought to be cytoplasmic, promoting ERK to reach the nucleus where it activates several transcription factors. In addition, H2O2 concentrations are known to modulate ERK intracellular translocation, which impacts on cellular proliferation. In this context, the objective of this work was to study the sequence of ERK phosphorylation under two redox conditions and to analyze a putative mitochondrial contribution to this process, in LP07 murine lung cells. A time-course of H2O2 administration was used and ERK phosphorylation was analyzed in cytosol, mitochondria and nuclei. At 1µM H2O2, a proliferative redox stimulus, immunoblot revealed a fast and transient increase in cytosol pTyr and a sustained increase in mitochondrial pTyr content. The detection for pThr/pTyrERK (2pERK) showed in cytosol a marked increase at 5 minutes with a fast dephosphorylation after that time, for both H2O2 concentrations. However, at 50 µM H2O2, an anti-proliferative condition, 2pERK was gradually retained in mitochondria. Interestingly, these results were confirmed by in vivo experiments using mice treated with a highly oxidizing agent [H2O2]. By the use of two ERK2 mutant constructions, where Tyr and Thr were replaced by alanine, we confirmed that 2pERK relied almost completely on pThr183. Confocal microscopy confirmed ERK subcellular distribution dependence on the incidence of cytosolic pTyr and mitochondrial pThr at 1µM H2O2. This work shows for the first time, both in vitro and in vivo, an ERK cycle involving a cross-talk between cytosol and mitochondria phosphorylation events, which may play a significant role in cell cycle progression, proliferation or differentiation under two different redox conditions.

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.

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.