Role of calcium signaling in the activation of mitochondrial nitric oxide synthase and citric acid cycle

An apparent discrepancy arises about the role of calcium on the rates of oxygen consumption by mitochondria: mitochondrial calcium increases the rate of oxygen consumption because of the activation of calcium-activated dehydrogenases, and by activating mitochondrial nitric oxide synthase (mtNOS), decreases the rates of oxygen consumption because nitric oxide is a competitive inhibitor of cytochrome oxidase. To this end, the rates of oxygen consumption and nitric oxide production were followed in isolated rat liver mitochondria in the presence of either L-Arg (to sustain a mtNOS activity) or N(G)-monomethyl-L-Arg (NMMA, a competitive inhibitor of mtNOS) under State 3 conditions. In the presence of NMMA, the rates of State 3 oxygen consumption exhibited a K(0.5) of 0.16 microM intramitochondrial free calcium, agreeing with those required for the activation of the Krebs cycle. By plotting the difference between the rates of oxygen consumption in State 3 with L-Arg and with NMMA at various calcium concentrations, a K(0.5) of 1.2 microM intramitochondrial free calcium was obtained, similar to the K(0.5) (0.9 microM) of the dependence of the rate of nitric oxide production on calcium concentrations. The activation of dehydrogenases, followed by the activation of mtNOS, would lead to the modulation of the Krebs cycle activity by the modulation of nitric oxide on the respiratory rates. This would ensue in changes in the NADH/NAD and ATP/ADP ratios, which would influence the rate of the cycle and the oxygen diffusion.     

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 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 Ca2+ and the heart

It is now well established that mitochondria accumulate Ca(2+) ions during cytosolic Ca(2+) ([Ca(2+)](i)) elevations in a variety of cell types including cardiomyocytes. Elevations in intramitochondrial Ca(2+) ([Ca(2+)](m)) activate several key enzymes in the mitochondrial matrix to enhance ATP production, alter the spatial and temporal profile of intracellular Ca(2+) signaling, and play an important role in the initiation of cell death pathways. Moreover, mitochondrial Ca(2+) uptake stimulates nitric oxide (NO) production by mitochondria, which modulates oxygen consumption, ATP production, reactive oxygen species (ROS) generation, and in turn provides negative feedback for the regulation of mitochondrial Ca(2+) accumulation. Controversy remains, however, whether in cardiac myocytes mitochondrial Ca(2+) transport mechanisms allow beat-to-beat transmission of fast cytosolic [Ca(2+)](i) oscillations into oscillatory changes in mitochondrial matrix [Ca(2+)](m). This review critically summarizes the recent experimental work in this field.     

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.     

Hypoxia induces production of nitric oxide and reactive oxygen species in glomus cells of rat carotid body

The carotid body is an arterial chemoreceptor organ that senses arterial pO(2) and pH. Previous studies have indicated that both reactive oxygen species (ROS) and nitric oxide (NO) are important potential mediators that may be involved in the response of the carotid body to hypoxia. However, whether their production by the chemosensitive elements of the carotid body is indeed oxygen-dependent is currently unclear. Thus, we have investigated their production under normoxic (20% O(2)) and hypoxic (1% O(2)) conditions in slice preparations of the rat carotid body by using fluorescent indicators and confocal microscopy. NO-synthesizing enzymes were identified by immunohistochemistry and histochemistry, and the subcellular localization of the NO-sensitive indicator diaminofluorescein was determined by a photoconversion technique and electron microscopy. Glomus cells of the carotid body responded to hypoxia by increases in both ROS and NO production. The hypoxia-induced increase in NO generation required (to a large extent, but not completely) extracellular calcium. Glomus cells were immunoreactive to endothelial NO synthase but not to the neuronal or inducible isoforms. Ultrastructurally, the NO-sensitive indicator was observed in mitochondrial membranes after exposure to hypoxia. The data show that glomus cells respond to exposure to hypoxia by the enhanced production of both ROS and NO. NO production by glomus cells is probably mediated by endothelial NO synthase, which is activated by calcium influx. The presence of NO indicator in mitochondria suggests the hypoxic regulation of mitochondrial function via NO in glomus cells.     

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 Nitric-Oxide Synthase: Enzyme Expression, Characterization, and Regulation

Nitric oxide is generated in vivo by nitric-oxide synthase (NOS) during the conversion of L-Arg to citrulline. Using a variety of biological systems and approaches emerging evidence has been accumulated for the occurrence of a mitochondrial NOS (mtNOS), identified as the alpha isoform of neuronal or NOS-1. Under physiological conditions, the production of nitric oxide by mitochondria has an important implication for the maintenance of the cellular metabolism, i.e. modulates the oxygen consumption of the organelles through the competitive (with oxygen) and reversible inhibition of cytochrome c oxidase. The transient inhibition suits the continuously changing energy and oxygen requirements of the tissue; it is a short-term regulation with profound pathophysiological consequences. This review describes the identification of mtNOS and the role of posttranslational modifications on mtNOS' activity and regulation.     

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).     

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.     

Differential requirements of calcium for oxoglutarate dehydrogenase and mitochondrial nitric-oxide synthase under hypoxia: impact on the regulation of mitochondrial oxygen consumption

Studies with isolated mitochondria are performed at artificially high pO(2) (220 to 250 microM oxygen), although this condition is hyperoxic for these organelles. It was the aim of this study to evaluate the effect of hypoxia (20-30 microM) on the calcium-dependent activation of 2-oxoglutarate dehydrogenase (or 2-ketoglutarate dehydrogenase; OGDH) and mitochondrial nitric-oxide synthase (mtNOS). Mitochondria had a P/O value 15% higher in hypoxia than that in normoxia, indicating that oxidative phosphorylation and electron transfer were more efficiently coupled, whereas the intramitochondrial free calcium concentrations were higher (2-3-fold) at lower pO(2). These increases were abrogated by ruthenium red indicating that the higher uptake via the calcium uniporter was involved in this process. Mitochondria at high calcium concentration microdomains may produce nitric oxide, given the K(0.5) of calcium for OGDH (0.16 microM) and mtNOS (approximately 1 microM). Nitric oxide, by binding to cytochrome oxidase in competition with oxygen, decreases the rate of oxygen consumption. This condition is highly beneficial for the following reasons: i, these mitochondria are still able to produce ATP and support calcium clearance; ii, it prevents the accumulation of ROS by slowing the rate of oxygen consumption (hence ROS production); iii, the onset of anoxia is delayed, allowing oxygen to diffuse back to these sites, thereby ameliorating the oxygen gradient between regions of high and low calcium concentration. In this way, oxygen depletion at the latter sites is prevented. This, in turn, assures continued aerobic metabolism which may involve the activated dehydrogenases.     

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.     

Functional evidence for nitric oxide production by skeletal-muscle mitochondria from lipopolysaccharide-treated mice

The possible existence of a mitochondrially localized nitric oxide (NO) synthase (mtNOS) is controversial. To clarify this, we studied the ability of intact mitochondria to generate NO and the effect of mitochondrial NO on respiration. Respiratory rates and oxygen kinetics (P(50) values) were determined by high-resolution respirometry in skeletal-muscle mitochondria from control mice and mice injected with Escherichia coli lipopolysaccharide (LPS). In the presence of the NOS substrate L-arginine, mitochondria from LPS-treated mice had lower respiration rates and higher P(50) values than control animals. These effects were prevented by the NOS inhibitor L-NMMA. Our results suggest that mitochondrially derived NO is generated by an LPS-inducible NOS protein other than iNOS and modulates oxygen consumption in mouse skeletal muscle.     

Mitochondrial ROS-K+ channel signaling pathway regulated secretion of human pulmonary artery endothelial cells

The objective was to investigate the molecular mechanism of mitochondrial reactive oxygen species (ROS) signaling regulation of pulmonary artery endothelial cell (HPAEC) secretion in the condition of oxidative stress. Acrolein (40 μM) induced HPAEC mitochondrial generation of ROS, rotenone (2 μmol/L) blocked mitochondrial respiratory chain complex I, cesium chloride (CsCl, 40 mmol/L)blocked K⁺channels, and saline (0.9 g/dl) were used as control. The generations of NOS, ET-1 and VEGF were determined with ELISA in the condition of different treatment reagents namely acrolein, acrolein plus rotenone, acrolein plus CsCl and saline. In the different reagent treatment of HPAECs, acrolein increased mitochondrial ROS, membrane potential, Kv1.5 mRNA and protein expression, intracellular calcium and the generation of NOS (determining NO production), ET-1 and VEGF, and those were reduced by rotenone. CsCl decreased the increment of membrane potential, the elevation of intracellular calcium and the upregulation of NOS, E-1 and VEGF expressions, which were induced by acrolein. The present study demonstrated that mitochondrial ROS-K⁺channel regulated HPAEC secretion of NO, ET-1 and VEGF in the condition of oxidative stress. Kv1.5 channel may be an important component of ROS-K+ channel signaling pathway, and intracellular calcium contributed to mitochondrial ROS-K⁺ channel signaling modulation of HPAEC secretion.     

Mitochondrial production of oxidants is necessary for physiological calcium oscillations

Mitochondrial involvement in Ca2+ signaling is thought to be due to the effect of mitochondrial Ca2+ removal from and Ca2+ release to cytosolic domains close to ryanodine and IP3 Ca2+ channels. However, mitochondria are a source of low levels of endogenous reactive oxygen species, and Ca2+ release channels are known to be redox-sensitive. In the present work, we studied the role of mitochondrial production of oxygen species in Ca2+ oscillations during physiological stimulation. Mitochondria-targeted antioxidants and mitochondrial inhibitors quickly inhibited calcium oscillations in pancreatic acinar cells stimulated by postprandial levels of the gut hormone cholecystokinin. Confocal microscopy using different redox-sensitive dyes showed that cholecystokinin-induced oscillations are associated with mitochondrial production of reactive oxygen species. This production is inhibited by application of mitochondria-targeted antioxidants and mitochondrial inhibitors. In addition, we found no correlation between inhibition of oscillations and mitochondrial depolarization. We conclude that low level production of reactive oxygen species by mitochondria is a necessary element in the development of Ca2+ oscillations during physiological stimulation. This study unveils a new and unexplored aspect of the participation of mitochondria in calcium signals.     

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