Brain mitochondria are primed by moderate Ca2+ rise upon hypoxia/reoxygenation for functional breakdown and morphological disintegration

In animal models, brain ischemia causes changes in respiratory capacity, mitochondrial morphology, and cytochrome c release from mitochondria as well as a rise in cytosolic Ca2+ concentration. However, the causal relationship of the cellular processes leading to mitochondrial deterioration in brain has not yet been clarified. Here, by applying various techniques, we used isolated rat brain mitochondria to investigate how hypoxia/reoxygenation and nonphysiological Ca2+ concentrations in the low micromolar range affect active (state 3) respiration, membrane permeability, swelling, and morphology of mitochondria. Either transient hypoxia or a micromolar rise in extramitochondrial Ca2+ concentration, given as a single insult alone, slightly decreased active respiration. However, the combination of both insults caused devastating effects. These implied almost complete loss of active respiration, release of both NADH and cytochrome c, and rupture of mitochondria, as shown by electron microscopy. Mitochondrial respiration deteriorated even in the presence of cyclosporin A, documenting that membrane permeabilization occurred independent of mitochondrial permeability transition pore. Ca2+ has to enter the mitochondrial matrix in order to mediate this mitochondrial injury, because blockade of the mitochondrial Ca2+-transport system by ruthenium red in combination with CGP37157 completely prevented damage. Furthermore, protection of respiration from Ca2+-mediated damage by the adenine nucleotide ADP, but not by AMP, during hypoxia/reoxygenation is consistent with the delayed susceptibility of brain mitochondria to prolonged hypoxia, which is observed in vivo.     

Ca(2+) rise within a narrow window of concentration prevents functional injury of mitochondria exposed to hypoxia/reoxygenation by increasing antioxidative defence

Injury of liver by ischaemia crucially involves mitochondrial damage. The role of Ca(2+) in mitochondrial damage is still unclear. We investigated the effect of low micromolar Ca(2+) concentrations on respiration, membrane permeability, and antioxidative defence in liver mitochondria exposed to hypoxia/reoxygenation. Hypoxia/reoxygenation caused decrease in state 3 respiration and in the respiratory control ratio. Liver mitochondria were almost completely protected at about 2 microM Ca(2+). Below and above 2 microM Ca(2+), mitochondrial function was deteriorated, as indicated by the decrease in respiratory control ratio. Above 2 microM Ca(2+), the mitochondrial membrane was permeabilized, as demonstrated by the sensitivity of state 3 respiration to NADH. Below 2 microM Ca(2+), the nitric oxide synthase inhibitor nitro-l-arginine methylester had a protective effect. The activities of the manganese superoxide dismutase and glutathione peroxidase after hypoxia showed maximal values at about 2 microM Ca(2+). We conclude that Ca(2+) exerts a protective effect on mitochondria within a narrow concentration window, by increasing the antioxidative defence.     

Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

Glibenclamide interferes with mitochondrial bioenergetics by inducing changes on membrane ion permeability

The interference of glibenclamide, an antidiabetic sulfonylurea, with mitochondrial bioenergetics was assessed on mitochondrial ion fluxes (H+, K+, and Cl-) by passive osmotic swelling of rat liver mitochondria in K-acetate, KNO3, and KCl media, by O2 consumption, and by mitochondrial transmembrane potential (Deltapsi). Glibenclamide did not permeabilize the inner mitochondrial membrane to H+, but induced permeabilization to Cl- by opening the inner mitochondrial anion channel (IMAC). Cl- influx induced by glibenclamide facilitates K+ entry into mitochondria, thus promoting a net Cl-/K+ cotransport, Deltapsi dissipation, and stimulation of state 4 respiration rate. It was concluded that glibenclamide interferes with mitochondrial bioenergetics of rat liver by permeabilizing the inner mitochondrial membrane to Cl- and promoting a net Cl-/K+ cotransport inside mitochondria, without significant changes on membrane permeabilization to H+.     

Mitochondrial permeability transition in neuronal damage promoted by Ca2+ and respiratory chain complex II inhibition

Changes in mitochondrial integrity, reactive oxygen species release and Ca2+ handling are proposed to be involved in the pathogenesis of many neurological disorders including methylmalonic acidaemia and Huntington's disease, which exhibit partial mitochondrial respiratory inhibition. In this report, we studied the mechanisms by which the respiratory chain complex II inhibitors malonate, methylmalonate and 3-nitropropionate affect rat brain mitochondrial function and neuronal survival. All three compounds, at concentrations which inhibit respiration by 50%, induced mitochondrial inner membrane permeabilization when in the presence of micromolar Ca2+ concentrations. ADP, cyclosporin A and catalase prevented or delayed this effect, indicating it is mediated by reactive oxygen species and mitochondrial permeability transition (PT). PT induced by malonate was also present in mitochondria isolated from liver and kidney, but required more significant respiratory inhibition. In brain, PT promoted by complex II inhibition was stimulated by increasing Ca2+ cycling and absent when mitochondria were pre-loaded with Ca2+ or when Ca2+ uptake was prevented. In addition to isolated mitochondria, we determined the effect of methylmalonate on cultured PC12 cells and freshly prepared rat brain slices. Methylmalonate promoted cell death in striatal slices and PC12 cells, in a manner attenuated by cyclosporin A and bongkrekate, and unrelated to impairment of energy metabolism. We propose that under conditions in which mitochondrial complex II is partially inhibited in the CNS, neuronal cell death involves the induction of PT.     

The effect of permeability transition pore opening on reactive oxygen species production in rat brain mitochondria

The influence of mitochondrial permeability transition pore (MPTP) opening on reactive oxygen species (ROS) production in the rat brain mitochondria was studied. It was shown that ROS production is regulated differently by the rate of oxygen consumption and membrane potential, dependent on steady-state or non-equilibrium conditions. Under steady-state conditions, at constant rate of Ca2+-cycling and oxygen consumption, ROS production is potential-dependent and decreases with the inhibition of respiration and mitochondrial depolarization. The constant rate of ROS release is in accord with proportional dependence of the rate of ROS formation on that of oxygen consumption. On the contrary, transition to non-equilibrium state, due to the release of cytochrome c from mitochondria and progressive respiration inhibition, results in the loss of proportionality in the rate of ROS production on the rate of respiration and an exponential rise of ROS production with time, independent of membrane potential. Independent of steady-state or non-equilibrium conditions, the rate of ROS formation is controlled by the rate of potential-dependent uptake of Ca2+ which is the rate-limiting step in ROS production. It was shown that MPTP opening differently regulates ROS production, dependent on Ca2+ concentration. At low calcium MPTP opening results in the decrease in ROS production because of partial mitochondrial depolarization, in spite of sustained increase in oxygen consumption rate by a cyclosporine A-sensitive component due to simultaneous work of Ca2+-uniporter and MPTP as Ca2+-influx and efflux pathways. The effect of MPTP opening at low Ca2+ concentrations is similar to that of Ca2+-ionophore, A-23187. At high calcium MPTP opening results in the increase of ROS release due to the rapid transition to non-equilibrium state because of cytochrome c loss and progressive gating of electron flow in respiratory chain. Thus, under physiological conditions MPTP opening at low intracellular calcium could attenuate oxidative damage and the impairment of neuronal functions by diminishing ROS formation in mitochondria.     

Mutant huntingtin expression induces mitochondrial calcium handling defects in clonal striatal cells: functional consequences

Huntington disease (HD) is caused by a pathological elongation of CAG repeats in the huntingtin protein gene and is characterized by atrophy and neuronal loss primarily in the striatum. Mitochondrial dysfunction and impaired Ca2+ homeostasis in HD have been suggested previously. Here, we elucidate the effects of Ca2+ on mitochondria from the wild type (STHdhQ7/Q7) and mutant (STHdhQ111/Q111) huntingtin-expressing cells of striatal origin. When treated with increasing Ca2+ concentrations, mitochondria from mutant huntingtin-expressing cells showed enhanced sensitivity to Ca2+, as they were more sensitive to Ca2+-induced decreases in state 3 respiration and DeltaPsim, than mitochondria from wild type cells. Further, mutant huntingtin-expressing cells had a reduced mitochondrial Ca2+ uptake capacity in comparison with wild type cells. Decreases in state 3 respiration were associated with increased mitochondrial membrane permeability. The DeltaPsim defect was attenuated in the presence of ADP and the decreases in Ca2+ uptake capacity were abolished in the presence of Permeability Transition Pore (PTP) inhibitors. These findings clearly indicate that mutant huntingtin-expressing cells have mitochondrial Ca2+ handling defects that result in respiratory deficits and that the increased sensitivity to Ca2+ induced mitochondrial permeabilization maybe a contributing mechanism to the mitochondrial dysfunction in HD.     

Mangiferin, a natural occurring glucosyl xanthone, increases susceptibility of rat liver mitochondria to calcium-induced permeability transition

Mitochondrial permeability transition (MPT) is a Ca(2+)-dependent, cyclosporine A-sensitive, non-selective inner membrane permeabilization induced by a wide range of agents or conditions, which has often been associated with necrotic or apoptotic cell death. When mitochondria isolated from livers of rats treated with the natural occurring glucosyl xanthone mangiferin (40 mg/kg body weight) were exposed in vitro to Ca(2+), they underwent CsA, NEM, and ADP-sensitive high amplitude swelling and associated membrane potential dissipation, release of pre-accumulated Ca(2+), oxidation of thiol groups, and depletion of GSH, without changes in the NAD(P)H redox state. The same treatment reduced the phosphorylation rate of mitochondria and the resting respiration by around 4 and 11%, respectively, as well as generation of reactive oxygen species (ROS) by organelle. The in vitro exposure of untreated mitochondria to mangiferin plus Ca(2+) also resulted in oxidation of thiol groups, in the same way that the compound inhibited the Ca(2+)-induced peroxidation of mitochondrial membrane lipids. The spectrum of mangiferin during its oxidation by the H(2)O(2)/HRP system showed a characteristic absorption peak at 380 nm, which decreased immediately after reaction was started; two isosbestic points at around 336 and 412 nm, with a blue shift in the position of the maxima absorption of mangiferin were observed, suggesting their conversion into one oxidation product. Glutathione abolished this decrease of absorbance, suggesting that the oxidation product of mangiferin forms adducts with GSH. We propose that Ca(2+) increases levels of mitochondria-generated ROS, which reacts with mangiferin producing quinoid derivatives, which in turn react with the most accessible mitochondrial thiol groups, thus triggering MPT. It seems probable that the free radical scavenging activity of mangiferin shifts its anti-oxidant protection to the thiol arylation. An interesting proposition is that accumulation of mangiferin quinoid products would take place in cells exposed to an overproduction of ROS, such as cancer cells, where the occurrence of MPT-mediated apoptosis may be a cellular defence mechanism against excessive ROS formation.     

Glutathione and thioredoxin peroxidases mediate susceptibility of yeast mitochondria to Ca(2+)-induced damage

The effect of thioredoxin peroxidases on the protection of Ca(2+)-induced inner mitochondrial membrane permeabilization was studied in the yeast Saccharomyces cerevisiae using null mutants for these genes. Since deletion of a gene can promote several other effects besides the absence of the respective protein, characterizations of the redox state of the mutant strains were performed. Whole cellular extracts from all the mutants presented lower capacity to decompose H(2)O(2) and lower GSH/GSSG ratios, as expected for strains deficient for peroxide-removing enzymes. Interestingly, when glutathione contents in mitochondrial pools were analyzed, all mutants presented lower GSH/GSSG ratios than wild-type cells, with the exception of DeltacTPxI strain (cells in which cytosolic thioredoxin peroxidase I gene was disrupted) that presented higher GSH/GSSG ratio. Low GSH/GSSG ratios in mitochondria increased the susceptibility of yeast to damage induced by Ca(2+) as determined by membrane potential and oxygen consumption experiments. However, H(2)O(2) removal activity appears also to be important for mitochondria protection against permeabilization because exogenously added catalase strongly inhibited loss of mitochondrial potential. Moreover, exogenously added recombinant peroxiredoxins prevented inner mitochondrial membrane permeabilization. GSH/GSSG ratios decreased after Ca(2+) addition, suggesting that reactive oxygen species (ROS) probably mediate this process. Taken together our results indicate that both mitochondrial glutathione pools and peroxide-removing enzymes are key components for the protection of yeast mitochondria against Ca(2+)-induced damage.     

Mitochondrial creatine kinase activity prevents reactive oxygen species generation: antioxidant role of mitochondrial kinase-dependent ADP re-cycling activity

As recently demonstrated by our group (da-Silva, W. S., Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846-39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H(2)O(2) production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H(2)O(2) generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H(2)O(2) production in a Cr-sensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both DeltaPsi(m) and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.     

H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (psi m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+],) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas.     

Ca2+-induced permeabilization promotes free radical release from rat brain mitochondria with partially inhibited complex I

Mitochondrial complex I dysfunction has been implicated in a number of brain pathologies, putatively owing to an increased rate of reactive oxygen species (ROS) release. However, the mechanisms regulating the ROS burden are poorly understood. In this study we investigated the effect of Ca2+ loads on ROS release from rat brain mitochondria with complex I partially inhibited by rotenone. The addition of 20 nm rotenone to brain mitochondria increased ROS release. Ca2+ (100 microm) alone had no effect on ROS release, but greatly potentiated the effects of rotenone. The effect of Ca2+ was decreased by ruthenium red. Ca2+-challenged mitochondria lose about 88% of their glutathione and 46% of their cytochrome c under these conditions, although this depends only on Ca2+ loading and not complex I inhibition. ADP in combination with oligomycin decreased the loss of glutathione and cytochrome c and free radical generation. Cyclosporin A alone was ineffective in preventing these effects, but augmented the protection provided by ADP and oligomycin. Non-specific permeabilization of mitochondria with alamethicin also increased the ROS signal, but only when combined with partial inhibition of complex I. These results demonstrate that Ca2+ can greatly increase ROS release by brain mitochondria when complex I is impaired.     

DJ‐1 plays an obligatory role in the cardioprotection of delayed hypoxic preconditioning against hypoxia/reoxygenation‐induced oxidative stress through maintaining mitochondrial complex I activity

DJ‐1 was recently reported to mediate the cardioprotection of delayed hypoxic preconditioning (DHP) by suppressing hypoxia/reoxygenation (H/R)‐induced oxidative stress, but its mechanism against H/R‐induced oxidative stress during DHP is not fully elucidated. Here, using the well‐established cellular model of DHP, we again found that DHP significantly improved cell viability and reduced lactate dehydrogenase release with concurrently up‐regulated DJ‐1 protein expression in H9c2 cells subjected to H/R. Importantly, DHP efficiently improved mitochondrial complex I activity following H/R and attenuated H/R‐induced mitochondrial reactive oxygen species (ROS) generation and subsequent oxidative stress, as demonstrated by a much smaller decrease in reduced glutathione/oxidized glutathione ratio and a much smaller increase in intracellular ROS and malondialdehyde contents than that observed for the H/R group. However, the aforementioned effects of DHP were antagonized by DJ‐1 knockdown with short hairpin RNA but mimicked by DJ‐1 overexpression. Intriguingly, pharmacological inhibition of mitochondria complex I with Rotenone attenuated all the protective effects caused by DHP and DJ‐1 overexpression, including maintenance of mitochondria complex I and suppression of mitochondrial ROS generation and subsequent oxidative stress. Taken together, this work revealed that preserving mitochondrial complex I activity and subsequently inhibiting mitochondrial ROS generation could be a novel mechanism by which DJ‐1 mediates the cardioprotection of DHP against H/R‐induced oxidative stress damage.     

Mitochondrial dysfunction in rat brain with aging Involvement of complex I, reactive oxygen species and cardiolipin

Reactive oxygen species (ROS) are considered a key factor in brain aging process. Mitochondrial respiration is an important site of ROS production and hence a potential contributor to brain functional changes with aging. In this study we examined the effect of aging on complex I activity, oxygen consumption, ROS production and phospholipid composition in rat brain mitochondria. The activity of complex I was reduced by 30% in brain mitochondria from 24 months aged rats relative to young animals. These changes in complex I activity were associated with parallel changes in state 3 respiration. H(2)O(2) generation was significantly increased in mitochondria isolated from aged rats. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 31% as function of aging, while there was a significant increase in the level of peroxidized cardiolipin. The age-related decrease in complex I activity in brain mitochondria could be reversed by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids. It is proposed that aging causes brain mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings may prove useful in elucidating the mechanism underlying mitochondrial dysfunction associated with brain aging.     

Involvement of Ca2+ release and activation of phospholipase A2 in mitochondrial dysfunction during anoxia

During anoxic incubation, depletion of mitochondrial ATP was followed by release of Ca2+ with concomitant increase in the rate of state 4 respiration due to disruption of the diffusion barrier against protons. The external addition of ATP and its non-metabolizable analog, beta,gamma-methylene adenosine 5'-triphosphate, prevented both the release of Ca2+ and increase in the rate of state 4 respiration. Addition of EGTA, which did not prevent release of the ion, resulted in little increase in the respiration rate. Addition of an inhibitor of mitochondrial phospholipase A2, such as quinacrine, dibucaine, or chlorpromazine, also prevented increase in the respiration rate without affecting Ca2+ release from mitochondria during anoxic incubation. Non-esterified polyunsaturated fatty acids were also found to be liberated from anoxic mitochondria. External addition of the ATP-analog, EGTA, and inhibitors of phospholipase A2 suppressed the liberation of non-esterified polyunsaturated fatty acids. Melittin and Ca2+, which activate phospholipase A2, increased the rate of state 4 respiration and the liberation of fatty acids. These findings support the hypothesis proposed previously that the following sequence changes occurs in mitochondria during anoxia; depletion of ATP, liberation of free calcium from mitochondria, and disruption of the diffusion barrier against H+ of the inner membrane. The results also indicate another event; activation of phospholipase A2 by release Ca2+ which results in H+ leakiness of the inner membrane.     

Role of alpha-tocopherol in the regulation of mitochondrial permeability transition

We previously showed that Ca2+-induced cyclosporin A-sensitive membrane permeability transition (MPT) of mitochondria occurred with concomitant generation of reactive oxygen species (ROS) and release of cytochrome c (Free Rad. Res.38, 29-35, 2004). To elucidate the role of alpha-tocopherol in MPT, we investigated the effect of alpha-tocopherol on mitochondrial ROS generation, swelling and cytochrome c release induced by Ca2+ or hydroxyl radicals. Biochemical analysis revealed that alpha-tocopherol suppressed Ca2+-induced ROS generation and oxidation of critical thiol groups of mitochondrial adenine nucleotide translocase (ANT) but not swelling and cytochrome c release. Hydroxyl radicals also induced cyclosporin A-sensitive MPT of mitochondria. alpha-Tocopherol suppressed the hydroxyl radical-induced lipid peroxidation, swelling and cytochrome c release from mitochondria. These results indicate that alpha-tocopherol inhibits ROS generation, ANT oxidation, lipid peroxidation and the opening of MPT, thereby playing important roles in the prevention of oxidative cell death.     

Reactive oxygen species generation is modulated by mitochondrial kinases: correlation with mitochondrial antioxidant peroxidases in rat tissues

Mitochondrial hexokinase (mt-HK) and creatine kinase (mt-CK) activities have been recently proposed to reduce the rate of mitochondrial ROS generation through an ADP re-cycling mechanism. Here, we determined the role of mt-HK and mt-CK activities in regulate mitochondrial ROS generation in rat brain, kidney, heart and liver, relating them to the levels of classical antioxidant enzymes. The activities of both kinases were significantly higher in the brain than in other tissues, whereas the activities of catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were higher in both liver and kidney mitochondria. In contrast, manganese superoxide dismutase (Mn-SOD) activity was not significantly different among these tissues. Activation of mitochondrial kinases by addition of their substrates increased the ADP re-cycling and thus the respiration by enhancing the oxidative phosphorylation. Succinate induced hydrogen peroxide (H(2)O(2)) generation was higher in brain than in kidney and heart mitochondria, and the lowest in liver mitochondria. Mitochondrial membrane potential (DeltaPsi(m)) and H(2)O(2) production, decreased with additions of 2-DOG or Cr to respiring brain and kidney mitochondria but not to liver. The inhibition of H(2)O(2) production by 2-DOG and Cr correspond to almost 100% in rat brain and about 70% in kidney mitochondria. Together our data suggest that mitochondrial kinases activities are potent preventive antioxidant mechanism in mitochondria with low peroxidase activities, complementing the classical antioxidant enzymes against oxidative stress.     

Effect of Peroxide, Sodium, and Calcium on Brain Mitochondrial Respiration In Vitro: Potential Role in Cerebral Ischemia and Reperfusion

Mitochondrial pyruvate-supported respiration was studied in vitro under conditions known to exist following ischemia, i.e., elevated extramitochondrial Ca2+, Na+, and peroxide. Ca2+ alone (7-10 nmol/mg) decreased state 3 and increased state 4 respiration to 81 and 141% of control values, respectively. Sodium (15 mM) and/or tert-butyl hydroperoxide (tBOOH; up to 2,000 nmol/mg protein) alone had no effect on respiration; however, Na+ or tBOOH in combination with Ca2+ dramatically altered respiration. Respiratory inhibition induced by Ca2+ and tBOOH does not involve pyruvate dehydrogenase (PDH) inhibition since PDH flux increased linearly with tBOOH concentration (R = 0.96). Calcium potentiated tBOOH-induced mitochondrial NAD(P)H oxidation and shifted the redox state of cytochrome b from 67 to 47% reduced. Calcium (5.5 nmol/mg) plus Na+ (15 mM) decreased state 3 and increased state 4 respiratory rates to 55 and 202% of control values, respectively. Sodium- as well as tBOOH-induced state 3 inhibition required mitochondrial Ca2+ uptake because ruthenium red addition before Ca2+ addition negated the effect. The increase in state 4 respiration involved Ca2+ cycling since ruthenium red immediately returned state 4 rates back to control values. The mechanisms for the observed Ca2(+)-, Na(+)-, and tBOOH-induced alterations in pyruvate-supported respiration in vitro are discussed and a multifactorial etiology for mitochondrial respiratory dysfunction following cerebral ischemia in vivo is proposed.     

Reverse electron flow-induced ROS production is attenuated by activation of mitochondrial Ca2+-sensitive K+ channels

Mitochondria generate reactive oxygen species (ROS) dependent on substrate conditions, O(2) concentration, redox state, and activity of the mitochondrial complexes. It is well known that the FADH(2)-linked substrate succinate induces reverse electron flow to complex I of the electron transport chain and that this process generates superoxide (O(2)(*-)); these effects are blocked by the complex I blocker rotenone. We demonstrated recently that succinate + rotenone-dependent H(2)O(2) production in isolated mitochondria increased mildly on activation of the putative big mitochondrial Ca(2+)-sensitive K(+) channel (mtBK(Ca)) by low concentrations of 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619). In the present study we examined effects of NS-1619 on mitochondrial O(2) consumption, membrane potential (DeltaPsi(m)), H(2)O(2) release rates, and redox state in isolated guinea pig heart mitochondria respiring on succinate but without rotenone. NS-1619 (30 microM) increased state 2 and state 4 respiration by 26 +/- 4% and 14 +/- 4%, respectively; this increase was abolished by the BK(Ca) channel blocker paxilline (5 microM). Paxilline alone had no effect on respiration. NS-1619 did not alter DeltaPsi(m) or redox state but decreased H(2)O(2) production by 73% vs. control; this effect was incompletely inhibited by paxilline. We conclude that under substrate conditions that allow reverse electron flow, matrix K(+) influx through mtBK(Ca) channels reduces mitochondrial H(2)O(2) production by accelerating forward electron flow. Our prior study showed that NS-1619 induced an increase in H(2)O(2) production with blocked reverse electron flow. The present results suggest that NS-1619-induced matrix K(+) influx increases forward electron flow despite the high reverse electron flow, and emphasize the importance of substrate conditions on interpretation of effects on mitochondrial bioenergetics.