Mechanism of induction of oxidative stress in liver mitochondria by low concentrations of tert-butyl hydroperoxide.

The mechanism of the effect of tert-butyl hydroperoxide (tBHP) on the kinetics of decrease in liver mitochondrial ΔΨ (transmembrane electric potential) in response to successive additions of tBHP in low concentrations has been studied. FeSO(4) was found to increase significantly the damaging effect of tBHP; this effect was shown to increase in the presence of low concentrations of Ca2+ starting from 2 µM CaCl(2). Cyclosporin A prevents these effects. The data show that the damaging effect of low concentrations of tBHP in the course of pyruvate oxidation in isolated liver mitochondria is caused by the opening of the nonspecific Ca2+-dependent cyclosporin A-sensitive pore in the inner mitochondrial membrane. Application of a method of studying oxidative stress regulators, developed in this work, is illustrated by an example of the prooxidant action of ascorbate. This method is proposed for studying mitochondria in hemochromatosis, a pathology caused by excessive accumulation of iron.

Mitochondrial models of pathologies with oxidative stress. Efficiency of alkalization to reduce mitochondrial damage.

Previously, we developed a method to monitor the development of oxidative stress in isolated liver mitochondria. The method is based on recording of membrane potential changes in response to sequential introduction of low concentrations (5-20 µM) of tert-butyl hydroperoxide (tBHP). It allows monitoring of the extent of amplification or attenuation of oxidative stress caused by external influences (changes in incubation conditions, additions of biologically active substances). Based on this method, we created a mitochondrial model for the study and improvement of treatment of pathologies associated with oxidative stress. The following two processes were simulated in the experiments: 1) introduction of desferal for treatment of serious diseases caused by cell overload with iron (high desferal concentrations were shown to suppress mitochondrial energetics); 2) efficiency of alkalization to reduce mitochondrial damage induced by oxidative stress. The experiments have shown that even a small increase in pH (alkalization) increases the amount of tBHP that can be added to mitochondria before the MPTP ("mitochondrial permeability transition pore") is induced. The effect of alkalization was shown to be close to the effect of cyclosporin A in the pH range 7.2-7.8. The mechanism of the similarities of these effects in the organism and in mitochondrial suspensions is explained by the increase in toxic reactive oxygen species in both systems under oxidative stress.

Features of mitochondrial energetics in living unicellular eukaryote Tetrahymena pyriformis. A model for study of mammalian intracellular adaptation.

Tetrahymena pyriformis is used in diverse studies as a non-mammalian alternative due to their resemblance in many main metabolic cycles. However, such basic features of mitochondrial energetics as Delta psi (electrical potential difference across the inner mitochondrial membrane) or maximal stimulation of respiration by uncouplers with different mechanisms of uncoupling, such as DNP (2,4-dinitrophenol) and FCCP (p-trifluoromethoxycarbonylcyanide phenylhydrazone), have not been studied in living ciliates. Tetrahymena pyriformis GL cells during stationary growth phase after incubation under selected conditions were used in this study. Maximal stimulation of cellular respiration by FCCP was about six-fold, thus the proton motive force was high. The DNP uncoupling effect was significantly lower. This suggests low activity of the ATP/ADP-antiporter, which performs not only exchange of intramitochondrial ATP to extramitochondrial ADP, but also helps in the uncoupling process. It participates by a similar mechanism in electrophoretic transport from matrix to cytosol of ATP(4-) and DNP anion, but not FCCP anion. Thus, in contrast with mammalian mitochondria, T. pyriformis mitochondria cannot rapidly supply the cytosol with ATP; possibly the cells need high intramitochondrial ATP. The difference between DNP and FCCP is hypothetically explained by low Delta psi value and/or an increase in concentration of long-chain acyl-CoAs, inhibitors of the ATP/ADP-antiporter. The first suggestion is confirmed by absence of mitochondria with bright fluorescence in T. pyriformis stained with the Delta psi-sensitive probe MitoTracker Red. These data suggest that T. pyriformis cells are useful as a model for study of mitochondrial role in adaptation at the intracellular level.

Cations SkQ1 and MitoQ accumulated in mitochondria delay opening of ascorbate/FeSO4-induced nonspecific pore in the inner mitochondrial membrane.

It is known that an addition of FeSO4 in the presence of ascorbic acid to cells or mitochondria can injure energy coupling and some other functions in mitochondria. The present study demonstrates that decrease in ascorbate concentration from 4 to 0.2 mM in the presence of the same low concentrations of FeSO4 accelerates the nonspecific pore opening, while cyclosporin A prevents and under some conditions reverses the pore opening. Hydrophobic cations SkQ1 and MitoQ (structural analogs of plastoquinone and coenzyme Q(10), respectively) delay pore opening, SkQ1 being more efficient. It is known that an increase in matrix ADP concentration delays pore opening, while an addition of carboxyatractylate to mitochondria accelerates the beginning of pore opening. Preliminary addition of SkQ1 into a mitochondrial suspension increased the effect of ADP and decreased the effect of carboxyatractylate. These results suggest that under the conditions used SkQ1 protects mitochondria from oxidative damage as an antioxidant when added at extremely low concentrations.

Involvement of mitochondrial inner membrane anion carriers in the uncoupling effect of fatty acids.

This paper considers stages of the search (initiated by V. P. Skulachev) for a receptor protein for fatty acids that is involved in their uncoupling effect. Based on these studies, mechanism of the ADP/ATP antiporter involvement in the uncoupling induced by fatty acids was proposed. New data (suppression by carboxyatractylate of the SDS-induced uncoupling, pH-dependence of the ADP/ATP and the glutamate/aspartate antiporter contributions to the uncoupling, etc.) led to modification of this hypothesis. During discussion of the uncoupling effect of fatty acids caused by opening of the Ca(2+)-dependent pore, special attention is given to the effects of carboxyatractylate added in the presence of ADP. The functioning of the uncoupling protein UCP2 in kidney mitochondria is considered, as well as the diversity observed by us in effects of 200 microM GDP on decrease in Deltapsi under the influence of oleic acid added after H(2)O(2) (in the presence of succinate, oligomycin, malonate). A speculative explanation of the findings is as follows: 1) products of lipid and/or fatty acid peroxidation (PPO) modify the ADP/ATP antiporter in such a way that its involvement in the fatty acid-induced uncoupling is suppressed by GDP; 2) GDP increases the PPO concentration in the matrix by suppression of efflux of fatty acid hydroperoxide anions through the UCP and/or of efflux of PPO anions with involvement of the GDP-sensitive ADP/ATP antiporter; 3) PPO can potentiate the oleate-induced decrease in Deltapsi due to inhibition of succinate oxidation.

Uncoupling effect of fatty acids in halo- and alkalotolerant bacterium Bacillus pseudofirmus FTU.

Natural uncouplers of oxidative phosphorylation, long-chain non-esterified fatty acids, cause uncoupling in the alkalo- and halotolerant bacterium Bacillus pseudofirmus FTU. The uncoupling effect in the bacterial cells was manifested as decrease of membrane potential and increase of respiratory activity. The membrane potential decrease was detected only in bacterial cells exhausted by their endogenous substrates. In proteoliposomes containing reconstituted bacterial cytochrome c oxidase, fatty acids caused a "mild" uncoupling effect by reducing membrane potential only at low rate of membrane potential generation. "Free respiration" induced by the "mild" uncouplers, the fatty acids, can be considered as possible mechanism responsible for adaptation of the bacteria to a constantly changed environment.

Cyclosporin A-sensitive cytochrome c release and activation of external pathway of NADH oxidation in liver mitochondria due to pore opening by acidification of phosphate-containing incubation medium.

Acidification of a high phosphate incubation medium from pH 7.4 to 6.5 promotes increase in rates of succinate oxidation and exogenous NADH oxidation via external (rotenone-and myxothiazol-resistant) pathway by factors 2 and 2.3 respectively. Cyclosporin A prevents these effects. To measure the cytochrome c release, mitochondrial cytochrome c concentration was calculated from absorption spectrum of alpha-band of cytochromes c + c1. The cytochrome c release is shown to be equal to 27 +/- 4%, 40 +/- 12%, 70 +/- 5% at pH 7.4, 7.0, 6.5, respectively, the last value being reduced by cyclosporin A to 10 +/- 3%. Immunoblot method gives the similar results. It is concluded that acidification of the high phosphate medium induces release of a large part of the cytochrome c pool from liver mitochondria due to opening the Ca(2+)-dependent cyclosporin A-sensitive permeability transition pore and subsequent high amplitude swelling.

Cyclosporin A-sensitive decrease in the transmembrane potential across the inner membrane of liver mitochondria induced by low concentrations of fatty acids and Ca2+.

At low Ca2+ concentrations the pore of the inner mitochondrial membrane can open in substates with lower permeability (Hunter, D. R., and Haworth, R. A. (1979) Arch. Biochem. Biophys., 195, 468-477). Recently, we showed that Ca2+ loading of mitochondria augments the cyclosporin A-dependent decrease in transmembrane potential (DeltaPsi) across the inner mitochondrial membrane caused by 10 micro M myristic acid but does not affect the stimulation of respiration by this fatty acid. We have proposed that in our experiments the pore opened in a substate with lower permeability rather than in the "classic" state (Bodrova, M. E., et al. (2000) IUBMB Life, 50, 189-194). Here we show that under conditions lowering the probability of "classic pore" opening in Ca2+-loaded mitochondria myristic acid induces the cyclosporin A-sensitive DeltaPsi decrease and mitochondrial swelling more effectively than uncoupler SF6847 does, though their protonophoric activities are equal. In the absence of P(i) and presence of succinate and rotenone (with or without glutamate) cyclosporin A either reversed or only stopped DeltaPsi decrease induced by 5 micro M myristic acid and 5 micro M Ca2+. In the last case nigericin, when added after cyclosporin A, reversed the DeltaPsi decrease, and the following addition of EGTA produced only a weak (if any) DeltaPsi increase. In P(i)-containing medium (in the presence of glutamate and malate) cyclosporin A reversed the DeltaPsi decrease. These data show that the cyclosporin A-sensitive decrease in DeltaPsi by low concentrations of fatty acids and Ca2+ cannot be explained by specific uncoupling effect of fatty acid. We propose that: 1) low concentrations of Ca2+ and fatty acid induce the pore opening in a substate with a selective cation permeability, and the cyclosporin A-sensitive DeltaPsi decrease results from a conversion of DeltaPsi to pH gradient due to the electrogenic cation transport in mitochondria; 2) the ADP/ATP-antiporter is involved in this process; 3) higher efficiency of fatty acid compared to SF6847 in the Ca2+-dependent pore opening seems to be due to its interaction with the nucleotide-binding site of the ADP/ATP-antiporter and higher affinity of fatty acids to cations.

Effects of cold exposure in vivo and uncouplers and recouplers in vitro on potato tuber mitochondria.

Effects of cold exposure in vivo and treatment with laurate, carboxyatractylate, atractylate, nucleotides, and BSA in vitro on potato tuber mitochondria have been studied. Cold exposure of tubers for 48-96 h resulted in some uncoupling that could be reversed completely by BSA and partially by ADP, ATP, UDP, carboxyatractylate, and atractylate. UDP was less effective than ADP and ATP, and atractylate was less effective than carboxyatractylate. The recoupling effects of nucleotides were absent when the nucleotides were added after carboxyatractylate. GDP, UDP, and CDP did not recouple mitochondria from either the control or the cold-exposed tubers. This indicates that the cold-induced fatty acid-mediated uncoupling in potato tuber mitochondria is partially due to the operation of the ATP/ADP antiporter. As to the plant uncoupling protein, its contribution to the uncoupling in tuber is negligible or, under the conditions used, somehow desensitized to nucleotides.

Ascorbate and low concentrations of FeSO4 induce Ca2+-dependent pore in rat liver mitochondria.

Oxidative stress is one of the most frequent causes of tissue and cell injury in various pathologies. The molecular mechanism of mitochondrial damage under conditions of oxidative stress induced in vitro with low concentrations of FeSO4 and ascorbate (vitamin C) was studied. FeSO4 (1-4 microM) added to rat liver mitochondria that were incubated in the presence of 2.3 mM ascorbate induced (with a certain delay) a decrease in membrane potential and high-amplitude swelling. It also significantly decreased the ability of mitochondria to accumulate exogenous Ca2+. All the effects of FeSO4 + ascorbate were essentially prevented by cyclosporin A, a specific inhibitor of the mitochondrial Ca2+-dependent pore (also known as the mitochondrial permeability transition). EGTA restored the membrane potential of mitochondria de-energized with FeSO4 + ascorbate. We hypothesize that oxidative stress induced in vitro with FeSO4 and millimolar concentrations of ascorbate damages mitochondria by inducing the cyclosporin A-sensitive Ca2+-dependent pore in the inner mitochondrial membrane.

Effect of the cationic detergent CTAB on the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria.

The influence of the positively charged amphiphilic compound cetyltrimethyl ammonium bromide (CTAB) on palmitate- and laurate-induced uncoupling and on carboxyatractylate and glutamate recoupling effects in liver mitochondria have been studied. CTAB (40 microM) in the presence of 3 mM MgCl2 had little (if any) effect on the palmitic acid-stimulated respiration of mitochondria; the glutamate recoupling effect increased, and the carboxyatractylate recoupling effect decreased to the same degree with the combined effect (about 80%) remaining unchanged. Thus, CTAB decreases the ADP/ATP antiporter involvement and increases to the same extent the aspartate/glutamate antiporter involvement in the fatty acid-induced uncoupling. The carboxyatractylate and glutamate recoupling effects were less pH dependent in the presence of CTAB than in its absence. These data could be interpreted with the assumption that fatty acid anions are more accessible to the ADP/ATP antiporter and their neutral forms are more accessible to the aspartate/glutamate antiporter, and that CTAB changes the relative anion carrier involvement in the fatty acid-induced uncoupling as it forms neutral complexes with fatty acid anions.

Comparative study on uncoupling effects of laurate and lauryl sulfate on rat liver and skeletal muscle mitochondria.

Uncoupling effects of laurate and lauryl sulfate have been studied in the isolated rat liver and skeletal muscle mitochondria. In the oligomycin-treated liver mitochondria, 0.02 mM laurate or 0.16 mM lauryl sulfate caused a two-fold stimulation of respiration, accompanied by a membrane potential decrease. Carboxyatractylate (CAtr) and glutamate (or aspartate) strongly decrease the effect of laurate and lauryl sulfate on respiratory rate and membrane potential (the recoupling effect). With both uncouplers, this effect is maximal for CAtr and glutamate (aspartate) at pH 7.8 and 7.0, respectively. Tetraphenyl phosphonium cations, which decrease negative membrane charges, cause an alkaline shift of these pH dependences. Small amounts of lauryl sulfate, which increase the membrane negative charge, induce the opposite shift when laurate is used as an uncoupler. ADP, but not GDP, partially recouple with both laurate and lauryl sulfate. We conclude that lauryl sulfate-induced uncoupling in rat liver, like the uncoupling induced by laurate, is mediated by the ATP/ ADP and glutamate/aspartate antiporters. In skeletal muscle mitochondria uncoupled by laurate, 200 microM GDP causes partial recoupling which can be enhanced by a subsequent additions of CAtr, glutamate and serum albumin. CAtr added before GDP promotes a larger recoupling than when added after GDP and prevents the subsequent effect of GDP. ADP is effective as recoupler at lower concentrations that GDP, whereas CDP is without influence. Lauryl sulfate uncoupling of skeletal muscle mitochondria is GDP-resistant but is sensitive to ADP, CAtr, glutamate and serum albumin. Our data suggest that in skeletal muscle mitochondria a GDP-sensitive mechanism is involved in uncoupling induced by laurate. This mechanism is absent in liver mitochondria. Possible mechanisms of laurate and lauryl sulfate-induced uncoupling are discussed.

Generation of transmembrane electrical potential during NADH oxidation via the external pathway and the fatty acid uncoupling effect after transient opening of the Ca2+-dependent cyclosporin A-sensitive pore in liver mitochondria.

The effects of transient pore opening on generation of the transmembrane gradient of electrical potential across the inner mitochondrial membrane (DeltaPsi) induced by NADH oxidation through the external pathway as well as on the uncoupling effect of fatty acids were studied. The pore opening was monitored by changes in the DeltaPsi value. The cycle of pore opening/closing was found to have only an insignificant effect on the sensitivity of DeltaPsi to fatty acid uncoupling. Once this cycle is over, NADH oxidation in the presence of exogenous cytochrome c results in generation of DeltaPsi. In the absence of cytochrome c, the generation of DeltaPsi induced by oxidation of exogenous NADH is observed if the incubation medium pH has been decreased from 7.4 to 7.0. The generation of DeltaPsi was inhibited by cyclosporin A. In isotonic salt medium containing 125 mM KCl, the maximum level of DeltaPsi generated by exogenous NADH after the cycle of pore opening/closing was significantly lower than the maximum level of DeltaPsi generated in hypotonic incubation medium. The data obtained in this work suggest that the cycle of pore opening/closing has little if any effect on the energy coupling in liver mitochondria, whereas the external pathway of NADH oxidation activated by this cycle may support the energy-dependent functions of liver mitochondria.

Role of the ADP/ATP-antiporter in fatty acid-induced uncoupling of Ca2+-loaded rat liver mitochondria.

We show that Ca2+ loading of mitochondria substantially augments the myristate-induced decrease in the transmembrane electric potential difference (deltapsi). Such a Ca2+ action is without effect on the respiration rate and is not accompanied by the high-amplitude swelling when low concentrations of Ca2+ and myristate are used. The myristate-induced deltapsi decrease is prevented and reversed by cyclosporin A (CsA); the decrease is prevented and transiently reversed by nigericin. To explain these effects, we suggest that myristate induces opening of the mitochondrial permeability transition pore at a low-conductance state. Addition of carboxyatractylate (CAtr) after myristate induces the CsA-sensitive uncoupling, but when added after myristate and CsA, CAtr produces a decrease in deltapsi, if the interval between myristate and CsA addition is sufficiently long. The CAtr effect is completely reversed by EGTA and transiently reversed by nigericin. This suggests that the ADP/ATP-antiporter participates in the CsA-sensitive uncoupling when present as a pore complex constituent. ADP/ATP-antiporter that does not take part in the pore complex formation is involved in the CsA-insensitive uncoupling.

Membrane potential generation coupled to oxidation of external NADH in liver mitochondria.

Oxidation of added NADH by rat liver mitochondria has been studied. It is found that exogenous NADH, when oxidized by rat liver mitochondria in sucrose hypotonic medium supplemented with Mg2+ and EGTA, generates a membrane potential (delta psi) even in the absence of added cytochrome c. ADP and phosphate decrease delta psi, the effect being reversed by oligomycin. Rotenone and myxothiazol do not inhibit delta psi generated by oxidation of exogenous NADH. Added cytochrome c increases the rate of the exogenous NADH oxidation and coupled delta psi formation. In sucrose isotonic medium, or in hypotonic medium without Mg2+, exogenous NADH fails to stimulate respiration and to form a membrane potential. In the presence of Mg2+, exogenous NADH appears to be effective in delta psi generation in isotonic sucrose medium if mitochondria were treated with digitonin. In isotonic KCl without Mg2+, oxidation of exogenous NADH is coupled to the delta psi formation and MgCl2 addition before mitochondria prevents this effect. In hypotonic (but not in isotonic) sucrose medium, Mg2+ makes a portion of the cytochrome c pool reducible by exogenous NADH or ascorbate. It is assumed that (i) hypotonic treatment or digitonin causes disruption of the outer mitochondrial membrane, and, as a consequence, desorption of the membrane-bound cytochrome c in a Mg2+-dependent fashion; (ii) incubation in isotonic KCI without Mg2+ results in swelling of mitochondrial matrix, disruption of the outer membrane and cytochrome c desorption whereas Mg2+ lowers the K+ permeability of the inner membrane and, hence, prevents swelling; (iii) desorbed cytochrome c is reduced by added NADH via NADH-cytochrome b5 reductase and cytochrome b5 or by ascorbate and is oxidized by cytochrome oxidase. The role of desorbed cytochrome c in oxidation of superoxide and cytoplasmic NADH as well as possible relations of these events to apoptosis are discussed.

Effect of palmitic and lauric acids on the phospholipid bilayer membrane conductivity.

Palmitic acid increased the conductivity of BLM from mitochondrial phospholipids when they were dissolved in a mixture of decane and chlorodecane, and was ineffective when phospholipids were dissolved in decane. Lauric acid produced an increase in the membrane conductivity independently of the phospholipid type in the membrane-forming solutions (mitochondrial phospholipids, asolectin, lecithin with cholesterol) and their solvents (decane or decane with chlorodecane). The results show that discrepancies between published data concerning fatty acid effects on the BLM conductivity may be explained by differences in phospholipids, their solvents and fatty acid used.

Involvement of aspartate/glutamate antiporter in uncoupling effect of fatty acids in heart mitochondria.

Earlier it was shown that fatty acid-induced uncoupling in liver mitochondria is suppressed by the substrates of the aspartate/glutamate antiporter (V. N. Samartsev, A. V. Smirnov, I. P. Zeldi, O. V. Markova, E. N. Mokhova, and V. P. Skulachev (1997) Biochim. Biophys. Acta, 1319, 251-257). In this study it is shown that in heart mitochondria aspartate, glutamate, and diethyl pyrocarbonate do not affect oxygen consumption and membrane potential in the presence of laurate at pH 7.4. These compounds have a weak (versus carboxyatractylate) coupling effect at pH 7.0. This effect is manifested only in the presence of carboxyatractylate, magnesium, and phosphate in the incubation medium. It is suggested that these tissue-specific effects are due not only to the specific characteristics of aspartate/glutamate antiporter, but also to the differences in the content of endogenous metabolites in heart mitochondria.

The pH-dependent reciprocal changes in contributions of ADP/ATP antiporter and aspartate/glutamate antiporter to the fatty acid-induced uncoupling.

The pH effect on carboxyatractylate-, glutamate- and aspartate-induced recoupling of palmitate-uncoupled rat liver mitochondria has been studied. Stimulation of respiration by low palmitate concentrations (5-20 microM) in the presence of 3 mM MgCl2 is shown to be pH-independent within the 7.0-7.8 range. The recoupling effect of glutamate (or aspartate) decreases and that of carboxyatractylate increases with increase in pH. The recoupling effect of a combination of carboxyatractylate and glutamate (aspartate) appears to be constant at these pH values, being as high as about 80%. It is concluded that uncoupling by low palmitate in liver mitochondria is mediated mainly by ATP/ADP and aspartate/glutamate antiporter.