Biomedical subjects

E N Mokhova

Publications and source records attributed to E N Mokhova.

At least 19 recordsLinked to original sources

PubMed2003

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.

Animals↗

PubMed2002

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.

Adaptation, Physiological↗

PubMed2001

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.

Animals↗

PubMed2001

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.

Amino Acid Transport Systems, Acidic↗

PubMed2000

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.

Adenosine Triphosphate↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Alkanes↗

PubMed1998

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.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

Involvement of aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria.

Effects of aspartate, glutamate and an inhibitor of the aspartate/glutamate antiporter, diethylpyrocarbonate (DEPC), on uncoupling of the energy transduction processes in rat liver mitochondria have been investigated. It is found that both the antiporter substrates and the antiporter inhibitor operate as recouplers when uncoupling is caused by free fatty acids (FFA). Recoupling consists in (1) partial inhibition of the FFA-stimulated respiration and (2) some increase in the membrane potential. Half-maximal effects are observed at concentrations of glutamate and aspartate close the K(m) values of the antiporter. Recouplings by glutamate (aspartate) and DEPC are not additive. On the other hand, recoupling by any of these compounds and carboxyatractylate or ADP appears to be additive. Uncoupling by dinitrophenol is less sensitive to the recouplers whereas that by FCCP is not sensitive at all. It is concluded that uncoupling by FFA in rat liver mitochondria is mediated not only by the ATP/ADP antiporter but also by the aspartate/glutamate antiporter.

Amino Acid Transport Systems, Acidic↗

PubMed1997

ATP/ADP antiporter- and aspartate/glutamate antiporter-mediated fatty acid-induced uncoupling of liver mitochondria in incubation media differing in ion composition.

Substitution of potassium cloride for sucrose in the incubation medium of liver mitochondria or addition of magnesium cloride to the sucrose medium produces a small (if any) effect on the palmitate-induced uncoupling and its sensitivity to carboxyatractylate and aspartate, but it exerts a pronounced increase in the recoupling effect of glutamate on this uncoupling. 20-40 microM cetyltrimethyl ammonium bromide increases the recoupling effect of glutamate and aspartate but decreases that of carboxyatractylate. The data are in line with suggestion that both ATP/ADP antiporter and aspartate/glutamate antiporters are involved in the fatty acid-induced uncoupling.

Amino Acid Transport Systems, Acidic↗

PubMed1997

Effect of cysteinesulfinate on fatty acid-dependent uncoupling: modulation of recoupling by substrates of the aspartate/glutamate antiporter and diethyl pyrocarbonate.

The action of cysteinesulfinate, the substrate of the aspartate/glutamate antiporter on the palmitate-induced uncoupling in rat liver mitochondria and the recoupling effect of glutamate, aspartate, and diethyl pyrocarbonate was studied. In the presence of palmitate and an inhibitor of the ADP/ATP-antiporter carboxyatractylate, cysteinesulfinate exerted a relatively week recoupling effect. However, it significantly decreased the recoupling action of glutamate, aspartate, and diethyl pyrocarbonate. In the presence of cysteinesulfinate, these compounds caused recoupling at higher concentrations. The data show that the recoupling action of glutamate, aspartate, and diethyl pyrocarbonate is due to their interaction with the aspartate/glutamate antiporter. The data also confirm the suggestion that this anion carrier is involved in the uncoupling action of fatty acids.

Animals↗

PubMed1997

Cytochrome c potentiates fatty acid-induced cyclosporin A-sensitive permeability transition in liver mitochondria.

Addition of 0.5-5 microM cytochrome c and 30 microM lauric acid to rat liver mitochondria stimulated the respiration rate. Stimulation of respiration by cytochrome c was prevented by cyclosporin A and was not observed in the absence of lauric acid. High amplitude swelling of mitochondria in the presence of lauric acid was also potentiated by cytochrome c; this effect was prevented by cyclosporin A or by EGTA. Polylysine (0.35 microM) induced cyclosporin A-insensitive high amplitude swelling of mitochondria. Polylysine-induced swelling was stimulated by lauric acid and cytochrome c; the effect was prevented by cyclosporin A. Incubation of mitochondria with 30 microM lauric acid resulted in an increase in cytochrome c concentration in the incubation medium. Added cytochrome c is supposed to potentiate the laurate-induced cyclosporin A-sensitive permeability transition in cytochrome c-depleted mitochondria.

Animals↗

PubMed1996

Zearalenone-induced uncoupling in plant mitochondria is sensitive to 6-ketocholestanol.

Zearalenone (F-2) is a mycotoxin which acts as a protonophoric uncoupler in plant mitochondria [Macri, F. and Vianello, A., (1990) J. Plant Physiol. 136, 754-757]. In the present paper, the mechanism of F-2-induced uncoupling in pea mitochondria was studied. The uncoupling by F-2 was partially reversed by 6-ketocholestanol (kCh) under conditions in which kCh completely reversed the FCCP-induced uncoupling and almost did not affect the palmitate-induced uncoupling. Recoupling effects of carboxyatractylate, ADP and cyclosporin A were small and could not essentially decrease the kCh-insensitive part of F-2-induced uncoupling. It is suggested that a protein, mediating kCh-sensitive uncoupling, is involved in the F-2 effect in plant mitochondria.

Atractyloside↗

PubMed1996

Carboxyatractylate- and cyclosporin A- sensitive uncoupling in liver mitochondria of ground squirrels during hibernation and arousal.

Oxygen consumption and transmembrane electrical potential difference in liver mitochondria from hibernating, arousing and active ground squirrels have been compared. It is found that arousal from hibernation is accompanied by uncoupling which is mediated by two different mechanisms, one sensitive to cyclosporin A and the other suppressed by carboxyatractylate. Both uncoupling effects reach their maxima at 20 - 25 degrees C body temperatures. Involvement of an increase in the free fatty acid concentration in the arousal-induced uncoupling mechanisms is discussed.

Animals↗

PubMed1995

Effect of cyclosporin A on energy coupling in pea stem mitochondria.

Effect of cyclosporin A on energy coupling in pea stem mitochondria is studied. It is found that incubation of mitochondria with 100 nM FCCP and/or CAtr, oligomycin, CaCl2, palmitate and ADP results, after some lag phase, in a collapse of delta psi generated by succinate oxidation in the presence of rotenone. Cyclosporin A (0.2-0.8 nmol/mg mitochondrial protein) markedly increases the lag phase. The cyclosporin A effect requires dithioerythritol to be added to the isolated medium. Metabisulphite fails to substitute for dithioerythritol. The relationships between these effects and cyclosporin A-sensitive mitochondrial permeability transition in animal mitochondria are discussed.

Animals↗