E. N. Mokhova

Penetrating cation/fatty acid anion pair as a mitochondria-targeted protonophore

A unique phenomenon of mitochondria-targeted protonophores is described. It consists in a transmembrane H+-conducting fatty acid cycling mediated by penetrating cations such as 10-(6’-plastoquinonyl)decyltriphenylphosphonium (SkQ1) or dodecyltriphenylphosphonium (C12TPP). The phenomenon has been modeled by molecular dynamics and directly proved by experiments on bilayer planar phospholipid membrane, liposomes, isolated mitochondria, and yeast cells. In bilayer planar phospholipid membrane, the concerted action of penetrating cations and fatty acids is found to result in conversion of a pH gradient (ΔpH) to a membrane potential (Δψ) of the Nernstian value (about 60 mV Δψ at ΔpH = 1). A hydrophobic cation with localized charge (cetyltrimethylammonium) failed to substitute for hydrophobic cations with delocalized charge. In isolated mitochondria, SkQ1 and C12TPP, but not cetyltrimethylammonium, potentiated fatty acid-induced (i) uncoupling of respiration and phosphorylation, and (ii) inhibition of H2O2 formation. In intact yeast cells, C12TPP stimulated respiration regardless of the extracellular pH value, whereas a nontargeted protonophorous uncoupler (trifluoromethoxycarbonylcyanide phenylhydrazone) stimulated respiration at pH 5 but not at pH 3. Hydrophobic penetrating cations might be promising to treat obesity, senescence, and some kinds of cancer that require mitochondrial hyperpolarization.

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.

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 μM) 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.

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 (Skulachev, V. P. (1991) FEBS Lett., 294, 158– 162). 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 Ca2+-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 µM GDP on decrease in Δψ under the influence of oleic acid added after H2O2 (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 (Goglia, F., and Skulachev, V. P. (2003) FASEB, 17, 1585–1591)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 Δψ due to inhibition of succinate oxidation.

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 (ΔΨ) even in the absence of added cytochrome c. ADP and phosphate decrease ΔΨ, the effect being reversed by oligomycin. Rotenone and myxothiazol do not inhibit ΔΨ generated by oxidation of exogenous NADH. Added cytochrome c increases the rate of the exogenous NADH oxidation and coupled ΔΨ 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 ΔΨ generation in isotonic sucrose medium if mitochondria were treated with digitonin. In isotonic KCl without Mg2+, oxidation of exogenous NADH is coupled to the ΔΨ 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 KCl 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 b 5 reductase and cytochrome b 5 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.

Uncoupling effect of fatty acids on heart muscle mitochondria and submitochondrial particles

The effect of ATP/ADP‐antiporter inhibitors on palmitate‐induced uncoupling was studied in heart muscle mitochondria and inside‐out submitochondrial particles. In both systems palmitate is found to decrease the respiration‐generated membrane potential. In mitochondria, this effect is specifically abolished by carboxyatractylate (CAtr) a non‐penetrating inhibitor of antiporter. In submitochondrial particles, CAtr does not abolish the palmitate‐induced potential decrease. At the same time, bongkrekic acid, a penetrating inhibitor of the antiporter, suppresses the palmitate effect on the potential both in mitochondria and particles. Palmitoyl‐CoA which is known to inhibit the antiporter in mitochondria as well as in particles decreases the palmitate uncoupling efficiency in both these systems. These data are in agreement with the hypothesis that the ATP/ADP‐antiporter is involved in the action of free fatty acids as natural uncouplers of oxidative phosphorylation.

Thermoregulatory, carboxyatractylate-sensitive uncoupling in heart and skeletal muscle mitochondria of the ground squirrel correlates with the level of free fatty acids

Thermoregulatory uncoupling of oxidative phosphorylation has been studied in heart and skeletal muscle mitochondria of ground squirrels. The respiratory rate of mitochondria in the presence of oligomycin was found to be much higher in winter (in hibernating, arousing, or aroused animals) than in summer. This additional respiration is strongly (arousing animals) or completely (hibernating and aroused animals) inhibited by carboxy‐atractylate (CAtr) and bovine serum albumin (BSA). The CAtr‐ and BSA‐induced decreases in the rate of respiration are accompanied by membrane potential increases. The rate of the CAtr‐ and BSA‐sensitive respiration is proportional to the content of free fatty acids which, in the heart, decreases in the order: arousing > aroused = hibernating > summer animals. Maximal respiratory rates observed in the presence of dinitrophenol (arousing > aroused > summer > hibernating animals) do not parallel the fatty acid level. It is assumed that some heat production in the winter animals is due to fatty acid‐induced, ATPIADP‐antiporter‐mediated uncoupling in heart and skeletal muscle mitochondria. The peak of heat production during arousal after hibernation also includes some other stimulatory effect on mitochondrial respiration.

Effect of 6-ketocholestanol on FCCP- and DNP-induced uncoupling in plant mitochondria

Effect of 6‐ketocholestanol on FCCP‐induced and DNP‐induced uncoupling in beef liver and pea stem mitochondria was studied, under experimental conditions at which this steroid abolished the effect of low concentrations of FCCP and other most potent uncouplers in rat mitochondria [Starkov et al. (1994) FEBS Lett., 355, 305–308]. It is shown that, in both types of mitochondria, 6‐ketocholestanol prevents or reverses the uncoupling induced by low concentrations of FCCP, but not that caused by high concentrations of FCCP or by any concentration of DNP. Progesterone and male sex hormones, showing recoupling capability in animal mitochondria, appear to be ineffective in the plant system. Cholesterol does not recouple in both animal and plant mitochondria. Plant steroids, such as β‐sitosterol and stigmasterol, are also without effect.

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.

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.