Effect of ethanol on the palmitate-induced uncoupling of oxidative phosphorylation in liver mitochondria

The effect of ethanol on the uncoupling activity of palmitate and recoupling activities of carboxyatractylate and glutamate was studied in liver mitochondria at various Mg²⁺ concentrations and medium pH values (7.0, 7.4, and 7.8). Ethanol taken at concentration of 0.25 M had no effect on the uncoupling activity of palmitic acid in the presence of 2 mM MgCl₂ and decreased the recoupling effects of carboxyatractylate and glutamate added to mitochondria either just before or after the fatty acid. However, ethanol did not modify the overall recoupling effect of carboxyatractylate and glutamate taken in combination. The effect of ethanol decreased as medium pH was decreased to 7.0. Elevated concentration of Mg²⁺ (up to 8 mM) inhibits the uncoupling effect of palmitate. Ethanol eliminates substantially the recoupling effect of Mg²⁺ under these conditions, but does not influence the recoupling effects of carboxyatractylate and glutamate. It is inferred that ADP/ATP and aspartate/glutamate antiporters are involved in uncoupling function as single uncoupling complex with the common fatty acid pool. Fatty acid molecules gain the ability to migrate under the action of ethanol: from ADP/ATP antiporter to aspartate/glutamate antiporter on addition of carboxyatractylate and in opposite direction on addition of glutamate. Possible mechanisms of fatty acid translocation from one transporter to another are discussed.     

Acetoacetate as regulator of palmitic acid-induced uncoupling involving liver mitochondrial ADP/ATP antiporter and aspartate/glutamate antiporter

The effect of acetoacetate on palmitate-induced uncoupling with the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter has been studied in liver mitochondria. The incubation of mitochondria with acetoacetate during succinate oxidation in the presence of rotenone, oligomycin, and EGTA suppresses the accumulation of conjugated dienes. This is considered as a display of antioxidant effect of acetoacetate. Under these conditions, acetoacetate does not influence the respiration of mitochondria in the absence or presence of palmitate but eliminates the ability of carboxyatractylate or aspartate separately to suppress the uncoupling effect of this fatty acid. The action of acetoacetate is eliminated by β-hydroxybutyrate or thiourea, but not by the antioxidant Trolox. In the absence of acetoacetate, the palmitate-induced uncoupling is limited by a stage sensitive to carboxyatractylate (ADP/ATP antiporter) or aspartate (aspartate/glutamate antiporter); in its presence, it is limited by a stage insensitive to the effect of these agents. In the presence of Trolox, ADP suppresses the uncoupling action of palmitate to the same degree as carboxyatractylate. Under these conditions, acetoacetate eliminates the recoupling effects of ADP and aspartate, including their joint action. This effect of acetoacetate is eliminated by β-hydroxybutyrate or thiourea. It is supposed that the stimulating effect of acetoacetate is caused both by increase in the rate of transfer of fatty acid anion from the inner monolayer of the membrane to the outer one, which involves the ADP/ATP antiporter and aspartate/glutamate antiporter, and by elimination of the ability of ADP to inhibit this transport. Under conditions of excessive production of reactive oxygen species in mitochondria at a high membrane potential and in the presence of small amounts of fatty acids, such effect of acetoacetate can be considered as one of the mechanisms of antioxidant protection.     

Role of the ADP/ATP and aspartate/glutamate antiporters in the uncoupling effect of fatty acids, lauryl sulfate, and 2, 4-dinitrophenol in liver mitochondria.

Study of the uncoupling effect of various saturated fatty acids (from caprylic to palmitic) revealed that the glutamate recoupling effect was more pronounced in the case of short chain fatty acids, whereas recoupling of mitochondria by carboxyatractylate was more effective in the case of long chain fatty acids. The overall recoupling effect, however, did not depend on the fatty acid chain length. Besides carboxyatractylate, glutamate and aspartate also exhibited a recoupling effect under uncoupling by lauryl sulfate. The uncoupling effect of lauryl sulfate was markedly weaker in the presence of DNP or laurate (but not FCCP) which were added in concentrations causing twofold increase in mitochondrial respiration. In the presence of lauryl sulfate the uncoupling action of laurate and DNP was insensitive to carboxyatractylate and glutamate. With laurate and DNP as uncouplers increasing the pH from 7.0 to 7.8 potentiated the recoupling effect of carboxyatractylate and attenuated the recoupling effect of glutamate. In the case of uncoupling by lauryl sulfate similar changes in the recoupling effect of carboxyatractylate and glutamate were observed only in the presence of 10 microM tetraphenylphosphonium. Thus, when uncoupling is induced by fatty acids, DNP, and lauryl sulfate, the ADP/ATP and aspartate/glutamate antiporters function as two parallel and independent pathways for mitochondrial membrane potential dissipation. We suggest that the role of the ADP/ATP antiporter in uncoupling includes proton capture from the intermembrane space with subsequent protonation of uncoupler anions, their transport as neutral molecules on the internal side, and deprotonation followed by proton release into the matrix and transfer of the uncoupler anion in the reverse direction. During uncoupling the aspartate/glutamate antiporter cyclically carries the uncoupler anion with simultaneous proton transfer from the intermembrane space into the matrix.     

Free fatty acids as inducers and regulators of uncoupling of oxidative phosphorylation in liver mitochondria with participation of ADP/ATP- and aspartate/glutamate-antiporter

In liver mitochondria fatty acids act as protonophoric uncouplers mainly with participation of internal membrane protein carriers — ADP/ATP and aspartate/glutamate antiporters. In this study the values of recoupling effects of carboxyatractylate and glutamate (or aspartate) were used to assess the degree of participation of ADP/ATP and aspartate/glutamate antiporters in uncoupling activity of fatty acids. These values were determined from the ability of these recoupling agents to suppress the respiration stimulated by fatty acids and to raise the membrane potential reduced by fatty acids. Increase in palmitic and lauric acid concentration was shown to increase the degree of participation of ADP/ATP antiporter and to decrease the degree of participation of aspartate/glutamate antiporter in uncoupling to the same extent. These data suggest that fatty acids are not only inducers of uncoupling of oxidative phosphorylation, but that they also act the regulators of this process. The linear dependence of carboxyatractylate and glutamate recoupling effects ratio on palmitic and lauric acids concentration was established. Comparison of the effects of fatty acids (palmitic, myristic, lauric, capric, and caprylic having 16, 14, 12, 10, and 8 carbon atoms, respectively) has shown that, as the hydrophobicity of fatty acids decreases, the effectiveness decreases to a greater degree than the respective values of their specific uncoupling activity. The action of fatty acids as regulators of uncoupling is supposed to consist of activation of transport of their anions from the internal to the external monolayer of the internal membrane with participation of ADP/ATP antiporter and, at the same time, in inhibition of this process with the participation of aspartate/glutamate antiporter.     

Oxidative stress as regulatory factor for fatty-acid-induced uncoupling involving liver mitochondrial ADP/ATP and aspartate/glutamate antiporters of old rats

Palmitate-induced uncoupling, which involves ADP/ATP and aspartate/glutamate antiporters, has been studied in liver mitochondria of old rats (22-26 months) under conditions of lipid peroxidation and inhibition of oxidative stress by antioxidants--thiourea, Trolox, and ionol. It has been shown that in liver mitochondria of old rats in the absence of antioxidants and under conditions of overproduction of conjugated dienes, the protonophoric uncoupling activity of palmitate is not suppressed by either carboxyatractylate or aspartate used separately. However, the combination of carboxyatractylate and aspartate decreased uncoupling activity of palmitate by 81%. In this case, palmitate-induced uncoupling is limited by a stage insensitive to both carboxyatractylate and aspartate. In the presence of antioxidants, the palmitate-induced protonophoric uncoupling activity is suppressed by either carboxyatractylate or aspartate used separately. Under these conditions, palmitate-induced uncoupling is limited by a stage sensitive to carboxyatractylate (ADP/ATP antiporter) or aspartate (aspartate/glutamate antiporter). In the absence of antioxidants, the uncoupling activity of palmitate is not suppressed by ADP either in the absence or in the presence of aspartate. However, in the presence of thiourea, Trolox, or ionol ADP decreased the uncoupling activity of palmitate by 38%. It is concluded that in liver mitochondria of old rats the development of oxidative stress in the presence of physiological substrates of ADP/ATP and aspartate/glutamate antiporters (ADP and aspartate) results in an increase of the protonophoric uncoupling activity of palmitate.     

Involvement of phosphate carrier as a part of complex with ADP/ATP and aspartate/glutamate antiporters in palmitic acid-induced uncoupling in liver mitochondria

In liver mitochondria, the phosphate carrier is involved in protonophoric uncoupling effect of fatty acids together with ADP/ATP and aspartate/glutamate antiporters (Samartsev et al. 2003. Biochemistry (Moscow). 68, 618–629). Liver mitochondria depleted of endogenous oxidation substrates (exhausted mitochondria) have been used in the present work. In these mitochondria, like in the intact liver mitochondria, the specific inhibitor of ADP/ATP antiporter (carboxyatractylate) and the substrate of aspartate/glutamate antiporter (aspartate) suppress the uncoupling activity of palmitic acid. It is shown that in exhausted mitochondria the substrate of phosphate carrier (inorganic phosphate) and its nonspecific inhibitor mersalyl partially suppress palmitic acid-induced uncoupling due to decrease in the component of uncoupling activity sensitive to carboxyatractylate and aspartate. In the presence of inorganic phosphate or mersalyl, carboxyatractylate and aspartate added separately subsequent to palmitic acid do not suppress its uncoupling activity. They are effective only when added jointly. In the presence of thiourea or pyruvate, such effects of inorganic phosphate and mersalyl are not observed. It is supposed that in the presence of inorganic phosphate or mersalyl and under the condition of oxidation of critical SH-groups in mitochondria, the phosphate carrier, ADP/ATP antiporter, and aspartate/glutamate antiporter are involved in uncoupling function together with the general fatty acid pool as an uncoupling complex. The role of phosphate carrier in this complex may consist in facilitation of lateral transfer of the fatty acid molecules from one antiporter to another.     

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 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 due to inhibition of succinate oxidation.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 197–202.Original Russian Text Copyright © 2005 by Mokhova, Khailova.This revised version was published online in April 2005 with corrections to the post codes.     

Simulation of the uncoupling activity of fatty acids with the participation of ADP/ATP and aspartate/glutamate antiporters in liver mitochondria

It has been found that the protonophoric specific uncoupling activity of palmitic acid in rat liver mitochondria does not change as its concentration increases from 5 to 40 μM. Under these conditions, the component of the specific uncoupling activity that describes the participation in uncoupling of the ADP/ATP antiporter (sensitive to carboxyatractylate) increases, and the component of specific uncoupling activity that characterizes the participation in the uncoupling of the aspartate/glutamate antiporter (sensitive to glutamate) decreases by the same value. A kinetic model of the fatty acid-induced uncoupling activity with the participation of ADP/ATP and aspartate/glutamate antiporters has been developed. According to the model, these carriers can exist in two forms: active, i.e., participating in the uncoupling, and inactive. The interaction of a fatty acid with the regulator site of the ADP/ATP antiporter translates it from the inactive to the active form, while the interaction of a fatty acid with the regulator site of the aspartate/glutamate antiporter, on the contrary, translates it from the active form to inactive. The velocity of transport of a fatty acid anion by the antiporter from the internal monolayer of the inner membrane to the external monolayer is proportional to the product of the concentration of the fatty acid and the active form of this carrier. A good conformity of the model to experimentally obtained data is shown provided that (a) ADP/ATP and aspartate/glutamate antiporters, being completely in active state, transfer fatty acid anions with the same velocity; (b) the equilibrium dissociation constants of a complex of the carrier with the fatty acid in these antiporters are equal.     

Simulation of the uncoupling activity of fatty acids with the participation of ADP/ATP and aspartate/glutamate antiporters in liver mitochondria

It has been found that the protonophoric specific uncoupling activity of palmitic acid in rat liver mitochondria does not change as its concentration increases from 5 to 40 microM. Under these conditions, the component of the specific uncoupling activity, which describes the participation in uncoupling of the ADP/ATP antiporter (sensitive to carboxyatractylate), increases, and the component of specific uncoupling activity, which characterizes the participation in the uncoupling of the aspartate/glutamate antiporter (sensitive to glutamate), decreases by the same value. A kinetic model of the fatty acid-induced uncoupling activity with the participation of ADP/ATP and aspartate/glutamate antiporters has been developed. According to the model, these carriers can exist in two forms: an active, i.e., participating in the uncoupling, and an inactive. The interaction of a fatty acid with the regulator site of the ADP/ATP antiporter translates it from the inactive to the active form, while the interaction of a fatty acid with the regulator site of the aspartate/glutamate antiporter, on the contrary, translates it from the active form to inactive. The velocity of transport of a fatty acid anion by the antiporter from the internal monolayer of the internal membrane to the external monolayer is proportional to the product of the concentration of the fatty acid and the active form of this carrier. A good conformity of the model to experimentally obtained data is shown provided that (a) ADP/ATP and aspartate/glutamate antiporters, being completely in an active state, transfer fatty acid anions with the same velocity; (b) the equilibrium dissociation constants of a complex of the carrier with the fatty acid in these antiporters are equal.     

Cyclosporin a inhibits the protonophoric uncoupling activity of laurate in liver mitochondria

The effects of cyclosporin A, carboxyatractylate, and glutamate on the protonophoric uncoupling activity of laurate in liver mitochondria have been studied. It was found that 5 μM cyclosporin A partly inhibits laurate-stimulated mitochondrial respiration, which is suggestive of its recoupling effect, i.e., the ability to suppress the protonophoric activity of this fatty acid. Under these conditions, cyclosporin A has no effect on the ability of carboxyatractylate and glutamate to inhibit the uncoupling effect of laurate. In their turn, these compounds do not influence the recoupling activity of cyclosporin A. The recoupling effects of cyclosporin A, carboxyatractylate, and glutamate are additive: acting simultaneously, they fully suppress the uncoupling activity of laurate. It is concluded that the protonophoric uncoupling activity of fatty acids in liver mitochondria is mediated not only by ADP/ATP and aspartate/glutamate antiporters, but also by a system that is sensitive to cyclosporin A, but is not related with cyclophilin D.     

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.     

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.     

Interaction of free fatty acids with mitochondria during uncoupling of oxidative phosphorylation

The activity of free saturated fatty acids (caprylic, capric, lauric, myristic, palmitic and stearic) as inducers and regulators of uncoupling of oxidative phosphorylation with participation of ADP/ATP antiporter, aspartate/glutamate antiporter and cyclosporin A-sensitive structure was investigated in experiments on rat liver mitochondria. It is established that at equal uncoupling activity of fatty acids the regulatory effect is minimal for caprylic acid and raised with increasing the hydrophobicity of fatty acids reaching the maximum value for stearic acid. There exists the linear dependence of the regulatory effect value of fatty acids on fatty acids content in the hydrophobic region of the inner membrane. The model that describes the interaction of fatty acids with the hydrophobic region of the mitochondrial inner membrane preserving functional activity of organelles is developed. It is established that if molecules of various fatty acids being in the hydrophobic region of the membrane are equally effective as uncoupling regulators, their specific uncoupling activity is different. Caprylic acid, a short-chain fatty acid, possesses the highest uncoupling activity. As the acyl chain length increases, the specific uncoupling activity of fatty acids reduces exponentially. Under these conditions components of the uncoupling activity sensitive to glutamate and carboxyatractylate and glutamate and insensitive to these reagents (but sensitive to cyclosporin A) change approximately equally.     

Participation of ADP/ATP- and Aspartate/Glutamate Antiporters in the Uncoupling Action of Fatty Acids in Liver Mitochondria of the Frog Rana temporaria

Data are presented on molecular mechanisms of uncoupling of oxidative phosphorylation by fatty acids (laurate) in liver mitochondria of one of the poikilothermal animals, the frog Rana temporaria. It has been shown that the uncoupling action of laurate in frog liver mitochondria, like in those of mammals, occurs with participation of protein carriers of anions of the inner mitochondrial membrane, ADP/ATP- and aspartate/glutamate antiporters. At the same time, in frog liver mitochondria the uncoupling activity of laurate is lower than in liver mitochondria of mammals (white mice). Seasonal differences in the laurate uncoupling activity in frog liver mitochondria are revealed: it is much lower in April, than in January, the season of metabolic depression. This difference is due to that in January the degree of participation of the aspartate/glutamate antiporter in the uncoupling is considerably decreased.     

Participation of ATP/ADP antiporter in oleate- and oleate hydroperoxide-induced uncoupling suppressed by GDP and carboxyatractylate

In experiments on isolated kidney and liver mitochondria, it is shown that oleate hydroperoxide induces a much smaller increase in the controlled respiration rate and DeltaPsi decrease than the same concentrations of oleate. Palmitate appears to be less efficient than oleate but more efficient than oleate hydroperoxide. In all cases, GDP and CAtr cause some recoupling, CAtr being more effective. Addition of 0.2 mM GDP before CAtr does not prevent further DeltaPsi increase by subsequent CAtr addition. On the other hand, GDP added after CAtr is without any effect. GDP partially prevents the DeltaPsi lowering by ADP at the State 4--State 3 transition if small amounts of CAtr are present. The data are consistent with the suggestion of F. Goglia and V.P. Skulachev (FASEB J. 17, 1585-1591, 2003) that fatty acid anions are translocated by mitochondrial anion carriers much better than their hydroperoxides. As to GDP recoupling, it cannot be regarded as a specific probe for uncoupling by UCPs since it can be mediated by the ATP/ADP antiporter.     

Inhibitors of the ATP/ADP antiporter suppress stimulation of mitochondrial respiration and H+ permeability by palmitate and anionic detergents

The action of ATP/ADP-antiporter inhibitors upon the uncoupling effect of palmitate, detergents and 'classical' uncouplers has been studied. The uncoupling effect was estimated by stimulation of succinate oxidation and of H+ permeability of rat liver mitochondria in the presence of oligomycin. It is shown that carboxyatractylate (CAtr) and pyridoxal 5-phosphate (PLP) suppress the uncoupling induced by palmitate and the anionic detergents SDS and cholate, but do not affect that induced by the cationic detergents CTAB, by the non-ionic detergent Triton X-100, as well as by the 'classical' uncouplers FCCP and DNP. The results are discussed in terms of a concept assuming that the ATP/ADP-antiporter facilitates the electrophoretic export of hydrophobic anions from mitochondria.     

Anion Carriers in Fatty Acid-Mediated Physiological Uncoupling

Physiological aspects of uncoupling of oxidation and phosphorylation are reviewed in thecontext of involvement of mitochondrial anion carriers. It is assumed that the carriers facilitateelectrophoretic translation of fatty acid anion, RCOO^-, from the inner to the outer leaflet ofthe mitochondrial membrane, whereas back movement of the protonated fatty acid, RCOOH,from the outer to the inner leaflet represents flip-flop of RCOOH via the phospholipid bilayerof the membrane. The RCOO^- transport seems to be catalyzed by the ATP/ADP and aspartate/glutamate antiporters, dicarboxylate carrier, and uncoupling proteins (UCP1, UCP2, UCP3^L,UCP3^s, and plant UCP). The fatty acid uncoupling is shown to be involved in thethermoregulatory heat production in animals and plants exposed to cold, as well as in performance ofrespiratory functions other than ATP synthesis, i.e., formation of useful substances,decomposition of unwanted substances, and antioxidant defense. Moreover, partial uncoupling might takepart in optimization of the rate of ATP synthesis in aerobic cells.     

Calcium-independent uncoupling activity of palmitic acid in liver mitochondria is regulated by the ion fluxes causing the interconversion of Δψ and ΔpH across the inner membrane

The effect of ion fluxes across the inner membrane on calcium-independent uncoupling activity of palmitic acid was investigated in experiments on rat liver mitochondria energized by the oxidation of succinate. The following compounds were used as the inductors of ion fluxes: the K⁺/H⁺ antiporter nigericin causing transformation of ΔpH into electrical potential difference (Δψ) across the inner membrane; tetraphenylphosphonium (TPP⁺) that freely crosses phospholipid membranes; protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) that induces a flow of H⁺ from the intermembrane space into the matrix and reduces Δψ and ΔpH. It was found that nigericin at a concentration of 20 nM, which causes an increase in maximal Δψ, partially inhibits the ability of palmitic acid to reduce Δψ and stimulates mitochondrial respiration. A specific inhibitor of the ATP/ADP antiporter (carboxyatractylate) and a substrate of the aspartate/glutamate antiporter (glutamate) increase Δψ and partially inhibit mitochondrial respiration in the presence of palmitic acid. Under these conditions, 10 μM cyclosporin A also inhibits respiration but has no effect on Δψ. The specific uncoupling activity of palmitic acid (V _U) and its specific components that characterize participation of the ATP/ADP antiporter (V _Catr), aspartate/glutamate antiporter (V _Glu), and cyclosporin-A-sensitive system (V _CsA) in the palmitic acid-induced uncoupling were estimated. It was shown that nigericin substantially reduces V _U, V _Catr and V _Glu but increases V _CsA. TPP⁺ at a concentration of 20 μM increases V _U and V _Glu, does not affect V _Catr and reduces V _CsA. FCCP at concentrations of 20 and 40 nM reduces Δψ by not more than 17% but does not affect V _U, V _Catr, V _Glu and V _CsA. The results suggest that the calcium-independent uncoupling effect of palmitic acid in liver mitochondria is caused by the return of protons to the matrix with participation of ADP/ATP and aspartate/glutamate antiporters and owing to activation of cyclosporin A-sensitive electron transport along the respiratory chain without affecting Δψ. The induced ion fluxes across the inner mitochondrial membrane can be considered as a factor of the calcium-independent regulation of uncoupling activity of palmitic acid in liver mitochondria with participation of the ADP/ATP and aspartate/glutamate antiporters and of the cyclosporin A-sensitive electron transport system.     

Uncoupling activity of fatty acids in liver mitochondria in the presence of substrates of ADP/ATP- and aspartate/glutamate antiporters is increased under oxidative stress

It is shown that upon oxidation of succinate in the presence of rotenone and antioxidant Trolox (or pyruvate) in liver mitochondria of mature rats (9–12-month old) the respiration stimulated by palmitate is suppressed by ADP (the substrate of ADP/ATP-antiporter) and aspartate (the substrate of aspartate/glutamate antiporter). However, it was found that in the presence of the oxidative agent tert-butylhydroperoxide neither ADP nor aspartate is effective even at their joint action. In the presence of ADP and aspartate, uncoupling activity of palmitate is minimal, since the lipid peroxidation is inhibited by Trolox or pyruvate, and rises as the accumulation rate of conjugated dienes increases, reaching the maximal value at the oxidative stress caused by tert-butylhydroperoxide. In liver mitochondria of senile rats (22–26-month old) at high intensity of lipid peroxidation, ADP and aspartate do not affect the uncoupling activity of palmitate (Samartsev and Kozhina, 2008, Biochemistry (Mosc.), vol. 73, no. 7, pp. 783–790). Comparative studies have shown that in liver mitochondria of mature and senile rats at the similar accumulation rate of the conjugated dienes in the presence of ADP and aspartate, the uncoupling activity of palmitate reaches the same level relative to the maximal activity. We conclude that an enhancement of free radical reactions and lipid peroxidation in liver mitochondria can result in an increase of protonophore uncoupling activity of fatty acids with the involvement of ADP/ATP- and aspartate/glutamate antiporters due to the suppression of the ability of physiological substrates of these carriers of ADP and aspartate to inhibit the uncoupling process.     

Mitochondrial porin regulates the sensitivity of anion carriers to inhibitors

In mitoplasts, respiratory stimulation by ADP, palmitate, DNP and CCCP and sensitivity of respiration to carboxyatractylate are considerably less pronounced than in mitochondria. Addition of porin-containing preparations (purified outer membranes or solubilized mitochondrial porin) to mitoplasts results in partial restoration of the oxygen consumption and sensitivity to carboxyatractylate (CAT). The uncoupling effect of FCCP in mitoplasts is CAT-resistant and does not depend on added porin. It is suggested that mitochondrial porin may be a natural activator of ADP/ATP antiporter and succinate carrier in mitochondria.