Quantitative relationship between protonophoric and uncoupling activities of substituted phenols

The protonophoric activity through liposomal membranes was measured and compared with the uncoupling activity with the oxidative phosphorylation of rat-liver mitochondria for 19 substituted phenols. Quantitative analyses of the protonophoric activity of the phenols in terms of physicochemical molecular parameters showed that the activity was mostly decided by two factors: the partition coefficient between the liposome and aqueous buffer phases and the acid dissociation constant. Correlation was excellent between protonophoric and uncoupling activities when the difference in the effect of acidity of phenols between liposomal and mitochondrial membranes was taken into account. The results were further evidence for the shuttle-type of mechanism of weakly acidic uncouplers based on the Mitchell chemiosmotic hypothesis.     

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.     

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.     

The anti-cancer agent nemorosone is a new potent protonophoric mitochondrial uncoupler

Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1-25 μM) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50-500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca(2+) from Ca(2+)-loaded mitochondria, decreased Ca(2+) uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells.     

A correlation between respiration and synthesis of ATP in mitochondria at different degree of uncoupling of oxidative phosphorylation

It is known that mitochondrial respiration in state 3 is due to three simultaneous and independent processes: synthesis of ATP (1), endogenous passive proton leakage (2), and proton leakage by protonophoric uncoupler (3). The total rate of processes (2) and (3) is equal to the product of respiration rate in state 4 and coefficient KR, which is defined as the ratio of the deltamuH+ value in state 3 to that in state 4. It is shown that it is possible to calculate both the rates of processes (1), (2) and (3) separately and the protonophoric activity of uncoupler using the coefficient KR and other coefficients, which are determined as the ratio of deltamuH+ values in state 3 or in state 4 to its maximal value. Simple methods of determination of these coefficients were developed, which are based on the study of the dependence of respiration rate in states 3 and 4 on the concentration of protonophoric uncoupler. It was found that the uncoupling action of palmitate, a natural uncoupler of oxidative phosphorylation, unlike classic uncoupler-protonophores DNP and FCCP, depends not only on its protonophoric activity but also on the inhibition of the process (1).     

Quantitative analyses of the uncoupling activity of substituted phenols with mitochondria from flight muscles of house flies

Uncoupling activity with flight-muscle mitochondria from house flies was measured for a series of weakly acidic uncouplers (substituted phenols) and compared with the protonophoric potency across lecithin liposomal membranes. The activity was linearly related to the protonophoric potency when such factors as the stability of anionic species in the membrane phase and the difference in the pH conditions of the extramembranous aqueous phase were taken into account. Relationships of the flight-muscle activity with activities measured previously with rat-liver mitochondria and spinach chloroplasts were linear. Our findings were further evidence for the shuttle-type mechanism of the uncoupling action of weakly acidic uncouplers.     

Quantitative relationship between protonophoric and uncoupling activities of analogs of SF6847 (2,6-di-t-butyl-4-(2',2'-dicyanovinyl)phenol)

Uncoupling activity with rat liver mitochondria and protonophoric activity across the lecithin liposomal membranes were measured for a series of non-classical uncouplers related to the most potent uncoupler known until now, SF6847 (2,6-di-t-butyl-4-(2',2'-dicyanovinyl)phenol). The correlation between uncoupling and protonophoric activities for a number of uncouplers, both non-classical and classical (simply substituted phenols), was examined quantitatively. Correlation was excellent when such factors as the stability of anionic species in the membrane phase and the difference in the pH conditions of the extramembranous aqueous phase were taken into account. Carbonylcyanide m-chlorophenylhydrazone (CCCP) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), which are structurally different, were correlated in a way that resembled the correlation of phenolic compounds, so we think that the mode of action of weakly acidic uncouplers was the same regardless of the structural type. Our findings were evidence for the shuttle-type mechanism of uncoupling action.     

Proton re-uptake partitioning between uncoupling protein and ATP synthase during benzohydroxamic acid-resistant state 3 respiration in tomato fruit mitochondria

The yield of oxidative phosphorylation in isolated tomato fruit mitochondria depleted of free fatty acids remains constant when respiratory rates are decreased by a factor of 3 by the addition of n-butyl malonate. This constancy makes the determination of the contribution of the linoleic acid-induced energy-dissipating pathway by the ADP/O method possible. No decrease in membrane potential is observed in state 3 respiration with increasing concentration of n-butyl malonate, indicating that the rate of ATP synthesis is steeply dependent on membrane potential. Linoleic acid decreases the yield of oxidative phosphorylation in a concentration-dependent manner by a pure protonophoric process like that in the presence of FCCP. ADP/O measurements allow calculation of the part of respiration leading to ATP synthesis and the part of respiration sustained by the dissipative H(+) re-uptake induced by linoleic acid. Respiration sustained by this energy-dissipating process remains constant at a given LA concentration until more than 50% inhibition of state 3 respiration by n-butyl malonate is achieved. The energy dissipative contribution to oxygen consumption is proposed to be equal to the protonophoric activity of plant uncoupling protein divided by the intrinsic H(+)/O of the cytochrome pathway. It increases with linoleic acid concentration, taking place at the expense of ADP phosphorylation without an increase in the respiration.     

Quantitative relationship between protonophoric and uncoupling activities of analogs of SF6847 (2,6-di-t-butyl-4-(2′,2′-dicyanovinyl)phenol)

Uncoupling activity with rat liver mitochondria and protonophoric activity across the lecithin liposomal membranes were measured for a series of non-classical uncouplers related to the most potent uncoupler known until now, SF6847 (2,6-di-t-butyl-4-(2′,2′-dicyanovinyl)phenol). The correlation between uncoupling and protonophoric activities for a number of uncouplers, both non-classical and classical (simply substituted phenols), was examined quantitatively. Correlation was excellent when such factors as the stability of anionic species in the membrane phase and the difference in the pH conditions of the extramembranous aqueous phase were taken into account. Carbonylcyanide m-chlorophenylhydrazone (CCCP) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), which are structurally different, were correlated in a way that resembled the correlation of phenolic compounds, so we think that the mode of action of weakly acidic uncouplers was the same regardless of the structural type. Our findings were evidence for the shuttle-type mechanism of uncoupling action.     

Mitochondrial protonophoric activity induced by a thyromimetic fatty acid analogue

Calcium-dependent uncoupling of liver mitochondrial oxidative phosphorylation by a non-metabolizable long chain fatty acyl analogue was compared with uncoupling induced by in vivo thyroid hormone treatment. beta,beta'-Methyl-substituted hexadecane alpha, omega-dioic acid (Medica 16) is reported here to induce a saturable 20-30% decrease in liver mitochondrial DeltaPsi, DeltapH and protonmotive force which proceeds in the presence of added Ca(2+) to cyclosporin A-sensitive mitochondrial permeabilization. Ca(2+)-dependent uncoupling by Medica 16 was accompanied by atractylate-enhanced, bongkrekic-inhibited activation of mitochondrial Ca(2+) efflux. The direct mitochondrial effect exerted in vitro by Medica 16 is similar to that induced by in vivo thyroid hormone treatment. Hence, the thyromimetic protonophoric activity of Medica 16 and the uncoupling activity of TH converge onto components of the mitochondrial permeabilization transition pore.     

Almitrine, a new kind of energy-transduction inhibitor acting on mitochondrial ATP synthase

At low concentrations, almitrine inhibits yeast cell multiplication by acting on oxidative metabolism. Studies on isolated mitochondria display the following features: (i) almitrine inhibits ATPase activity and decreases ATP/O ratio during oxidative phosphorylation; (ii) no direct effect on respiration can be evidenced; (iii) ATP/O value decreases without any change in the magnitude of delta p; (iv) the higher the ATP synthesis and respiratory fluxes, the larger is the decrease in ATP/O ratio induced by almitrine. These results indicate that almitrine does not act as a classical protonophoric uncoupler nor as previously studied non protonophoric uncouplers (e.g., general anesthetics). Our data show a direct inhibitory effect of almitrine on ATPase-ATP synthase complex. But, in contrast to the classical inhibitors of this complex, almitrine decreases the ATP/O ratio in a flux-dependent manner. Thus, almitrine could induce either an intrinsic uncoupling of H+/-ATPase (i.e., slip in this proton pump) or a change in the mechanistic H+/ATP stoichiometry at the ATPase level.     

Energy conservation and uncoupling in mitochondria

Energy conservation and uncoupling in mitochondria are examined in the light of three important new findings: (a) Studies with the photoaffinity-labeling uncoupler 2-azido-4-nitrophenol have shown that mitochondria contain a specific uncoupler binding site (apparently a polypeptide of M_r = 30,000 ± 10%). (b) This site fractionates into an enzyme complex (complex V), which is capable of oligomycin- and uncoupler-sensitive ATP-Pi exchange. It is absent from electron transfer complexes I, III, and IV, which represent segments of the respiratory chain containing coupling sites 1, 2, and 3, respectively. (c) Trinitrophenol is a membrane-impermeable uncoupler (uncouples submitochondrial particles, but not mitochondria) and a poor protonophore. There is an excellent correlation between the uncoupling potencies and the affinities of uncouplers for the mitochondrial uncoupler-binding site. There is no correlation between uncoupling potency and protonophoric activity of uncouplers when a membrane-permeable uncoupler is compared with a membrane-impermeable one.     

Uncoupling activity and physicochemical properties of derivatives of fluazinam.

The physico-chemical properties and uncoupling activity of eight derivatives of N-phenyl-2-pyridinamines related to the fungicide fluazinam were analyzed using rat liver mitochondria. The uncoupling activity of these compounds relies on the deprotonable secondary amino group. One of the derivatives tested (B-3) was slightly more efficient than fluazinam. By phase-distribution analysis we could show that the N-phenyl-2-pyridinamines are chemicals with moderate hydrophobicity. Deprotonation of the compound reduces the water/octanol partition coefficient by about one order of magnitude. The pKA value of the deprotonable group is affected equally by electron withdrawing substituents of the phenyl- and the pyridinyl-ring, and could be predicted simply from the sum of the Hammett coefficients. The uncoupling efficiency was not dependent on the hydrophobicity of the compound, but appeared to be governed by the pKA of the deprotonable group. This structure/uncoupling characteristic is different from that of the generally more hydrophobic uncouplers of the salicylanilide-type. The pKA resulting in the most efficient uncoupling was found to lie in the range of the pH of the reaction medium. A model based on a solution complexation mechanism, which describes this behaviour, is presented. We conclude that the N-phenyl-2-pyridinamines uncoupled the mitochondria by a simple protonophoric cycle involving protonation/deprotonation in the bulk phase, and that the kinetics of uncoupling were primarily governed by the total concentration of the limiting uncoupler species.     

Protonophoric action of triclosan causes calcium efflux from mitochondria,plasma membrane depolarization and bursts of miniature end-plate potentials

The formerly widely used broad-spectrum biocide triclosan (TCS) has now become a subject of special concern due to its accumulation in the environment and emerging diverse toxicity. Despite the common opinion that TCS is an uncoupler of oxidative phosphorylation in mitochondria, there have been so far no studies of protonophoric activity of this biocide on artificial bilayer lipid membranes (BLM). Yet only few works have indicated the relationship between TCS impacts on mitochondria and nerve cell functioning. Here, we for the first time report data on a high protonophoric activity of TCS on planar BLM. TCS proved to be a more effective protonophore on planar BLM, than classical uncouplers. Correlation between a strong depolarizing effect of TCS on bacterial membranes and its bactericidal action on Bacillus subtilis might imply substantial contribution of TCS protonophoric activity to its antimicrobial efficacy. Protonophoric activity of TCS, monitored by proton-dependent mitochondrial swelling, resulted in Ca²⁺ efflux from mitochondria. A comparison of TCS effects on molluscan neurons with those of conventional mitochondrial uncouplers allowed us to ascribe the TCS-induced neuronal depolarization and suppression of excitability to the consequences of mitochondrial deenergization. Also similar to the action of common uncouplers, TCS caused a pronounced increase in frequency of miniature end-plate potentials at neuromuscular junctions. Thus, the TCS-induced mitochondrial uncoupling could alter neuronal function through distortion of Ca²⁺ homeostasis.     

Within brown-fat cells, UCP1-mediated fatty acid-induced uncoupling is independent of fatty acid metabolism

In the present investigation, we have utilized the availability of UCP1(-/-) mice to examine a wide range of previously proposed lipid activators of Uncoupling Protein 1 (UCP1) in its native environment, i.e. in the brown-fat cells. A non-metabolizable fatty acid analogue, beta,beta cent-methyl-substituted hexadecane alpha,omega-dicarboxylic acid (Medica-16) is a potent UCP1 (re)activator in brown-fat cells, despite its bipolar structure. All-trans-retinoic acid activates UCP1 within cells, whereas beta-carotene only does so after metabolism. The UCP1-dependent effects of fatty acids are positively correlated with their chain length. Medium-chain fatty acids are potent UCP1 activators in cells, despite their lack of protonophoric properties in mitochondrial membranes. Thus, neither the ability to be metabolized nor an innate uncoupling/protonophoric ability is a necessary property of UCP1 activators within brown-fat cells.     

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.     

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.     

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.     

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.