A short-chain alkyl derivative of Rhodamine 19 acts as a mild uncoupler of mitochondria and a neuroprotector

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C₄R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C₁₂R1). Surprisingly, C₁₂R1 increased H⁺ conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C₄R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C₄R1. In experiments with HeLa cells, C₄R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C₄R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C₁₂R1. Thus, C₄R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.     

The effect of the homogenates from different developmental stages of the nematode Protostrongylus rufescens (Leuckart, 1895) on mitochondrial and lipid bilayer membranes

The effect of the homogenates from different developmental stages of the nematode Protostrongylus rufescens on mitochondrial and lipid bilayer membranes has been studied. The homogenate of P. rufescens affects efficiently the cell energy by the inhibition of the mitochondrial respiration in the metabolic state V3, uncouples oxidative phosphorylation and affects the functions of mitochondria at the level of cyclosporine A-sensitive pore by making it highly permeable. Moreover, the nematode homogenate at the concentration of 1 mkg/ml increases efficiently the integral permeability of lipid bilayer membranes. An increase in this permeability is connected apparently with the formation of single ion channels. The channels of lipid bilayer membranes induced by the nematode homogenate show cation selectivity.     

The kinetic mechanism of action of an uncoupler of oxidative phosphorylation

Summary The chemiosmotic hypothesis predicts that the mechanism by which weak acids uncouple oxidative phosphorylation in mitochondria is identical to the mechanism by which they transport hydrogen ions across artificial bilayer membranes. We report here the results of a kinetic study of uncoupler-mediated hydrogen ion transport across bilayer membranes. We made electrical relaxation measurements on black lipid membranes exposed to the substituted benzimidazole 5,6-dichloro-2-trifluoromethylbenzimidazole. The simplest model consistent with our experimental data allowed us to deduce values for adsorption coefficients and rate constants. Our major conclusions are that the back diffusion of the neutral species is the rate limiting step for the steady state transport of hydrogen ions, that both the neutral and charged forms of the uncoupler adsorb strongly to the interfaces, and that the reactions at the membrane-solution interfaces occur sufficiently rapidly for equilibrium to be maintained. Independent measurements of the adsorption coefficients of both the neutral and anionic forms of the weak acid and also of the permeability of the membrane to the neutral form agreed well with the values deduced from the kinetic study.     

The kinetic mechanism of action of an uncoupler of oxidative phosphorylation.

The chemiosmotic hypothesis predicts that the mechanism by which weak acids uncouple oxidative phosphorylation in mitochondria is identical to the mechanism by which they transport hydrogen ions across artificial bilayer membranes. We report here the results of a kinetic study of uncoupler-mediated hydrogen ion transport across bilayer membranes. We made electrical relaxation measurements on black lipid membranes exposed to the substituted benzimidazole 5,6-dichloro-2-trifluoromethylbenzimidazole. The simplest model consistent with our experimental data allowed us to deduce values for adsorption coefficients and rate constants. Our major conclusions are that the back diffusion of the neutral species is the rate limiting step for the steady state transport of hydrogen ions, that both the neutral and charged forms of the uncoupler adsorb strongly to the interfaces, and that the reactions at the membrane-solution interfaces occur sufficiently rapidly for equilibrium to be maintained. Independent measurements of the adsorption coefficients of both the neutral and anionic forms of the weak acid and also of the permeability of the membrane to the neutral form agreed well with the values deduced from the kinetic study.     

The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) uncouples mitochondrial oxidative phosphorylation in both sea lamprey (Petromyzon marinus) and TFM-tolerant rainbow trout (Oncorhynchus mykiss)

The toxicity of 3-trifluoromethyl-4-nitrophenol (TFM) appears to be due to a mismatch between ATP supply and demand in lamprey, depleting glycogen stores and starving the nervous system of ATP. The cause of this TFM-induced ATP deficit is unclear. One possibility is that TFM uncouples mitochondrial oxidative phosphorylation, thus impairing ATP production. To test this hypothesis, mitochondria were isolated from the livers of sea lamprey and rainbow trout, and O(2) consumption rates were measured in the presence of TFM or 2,4-dinitrophenol (2,4-DNP), a known uncoupler of oxidative phosphorylation. TFM and 2,4-DNP markedly increased State IV respiration in a dose-dependent fashion, but had no effect on State III respiration, which is consistent with uncoupling of oxidative phosphorylation. To determine how TFM uncoupled oxidative phosphorylation, the mitochondrial transmembrane potential (TMP) was recorded using the mitochondria-specific dye rhodamine 123. Mitochondrial TMP decreased by 22% in sea lamprey, and by 28% in trout following treatment with 50μmolL(-1) TFM. These findings suggest that TFM acted as a protonophore, dissipating the proton motive force needed to drive ATP synthesis. We conclude that the mode of TFM toxicity in sea lamprey and rainbow trout is via uncoupling of oxidative phosphorylation, leading to impaired ATP production.     

Organizational changes in phospholipid multibilayers induced by uncouplers of oxidative phosphorylation: a spin label study

A cholestane spin probe was used to study the effect of uncouplers of oxidative phosphorylation (2,4-dinitrophenol, pentachlorophenol and dicumarol) on the degree of organization of phospholipids in hydrated multibilayers. Disruptive effects were observed—their magnitude depending on pH, time and the presence of cholesterol. A correlation between changes in probe organization and ion conductivity, with maximum effects at the pH corresponding to the pK of the uncoupler, could be demonstrated in the films containing cholesterol. Egg lecithin films containing no cholesterol were disordered maximally at pH 4.0 irrespective of the uncoupler used. The effect of uncouplers on the probe disorganization varied with time after exposure. These time effects indicated that relative movement of uncoupler, probe and lipid molecules occur to produce lipid organizations differing from those after initial exposure to uncoupler. The results show that even in a simple model system uncoupler effects may be complex, and suggest that changes in bilayer lipid organization parameters may play a role in uncoupling oxidative phosphorylation.     

Interaction of respiration and luminescence in a common marine bacterium

Investigators have proposed for some time that bacterial luciferase forms a shunt around the pathway of respiratory electron transport. Certain physiologic evidence for coupling between luminescence and respiration has supported such a view. In this study, Vibrio harveyi cells were monitored for luminescent responses to artificial manipulation of respiratory electron flow. The effects of cyanide under aerobic and anaerobic conditions confirmed that luminescence and respiration compete for oxygen. The effects of an uncoupler of oxidative phosphorylation indicated that luminescence and respiration compete for a common reductant. Treatment with uncoupler also induced aldehyde deficiency in vivo.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - Tris tris(hydroxymethyl) aminomethane     

Interactions of the channel forming peptide alamethicin with artificial and natural membranes

Alamethicin and related α-aminoisobutyric acid peptides form transmembrane channels across lipid bilayers. This article briefly reviews studies on the effect of alamethicin on lipid phase transitions in lipid bilayers and on mitochondrial oxidative phosphorylation. Fluorescence polarization studies, employing 1,6-diphenyl-1,3,5-hexatriene as a probe, suggest that alamethicin fluidizes lipid bilayers below the phase transition t-emperature, but has little effect above the gel-liquid crystal transition point. Alamethicin is shown to function as an uncoupler of oxidative phosphorylation in rat liver mitochondria. The influence of alamethicin on mitochondrial respiration is modulated by the phosphate ion concentration in the medium. Classical uncoupling activity is evident at low phosphate levels while inhibitory effects set in at higher phosphate concentrations. Time-dependent changes in respiration rates following peptide addition are rationalized in terms of alamethicin interactions with mitochondrial membrane components.     

Localization of coenzyme Q10 in the center of a deuterated lipid membrane by neutron diffraction

Quinones (e.g., coenzyme Q, CoQ10) are best known as carriers of electrons and protons during oxidative phosphorylation and photosynthesis. A myriad of mostly more indirect physical methods, including fluorescence spectroscopy, electron-spin resonance, and nuclear magnetic resonance, has been used to localize CoQ10 within lipid membranes. They have yielded equivocal and sometimes contradictory results. Seeking unambiguous evidence for the localization of ubiquinone within lipid bilayers, we have employed neutron diffraction. CoQ10 was incorporated into stacked bilayers of perdeuterated dimyristoyl phosphatidyl choline doped with dimyristoyl phosphatidyl serine containing perdeuterated chains in the natural fluid-crystalline state. Our data show CoQ10 at the center of the hydrophobic core parallel to the membrane plane and not, as might be expected, parallel to the lipid chains. This localization is of importance for its function as a redox shuttle between the respiratory complexes and, taken together with our recent result that squalane is in the bilayer center, may be interpreted to show that all natural polyisoprene chains lie in the bilayer center. Thus ubiquinone, in addition to its free radical scavenging and its well-known role in oxidative phosphorylation as a carrier of electrons and protons, might also act as an inhibitor of transmembrane proton leaks.     

The effect of the polar moiety of lipids on bilayer conductance induced by uncouplers of oxidative phosphorylation

Summary Bilayer membranes were formed from decane, cholesterol, and three lipids isolated fromStaphylococcus aureus: positively charged lysyl phosphatidylglycerol (LysPG), negatively charged phosphatidylglycerol (PG), and neutral diglucosyldiglyceride (DiGluDiGly). The uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP) and 3-t-butyl,5-chloro,2-chloro,4-nitrosalicylanilide (S 13), increased the electrical conductance of all three differently charged bilayers. S 13 was found to be the most effective reagent of the known uncouplers in increasing conductance of the bilayers. The conductance induced by uncouplers was investigated as a function of pH and uncoupler concentration. The pH of maximum conductance for each uncoupling agent was dependent on both the uncoupler and the lipid; it was lower for each uncoupler in LysPG and higher in PG compared to DiGluDiGly bilayers. At a pH below the optimum for LysPG, the conductance of the positively charged membrane was 500 times and of the neutral one 10 times higher than that of the negatively charged bilayer at equal uncoupler concentration and pH. Above the pH optimum for DiGluDiGly, the conductance was approximately equal for the positive and neutral membranes, but was lower in PG bilayers. Conductance depended linearly on uncoupler concentration. The bilayer conductance induced by S 13 was entirely due to increased proton permeability in all three lipids. The findings are consistent with the role of uncouplers as carriers for protons across the hydrocarbon interior of lipid membranes. The differences in conductance of differently charged lipid bilayers at equal uncoupler concentration, as well as the change of pH optimum of conductance with lipid charge, can be explained in terms of an electrostatic energy contribution of the fixed lipid charges to the distribution of the uncoupler anion between the aqueous and the membrane phases.     

Mitochondrial bioenergetics during exposure of rats to perfluidone,a fluorinated arylalkylsulphonamide

Apart from the symptoms of poisoning which the fluorinated arylalkylsulphonamides share with the classical protonphore and uncoupler of oxidative phosphorylation, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), the direct correlation between the lipophilic weak acid properties of these chemicals and their biological activity suggests that permeation of the inner mitochondrial membrane could be the initial step in the molecular mechanism of their biological activity. Mitochondria isolated from the livers of rats intraperitoneally exposed to varying doses (0–80 mg/kg body wt.) of perfluidone (1,1,1-trifluoro-N-(2 methyl-4-(phenylsulphonyl)phenyl methanesulphonamide), a fluorinated arylalkylsulphonamide pesticide, exhibit the following dose-dependent features: (i) increased state-4 respiration: stimulation being maximal (≥400%) at 80 mg perfluidone per kg body wt.), (ii) release of respiratory control by ADP: least respiratory control ratios (RCRs) (≤1.2) were obtained at 80 mg perfluidone per kg body wt., (iii) reduced ADP/O ratios, (iv) increased mitochondrial passive swelling, (vi) reduced rates of mitochondrial proton ejection during succinate oxidation, (vi) reduced rates of respiration-dependent Ca²⁺ accumulation and (vii) an enhanced oligomycin-sensitive ATPase action. These features which are qualitatively identical to those of the classical protonophore FCCP, suggest that permeation of the inner mitochondrial membrane by perfluidone is accompanied by a movement of protons into the matrix such that the proton motive force required for ATP synthesis and ion transport becomes small or not formed at all.     

Uncoupler antagonism of valinomycin induced bilayer membrane conductance

We have found that an uncoupler of oxidative phosphorylation, tetrachloro-2-trifluoromethylbenzimidazole (TTFB), can block valinomycin induced potassium ion, (K⁺), conductance through bilayer membranes. Blocking is most pronounced at high K⁺ concentration, (≥ 0.1 M), and at a pH greater than the pK of TTFB. A blocking mechanism involving competition for membrane sites, together with association of oppositely charged species within the membrane, is proposed.     

Mitochondrial superoxide and aging: uncoupling-protein activity and superoxide production

Mitochondria are a major source of superoxide, formed by the one-electron reduction of oxygen during electron transport. Superoxide initiates oxidative damage to phospholipids, proteins and nucleic acids. This damage may be a major cause of degenerative disease and aging. In isolated mitochondria, superoxide production on the matrix side of the membrane is particularly high during reversed electron transport to complex I driven by oxidation of succinate or glycerol 3-phosphate. Reversed electron transport and superoxide production from complex I are very sensitive to proton motive force, and can be strongly decreased by mild uncoupling of oxidative phosphorylation. Both matrix superoxide and the lipid peroxidation product 4-hydroxy-trans-2-nonenal can activate uncoupling through endogenous UCPs (uncoupling proteins). We suggest that superoxide releases iron from aconitase, leading to a cascade of lipid peroxidation and the release of molecules such as hydroxy-nonenal that covalently modify and activate the proton conductance of UCPs and other proteins. A function of the UCPs may be to cause mild uncoupling in response to matrix superoxide and other oxidants, leading to lowered proton motive force and decreased superoxide production. This simple feedback loop would constitute a self-limiting cycle to protect against excessive superoxide production, leading to protection against aging, but at the cost of a small elevation of respiration and basal metabolic rate.     

Blocking phenomena and charge transport through membranes

Summary Substances which uncouple oxidative phosphorylation in mitochondrial membranes usually increase the electrical conductivity of synthetic bimolecular phospholipid membranes. Among these uncouplers is a group characterized chemically as weak acids. For this group the conductivity of synthetic membranes, when measured versus pH at fixed uncoupler concentration, shows a maximum at a pH approximately equal to the pK value of the uncoupler used. Corresponding maxima in membrane electrical potential arising from ion concentration gradients are also observed. To explain such phenomena a model is proposed which assumes charge transport by the direct transfer of either protons or anions of the uncoupler between binding sites located on the membrane boundaries. A fixed surface density of such sites is assumed. The transfer of an ion requires both its presence on an initiating site and the availability of a terminal site which is not already occupied by an ion of the same species. Failure to satisfy both criteria leads to blockage of current flow at both low and high concentrations of the transported ion.On sabbatical leave for the academic year 1968–69 from the University of California, Riverside, California, USA.     

Energy metabolism of isolated hepatocytes at various levels of oxidative phosphorylation uncoupling]

The energy metabolism changes in isolated hepatocytes at different levels of proton conductivity of cellular membranes were studied. The low doses of the uncoupler which increased hepatocyte respiration rate but did not markedly affect the mitochondrial potential caused: the reduction in total adenine nucleotide contents (ATP + ADP + AMP), the oxidation of mitochondrial NADH, the increase in the rates of glycogenolysis and net flux via phosphofructokinase without any changes in the rates of glucose, lactate and pyruvate accumulation. High doses of the uncoupler which eliminated completely oxidative phosphorylation decreased Atkinson's energy charge down to 0.5, reduced cytoplasmic NADH, induced a further increase in the glycogenolysis rate, increased the rates of glucose and lactate accumulation, heightened glucose-6-phosphate content and lowered contents of 3-phosphoglycerate and 2-oxoglutarate.     

The mechanism of action of DNP on phospholipid bilayer membranes

Summary The weak acid 2,4-dinitrophenol (DNP) acts as an uncoupler of oxidative phosphorylation in biological systems and, in consonance with the Mitchell hypothesis, also enhances the conductance of phospholipid bilayer membranes. Several models have been proposed in the literature to explain the molecular mechanism by which DNP exerts its electrical effects on the model membranes, none of which accounts for all of the data, and all of which ignore the possibility that the anion of DNP is also binding to the surface of the bilayer and modifying the charge density. Experimental evidence is presented in this report which suggests that when a bilayer membrane is formed from a neutral lipid, DNP does in fact adsorb to its surface and produce a substantial negative surface potential. When this phenomenon is taken into account, the model proposed by Lea and Croghan and by Finkelstein is capable of describing all of the effects of DNP on bilayer membranes. In this model, the permeant species is a negatively charged complex formed from the undissociated acid and its anion.     

Characterisation of the control of respiration in potato tuber mitochondria using the top-down approach of metabolic control analysis.

Control over oxidative phosphorylation by purified potato mitochondria was determined using the top-down approach of metabolic control analysis. The control over the respiration rate, phosphorylation rate, proton-leak rate and proton motive force exerted by the respiratory chain, phosphorylation reactions and the proton leak were measured over a range of phosphorylation rates from resting (state 4) to maximal (state 3). These rates were obtained by adding different amounts of hexokinase in the presence of glucose, or different amounts of oligomycin in the presence of ADP. The respiratory substrate was NADH or succinate, both of which feed electrons directly to ubiquinone. The rate of oxygen consumption by the alternative oxidase pathway was negligible with NADH as substrate but was measurable with succinate and was subtracted. Control over the respiration rate in potato mitochondria was predominantly exerted by the respiratory chain at all rates except close to state 4, where control by the proton leak was equally or more important. For oxidation of NADH, the flux control coefficient over the respiration rate exerted by the respiratory chain in state 3 was between 0.8 and 1.0, while in state 4, control over the respiration rate was shared about equally between the chain and the proton leak. The control over the phosphorylation rate was predominantly exerted by the respiratory chain, although at low rates control by the phosphorylation system was also important. For oxidation of NADH, the flux control coefficient over the phosphorylation rate exerted by the respiratory chain in state 3 was 0.8-1.0, while near state 4 the flux control coefficients over the phosphorylation rate were about 0.8 for the phosphorylation system and 0.25 for the chain. Control over the proton leak rate was shared between the respiratory chain and the proton leak; the phosphorylation system had negative control. For oxidation of NADH, the flux control coefficients over the leak rate in state 3 were 1.0 for the leak, 0.4 for the chain and -0.4 for the phosphorylation system, while in state 4 the flux control coefficients over leak rate were about 0.5 for the leak and 0.5 for the chain. Control over the magnitude of the protonmotive force was small, between -0.2 and +0.2, reflecting the way the system operates to keep the protonmotive force fairly constant; the respiratory chain and the phosphorylation system had equal and opposite control and there was very little control by the proton leak except near state 4.     

Perfluorooctane sulfonamide: a structurally novel uncoupler of oxidative phosphorylation

The effects of sulfluramide (N-ethylperfluorooctane sulfonamide) and perfluorooctane sulfonamide (DESFA) on isolated rabbit renal cortical mitochondria (RCM) were examined. Sulfluramid (1-100 microM) and DESFA (0.5-50 microM) increased state 4 respiration of RCM respiring on pyruvate/malate or succinate in a concentration dependent manner in the absence of a phosphate acceptor. In addition, both sulfluramid and DESFA increased state 4 respiration in the presence of oligomycin, an inhibitor of F0F1-ATPase. The effects of sulfluramid (200 microM), DESFA (100 microM), and the known protonophore and uncoupler of oxidative phosphorylation, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (1 microM), on RCM proton movement were examined directly by monitoring extramitochondrial pH and indirectly by monitoring passive mitochondrial swelling. Immediately upon addition, DESFA and FCCP, but not sulfluramid, dissipated the RCM proton gradient and caused RCM to swell in solutions of NaCl or NH4Cl. These results show that DESFA uncouples oxidative phosphorylation by acting as a protonophore. RCM were shown to metabolize sulfluramid to DESFA which suggests that the increase in state 4 respiration observed with sulfluramid is due to DESFA. DESFA is unique in that it is one of two uncouplers that does not contain a ring structure and thus may be a useful model in the study of oxidative phosphorylation.     

Respiratory activity of isolated hepatocytes

Oligomycin and uncoupler of oxidative phosphorylation have been studied for their effect on the respiration activity of hepatocytes in rats. The respiration rate in the presence of oligomycin and uncoupler is higher than it is with the respiration uncoupled in the absence of oligomycin. Exogenic succinate makes endogenic respiration of hepatocytes in the presence of digitonin 5 times more intensive. The obtained results evidence for the fact that the uncoupled respiration is limited by the concentration of substrates able to be oxidized in the respiration chain of mitochondria. Oligomycin induces accumulation of substrates and following addition of the disconnector evokes their fast oxidation.     

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).