oligomycins and triphenylmethylphosphonium

The plasma-membrane potential (Delta(psi)p) in bloodstream forms of Trypanosoma brucei was studied using several different radiolabelled probes: 86Rb+ and [14C]SCN- were used to report Delta(psi)p directly because they distribute in easily measured quantities across the plasma membrane only, and [3H]methyltriphenylphosphonium (MePh3P+) was used to report Delta(psi)p only when Delta(psi)m had been abolished with FCCP because it reports the algebraic sum of the two potentials when used alone. The unperturbed Delta(psi)p had a value of -82 mV and was found to be essentially identical with, and determined almost completely by, the potassium diffusion potential, as evidenced by: (a) the lack of effect of valinomycin on the value obtained under appropriate conditions when any of these probes were used; (b) the close agreement of this measured value with that predicted from the measured distribution of K+ across the plasma membrane (-76 mV); (c) the large effect of changes in the extracellular K+ concentration by substitution with Na+ on Delta(psi)p together with the complete lack of effect of substitution of extracellular Na+ by the choline cation or substitution of extracellular Cl- by the gluconate anion on Delta(psi)p. The contribution to Delta(psi)p by electrogenic pumping of Na+/K+-ATPase was found to be small (of the order of 6 mV). H+ was not found to be pumped across the plasma membrane or to contribute to Delta(psi)p.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Chlorine; Gluconates; Glucose; Glycerol; Ionophores; Ions; Membrane Potentials; Oligomycins; Onium Compounds; Potassium; Rubidium Radioisotopes; Sodium; Sodium-Potassium-Exchanging ATPase; Time Factors; Trityl Compounds; Trypanosoma brucei brucei; Uncoupling Agents; Valinomycin

In the present study we investigated the changes in the hepatic mitochondrial respiratory system in the transition from weaning to adulthood in the rat. We conceptually divided the system into blocks of reactions that produced or consumed mitochondrial membrane potential and then measured the kinetic responses of these blocks of reactions to changes in this potential in isolated liver mitochondria from 25- and 60-day-old rats using succinate as substrate. Moreover, we considered the mitochondrial membrane potential producers to be divided into blocks of reactions that reduced or oxidized ubiquinone-2 (Q-2) and then measured the kinetic responses of these two blocks to changes in Q-2 redox state as well as the flux control coefficients and the cytochrome content. We found that adult rats exhibited significantly higher state 3 respiratory rates with increased kinetic response of the substrate oxidation pathway to the mitochondrial membrane potential, slightly decreased activity of the phosphorylating system, increased kinetic responses of both Q-2 reducers and oxidizers to Q-2 redox state, and increased cytochrome content. Our results indicate that important changes in the hepatic mitochondrial respiratory system occur in the transition from weaning to adulthood in rats.

Topics: Age Factors; Animals; Cell Respiration; Cytochromes; Kinetics; Male; Membrane Potentials; Mitochondria, Liver; Oligomycins; Onium Compounds; Oxidation-Reduction; Oxygen Consumption; Phosphorylation; Rats; Rats, Wistar; Rubidium; Trityl Compounds; Ubiquinone

We investigated the effects of ischemia on the kinetics and control of mitochondria isolated from normal and ischemic heart. The dependence of the respiratory chain, phosphorylation system and proton leak on the mitochondrial membrane potential were measured in mitochondria from hearts after 0, 30 min and 45 min of in vitro ischemia. Data showed that during the development of ischemia from the reversible (30 min) to the irreversible (45 min) phase, a progressive decrease in activity of the respiratory chain occurs. At the same time an increase in proton leak across the mitochondrial inner membrane was observed. Phosphorylation is inhibited but seems to be less affected by ischemia than respiratory chain or proton leak. Control coefficients of the 3 blocks of reactions over respiration rate were determined in different respiratory states between state 4 and state 3. Ischemia caused the control exerted by the proton leak to increase in state 3 and the intermediate state and caused the control by the phosphorylation system to decrease in the intermediate state. Taken together, these results indicate that the main effects of ischemia on mitochondrial respiration are an inhibition of the respiratory chain and an increase of the proton leak.

Topics: Animals; Electron Transport; In Vitro Techniques; Kinetics; Membrane Potentials; Mitochondria, Heart; Myocardial Ischemia; Oligomycins; Onium Compounds; Oxidative Phosphorylation; Oxygen Consumption; Phosphorylation; Protons; Rats; Rubidium; Trityl Compounds

Treatment of isolated mitochondria with calcium and inorganic phosphate induces inner membrane permeability that is thought to be mediated through a non-selective, calcium-dependent pore. The inner membrane permeability results in the rapid efflux of small matrix solutes such as glutathione and calcium, loss of coupled functions, and large amplitude swelling. We have identified conditions of permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing calcium and inorganic phosphate abolished large-amplitude swelling of mitochondria but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial glutathione and calcium. The release of both glutathione and calcium was inhibited by the addition of cyclosporin A, a potent inhibitor of permeability transition. Transmission electron microscopy analysis, combined with the glutathione and calcium release data, indicate that permeability transition can be observed in the absence of large-amplitude swelling. Permeability transition occurring both with and without large-amplitude swelling was accompanied by a collapse of the membrane potential. We conclude that cyclosporin A-sensitive permeability transition can occur without obvious morphological changes such as large-amplitude swelling. Monitoring the cyclosporin A-sensitive release of concentrated endogenous matrix solutes, such as GSH, may be a sensitive and useful indicator of permeability transition.

Topics: Animals; Antimycin A; Biological Transport; Calcium; Cyclosporine; Glutathione; Male; Microscopy, Electron; Mitochondria, Liver; Mitochondrial Swelling; Oligomycins; Onium Compounds; Permeability; Phosphates; Rats; Rats, Sprague-Dawley; Sulfides; Time Factors; Trityl Compounds

Bloodstream forms of Trypanosoma brucei were found to maintain a significant membrane potential across their mitochondrial inner membrane (delta psi m) in addition to a plasma membrane potential (delta psi p). Significantly, the delta psi m was selectively abolished by low concentrations of specific inhibitors of the F1F0-ATPase, such as oligomycin, whereas inhibition of mitochondrial respiration with salicylhydroxamic acid was without effect. Thus, the mitochondrial membrane potential is generated and maintained exclusively by the electrogenic translocation of H+, catalysed by the mitochondrial F1F0-ATPase at the expense of ATP rather than by the mitochondrial electron-transport chain present in T. brucei. Consequently, bloodstream forms of T. brucei cannot engage in oxidative phosphorylation. The mitochondrial membrane potential generated by the mitochondrial F1F0-ATPase in intact trypanosomes was calculated after solving the two-compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh3P+) and was shown to have a value of approximately 150 mV. When the value for the delta psi m is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton-motive force was calculated to be greater than 190 mV. It seems likely that this mitochondrial proton-motive force serves a role in the directional transport of ions and metabolites across the promitochondrial inner membrane during the bloodstream stage of the life cycle, as well as promoting the import of nuclear-encoded protein into the promitochondrion during the transformation of bloodstream forms into the next stage of the life cycle of T. brucei.

Topics: Animals; Energy Metabolism; Intracellular Membranes; Kinetics; Membrane Potentials; Mitochondria; Oligomycins; Onium Compounds; Proton Pumps; Proton-Translocating ATPases; Protons; Rubidium; Salicylamides; Trityl Compounds; Trypanocidal Agents; Trypanosoma brucei brucei

The mitochondrial membrane potential in isolated hepatocytes was measured using the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP+) with appropriate corrections for plasma membrane potential, cytoplasmic and mitochondrial binding of TPMP+, and other factors. The relationship between mitochondrial membrane potential and respiration rate in hepatocytes was examined as the respiratory chain was titrated with myxothiazol in the presence of oligomycin. This relationship was nonproportional and similar to results with isolated mitochondria respiring on succinate. This shows that there is an increased proton conductance of the mitochondrial inner membrane in situ at high values of membrane potential. From the respiration rate and mitochondrial membrane potential of hepatocytes in the absence of oligomycin, we estimate that the passive proton permeability of the mitochondrial inner membrane accounts for 20-40% of the basal respiration rate of hepatocytes. The relationship between log[TPMP+]tot/[TPMP+]e and respiration rate in thymocytes was also nonproportional suggesting that the phenomenon is not peculiar to hepatocytes. There is less mitochondrial proton leak in hepatocytes from hypothyroid rats. A large proportion of the difference in basal respiration rate between hepatocytes from normal and hypothyroid rats can be accounted for by differences in the proton permeability characteristics of the mitochondrial inner membrane.

Topics: Animals; Cell Membrane; Cells, Cultured; Electric Conductivity; Female; Hydrogen-Ion Concentration; Hypothyroidism; Intracellular Membranes; Kinetics; Male; Membrane Potentials; Mitochondria, Liver; Nigericin; Oligomycins; Onium Compounds; Oxygen Consumption; Rats; Rats, Inbred Strains; Reference Values; Submitochondrial Particles; Thymus Gland; Trityl Compounds

Cultured rat hepatocytes were treated with potassium cyanide, an inhibitor of cytochrome oxidase; valinomycin, a K+ ionophore; carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophore; and the ATP synthetase inhibitor oligomycin. The effect of these agents on the viability of the cells was related to changes in ATP content and the deenergization of the mitochondria. The ATP content was reduced by over 90% by each inhibitor. All of the agents except oligomycin killed the cells within 4 h. With the exception of oligomycin, the mitochondrial membrane potential as measured by the distribution of [3H]triphenylmethylphosphonium collapsed with each of the agents. Monensin, a H+/Na+ ionophore, potentiated the toxicity of cyanide and CCCP, whereas the toxicity of valinomycin was reduced. The effect of cyanide and monesin on the cytoplasmic pH of cultured hepatocytes was measured with the fluorescent probe, 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Cyanide promptly acidified the cytosol, and the addition of 10 microM monensin caused a rapid alkalinization of the cytosol. A reduction of pH of the culture medium from 7.4 to 6.6 and 6.0 prevented the cell killing both by cyanide alone and by cyanide in the presence of monensin. However, neither monensin nor extracellular acidosis had any effect on the loss of mitochondrial energization in the presence of cyanide. It is concluded that ATP depletion per se is insufficient to explain the cell killing with cyanide, CCCP, and valinomycin. Rather, cell killing is better correlated with a loss of mitochondrial energization. With cyanide an intracellular acidosis interferes with the mechanism that couples collapse of the mitochondrial membrane potential to lethal cell injury.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Survival; Cells, Cultured; Cytoplasm; Drug Interactions; Energy Metabolism; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Liver; Male; Membrane Potentials; Mitochondria, Liver; Monensin; Oligomycins; Onium Compounds; Potassium Cyanide; Rats; Rats, Inbred Strains; Trityl Compounds; Valinomycin

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.

Topics: Adenine Nucleotides; Animals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Energy Metabolism; Gluconeogenesis; Indicators and Reagents; Intracellular Membranes; Liver; Male; Membrane Potentials; Mitochondria, Liver; Oligomycins; Onium Compounds; Rats; Rats, Inbred Strains; Staining and Labeling; Thermodynamics; Trityl Compounds; Trypan Blue; Urea; Valinomycin