oligomycins and tetraphenylphosphonium

Isolated brain mitochondria were examined for their responses to calcium challenges under varying conditions. Mitochondrial membrane potential was monitored by following the distribution of tetraphenylphosphonium ions in the mitochondrial suspension, mitochondrial swelling by observing absorbance changes, calcium accumulation by an external calcium electrode, and oxygen consumption with an oxygen electrode. Both the extent and rate of calcium-induced mitochondrial swelling and depolarization varied greatly depending on the energy source provided to the mitochondria. When energized with succinate plus glutamate, after a calcium challenge, CNS mitochondria depolarized transiently, accumulated substantial calcium, and increased in volume, characteristic of a mitochondrial permeability transition. When energized with 3 mM succinate, CNS mitochondria maintained a sustained calcium-induced depolarization without appreciable swelling and were slow to accumulate calcium. Maximal oxygen consumption was also restricted under these conditions, preventing the electron transport chain from compensating for this increased proton permeability. In 3 mM succinate, cyclosporin A and ADP plus oligomycin restored potential and calcium uptake. This low conductance permeability was not effected by bongkrekic acid or carboxyatractylate, suggesting that the adenine nucleotide translocator was not directly involved. Fura-2FF measurements of [Ca(2+)](i) suggest that in cultured hippocampal neurons glutamate-induced increases reached tens of micromolar levels, approaching those used with mitochondria. We propose that in the restricted substrate environment, Ca(2+) activated a low-conductance permeability pathway responsible for the sustained mitochondrial depolarization.

Topics: Adenosine Diphosphate; Animals; Antineoplastic Agents; Atractyloside; Brain; Calcium; Electric Conductivity; Fluorescent Dyes; Fura-2; Glutamic Acid; Membrane Potentials; Mitochondria; Mitochondrial Swelling; Nerve Degeneration; Oligomycins; Onium Compounds; Organophosphorus Compounds; Oxidation-Reduction; Oxygen Consumption; Rats; Strontium; Succinic Acid; Uncoupling Agents

Activation of the mitochondrial permeability transition may contribute to excitotoxic neuronal death (Ankarcrona et al., 1996; Dubinsky and Levi, 1998). However, cyclosporin A (CsA), a potent inhibitor of the permeability transition in liver mitochondria, only protects against neuronal injury by limited doses of glutamate and selected ischemic paradigms. The lack of consistent CsA inhibition of the mitochondrial permeability transition was analyzed with the use of isolated brain mitochondria. Changes in the permeability of the inner mitochondrial membrane were evaluated by monitoring mitochondrial membrane potential (Deltapsi), using the distribution of tetraphenylphosphonium, and by monitoring mitochondrial swelling, using light absorbance measurements. Metabolic impairments, large Ca(2+) loads, omission of external Mg(2+), or low doses of palmitic acid or the protonophore FCCP exacerbated Ca(2+)-induced sustained depolarizations and swelling and eliminated CsA inhibition. BSA restored CsA inhibition in mitochondria challenged with 50 microm Ca(2+), but not with 100 microm Ca(2+). CsA failed to prevent Ca(2+)-induced depolarization or to repolarize mitochondria when mitochondria were depolarized excessively. Similarly, CsA failed to prevent mitochondrial swelling or PEG-induced shrinkage after swelling when the Ca(2+) challenge produced a strong, sustained depolarization. Thus in brain mitochondria CsA may be effective only as an inhibitor of the permeability transition and the Ca(2+)-activated low permeability state under conditions of partial depolarization. In contrast, ADP plus oligomycin inhibited both permeabilities under all of the conditions that were tested. In situ, the neuroprotective action of CsA may be limited to glutamate challenges sufficiently toxic to induce the permeability transition but not so severe that mitochondrial depolarization exceeds threshold.

Topics: Adenosine Diphosphate; Animals; Brain; Brain Chemistry; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cyclophilins; Cyclosporine; Enzyme Inhibitors; Glutamic Acid; Intracellular Membranes; Magnesium; Membrane Potentials; Mitochondria; Mitochondrial Swelling; Oligomycins; Onium Compounds; Organophosphorus Compounds; Palmitic Acid; Permeability; Polyethylene Glycols; Rats; Serum Albumin, Bovine

The effect of TPP+ on the fatty acid or FCCP-induced uncoupling in rat heart mitochondria was studied. It was found that (a) TPP+ increases the stimulation of oxygen consumption by palmitic acid or FCCP in the presence of oligomycin, (b) TPP+ greatly enhances the palmitic acid or FCCP-induced delta psi decrease. Both effects of TPP+ were strongly suppressed by carboxyatractylate in the case of palmitate but were not in the case of FCCP. The role of ATP/ADP-antiporter in the TPP+ and palmitic acid effects is discussed.

Topics: Animals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Fluorescent Dyes; In Vitro Techniques; Mitochondria, Heart; Oligomycins; Onium Compounds; Organophosphorus Compounds; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Phenazines; Rats; Spectrometry, Fluorescence; Uncoupling Agents

Evidence for a membrane potential (delta psi) generating capacity of inorganic pyrophosphate (PPi) with inorganic pyrophosphatase in mitochondria from the yeast Saccharomyces cerevisiae is presented. Addition of PPi increases the accumulation of the permeant cation tetraphenylphosphonium (TTP+) in mitochondria, as measured with a TPP(+)-selective electrode or using [3H]TPP+. This accumulation is strongly inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) but not by oligomycin, whereas accumulation of TPP+ induced by ATP is strongly inhibited by both FCCP and oligomycin. The values of delta psi obtained upon addition of PPi were similar to those obtained when yeast mitochondria were energized by NADH or ATP. Energization of the wild type yeast mitochondria with ATP resulted in higher delta psi values than those achieved when PPi was hydrolyzed, whereas the effect of PPi was more pronounced in mitochondria from a pyrophosphatase (PPase) overproducing yeast strain. These results clearly indicate that PPi is significant as an energy donor and that the yeast mitochondrial PPase, the gene for which we have recently cloned and sequenced, may participate in energy transfer reactions.

Topics: Adenosine Triphosphate; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Diphosphates; Membrane Potentials; Mitochondria; NAD; Oligomycins; Onium Compounds; Organophosphorus Compounds; Pyrophosphatases; Saccharomyces cerevisiae

The membrane electric effects of N,N'-dicyclohexyl-carbodiimide (DCCD) and vanadate were studied in murine erythroleukemia cells (MELC), comparing the patch-clamp technique and the accumulation ratio (ARexp) of [3H]-tetraphenylphosphonium (TPP+). Electrophysiological measurements showed that both these inhibitors produce, at micromolar concentrations, a 20-30 mV hyperpolarization of resting potential (delta psi p) of MELC, which is abolished when the electrochemical equilibrium potential of K+ (EK) is brought close to zero. DCCD and vanadate turned out to have distinct targets on the plasma membrane of MELC (an H+ pump and the Na+,K(+)-ATPase, respectively). Measurements of ARexp showed that: (i) patch-clamp measurements of delta psi p were equivalent to those based on ARexp of antimycin-pretreated cells (ARANT); (ii) DCCD produced a strong increase in ARANT, that was antagonized by carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and diethylstilbestrol (DES); (iii) vanadate determined a marked increase in ARANT that was insensitive to FCCP, but antagonized by ouabain; (iv) incubation in high K+ medium (HK) brought ARANT to 1.0 in the controls, but did not lower this ratio below 3.0 in the presence of DCCD or vanadate; (v) the total amount of TPP+ taken up by the cells was in any case water extractable by a freezing and thawing procedure. On the whole, our data indicate that DCCD and vanadate hyperpolarize the MELC by increasing the K+ conductance and, at the same time, enhance the TPP+ binding, probably by changing the electrostatic potential profile of the plasma membrane. These effects seem to involve functional modifications of the target pumps, apparently related to the ion-occluding state of these enzymes.

Topics: Adenosine Triphosphatases; Animals; Cell Membrane; Dicyclohexylcarbodiimide; Diethylstilbestrol; Leukemia, Erythroblastic, Acute; Membrane Potentials; Mice; Oligomycins; Onium Compounds; Organophosphorus Compounds; Ouabain; Potassium; Tumor Cells, Cultured; Vanadates

In state 4, the cumene hydroperoxide-induced decrease of mitochondrial transmembrane potential is prevented by the free radical scavenger, butylhydroxytoluene. This decrease is accompanied by accumulation of lipid hydroperoxides. Oligomycin suppresses the cumene hydroperoxide-induced uncoupling of mitochondria, but has no significant effect on lipid peroxidation. The transition of mitochondria from state 4 to state 3 by ADP addition leads to the suppression of both lipid peroxidation and mitochondrial uncoupling. This effect on lipid peroxidation is prevented by carboxyatractyloside and oligomycin.

Topics: Adenosine Diphosphate; Animals; Benzene Derivatives; Butylated Hydroxytoluene; Dicyclohexylcarbodiimide; Electrochemistry; Lipid Peroxidation; Membrane Potentials; Mitochondria, Liver; Oligomycins; Onium Compounds; Organophosphorus Compounds; Proton-Translocating ATPases; Rats

Presumptive evidence suggests that the brown fat mitochondrial uncoupling protein, thermogenin, is involved in the mechanism of stimulation of respiration by norepinephrine in the intact tissue. Conflicting data have been reported which suggest involvement of either adenine nucleotides, or fatty acids, or long chain acyl-CoA, or protons in the physiological regulation. We measured the electrical potential gradient across the mitochondrial membrane (delta psi m) in control cells and in cells stimulated with norepinephrine, using the accumulation of lipophilic cation, tetraphenylphosphonium, as an indicator of the potential gradient. The value of delta psi m in the cells in the control state is 116 mV, and in the hormonally stimulated state it is 56.6 mV. This supports the view that the protein is involved in the mechanism of hormone action. Other studies were designed to distinguish between the effects of fatty acids and ATP levels on the uncoupling protein in isolated mitochondria and in the adipocytes. ATP levels and fatty acid levels inside intact cells were independently varied using oligomycin or external fatty acids. Their effect on thermogenin was monitored as the capacity of the cells for reverse electron transport from durohydroquinone. The results suggest that ATP modulates the activity of thermogenin, while fatty acids can alter the relationship between ATP and thermogenin activity such that the protein appears to be activated at a higher cellular ATP level in the presence of fatty acids than in their absence.

Topics: Adenosine Triphosphate; Adipose Tissue, Brown; Animals; Carrier Proteins; Fatty Acids; Fatty Acids, Nonesterified; Intracellular Membranes; Ion Channels; Mathematics; Membrane Potentials; Membrane Proteins; Mitochondrial Proteins; Norepinephrine; Oligomycins; Onium Compounds; Organophosphorus Compounds; Oxygen Consumption; Serum Albumin, Bovine; Uncoupling Protein 1

Accumulation of the permeant lipophilic cation [(3)H]tetraphenylphosphonium (TPP(+)) by synaptosome preparations from guinea pig brain cerebral cortex is inhibited 1:10 by medium containing 193 mM K(+) and by veratridine. A further 1:10 to 1:15 decrease in TPP(+) uptake occurs under nitrogen and in the presence of mitochondrial inhibitors such as oligomycin, whereas starvation and succinate supplementation have no effect. These data indicate that, in analogy to intact neurons, there is an electrical potential (DeltaPsi, interior negative) of -60 to -80 mV across the synaptosomal membrane that is due primarily to a K(+) diffusion gradient (K(+) (in)-->K(+) (out)). The data also indicate that mitochondria entrapped within the synaptosome but not free mitochondria make a large contribution to the TPP(+) concentration gradients observed. Conditions are defined in which tetanus toxin binds specifically and immediately to synaptosomes in media used to measure TPP(+) uptake. Under these conditions tetanus toxin induces dose-dependent changes in TPP(+) uptake that are blocked by antitoxin and not mimicked by biologically inactivated toxin preparations. The effect of tetanus toxin on TPP(+) uptake is not evident in the presence of 193 mM K(+) or veratridine but remains under conditions known to abolish the mitochondrial DeltaPsi. Moreover, tetanus toxin has no effect on TPP(+) uptake by isolated synaptosomal mitochondria. The results thus define an in vitro action of tetanus toxin on the synaptosomal membrane that can be correlated with biological potency in vivo and is consistent with the in vivo effects of tetanus toxin on neuronal transmission.

Topics: Anaerobiosis; Animals; Biological Transport; Brain; Guinea Pigs; Kinetics; Male; Oligomycins; Onium Compounds; Organophosphorus Compounds; Potassium; Synaptosomes; Tetanus Toxin