atractyloside 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

Alzheimer's disease (AD) brains exhibit oxidative stress and a biochemical defect of complex IV (cytochrome oxidase, COX) of the mitochondrial electron transport chain (ETC). This defect can be transferred through mitochondrial DNA (mtDNA) into clonal SY5Y cells depleted of their mtDNA. The resulting cytoplasmic hybrids or "cybrids" retain the complex IV defect and exhibit oxidative stress. We measured the mitochondrial membrane potential (delta psi m) in AD and control cybrids via H3-tetraphenylphosphonium ion (H3-TPP+) accumulation. AD cybrids exhibited a significant (about 30%) decrease in H3-TPP+ accumulation relative to controls. Acute treatment of normal SY5Ys with azide, a COX inhibitor, moderately decreased H3-TPP+ retention and strongly inhibited COX activity in a dose-dependent manner. As the mitochondrial transition pore (MTP) can be activated by reactive oxygen species and ETC inhibitors, and its opening causes delta psi m dissipation, we tested the effects of the MTP inhibitor cyclosporin A (CsA) on TPP+ accumulation. 5mM CsA increased basal H3-TPP+ accumulation in SY5Y cells about 10-fold, corresponding to about a 2-fold increase in delta psi m. In the AD cybrids, CsA increased the apparent delta psi m to the same final levels as it did in controls. These results indicate that low-conductance MTP activity contributes significantly to resting delta psi m in SY5Y cells. We propose the novel hypothesis that the COX defect and resulting oxidative stress in AD may pathologically activate the MTP, resulting in lower delta psi m and the release of mitochondrial factors involved in apoptosis.

Topics: Alzheimer Disease; Atractyloside; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cyclosporine; DNA, Mitochondrial; Electron Transport Complex IV; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Monoamine Oxidase; Onium Compounds; Organophosphorus Compounds; Sodium Azide; Tumor Cells, Cultured

We studied the relative potencies of cyclosporin A and endogenous effectors (Mg2+ and ADP) to recouple rat liver mitochondria permeabilized by different Ca(2+)-loading in a P(i)-containing medium. Recoupling efficiency of cyclosporin A dramatically decreased at high Ca(2+)-loading (approx. 100 nM of Ca2+/mg protein and more). Mitochondria permeabilized by high Ca2+ were recoupled with approximately equal efficiency by higher cyclosporin A concentrations or by adding 1-5 mM Mg2+ together with low concentrations of cyclosporin A while potentiating effect of ADP on the cyclosporin A recoupling potency was insignificant. Mg2+ ions at concentrations of 3 mM and higher also prevented the carboxyatractylate-induced reversion of cyclosporin A recoupling effect. The data point to competitive relationships between cyclosporin A and/or Mg2+ ions and Ca2+ ions for the site(s) regulating permeability state of the pore.

Topics: 2,4-Dinitrophenol; Adenosine Diphosphate; Animals; Atractyloside; Calcium Chloride; Cyclosporine; Dinitrophenols; Egtazic Acid; Magnesium Sulfate; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Onium Compounds; Organophosphorus Compounds; Permeability; Rats

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