valinomycin and oxophenylarsine

The endogenous divalent cations, Ca(2+) and Zn(2+), are both highly toxic upon excessive glutamate triggered intracellular accumulation. Given apparent parallels in their neurotoxic mechanisms, the present study aimed to explore interactions between these cations, by examining effects of moderate intracellular Zn(2+) loading on responses to subsequent Ca(2+) influx. Cortical cultures were briefly exposed to high-K(+) buffer in the presence or absence of Zn(2+) (50-100 microM), to activate and permit a modestly toxic amount of Zn(2+) to enter through VSCC. After 1 h, the cultures were loaded with fluorescent probes, and 2 h after the Zn(2+) exposure, imaged before and after induction of Ca(2+) entry or addition of other drugs. In Zn(2+) preexposed cultures loaded with the Zn(2+) probe, Newport Green, induction of Ca(2+) entry through either VSCC or NMDA channels induced cytoplasmic release of sequestered Zn(2+). The source of this Ca(2+) dependent intracellular Zn(2+) release appears largely to be mitochondria, as indicated by the ability of the mitochondrial protonophore, FCCP, the mitochondrial uncoupler, dinitrophenol with the K(+) ionophore, valinomycin, or the inducer of mitochondrial permeability transition (mPT), phenylarsine oxide (PAO), to substitute for NMDA in triggering Zn(2+) release. Suggesting functional consequences of mitochondrial Zn(2+) uptake, Zn(2+) preexposures resulted in long-lasting mitochondrial depolarization (assessed with rhodamine 123), and reduced mitochondrial reactive oxygen species generation (assessed with hydroethidine) in response to subsequent NMDA triggered Ca(2+) influx.

Topics: Animals; Arsenicals; Calcium; Calcium Channels; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Compartmentation; Cerebral Cortex; Cytosol; Dinitrophenols; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Intracellular Membranes; Ion Channel Gating; Ion Transport; Ionophores; Membrane Potentials; Mice; Mitochondria; Nerve Tissue Proteins; Neurons; Potassium; Reactive Oxygen Species; Receptors, N-Methyl-D-Aspartate; Synaptic Vesicles; Thapsigargin; Uncoupling Agents; Valinomycin; Zinc

We studied the modulation of the permeability transition pore (MTP), a cyclosporin-A-sensitive channel, in deenergized mitochondria. Rat liver mitochondria were incubated in a potassium gluconate medium and treated with uncoupler, valinomycin, oligomycin and A23187. Under these conditions the membrane and Donnan potentials are collapsed, and no ion gradients are maintained, allowing the study of the dependence of MTP opening on the Ca2+ concentration under a variety of oxidative conditions without the complexities arising from changes of the membrane potential and matrix pH, and from secondary-ion redistribution. Cross-linking of mitochondrial dithiols with arsenite or phenylarsine oxide, or treatment with tert-butylhydroperoxide leading to complete oxidation of glutathione, increased the sensitivity of MTP opening to Ca2+. This effect could be fully prevented by prior treatment of mitochondria with monobromobimane and restored by reduction with dithiothreitol. The effect of tert-butylhydroperoxide was not additive with that of AsO, and it was completely blocked by modification of reduced glutathione with 1-chloro 2,4-dinitrobenzene through glutathione-S-transferase, indicating that oxidized glutathione affects the pore through the AsO-reactive and PhAsO-reactive dithiol. Oxidation of mitochondrial pyridine nucleotides by a variety of treatments also increased the sensitivity of MTP opening to Ca2+ under conditions where glutathione was maintained in the reduced state. This effect could be fully prevented when reduced pyridine nucleotides levels were reestablished with 2-hydroxybutyrate but not by treatment with monobromobimane or dithiothreitol. The effects of dithiol cross-linking or oxidation, and of pyridine nucleotide oxidation on the MTP were additive. The contribution of each of these two oxidative events, when they were induced at the same time, could still be selectively blocked by monobromobimane and dithiothreitol. The effects of dithiol cross-linking or oxidation, and of pyridine nucleotide oxidation on the MTP were additive. The contribution of each of these two oxidative events,when they were induced at the same time, could still be selectively blocked by monobromobimane and dithiothreitol, or by 2-hydroxybutyrate, respectively. Complete oxidation of pyridine nucleotides did not affect the reactivity of the dithiol towards monobromobimane, indicating that it remained in the reduced state. After transient opening of the MTP, sensitivity to py

Topics: Animals; Arsenicals; Calcimycin; Calcium; Cross-Linking Reagents; Glutathione; Intracellular Membranes; Ion Channel Gating; Mitochondria, Liver; NAD; NADP; Nucleotides; Oligomycins; Oxidants; Oxidation-Reduction; Oxidative Stress; Permeability; Pyridines; Rats; Sulfhydryl Compounds; Valinomycin