oligomycins and oxophenylarsine

Alternatives for the treatment of amyotrophic lateral sclerosis (ALS) are scarce and controversial. The etiology of neuronal vulnerability in ALS is being studied in motor neuron-like NSC-34 cells to determine the underlying mechanisms leading to selective loss of motor neurons. One such mechanism is associated with mitochondrial oxidative stress, Ca(2+) overload, and low expression of Ca(2+)-buffering proteins. Therefore, in order to elicit neuronal death in ALS, NSC-34 cells were exposed to the following cytotoxic agents: (1) a mixture of oligomycin 10 µM and rotenone 30 µM (O/R), or (2) phenylarsine oxide 1 µM (PAO) (to mimic excess free radical production during mitochondrial dysfunction), and (3) veratridine 100 µM (VTD) (to induce overload of Na(+) and Ca(2+) and to alter distribution of Ca(2+)-buffering proteins [parvalbumin and calbindin-D28k]). Thus, the aim of the study was to test the novel neuroprotective compound ITH33/IQM9.21 (ITH33) and to compare it with riluzole on in vitro models of neurotoxicity. Cell viability measured with MTT showed that only ITH33 protected against O/R at 3 μM and PAO at 10 μM, but not riluzole. ITH33 and riluzole were neuroprotective against VTD, blocked the maximum peak and the number of [Ca(2+)]c oscillations per cell, and restored the effect on parvalbumin. However, only riluzole reversed the effect on calbindin-D28k levels. Therefore, ITH33 was neuroprotective against oxidative stress and Na(+)/Ca(2+) overload, both of which are involved in ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Arsenicals; Benzamides; Calbindin 1; Calcium; Cell Survival; Drug Evaluation, Preclinical; Glutamates; Mice; Mitochondria; Motor Neurons; Neuroprotective Agents; Oligomycins; Oxidative Stress; Parvalbumins; Riluzole; Rotenone; Sodium; Veratridine

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