Oxidative and nitrative stress in relation to mitochondrial dysfunction in liver and heart cells

The immunosuppressant, cyclosporin A (CsA), inhibits mitochondrial injury caused by oxidative and nitrative stress by preventing the mitochondrial permeability transition (MPT) from occurring. However, the usefulness of CsA as an inhibitor of oxidative and nitrative stress-induced mitochondrial injury is unclear due to its own toxicity and ineffectiveness against all types of injury. Therefore, the goal of this research project was to study mechanisms for how oxidative and nitrative stresses induce mitochondria dysfunction that results in cell death to liver and heart cells.; The first aim was to develop assays to assess mitochondrial dysfunction following oxidative and nitrative stress. Isolated rat liver mitochondria were used to develop high throughput screening assays to assess changes in mitochondrial swelling, inner membrane depolarization and permeabilization, all changes associated with the MPT. Using a multiwell fluorescence plate reader, changes in fluorescence and light scattering were measured following addition of Ca2+ and oxidants. In these assays, CsA and the non-immunosuppressive cyclosporin, NIM811, inhibited the MPT with similar efficacy. Furthermore, the intracellular Ca2+ modulator, 2-aminoethoxydiphenyl borate delayed the CsA-insensitive MPT induced by high-doses of the oxidants, phenylarsine oxide and mercuric chloride.; Next studied was the role of reactive nitrogen species (RNS) formation during hypoxia and ischemia. Increased RNS formation was observed by nitrotyrosine staining in liver and heart. However, only RNS produced during hypoxia was prevented by nitric oxide synthase (NOS) inhibition. Hepatocellular RNS production was increased during ischemia as assessed by an increase in DAF-FM fluorescence. This increase was pH-dependent, only occurring at acidic pH, and was unaffected by NOS or xanthine oxidase modulators or mitochondrial respiratory inhibitors.; Finally, mitochondrial dysfunction was studied using a model of pH-dependent ischemia/reperfusion in neonatal myocytes. Mitochondria dysfunction was observed by a decrease in mitochondrial membrane potential using TMRM fluorescence. However, acidic reperfusion and pyruvate improved repolarization and cell viability. Glycine also improved viability, but independent of mitochondrial function. MPT inhibitors offered further protection when added in combination with a respiratory substrate suggesting the MPT is involved. Taken together, these data suggest the MPT is an important mechanism of mitochondrial injury resulting from oxidative and nitrative stress.