myxothiazol and Necrosis

We tested whether nitric oxide (NO) could synergize with hypoxia to induce damage to the aorta isolated from rat. We found that 4 h of mild hypoxia (5% O2) caused substantial necrosis of isolated rat aortae (measured as lactate dehydrogenase release) if inducible NO synthase (iNOS) had previously been induced by endotoxin plus interferon-gamma. Mild hypoxia caused no significant necrosis in the absence of this inflammatory activation, and inflammatory activation caused little damage at a higher oxygen levels (21% oxygen). An iNOS inhibitor (1400W) prevented the necrosis induced by inflammation plus mild hypoxia, whereas the NO donor diethylenetriamine (DETA)/NO adduct, 0.5 mM) greatly sensitized the noninflammed aorta to necrosis induced by mild hypoxia. NO inhibited aortic respiration to a greater degree at lower oxygen concentrations, consistent with NO inhibition of cytochrome oxidase in competition with oxygen. A specific inhibitor of mitochondrial respiration, myxothiazol, caused necrosis of aortae over a similar time course to NO. DETA/NO plus mild hypoxia-induced cell death was substantially reduced by a glycolytic intermediate 3-phosphoglycerate, suggesting that necrosis resulted from energy depletion secondary to respiratory inhibition. This NO-induced sensitization of aorta to mild hypoxia may be important in sepsis and other pathologies where iNOS is expressed.

Topics: Animals; Antifungal Agents; Aorta; Enzyme Activation; Glyceric Acids; Glycolysis; Hypoxia; Inflammation; Male; Methacrylates; Mitochondria; Necrosis; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxygen; Polyamines; Rats; Rats, Wistar; Respiration; Thiazoles; Time Factors

Nitric oxide (NO) can trigger either necrotic or apoptotic cell death. We have used PC12 cells to investigate the extent to which NO-induced cell death is mediated by mitochondria. Addition of NO donors, 1 mM S-nitroso-N-acetyl-DL-penicillamine (SNAP) or 1 mM diethylenetriamine-NO adduct (NOC-18), to PC12 cells resulted in a steady-state level of 1-3 microM: NO, rapid and almost complete inhibition of cellular respiration (within 1 min), and a rapid decrease in mitochondrial membrane potential within the cells. A 24-h incubation of PC12 cells with NO donors (SNAP or NOC-18) or specific inhibitors of mitochondrial respiration (myxothiazol, rotenone, or azide), in the absence of glucose, caused total ATP depletion and resulted in 80-100% necrosis. The presence of glucose almost completely prevented the decrease in ATP level and the increase in necrosis induced by the NO donors or mitochondrial inhibitors, suggesting that the NO-induced necrosis in the absence of glucose was due to the inhibition of mitochondrial respiration and subsequent ATP depletion. However, in the presence of glucose, NO donors and mitochondrial inhibitors induced apoptosis of PC12 cells as determined by nuclear morphology. The presence of apoptotic cells was prevented completely by benzyloxycarbonyl-Val-Ala-fluoromethyl ketone (a nonspecific caspase inhibitor), indicating that apoptosis was mediated by caspase activation. Indeed, both NO donors and mitochondrial inhibitors in PC12 cells caused the activation of caspase-3- and caspase-3-processing-like proteases. Caspase-1 activity was not activated. Cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) decreased the activity of caspase-3- and caspase-3-processing-like proteases after treatment with NO donors, but was not effective in the case of the mitochondrial inhibitors. The activation of caspases was accompanied by the release of cytochrome c from mitochondria into the cytosol, which was partially prevented by cyclosporin A in the case of NO donors. These results indicate that NO donors (SNAP or NOC-18) may trigger apoptosis in PC12 cells partially mediated by opening the mitochondrial permeability transition pores, release of cytochrome c, and subsequent caspase activation. NO-induced apoptosis is blocked completely in the absence of glucose, probably due to the lack of ATP. Our findings suggest that mitochondria may be involved in both types of cell death induced by NO donors: necrosis by respiratory in

Topics: Adenosine Triphosphate; Animals; Apoptosis; Azides; Caspase Inhibitors; Caspases; Cell Respiration; Cyclosporine; Cytochrome c Group; Cytosol; Enzyme Inhibitors; Glucose; Lactic Acid; Membrane Potentials; Methacrylates; Mitochondria; Necrosis; Nitric Oxide; Nitric Oxide Donors; Oxygen Consumption; PC12 Cells; Rats; Thiazoles

Topics: Animals; Apoptosis; Azides; Caspases; Cysteine Proteinase Inhibitors; Glucose; Methacrylates; Mitochondria; Necrosis; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Oxygen Consumption; PC12 Cells; Rats; Thiazoles