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 inhibition and apoptosis by opening the permeability transition pore. Further, our results indicate that the mode of cell death (
necrosis versus apoptosis) induced by either NO or mitochondrial inhibitors depends critically on the glycolytic capacity of the cell.