Mechanism of nitric oxide-induced apoptosis in human neuroblastoma SH-SY5Y cells.
We have attempted to elucidate the precise mechanism of nitric oxide (NO)-induced apoptotic neuronal cell death. Enzymatic cleavages of DEVD-AFC, VDVAD-AFC, and LEHD-AFC (specific substrates for caspase-3-like protease (caspase-3 and -7), caspase-2, and caspase-9, respectively) were observed by treatment with NO. Western blot analysis showed that pro-forms of caspase-2, -3, -6, and -7 are decreased during apoptosis. Interestingly, Ac-DEVD-CHO, a caspase-3-like protease inhibitor, blocked not only the decreases in caspase-2 and -7, but also the formation of p17 from p20 in caspase-3 induced by NO, suggesting that caspase-3 exists upstream of caspase-2 and -7. Bongkrekic acid, a potent inhibitor of mitochondrial permeability transition, specifically blocked both the loss of mitochondrial membrane potential and subsequent DNA fragmentation in response to NO. Thus, NO results in neuronal apoptosis through the sequential loss of mitochondrial membrane potential, caspase activation, and degradation of inhibitor of caspase-activated DNase (CAD) (CAD activation).
Moriya
R
R
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, 060-0812, Sapporo, Japan.
Uehara
T
T
Nomura
Y
Y
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001865:Bongkrekic Acid / Q000494:pharmacology; D053143:Caspase 2; D053148:Caspase 3; D053179:Caspase 7; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D003374:Coumarins / Q000494:pharmacology; D053938:DNA Fragmentation; D004789:Enzyme Activation; D006801:Humans; D007425:Intracellular Membranes / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000502:physiology; D009447:Neuroblastoma; D009569:Nitric Oxide / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D003374:Coumarins; D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C115495:aspartyl-glutamyl-valyl-aspartyl-7-amino-4-trifluoromethylcoumarin; D001865:Bongkrekic Acid; D009569:Nitric Oxide; C505407:CASP3 protein, human; C505520:CASP7 protein, human; C506433:CASP9 protein, human; D053143:Caspase 2; D053148:Caspase 3; D053179:Caspase 7; D053453:Caspase 9; D020169:Caspases