title: Caspase-independent cell death by low concentrations of nitric oxide in PC12 cells: involvement of cytochrome C oxidase inhibition and the production of reactive oxygen species in mitochondria.
abstract: We reported previously that low levels of nitric oxide (NO) induced cell death with properties of apoptosis, including chromatin fragmentation and condensation in undifferentiated PC12 pheochromocytoma cells. The present study demonstrates that cytotoxicity of low concentrations of NO is mediated by inhibition of mitochondrial cytochrome c oxidase and generation of reactive oxygen species (ROS). An NO donor, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3) induced cell death even at low concentrations (10-100 microM), whereas peroxynitrite and a peroxynitrite generator, 3-(4-morpholinyl)-sydnonimine (SIN-1), did not have a significant effect on cell viability up to a concentration of 0.5 mM. The NOR3-induced cell death was unaffected by pretreatment with superoxide dismutase (SOD) or its mimetic peroxynitrite scavenger, manganese(III) tetrakis(benzoic acid)porphyrin chloride (Mn-TBAP), or with uric acid. These findings indicate that peroxynitrite does not contribute to this cell death. Furthermore, neither the release of cytochrome c from mitochondrial membranes, the cleavage of poly-ADP ribose polymerase (PARP), nor the activation of caspase-3-like activities was observed. Inhibitors of PARP, benzamide, and aminobenzamide, had no effect on the NOR3-induced cell death. In addition, pretreatment with general or selective caspase inhibitors, benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), and benzyloxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-Ch(2)-DCB) did not prevent NOR3-induced cell death. Taken together, these findings suggest that cell death induced by NOR3 occurs by a caspase-independent mechanism. In contrast, we found an early increase in mitochondrial H(2)O(2) production during NOR3 exposure using the fluorescent dye 2',7'-dichlorofluorescin-diacetate (DCFH-DA) and dihydrorohdamine123 (DHR123), and these events were accompanied by strong inhibition of cytochrome c oxidase activity in the cells. Furthermore, we observed that several antioxidants, such as ascorbate, glutathione (GSH), cysteine, tetrahydrobiopterin, and dithiothreitol (DTT), all effectively prevented the NOR3-induced cell death. NOR3 treatment decreased the level of total intracellular GSH, but did not affect the activities of antioxidant enzymes SOD, GSH-peroxidase (GPX), and catalase. These results suggest that cell death induced at physiologically low concentrations of NO is mediated by ROS production in mitochondria, most likely resulting from the inhibition of cytochrome c oxidase, with ROS acting as an initiator of caspase-independent cell death.
authors.affiliation: Laboratory of Molecular Recognition, Graduate School of Integrated Science, Yokohama City University, Yokohama, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Yuyama
authors.forename: Hideko
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Itone
authors.initials: I
authors.lastname: Nishizaki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kato
authors.forename: Ichiro
authors.initials: I
authors.lastname: Sora
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009574:Nitro Compounds / Q000494:pharmacology; D016716:PC12 Cells; D030421:Peroxynitrous Acid / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000975:Antioxidants; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D020030:Nitric Oxide Donors; D009574:Nitro Compounds; D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C061872:FK 409; D003576:Electron Transport Complex IV; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases