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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.
J Neurosci Res. 2003 Aug 01; 73(3):351-63.JN

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+Show Affiliations

Laboratory of Molecular Recognition, Graduate School of Integrated Science, Yokohama City University, Yokohama, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

12868069

Citation

Yuyama, Kohei, et al. "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." Journal of Neuroscience Research, vol. 73, no. 3, 2003, pp. 351-63.
Yuyama K, Yamamoto H, Nishizaki I, et al. 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. J Neurosci Res. 2003;73(3):351-63.
Yuyama, K., Yamamoto, H., Nishizaki, I., Kato, T., Sora, I., & Yamamoto, T. (2003). 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. Journal of Neuroscience Research, 73(3), 351-63.
Yuyama K, et al. 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. J Neurosci Res. 2003 Aug 1;73(3):351-63. PubMed PMID: 12868069.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Yuyama,Kohei, AU - Yamamoto,Hideko, AU - Nishizaki,Itone, AU - Kato,Takeshi, AU - Sora,Ichiro, AU - Yamamoto,Toshifumi, PY - 2003/7/18/pubmed PY - 2003/9/5/medline PY - 2003/7/18/entrez SP - 351 EP - 63 JF - Journal of neuroscience research JO - J Neurosci Res VL - 73 IS - 3 N2 - 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. SN - 0360-4012 UR - https://www.unboundmedicine.com/medline/citation/12868069/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_ L2 - https://doi.org/10.1002/jnr.10669 DB - PRIME DP - Unbound Medicine ER -