Yuyama Kohei, Yamamoto Hideko, Nishizaki Itone, Kato Takeshi, Sora Ichiro, Yamamoto Toshifumi
Laboratory of Molecular Recognition, Graduate School of Integrated Science, Yokohama City University, Yokohama, Japan.
J Neurosci Res. 2003 Aug 1;73(3):351-63. doi: 10.1002/jnr.10669.
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.
我们之前报道过，低水平的一氧化氮（NO）会诱导未分化的PC12嗜铬细胞瘤细胞发生具有凋亡特征的细胞死亡，包括染色质碎片化和凝聚。本研究表明，低浓度NO的细胞毒性是通过抑制线粒体细胞色素c氧化酶和产生活性氧（ROS）介导的。一种NO供体，（±）-（E）-4-乙基-2-[(E)-羟基亚氨基]-5-硝基-3-己烯酰胺（NOR3）即使在低浓度（10 - 100 microM）时也能诱导细胞死亡，而过氧亚硝酸盐和过氧亚硝酸盐生成剂3-(4-吗啉基)-西多胺（SIN-1）在浓度高达0.5 mM时对细胞活力没有显著影响。NOR3诱导的细胞死亡不受超氧化物歧化酶（SOD）或其模拟过氧亚硝酸盐清除剂四（苯甲酸）氯化锰（III）卟啉（Mn-TBAP）或尿酸预处理的影响。这些发现表明过氧亚硝酸盐对这种细胞死亡没有作用。此外，未观察到细胞色素c从线粒体膜释放、聚ADP核糖聚合酶（PARP）的裂解或caspase-3样活性的激活。PARP抑制剂苯甲酰胺和氨基苯甲酰胺对NOR3诱导的细胞死亡没有影响。此外，用一般或选择性caspase抑制剂苄氧羰基-Val-Ala-Asp-氟甲基酮（Z-VAD-fmk）、N-乙酰-Asp-Glu-Val-Asp-醛（Ac-DEVD-CHO）和苄氧羰基-Asp-2,6-二氯苯甲酰氧基甲基酮（Z-Asp-Ch(2)-DCB）预处理并不能阻止NOR3诱导的细胞死亡。综上所述，这些发现表明NOR3诱导的细胞死亡是通过一种不依赖caspase的机制发生的。相比之下，我们发现使用荧光染料2',7'-二氯荧光素二乙酸酯（DCFH-DA）和二氢罗丹明123（DHR123）在NOR3暴露期间线粒体H(2)O(2)产生早期增加，并且这些事件伴随着细胞中细胞色素c氧化酶活性的强烈抑制。此外，我们观察到几种抗氧化剂，如抗坏血酸、谷胱甘肽（GSH）、半胱氨酸、四氢生物蝶呤和二硫苏糖醇（DTT），都能有效阻止NOR3诱导的细胞死亡。NOR3处理降低了细胞内总GSH水平，但不影响抗氧化酶SOD、谷胱甘肽过氧化物酶（GPX）和过氧化氢酶的活性。这些结果表明，生理低浓度NO诱导的细胞死亡是由线粒体中ROS产生介导的，最有可能是由于细胞色素c氧化酶的抑制，ROS作为不依赖caspase的细胞死亡的启动因子。
