Yoshimura S, Banno Y, Nakashima S, Takenaka K, Sakai H, Nishimura Y, Sakai N, Shimizu S, Eguchi Y, Tsujimoto Y, Nozawa Y
Department of Neurosurgery, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500-8705, Japan.
J Biol Chem. 1998 Mar 20;273(12):6921-7. doi: 10.1074/jbc.273.12.6921.
PC12 cells undergo apoptosis as well as necrosis following exposure to hypoxia. Following a 6-h hypoxic treatment, a time-dependent increase in intracellular ceramide level was observed with a concurrent decrease in sphingomyelin. It was also shown that the hypoxia-induced ceramide accumulation resulted from activation of neutral magnesium-dependent sphingomyelinase. Comparative kinetic analyses of the neutral sphingomyelinase in the cells under normoxia and hypoxia showed that hypoxia increased Vmax but did not affect Km of the enzyme. In PC12 cells overexpressing Bcl-2 which show strong resistance to hypoxia, sphingomyelin hydrolysis was decreased and activation of neutral sphingomyelinase was reduced. Addition of exogenous C2-ceramide induced cell death and activated caspase-3 as markedly as the hypoxia treatment. On the other hand, in PC12 cells overexpressing Bcl-2, significant decreases in cell death and inhibition of caspase-3 activation were observed after exogenous addition of C2-ceramide. The inhibitors of caspase-3 prevented cell death by either hypoxia or C2-ceramide. These results suggest that ceramide generated by activation of neutral magnesium-dependent sphingomyelinase mediates hypoxic cell death and that Bcl-2 has inhibitory effects on ceramide formation and caspase activation.
PC12细胞在暴露于缺氧环境后会发生凋亡以及坏死。经过6小时的缺氧处理后，观察到细胞内神经酰胺水平随时间增加，同时鞘磷脂减少。研究还表明，缺氧诱导的神经酰胺积累是由中性镁依赖性鞘磷脂酶的激活引起的。对常氧和缺氧条件下细胞中的中性鞘磷脂酶进行的比较动力学分析表明，缺氧增加了该酶的Vmax，但不影响其Km。在过表达Bcl-2且对缺氧具有强抗性的PC12细胞中，鞘磷脂水解减少，中性鞘磷脂酶的激活也降低。添加外源性C2-神经酰胺诱导细胞死亡并激活caspase-3，其程度与缺氧处理一样显著。另一方面，在过表达Bcl-2的PC12细胞中，外源性添加C2-神经酰胺后，细胞死亡显著减少，caspase-3激活受到抑制。caspase-3抑制剂可防止缺氧或C2-神经酰胺诱导的细胞死亡。这些结果表明，中性镁依赖性鞘磷脂酶激活产生的神经酰胺介导了缺氧诱导的细胞死亡，并且Bcl-2对神经酰胺形成和caspase激活具有抑制作用。
