Kirkland R A, Franklin J L
Department of Neurological Surgery, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
J Neurosci. 2001 Mar 15;21(6):1949-63. doi: 10.1523/JNEUROSCI.21-06-01949.2001.
Sympathetic neurons die by apoptosis when they are deprived of nerve growth factor (NGF). Activation of caspases by cytochrome c released from mitochondria is central to this death. In this report we present evidence that cellular redox state regulates cytochrome c redistribution in these neurons. An increase of mitochondrial-produced reactive oxygen species (ROS) occurred in rat sympathetic neurons in cell culture within 3 hr of NGF withdrawal. Caspase inhibitors blocked this ROS burst. By 6 hr after NGF deprivation, glutathione (GSH) levels had increased, neutralizing elevated hydrogen peroxide levels and returning cellular redox state to basal levels. By 12 hr after deprivation, ROS levels had again increased and remained elevated during the rest of the apoptotic process. The later ROS burst appeared to have both caspase-dependent and caspase-independent components and was coincident with the period of cytochrome c release. Inhibition of protein synthesis with cycloheximide (CHX) and treatment with the antioxidant compound, N-acetyl-l-cysteine (l-NAC), blocked both the early and late ROS bursts by increasing cellular GSH levels (Ratan et al., 1994; Tan et al., 1998). Both compounds, and a membrane-permeant form of GSH, also inhibited cytochrome c release and death. Treatment of NGF, CHX-, l-NAC-, and GSH-saved cells with hydrogen peroxide caused rapid cytochrome c release. These data suggest a role for cellular redox state in regulating cytochrome c release during apoptosis induced by NGF withdrawal.
当交感神经元被剥夺神经生长因子（NGF）时，会通过凋亡而死亡。线粒体释放的细胞色素c激活半胱天冬酶是这种死亡的核心机制。在本报告中，我们提供证据表明细胞氧化还原状态调节这些神经元中细胞色素c的重新分布。在细胞培养中，大鼠交感神经元在NGF撤除后3小时内线粒体产生的活性氧（ROS）增加。半胱天冬酶抑制剂可阻断这种ROS爆发。在NGF剥夺后6小时，谷胱甘肽（GSH）水平升高，中和了升高的过氧化氢水平，并使细胞氧化还原状态恢复到基础水平。在剥夺后12小时，ROS水平再次升高，并在凋亡过程的其余时间保持升高。后期的ROS爆发似乎具有半胱天冬酶依赖性和半胱天冬酶非依赖性成分，并且与细胞色素c释放的时期一致。用放线菌酮（CHX）抑制蛋白质合成以及用抗氧化剂化合物N-乙酰-L-半胱氨酸（L-NAC）处理，通过增加细胞GSH水平来阻断早期和晚期的ROS爆发（拉坦等人，1994年；谭等人，1998年）。这两种化合物以及一种膜渗透性形式的GSH也抑制细胞色素c的释放和细胞死亡。用过氧化氢处理NGF、CHX、L-NAC和GSH挽救的细胞会导致细胞色素c迅速释放。这些数据表明细胞氧化还原状态在调节NGF撤除诱导的凋亡过程中细胞色素c释放方面发挥作用。
