Takai N, Nakanishi H, Tanabe K, Nishioku T, Sugiyama T, Fujiwara M, Yamamoto K
Department of Pharmacology, Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
J Neurosci Res. 1998 Oct 15;54(2):214-22. doi: 10.1002/(SICI)1097-4547(19981015)54:2<214::AID-JNR9>3.0.CO;2-H.
Activation of proteolytic enzymes, including the caspase family of proteinases, is a feature characteristic of the apoptotic program. In the present study, we examined a potential role of intracellular proteinases in the death of neuronal PC12 and primary cultured rat microglial cells induced by 6-hydroxydopamine (6-OHDA). In both neuronal PC12 and microglial cells, 6-OHDA (10-200 microM) induced apoptosis in a dose-dependent manner as judged by the DNA break. The 6-OHDA was ineffective in Bcl-2-overexpressing neuronal PC12 cells. Pretreatment of these cells with two caspase inhibitors, acetyl-Try-Val-Ala-Asp-aldehyde and acetyl-Asp-Glu-Val-Asp-aldehyde, prevented the 6-OHDA-induced apoptosis. Pepstatin A and leupeptin, potent inhibitors of aspartic and cysteine proteinases, respectively, partly inhibited the apoptosis of microglia but not neuronal PC12 cells. In contrast, GBR12935, a dopamine uptake inhibitor, significantly inhibited the apoptotic death of neuronal PC12 cells but not microglia. These results suggest that mechanisms by which 6-OHDA induces apoptosis in these two cell types are distinct; 6-OHDA incorporated into neuronal PC12 cells and its metabolites may activate the caspase-like enzymes, whereas oxidative metabolites of the agent produced extracellularly may activate the caspase and the endosomal/lysosomal proteolytic systems in microglia.
包括半胱天冬酶蛋白酶家族在内的蛋白水解酶的激活是凋亡程序的一个特征。在本研究中，我们研究了细胞内蛋白酶在6-羟基多巴胺（6-OHDA）诱导的神经元PC12细胞和原代培养的大鼠小胶质细胞死亡中的潜在作用。在神经元PC12细胞和小胶质细胞中，6-OHDA（10-200 microM）以剂量依赖的方式诱导凋亡，这通过DNA断裂来判断。6-OHDA对过表达Bcl-2的神经元PC12细胞无效。用两种半胱天冬酶抑制剂乙酰-色氨酸-缬氨酸-丙氨酸-天冬氨酸醛和乙酰-天冬氨酸-谷氨酸-缬氨酸-天冬氨酸醛预处理这些细胞可防止6-OHDA诱导的凋亡。胃蛋白酶抑制剂A和亮抑蛋白酶肽分别是天冬氨酸和半胱氨酸蛋白酶的有效抑制剂，它们部分抑制小胶质细胞的凋亡，但不抑制神经元PC12细胞的凋亡。相反，多巴胺摄取抑制剂GBR12935显著抑制神经元PC12细胞的凋亡性死亡，但不抑制小胶质细胞的凋亡。这些结果表明，6-OHDA在这两种细胞类型中诱导凋亡的机制是不同的；进入神经元PC12细胞的6-OHDA及其代谢产物可能激活半胱天冬酶样酶，而细胞外产生的该药物的氧化代谢产物可能激活小胶质细胞中的半胱天冬酶和内体/溶酶体蛋白水解系统。
