Protein kinase C expression and subcellular distributions by hypoxia

缺氧/缺血后神经元变性的机制尚不清楚，但其过程包括神经递质释放增加，胞浆游离钙浓度升高，以及信号转导途径的改变。蛋白激酶C(PKC)多基因家族的激活与神经递质的释放和神经元的存活密切相关。然而，特定的PKC同工酶在调节这种反应中的作用还不是很清楚。本研究的目的是研究PKC同工酶的表达及其在调节细胞损伤中的作用。因此，为了了解哪些PKC同工酶参与了缺氧/缺血诱导的神经元变性，我们检测了低氧(1%02)或KCN暴露后的大鼠嗜铬细胞瘤细胞(PC12细胞)中的PKC同工酶。PC12细胞系在含10%(v/v)胎牛血清、5%(v/v)马血清的RPMI1640培养液中培养37℃。用KCN或多气体培养箱(APMW-36，ASTEC株式会社，福冈)诱导缺氧。乳酸脱氢酶(LDH)法检测细胞活性。免疫印迹法检测各亚细胞组份中PKCs的蛋白水平。PC12细胞暴露于低氧(1%02)和KCN后，细胞存活率呈时间依赖性下降。KCN对无糖RPMI1640细胞的毒性作用明显增强。结果表明，在低氧条件下，PC12细胞膜和线粒体发生选择性易位，只有钙依赖的γ同工酶显著增加，其他同工酶无明显增加。因此，我们认为低氧诱导的PKC-γ的激活/转位在低氧诱导的PC12细胞损伤的调控中具有重要作用。

The mechanisms of neuronal degeneration following hypoxia/ischemia remain undefined, but the processes include increases in neurotransmitter release, elevation of cytosolic-free calcium concentration, and changes in signal transduction pathways. Activation of the multigene family of protein kinase C (PKC) has been associated with the release of neurotransmitter and the survival of neurons. The roles for specific PKC isozymes in regulating this response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC isozymes expression in regulating cellular damage. Therefore, to understand which PKC isozymes are involved in hypoxia/ischemia-induced neuronal degeneration, we examined PKC isozymes after hypoxia (1 % 02) or KCN exposure in rat pheochromocytoma cells (PC12 cells). A PC12 cell line was maintained in RPMI 1640 medium supplemented with 10 % (v/v) FBS, 5 % (v/v) horse serum in a humidified atmosphere containing 5 % CO2 at 37℃. Hypoxia was induced with KCN or a multigas incubator (APMW-36, ASTEC Co. Ltd, Fukuoka). The cell viability was assessed by the lactate dehydrogenase (LDH) assay. The protein levels of PKCs in the subcellular fractions were measured by immunoblot analysis. PC12 cells underwent a time-dependent decrease in cell viability after exposure to hypoxia (1 % 02) and KCN. Cell toxicity was increased significantly by KCN in glucose free RPMI 1640. Data shows that selective translocation of specific PKC isozymes occurs during hypoxia, with only calcium-dependent PKC-γ and not the other PKC isozymes increasing significantly hi the membrane and mitochondria fractions after KCN treatment of PC12 cells. Therefore, we proposed that activation/translocation of PKC-γ by hypoxia is important in the regulation of hypoxia-induced cell injury in PC 12 cells.