Protein kinase C expression and subcellular distributions by hypoxia

缺氧时蛋白激酶 C 的表达和亚细胞分布

• 批准号: 11671508
• 负责人: TODOROKI Sachiko
• 金额: $ 2.24万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671508/
• 关键词: hypoxia; protein kinase C; translocation; KCN; PC12 cell; mitochondria; 虚血; 虚血性神経障害; プロテインカイネースC(PKC); PKCサブタイプ; ラット副腎髄質由来細胞; 転写調節因子

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.

缺氧/缺血后神经元变性的机制仍然不确定，但这些过程包括神经递质释放的增加，无细胞胞质钙浓度的升高以及信号转导途径的变化。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有关。但是，特定PKC同工酶在确定这一响应中的作用尚不很好地理解。这项研究的目的是表征PKC同工酶的表达以及PKC同工酶在调节细胞损伤中的作用。因此，为了了解哪些PKC同工酶参与缺氧/缺血诱导的神经元变性，我们检查了低氧（1％02）或大鼠嗜铬细胞瘤细胞（PC12细胞）中缺氧（1％02）或KCN暴露后的PKC同工酶。在补充10％（v/v）FBS的RPMI 1640培养基中，将PC12细胞系保持在37℃的加湿气氛中，其中5％CO2。通过KCN或多式孵化器（APMW-36，ASTEC Co. Ltd，Fukuoka）诱导缺氧。通过乳酸脱氢酶（LDH）测定法评估细胞活力。通过免疫印迹分析测量了亚细胞级分中PKC的蛋白质水平。暴露于缺氧（1％02）和KCN后，PC12细胞的细胞活力降低了时间依赖性。 KCN在不含葡萄糖的RPMI 1640中显着增加了细胞毒性。数据表明，在缺氧期间，特定PKC同工酶的选择性转运仅发生，只有钙依赖性PKC-γ，而不是其他PKC同酶在KCN治疗PC12细胞后均显着增加了膜和线粒体裂纹的高度增加。因此，我们提出，缺氧对PKC-γ的激活/易位对于调节PC 12细胞中缺氧诱导的细胞损伤很重要。