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缺氧肺动脉平滑肌细胞中线粒体ATP敏感钾通道开放对细胞色素C的分布及细胞增殖的作用

胡红玲, 张珍祥, 赵建平, 汪涛, 徐永健

华中科技大学同济医学院附属同济医院呼吸疾病研究所.湖北,武汉 430030

摘要

为了探讨线粒体ATP敏感钾通道(mitochondrial ATP--sensitive K~(+) channel,mitoK_(ATP))和线粒体膜电位(#DELTA##phi#m)在细胞缺氧信号转导中的作用以及对缺氧肺动脉平滑肌细胞中细胞色素C在细胞内的分布及细胞增殖的影响,该实验将人肺动脉平滑肌细胞进行常氧或24h缺氧培养,并将标本分为六组：(1)对照组；(2)mitoK_(ATP)开放剂diazoxide组；(3)mitoK_(ATP)阻断剂5--HD组：(4)24h缺氧组：(5)24h缺氧+diazoxide组；(6)24h缺氧+5--HD组。利用激光共聚焦显微镜成像法检测#DELTA##phi#m：线粒体/胞浆成分分离试剂盒(BioVision)分离线粒体和胞浆成分后,Western blot检测两者细胞色素C：Western blot检测细胞中caspase--9的蛋白表达量；MTT法及PI染色后流式细胞仪检测细胞增殖情况。结果显示：(1)diazoxide作用24h后,R--123荧光明显增强,胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase--9的蛋白表达显著减少,细胞增殖明显增多、凋亡减少,与正常对照组相比较,均{sl P}＜0.05；而5--HD作用24 h与正常对照组比较，上述指标无明显变化({sl P}>0.05)。(2)缺氧24 h后，结果与diazoxide组相似，R--123荧光明显增强，细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低，caspase--9的蛋白表达显著减少，细胞明显增殖增多、凋亡减少，与正常对照组相比较，均{sl P}<0.05；24 h缺氧+diazoxide组与缺氧组相比较，R--123荧光明显增强，细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低，caspase--9的蛋白表达显著减少，细胞明显增殖增多、凋亡减少({sl P}<0.05)；而24 h缺氧+5--HD组与缺氧组比较，R--123荧光明显降低，细胞胞浆细胞色素C与线粒体细胞色素C的比值明显升高，caspase--9的蛋白表达显著增加，细胞明显增殖减少、凋亡增多({sl P}<0.05)。上述结果提示，缺氧可以引起mitoK_(ATP)的开放以及#DELTA##phi#m的去极化，并进而抑制细胞色素C从细胞线粒体释放到细胞浆，抑制线粒体凋亡途径，从而参与并影响肺动脉高压的发生发展。

关键词： 肺动脉平滑肌细胞; 缺氧; 线粒体膜电位; 线粒体膜ATP敏感钾通道; 细胞色素C

Effect of opening of mitochondrial ATP--sensitive K~(+) channel on the distribution of cytochrome C and on proliferation of human pulmonary arterial smooth muscle cells in hypoxia

Hu Hongling, Zhang Zhenxiang, Zhao Jianping, Jiang Tao, Xu Yongjian

Department of Respiratory Medicine,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology.Wuhan 430030,Hubei

Abstract

The objective of this paper was to investigate the contribution of mitochondrial ATP-sensitive K~(+) channel (mitoK_(ATP)) and mitochondrial membrane potential (#DELTA##phi#m) to distribution of cytochrome C in human pulmonary arterial smooth muscle cells (HPASMCs) and to the proliferation of HPASMCs induced by hypoxia. HPASMCs were divided into several groups, as follow: (1) control group: cultured under normoxia; (2) diazoxide group: cultured in normoxia with diazoxide, an opener of mitoK_(ATP); (3) 5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of mitoK_(ATP); (4) 24-hour hypoxia group: cultured in hypoxia for 24 h; (5) 24-hour hypoxia + diazoxide group: cultured in hypoxia with diazoxide for 24 h; (6) 24-hour hypoxia + 5-HD group: cultured in hypoxia with 5-HD for 24 h. The relative changes in mitochondrial potential were tested with rhodamine fluorescence (R-123) technique. Western blot technique was used to detect the expression of cytochrome C protein in cell plasma and mitochondria, respectively. The expression of cell caspase-9 protein was determined with western blot technique, too. The proliferation of HPASMCs was examined by cell cycle analysis and MTT colorimetric assay. The results were as follow: after exposed to diazoxide for 24 h, the intensity of R-123 fluorescence in normoxic HPASMCs was significantly increased as compared with that of the control group (P<0.05), but there was no significant change of the intensity of R-123 fluorescence after the HPASMCs had been exposed to 5-HD for 24 h; 24-hour hypoxia or 24-hour hypoxia + diazoxide could markedly increase the intensity of R-123 fluorescence in HPASMC as compared with that of the control group (P<0.05), the change was more significant in 24-hour hypoxia + diazoxide group than that of 24-hour hypoxia group (P<0.05); 5-HD could weaken the effect of 24-hour hypoxia on the intensity of R-123 fluorescence. After exposed to diazoxide for 24 h, the rate of the expression of cytosolic cytochrome C protein to that of mitochondrial cytochrome C protein was significantly decreased as compared with control group (P<0.05), the expression of caspase-9 protein was significantly decreased as compared with that of the control group (P<0.05). The percentage of S phase and A value of MTT were significantly increased as compared with those of the control group (P<0.05). But there were no significant changes of these tests after HPASMCs had been exposed to 5-HD for 24 h (P>0.05). After exposed to hypoxia or hypoxia + diazoxide for 24 h, the rate of the expression of cytosolic cytochrome C protein to that of mitochondrial cytochrome C protein and the expression of caspase-9 protein were significantly decreased as compared with those of the control group (P<0.05). The percentage of S phase and A value of MTT were significantly increased as compared with those of the control group (P<0.05). These changes were more significant in 24-hour hypoxia + diazoxide group than those of 24-hour hypoxia group (P<0.05). 5-HD could weaken the effect of hypoxia on the changes of the distribution of cytochrome C, the expression of caspase-9 in human pulmonary arterial smooth muscle cells and the proliferation of HPASMCs induced by hypoxia (P<0.05). All these results suggest that the opening of mitoK_(ATP) followed by a depolarization of #DELTA##phi#m induced by hypoxia might contribute to the inhibition of the release of cytochrome C from cell mitochondria to cell plasma in HPASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension. The signal transduction pathway of mitochondria might play an important role in the relationship between #DELTA##phi#m and apoptosis of HPASMCs.

Key words: pulmonary arterial smooth muscle cell;Hypoxia;Mitochondrial membrane potential;mitochondrial ATP-sensitive potassium channel;cytochrome C

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引用本文：

胡红玲, 张珍祥, 赵建平, 汪涛, 徐永健. 缺氧肺动脉平滑肌细胞中线粒体ATP敏感钾通道开放对细胞色素C的分布及细胞增殖的作用[J]. 生理学报 2006; 58 (3): .

Hu Hongling, Zhang Zhenxiang, Zhao Jianping, Jiang Tao, Xu Yongjian. Effect of opening of mitochondrial ATP--sensitive K~(+) channel on the distribution of cytochrome C and on proliferation of human pulmonary arterial smooth muscle cells in hypoxia. Acta Physiol Sin 2006; 58 (3): (in Chinese with English abstract).