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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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Citing This Article：

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).