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Effect of opening of mitochondrial ATP--sensitive K~(+) channels on the expression of hypoxia inducible factor--1#alpha# and cell proliferation in pulmonary arterial smooth muscle cells of rats

Zhao Jianping, Guo Zhi, Zhou Zhigang, Chen Jun, Hu Hongling, Wang Tao, Zhang Zhenxiang

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 effect of mitochondrial ATP-sensitive K~(+) (MitoK_(ATP) channel on the expression of HIF-1#alpha# and cell proliferation in pulmonary arterial smooth muscle cells (PASMCs) of rats. Cultured PASMCs were divided into six groups as follows: (1) normoxia group: cultured under normoxia; (2) normoxia + diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_(ATP); (3) normoxia + 5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_(ATP); (4) hypoxia group: cultured under hypoxia (37℃, 5% O_(2), 5% CO_(2), 90% N_(2)) for 24 h; (5) hypoxia + diazoxide group, cultured under hypoxia (37℃, 5% O_(2), 5% CO_(2), 90% N_(2)) with diazoxide for 24 h; (6) hypoxia + 5-HD group, cultured under hypoxia (37℃, 5% O_(2), 5% CO_(2), 90% N_(2)) with 5-HD for 24 h. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. Immunohistochemical method was used to trace the expression of HIF-1#alpha#. The proliferation of PASMCs was examined by MTT colorimetric assay. The results were as follows: The intensity of R-123 fluorescence in normoxia + diazoxide group was significantly increased as compared with that in normoxia group (P<0.05), and the intensity of R-123 fluorescence in hypoxia + diazoxide group was also significantly increased as compared with that in hypoxia group (P<0.05). 24-hour hypoxia or 24-hour hypoxia + diazoxide markedly increased the intensity of R-123 fluorescence in PASMCs as compared with normoxia (P<0.05), and the change was more prominant in hypoxia + diazoxide group than that of hypoxia group (P<0.05). There was no significant difference in the intensity of R-123 fluorescence between normoxia group and normoxia + 5-HD group (P>0.05). However, 5-HD weakened the effect of 24-hour hypoxia on the intensity of R-123 fluorescence. The intensity of R-123 fluorescence in hypoxia + 5-HD group was significantly decreased as compared with that in hypoxia group (P<0.05). After exposure to hypoxia or hypoxia + diazoxide for 24 h, the expression of HIF-1#alpha# and the proliferation of PASMCs were significantly increased as compared with that in normoxia or normoxia + diazoxide group (P<0.05), and the change was more significant in hypoxia + diazoxide group than that in hypoxia group (P<0.05). There was no significant difference in the expression of HIF-1#alpha# and the proliferation of PASMCs between normoxia group and normoxia + 5-HD group (P>0.05). Since 5-HD could weaken the effect of 24-hour hypoxia, the expression of HIF-1#alpha# and the proliferation of PASMCs in hypoxia + 5-HD group were significantly decreased as compared with that in hypoxia group (P<0.05). All these results suggest that the opening of MitoK_(ATP) followed by a depolarization of mitochondrial membrane might contribute to the increase of the expression of HIF-1#alpha# and the proliferation of PASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension.

Key words: Hypoxia;hypoxia inducible factor-1#alpha#;Mitochondrial membrane potential;smooth muscle

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Citing This Article：

Zhao Jianping, Guo Zhi, Zhou Zhigang, Chen Jun, Hu Hongling, Wang Tao, Zhang Zhenxiang. Effect of opening of mitochondrial ATP--sensitive K~(+) channels on the expression of hypoxia inducible factor--1#alpha# and cell proliferation in pulmonary arterial smooth muscle cells of rats. Acta Physiol Sin 2007; 59 (2): 157-162 (in Chinese with English abstract).