[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].

The objective of this paper was to investigate the contribution of mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) and mitochondrial membrane potential (Deltapsim) to the 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 6 groups, as following: (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 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. The proliferation of HPASMCs was examined by cell cycle analysis and MTT colorimetric assay. The results were as following: after exposure to diazoxide for 24 h, the intensity of R-123 fluorescence in normoxic HPASMCs was significantly increased compared with that in 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 HPASMCs compared with normoxia (P<0.05), and the change was more significant in 24-hour hypoxia + diazoxide group than that in 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 exposure to diazoxide for 24 h, the ratio of the expression of cytosolic cytochrome C protein to that of mitochondrial cytochrome C protein was significantly decreased compared with that in the control group (P<0.05), and the expression of caspase-9 protein was significantly decreased compared with that in the control group (P<0.05). The percentage of S phase and A value of MTT were significantly increased compared with those in the control group (P<0.05). But there were no significant changes in these tests after HPASMCs had been exposed to 5-HD for 24 h (P>0.05). After exposure to hypoxia or hypoxia + diazoxide for 24 h, the ratio 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 compared with those in the control group (P<0.05). The percentage of S phase and A value of MTT were significantly increased compared with those in the control group (P<0.05). These changes were more significant in 24-hour hypoxia + diazoxide group than those in 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 HPASMCs 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 Deltapsim induced by hypoxia might contribute to the inhibition of the release of cytochrome C from mitochondria to 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 Deltapsim and apoptosis of HPASMCs.