Effect of mitochondrial KATP channel on voltage-gated K+ channel in 24 hour-hypoxic human pulmonary artery smooth muscle cells.
BACKGROUND
Hypoxic pulmonary hypertension (HPH) is initiated by inhibition of O2-sensitive, voltage-gated (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs). The mechanism of hypoxic pulmonary hypertension has not yet been fully elucidated. The mitochondrial ATP-sensitive K+ channel (MitoKATP) is extremely sensitive to hypoxia, and is a decisive factor in the control of mitochondrial membrane potential (DeltaPsim). This study investigated the changes of cell membrane potential and Kv channel in cultured human pulmonary artery smooth muscle cell (hPASMC) exposed to 24 hour-hypoxia, and explored the role of MitoKATP and DeltaPsim in this condition.


METHODS
Fresh human lung tissues were obtained from the patients undergoing a chest operation. hPASMCs were isolated, cultured, and divided into 6 groups: (1) control group, cultured under normoxia; (2) diazoxide group, cultured in normoxia with diazoxide, an opener of MitoKATP; (3) 5-HD group, cultured in normoxia with sodium 5-hydroxydecanoate (5-HD), an antagonist of MitoKATP; (4) 24 hour-hypoxia group; (5) 24 hour-hypoxia + diazoxide group; and (6) 24 hour-hypoxia + 5HD group. Whole-cell patch-clamp technique was used to trace the cell membrane K+ currents. The expressions of cell membrane Kv1.5 mRNA and protein were determined by RT-PCR and Western blot technique, respectively. The relative changes in mitochondrial potential were tested with rhodamine fluorescence (R-123) technique.


RESULTS
After exposure to diazoxide for 24 hours, the intensity of R-123 fluorescence in normoxic hPASMCs was significantly increased compared with control group (P < 0.05), but there were no significant changes in these tests after the hPASMCs had been exposed to 5-HD for 24 hours. Twenty-four hour-hypoxia or 24 hour-hypoxia + diazoxide could markedly increase the intensity of R-123 fluorescence in hPASMC and the changes were more significant in 24 hour-hypoxia +diazoxide group than in 24 hour-hypoxia group (P < 0.05) although 5-HD could partly weaken the effect of 24 hour-hypoxia on the intensity of R-123 fluorescence. After exposure to diazoxide for 24 hours, the cell membrane K+ currents and the expression of cell membrane Kv1.5 mRNA and protein in normoxic hPASMCs were significantly decreased compared with control group (P < 0.05), but there were no significant changes in these tests after the hPASMCs had been exposed to 5-HD for 24 hours. Also, 24 hour-hypoxia or 24 hour-hypoxia + diazoxide decreased the cell membrane K+ currents and the expression of Kv1.5 mRNA and protein (P < 0.05) but the changes were more significant in 24 hour-hypoxia + diazoxide group than in 24 hour-hypoxia group (P < 0.05). Again, 5-HD could partly weaken the inhibitory effect of 24 hour-hypoxia on the cell membrane K+ currents and the expression of Kv1.5 mRNA or protein (P < 0.05).


CONCLUSIONS
The opening of MitoKATP followed by a depolarization of DeltaPsim in hypoxia might contribute to the alterations in the expression of cell membrane Kv1.5 mRNA and protein leading to change in the cell membrane potential of hypoxic hPASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension.
Wang
Tao
T
Department of Respiratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. wt7636@sina.com.cn
Zhang
Zhen-xiang
ZX
Xu
Yong-jian
YJ
D015687:Cell Hypoxia; D002478:Cells, Cultured; D003981:Diazoxide / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000502:physiology*; D008928:Mitochondria / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated / Q000502:physiology*; D011651:Pulmonary Artery / Q000166:cytology* / Q000502:physiology
D008565:Membrane Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; C117780:mitochondrial K(ATP) channel; D003981:Diazoxide