The Role Of Mitochondrial ATP-sensitive K~+ Channel On The Unbalance Between Proliferation And Apoptosis Of Hypoxic Pulmonary Artery Smooth Muscle Cells

BackgroudSo far,the incidence and mortality of pulmonary hypertension and pulmonary heart disease still remain high. Most of pulmonary hypertension is hypoxic pulmonary hypertension (HPH), and the mechanism of hypoxic pulmonary hypertension has still not been fully elucidated. HPH has two main pathophysiological characters: hypoxic pulmonary artery vasoconstriction (HPV) and hypoxic pulmonary vascular remodeling. HPV has been demonstrated not only in perfused lungs but also in pulmonary arterial rings denuded of endothelium and in single pulmonary arterial smooth muscle cells (PASMCs). Recently, the potassium channel and the cytoplasmic free calcium concentration have been considered to play very important role in the regulation of vascular tone. The activity of voltage-gated potassium channels (Kv) controls the cell membrane potential, which subsequently regulates the cytoplasmic free calcium concentration and the proliferation of PASMCs. Studies have shown that acute or chronic hypoxia can inhibit the function of Kv in PASMCs, inducing membrane depolarization and a rise in intracellular free Ca2+ concentration ([Ca²⁺]_i), that triggers vasoconstriction and stimulates the proliferation of PASMCs.The mitochondrial ATP-sensitive K+ channel has been implicated in cellular protection against metabolic stress in a variety of tissues, including liver, gut, brain, and kidney, and has been shown to be an essential component of the mechanism of ischemic preconditioning in the heart. It is unclear how the opening of a K+ channel in the mitochondria would lead to cardioprotection. However three hypotheses have emerged to explain the link between mitoK_ATP channel opening and cardioprotection: (1) A decrease in the mitochondrial Ca²⁺ uptake. Holmuhamedov et al showed that diazoxide and pinacidil could reduce the magnitude of mitochondrial Ca2+ uptake, and the effect of both can be reversed by 5-HD. This change in mitochondrial Ca2+ uptake was thought to be mediated by a partial depolarization of mitochondrial membrane potential ( m) in response to mitoK_ATP opening. (2) Swelling of the mitochondrial matrix and changes in ATP synthesis. It has been known for some time that the opening of mitoK_ATP causes mitochondrial matrix swelling, and that this in turn activates the respiratory chain providing more ATP to support the recovering myocardium. (3) Changes in the levels of reactive oxygen species (ROS). Reactive oxygen species are a double-edge sword when it comes to cardioprotection. The ROS generated during the preconditioning period is thought to be protective. However, the ROS that is produced during reperfusion, is detrimental and causes cell death. It is thought that the opening of mitoK_ATP results in an increase in the protective ROS produced during preconditioning phase and a decrease in the levels of ROS generated during reperfusion phase.A study of our lab suggested that the opening of mitoK_ATP followed by a depolarization of m in hypoxia might contribute to the alterations in the expression of cell membrane Kv1.5 mRNA and protein leading to the change in the cell membrane potential of hypoxic HPASMCs. This study continue to investige the detail mechanism about the role of mitochondrial ATP-sensitive K+ channel on the unbalance between proliferation and apoptosis of hypoxic rat and human pulmonary artery smooth muscle cells.Part 1 The role of mitochondrial ATP-sensitive K+ channel on the balance between proliferation and apoptosis of hypoxic rat pulmonary artery smooth muscle cellsSubject 1 The effect of mitochondrial ATP-sensitive K+ channel on distributing of cytochrome C in rat pulmonary artery smooth muscle cells and on proliferation of hypoxic rat pulmonary artery smooth muscle cellsObjective : The objective of this paper is to investigate the contribution of mitochondrial ATP-sensitive K+ channel (MitoK_ATP) and mitochondrial membrane potential ( m) to distributing of cytochrome C in rat pulmonary artery smooth muscle cells and on proliferation of rat pulmonary artery smooth muscle cells induced by hypoxia.Methods:Fresh normal rat lung tissues were obtained. Then rat PASMCs were isolated and cultured, which were divided into 6 groups, as follow:â‘ control group: cultured under normoxia;â‘¡diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_ATP;â‘¢5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_ATP;â‘£c hronic hypoxia group: cultured under hypoxia;⑤chronic hypoxia+diazoxide group;â‘¥chronic hypoxia +5-HD group. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. Western blot technique was used to trace the expression of cytochrome C protein in cell plasma and mitochondria respectively. The proliferation of rat PASMCs was examined by cell cycle analysis and MTT colorimetric assay.Results:After exposed to diazoxide for 24h, the intensity of R-123 fluorescence in normoxic rat PASMCs were significantly increased as compared with control group (P<0.05), but there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24h (P>0.05); chronic hypoxia or chronic hypoxia+diazoxide could markedly increase the intensity of R-123 fluorescence in rat PASMC as compared with control group (P<0.05), the changes were more significant in chronic hypoxia +diazoxide group than those of chronic hypoxia group (P<0.05); 5-HD could partly weaken the effect of hypoxia on the intensity of R-123 fluorescence (P<0.05) too, After exposed to diazoxide for 24h, the rate of the expression of cytochrome C protein in cell plasma to that in cell mitochondria were significantly decreased as compared with control group (P<0.05), The A value were significantly increased as compared with control group(P<0.05),and the apoptosis of rat PASMCs were significantly decreased as compared with control group(P<0.05) . But there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24h; After exposed to hypoxia or hypoxia+diazoxide for 24h, the rate of the expression of cytochrome C protein in cell plasma to that in cell mitochondria were significantly decreased as compared with control group (P<0.05), the A value were significantly increased as compared with control group (P<0.05), and the apoptosis of rat PASMCs were significantly decreased as compared with control group (P<0.05); The changes were more significant in hypoxia +diazoxide group than those of hypoxia group (P<0.05). 5-HD could partly weaken the effect of hypoxia on the changes of the rate of the expression of cytochrome C protein in cell plasma to that in cell mitochondria in rat pulmonary artery smooth muscle cells and on the proliferation of rat PASMCs induced by hypoxia (P<0.05).Conclusion:The results suggested that the opening of MitoK_ATP followed by a depolarization of m might contribute to the inhibition of the release of cytochrome C from cell mitochondria to cell plasma in rat PASMCs and proliferation of rat PASMCs induced by hypoxia. This might be a mechanism of the development of hypoxic pulmonary hypertension.Subject 2 The effect of mitochondrial ATP-sensitive K+ channel on changes of reactive oxygen species in rat pulmonary artery smooth muscle cells and on proliferation of hypoxic rat pulmonary artery smooth muscle cellsObjective : The objective of this paper is to investigate the contribution of mitochondrial ATP-sensitive K+ channel (MitoK_ATP) and mitochondrial membrane potential ( m) to changes of H2O2 in rat pulmonary artery smooth muscle cells and on proliferation of hypoxic rat pulmonary artery smooth muscle cells.Methods:Fresh normal rat lung tissues were obtained. Then rat PASMCs were isolated and cultured, which were divided into 6 groups, as follow:â‘ control group: cultured under normoxia;â‘¡diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_ATP;â‘¢5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_ATP;â‘£c hronic hypoxia group: cultured under hypoxia;⑤chronic hypoxia+diazoxide group;â‘¥chronic hypoxia +5-HD group. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. The level of H2O2 in rat PASMCs were tested with chemiluminescence method. The proliferation of rat PASMCs was examined by cell cycle analysis and MTT colorimetric assay.Results:After exposed to diazoxide for 24h, the intensity of R-123 fluorescence in normoxic rat PASMCs were significantly increased as compared with control group (P<0.05), but there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24h (P>0.05); chronic hypoxia or chronic hypoxia+diazoxide could markedly increase the intensity of R-123 fluorescence in rat PASMC as compared with control group (P<0.05), the changes were more significant in chronic hypoxia +diazoxide group than those of chronic hypoxia group (P<0.05); 5-HD could partly weaken the effect of hypoxia on the intensity of R-123 fluorescence (P<0.05) too, After exposed to diazoxide for 24h, the level of H2O2 in rat PASMCs were significantly increased as compared with control group (P<0.05), The A value were significantly increased as compared with control group(P<0.05),and the apoptosis of rat PASMCs were significantly decreased as compared with control group(P<0.05) . But there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24h; After exposed to hypoxia or hypoxia+diazoxide for 24h, the level of H2O2 in rat PASMCs were significantly increased as compared with control group (P<0.05), the A value were significantly increased as compared with control group (P<0.05), and the apoptosis of rat PASMCs were significantly decreased as compared with control group (P<0.05); The changes were more significant in hypoxia +diazoxide group than those of hypoxia group (P<0.05). 5-HD could partly weaken the effect of hypoxia on the changes of the level of H2O2 in rat pulmonary artery smooth muscle cells and on the proliferation of rat PASMCs induced by hypoxia (P<0.05).Conclusion:The results suggested that the opening of MitoK_ATP followed by a depolarization of m might contribute to the the increasing of the level of H2O2 in rat PASMCs and proliferation of rat PASMCs induced by hypoxia. This might be a mechanism of the development of hypoxic pulmonary hypertension. Part 2 The role of mitochondrial ATP-sensitive K+ channel on the balance between proliferation and apoptosis of hypoxic human pulmonary artery smooth muscle cellsSubject 1 The effect of mitochondrial ATP-sensitive K+ channel on distributing of cytochrome C in human pulmonary artery smooth muscle cells and on proliferation of hypoxic human pulmonary artery smooth muscle cellsObjective : The objective of this paper is to investigate the contribution of mitochondrial ATP-sensitive K+ channel (MitoK_ATP) and mitochondrial membrane potential (Δψ_m) to distributing of cytochrome C in human pulmonary arterial smooth muscle cells (HPASMCs) and to proliferation of HPASMCs induced by hypoxia.Methods:HPASMCs were divided into several groups, as following:â‘ control group: cultured under normoxia;â‘¡diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_ATP;â‘¢5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_ATP;â‘£2 4-hours hypoxia group: cultured under hypoxia for 24 hours;⑤2 4-hours hypoxia+diazoxide group, cultured under hypoxia with diazoxide for 24 hours;â‘¥2 4-hours hypoxia+5-HD group, cultured under hypoxia with 5-HD for 24 hours. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. Western blot technique was used to trace 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.Results:After exposed to diazoxide for 24 h, the intensity of R-123 fluorescence in normoxic HPASMCs was significantly increased as compared with 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-hours hypoxia or 24-hours hypoxia+diazoxide could markedly increase the intensity of R-123 fluorescence in HPASMC as compared with control group (P<0.05), the change was more significant in 24-hours hypoxia +diazoxide group than that of 24-hours hypoxia group (P<0.05); 5-HD could weaken the effect of 24-hours hypoxia on the intensity of R-123 fluorescence. After exposed 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 as compared with control group (P<0.05), the expression of caspase-9 protein was significantly decreased as compared with control group (P<0.05), the percentage of S phase and A value of MTT were significantly increased as compared with control group (P<0.05). But there were no significant changes of these tests after the 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 control group (P<0.05), the percentage of S phase and A value of MTT were significantly increased as compared with control group (P<0.05). These changes were more significant in 24-hours hypoxia +diazoxide group than those of 24-hours hypoxia group (P<0.05). 5-HD could weaken the effect of hypoxia on the changes of distributing 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).Conclusion:All these results suggested that the opening of MitoK_ATP followed by a depolarization ofΔψ_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 betweenΔψ_m and apoptosis of HPASMCs. Subject 2 The effect of mitochondrial ATP-sensitive K+ channel on changes of reactive oxygen species in human pulmonary artery smooth muscle cells and on proliferation of hypoxic human pulmonary artery smooth muscle cellsObjective:The objective of this paper is to investigate the contribution of diazoxide, an opener of mitochondrial ATP-sensitive K+ channel (MitoK_ATP), and mitochondrial membrane potential(Δψ_m) to changes of reactive oxygen species in human pulmonary arterial smooth muscle cells (HPASMCs) and to proliferation of HPASMCs induced by hypoxia.Methods:HPASMCs were divided into several groups, as follow:â‘ control group: cultured under normoxia;â‘¡diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_ATP;â‘¢5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_ATP;â‘£chronic hypoxia group: cultured under hypoxia for 24 hours;⑤chronic hypoxia+diazoxide group;â‘¥chronic hypoxia+5-HD group. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. The level of H2O2 in HPASMCs was tested with chemiluminescence method. The proliferation of HPASMCs was examined by examing the expression of PCNA, c-fos and c-jun proteins, and by MTT colorimetric assay.Results:After exposed to diazoxide for 24h, the intensity of R-123 fluorescence in normoxic HPASMCs(105.45±4.38) was significantly increased as compared with control group (74.67±7.02) (P<0.05), but there was no significant change in this test after the HPASMCs had been exposed to 5-HD for 24h; chronic hypoxia (95.24±12.92)or chronic hypoxia +diazoxide (126.47±7.63)could markedly increase the intensity of R-123 fluorescence in HPASMC as compared with control group (P<0.05), the change was more significant in chronic hypoxia +diazoxide group than that of hypoxia group (P<0.05); CH+5-HD(70.69±3.73) could weaken the effect of hypoxia on the intensity of R-123 fluorescence. After exposed to diazoxide for 24h, the level of H2O2(3044.69±126.34)in HPASMCs was significantly increased as compared with control group (2771.69±48.66) (P<0.05), the expression of PCNA(53.52±2.28%), c-fos(164.47±53.47) and c-jun(130.25±10.39 ) proteins were significantly increased as compared with control group(PCNA 42.55±1.97%,c-fos 68.58±21.54 and c-jun 76.18±12.65 ) (P<0.05) too, The A value ( 0.3048±0.022 ) was significantly increased as compared with control group(0.2368±0.013) (P<0.05). But there were no significant changes in these tests after the HPASMCs had been exposed to 5-HD for 24h; After exposed to chronic hypoxia for 24h, the level of H2O2(3115.88±34.12) in HPASMCs was significantly increased as compared with control group (P<0.05), the expression of PCNA(54.55±2.14%), c-fos (160.95±47.49)and c-jun (127.87±17.44)proteins were significantly increased as compared with control group (P<0.05) too, The A value (0.3282±0.078)was significantly increased as compared with control group(P<0.05). These changes were more significant in chronic hypoxia +diazoxide group( the level of H2O2 3236.01±30.86,PCNA 66.13±2.59%, C-fos 370.65±90.85,C-jun 275.97±49.77 and A value 0.4398±0.023) than those of hypoxia group (P<0.05); 5-HD could weaken the effect of hypoxia on the changes of the level of H2O2 (2863.08±132.06) in human pulmonary arterial smooth muscle cells and on the proliferation of HPASMCs ( PCNA 43.48±1.23%,C-fos 70.87±21.46 , C-jun 79.87±12.75 and A value 0.2637±0.045) induced by hypoxia (P<0.05).Conclusion:The results suggested that the opening of MitoK_ATP followed by a depolarization ofΔψ_m might contribute to the increasing of the level of ROS in HPASMCs. The ROS in HPASMCs play an important role in the prolifeation of HPASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension.