Objective: To investigate the contribution of the opening of mitochondrial ATP-sensitive K+ channel (MitoK(ATP)) and mitochondrial membrane potential (delta psi m) to changes of reactive oxygen species (ROS) and to the imbalance of proliferation/apoptosis of human pulmonary arterial smooth muscle cells (HPASMC) induced by hypoxia.
Methods: HPASMC were divided into the following groups: (1) control group (A group): cultured under normoxia; (2) 5-HD group (B group): cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK(ATP), for 24 h; (3) diazoxide group (C group): cultured in normoxia with diazoxide, an opener of MitoK(ATP), for 24 h; (4) chronic hypoxia group (D group): cultured under hypoxia for 24 h; (5) chronic hypoxia + diazoxide group (E group): cultured in hypoxia with diazoxide for 24 h; (6) chronic hypoxia + 5-HD group (F group): cultured in hypoxia with 5-HD for 24 h. The relative changes in mitochondrial potential were tested with rhodamine fluorescence (R-123) technique. The level of ROS in HPASMC was tested with chemiluminescence method. The proliferation of HPASMC was examined by the expression of PCNA, c-fos and c-jun proteins, and by MTT colorimetric assay.
Results: The mitochondrial membrane potentials (expressed by intensity of R-123) of C group (105 +/- 4), D group (95 +/- 13) and E group (126 +/- 8) were significantly depolarized as compared with A group (75 +/- 7, q = 5.474, 3.659, 9.213, all P < 0.05). The levels of ROS of C group (3045 +/- 126), D group (3116 +/- 34) and E group (3236 +/- 31) were significant increased than that of A group (2772 +/- 49) (q = 6.882, 7.448, 16.289, all P < 0.05). The cell viability of C group (0.305 +/- 0.022), D group (0.328 +/- 0.078) and E group (0.440 +/- 0.023) were significant increased than that of A group (0.237 +/- 0.013) in HPASMC (q = 2.993, 4.017, 8.919, all P < 0.05). The mitochondrial membrane potential, the level of ROS and the cell viability in HPASMC of E group were significantly different as compared with D group (q = 5.554, 8.841, 4.902, all P < 0.05). The mitochondrial membrane potential (71 +/- 4), the level of ROS (2863 +/- 132) and the cell viability (0.264 +/- 0.045) of F group were significantly decreased than those of D group (95 +/- 13, 3 116 +/- 34, 0.328 +/- 0.078, q = 4.367, 5.907, 2.832, all P < 0.05).
Conclusions: The results suggested that the opening of MitoK(ATP) followed by a depolarization of delta psi m might contribute to the increasing of the level of ROS in HPASMC, and play an important role in the proliferation of HPASMC. This might be a mechanism for the development of hypoxic pulmonary hypertension.