Iptakalim induces mitochondria-dependent apoptosis in hypoxic rat pulmonary arterial smooth muscle cells.

Abstract

Pulmonary vascular medial hypertrophy in hypoxic pulmonary arterial hypertension (HPH) is caused in part by decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Iptakalim (Ipt), an ATP sensitive potassium channel opener, ameliorates HPH in animal models. Here we investigated the effects of Ipt on proliferation and apoptosis of hypoxic rat PASMCs, and to determine the possible underlying mechanisms. Primary rat PASMCs were isolated and cultured. PASMCs were cultured for 24h in normoxia or hypoxia (5% O2) conditions with and without Ipt. Cell proliferation and cycle were determined by MTT assay and flow cytometry, respectively. Mitochondrial membrane potential (Δym) was detected by fluorescence microscope Western blot assays were used to examine the expression of cyclin D, CDK4, endothelin-1 (ET-1), hypoxia-inducible factor-1 (HIF-1), platelet-derived growth factor-BB (PDGF-BB), Bax, Bcl-2, cytochrome c (Cyt c), caspase-9, and caspase-3 in PASMCs. We found that hypoxia significantly stimulated proliferation and rendered resistance to apoptosis in PASMCs. Ipt suppressed proliferation and induced cell cycle arrest in hypoxia PASMCs. Ipt decreased the expression of cyclin D, CDK4, HIF-1, ET-1, and PDGF-BB in hypoxia PASMCs. It reversed the depolarization of Δψm in hypoxia PASMCs too. Ipt significantly upregulated Bax expression and downregulated Bcl-2 expression, and promoted the release of Cyt c from mitochondria to cytoplasm in hypoxia PASMCs. Furthermore, Ipt significantly activated the caspase cascades evidenced by increased expression of caspase-9 and caspase-3 in hypoxia PASMCs. Ipt could inhibit cell proliferation and induce apoptosis associated with cell cycle arrest, decreased ET-1, HIF-1, cyclin D, CDK4, PDGF-BB and Δψm, increased Bax/Bcl-2 ratio, enhanced Cyt c release, and activation of caspases in PASMCs under hypoxia status. Our data indicated that Ipt could be a therapeutic candidate for treatment of HPH.