Two-pore channels mediated receptor-operated Ca2+ entry in pulmonary artery smooth muscle cells in response to hypoxia

Abstract

The aim of this study was to investigate the influence of two-pore channels mediated receptor-operated Ca²⁺ entry on pulmonary arterial smooth muscle cell (PASMC) under hypoxia conditions. PASMCs were separated using the direct adherent culture method. The cultured cells were observed under optic microscope and the phenotypes of cells were identified by immunohistochemistry. The expression of NAADP was examined by ELISA. CaN, TPC1, TPC2 and NFATc3 protein levels were examined using Western blotting. Real-time PCR was utilized to detect the level of TPC1 and TPC2 mRNA. Fluorescent probe technique was used to explore the [Ca²⁺]i in PASMCs. Proliferation and migration of PASMCs were examined by MTT assay and Transwell, respectively. The results showed that cells displayed a typical "peak-valley" growth pattern and positive for α-actin staining. Expression of NAADP, CaN, NFATc3, TPC1 and TPC2 under PASMCs exposed to hypoxia after 24 h and 48 h were higher than control, however, cells treated with Ned-19 were significantly decreased compared with control. Levels of CaN and NFATc3 protein collected from RPASMCs transfected with TPCs siRNA were observably decreased than scrambled siRNA. Under hypoxia condition for 12 h, 24 h and 48 h, TPC1 and TPC2 mRNA levels were higher in PASMCs compared as control. The [Ca²⁺]i evoked by hypoxia significantly increased than normoxia group. Nevertheless, the [Ca²⁺]i of the groups treated with Ned-19 and transfected with TPCs siRNA were markedly lower compared with control. In conclusion, the TPCs influence on function of pulmonary artery smooth muscle cells by mediated Ca²⁺ Signals under hypoxia condition.

Keywords: Hypoxia; PAH; PASMCs; SOCE; Two-pore channels.