Two-pore channels mediated receptor-operated Ca2+ entry in pulmonary artery smooth muscle cells in response to hypoxia
Two-pore channels mediated receptor-operated Ca2+ entry in pulmonary artery smooth muscle cells in response to hypoxia
复制标题
双孔通道介导受体操作的 Ca2+ 进入肺动脉平滑肌细胞以应对缺氧
DOI：
10.1016/j.biocel.2018.01.012
复制
发表时间：
2018-04-01
影响因子：
4
通讯作者：
Dai, Aiguo
中科院分区：
文献类型：
--
作者：
Jiang, Yongliang;Zhou, Yumin;Dai, Aiguo
本研究旨在探讨低氧条件下双孔通道介导的受体操纵性钙离子内流对肺动脉平滑肌细胞(PASMC)的影响。采用直接贴壁培养法分离PASMC。光镜下观察细胞形态，免疫组织化学方法鉴定细胞表型。用酶联免疫吸附试验检测NAADP的表达。Western blotting检测CaN、TPC1、TPC2和NFATc3蛋白水平。实时荧光定量聚合酶链式反应检测TPC1、TPC2基因的表达水平。用荧光探针技术检测PASMCs内[Ca~(2+)]i。分别用四甲基偶氮唑盐比色法和Transwell法检测PASMCs的增殖和迁移。结果显示，细胞呈典型的“峰-谷”生长模式，α-肌动蛋白染色阳性。PASMCs缺氧24 h和48 h后，NAADP、CaN、NFATc3、TPC1和TPC2的表达均高于对照组，而Ned-19处理组的表达明显低于对照组。转导TPCS siRNA的RPASMCs的CaN和NFATc3蛋白表达水平明显低于杂交瘤siRNA。缺氧12 h、24 h和48 h时，PASMCs TPC1和TPC2的mRNA水平均高于对照组。低氧引起的[Ca~(2+)]i显著高于常氧组。然而，Ned-19处理组和TPC siRNA转染组的[Ca~(2+)]i明显低于对照组。综上所述，低氧条件下，TPC通过介导钙信号影响肺动脉平滑肌细胞的功能。
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 a-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.