关键词: 关键词：Orai2, 一氧化氮, 钙离子, 人脐静脉内皮细胞

Abstract: Objective To study the roles of calcium release-activated calcium modulator 2(Orai2) in extracellular Ca2+-sensing receptor (CaR)-induced extracellular Ca2+ influx and the production of nitric oxide (NO) in human umbilical vein endothelial cells (HUVEC). Methods 1) We silenced the expression of Orai2 genes in HUVEC by transfection constructed Orai2 RNA interference plasmids. The expression of Orai2 protein and mRNA levels were determined by Western blotting and real time RT-PCR, respectively. 2) The second to fifth passage of HUVEC were incubated with CaR agonist spermine(activating store-operates cation channels (SOC) and receptor-operated channels (ROC)), CaR negative allosteric modulator Calhex231(blocking SOC and activating ROC) and ROC analogue TPA (activating ROC and blocking SOC), protein kinase C (PKC) inhibitor Ro31-8220, PKCs and PKCμ inhibitor Go6967 (activate SOC and blocking ROC ), respectively .Those cell models were divided into three groups: Orai2-71 short hairpin RNA group (Orai2shRNA); control group and vehicle group. Intracellular Ca2+ concentration ([Ca2+]i) was detected using the fluorescence Ca2+ indicator Fura-2/AM, the production of NO was determined by DAF-FM (NO fluorescent probe) of every group in HUVEC. Results 1) Compared with the control group, the results of transfection constructed Orai2 RNA interference plasmids demonstrated that shRNA targeted to the Orai2 genes decreased Orai2 protein and mRNA levels by 70.58% and 75.75%, respectively (P<0.05). (2) Compared with the control group and vehicle group, the [Ca2+]i ratio and the net NO fluorescence intensity values of Orai2shRNA group in four different treatments were significantly reduced (P<0.05). Conclusions Orai2 participates in CaR-mediated Ca2+ influx and NO production by SOC and ROC activation in HUVEC.

Key words: [Key words]: Orai2, Nitric oxide, Ca2+, Human umbilical vein endothelial cells