Orai3参与了HUVECs外钙敏感受体介导的钙内流和NO生成

 目的：研究Ca²⁺通道蛋白Orai3在人脐静脉内皮细胞(HUVECs)外钙敏感受体(CaR)介导的钙内流和一氧化氮(NO)生成中的作用和机制。方法：(1)利用转染技术将构建的Orai3干扰质粒(Orai3 shRNA)转染HUVECs，采用激光共聚焦显微镜观察细胞转染效果，实时定量RT-PCR和Western blotting检测Orai3shRNA转染后Orai3 mRNA和蛋白的表达以及抑制效率。(2)取第2~5代细胞随机分组：特异性质粒转染组即实验组(Orai3 shRNA组)、未转染组即空白对照组(control组)和空质粒组(vehicle组)，将上述3组细胞分别与CaR激动剂精胺［钙池操纵性钙通道(SOC)和受体操纵性钙通道(ROC)均激活］、ROC模拟剂12-O-十四烷酰佛波醇-13-乙酸酯(TPA)+CaR负性变构调节剂Calhex 231(激活ROC、阻断SOC)、蛋白激酶C(PKC)抑制剂Ro31-8220及经典型PKCs和PKCμ抑制剂Go6967(阻断ROC、激活SOC)孵育，用荧光探针Fura-2/AM和DAF-FM负载方法同步检测［Ca 2+］ i和NO生成的变化。结果：(1)实时定量RT-PCR及Western blotting检测结果显示：与control组相比较，shOrai3-77干扰后，Orai3 mRNA的表达明显降低(抑制率为84.5%,P<0.05),Orai3蛋白的表达亦明显降低(抑制率为70.68%,P<0.05)。(2)［Ca 2+］ i Δratio值和NO净荧光强度值的测定结果显示：与control组及vehicle组相比，在4种不同处理因素作用下，Orai3 shRNA转染组中［Ca 2+］ i Δratio值和NO净荧光强度值均明显降低(P<005)，而vehicle组无显著差异(P>0.05)。结论：在HUVECs中Orai3参与了CaR经SOC和ROC激活介导的钙内流和NO生成。

Orai3 participates in CaR-mediated Ca2+ entry and NO generation in human umbilical veinendothelial cells

AIM：To study the function and mechanism of Orai3 in extracellular Ca 2+-sensing receptor (CaR)-induced extracellular Ca 2+ influx and the production of nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs). METHODS：The expression Orai3 gene in HUVECs was silenced by transfection of constructed Orai3 RNA interference plasmids. The interference efficiency at protein and mRNA levels was determined by Western blotting and real-time RT-PCR, respectively. The HUVECs in the 2nd to 5th passage was divided into Orai3-77 short hairpin RNA group, control group and vehicle group, which were incubated with CaR agonist spermine ［activating store-operated Ca 2+ channels (SOC) and receptor-operated Ca 2+ channels (ROC)］, ROC analogue 12-O-tetradecanoylphorbol-13-acetate (TPA) plus CaR negative allosteric modulator Calhex 231 (activating ROC, blocking SOC), protein kinase C (PKC) inhibitor Ro31-8220, and PKCs and PKCμ inhibitor Go6967 (activating SOC, blocking ROC), respectively. Intracellular Ca 2+ concentration (［Ca 2+］ i) was detected using the fluorescence Ca 2+ indicator Fura-2/AM. The production of NO was determined by DAF-FM (NO fluorescent probe) in HUVECs. RESULTS：Transfection of Orai3 RNA interference plasmids demonstrated that shRNA targeting  Orai3 gene decreased Orai3 mRNA level by 84.5%, and decreased the protein level of Orai3 by 70.68%. Compared with control group, the ［Ca 2+］ i ratio and the net NO fluorescence intensity value in Orai3 shRNA group with 4 different treatments were significantly reduced, and those in vehicle group were not changed. CONCLUSION：Orai3 participates in CaR-mediated Ca 2+ influx and NO production by SOC and ROC activation in HUVECs.