关键词: TRPC1, Orai1, 一氧化氮, 钙离子, 人脐静脉内皮细胞

Abstract: Objective To study the function of TRPC1/ORAI1 in store and receptor-operated Ca2+ entry and nitric oxide generation by SOC and ROC in human umbilical vein endothelial cells. Methods HUVECs were collected and cultured to the second~third passage. We silenced the expression of their genes in HUVEC by transfection constructed TRPC1 or ORAI1 RNA interference plasmids. The interference efficiency of their protein and mRNA levels were determined by Western blotting and real-time PCR, respectively. The cells were divided into: short hairpin RNA(shRNA) group; control group and vehicle group. The cells were incubated with CaR agonist, CaR negative allosteric modulator Calhex231 and receptor-operated channels (ROC) analogue TPA, Protein kinase C (PKC) inhibitor Ro31-8220, PKCs and PKCμ inhibitor Go6967 incubate, intracellular Ca2+ concentration ([Ca2+]i) was detected using the fluorescence Ca2+ indicator Fura-2/AM, the production of NO was determined by DAF-FM of every group in HUVEC. HUVECs were transduced with shRNA-TRPC1 and shRNA-ORAI1 at same time, after cultured with CaR agonist, [Ca2+]i and the production of NO was determined. The interaction between ORAI1 and TRPC1 were examined by Co-immunoprecipitation. Results 1) Compared with control group, shRNA targeted to the TRPC1 or ORAI1 genes decreased their mRNA and protein levels, respectively (p＜0.05); 2) In four different treatment under the action of factors, the [Ca2+]i and the net NO fluorescence intensity ratio values of transfection of TRPC1 or ORAI1 shRNA group were significantly reduced (p＜0.05). 3) Compared with the control and single shRNA group, the[Ca2+]i and the net NO fluorescence intensity ratio values of transfection of TRPC1 and ORAI1 shRNA group were significantly reduced (p＜0.05). 4) ORAI1 co-precipitates with TRPC1, indicating internation of a molecular complex had enhanced by CaR agonist. Conclusions TRPC1, ORAI1 are components of SOCE and ROCE channels in store and receptor-operated Ca2+ entry and nitric oxide generation in human umbilical vein endothelial cells.

Key words: TRPC1, ORAI1, Nitric Oxide, Ca2+, Human umbilical vein endothelial cells