TRPC1/STIM1复合体调节人脐静脉内皮细胞钙池操纵性钙通道和受体操纵性钙通道介导的钙内流和NO生成

TRPC1/STIM1 regulates Ca^(2+) entry and NO generation mediated by human umbilical vein endothelial cell store-and receptor-operated calcium channels

目的研究瞬时受体电位通道1(TRPC1)/基质交联分子1(STIM1)复合体在人脐静脉内皮细胞(HUVEC)钙池操纵性钙通道(SOCC)和受体操纵性钙通道(ROCC)介导的钙内流和NO生成中的作用。方法取2~3代HUVEC,将构建的TRPC1和STIM1干扰质粒分别转染HUVEC,观察细胞转染效果的同时采用real-time PCR和Western blot检测TRPC1、STIM1 mRNA和蛋白的表达。将细胞分别与钙敏感受体(CaR)激动剂精胺、ROCC模拟剂(TPA)+CaR负性变构调节剂Calhex231、蛋白激酶C(PKC)抑制剂Ro31-8220及经典型PKCs和PKCμ抑制剂Go6967孵育后,用荧光探针Fura-2/AM及DAF-FM负载方法同步检测[Ca2+]i和NO生成的变化;随后用TRPC1和STIM1干扰质粒同时转染HUVEC,与精胺孵育后检测[Ca2+]i和NO生成,免疫共沉淀法检测TRPC1和STIM1的相互作用。结果与Control组相比,TRPC1转染组和STIM1转染组TRPC1、STIM1 mRNA和蛋白表达均明显降低(P<0.05);在四种不同处理作用下,TRPC1转染组和STIM1转染组[Ca2+]i△ratio值和NO净荧光强度值均明显降低(P<0.05);与Control组、TRPC1转染组及STIM1转染组相比,TRPC1和STIM1共转染组[Ca2+]i△ratio值和NO净荧光强度值均明显降低(P<0.05);TRPC1与STIM1相互作用形成复合体,且在CaR激动剂的剌激下作用增强。结论 TRPC1/STIM1复合体共同调节CaR经SOCC和ROCC激活介导的钙内流和NO生成。

Aim To study the function of TRPC1/STIM1 in Ca^（2＋）entry and nitric oxide（ NO） generation mediated by store-operated calcium channel（ SOCC） and receptor-operated calcium channel（ ROCC） in human umbilical vein endothelial cells. Methods Human umbilical vein endothelial cells were collected and cultured to the second ~ third passage. The constructed TRPC1 and STIM1 interference plasmids were transfected into human umbilical vein endothelial cells respectively,and the transfection efficiency was observed. The expressions of TRPC1 and STIM1 mRNA and protein were detected by real-time PCR and Western blot. The cells were incubated with CaR agonist spermine,CaR negative allosteric modulator Calhex231 and ROCC analogue TPA,protein kinase C（ PKC） inhibitor Ro31-8220,PKCs and PKCμinhibitor Go6967. 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 of every group in HUVEC. The constructed TRPC1 and STIM1 interference plasmids were simultaneously transfected with human umbilical vein endothelial cells and incubated with CaR agonists to detect [Ca^（2＋）]iand NO production,and the interaction between STIM1 and TRPC1 was examined by co-immunoprecipitation. Results Compared with the control group,the expression of TRPC1 and STIM1 mRNA and protein in TRPC1 transfection group and STIM1 transfection group decreased obviously（ P0.05）. In four different treatment under the action of factors,the [Ca^（2＋）]iand the net NO fluorescence intensity ratio values of TRPC1 transfection group and STIM1 transfection group were significantly reduced（ P〈0. 05）. Compared with control group,TRPC1 transfection group and STIM1 transfection group,the [Ca^（2＋）]iand the net NO fluorescence intensity ratio values of co-transfection group were significantly reduced（ P〈0. 05）. TRPC1 and STIM1 interact to form a complex,and in the stimulation of CaR agonists under enhanced interaction. Conclusion TRPC1,STIM1 are components of SOCC and ROCC in Ca^（2＋）entry and NO generation in human umbilical vein endothelial cells.