Department of Pathophysiology and Key Laboratory of Education Ministry of Xinjiang Endemic and Ethnic Diseases, Medical College of Shihezi University, Shihezi, Xinjiang 832002, P.R. China.
Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology and Key Laboratory for Respiratory Diseases, Health Ministry of China, Wuhan, Hubei 430030, P.R. China.
Mol Med Rep. 2017 Oct;16(4):4613-4619. doi: 10.3892/mmr.2017.7164. Epub 2017 Aug 4.
Store‑operated Ca2+ entry (SOCE) via store‑operated Ca2+ channels (SOCC), encoded by transient receptor potential canonical (TRPC) channel proteins, is an important underlying mechanism regulating intracellular Ca2+ concentration ([Ca2+]i) and various intracellular functions in endothelial cells (ECs). TRPC1, the probable candidate for SOCC, is expressed in ECs. Ca2+‑sensing receptor (CaSR) is functionally expressed in vascular endothelium and is important in Ca2+ mobilization and cardiovascular functions. To date, there have been no reports demonstrating an association between CaSR and TRPC1 in ECs. The present study investigated the effects of TRPC1 on CaSR‑induced Ca2+ influx and nitric oxide (NO) production in human umbilical vein ECs (HUVECs). TRPC1 and CaSR proteins in HUVECs were measured by immunostaining and western blot analysis. [Ca2+]i levels were measured using the Fura‑2‑acetoxymethyl ester method. The indicator 3‑amino, 4‑aminomethyl‑2, 7‑difluorescein diacetate was used to measure NO production in HUVECs. The expression of TRPC1 protein in HUVECs was silenced by transfecting HUVECs with small interfering RNA (siRNA) against TRPC1. Although changes in extracellular Ca2+ failed to alter [Ca2+]i in HUVECs, the CaSR agonist spermine increased [Ca2+]i and NO production in HUVECs. NO production in HUVECs was diminished in Ca2+‑free medium or following treatment with a CaSR negative allosteric modulator (Calhex231), SOCC inhibitor (MRS1845) or TRPC inhibitor (SKF96365). The spermine‑induced increases in [Ca2+]i and NO production were reduced in HUVECs transfected with TRPC1 siRNA. These results suggested that TRPC1 is a primary candidate in forming SOCC that stimulates CaSR‑induced SOCE and NO production in HUVECs and is a potential therapeutic target for vascular diseases.
钙库操纵性钙内流（SOCE）通过钙库操纵性钙通道（SOCC）进行，其编码由瞬时受体电位经典（TRPC）通道蛋白组成，是调节内皮细胞（ECs）细胞内钙离子浓度([Ca2+]i)和各种细胞内功能的重要基础机制。TRPC1 是 SOCC 的可能候选物，在 ECs 中表达。钙敏感受体（CaSR）在血管内皮中功能性表达，对于 Ca2+动员和心血管功能非常重要。迄今为止，尚无报道表明 CaSR 与 ECs 中的 TRPC1 之间存在关联。本研究探讨了 TRPC1 对人脐静脉内皮细胞（HUVECs）中 CaSR 诱导的 Ca2+内流和一氧化氮（NO）产生的影响。通过免疫染色和 Western blot 分析测量 HUVEC 中的 TRPC1 和 CaSR 蛋白。使用 Fura-2-乙酰氧基甲酯法测量 [Ca2+]i 水平。使用 3-氨基，4-氨甲基-2,7-二氟荧光素二乙酸酯作为指示剂测量 HUVEC 中的 NO 产生。通过用针对 TRPC1 的小干扰 RNA（siRNA）转染 HUVEC 来沉默 HUVEC 中 TRPC1 蛋白的表达。尽管细胞外 Ca2+的变化未能改变 HUVEC 中的 [Ca2+]i，但 CaSR 激动剂 spermine 增加了 HUVEC 中的 [Ca2+]i 和 NO 产生。在无钙培养基中或在用 CaSR 负变构调节剂（Calhex231）、SOCC 抑制剂（MRS1845）或 TRPC 抑制剂（SKF96365）处理后，HUVEC 中的 NO 产生减少。在转染了 TRPC1 siRNA 的 HUVEC 中，spermine 诱导的 [Ca2+]i 和 NO 产生的增加减少。这些结果表明，TRPC1 是形成 SOCC 的主要候选物，该 SOCC 刺激 CaSR 诱导的 SOCE 和 HUVEC 中的 NO 产生，是血管疾病的潜在治疗靶标。
