Acetylcholine attenuated TNF-alpha-induced intracellular Ca2+ overload by inhibiting the formation of the NCX1-TRPC3-IP3R1 complex in human umbilical vein endothelial cells
Acetylcholine attenuated TNF-alpha-induced intracellular Ca2+ overload by inhibiting the formation of the NCX1-TRPC3-IP3R1 complex in human umbilical vein endothelial cells
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乙酰胆碱通过抑制人脐静脉内皮细胞中NCX1-TRPC3-IP3R1复合物的形成来减轻TNF-α诱导的细胞内Ca2超载
DOI：
10.1016/j.yjmcc.2017.04.001
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发表时间：
2017
影响因子：
5
通讯作者：
Zang Wei-Jin
中科院分区：
文献类型：
--
作者：
Zhao Ming;Jia Hang-Huan;Liu Long-Zhu;Bi Xue-Yuan;Xu Man;Yu Xiao-Jiang;He Xi;Zang Wei-Jin
内质网（ER）与质膜（PM）形成离散连接，其在细胞生物能量学、凋亡和自噬期间的Ca 2+信号调节中起关键作用。我们先前已经证实，乙酰胆碱可以抑制炎症损伤后的ER应激和细胞凋亡。然而，有限的研究集中在乙酰胆碱对ER-PM连接的影响。在这项工作中，我们评估了超分子钠钙交换器1（NCX 1）-瞬时受体电位典型3（TRPC 3）-肌醇1，4，5-三磷酸受体1（IP 3R 1）复合物的结构和功能，该复合物参与调节炎症损伤期间的Ca 2+稳态。用透射电镜和Ca 2+荧光探针测量人脐静脉内皮细胞（HUVECs）ER-PM连接的宽度（单位：纳米）。通过免疫沉淀法评估蛋白质-蛋白质相互作用。使用共聚焦显微镜测量Ca 2+浓度。采用siRNA测定来沉默特定蛋白质。我们的结果表明，当肿瘤坏死因子-α（TNF-α）诱导时，外周ER移位到PM连接位点，并在这些位点形成NCX 1-TRPC 3-IP 3R 1复合物。下调NCX 1或IP 3R 1蛋白表达后，发现TNF-α处理的细胞中NCX 1介导的Ca 2+内流和胞内Ca 2+库的释放减少。我们还观察到乙酰胆碱减弱了TNF-α处理的细胞中NCX 1-TRPC 3-IP 3R 1复合物的形成，并维持了钙稳态。有趣的是，乙酰胆碱的积极作用被取消的选择性M3 AChR拮抗剂达非那新和AMPK siRNA。这些结果表明，乙酰胆碱保护内皮细胞免受TNF-α诱导的损伤，[Ca ~（2+）]细胞超载和ER-PM相互作用，这依赖于M受体/AMPK途径，乙酰胆碱可能是一种新的抑制[Ca ~（2+）]细胞超载的抑制剂。
The endoplasmic reticulum (ER) forms discrete junctions with the plasma membrane (PM) that play a critical role in the regulation of Ca2 +signaling during cellular bioenergetics, apoptosis and autophagy. We have previously confirmed that acetylcholine can inhibit ER stress and apoptosis after inflammatory injury. However, limited research has focused on the effects of acetylcholine on ER-PM junctions. In this work, we evaluated the structure and function of the supramolecular sodium-calcium exchanger 1 (NCX1)-transient receptor potential canonical 3 (TRPC3)-inositol 1,4,5-trisphosphate receptor 1 (IP3R1) complex, which is involved in regulating Ca2 +homeostasis during inflammatory injury. The width of the ER-PM junctions of human umbilical vein endothelial cells (HUVECs) was measured in nanometres using transmission electron microscopy and a fluorescent probe for Ca2 +. Protein-protein interactions were assessed by immunoprecipitation. Ca2 +concentration was measured using a confocal microscope. An siRNA assay was employed to silence specific proteins. Our results demonstrated that the peripheral ER was translocated to PM junction sites when induced by tumour necrosis factor-alpha (TNF-α) and that NCX1-TRPC3-IP3R1 complexes formed at these sites. After down-regulating the protein expression of NCX1 or IP3R1, we found that the NCX1-mediated inflow of Ca2 +and the release of intracellular Ca2 +stores were reduced in TNF-α-treated cells. We also observed that acetylcholine attenuated the formation of NCX1-TRPC3-IP3R1 complexes and maintained calcium homeostasis in cells treated with TNF-α. Interestingly, the positive effects of acetylcholine were abolished by the selective M3AChR antagonist darifenacin and by AMPK siRNAs. These results indicate that acetylcholine protects endothelial cells from TNF-alpha-induced injury, [Ca2 +]cytoverload and ER-PM interactions, which depend on the muscarinic 3 receptor/AMPK pathway, and that acetylcholine may be a new inhibitor for suppressing [Ca2 +]cytoverload.