Acetylcholine attenuated TNF-α-induced intracellular Ca(2+) overload by inhibiting the formation of the NCX1-TRPC3-IP3R1 complex in human umbilical vein endothelial cells.

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Acetylcholine attenuated TNF-α-induced intracellular Ca(2+) overload by inhibiting the formation of the NCX1-TRPC3-IP3R1 complex in human umbilical vein endothelial cells.

J Mol Cell Cardiol. 2017 Apr 07;:

Authors: Zhao M, Jia HH, Liu LZ, Bi XY, Xu M, Yu XJ, He X, Zang WJ

Abstract
The endoplasmic reticulum (ER) forms discrete junctions with the plasma membrane (PM) that play a critical role in the regulation of Ca(2+) 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 Ca(2+) 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 Ca(2+). Protein-protein interactions were assessed by immunoprecipitation. Ca(2+) 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 Ca(2+) and the release of intracellular Ca(2+) 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, [Ca(2+)]cyt overload and ER-PM interactions, which depend on the muscarinic 3 receptor/AMPK pathway, and that acetylcholine may be a new inhibitor for suppressing [Ca(2+)]cyt overload.

PMID: 28395930 [PubMed - as supplied by publisher]

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