RESUMEN
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+]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 [Ca2+]cyt overload.
Asunto(s)
Inflamación/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Intercambiador de Sodio-Calcio/genética , Canales Catiónicos TRPC/genética , Factor de Necrosis Tumoral alfa/metabolismo , Acetilcolina/metabolismo , Apoptosis/genética , Calcio/metabolismo , Señalización del Calcio/genética , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Homeostasis/genética , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Receptores de Inositol 1,4,5-Trifosfato/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , ARN Interferente Pequeño/genética , Intercambiador de Sodio-Calcio/química , Canales Catiónicos TRPC/químicaRESUMEN
Calcium overload is one of the important mechanisms of cardiovascular disease. Endoplasmic reticulum is an important organelle which regulates intracellular calcium homeostasis by uptake, storage and mobilization of calcium. So it plays a critical role in regulation of intracellular calcium homeostasis. Endoplasmic reticulum, which is widely distributed in cytoplasm, has a large number of membrane junction sites. Recent studies have reported that these junction sites are distributed on plasma membrane and organelle membranes (mitochondria, lysosomes, Golgi apparatus, etc.), separately. They could form complexes to regulate calcium transport. In this review, we briefly outlined the recent research progresses of endoplasmic reticulum-plasma membrane junctions in intracellular calcium homeostasis and cardiovascular disease, which may offer a new strategy for prevention and treatment of cardiovascular disease.