title: Acetylcholine ameliorates endoplasmic reticulum stress in endothelial cells after hypoxia/reoxygenation via M3 AChR-AMPK signaling.
abstract: Endoplasmic reticulum (ER) stress is associated with various cardiovascular diseases. However, its pathophysiological relevance and the underlying mechanisms in the context of hypoxia/reoxygenation (H/R) in endothelial cells are not fully understood. Previous findings have suggested that acetylcholine (ACh), the major vagal nerve neurotransmitter, protected against cardiomyocyte injury by activating AMP-activated protein kinase (AMPK). This study investigated the role of ER stress in endothelial cells during H/R and explored the beneficial effects of ACh. Our results showed that H/R triggered ER stress and apoptosis in endothelial cells, evidenced by the elevation of glucose-regulated protein 78, cleaved caspase-12 and C/EBP homologous protein expression. ACh significantly decreased ER stress and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling positive cells and restored ER ultrastructural changes induced by H/R, possibly via protein kinase-like ER kinase and inositol-requiring kinase 1 pathways. Additionally, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3 AChR) inhibitor, abolished ACh-mediated increase in AMPK phosphorylation during H/R. Furthermore, M3 AChR or AMPK siRNA abrogated the ACh-elicited the attenuation of ER stress in endothelial cells, indicating that the salutary effects of ACh were likely mediated by M3 AChR-AMPK signaling. Overall, ACh activated AMPK through M3 AChR, thereby inhibited H/R-induced ER stress and apoptosis in endothelial cells. We have suggested for the first time that AMPK may function as an essential intermediate step between M3 AChR stimulation and inhibition of ER stress-associated apoptotic pathway during H/R, which may help to develop novel therapeutic approaches targeting ER stress to prevent or alleviate ischemia/reperfusion injury.
authors.affiliation: a Department of Pharmacology ; Xi'an Jiaotong University Health Science Center , Xi'an , P.R. China.
authors.forename: Xueyuan
authors.initials: X
authors.lastname: Bi
authors.forename: Xi
authors.initials: X
authors.lastname: He
authors.forename: Man
authors.initials: M
authors.lastname: Xu
authors.forename: Ming
authors.initials: M
authors.lastname: Zhao
authors.forename: Xiaojiang
authors.initials: X
authors.lastname: Yu
authors.forename: Xingzhu
authors.initials: X
authors.lastname: Lu
authors.forename: Weijin
authors.initials: W
authors.lastname: Zang
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000109:Acetylcholine / Q000378:metabolism*; D017209:Apoptosis; D053454:Caspase 12 / Q000378:metabolism; D015687:Cell Hypoxia / Q000502:physiology*; D002460:Cell Line; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000502:physiology*; D004722:Endoribonucleases / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000473:pathology*; D006801:Humans; D010880:Piperidines / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D043587:Receptor, Muscarinic M3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015427:Reperfusion Injury / Q000473:pathology; D015398:Signal Transduction; D051742:Transcription Factor CHOP / Q000378:metabolism
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D010880:Piperidines; D034741:RNA, Small Interfering; D043587:Receptor, Muscarinic M3; D051742:Transcription Factor CHOP; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D004253:DNA Nucleotidylexotransferase; D004722:Endoribonucleases; C506444:CASP12 protein, human; D053454:Caspase 12; D000109:Acetylcholine
keywords: 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; 4-PBA, 4-phenyl butyric acid; ACh, acetylcholine; AMPK; AMPK, AMP-activated protein kinase; ATF6, activating transcription factor 6; CHOP, C/EBP homologous protein; DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; GAPDH, glyceraldehyde 3-phospharte dehydrogenase; GRP78, glucose-regulated protein 78; H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; IRE1, inositol-requiring kinase 1; M3 AChR; MAChR, muscarinic acetylcholine receptor; PBS, phosphate-buffered saline; PERK, protein kinase-like ER kinase; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling; acetylcholine; apoptosis; endoplasmic reticulum stress; endothelial cells; ischemia/reperfusion injury