title: Acetylcholine attenuates hypoxia/ reoxygenation-induced mitochondrial and cytosolic ROS formation in H9c2 cells via M2 acetylcholine receptor.
abstract: BACKGROUND
  The anti-infammatory and cardioprotective effect of acetylcholine (ACh) has been reported; nevertheless, whether and how ACh exhibits an antioxidant property against ischemia/reperfusion (I/R)-induced oxidative stress remains obscure.
  
  
  METHODS
  In the present study, H9c2 rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) to mimic I/R injury. We estimated intracellular different sources of reactive oxygen species (ROS) by measuring mitochondrial ROS (mtROS), mitochondrial DNA (mtDNA) copy number, xanthine oxidase (XO) and NADPH oxidase (NOX) activity and expression of rac 1. Cell injury was determined by lactate dehydrogenase (LDH) release and cleaved caspase-3 expression. The siRNA transfection was performed to knockdown of M2 acetylcholine receptor (M2 AChR) expression.
  
  
  RESULTS
  12-h hypoxia followed by 2-h reoxygenation resulted in an abrupt burst of ROS in H9c2 cells. Administration of ACh reduced the levels of ROS in a concentration-dependent manner. Compared to the H/R group, ACh decreased mtROS, recovered mtDNA copy number, diminished XO and NOX activity, rac 1 expression as well as cell injury. Co- treatment with atropine rather than hexamethonium abolished the antioxidant and cardioprotective effect of ACh. Moreover, knockdown of M2 AChR by siRNA showed the similar trends as atropine co-treatment group.
  
  
  CONCLUSIONS
  ACh inhibits mitochondria-, XO- and NOX-derived ROS production thus protecting H9c2 cells against H/R-induced oxidative stress, and these benefcial effects are mainly mediated by M2 AChR. Our findings suggested that increasing ACh release could be a potential therapeutic strategy for treatment and prevention of I/R injury.
authors: Department of Pharmacology, Xi'an Jiaotong University, College of Medicine, Xi'an, P R China.
authors: Yi
authors: Y
authors: Miao
authors: Jun
authors: J
authors: Zhou
authors: Mei
authors: M
authors: Zhao
authors: Jinjun
authors: J
authors: Liu
authors: Lei
authors: L
authors: Sun
authors: Xiaojiang
authors: X
authors: Yu
authors: Xi
authors: X
authors: He
authors: Xiaoyue
authors: X
authors: Pan
authors: Weijin
authors: W
authors: Zang
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D043585:Receptor, Muscarinic M2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology; D014969:Xanthine Oxidase / Q000378:metabolism
chemical_list: D000975:Antioxidants; D004272:DNA, Mitochondrial; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D043585:Receptor, Muscarinic M2; D014969:Xanthine Oxidase; D019255:NADPH Oxidases; D053148:Caspase 3; D000109:Acetylcholine