title: Role of reactive oxygen species in acetylcholine-induced preconditioning in cardiomyocytes.
abstract: We examined the ability of ACh to mimic ischemic preconditioning in cardiomyocytes and the role of ATP-sensitive potassium (KATP) channels and mitochondrial reactive oxygen species (ROS) in mediating this effect. Chick embryonic ventricular myocytes were studied in a flow-through chamber while flow rate, pH, PO2, and PCO2 were controlled. Cell viability was quantified with propidium iodide (5 microM), and production of ROS was measured using 2', 7'-dichlorofluorescin diacetate. Data were expressed as means +/- SE. Preconditioning with 10 min of ischemia followed by 10 min of reoxygenation or 10 min of ACh (1 mM) followed by a drug-free period before 1 h of ischemia and 3 h of reoxygenation reduced cell death to the same extent [preconditioning 19 +/- 2% (n = 6, P < 0.05) ACh 21 +/- 5% (n = 6, P < 0.05) vs controls 42 +/- 5% (n = 9)]. Like preconditioning, ACh increased ROS production threefold before ischemia [0.60 +/- 0.16 (n = 7, P < 0.05) vs. controls, 0.16 +/- 0. 03 (n = 6); arbitrary units]. Protection and increased ROS production during ACh preconditioning were abolished with 5-hydroxydecanoate (5-HD, 100 microM), a selective mitochondrial K(ATP) channel antagonist, and the thiol reductant 2-mercaptopropionyl glycine (2-MPG, 1 mM), an antioxidant [cell death: 5-HD+ACh 37 +/- 7% (n = 5), 2-MPG+ACh 47 +/- 6% (n = 6); ROS signals: 5-HD+ACh 0.09 +/- 0.03 (n = 5), 2-MPG+ACh 0.01 +/- 0.04 (n = 4)]. In addition, ACh-induced ROS signaling was blocked by the mitochondrial site III electron transport inhibitor myxothiazol (0.02 +/- 0.07, n = 5). These results demonstrate that activation of mitochondrial K(ATP) channels and increased ROS production from mitochondria are important intracellular signals that participate in ACh-induced preconditioning in cardiomyocytes.
authors.affiliation: Department of Anesthesia and Critical Care, University of Chicago, Chicago, Illinois 60637, USA. zyao@airway2.uchicago.edu
authors.forename: Z
authors.initials: Z
authors.lastname: Yao
authors.forename: J
authors.initials: J
authors.lastname: Tong
authors.forename: X
authors.initials: X
authors.lastname: Tan
authors.forename: C
authors.initials: C
authors.lastname: Li
authors.forename: Z
authors.initials: Z
authors.lastname: Shao
authors.forename: W C
authors.initials: WC
authors.lastname: Kim
authors.forename: T L
authors.initials: TL
authors.lastname: vanden Hoek
authors.forename: L B
authors.initials: LB
authors.lastname: Becker
authors.forename: C A
authors.initials: CA
authors.lastname: Head
authors.forename: P T
authors.initials: PT
authors.lastname: Schumacker
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D002316:Cardiotonic Agents / Q000494:pharmacology; D002478:Cells, Cultured; D002642:Chick Embryo; D003652:Decanoic Acids / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D006352:Heart Ventricles / Q000196:embryology; D006863:Hydrogen-Ion Concentration; D006880:Hydroxy Acids / Q000494:pharmacology; D019194:Ischemic Preconditioning / Q000379:methods*; D008689:Methacrylates; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism; D017202:Myocardial Ischemia; D015425:Myocardial Reperfusion; D009206:Myocardium / Q000166:cytology; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology; D017382:Reactive Oxygen Species / Q000502:physiology*; D015398:Signal Transduction; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D008625:Tiopronin / Q000494:pharmacology
chemical_list: D000889:Anti-Arrhythmia Agents; D002316:Cardiotonic Agents; D003652:Decanoic Acids; D006880:Hydroxy Acids; D008689:Methacrylates; D015221:Potassium Channels; D017382:Reactive Oxygen Species; D013844:Thiazoles; C052853:5-hydroxydecanoic acid; C030517:myxothiazol; D008625:Tiopronin; D000109:Acetylcholine