title: ROS-mediated ERK activation in delayed protection from anoxic preconditioning in neonatal rat cardiomyocytes.
abstract: BACKGROUND
  The activation of extracellular signal-regulated kinase1/2 (ERK1/2) has been shown to be important signaling pathway in the ischemic preconditioning (IPC) response. Recently, some studies suggest a key role for the mitochondrial ATP-sensitive potassium channel (mKATP) as both a trigger and an end effector of acute and delayed protection of IPC. Hence, this study was undertaken to elucidate the relationship between mKATP and ERK1/2 in the delayed protection mechanism of anoxic preconditioning (APC).
  
  
  METHODS
  An APC model was established using cultured neonatal rat cardiomyocytes. Pharmacological agents [diazoxide, 5-hydroxydecanoate (5-HD), 2-mercaptopropionylglycine (MPG), and PD98059] were used to modulate mKATP and ERK1/2 activation. Cellular injury was evaluated by measuring cellular superoxide dismutase (SOD) activity, cell viability, and lactate dehydrogenase (LDH) release. The generation of cellular reactive oxygen species (ROS) and the activation of ERK1/2 were determined at different time points starting from the beginning of preconditioning with anoxia or diazoxide (an mKATP opener).
  
  
  RESULTS
  Cell viability and SOD activity in the APC [(81.9 +/- 11.4)%, (13.6 +/- 3.7) U/L] and diazoxide [(79.2 +/- 12.4)%, (16.5 +/- 4.6) U/L] groups were significantly higher than in the anoxia/reoxygenation (A/R) [(42.2 +/- 7.3)%, (8.8 +/- 2.8) U/L] group (all P < 0.01). LDH activity in the APC group [(101.9 +/- 18.9) U/L] and diazoxide group [(97.5 +/- 17.7) U/L] was significantly lower than in the A/R group [(250.5 +/- 43.6) U/L] (all P < 0.01). Both APC and diazoxide simultaneously facilitated intracellular ROS generation and rapid ERK1/2 activation. But the effects of APC and diazoxide were remarkedly attenuated by 5-HP (an mKATP blocker) and by MPG (a free radical scavenger). In addition, the ERK1/2 inhibitor PD98059 also abolished the cellular protective effects induced by diazoxide.
  
  
  CONCLUSIONS
  mKATP may mediate ERK1/2 activation during anoxia preconditioning by generating ROS, which then triggers the delayed protection of APC in rat cardiomyocytes.
authors.affiliation: Department of Cardiology, First People's Hospital of Yangzhou, Yangzhou 225001, China. yungkzh@126.com
authors.forename: Kai-zheng
authors.initials: KZ
authors.lastname: Gong
authors.forename: Zhen-gang
authors.initials: ZG
authors.lastname: Zhang
authors.forename: Ai-hua
authors.initials: AH
authors.lastname: Li
authors.forename: Yi-feng
authors.initials: YF
authors.lastname: Huang
authors.forename: Ping
authors.initials: P
authors.lastname: Bu
authors.forename: Feng
authors.initials: F
authors.lastname: Dong
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D004789:Enzyme Activation; D019194:Ischemic Preconditioning; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species; D020928:Mitogen-Activated Protein Kinases