title: Early ischaemic preconditioning requires Akt- and PKA-mediated activation of eNOS via serine1176 phosphorylation.
abstract: OBJECTIVE
  The role of endothelial nitric oxide synthase (eNOS)/NO signalling is well documented in late ischaemic preconditioning (IPC); however, the role of eNOS and its activation in early IPC remains controversial. This study investigates the role of eNOS in early IPC and the signalling pathways and molecular interactions that regulate eNOS activation during early IPC.
  
  
  RESULTS
  Rat hearts were subjected to 30-min global ischaemia and reperfusion (I/R) with or without IPC (three cycles 5-min I and 5-min R) in the presence or absence of the NOS inhibitor l-NAME, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY), and protein kinase A (PKA) inhibitor H89 during IPC induction or prior endothelial permeablization. IPC improved post-ischaemic contractile function and reduced infarction compared with I/R with this being abrogated by l-NAME or endothelial permeablization. eNOS(Ser1176), Akt(Ser473), and PKA(Thr197) phosphorylation was increased following IPC. I/R decreased eNOS(Ser1176) phosphorylation, whereas IPC increased it. Mass spectroscopy confirmed eNOS(Ser1176) phosphorylation and quantitative Western blots showed ∼24% modification of eNOS(Ser1176) following IPC. Immunoprecipitation demonstrated eNOS, Akt, and PKA complexation. Immunohistology showed IPC-induced Akt and PKA phosphorylation in cardiomyocytes and endothelium. With eNOS activation, IPC increased NO production as measured by electron paramagnetic resonance spin trapping and fluorescence microscopy. LY or H89 not only decreased Akt(Ser473) or PKA(Thr197) phosphorylation, respectively, but also abolished IPC-induced preservation of eNOS and eNOS(Ser1176) phosphorylation as well as cardioprotection.
  
  
  CONCLUSIONS
  Thus, Akt- and PKA-mediated eNOS activation, with phosphorylation near the C-terminus, is critical for early IPC-induced cardioprotection, with eNOS-derived NO from the endothelium serving a critical role.
authors.affiliation: Division of Cardiovascular Medicine, Department of Internal Medicine, Davis Heart and Lung Research Institute, The Ohio State University, 473 W. 12th Ave, Columbus, OH 43210, USA.
authors.forename: Changjun
authors.initials: C
authors.lastname: Yang
authors.forename: M A Hassan
authors.initials: MA
authors.lastname: Talukder
authors.forename: Saradhadevi
authors.initials: S
authors.lastname: Varadharaj
authors.forename: Murugesan
authors.initials: M
authors.lastname: Velayutham
authors.forename: Jay L
authors.initials: JL
authors.lastname: Zweier
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003331:Coronary Vessels / Q000187:drug effects / Q000201:enzymology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004195:Disease Models, Animal; D004578:Electron Spin Resonance Spectroscopy; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047468:Immunoprecipitation; D019194:Ischemic Preconditioning; D008297:Male; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009200:Myocardial Contraction; D009203:Myocardial Infarction / Q000201:enzymology / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control*; D015428:Myocardial Reperfusion Injury / Q000201:enzymology / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control*; D009206:Myocardium / Q000201:enzymology* / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010477:Perfusion; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine; D015398:Signal Transduction; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D009569:Nitric Oxide; D012694:Serine; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases