title: Activation of cGMP/protein kinase G pathway in postconditioned myocardium depends on reduced oxidative stress and preserved endothelial nitric oxide synthase coupling.
abstract: BACKGROUND
  The cGMP/protein kinase G (PKG) pathway is involved in the cardioprotective effects of postconditioning (PoCo). Although PKG signaling in PoCo has been proposed to depend on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt cascade, recent data bring into question a causal role of reperfusion injury signaling kinase (RISK) in PoCo protection. We hypothesized that PoCo increases PKG activity by reducing oxidative stress-induced endothelial nitric oxide synthase (NOS) uncoupling at the onset of reperfusion.
  
  
  RESULTS
  Isolated rat hearts were submitted to 40 minutes of ischemia and reperfusion with and without a PoCo protocol. PoCo reduced infarct size by 48% and cGMP depletion. Blockade of cGMP synthesis (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and inhibition of PKG (KT5823) or NOS (l-NAME) abolished protection, but inhibition of PI3K/Akt cascade (LY294002) did not (n=5 to 7 per group). Phosphorylation of the RISK pathway was higher in PoCo hearts. However, this difference is due to increased cell death in control hearts because in hearts reperfused with the contractile inhibitor blebbistatin, a drug effective in preventing cell death at the onset of reperfusion, RISK phosphorylation increased during reperfusion without differences between control and PoCo groups. In these hearts, PoCo reduced the production of superoxide (O2(-)) and protein nitrotyrosylation and increased nitrate/nitrite levels in parallel with a significant decrease in the oxidation of tetrahydrobiopterin (BH4) and in the monomeric form of endothelial NOS.
  
  
  CONCLUSIONS
  These results demonstrate that PoCo activates the cGMP/PKG pathway via a mechanism independent of the PI3K/Akt cascade and dependent on the reduction of O2(-) production at the onset of reperfusion, resulting in attenuated oxidation of BH4 and reduced NOS uncoupling.
authors.affiliation: Laboratory of Experimental Cardiology, Vall d'Hebron University Hospital and Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain. jinserte@ir.vhebron.net
authors.forename: Javier
authors.initials: J
authors.lastname: Inserte
authors.forename: Victor
authors.initials: V
authors.lastname: Hernando
authors.forename: Úrsula
authors.initials: Ú
authors.lastname: Vilardosa
authors.forename: Elena
authors.initials: E
authors.lastname: Abad
authors.forename: Marcos
authors.initials: M
authors.lastname: Poncelas-Nozal
authors.forename: David
authors.initials: D
authors.lastname: Garcia-Dorado
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D016923:Cell Death; D006152:Cyclic GMP / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004195:Disease Models, Animal; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057775:Ischemic Postconditioning; D008297:Male; D009203:Myocardial Infarction / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D015428:Myocardial Reperfusion Injury / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D009206:Myocardium / Q000201:enzymology* / Q000473:pathology; D009566:Nitrates / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D009566:Nitrates; D009573:Nitrites; D013481:Superoxides; D001708:Biopterins; C002744:3-nitrotyrosine; D014443:Tyrosine; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D017869:Cyclic GMP-Dependent Protein Kinases; C003402:sapropterin; D006152:Cyclic GMP