Activation of protein kinase B by adenosine A1 and A3 receptors in newborn rat cardiomyocytes.
It is well established that adenosine receptors are involved in cardioprotection and that protein kinase B (PKB) is associated with cell survival. Therefore, in this study we have investigated whether adenosine receptors (A(1), A(2A) and A(3)) activate PKB by Western blotting and determined the involvement of phosphatidylinositol 3-kinase (PI-3K)/PKB in adenosine-induced preconditioning in cultured newborn rat cardiomyocytes. Adenosine (non-selective agonist), CPA (A(1) selective agonist) and Cl-IB-MECA (A(3) selective agonist) all increased PKB phosphorylation in a time- and concentration-dependent manner. The combined maximal response to CPA and Cl-IB-MECA was similar to the increase in PKB phosphorylation induced by adenosine alone. CGS 21680 (A(2A) selective agonist) did not stimulate an increase in PKB phosphorylation. Adenosine, CPA and Cl-IB-MECA-mediated PKB phosphorylation were inhibited by pertussis toxin (PTX blocks G(i)/G(o)-protein), genistein (tyrosine kinase inhibitor), PP2 (Src tyrosine kinase inhibitor) and by the epidermal growth factor (EGF) receptor tyrosine kinase inhibitor AG 1478. The PI-3K inhibitors wortmannin and LY 294002 blocked A(1) and A(3) receptor-mediated PKB phosphorylation. The role of PI-3K/PKB in adenosine-induced preconditioning was assessed by monitoring Caspase 3 activity and lactate dehydrogenase (LDH) release induced by exposure of cardiomyocytes to 4 h hypoxia (0.5% O(2)) followed by 18 h reoxygenation (HX4/R). Pre-treatment with wortmannin had no significant effect on the ability of adenosine-induced preconditioning to reduce the release of LDH or Caspase 3 activation following HX4/R. In conclusion, we have shown for the first time that adenosine A(1) and A(3) receptors trigger increases in PKB phosphorylation in rat cardiomyocytes via a G(i)/G(o)-protein and tyrosine kinase-dependent pathway. However, the PI-3K/PKB pathway does not appear to be involved in adenosine-induced cardioprotection by preconditioning.
Germack
Renée
R
Interdisciplinary Biomedical Research Centre, School of Science, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK. renee.germack@ntu.ac.uk
Griffin
Martin
M
Dickenson
John M
JM
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D058907:Adenosine A1 Receptor Agonists; D058909:Adenosine A3 Receptor Agonists; D000818:Animals; D000831:Animals, Newborn; D002316:Cardiotonic Agents / Q000494:pharmacology; D015687:Cell Hypoxia / Q000187:drug effects; D002478:Cells, Cultured; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000502:physiology; D017202:Myocardial Ischemia / Q000517:prevention & control; D015428:Myocardial Reperfusion Injury / Q000517:prevention & control; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D037342:Pertussis Toxin / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000819:agonists / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D043682:Receptor, Adenosine A1 / Q000502:physiology*; D043684:Receptor, Adenosine A3 / Q000502:physiology*
D000241:Adenosine; D058907:Adenosine A1 Receptor Agonists; D058909:Adenosine A3 Receptor Agonists; D002316:Cardiotonic Agents; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D048052:Mitogen-Activated Protein Kinase 3; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D043682:Receptor, Adenosine A1; D043684:Receptor, Adenosine A3; C048599:N(6)-cyclopentyladenosine; C090034:2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide