Activation of protein kinase B by adenosine A1 and A3 receptors in newborn rat cardiomyocytes

• Published in: Journal of molecular and cellular cardiology 2004-11, Vol.37 (5), p.989-999
• Main Authors: Germack, Renée, Griffin, Martin, Dickenson, John M
• Format: Article
• Language: English
• Subjects: Adenosine - analogs & derivatives; Adenosine - pharmacology; Adenosine A1 Receptor Agonists; Adenosine A3 Receptor Agonists; Animals; Animals, Newborn; Cardiotonic Agents - pharmacology; Cell Hypoxia - drug effects; Cells, Cultured; GTP-Binding Protein alpha Subunits, Gi-Go - antagonists & inhibitors; Mitogen-Activated Protein Kinase 3 - antagonists & inhibitors; Mitogen-Activated Protein Kinase 3 - physiology; Myocardial Ischemia - prevention & control; Myocardial Reperfusion Injury - prevention & control; Myocytes, Cardiac - drug effects; Myocytes, Cardiac - enzymology; Pertussis Toxin - pharmacology; Phosphatidylinositol 3-Kinases - antagonists & inhibitors; Phosphatidylinositol 3-Kinases - physiology; Phosphorylation - drug effects; Protein Kinase Inhibitors - pharmacology; Protein-Serine-Threonine Kinases - metabolism; Proto-Oncogene Proteins - agonists; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-akt; Rats; Receptor, Adenosine A1 - physiology; Receptor, Adenosine A3 - physiology
• ISSN: 0022-2828

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.