Activation of protein kinase B by adenosine A(1) and A(3) receptors in newborn rat cardiomyocytes

作者：Germack R^*; Griffin M; Dickenson JM

来源：Journal of Molecular and Cellular Cardiology, 2004, 37(5): 989-999.

DOI：10.1016/j.yjmcc.2004.08.001

• adenosine A(1) receptor
• adenosine A(3) receptor
• cardiomyocytes protein kinase B Ak(1)
• hypoxia
• ischaemia
• reoxygenation
• caspase 3
• lactate dehydrogenase

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 (Pl-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 Gi/G.-protein and tyrosine kinase-dependent pathway. However, the PI-3K/PKB pathway does not appear to be involved in adenosine-induced cardioprotection by preconditioning.