Germack Renée, Griffin Martin, Dickenson John M
Interdisciplinary Biomedical Research Centre, School of Science, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK.
J Mol Cell Cardiol. 2004 Nov;37(5):989-99. doi: 10.1016/j.yjmcc.2004.08.001.
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.
腺苷受体参与心脏保护以及蛋白激酶B（PKB）与细胞存活相关，这一点已得到充分证实。因此，在本研究中，我们通过蛋白质印迹法研究了腺苷受体（A(1)、A(2A)和A(3)）是否激活PKB，并确定了磷脂酰肌醇3激酶（PI - 3K）/PKB在培养的新生大鼠心肌细胞中腺苷诱导的预处理中的作用。腺苷（非选择性激动剂）、CPA（A(1)选择性激动剂）和Cl - IB - MECA（A(3)选择性激动剂）均以时间和浓度依赖性方式增加PKB磷酸化。CPA和Cl - IB - MECA的联合最大反应类似于单独腺苷诱导的PKB磷酸化增加。CGS 21680（A(2A)选择性激动剂）未刺激PKB磷酸化增加。腺苷、CPA和Cl - IB - MECA介导的PKB磷酸化被百日咳毒素（PTX阻断G(i)/G(o)蛋白）、染料木黄酮（酪氨酸激酶抑制剂）、PP2（Src酪氨酸激酶抑制剂）以及表皮生长因子（EGF）受体酪氨酸激酶抑制剂AG 1478抑制。PI - 3K抑制剂渥曼青霉素和LY 294002阻断A(1)和A(3)受体介导的PKB磷酸化。通过监测心肌细胞暴露于4小时缺氧（0.5% O(2)）随后18小时复氧（HX4/R）诱导的半胱天冬酶3活性和乳酸脱氢酶（LDH）释放，评估了PI - 3K/PKB在腺苷诱导的预处理中的作用。渥曼青霉素预处理对腺苷诱导的预处理减少HX4/R后LDH释放或半胱天冬酶3激活的能力没有显著影响。总之，我们首次表明腺苷A(1)和A(3)受体通过G(i)/G(o)蛋白和酪氨酸激酶依赖性途径触发大鼠心肌细胞中PKB磷酸化增加。然而，PI - 3K/PKB途径似乎不参与腺苷通过预处理诱导的心脏保护作用。
