Germack R, Dickenson J M
Biomedical Research Centre, School of Biomedical and Natural Sciences, Nottingham-Trent-University, UK.
J Mol Cell Cardiol. 2005 Sep;39(3):429-42. doi: 10.1016/j.yjmcc.2005.06.001.
Three subtypes of adenosine receptors (A(1), A(2A) and A(3) ARs) are functionally expressed in cardiomyocytes. Adenosine released during ischemia and ischemia/reperfusion plays a major role in cardioprotection. Phosphatidylinositol 3-kinase (PI-3K)/protein kinase B (PKB) and MEK/ERK1/2 pathways are involved in cell survival. Since the role of these pathways in AR-mediated preconditioning is poorly understood, we have investigated whether PI-3K/PKB and/or MEK1/ERK1/2 pathways are involved in AR-induced cardioprotection in neonatal rat cardiomyocytes. Cells were pre-treated (15 min) with adenosine (non-selective), CPA (A(1)), CGS 21680 (A(2A)) or Cl-IB-MECA (A(3)) before 4 h hypoxia (0.5% O(2)) and 18 h reoxygenation (HX4/R). HX4/R-induced increase in LDH release was significantly reduced by adenosine (70%), CPA (59%) and Cl-IB-MECA (46%). The MEK1 inhibitor PD 98059 suppressed the effects of adenosine, CPA, and Cl-IB-MECA on LDH release, whereas the PI-3K inhibitor wortmannin did not reverse this cardioprotection. Western blotting of phosphorylated ERK1/2 and PKB during HX4/R supported the involvement of ERK1/2 and not PKB in A(1) and A(3) agonist-mediated cardioprotection. In addition, adenosine, CPA and Cl-IB-MECA inhibited HX4/R-induced caspase 3 activity by 75%, 70% and 59%, respectively, and this inhibition was abolished by PD 98059. Interestingly, wortmannin inhibited by 66% the anti-apoptotic response triggered by Cl-IB-MECA but had no effect on adenosine or CPA-induced inhibition of caspase 3. CGS 21680 did not modify cell survival or caspase 3 activity. In conclusion, these data show that the preconditioning effect of adenosine requires A(1) and A(3) but not A(2A) ARs and involves an anti-apoptotic effect via MEK1/ERK1/2 pathway in neonatal rat cardiomyocytes. In addition, A(3)AR-induced preconditioning also involves a PI-3K dependent pathway.
三种亚型的腺苷受体（A(1)、A(2A) 和 A(3) 腺苷受体）在心肌细胞中功能性表达。缺血及缺血/再灌注过程中释放的腺苷在心脏保护中起主要作用。磷脂酰肌醇 3-激酶（PI-3K）/蛋白激酶 B（PKB）和 MEK/ERK1/2 信号通路参与细胞存活。由于这些信号通路在腺苷受体介导的预处理中的作用尚不清楚，我们研究了 PI-3K/PKB 和/或 MEK1/ERK1/2 信号通路是否参与新生大鼠心肌细胞中腺苷受体诱导的心脏保护作用。在 4 小时缺氧（0.5% O₂）和 18 小时复氧（HX4/R）之前，细胞先用腺苷（非选择性）、CPA（A(1)）、CGS 21680（A(2A)）或 Cl-IB-MECA（A(3)）预处理（15 分钟）。腺苷（70%）、CPA（59%）和 Cl-IB-MECA（46%）显著降低了 HX4/R 诱导的乳酸脱氢酶（LDH）释放增加。MEK1 抑制剂 PD 98059 抑制了腺苷、CPA 和 Cl-IB-MECA 对 LDH 释放的作用，而 PI-3K 抑制剂渥曼青霉素并未逆转这种心脏保护作用。HX4/R 期间磷酸化 ERK1/2 和 PKB 的蛋白质印迹分析支持 ERK1/2 而非 PKB 参与 A(1) 和 A(3) 激动剂介导的心脏保护作用。此外，腺苷、CPA 和 Cl-IB-MECA 分别将 HX4/R 诱导的半胱天冬酶 3 活性抑制了 75%、70% 和 59%，而这种抑制作用被 PD 98059 消除。有趣的是，渥曼青霉素抑制了 Cl-IB-MECA 触发的 66% 的抗凋亡反应，但对腺苷或 CPA 诱导的半胱天冬酶 3 抑制作用没有影响。CGS 21680 未改变细胞存活或半胱天冬酶 3 活性。总之，这些数据表明，腺苷的预处理作用需要 A(1) 和 A(3) 腺苷受体而非 A(2A) 腺苷受体，并且在新生大鼠心肌细胞中通过 MEK1/ERK1/2 信号通路涉及抗凋亡作用。此外，A(3) 腺苷受体诱导的预处理还涉及一条 PI-3K 依赖性信号通路。
