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

https://doi.org/10.1016/j.yjmcc.2004.08.001Get rights and content

Abstract

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 (A1, A2A and A3) 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 (A1 selective agonist) and Cl-IB-MECA (A3 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 (A2A 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 Gi/Go-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 A1 and A3 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% O2) 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 A1 and A3 receptors trigger increases in PKB phosphorylation in rat cardiomyocytes via a Gi/Go-protein and tyrosine kinase-dependent pathway. However, the PI-3K/PKB pathway does not appear to be involved in adenosine-induced cardioprotection by preconditioning.

Introduction

It is well established that the nucleoside adenosine, which is released from myocardial tissue during ischaemia, exerts a protective role against cell injury via the activation of specific cell surface receptors [1]. To date, four adenosine receptors (A1, A2A, A2B and A3), belonging to the G-protein coupled receptor superfamily, have been cloned and pharmacologically characterised [2]. Adenosine A2A and A2B receptors are positively coupled to adenylyl cyclase via Gs-proteins, whereas adenosine A1 and A3 receptors are negatively coupled to adenylyl cyclase via PTX-sensitive Gi/Go-proteins.

The cardioprotective effects of adenosine have been widely explored using in vivo models of reperfusion injury, isolated heart preparations and cardiomyocytes in culture (see [1], [3] for reviews) The involvement of the adenosine A1 receptor in protecting myocardial tissue from ischaemia-induced cell injury is well documented [1], [3]. The adenosine A3 receptor has also been shown to exert cardioprotection against ischaemia-induced injury both in vivo and in isolated cardiomyocytes [4], [5], [6]. Finally, there is also evidence of the involvement of the adenosine A2A receptor in cardioprotection possibly through anti-inflammatory effects rather than a direct effect on cardiomyocytes [7], [8], [9].

Protein kinase B (PKB; also known as Akt) is a downstream target of phosphatidylinositol 3-kinase (PI-3K), which mediates cell survival of many cell types including cardiomyocytes [10], [11], [12], [13]. For example, adenoviral mediated expression of constitutively active PKB protects against ischaemia/reperfusion-induced injury in mouse and rat hearts [14], [15] and hypoxia-induced apoptosis in isolated rat cardiomyocytes [16]. In addition, PKB is activated during simulated ischaemia/reperfusion in isolated rat cardiomyocytes via a PI-3K and Src tyrosine kinase-dependent pathway [17]. PKB promotes cell survival by either activating anti-apoptotic targets or inactivating pro-apoptotic factors [18], [19]. Growth factors such as insulin and insulin-like growth factor-1 prevent apoptotic cell death in isolated heart preparations and in cultured cardiomyocytes via activation of the PI-3K/PKB pathway [20], [21], [22], [23]. Similarly, members of the G-protein coupled receptor family (namely the β2-adrenergic receptor and corticotropin-releasing factor type 2 receptor) have also been shown to trigger cardioprotection via activation of the PI-3K/PKB pathway [24], [25]. Furthermore, previous studies have shown that adenosine A1 and A3 receptors activate PKB in smooth muscle DDT1MF-2 cells and RBL-2H3 mast cells, respectively [26], [27]. More recently, it has been reported that adenosine stimulates PKB phosphorylation in isolated rabbit hearts [28]. Our previous studies have shown that isolated rat newborn cardiomyocytes express functional adenosine A1, A2A and A3 receptors [29]. Therefore, given the prominent roles of adenosine receptors and PKB in cardioprotection, the aim of the present study was to determine whether adenosine A1, A2A and A3 receptors activate PKB signalling in cultured rat neonatal cardiomyocytes.

Section snippets

Materials

Adenosine, bovine serum albumin, N6-cyclopentyladenosine (CPA), Dulbecco’s modified Eagles medium (DMEM), 1,3-dipropylcyclopentylxanthine (DPCPX), epidermal growth factor (EGF), foetal calf serum, forskolin, IGEPAL CA-630 ((octylphenoxy)polyethoxyethanol), leupeptin, PTX were obtained from Sigma Chemical Co. (Poole, Dorset, UK). AG 1478 ([4-(3-chloroanilino)-6,7-dimethoxyquinzoline]), genistein, LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), PP2 (4-amino-5-(4-chlorophenyl)-7-(t

Effect of adenosine receptor agonists on PKB phosphorylation

Since PKB activation requires the phosphorylation of amino acid residues Thr308 and Ser473, the activation of PKB in isolated rat neonatal cardiomyocytes was detected by Western blotting using an anti-phospho-PKB (Ser473) antibody that detects PKB only when phosphorylated (and therefore activated) at Ser473 [30]. As shown in Fig. 1 adenosine (non-selective agonist; 1 μM), N6-cyclopentyladenosine (CPA; adenosine A1 receptor selective agonist; 1 μM) and the selective adenosine A3 receptor agonist

Discussion

As outlined in Section 1, PKB (also known as Akt) is an important mediator of cell survival in many cell types including cardiomyocytes [10], [11], [12], [13]. Likewise, adenosine is well known to mediate cardioprotection through the activation of adenosine receptors [1], [3]. However, at present it is not known whether adenosine receptors are involved the activation of PKB. In this study we therefore determined whether selective adenosine receptor agonists trigger PKB activation in newborn rat

Acknowledgements

This work was funded by the Wellcome Trust (grant reference 058137/z/99/z).

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