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In human coronary smooth muscle cells adenosine A(2B) receptors mediate the inhibition of platelet-derived growth factor (PDGF)-induced proliferation via induction of the transcription factor nuclear receptor subfamily 4, group A, member 1 (NR4A1). In the absence of PDGF, adenosine analogues increased proliferation. In the present study we characterised the adenosine receptor mediating the increase in proliferation of these cells and identified involved transcription factors. Cultured human coronary smooth muscle cells were treated with selective A(3) receptor ligands. Effects on proliferation were determined by counting cells and measuring changes in impedance. The induction of transcription factors was assessed by qPCR. The A(3) receptor agonist 2-chloro-IB-MECA (2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide) enhanced the number of human coronary smooth muscle cells with a half-maximal concentration of only 1 nM. 2-chloro-IB-MECA also increased the expression of the transcription factors early growth response protein (EGR)2 and EGR3, but not of EGR1, NR4A1, NR4A2 and NR4A3. The responses to 2-chloro-IB-MECA were blocked by two A(3) receptor antagonists, MRS1523 (3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate; 10-300 μM) and VUF 5574 (N-(2-methoxyphenyl)-N'-[2-(3-pyridinyl)-4-quinazolinyl]-urea; 1-100 nM, as well as by the phospholipase C-inhibitor U73343 (0.2 μM). Small interfering RNA directed against EGR2 and EGR3 abolished the increases in proliferation induced by 2-chloro-IB-MECA. In summary, this is the first report demonstrating a coupling of smooth muscle adenosine A(3) receptors to increases in proliferation of human coronary smooth cells by the activation of phospholipase C and an induction of the transcriptions factors EGR2 and EGR3. The results facilitate the understanding of the role of adenosine A(3) receptors in the cardiovascular system., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.