A2B receptors mediate the induction of early genes and inhibition of arterial smooth muscle cell proliferation via Epac

Aims Extracellular adenosine and adenine nucleotides play important roles in the regulation of the blood vessel tonus and platelet aggregation. Less is known about the effects of these extracellular signalling molecules on gene expression in vascular smooth muscle cells involved in long-term vascular effects. In the present study, we therefore searched for adenosine-induced changes in the expression of early genes in cultured human coronary artery smooth muscle cells (HCASMCs). Methods and results Whole-genome DNA array hybridization revealed that adenosine induced a set of early genes including the nuclear receptor subfamily 4, group A, member 1 (NR4A1/Nur77/TR3). The pattern of the effects of adenosine on gene expression resembles the change in expression induced by the direct activator of adenylate cyclase forskolin. Real-time reverse-transcriptase PCR confirmed that adenosine and its analogue N-ethyl-carboxamidoadenosine elicited a strong induction of NR4A1. These effects were markedly attenuated by A(2B) receptor antagonists including 8-[4-(4-benzylpiperazide-1-sulfonyl)phenyl]-1-propylxanthine (PSB-601) and were mimicked by a cyclic AMP (cAMP) analogue [8-(4-chlorophenylthio)-2'-O-methyl-cAMP, 8CPT] acting on the exchange protein activated by cAMP (Epac). Long-term experiments over 5 days showed that 2-chloroadenosine decreased cell proliferation in the presence of platelet-derived growth factor. This effect of 2-chloroadenosine was also attenuated by PSB-601 and mimicked by 8CPT. Treatment with small interfering RNA directed against NR4A1 attenuated the inhibitory effect of 8CPT on proliferation. Conclusions In summary, our results demonstrate the operation of adenosine A(2B) receptors mediating an early induction of NR4A1 and a decrease in cell proliferation via the cAMP/Epac pathway in HCASMCs.