Activation of Gs-coupled receptors enhances the increase in cyclic AMP mediated by adenylate cyclases. As it has been shown that cyclic AMP inhibits the epidermal growth factor-activated mitogen-activated protein kinase (MAPK) signalling pathway, stimulation of Gs-coupled receptors may lead to the inhibition of MAPK activation. To investigate the effect of a Gs-coupled receptor on the MAPK cascade, we cloned the adenosine (Ado) A2a receptor from a guinea-pig leucocyte cDNA library, and established Chinese hamster ovary (CHO) cells stably expressing the receptor (CHOAdoA2R). The [3H]5'-N-ethylcarbamoyladenosine (NECA) binding characteristics (Kd = 91.0 +/- 5.4 nM, Bmax = 707 +/- 11 fmol/mg of protein, n = 3) and NECA-induced cyclic AMP production indicate that the cloned Ado A2a receptor was functionally expressed in the cells. In CHO cells, thrombin induced intracellular Ca2+ increase and MAPK activation through the intrinsic G-coupled receptor. In CHOAdoA2R cells, NECA partially inhibited thrombin-elicited MAPK activation. When combining NECA-treatment with 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) loading, a nearly complete inhibition of the MAPK activation occurred. Forskolin also partially inhibited the MAPK activation and synergized with BAPTA-AM, suggesting that partial inhibition of MAPK activation by NECA results from cyclic AMP production via Ado A2a receptor activation. The same synergism of MAPK inhibition between wortmannin and BAPTA-AM was observed, but not between wortmannin and NECA. These results suggest that cyclic AMP production through Ado A2a receptor inhibits thrombin-elicited MAPK activation by a Ca(2+)-independent/wortmannin-sensitive pathway in CHO cells.