FUNCTIONAL COUPLING OF ADENOSINE A(2A) RECEPTOR TO INHIBITION OF THE MITOGEN-ACTIVATED PROTEIN-KINASE CASCADE IN CHINESE-HAMSTER OVARY CELLS

Activation of G(s)-coupled receptors enhances the increase in cyclic A MP mediated by adenylate cyclases. As it has been shown that cyclic AM P inhibits the epidermal growth factor-activated mitogen-activated pro tein kinase (MAPK) signalling pathway, stimulation of G(s)-coupled rec eptors may lead to the inhibition of MAPK activation. To investigate t he effect of a G(s)-coupled receptor on the MAPK cascade, we cloned th e adenosine (Ado) A(2a) receptor from a guinea-pig leucocyte cDNA libr ary, and established Chinese hamster ovary (CHO) cells stably expressi ng the receptor (CHOAdoA2R). The [H-3]5'-N-ethylcarbamoyladenosine (NE CA) binding characteristics (K-d = 91.0 +/- 5.4 nM, B-max = 707 +/- 11 fmol/mg of protein, n = 3) and NECA-induced cyclic AMP production ind icate that the cloned Ado A(2a) 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 CHOA doA2R cells, NECA partially inhibited thrombin-elicited MAPK activatio n. When combining NECA-treatment with -bis-(o-aminophenoxy)ethane-N,N, N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) loading, a near ly 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 A(2a) receptor activation. The sam e synergism of MAPK inhibition between wortmannin and BAPTA-AM was obs erved, but not between wortmannin and NECA. These results suggest that cyclic AMP production through Ado A(2a) receptor inhibits thrombin-el icited MAPK activation by a Ca2+-independent/wortmannin-sensitive path way in CHO cells.