INVERSE AGONISTS AND NEUTRAL ANTAGONISTS OF RECOMBINANT HUMAN A(1) ADENOSINE RECEPTORS STABLY EXPRESSED IN CHINESE-HAMSTER OVARY CELLS

Receptor antagonists can be classified as neutral antagonists or antag onists with inverse agonist activity based on their effectiveness to r educe the spontaneous agonist-independent activity of receptors. The g oals of this study were to (1) demonstrate that A(1)-adenosine recepto rs (A(1)AdoRs) expressed at high density (4000-8000 fmol/mg of protein ) in Chinese hamster ovary (CHO) cells cause constitutive activation o f inhibitory G proteins and inhibition of adenylyl cyclase activity an d (2) identify both neutral A(1)AdoR antagonists and antagonists with inverse agonist activity. The activity of A(1)AdoR agonists and antago nists was determined by assays of both specific binding of [S-35]guano sine-5'-O-(3-thio)triphosphate ([S-35]GTP gamma S) to membranes and cA MP content of intact cells in the presence of adenosine deaminase (2-5 units/ml). The A(1)AdoR agonist N-6-cyclopentyladenosine (CPA) signif icantly increased binding of [S-35]GTP gamma S by 241 +/- 7% compared with control. The A(1)AdoR antagonists N-0861, N-0840, and WRC-0342 di d not alter binding of [S-35]GTP gamma S, whereas the antagonists 8-cy clopentyl-1,3-dipropylxanthine (CPX), CGS-15943, xanthine amine congen er, and WRC-0571 significantly reduced binding of [S-35]GTP gamma S by 28-53% from control, respectively. The effects of both the agonist N- 6-cyclopentyladenosine (CPA) and the antagonist CPX to alter binding o f [S-35]GTP gamma S were attenuated by 1 mu M N-0861. CPA reduced cAMP content of forskolin-stimulated CHO:A(1)AdoR cells, and N-0861 and WR C-0342 did not alter cAMP content, but the antagonists CPX and WRC-057 1 increased the cAMP content of CHO:A(1)AdoR cells. The effects of bot h CPX and WRC-0571 to increase cAMP content of forskolin-stimulated CH O:A(1)AdoR cells were attenuated by either N-0861 or WRC-0342, The res ults indicate that both N-0861 and WRC-0342 are neutral antagonists, w hereas both CPX and WRC-0571 are antagonists with inverse agonist acti vity.