Abstract

Receptor antagonists can be classified as neutral antagonists or antagonists with inverse agonist activity based on their effectiveness to reduce the spontaneous agonist-independent activity of receptors. The goals of this study were to (1) demonstrate that A₁-adenosine receptors (A₁AdoRs) expressed at high density (4000–8000 fmol/mg of protein) in Chinese hamster ovary (CHO) cells cause constitutive activation of inhibitory G proteins and inhibition of adenylyl cyclase activity and (2) identify both neutral A₁AdoR antagonists and antagonists with inverse agonist activity. The activity of A₁AdoR agonists and antagonists was determined by assays of both specific binding of [³⁵S]guanosine-5′-O-(3-thio)triphosphate ([³⁵S]GTPγS) to membranes and cAMP content of intact cells in the presence of adenosine deaminase (2–5 units/ml). The A₁AdoR agonist N⁶-cyclopentyladenosine (CPA) significantly increased binding of [³⁵S]GTPγS by 241 ± 7% compared with control. The A₁AdoR antagonists N-0861, N-0840, and WRC-0342 did not alter binding of [³⁵S]GTPγS, whereas the antagonists 8-cyclopentyl-1,3-dipropylxanthine (CPX), CGS-15943, xanthine amine congener, and WRC-0571 significantly reduced binding of [³⁵S]GTPγS by 28–53% from control, respectively. The effects of both the agonist N⁶-cyclopentyladenosine (CPA) and the antagonist CPX to alter binding of [³⁵S]GTPγS were attenuated by 1 μm N-0861. CPA reduced cAMP content of forskolin-stimulated CHO:A₁AdoR cells, and N-0861 and WRC-0342 did not alter cAMP content, but the antagonists CPX and WRC-0571 increased the cAMP content of CHO:A₁AdoR cells. The effects of both CPX and WRC-0571 to increase cAMP content of forskolin-stimulated CHO:A₁AdoR cells were attenuated by either N-0861 or WRC-0342. The results indicate that both N-0861 and WRC-0342 are neutral antagonists, whereas both CPX and WRC-0571 are antagonists with inverse agonist activity.