Abstract

The allosteric enhancer PD 81,723, a 2-amino-3-benzoylthiophene derivative, has been shown to potentiate agonist binding to A₁ adenosine receptors (A₁AdoRs) and to enhance the functional effects of adenosine and adenosine analogs. The objective of this study was to determine whether the apparent agonist-independent effect of PD 81,723 observed in CHO cells stably expressing the recombinant human A₁AdoR was due to the potentiation of the action of endogenous adenosine, to the presence of constitutive receptor activity and/or to the binding of PD 81,723 to the agonist binding site of the A₁AdoR. The allosteric enhancer PD 81,723, the A₁AdoR agonist (R)-N⁶-(2-phenylisopropyl)adenosine and adenosine all significantly inhibited forskolin-stimulated cAMP accumulation in intact cells and increased [³⁵S]-5′-(γ-thio)triphosphate binding to cell membranes. The effects of adenosine on cAMP formation and [³⁵S]-5′-(γ-thio)triphosphate binding were attenuated by adenosine deaminase, but the effects of PD 81,723 were not. In the presence of ADA, the A₁AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine increased forskolin-stimulated cAMP accumulation in cells expressing the recombinant human A₁AdoR but not in nontransfected CHO cells. In binding experiments, the agonist (R)-N⁶-(2-phenylisopropyl)adenosine, but not PD 81,723, significantly displaced the specific binding of the A₁AdoR agonist [³H]-N⁶-cyclohexyladenosine and the antagonist [³H]-8-cyclopentyl-1,3-dipropylxanthine. The results of this study demonstrate that in CHO cells stably expressing the recombinant human A₁AdoR, the agonist-independent effect of PD 81,723 is not due to potentiation of the action of endogenous adenosine or mediated by the binding of the allosteric enhancer to the agonist binding site of the recombinant human A₁AdoR. It is possible that these effects are due to potentiation of constitutive receptor activity by PD 81,723.