Agonist and antagonist affinities for inhibitory adenosine receptors are reciprocally affected by 5'-guanylylimidodiphosphate or N-ethylmaleimide.

Adenosine analogs such as P-(L-phenylisopropyl) adenosine and 5'-N-ethylcarboxamide adenosine inhibit the basal adenylate cyclase activity in membrane preparations of rat hippocampus. In the same preparations three affinity states of the adenosine receptor for agonist ligands can be detected. In the absence of added guanine nucleotide the agonist [3HJW-cyclohexyladenosine binds to a single high affinity state of the inhibitory receptor (Ri receptor) with a KO of 1.8 n~ and a B,, of approximately 500 fmol/mg of protein. The KO increases to 15 n~ and the B,, decreases to 300 fmol/mg in the presence of 5'-guanylylimidodiphosphate (Gpp(NH)p). A third affinity state can be indirectly demonstrated in N-ethylmaleimide (NEM)-pretreated membranes by measuring the ability of N6-cyclohexyladenosine to inhibit the binding of the antagonist radioligand [3H]1,3-diethyl-8-phenylxanthine. N6Cyclohexyladenosine inhibits [3H]diethylphenylxanthine binding in NEM-pretreated membranes with a Ki of 430 n ~ . Thus w-cyclohexyladenosine binds to three distinct states of the Ri receptor (KO or Ki of 1.8,15, and 430 IIM). The B,, for the antagonist [3H]diethylphenylxanthine is increased by Gpp(NH)p or NEM pretreatment while the KO (60 m) is unaffected. However, the Ki of diethylphenylxanthine for inhibiting the binding of [3H] p-cyclohexyladenosine both in the absence and presence of Gpp(NH)p is 300 nM. These results suggest that the filtration binding assay used measures the binding of [3H]diethylphenylxanthine to a high antagonist affinity state while binding to a low antagonist affinity state(s) is undetected. The increase in the B,, of ['HI diethylphenylxanthine in the presence of Gpp(NH)p or NEM pretreatment occurs because these treatments shift the state of the receptor from a low antagonist affinity state(s) that is not detected with the filtration binding assay to a high antagonist affinity state that is detected. As Gpp(NH)p and NEM pretreatment decrease the affinity of the receptor for the agonist N6cyclohexyladenosine it appears that the states of the receptor with affinity constants of 1.8, 15, and 430 n~ for h@-cyclohexyladenosine correspond to those with affinity constants for the antagonist diethylphenylxanthine of 300, 300, and 60 IIM, respectively. Thus the receptor exists in high agonist/low antagonist and high antagonist/low agonist affinity states.