Pharmacology of a unique adenosine binding site in rat brain using a selective ligand

1. In order to further characterize the adenosine binding sites that we previously purified and termed P3 purinergic receptor-like protein (P3LP), a reliable binding assay method was developed using [3H]-5'-N-ethylcarboxamidoadenosine (NECA) as a radioligand and the newly developed high-affinity selective ligand 9-(6,7-dideoxy-beta-D-allo-hept-5-ynofuranosyl) adenine (HAK2701) as a selective displacer. 2. Using this assay method, it was found that rat brain membranes possess high- and low-affinity [3H]-NECA binding sites. The high-affinity binding site showed KD and Bmax values of 19.7+/-2.5 nmol/L and 0.192+/-0.05 pmol/mg protein, respectively, and the KD value for the low-affinity binding site was 4260+/-330 nmol/L. The KD value for the high-affinity site agreed well with that of the [3H]-NECA binding site determined with the partially purified P3LP preparation described previously. 3. The distribution of P3LP in rat tissues was determined using the [3H]-NECA binding method described above. The highest level of P3LP was in the cerebellum followed by the olfactory bulb and the spinal cord. 4. The order of the affinity for various purinergic or related compounds to P3LP in rat brain preparations was also determined by the [3H]-NECA binding method to be HAK2701 > NECA = adenosine 5'-O-(2-thiodiphosphate) > cAMP = beta,gamma-methyleneadenosine 5'-triphosphate > diadenosine tetraphosphate > alpha,beta-methyleneadenosine 5'-triphosphate > 5'-deoxy-5'-methylthioadenosine > N6-cyclopentyladenosine. 5. These studies reveal that the [3H]-NECA binding assay in combination with HAK2701 is successful in the characterization of P3LP, especially the membrane-bound form.