Photoaffinity Labeling with 2‐[2‐(4‐Azido‐3‐[125I]‐Iodophenyl)ethylamino]adenosine and Autoradiography with 2‐[2‐(4‐Amino‐3‐[125I]Iodophenyl)ethylamino]adenosine of A2a Adenosine Receptors in Rat Brain

Abstract: The A2a adenosine receptor agonist 2‐[2‐(4‐amino‐3‐iodophenyl)ethylamino]adenosine is a potent coronary vasodilator. The corresponding radioiodinated ligand, [125I]APE, discriminates between high‐ and low‐affinity conformations of A2a adenosine receptors. In this study, [125I]APE was used for rapid (24‐h) autoradiography in rat brain sections. The pattern of [125I]APE binding is consistent with that expected of an A2a‐selective radioligand. It is highest in striatum, nucleus accumbens, and olfactory tubercle, with little binding to cortex and septal nuclei. Specific [125I]APE binding to these brain regions is abolished by 1 µM 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS‐21680) but is little affected by 100 nM 8‐cyclopentyl‐1,3‐dipropylxanthine. Conversion of [125I]APE to the corresponding arylazide results in [125I]AzPE. The rank‐order potency of compounds to compete for [125I]AzPE binding in the dark is CGS‐21680 > d‐(R)‐N6‐phenylisopropyladenosine > N6‐cyclopentyladenosine, indicating that it also is an A2a‐selective ligand. Specific photoaffinity labeling by [125I]AzPE of a single polypeptide (42 kDa) corresponding to A2a adenosine receptors is reduced 55 ± 4% by 100 µM guanosine 5′‐O‐(3‐thiotriphosphate) and 91 ± 1.3% by 100 nM CGS‐21680. [125I]APE and [125I]AzPE are valuable new tools for characterizing A2a adenosine receptors and their coupling to GTP‐binding proteins by autoradiography and photoaffinity labeling.