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

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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 microM 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 microM 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.

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