Characterization of Human Striatal A₂-Adenosine Receptors Using Radioligand Binding and Photoaffinity Labeling

Abstract

The adenosine agonist [³H]CGS21680 (2-[4-[[2-carboxyethyl]phenyl]ethyl-amino]-5′-N-ethylcarboxamidoadenosine) bound to A₂ receptors in human striatal membranes with a K_d of 17.8±1.1 nM and a B_max of 313± 10 fmol/mg protein. The addition of 100 μM GTP diminished both the affinity of agonist radioligand for A₂ adenosine binding sites and the total binding, resulting in K_d and B_max values of 28.6±1.0 nM and 185±22 fmol/mg of protein. Adenosine ligands competed for [³H]CGS21680 with the expected potency order. The adenosine antagonist [³H]XAC (8-[4-[[[[(2-aminoethyl)-amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine), although A₁-selective in the rat, binds to human striatal A₂ receptors with high affinity. 25 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), an A₁-selective antagonist, was added to the incubation medium and effectively eliminated 91% of [³H]XAC (1 nM) binding to human A₁ receptors, yet preserved 90% of binding to A₂ receptors. [³H]XAC exhibited saturable, specific binding (50% of total) to A₂ sites with a K_d of 2.98±0.54 nM and a B_max of 0.71±0.23 pmol/mg protein (25°C, non-specific binding defined with 100 μM NECA). The potency order for antagonists against 1 nM [³H]XAC was CGS15943A > XAC ± PD115,199 > PAPA-XAC > CPX > HTQZ ± XCC ± CP-66,713 > theophylline ± caffeine, indicative of an A₂-type binding site. A_2a-receptors were found to be present in the human cortex, albeit at a much lower density than in the striatum. Photoaffinity labeling using ¹²⁵I-PAPA-APEC revealed a molecular weight of 45K, but proteolytic cleavage was observed, resulting in fragments of MW 43K and 37K. In the absence of proteolytic inhibitors the 37K fragment, which still bound ¹²⁵I-PAPA-APEC, was predominant.