Characterization of two affinity states of adenosine A2a receptors with a new radioligand, 2-[2-(4-amino-3-[125I]iodophenyl)ethylamino]adenosine.

Adenosine analogs substituted in the 2-position with arylamino groups have been found to have high affinity and selectivity for A2a adenosine receptors. Two such compounds, 2-[2-(4-aminophenyl)ethylamino]adenosine and 2-[2-4-amino-3-iodophenyl)ethylamino]adenosine (I-APE), were synthesized and found to be potent coronary vasodilators (ED50 < 3 nm). These compounds bind weakly to A1 adenosine receptors of rat cortex (Ki > 150 nM). 125I-APE was synthesized and the new radioligand was found to bind to two affinity states of rat striatal A2a adenosine receptors (Kd = 1.3 +/- 0.1 nM and 19 +/- 4.5 nM). The high affinity site represents a previously unrecognized small (15-20%) fraction of A2a adenosine receptors coupled to G proteins. Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) reduces specific binding of 125I-APE half-maximally at a concentration of 45 +/- 2 nM. [3H]CGS21680 also binds to two affinity states of A2a receptors on striatal membranes (Kd = 3.9 +/- 0.9 and 51 +/- 5.5 nM), although in previous studies single Kd values ranging from 5 to 15 nM have been reported. This high affinity site is substantiated by the finding that the IC50 of CGS21680 in competition with 125I-APE binding to striatal membranes is shifted leftward in membranes diluted for 4 min before filtration, to selectively dissociate radioligand from low affinity receptors. Assuming that agonist radioligands bind to both coupled and uncoupled forms of striatal A2a adenosine receptors, we could simulate with the computer the finding that the decrease in specific binding induced by GTP gamma S (100 microM) is variable and depends on radioligand concentration, ranging from 20 to 90%. Unlike 125I-APE, [3H]CGS21680 is charged at physiological pH, and treatment of membranes with the pore-forming antibiotic alamethicin uncovers cryptic [3H]CGS21680 but not 125I-APE binding sites. We conclude that the GTP gamma S-sensitive high affinity form of the A2a adenosine receptor can be preferentially labeled by 125I-APE, due to both its high specific activity and its physicochemical properties. Possible functional manifestations of poor coupling of A2a adenosine receptors to G proteins are discussed.