CHARACTERIZATION OF 2 AFFINITY STATES OF ADENOSINE-A(2A) RECEPTORS WITH A NEW RADIOLIGAND, -(4-AMINO-3-[I-125]IODOPHENYL)ETHYLAMINO]ADENOSINE

Adenosine analogs substituted in the 2-position with arylamino groups have been found to have high affinity and selectivity for A(2a) adenos ine receptors. Two such compounds, 2-[2-(4-aminophenyl)ethylamino]aden osine and 2-[2-(4-amino-3-iodophenyl)ethylamino]adenosine (I-APE), wer e synthesized and found to be potent coronary vasodilators (ED(50) < 3 nM). These compounds bind weakly to Al adenosine receptors of rat cor tex (K-i > 150 nM). I-125-APE,as synthesized and the new radioligand w as found to bind to two affinity states of rat striatal A(2a) adenosin e receptors (K-d = 1.3 +/- 0.1 nM and 19 a 4.5 nM), The high affinity site represents a previously unrecognized small (15-20%) fraction of A pa adenosine receptors coupled to G proteins. Guanosine 5'-O-(3-thio)t riphosphate (GTP gamma S) reduces specific binding of I-125-APE half-m aximally at a concentration of 45 +/- 2 nM, [H-3]CGS21680 also binds t o two affinity states of A(2a) receptors on striatal membranes (K-d = 3.9 +/- 0.9 and 51 +/- 5.5 nM), although in previous studies single K- d values ranging from 5 to 15 nM have been reported. This high affinit y site is substantiated by the finding that the IC50 of CGS21680 in co mpetition with I-125-APE binding to striatal membranes is shifted left ward in membranes diluted for 4 min before filtration, to selectively dissociate radioligand from low affinity receptors. Assuming that agon ist radioligands bind to both coupled and uncoupled forms of striatal A(2a) adenosine receptors, we could simulate with the computer the fin ding that the decrease in specific binding induced by GTP gamma S (100 mu M) is variable and depends on radioligand concentration, ranging f rom 20 to 90%. Unlike I-125-APE, [H-3]CGS21680 is charged at physiolog ical pH, and treatment of membranes with the pore-forming antibiotic a lamethicin uncovers cryptic [H-3]CGS21680 but not I-125-APE binding si tes. We conclude that the GTP gamma S-sensitive high affinity form of the A(2a) adenosine receptor can be preferentially labeled by I-125-AP E, due to both its high specific activity and its physicochemical prop erties. Possible functional manifestations of poor coupling of A(2a) a denosine receptors to G proteins are discussed.