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

Luthin,Olsson,Thompson,Sawmiller,Linden

, p. 307 - 313 (2007/10/03)

Adenosine analogs substituted in the 2-position with arylamino groups have been found to have high affinity and selectivity for A(2a) 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 1 adenosine receptors of rat codex (K(i) > 150 nM). 125I- APE was synthesized and the new radioligand was found to bind to two affinity states of rat striatal A(2a) adenosine receptors (K(d) = 1.3 ± 0.1 nM and 19 ± 4.5 nM). The high affinity site represents a previously unrecognized small (15-20%) fraction of A(2a) adenosine receptors coupled to G proteins. Guanosine 5'-O-(3-thio)triphosphate (GTPγS) reduces specific binding of 125I-APE half-maximally at a concentration of 45 ± 2 nM. [3H]CGS21680 also binds to 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 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 A(2a) adenosine receptors, we could simulate with the computer the finding that the decrease in specific binding induced by GTPγS (100 μM) 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 GTPγS-sensitive high affinity form of the A(2a) 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 A(2a) adenosine receptors to G proteins are discussed.