Radioligand binding and functional responses of ligands for human recombinant adenosine A3receptors

Summary1 The binding and functional properties of adenosine receptor ligands were compared in Chinese hamster ovary cells transfected with human adenosine A3receptors. Inhibition of [125I]‐aminobenzyl‐5′‐N‐methylcarboamidoadenosine ([125I]‐AB‐MECA) binding by adenosine receptor ligands was examined in membrane preparations. Inhibition of forskolin‐induced cAMP accumulation by agonists was measured using a cAMP enzyme immunoassay.2 The rank order of agonist potency for both assays wasN6‐(3‐iodobenzyl)‐adenosine‐5′‐N‐methyluronamide (IB‐MECA) > 5′‐N‐ethylcarboxamidoadenosine (NECA) > (−)‐N6‐[(R)‐phenylisopropyl] adenosine (R‐PIA) > 4‐aminobenzyl‐5′‐N‐methylcarboxamidoadenosine (AB‐MECA) > N6‐cyclopentyl adenosine (CPA) > adenosine. The radioligand binding rank order of antagonist potency wasN‐[9‐chloro‐2‐(2‐furanyl)[1,2,4]‐triazolo[1,5‐c]quinazolin‐5‐benzeneacetamide (MRS1220) > 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX) > 8‐phenyltheophylline (8‐PT) > 8‐(p‐sulfophenyl)‐theophylline (8‐SPT). MRS1220 competitively inhibited the effect of IB‐MECA on cAMP production, with aKBvalue of 0.35 nm. These data are characteristic of adenosine A3receptors.3 The absence of Mg2+and presence of guanosine 5′‐(γ‐thio)triphosphate (GTPγS) significantly reduced agonist binding inhibition potency, indicating binding to high‐ and low‐affinity states. The IB‐MECA, NECA and R‐PIA IC50values were greater for the cAMP assay than for radioligand binding, suggesting an efficient stimulus–response transduction pathway.