This study demonstrates quantification of A(2A) adenosine receptors (A(2A)AdoRs) in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells. Radioligand binding assays were performed using the new selective A(2A)AdoR antagonist radioligand [H-3]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-epsilon]-1,2,4-triazolo[1,5 c)pyrimidine ([H-3]SCH58261). Binding of the radioligand to membranes was rapid, reversible, and saturable. The densities of A(2A)AdoRs in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells were 900+/-61, 892+/-35, and 959+/-76 fmol/mg protein, respectively. Equilibrium dissociation constants (K-d values) calculated from results of saturation binding assays were 2.19, 1.20, and 0.81 nmol/L, and K-d values calculated from results of association and dissociation assays were 2.42, 1.01, and 0.40 nmol/L for [H-3]SCH58261 binding to membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells, respectively. The specific binding of [H-3]SCH58261 as a percentage of total binding at a radioligand concentration equal to the K-d value was 65% to 90% in the three membrane preparations. The order of ligand potencies determined by assay of competition binding to sites in porcine coronary membranes using [H-3]SCH58261, unlabeled antagonists (SCH58261, 8-(3-chlorostyryl)caffeine [CSC], and xanthine amine congener [XAC]), and unlabeled agonists ([H-3] 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine [CGS 21680], 2-hexynyl-5'-N-ethylcarboxamidoadenosine [HE-NECA], [H-3]5'-N-ethylcarboxamidoadenosine [NECA], and R(-)N-6-(2-phenylisopropyl)adenosine [R-PIA]) was SCH58261>HE-NECA=CSC =CGS 21680=XAC>NECA=R-PIA. The Hill coefficients of displacement by A(2A)AdoR ligands of [H-3]SCH58261 binding were not significantly different from unity, indicating that [H-3]SCH58261 bound to a group of homogeneous noninteracting sites in all membrane preparations. The order of ligand potencies to compete for [H-3]SCH58261 binding sites in porcine striatal and PC12 cell membranes was, in part different from that for porcine coronary arterial membranes. The different rank orders of potencies for agonists and antagonists at A(2A) receptors of porcine coronary arteries, striatum, and PC12 cells and significant differences in absolute values of potency of ligands in the three preparations may indicate the existence of different subtypes of A(2A)AdoRs. The antagonist radioligand [H-3]SCH58261 should be of value for pharmacological characterization of A(2A) adenosine receptors in other preparations.
