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(Circulation Research. 1996;79:1153-1160.)
© 1996 American Heart Association, Inc.

Articles

Binding of the Novel Nonxanthine A_2A Adenosine Receptor Antagonist [³H]SCH58261 to Coronary Artery Membranes

Luiz Belardinelli, John C. Shryock, Jackie Ruble, Angela Monopoli, Silvio Dionisotti, Ennio Ongini, Donn M. Dennis, Stephen P. Baker

the Departments of Medicine (L.B., J.C.S., J.R.), Pharmacology (L.B., D.M.D., S.P.B.), and Anesthesiology (D.M.D.), College of Medicine, University of Florida, Gainesville, and Schering-Plough Research Institute (A.M., S.D., E.O.), San Raffaele Science Park, Milan, Italy.

Correspondence to Luiz Belardinelli, MD, Professor of Medicine, University of Florida, PO Box 100277, Gainesville, FL 32610-0277. E-mail ramsey.med@shands.ufl.edu.

This study demonstrates quantification of A_2A adenosine receptors (A_2AAdoRs) in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells. Radioligand binding assays were performed using the new selective A_2AAdoR antagonist radioligand [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-]-1,2,4-triazolo[1,5-c)pyrimidine ([³H]SCH58261). Binding of the radioligand to membranes was rapid, reversible, and saturable. The densities of A_2AAdoRs 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 [³H]SCH58261 binding to membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells, respectively. The specific binding of [³H]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 [³H]SCH58261, unlabeled antagonists (SCH58261, 8-(3-chlorostyryl)caffeine [CSC], and xanthine amine congener [XAC]), and unlabeled agonists ([³H]2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine [CGS 21680], 2-hexynyl-5'-N-ethylcarboxamidoadenosine [HE-NECA], [³H]5'-N-ethylcarboxamidoadenosine [NECA], and R(-)N⁶-(2-phenylisopropyl)adenosine [R-PIA]) was SCH58261>HE-NECA=CSC=CGS 21680=XAC>NECA=R-PIA. The Hill coefficients of displacement by A_2AAdoR ligands of [³H]SCH58261 binding were not significantly different from unity, indicating that [³H]SCH58261 bound to a group of homogeneous noninteracting sites in all membrane preparations. The order of ligand potencies to compete for [³H]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_2AAdoRs. The antagonist radioligand [³H]SCH58261 should be of value for pharmacological characterization of A_2A adenosine receptors in other preparations.

Key Words: A_2A adenosine receptor • coronary artery • radioligand • A_2A receptor antagonist • SCH58261

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