[ 3 H]MRE 3008F20: A Novel Antagonist Radioligand for the Pharmacological and Biochemical Characterization of Human A 3 Adenosine Receptors KATIA VARANI, STEFANIA MERIGHI, STEFANIA GESSI, KARL-NORBERT KLOTZ, EDWARD LEUNG, PIER GIOVANNI BARALDI, BARBARA CACCIARI, ROMEO ROMAGNOLI, GIAMPIERO SPALLUTO, and PIER ANDREA BOREA Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, Italy (K.V., S.M., S.G., P.A.B.); Institut fu ¨r Pharmakologie und Toxikologie, Universita ¨ t Wu ¨ rzburg, Germany (K.-N.K.); Medco Research, Research Triangle Park, North Carolina (E.L.); Department of Pharmaceutical Sciences, University of Ferrara, Italy (P.G.B., B.C., R.R.); and Department of Pharmaceutical Sciences, University of Trieste, Italy (G.S.) Received October 1, 1999; accepted January 10, 2000 This paper is available online at http://www.molpharm.org ABSTRACT The lack of a radiolabeled selective A 3 adenosine receptor antagonist is a major drawback for an adequate characteriza- tion of this receptor subtype. This paper describes the phar- macological and biochemical characterization of the tritiated form of a new potent A 3 adenosine receptor antagonist, the pyrazolo triazolo pyrimidine derivative [ 3 H]5N-(4-methoxyphe- nylcarbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e] -1,2,4- triazolo[1,5-c]pyrimidine ([ 3 H]MRE 3008F20). [ 3 H]MRE 3008F20 bound specifically to the human adenosine A 3 re- ceptor expressed in CHO cells (hA 3 CHO), and saturation analysis revealed a single high affinity binding site, K D = 0.80  0.06 nM, with a B max = 300  33 fmol/mg protein. This new ligand displayed high selectivity (1294-, 165-, and 2471-fold) in binding assay to human A 3 versus A 1 ,A 2A , and A 2B receptors, respectively, and binds to the rat A 3 receptors with a K i  10 M. The pharmacological profile of [ 3 H]MRE 3008F20 binding to hA 3 CHO cells was evaluated using known adenosine receptor agonists and antagonists with a rank order of potency consistent with that typically found for interactions with the A 3 adenosine receptors. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency identical with that observed in binding experiments. Thermodynamic data indicated that [ 3 H]MRE 3008F20 bind- ing to hA 3 CHO is entropy- and enthalpy-driven in agreement with the typical behavior of other adenosine antagonists to A 1 and A 2A receptors. These results show that [ 3 H]MRE 3008F20 is the first antagonist radioligand with high affinity and selectivity for the human A 3 adenosine receptor and may be used to investigate the physiopathological role of A 3 adenosine receptors. Adenosine, an endogenous modulator of a wide range of biological functions, interacts with at least four cell surface receptor subtypes classified as A 1 ,A 2A ,A 2B , and A 3 . These receptor subtypes belong to the superfamily of G protein- coupled receptors and have been cloned in several animal species (Fredholm et al., 1994). Typically, G protein-coupled receptors show sequence homologies ranging from 85% to 95% among different species (Ralevic and Burnstock, 1998). On the contrary, the A 3 subtype exhibits a lower degree of homology that is only 74% between rats and humans or sheep and 85% between sheep and humans (Zhou et al., 1992; Linden, 1994). Moreover no considerable changes in binding affinity of several agonists and antagonists have been found between rat and human A 1 and A 2A receptors (Dionisotti et al., 1997), whereas the rat A 3 receptor differs from the hu- man in the antagonist binding (Salvatore et al., 1993). Fur- thermore, A 3 adenosine receptors have large interspecies differences in peripheral distribution. The rat transcript has been detected in testis, lung, kidneys, heart, and brain (Hill et al., 1997; Jacobson et al., 1998b). The human A 3 receptor transcript is widespread, with the most abundant expression being found in the lung and liver. This suggests that numer- ABBREVIATIONS: AB-MECA, 4-aminobenzyl-5'-N-methylcarboxamidoadenosine; MRE 3008F20, 5-N-(4-methoxyphenylcarbamoyl)amino-8- propyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine; NECA, 5'-N-ethylcarboxamidoadenosine; DPCPX, 1,3-dipropyl-8-cyclopentyl- xanthine; SCH 58261, 5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine; R-PIA, R(-)-N 6 -(2-phenylisopropyl)ad- enosine; S-PIA, S(-)-N 6 -(2-phenylisopropyl)adenosine; CGS 21680, 2-[p-(2-carboxyethyl)phenetylamino]-5'-N-ethylcarboxamidoadenosine; IB- MECA, N 6 -(3-iodobenzyl)adenosine-5'-N-methyluronamide; CHO, Chinese hamster ovary; CGS 15943, 5-amino-9-chloro-2-(furyl)-1,2,4-tria- zolo[1,5-c]quinazoline; XAC, 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine; MPC-NECA, N 6 -(4-methoxyphenylcarbamoyl)- adenosine-5'-N-ethyluronamide; MPC-MECA, N 6 -(4-methoxyphenylcarbamoyl)adenosine-5'-N-methyluronamide; MRE 3010F20, 5-N-(3-chlorophenylcar- bamoyl)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine; hA 3 CHO, human adenosine A 3 receptor expressed in CHO cells. 0026-895X/00/050968-08$3.00/0 MOLECULAR PHARMACOLOGY Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics MOL 57:968–975, 2000 /13009/817853 968 at ASPET Journals on February 27, 2018 molpharm.aspetjournals.org Downloaded from