[3H]MRE 3008F20: A Novel Antagonist Radioligand for the Pharmacological and Biochemical Characterization of Human A3 Adenosine Receptors
- Katia Varani1,
- Stefania Merighi1,
- Stefania Gessi1,
- Karl-Norbert Klotz2,
- Edward Leung3,
- Pier Giovanni Baraldi4,
- Barbara Cacciari4,
- Romeo Romagnoli4,
- Giampiero Spalluto5 and
- Pier Andrea Borea1
- 1Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, Italy (K.V., S.M., S.G., P.A.B.); 2Institut für Pharmakologie und Toxikologie, Universität Würzburg, Germany (K.-N.K.); 3Medco Research, Research Triangle Park, North Carolina (E.L.); 4Department of Pharmaceutical Sciences, University of Ferrara, Italy (P.G.B., B.C., R.R.); and 5Department of Pharmaceutical Sciences, University of Trieste, Italy (G.S.)
Abstract
The lack of a radiolabeled selective A3 adenosine receptor antagonist is a major drawback for an adequate characterization of this receptor subtype. This paper describes the pharmacological and biochemical characterization of the tritiated form of a new potent A3 adenosine receptor antagonist, the pyrazolo triazolo pyrimidine derivative [3H]5N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e] -1,2,4- triazolo[1,5-c]pyrimidine ([3H]MRE 3008F20). [3H]MRE 3008F20 bound specifically to the human adenosine A3 receptor expressed in CHO cells (hA3CHO), and saturation analysis revealed a single high affinity binding site, KD = 0.80 ± 0.06 nM, with a Bmax = 300 ± 33 fmol/mg protein. This new ligand displayed high selectivity (1294-, 165-, and 2471-fold) in binding assay to human A3 versus A1, A2A, and A2B receptors, respectively, and binds to the rat A3 receptors with a Ki > 10 μM. The pharmacological profile of [3H]MRE 3008F20 binding to hA3CHO 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 A3 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 [3H]MRE 3008F20 binding to hA3CHO is entropy- and enthalpy-driven in agreement with the typical behavior of other adenosine antagonists to A1 and A2A receptors. These results show that [3H]MRE 3008F20 is the first antagonist radioligand with high affinity and selectivity for the human A3 adenosine receptor and may be used to investigate the physiopathological role of A3 adenosine receptors.
Footnotes
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Send reprint requests to: Prof. Dr. Pier Andrea Borea, Faculty of Medicine, University of Ferrara, Department of Clinical and Experimental Medicine, Pharmacology Unit, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy. E-mail: bpa{at}dns.unife.it
- 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-cyclopentylxanthine
- 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(−)-N6-(2-phenylisopropyl)adenosine
- S-PIA
- S(−)-N6-(2-phenylisopropyl)adenosine
- CGS 21680
- 2-[p-(2-carboxyethyl)phenetylamino]-5′-N-ethylcarboxamidoadenosine
- IB-MECA
- N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide
- CHO
- Chinese hamster ovary
- CGS 15943
- 5-amino-9-chloro-2-(furyl)-1,2,4-triazolo[1,5-c]quinazoline
- XAC
- 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine
- MPC-NECA
- N6-(4-methoxyphenylcarbamoyl)- adenosine-5′-N-ethyluronamide
- MPC-MECA
- N6-(4-methoxyphenylcarbamoyl)adenosine-5′-N-methyluronamide
- MRE 3010F20
- 5-N-(3-chlorophenylcarbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine
- hA3CHO
- human adenosine A3 receptor expressed in CHO cells
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- Received October 1, 1999.
- Accepted January 10, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
