title: [(3)H]MRE 3008F20: a novel antagonist radioligand for the pharmacological and biochemical characterization of human A(3) adenosine receptors.
abstract: The lack of a radiolabeled selective A(3) 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 A(3) adenosine receptor antagonist, the pyrazolo triazolo pyrimidine derivative [(3)H]5N-(4-methoxyphenylcarbamoyl)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) receptor 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 microM. 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 binding 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.
authors.affiliation: Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, Italy.
authors.forename: K
authors.initials: K
authors.lastname: Varani
authors.forename: S
authors.initials: S
authors.lastname: Merighi
authors.forename: S
authors.initials: S
authors.lastname: Gessi
authors.forename: K N
authors.initials: KN
authors.lastname: Klotz
authors.forename: E
authors.initials: E
authors.lastname: Leung
authors.forename: P G
authors.initials: PG
authors.lastname: Baraldi
authors.forename: B
authors.initials: B
authors.lastname: Cacciari
authors.forename: R
authors.initials: R
authors.lastname: Romagnoli
authors.forename: G
authors.initials: G
authors.lastname: Spalluto
authors.forename: P A
authors.initials: PA
authors.lastname: Borea
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D010671:Phenylurea Compounds / Q000737:chemistry / Q000494:pharmacology*; D058915:Purinergic P1 Receptor Antagonists; D011869:Radioligand Assay; D051381:Rats; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D014230:Triazoles / Q000737:chemistry / Q000494:pharmacology*; D014316:Tritium
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; C405605:MRE 3008-F20; D010671:Phenylurea Compounds; D058915:Purinergic P1 Receptor Antagonists; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1; D014230:Triazoles; D014316:Tritium; C084956:N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine; D000242:Cyclic AMP; D000241:Adenosine