[H-3]MRE 3008F20: A novel antagonist radioligand for the pharmacological and biochemical characterization of human A(3) adenosine receptors

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, t he pyrazolo triazolo pyrimidine derivative [H-3]5N-(4-methoxyphenylcarbamoy l)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([H-3]MRE 3008F20). [H-3]MRE 3008F20 bound specifically to the human adeno sine A(3) receptor expressed in CHO cells (hA(3) CHO), and saturation analy sis revealed a single high affinity binding site, K-D = 0.80 +/- 0.06 nM, w ith a B-max = 300 +/- 33 fmol/mg protein. This new ligand displayed high se lectivity (1294-, 165-, and 2471-fold) in binding assay to human A(3) versu s A(1), A(2A), and A(2B) receptors, respectively, and binds to the rat A(3) receptors with a K-i > 10 mu M. The pharmacological profile of [H-3]MRE 30 08F20 binding to hA(3)CHO cells was evaluated using known adenosine recepto r agonists and antagonists with a rank order of potency consistent with tha t typically found for interactions with the A(3) adenosine receptors. In th e adenylyl cyclase assay the same compounds exhibited a rank order of poten cy identical with that observed in binding experiments. Thermodynamic data indicated that [H-3]MRE 3008F20 binding to hA(3)CHO is entropy- and enthalp y-driven in agreement with the typical behavior of other adenosine antagoni sts to A(1) and A(2A) receptors. These results show that [H-3]MRE 3008F20 i s the first antagonist radioligand with high affinity and selectivity for t he human A(3) adenosine receptor and may be used to investigate the physiop athological role of A(3) adenosine receptors.