Studies on the adenosine A 2 receptor mediating pulmonary surfactant phosphatidylcholine secretion in primary cultures of type II pneumocytes isolated from adult rats

Abstract

MRS 1754 [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide] is a selective antagonist ligand of A2B adenosine receptors. This is the least well-defined adenosine receptor subtype, and A2B antagonists have potential as antiasthmatic drugs. For use as a radioligand, MRS 1754, a p-cyanoanilide xanthine derivative, was tritiated on the propyl groups in a two-step reaction using a p-carboxamido precursor, which was dehydrated to the cyano species using trifluoroacetic anhydride. [3H]MRS 1754 (150 Ci/mmol) bound to recombinant human A2B adenosine receptors in membranes of stably transfected HEK-293 cells. Specific binding was saturable, competitive, and followed a one-site model, with a KD value of 1.13 ± 0.12 nM and a Bmax value of 10.9 ± 0.6 pmol/mg protein. Specific binding utilizing 0.7 nM [3H]MRS 1754 was > 70% of total binding. The affinity calculated from association and dissociation binding constants was 1.22 nM (N = 4). Binding to membranes expressing rat and human A1 and A3 adenosine receptors was not significant, and binding in membranes of HEK-293 cells expressing human A2A receptors was of low affinity (KD > 50 nM). The effects of cations and chelators were explored. Specific binding was constant over a pH range of 4.5 to 6.5, with reduced binding at higher pH. The pharmacological profile in competition experiments with [3H]MRS 1754 was consistent with the structure–activity relationship for agonists and antagonists at A2B receptors. The Ki values of XAC (xanthine amine congener) and CPX (8-cyclopentyl-1,3-dipropylxanthine) were 16 and 55 nM, respectively. NECA (5′-N-ethylcarboxamidoadenosine) competed for [3H]MRS 1754 binding with a Ki of 570 nM, similar to its potency in functional assays. Thus, [3H]MRS 1754 is suitable as a selective, high-affinity radioligand for A2B receptors.