Molecular Probes for Adenosine Receptors

• Published in: Japanese Journal of Pharmacology 1990, Vol.52 (suppl.2), p.8-8
• Main Authors: Kenneth A. Jacobson, Daniel L. Boring, Xiao-duo Ji, William Barrington, Vickram Ramkumar, Gary L. Stiles
• Format: Article
• Language: Japanese
• ISSN: 0021-5198

We have developed novel agonist and antagonist ligands as probes for characterizing adenosine receptors by chemical and physical methods. XAC (xanthine amine congener, antagonist), ADAC (adenosine amine congener, A_1 -agonist), and APEC (A_2 -agonist) were designed as functionalized congeners, i.e. they contain a chemical functional group that can be coupled covalently to other molecules resulting in biologically active conjugates. The utility of functionalized congeners is two-fold, both as high affinity radioligands and pharmacological standards and as synthetic intermediates. Conjugates have been prepared that contain prosthetic groups for the incorporation of radioisotopes, or fluorescent groups for spectrophotometric detection. Conjugates may be coupled to an agarose matrix for receptor isolation and to bifunctional cross-linking reagents for irreversible receptor binding. XAC and APEC were condensed to p-aminophenylacetic acid (PAPA) to form PAPAXAC and PAPA-APEC, respectively. These aryl amines served as substrates for iodination with ^^125 I, for the labeling of A_1 and A_2 -adenosine receptors, respectively. The resulting radioligands bound to bovine brain adenosine receptors with a K_d of 0.1 nM (PAPAXAC) and 1.5 nM (PAPA-APEC). The aryl amine was crosslinked to the receptor, either through photoaffinity crosslinking, using a photoactivatable azido (N_3 ) compound containing an active ester group (SANPAH) or by conversion of the amine to an azide. For chemical crosslinking to A_1 receptors, XAC was coupled to m- and p-phenylene diisothiocyanates. The resulting conjugates, m- and p-DITC-XAC, selectively inactivated adenosine receptors during incubations of brain membranes with submicromolar concentrations of the xanthine. When the DITC-XAC isomers were synthesized using [^^3 H]XAC (a simple, single step reaction), the xanthine specifically labeled the receptor protein for detection on SDS gel electrophoresis. We now report highly water soluble (and thus useful for physiological studies) purine isothiocyanate analogs that irreversibly inhibit adenosine receptors. Using selective probes it is now possible to study regulation and signal transduction of both A_1 and A_2 -receptors in same tissue. DDT_1 MF-2 clonal cells from smooth muscle of the Syrian hamster were found to contain A_1 - and A_2 - receptors in densities of 0.8 and 0.2 pmol/mg protein. The receptor proteins were found to be of distinct molecular weights (38 and 42 kDa, respectively).