RECOMBINANT HUMAN ALPHA(2)-ADRENOCEPTOR SUBTYPES - COMPARISON OF [H-3] RAUWOLSCINE, [H-3] ATIPAMEZOLE AND [H-3] RX821002 AS RADIOLIGANDS

Kinetic, saturation and competition binding assays were employed to optimize and validate radioligand binding methods for characterization of recombinant human alpha(2)-adrenoceptor subtypes and for screening of new subtype-selective ligands. Stable transfected lines of Shionogi 115 mouse mammary tumour cells (S115) and three structurally different antagonist radioligands, [H-3]rauwolscine, [H-3]atipamezole and [H-3]RX821002, were used. Specificity of alpha(2)-adrenergic binding was defined with 100 mu M (-)-adrenaline. Steady-state was leached with all three radioligands within 15-30 min at 25 degrees C, and the binding was rapidly reversible. The receptor affinities (alpha(2)-C10) were highest in glycylglycine, almost equally high in K+-phosphate, and lowest in Tris buffer for all three [H-3]-ligands. This was mainly caused by different association rates. [H-3]RX821002 was bound with high affinity and similar kinetic properties to all three a,-adrenoceptor subtypes in K+-phosphate buffer, and had the highest proportion of specific binding (96-98%). [H-3]RX821002 and K+-phosphate buffer were subsequently used in competition assays. The rank order of affinity of compounds selective for alpha(2)-adrenoceptor subtypes was alpha(2)-C10 > alpha(2)-C4 > alpha(2)-C2 for oxymetazoline, alpha(2)-C4 > alpha(2)-C2 > alpha(2)-C10 for prazosin and alpha(2)-C2 > alpha(2)-C4 > alpha(2)-C10 for chlorpromazine. The drug affinities (K-i values) determined in this system were in close agreement with earlier results with [H-3]rauwolscine in Tris buffer (r = 0.94). Agonist competition for [H-3]RX821002 binding was biphasic in K+-phosphate buffer supplemented with 10 mM MgCl2, indicating functional coupling of receptors to G-proteins. Accordingly high-affinity binding of the agonists (-)-noradrenaline and UK14,304 was eliminated by 10 mu M Gpp(NH)p in the assays. Our results confirm that [H-3]RX821002 is a suitable radioligand for the characterization of all three human (alpha(2)-adrenoceptor subtypes and for the determination of the subtype-selectivity of new alpha(2)-adrenoceptor agonists and antagonists.