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 op timize and validate radioligand binding methods for characterization o f recombinant human alpha(2)-adrenoceptor subtypes and for screening o f 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 de fined with 100 mu M (-)-adrenaline. Steady-state was leached with all three radioligands within 15-30 min at 25 degrees C, and the binding w as rapidly reversible. The receptor affinities (alpha(2)-C10) were hig hest 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 affini ty and similar kinetic properties to all three a,-adrenoceptor subtype s in K+-phosphate buffer, and had the highest proportion of specific b inding (96-98%). [H-3]RX821002 and K+-phosphate buffer were subsequent ly 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 > al pha(2)-C10 for prazosin and alpha(2)-C2 > alpha(2)-C4 > alpha(2)-C10 f or chlorpromazine. The drug affinities (K-i values) determined in this system were in close agreement with earlier results with [H-3]rauwols cine in Tris buffer (r = 0.94). Agonist competition for [H-3]RX821002 binding was biphasic in K+-phosphate buffer supplemented with 10 mM Mg Cl2, indicating functional coupling of receptors to G-proteins. Accord ingly high-affinity binding of the agonists (-)-noradrenaline and UK14 ,304 was eliminated by 10 mu M Gpp(NH)p in the assays. Our results con firm that [H-3]RX821002 is a suitable radioligand for the characteriza tion of all three human (alpha(2)-adrenoceptor subtypes and for the de termination of the subtype-selectivity of new alpha(2)-adrenoceptor ag onists and antagonists.