Recombinant human α2-adrenoceptor subtypes: comparison of [3H]rauwolscine, [3H]atipamezole and [3H]RX821002 as radioligands

AbstractKinetic, saturation and competition binding assays were employed to optimize and validate radioligand binding methods for characterization of recombinant human α2-adrenoceptor subtypes and for screening of new subtype-selective ligands. Stable transfected lines of Shionogi 115 mouse mammary tumour cells (5115) and three structurally different antagonist radioligands, [3H]rauwolscine, [3H]atipamezole and [3H]RX821002, were used. Specificity of α2-adrenergic binding was defined with 100 μM (−)-adrenaline. Steady-state was reached with all three radioligands within 15–30 min at 25°C, and the binding was rapidly reversible. The receptor affinities (α2-C10) were highest in glycylglycine, almost equally high in K+-phosphate, and lowest in Tris buffer for all three [3H]-ligands. This was mainly caused by different association rates. [3H]RX821002 was bound with high affinity and similar kinetic properties to all three αa2adrenoceptor subtypes in K+-phosphate buffer, and had the highest proportion of specific binding (96–98%). [3H]RX821002 and K+-phosphate buffer were subsequently used in competition assays. The rank order of affinity of compounds selective for α2-adrenoceptor subtypes was α2-C1O > α2-C4 > α2-C2 for oxymetazoline, α2-C4 > α2-C2 > α2-C10 for prazosin and α2-C2 > α2-C4 > α2-C10 for chlorpromazine. The drug affinities (Ki values) determined in this system were in close agreement with earlier results with [3H]rauwolscine in Tris buffer (r = 0.94). Agonist competition for [3H]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 μM Gpp(NH)p in the assays. Our results confirm that [3H]RX821002 is a suitable radioligand for the characterization of all three human α2-adrenoceptor subtypes and for the determination of the subtype-selectivity of new α2-adrenoceptor agonists and antagonists