title: Recombinant human alpha 2-adrenoceptor subtypes: comparison of [3H]rauwolscine, [3H]atipamezole and [3H]RX821002 as radioligands.
abstract: 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, [3H]rauwolscine, [3H]atipamezole and [3H]RX821002, were used. Specificity of alpha 2-adrenergic binding was defined with 100 microM (-)-adrenaline. Steady-state was reached 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 [3H]-ligands. This was mainly caused by different association rates. [3H]RX821002 was bound with high affinity and similar kinetic properties to all three alpha 2-adrenoceptor 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 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 (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 microM Gpp(NH)p in the assays. Our results confirm that [3H]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.
authors.affiliation: Department of Pharmacology, University of Turku, Finland.
authors.forename: M
authors.initials: M
authors.lastname: Halme
authors.forename: B
authors.initials: B
authors.lastname: Sjöholm
authors.forename: J M
authors.initials: JM
authors.lastname: Savola
authors.forename: M
authors.initials: M
authors.lastname: Scheinin
mesh_terms: D058647:Adrenergic alpha-2 Receptor Agonists; D058669:Adrenergic alpha-2 Receptor Antagonists; D000818:Animals; D001667:Binding, Competitive; D002021:Buffers; D002460:Cell Line; D004146:Dioxanes / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006801:Humans; D019329:Idazoxan / Q000031:analogs & derivatives; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors; D015016:Yohimbine / Q000494:pharmacology*
chemical_list: D058647:Adrenergic alpha-2 Receptor Agonists; D058669:Adrenergic alpha-2 Receptor Antagonists; D002021:Buffers; D004146:Dioxanes; D007093:Imidazoles; D011994:Recombinant Proteins; C050701:atipamezole; D015016:Yohimbine; C058525:2-methoxyidazoxan; D019329:Idazoxan