CHARACTERIZATION OF [H-3] RX821002 BINDING TO ALPHA-2-ADRENERGIC RECEPTOR SUBTYPES

Alpha-2 adrenergic receptors have been divided into four pharmacologic al subtypes based on their differences in affinity for several drugs. Previous studies showed that [H-3]RX821002 has a high affinity for the alpha-2A subtype. The current study characterized the binding propert ies of [H-3]RX821002 [2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline] to the alpha-2A receptor in CHO-C10 cells, alpha-2B in neonatal rat l ung, alpha-2C in OK cells and alpha-2D in bovine pineal gland. Membran e binding studies of [H-3]RX821002 were done in 25 mM glycylglycine bu ffer at room temperature. The nonspecific binding rates at the K-D con centration were 4.9%, 20%, 14% and 8.3% of the total for CHO-C10, neon atal rat lung, OK cells and bovine pineal, respectively, which were de termined by adding 100 mu M norepinephrine. Saturation curves indicate that [H-3]RX821002 has a high affinity for all alpha-2 adrenergic sub types. The K-D values were 0.29, 1.05, 0.37 and 0.19 nM for CHO-C10, n eonatal rat lung, OK cells and bovine pineal, respectively. [H-3]Rauwo lscine has affinities of 0.34, 0.55 and 0.24 nM for the alpha-2A, -2B and -2C subtypes. By contrast, [H-3]rauwolscine has a much lower affin ity for alpha-2D subtype with a K-D value of 5.2 nM. The binding site density for [H-3]RX821002 was significantly lower in the neonatal rat lung compared with [H-3]rauwolscine. The correlation coefficients of p K(i) values of adrenergic compounds against [H-3]RX821002 versus [H-3] rauwolscine were close to unity for each tissue. These data clearly sh ow that the two ligands label the same alpha-2 adrenergic receptor pop ulation. [H-3]RX821002 appears to be relatively nonselective and thus an appropriate radioligand for the study of all alpha-2 adrenergic rec eptor subtypes.