BUFFERS DIFFERENTIALLY ALTER THE BINDING OF [H-3] RAUWOLSCINE AND [H-3] RX821002 TO THE ALPHA-2-ADRENERGIC RECEPTOR SUBTYPES

H-3-antagonists are known to bind to the alpha-2A adrenergic receptor with higher affinity in glycylglycine buffer than in Tris buffer. The purpose of this study was to examine the effect of buffers on the bind ing of antagonists to all four subtypes of the alpha-2 adrenergic rece ptor. Our approach was to examine the effects of glycylglycine, Tris, sodium phosphate (NaPO4) and potassium phosphate buffers on the bindin g of [H-3]rauwolscine, [H-3]RX821002, prazosin and oxymetazoline. We f ound that the affinities for the different subtypes varied with the bu ffer and the ligands used. Although the B-max values varied somewhat w ith the buffers, they were similar for both radioligands for a specifi c subtype. The highest affinities and B-max values for both radioligan ds were generally obtained with NaPO4 buffer, The affinities of antago nists in Tris buffer were always significantly lower than in either Na PO4 or glycylglycine buffer, and the affinities decreased as the conce ntration of Tris increased. In contrast, the affinity of norepinephrin e for the alpha-2B subtype was higher in Tris than in NaPO4 buffer. Th e buffer effects did not appear to be dependent on the cell membrane c omposition. There appeared to be some species differences in the effec ts of buffers on the alpha-2C subtype. These results indicate that buf fers affect the binding of antagonists to alpha-2 adrenergic receptors , that not all subtypes are altered in the same manner and that buffer s alter the binding of different antagonists differently. It is genera lly recommended that NaPO4 buffer be used, and that Tris be avoided, w hen measuring the binding of antagonists to the alpha-2 adrenergic rec eptor.