Buffers differentially alter the binding of [3H]rauwolscine and [3H]RX821002 to the alpha-2 adrenergic receptor subtypes.
3H-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 binding of antagonists to all four subtypes of the alpha-2 adrenergic receptor. Our approach was to examine the effects of glycylglycine, Tris, sodium phosphate (NaPO4) and potassium phosphate buffers on the binding of [3H]rauwolscine, [3H]RX821002, prazosin and oxymetazoline. We found that the affinities for the different subtypes varied with the buffer and the ligands used. Although the Bmax values varied somewhat with the buffers, they were similar for both radioligands for a specific subtype. The highest affinities and Bmax values for both radioligands were generally obtained with NaPO4 buffer. The affinities of antagonists in Tris buffer were always significantly lower than in either NaPO4 or glycylglycine buffer, and the affinities decreased as the concentration of Tris increased. In contrast, the affinity of norepinephrine for the alpha-2B subtype was higher in Tris than in NaPO4 buffer. The buffer effects did not appear to be dependent on the cell membrane composition. There appeared to be some species differences in the effects of buffers on the alpha-2C subtype. These results indicate that buffers affect the binding of antagonists to alpha-2 adrenergic receptors, that not all subtypes are altered in the same manner and that buffers alter the binding of different antagonists differently. It is generally recommended that NaPO4 buffer be used, and that Tris be avoided, when measuring the binding of antagonists to the alpha-2 adrenergic receptor.