Molecular Pharmacology, Vol 20, 28-34, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Determination of Subtype Selectivity of Alpha-Adrenergic Antagonists

Comparison of Selective and Nonselective Radioligands

¹ Howard Hughes Medical Institute, Departments of Medicine (Division of Cardiology) and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

The alpha₁/alpha₂-adrenergic receptor subtype selectivity of twelve adrenergic antagonists was assessed by two different radioligand binding approaches in rabbit uterine membranes which contain both receptor subtypes. In the first approach, a nonsubtype selective antagonist radioligand, [³H]dihydroergocryptine ([³H]DHE), was used to label all of the alpha-receptors. Selective competing ligands produced complex competition curves which could be analyzed by nonlinear least-squares curve-fitting methods to yield the K_d values of the competitor for the alpha₁- and alpha₂-receptors, respectively. The second approach utilized the radioligand antagonists [³H]prazosin and [³H]yohimbine. [³H]Prazosin was found to label alpha₁-receptors, whereas [³H]yohimbine labeled alpha₂-receptors. Competition experiments performed with subtype selective antagonists with these radioligands produced steep uniphasic competition curves in all cases. Dissociation constants of drugs for the [³H]prazosin and [³H]yohimbine sites correlated highly with the alpha₁ and alpha₂ components of the complex [³H]DHE competition curves, respectively. Good quantitative agreement between the two sets of data was obtained, indicating the validity of both approaches for the determination of receptor subtype affinities. Nonetheless, use of subtype selective radioligands offered several advantages. When the nonsubtype selective radioligand [³H]DHE was used in this system, 100-fold selectivity of a competitor was required in order to determine reliably alpha₁ and alpha₂ affinities. In contrast, use of the selective radioligands discriminated much smaller degrees of selectivity without the necessity for sophisticated computer analysis of the data.

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ACKNOWLEDGMENTS We would like to thank Drs. Frank E. Harrel and Andre De Lean for their helpful suggestions and statistical advice.

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