Antibodies to the alpha 1- and alpha 2-selective antagonists prazosin and yohimbine as probes of the alpha-adrenergic binding sites.

Abstract

SUMMARY Antibodies were raised against a newly synthesized analog (CP57.609) of the o,-selective antagonist prazosin, and against the a.-selective antagonist, yohimbine, by Immunization of rabbits with antigens prepared by covalent linkage of these Uganda to albumin. Competitive Inhibition of [ a H]prazosln binding to anti-CP57,609 antiserum by a variety of nnlabeled Uganda revealed a spectrum of antibody specificity, with a r selective agents competing more potently than a,-seiectiTe llgands. In contrast, a,-selective ligands competed more potently with the binding of ['H]yofaimbine to the anti-yohlmbine antiserum than a^-selective agents. These respective antisera were subjected to affinity fractionation on a CP57,609-or yohimbine-Sepharose 4B resin. Fractions from the CP57,609 resin were eluted successively with phentolamine (10~'M), prazosin (10 4 M), and guanidine (5M), and from the yohimbine resin with prazosin (10^M), yohimbine (HMM), and guanldlne (SM). The binding profiles of these fractions differed, and in certain fractions the relative order of potency of adrenergic agents was almost identical to that observed with membrane a-adrenergic receptors. Moreover, using these eluted fractions as immunogens, antisera have been obtained which, in the initial bleeds, already possess antiidiotypic activity. These findings therefore suggest that affinity fractionation of antibodies raised against a r and a t -selective antagonists may provide useful analogs for the further study of the ligand recognition properties of a-adrenergic receptors. Additionally, it is probable that antiidiotypic antisera will be developed which will recognize the a-adrenergic binding sites. A S with the /?-adrenergic system, the binding of pharmacologically active agents to a membrane receptor is the requisite first step in aadrenergic modulation of cellular processes. On the basis of pharmacologic and radioligand binding studies, these receptors have been identified in a number of tissues 1 and subclassified according to their relative affinities for a variety of a-adrenergic agonists and antagonists into a x -and at-subtypes. J However, little is known about the molecular interaction of aadrenergic agents with these membrane-binding sites, as the purification of the receptor proteins and their structural characterization have not been performed. As an alternative indirect approach to studies of the /8-adrenergic receptor, we have developed /8-antagonistspecific antiserum and demonstrated that its component antibodies can be used as probes to study the ligand recognition properties of the /J-adrenergic receptor. 8 In the present paper, a similar approach has been applied to studies of the recognition properties of a-adrenergic receptors by the development and affinity fractionation of a,-and a a -selective antagonist-specific antibodies.