Kinetic Analysis of Estrogen and Antiestrogen Competition for Hypothalamic Cytosol Estrogen Receptors. Evidence for Noncompetitive Ligand-Receptor Interactions

Abstract

The binding of (³H)-estradiol (E₂) to cytoplasmic estrogen receptors isolated from female rat hypothalamus-preoptic area by unlabeled estrogen and antiestrogens (nafoxidine and tamoxifen) was examined when the concentrations of both the agonist (³H)-E₂) and unlabeled competitors were varied over a wide range. Concentrations of unlabeled E₂ up to 10^-10M decreased the affinity of the labeled steroid for its receptor; at higher E₂ concentrations, the apparent number of binding sites (B_max) began to decline. Thus at some concentrations, E₂ may act as a mixed competitive and noncompetitive inhibitor of its own binding to hypothalamic cytosol receptors. The antiestrogens differed markedly from E2 in their interactions with hypothalamic estrogen receptors. Only at relatively high competitor concentrations (e.g., > 10^-9M) did the antagonists appear to competitively inhibit (³H)-E₂-receptor binding. The most striking observation was that tamoxifen and nafoxidine significantly inhibited (³H)-E₂-receptor binding at very low competitor concentrations (e.g., 1 pM), but only slightly inhibited estrogen binding at intermediate concentrations (e.g., 10^-10M). It was proposed that the non-linear, concentration-dependent effects of antiestrogens on the neural estrogen receptors might be due to complex interactions of the antagonists with a non-estrogen binding site.