Interaction of a high affinity anti-estrogen (α-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-α'-nitrostilbene, CI628M) with uterine estrogen receptors

The polar metabolite of the anti-estrogen α-[4-pyrrolidinoethoxy]phenyl-4-methoxy-α'-nitrostilbene (CI628), that is selectively accumulated by the nuclear estrogen receptor in vivo, is identified as the corresponding free phenol (α-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-α'-nitrostilbene (CI628M) and has been prepared in high specific activity tritium-labeled form to study its interaction with the rat uterine estrogen receptor. CI628M has an affinity for the cytosol receptor that is comparable to that of estradiol (K(d) (CI628M)=0.16 nM; K(d) (estradiol) = 0.28 nM), and the binding of these two compounds is mutually competitive. Dissociation of the CI628M complex with the estrogen receptor is somewhat slower than that of the estradiol complex, and prewarming the complexes at 28°C causes a characteristic decrease in the rate of dissociation. On low salt sucrose gradients, the CI628M and estradiol cytoplasmic receptor complexes both sediment as 8S species; their sedimentation profiles on high salt gradients are somewhat different, however. While the estradiol receptor complex shows mainly a 4.6S species that shifts to a 5.8S species upon warming, the unwarmed CI628M complex is predominantly 5.8S and becomes somewhat more disperse upon warming. Under high salt conditions, there is a progressive shift of the 4.6S to 5.8S species as cytosol concentration increases; the CI628M complex shows a greater tendency to form the more rapidly sedimenting form at all cytosol concentrations. After in vivo injection of [³H]CI628M, the uterine nuclear receptor sediments as a characteristic 5S species. Cytosol receptor complexes with estradiol and CI628M, maintained at 0°C, show very little binding to ATP-Sepharose, while after warming, 50-60% of both complexes are bound by the resin. Both complexes also show similar elution profiles from DNA-cellulose columns; however, the extent of binding of the [³H]CI628M receptor complex to the column is consistently only 40-50% that of the estradiol receptor complex. These results indicate that the properties of the uterine estrogen receptor complexed with estradiol and with the high affinity anti-estrogen CI628M are very similar. The differences observed in the behavior of the CI628M cytosol receptor complex on high salt sucrose gradients and on DNA-cellulose columns, though quantitative rather than qualitative, may be indicative of differences in receptor properties that are important to the antagonist activity of this compound.