Antiestrogens are known to interact with the estrogen receptor system of target cells. In addition, it is possible that recently identified estrogen-noncompetable, antiestrogenspecific binding sites may be of importance in mediating and/or modulating the actions of antiestrogens, and hence, we have examined their nature and properties. We find these sites to fractionate with what is likely the microsomal fraction of rat uterus with only very small amounts in high speed cytosol. Antiestrogen-specific binding sites are present in the 800 ×g - 10 min or 12, 000 or 20, 000 - g - 30 min supernatants, but are pelleted upon centrifugation at 100, 000 or 180, 000 - g - 60 min. These sites are present in the 12, 000 - g supernatant fraction of almost all rat tissues examined. They are highest in concentration in the liver, uterus, esophagus, ovary, brain, and kidney; lower levels are found in lung and spleen, and negligible amounts are seen in muscle, heart, and serum. Hence, these sites are not localized exclusively or primarily in estrogen target tissues and their level does not parallel that of the estrogen receptor, which is high only in the uterus and ovary and at lower levels in liver and kidney. Affinity for this binding site follows the order trans-tamoxifen (1-[4-(2-dimethylaminoethoxy) phenyl]-l, 2-diphenylbut-l(Z)-ene) (set at 100%) ≥ cis-tamoxifen > CI628 (α-(p-[2-(l-pyrrolidino)ethoxy]phenyl)-4-methoxy-α'-nitrostilbene) (50%) > trans-hydroxytamoxifen (l, 4-(2-dimethylaminoethoxy)phenyl-l-(4-hydroxyphenyl)-2-phenylbut-1(Z)-ene) (40%) > CI628M (α-(p-[2-(l-pyrrolidino) ethoxy]phenylj-4-hydroxy-α′-nitrostilbene) (20%) > LY117018 (6-hydroxy-2-(p-hydroxyphenyl)benzo[b]thien-3-yl p-[2-(l-pyrrolidinyl)ethoxy]phenyl ketone) (2%). Triphenylethylene antiestrogens lacking the amine side chain do not bind to these antiestrogen-specific sites, but several tamoxifen analogs with altered basic side chains have affinities for these sites 4- to 10-fold greater than that of tamoxifen. Hence, the affinity of different antiestrogens for this site does not parallel the potency of these compounds as antiestrogens or their affinity for the estrogen receptor. Antiestrogen-specific binding sites and estrogen receptor sites differ in their thermal stability, pH stability, and protease sensitivity, and in their pattern of depletion from the 12, 000 - g supernatant fraction following the administration of estradiol. From these findings, we believe that these sites may not be directly involved in mediation of the classically recognized estrogen antagonism of antiestrogens. Nevertheless, the high affinity (dissociation constant of 1–3 nM) and wide distribution of these sites suggest that they might influence the action of antiestrogens in vivo, possibly in a passive manner, by altering the apparent distribution volume of antiestrogens and, hence, their biological potency and pharmacokinetics.
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