Anti-estrogen Interaction with Uterine Estrogen Receptors

Radiolabeled anti-estrogen cu-[4-pyrrolidinoethoxylphenyl4-methoxy-cY’-nitrostilbene ([3HlCI-628) of high specific activity (16 Cilmmol) and radiochemical purity (>95%) has been prepared by catalytic tritium-iodine exchange and purified by alumina column chromatography. Its interaction with estrogen receptors in immature (day 20 to 24) rat uterus is studied in vitro and in ho. [3H]CI-628 (CI) competes with estra-1,3,5(10)-triene3,17&diol (estradiol (E,)) for binding to specific cytoplasmic estrogen receptors, but has a lower binding affinity (CI: I(d = 1.7 x 10e9 M; E,: K, = 1 x lo-lo M, at O-4”, determined at equilibrium by charcoalldextran adsorption). Both PHICI and [3HIE, have similar rates of association with the receptor (CI: k,, = 5.5 X lo5 M-' s-l; E,: k,, = 4.4 X lo5 M -I s-l, at O-4”), but 13H]CI dissociates much more rapidly (CI: k-, = 2.6 x 10m4 s-l, t, = 44 min; E,: k --1 = 2.4 x 10e6 s-l, t, = 80 h, at O-4”). Different sedimentation profiles are seen for the cytoplasmic receptor complexes with C3HICI and with L3H1E, on low salt sucrose density gradients: over 90% of the [3H1E,.receptor complexes sediment at 8.0 S, whereas 70% of the [3HlCI.receptor complexes (estrogen-competible sites) sediment at 4.5 S, only 30% sedimenting at 8.0 S. After administration of C3H]C!I in u&o, radioactivity can be found associated with specific estrogen-receptor sites in uterine nuclei. Salt-extracted nuclear receptor. 13HlCI complexes sediment at 5.4 S on 0.4 M KC1 sucrose gradients as do C3H]E, * nuclear receptor complexes. Thin layer chromatographic analysis of ethyl acetate extracts of the nuclear receptor peak fractions from sucrose gradients shows that a polar metabolite of CL628 is selectively bound to the 5.4 S receptor. Pharmacokinetic studies reveal that high levels of unmetabolized CI persist in serum and uterus for