Binding properties of an anti‐estrogen to the estradiol receptor of uterine cytosol

Rochefort, Henri; Capony, Françoise

doi:10.1016/0014-5793(72)80004-8

Cited by 37 publications

(7 citation statements)

References 12 publications

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“…However, rat AFP or the 4-5S complex was less effective in inhibiting the rate of estradiol binding in the presence of nafoxidine. In contrast, Rochefort et al (21), using a different technique than ours, have reported identical effects of nafoxidine in competitive assays with estradiol on both the 8S and 4-5S entities. Table 1 summarizes the results presented here and incorporates some physiochemical data taken from the literature.…”

contrasting

confidence: 78%

“…After the 8S -4-5S transformation in 0.4 M KC1, the affinity order changes to estrone > estradiol > diethylstilbestrol 0 estriol, a sequence similar to that found for rat AFP. Competitive assays of the type described above were also carried out with the nonsteroidal anti-estrogen nafoxidine, which is known to compete with estradiol for binding sites in uterine cytosols, both in vivo (20) and in vitro (21,22). The results obtained (Table 1) confirm the great effectiveness of nafoxidine in reducing estradiol uptake by the 8S complex.…”

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confidence: 73%

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Alpha-fetoprotein: the major high-affinity estrogen binder in rat uterine cytosols.

Uriel¹,

Bouillon²,

Aussel³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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Evidence is presented that alpha-fetoprotein (AFP), a serum globulin, accounts mainly, if not entirely, for the high estrogen-binding properties of uterine cytosols from immature rats. By the use of specific immunoadsorbents to AFP and by competitive assays with unlabeled steroids and pure AFP, it has been demonstrated that in hypotonic cytosols AFP is present partly as free protein with a sedimentation coefficient of about 4-5 S and partly in association with some intracellular constituent(s) to form an 8S estrogen-binding entity. The (7,8) and in adult animals bearing primary liver cancer (9). In immature rats, serum AFP ranges from about 1 mg/ml (21-day-old animals) to several hundred ,ug/ml (25-day-old animals).Numerous studies, reviewed recently (10, 11), have demonstrated the presence of high-affinity hormone binders, called "receptor" proteins, in soluble homogenates of estrogen-responsive tissues. Rat-uterine cytoplasmic extracts (cytosols) incubated with tritiated estrogens and subjected to density gradient centrifugation have revealed two major macromolecular complexes with sedimentation coefficients close to 4-5 S and 8-9 S; these are currently referred to as the 4-5S and the 8S "receptors." The 8S complex predomiAbbreviation: AFP, alpha-fetoprotein. nates in hypotonic solutions, whereas in salt concentrations above 0.2 M the 4S complex is by far the major binding entity.The relatively high levels of serum AFP in immature rats prompted us to explore the contribution of AFP to the estrogen-binding capacity of uterine homogenates. The results obtained with specific anti-AFP immunoadsorbents (12,13) provided evidence that at low salt concentrations,'AFP ac-' counts for most of the estrogen-binding capacity associated with the 4-5S macromolecular complex. It was concluded that the 4-5S entity and AFP were identical and that the presence of AFP in uterine cytosols was probably due to serum contamination.We report here further observations which strongly suggest that the other macromolecular complex, the 8S entity, is also made up of AFP in reversible association with some intracellular constituent(s). Immunologic data and competitive assays with unlabeled estrogens and anti-estrogens have also enabled us to demonstrate significant changes in antigenicity and binding specificity of native serum AFP upon its transition to the 8S form and the reversion to the original properties of native AFP after the 8S --4-5S transformation in hypertonic solutions.MATERIAL AND METHODS Animals. Female Wistar rats were used in all experiments. Amniotic fluids were obtained by puncturing, the amniotic sacs of animals on the 17th to 19th day of pregnancy. The fluids were pooled, treated with charcoal (2 mg/ml) for 20 min at 370 with mild agitation, centrifuged at 5000 rpm (4000 X g), and stored at -80°. Uteri

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confidence: 78%

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confidence: 73%

Alpha-fetoprotein: the major high-affinity estrogen binder in rat uterine cytosols.

Uriel¹,

Bouillon²,

Aussel³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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“…We have shown [31] by using rat or calf uterine cytosol that nafoxidine was bound reversibly to Rc and that it was a complete inhibitor of estradiol binding, since totality of the bound estradiol was displaced by high concentrations of this antagonist ( fig. 2) similar inhibition was obtained on the 8S Rc, before and after its dissociation by the KC1 (fig.…”

Section: Binding To the Cytosol Receptormentioning

confidence: 99%

“…supernatant (cytosol) were incubated together at 0-2° C. The specifically bound estradiol was assayed using the dextran-coated charcoal technique or sucrose gradient ultracentrifugation, as indicated previously [30,31].-The binding of estradiol to Rc was not modified by androgen or pro gesterone at concentrations up to 1 fiu, confirming results found on cytosol [4,18] and on the whole uteri at 0° C [28]. When using 3H-testosterone or 3H-dihydrotestosterone, specific binding has been demonstrated in rat [12] and calf [17] uteri indicating the presence of specific receptors for androgen in the uterus.…”

Section: Binding To the Cytosol Receptormentioning

confidence: 99%

Effect of Antiestrogens on Uterine Estradiol Receptors

Rochefort¹,

Lignon²,

Capony³

1972

Gynecol Obstet Invest

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Synthetic and natural antiuterotrophic and uterotrophic compounds were tested invitro in their ability to bind to the uterine cytosol receptor for estradiol (Rc) and to favor the formation of the nuclear receptor. These compounds were compared to estradiol in their capacity to decrease the number of specifically bound estradiol in cytosol and increase those in nuclear extracts when incubated with uteri invitro. For each compound, a ‘nuclear transfer activity’ was defined and compared on the one hand to its binding affinity for Rc and on the other hand to its invivo uterotrophic activity. For estrogens devoided of antiestrogenic activity, estradiol and estrone, the nuclear transfer activity was parallel to the affinity for Re and the biological activity. The antiestrogen nafoxidine (U11,100) when added to the cytosol was found to be a reversible and competitive inhibitor of estradiol for Re with an affinity about 30-fold less than estradiol (Ki 7 nM). A complete inhibition could be obtained both before and after the dissociation of Rc by KCl. However, these binding properties of nafoxidine were not in agreement with its partial nuclear transfer activity and its partial uterotrophic activity. The androgens do not bind to Rc; however, they were shown to activate the transfer of the estrogen Rc to the nucleus whereas progesterone did not. These results are discussed in relation to the mechanism of action of uterotrophic and antiuterotrophic compounds.

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“…It has been generally accepted that the initial response of estrogen sensitive tissue to estradiol is an accumulation of estrogen by the specific binding to macromolecular receptor of the cells (Jensen et al, 1968 andGorski et al, 1969). A variety of non-steroidal anti-estrogens, especially amino-ether derivatives of polycyclic phenols have been known to act by competing with specific estrogen binding sites of the target tissue (Jensen et al, 1966;Wyss et al, 1968;Korenman, 1969b;Terenius, 1970;Rochefort and Capony, 1971;Skidmore et al, 1972 …”

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confidence: 99%