Analysis of tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen levels in cytosol and KCl-nuclear extracts of breast tumours from tamoxifen treated patients by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring (SIM)

Tamoxifen, 4-hydroxytamoxifen and desmethyltamoxifen levels were measured in cytosolic and 0.5 M KCl extracted nuclear fractions from a small series of breast tumours from tamoxifen treated patients by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring (SIM). Tamoxifen and desmethyltamoxifen were the most abundant metabolites. There was a small increment in the relative abundance of 4-hydroxytamoxifen in the nuclear extract over cytosol relative to both tamoxifen and desmethyltamoxifen. Further, there was a selective retention of tamoxifen relative to desmethyltamoxifen in the nuclear extract relative to the cytosol. It is concluded that all three compounds could potentially contribute to estrogen receptor mediated antiestrogenic effects in this target tissue.     

Effect of tamoxifen and tamoxifen derivatives on the conversion of estrone-sulfate to estradiol in the R-27 cells, a tamoxifen-resistant line derived from MCF-7 human breast cancer cells

R-27 cells, a tamoxifen-resistant clone of MCF-7 mammary cancer cells, were used to study the effect of tamoxifen and its derivatives (4-hydroxytamoxifen, N-desmethyltamoxifen and cis-tamoxifen) on the conversion of estrone sulfate to estradiol. The present data indicate that (1) tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen and cis-tamoxifen inhibit the uptake of the radioactivity after incubation of these triphenylethylene derivatives with [3H]-estrone sulfate; (2) there is a significant decrease of the conversion of estrone sulfate to estradiol by these antiestrogens; (3) the concentrations of estradiol (cytosol + 0.6 M KCl nuclear extract) which are 293 +/- 50 pg/mg DNA in the control studies (estrone sulfate alone), diminish to 26 +/- 5 pg/mg DNA after addition of tamoxifen, to 9 +/- 2 with 4-hydroxytamoxifen, to 24 +/- 7 with N-desmethyltamoxifen and to 32 +/- 6 with cis-tamoxifen. It is concluded that estrone sulfate can play an important role in the biological responses to estrogens in this breast cancer cell line and tamoxifen and its derivatives block the conversion of estrone sulfate to estradiol. The decrease in concentration of estradiol could be explained by the presence of the estrogen receptor system but other ways of the action of antiestrogens remain to be explored.     

Effect of ATP on the regulation of the steroid binding activity of the oestradiol receptor

We have observed that ATP induces a second type of oestradiol binding site with slightly lower affinity (Ka 3.3 x 10(8) M-1) and lower sedimentation coefficient (4 S) in cytosol from immature lamb uterus and MCF-7 cells. A factor isolated from immature lamb uterine nuclear extract was found to decrease the steroid binding activity of oestradiol receptor that had been purified by heparin Sepharose and oestradiol-Sepharose chromatography. Inhibition of this factor by known phosphatase inhibitors, indicated that this factor may be a phosphatase. Another factor isolated from immature lamb uterine cytosol was found to enhance the effect of ATP on receptor binding in cytosol from immature lamb uterus and MCF-7 cells. The ability of this factor to phosphorylate a partially purified cytosol receptor from immature lamb uterus when incubated with [gamma 32P]ATP, indicates that this factor is a phosphokinase. The phosphorylated products after labeling with [3H]tamoxifen aziridine were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Three phosphorylated proteins with molecular weights 150, 97, and 67 kDa bound [3H]tamoxifen aziridine. Ammonium sulphate precipitated cytosol oestradiol receptor from immature lamb uterus was inactivated with receptor inactivating factor and then reactivated with receptor activating factor in the presence of [gamma 32P]ATP and substantially affinity labelled with [3H]tamoxifen aziridine. The affinity labelled oestradiol receptor was immunopurified with the monoclonal antibody JS 34/32. Three proteins with molecular weights 67, 50 and 43 kDa specifically bound [3H]tamoxifen aziridine and only 43 kDa receptor fragment was phosphorylated. The relevance of inactivation/reactivation of oestradiol receptor to the dephosphorylation/phosphorylation of receptor is discussed.     

Liquid chromatography-mass spectrometry method for the quantification of tamoxifen and its metabolite 4-hydroxytamoxifen in rat plasma: application to interaction study with biochanin A (an isoflavone)

Tamoxifen is the agent of choice for the treatment of estrogen receptor-positive breast cancer. Tamoxifen is a substrate of P-glycoprotein (P-gp) and microsomal cytochrome P450 (CYP) 3A, and biochanin A (BCA) is an inhibitor of P-gp and CYP3A. Hence, it could be expected that BCA would affect the pharmacokinetics of tamoxifen. In the present study we have developed and validated a simple, sensitive and specific LC-ESI-MS/MS method for the simultaneous quantification of tamoxifen and its metabolite 4-hydroxytamoxifen with 100 μL rat plasma using centchroman as an internal standard (IS). Tamoxifen, 4-hydroxytamoxifen and IS were separated on a Supelco Discovery C18 (4.6 mm × 50 mm, 5.0 μm) column under isocratic condition using 0.0 1M ammonium acetate (pH 4.5):acetonitrile (10:90, v/v) as a mobile phase. The mobile phase was delivered at a flow rate of 0.8 mL/min. The method was proved to be accurate and precise at linearity range of 0.78-200 ng/mL with a correlation coefficient (r) of ≥ 0.996. The intra- and inter-day assay precision ranged from 1.89 to 8.54% and 3.97 to 10.26%, respectively; and intra- and inter-day assay accuracy was between 87.63 and 109.06% and 96 and 103.89%, respectively for both the analytes. The method was successfully applied to study the effect of oral co-administration of BCA (an isoflavone) on the pharmacokinetics of tamoxifen and 4-hydroxytamoxifen in female rats. The coadministration of BCA caused no significant changes in the pharmacokinetics of tamoxifen and 4-hydroxytamoxifen. However, the peak plasma concentration (C(max)) of 4-hydroxytamoxifen in BCA pretreated rats was significantly (P<0.05) lower than those from control group.     

Interactions of tamoxifen in the chicken

The triphenylethylene antiestrogens are very potent antagonists of estrogen action in the chicken and manifest little agonist activity compared to their action in other species. The estrogen antagonism is most probably mediated by the estrogen receptor, to which tamoxifen binds with a Ki of 2.6 nM. Tamoxifen is readily metabolized by liver to 4-hydroxytamoxifen, which binds the liver nuclear estrogen receptor with a Ki of 0.1 nM. The Kd of the receptor is 0.7 nM. Estrogen receptor concentrations in liver from immature chickens are relatively low both in nuclear and cytosol fractions. Treatment with estradiol results in 10-fold up-regulation of the nuclear levels to give a total receptor concentration of about 2 pmol/g tissue. Tamoxifen can promote this up-regulation to a limited extent, but interpretation of experimental results is compromised by difficulties with exchange assays in the face of the very high binding affinity of 4-hydroxytamoxifen. Tamoxifen also binds with high affinity (Kd 2-4 nM) and distinctive specificity to antiestrogen binding sites (AEBS) present in a wide variety of chicken tissues and in the highest concentration in the liver (800 pmol/g tissue). Liver and serum contain ether-soluble components which can compete for binding of [3H]tamoxifen to the AEBS. The serum AEBS inhibitory activity is chromatographically heterogeneous and is associated with a sterol-like fraction as well as with a fatty-acid-containing fraction. Tamoxifen treatment of cockerels results in dose- and time-dependent decreases in serum free and esterified cholesterol, and in phospholipids and triglycerides. These changes may reflect estrogen-receptor-independent interactions of tamoxifen.     

Analysis of tamoxifen and its metabolites in human plasma by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring (SIM)

A method for the analysis of tamoxifen and its metabolites in plasma from tamoxifen treated breast cancer patients, by capillary GC-MS using selected ion monitoring has been developed. Metabolite extraction was carried out on a Sep-pak C18 cartridge and metabolite purification by selective ion exchange chromatographic steps. Satisfactory recovery of radioactive standards through the extraction and purification steps was obtained. The method was shown to be accurate and precise with precision coefficient of variation values ranging from 4.3-11% for tamoxifen and its metabolites. Tamoxifen, 4-hydroxytamoxifen, metabolite Y and N-desmethyltamoxifen were identified with certainty in patient plasma on the basis of GC relative retention times and mass spectral comparison with authentic standards; because of their low abundance in plasma cis-metabolite E and 3,4-dihydroxytamoxifen could only be tentatively identified but identical GC behaviour and a satisfactory comparison of the abundance of key fragment ions was achieved. The tamoxifen and metabolite concentration ranges (ng X ml-1) in the group of patients who received 40 or 80 ng tamoxifen for 14 days were tamoxifen, 307-745; N-desmethyltamoxifen, 185-491; 4-hydroxytamoxifen, 1.4-2.5; 3,4-dihydroxytamoxifen, 0.7-2.0; metabolite Y, 19.0-112; and metabolite E1, 0.9-2.0.     

Antagonistic effect of the antiestrogen 4-hydroxytamoxifen on estradiol-stimulated acetylation of nuclear high mobility group (HMG) proteins in the uterus of newborn guinea-pigs

The effect of the antiestrogen 4-hydroxytamoxifen on the estradiol-stimulated acetylation of nuclear high mobility group (HMG) proteins was studied in the uterus of newborn (3-day-old) guinea-pig. 4-Hydroxytamoxifen (10(-6) M) selectively inhibits the stimulatory effect of estradiol (5 x 10(-8) M) on the acetylation of HMG-14 proteins 30 min after incubation with uterine tissue slices. No effect of 4-hydroxytamoxifen was observed on HMG-1 + HMG-2 or HMG-17 proteins. The data suggest that the blockage of HMG-14 acetylation is an early event in gene expression which is in relation to the antagonistic effect of the antiestrogen.     

Uterine bioassay of tamoxifen,trioxifene and a new estrogen antagonist (LY117018) in rats and mice

Dose response uterotrophic and antiuterotrophic activity of antiestrogens was examined in immature rats, immature mice and adult ovariectomized mice. LY117018 was the most active antagonist and the least estrogenic, while tamoxifen induced the greatest uterine growth and the weakest antagonism. The reported estrogenic activity of tamoxifen in mice (1) was found to be related to maturity. All compounds caused uterotrophic changes in immature mice similar to those observed in immature rats. However, in adult mice tamoxifen was devoid of antagonism, and trioxifene was active only at a very high dose as both were extremely estrogenic in this model. LY117018 activity in adult mice was comparable to that observed in immature rats and mice. Results depict significant agonist and antagonist advantages of LY117018 over tamoxifen and trioxifene.     

In vitro and in vivo binding of toremifene and its metabolites in rat uterus

The in vitro binding affinities of toremifene (TOR), 4-hydroxy toremifene (4-OH-TOR) and several other metabolites for the rat uterine cytosolic estrogen receptor were compared with those of tamoxifen (TAM) and 4-hydroxy tamoxifen (4-OH-TAM). Only small differences were observed and the binding affinities of both 4-hydroxy metabolites were similar to that of estradiol (E2). Uterine uptake and subcellular distribution of [3H]TOR and [3H]TAM were then compared at 1, 8 and 72 h after administration to castrated rats. The uptake and retention of both antiestrogens were similar at all times. In each case the amount of nuclear bound radioactivity declined to low levels at 8 and 72 h but the ratios of 4-OH-TAM/TAM and 4-OH-TOR/TOR determined by HPLC analysis increased dramatically at 72 h. The level of radioactivity in both plasma and uterine cytosol at 72 h was significantly higher following [3H]TAM administration. However, most of the radioactivity appeared to be in a conjugated form since it was not extractable with solvent. Finally, the ability of prior administration of each antiestrogen (100 mg/kg) to block uterine [3H]estradiol uptake was examined at 3 and 7 days. It was found that uterine wet weights were higher than control one week after administration of both compounds. Prior administration of TOR increased nuclear uptake of [3H]E2 whereas TAM had no effect. The results of these experiments suggest that toremifene and tamoxifen have very similar in vitro and in vivo binding properties but differences in metabolism exist that may be important.     

Estradiol-17β receptors in the immature rat ovary

Estradiol binding components in the cytosol and nuclear fractions of the ovary from immature rats (22–28 days old) were characterized by $\text{in vitro}$ methods. Several of the biochemical characteristics of the estradiol binding components in the ovarian tissue were compared with the estradiol receptor from the uterus. The results suggest that the ovarian estradiol binding components are similar to the specific high affinity estradiol receptors in the uterus. In the cytosol of intact rat ovary a significant fraction of the total binding sites was found to be occupied, presumably by the endogenous estrogen. Following hypophysectomy there was a significant increase in the available cytosol binding sites. Evidence for translocation of cytosol receptor-estrogen (RE) complex to the nucleus was obtained for the ovary. The sedimentation properties of the RE complex of the ovary and the uterus are similar. The ovarian cytosol RE complex sediments at 7-8S in glycerol gradients at low ionic strength and at 4S in sucrose gradients at high ionic strength. Following extraction with 0.4 M KCl the ovarain nuclear RE complex sediments at 5S in sucrose gradients which is identical to that of the uterine nuclear receptor.     

Oestrogen receptors in chick oviduct. Characterization and subcellular distribution.

Macromolecular components with properties of oestrogen receptors have been identified in the 0.5 M KCl nuclear soluble, the nuclear insoluble and the cytosol fractions of laying hen and immature (2--4 weeks, untreated by hormone) chicken oviduct. 7n the 0.5 M KCl extract of laying hen oviduct nuclei, a receptor, of protein nature according to the effects of enzymic treatments, has been identified. It exhibits high affinity for oestradiol with an apparent equilibrium association constant KA = 4 - 109 M-1 at 4 degrees C. The binding of [3H] oestradiol is abolished by 1 muM oestriol, oestrone and diethylstilboestrol, but not by the same concentration of progesterone, testosterone, and cortisol. Sucrose gradient ultracentrifugation studies in the presence of 0.5 M KCl indicate a sedimentation coefficient of 4.3 S, and there is partial aggregation in low-ionic-strength medium. The estimated number of binding sites per nucleus is about 5000, as calculated from DNA content of chick diploid genome. Most of the binding sites were found to be occupied by endogenous oestrogen(s). Oestradiol dissociates from the receptor according to an apparent two-step mechanism. The half-life time for the faster dissociation step is 18 h at 0 degrees C, 25 min at 20 degrees C and 10 min at 30 degrees C, and for the slower one is 180 h, 115 min and 60 min, respectively. In the 0.5 M KCl extract of immature chicken oviduct nuclei, there are approximately 500 receptor sites per nucleus; their affinity for oestradiol is the same as in the case of laying hen soluble nuclear receptor. After repeated extractions of nuclei with 0.5 M KCl medium, a substantial quantity of oestrogen binding sites remains in the residual fraction. Binding characteristics of this insoluble nuclear receptor resemble those of the soluble nuclear receptor: high affinity for oestradiol (KA = 7 - 10(8) M-1 at 37 degrees C) and specificity for oestrogens. The estimated number of binding sites are approximately 2000/cell for laying hen, and approximately 1000/cell for immature chicken. In the high-speed supernatant fraction of laying hen oviduct homogenates, an oestrogen receptor is also present, but its concentration is low (less than or equal to 100 sites/cell) and at the limits of sensitivity of the methods used. In the cytosol of immature chicken oviduct, there are approximately 2500 oestradiol receptor sites per cell.     

(3H)-estradiol binding by chick liver nuclear extracts: mechanism of increase in binding following estradiol injection.

Specific high affinity binding of [3H]-estradiol by 0.5 M KCl extracts of chick liver nuclei is substantially increased by estradiol injection of the immature chick. The effect is observed shortly after estradiol injection, while the estradiol-induced production of serum phosphoproteins (vitellogenic response) is not detectable until about 24 hr. Cycloheximide given 90 min before estradiol inhibits the increase in nuclear binding for 12-15 hr. At 24-48 hr the levels of nuclear binding are similar to those in the estradiol-treated animals not given cycloheximide, but serum phosphoprotein levels are depressed by about 80% at 48 hr. By 75 hr however the serum of the cycloheximide-treated estrogenized chicks contains about twice as much phosphoprotein as does serum of chicks given estradiol alone. It is suggested that the inhibition of protein synthesis for 12-15 hr delays the vitellogenic response until sufficient levels of nuclear [3H]-estradiol binding protein can be synthesized. A correlation between the levels of nuclear [3H]-estradiol binding at 24 hr and phosphoprotein at 48 hr is shown in a dose-response experiment. In vitro, nafoxidine-HCl (Upjohn 11,100 A) inhibits binding of [3H]-estradiol by the chick liver nuclear extracts. In vivo, a single injection of nafoxidine with estradiol inhibits phosphoprotein production. Injection of nafoxidine alone results in a small but significant increase in [3H]-estradiol binding by nuclear extracts, but it is not estrogenic. A possible interpretation is that nafoxidine transfers low levels of a putative cytosol receptor to the nucleus, but is unable to induce the amplification mechanism required to give the levels of nuclear estradiol-binding protein needed for the vitellogenic response.     

Study of the in-vivo antioestrogenic action of N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE), a novel intracellular histamine antagonist and antioestrogen binding site ligand

N,N-Diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE) binds with high affinity to the antioestrogen binding site (AEBS), but not to the oestrogen receptor. There is an association of AEBS with a novel intracellular histamine receptor (H1C) of micromolar affinity through which histamine acts as a second messenger. An optimal dose of 4 mg DPPE/kg antagonized the uterine growth-stimulating effects of oestradiol in immature oophorectomized rats. Unlike tamoxifen, DPPE alone was not a partial agonist, but decreased uterine size and weight below control values at concentrations between 0.1 and 75 mg/kg. DPPE also antagonized oestradiol-stimulated uterine growth at 72 h; the inhibition observed was not significantly different from that seen with tamoxifen. Oestradiol-treated animals receiving the combination of DPPE (4 mg/kg) + low dose tamoxifen (0.04 mg/kg) for 72 h had significantly smaller uteri than did those receiving the same dose of DPPE or tamoxifen alone. Histologically, either DPPE or tamoxifen antagonized oestradiol stimulation of eosinophil migration and glandular epithelial proliferation; the latter inhibition was significantly greater for DPPE + tamoxifen (0.04 mg/kg) than for the same dose of DPPE or tamoxifen alone. Unlike tamoxifen, DPPE did not antagonize oestradiol stimulation of luminal epithelial proliferation, but in the presence of oestradiol, DPPE significantly decreased tamoxifen (0.65 mg/kg)-induced hypertrophy of the luminal epithelium. Based on these findings, we suggest that binding to the AEBS/intracellular histamine receptor is important to the action of antioestrogens.     

Non-stoichiometric nuclear-cytoplasmic redistribution of estrogen receptor in adult rat uterus, following estradiol injection

In immature and ovariectomized rats acutely injected with estradiol (E2), accumulation of estradiol receptor complexes (E2R) from the uterine cytosol to the nucleus has been shown to be quantitative by numerous investigators. In the present study, translocation of E2R from the cytosol to the nuclear fraction in adult and ovariectomized estrogen prestimulated rats was analyzed. Twenty micrograms of E2, dissolved in saline containing 10% ethanol and 1 g% bovine serum albumin (B.S.A.) were injected intraperitoneally to the animals and 2 h later E2R in the cytosol and crude nuclear fractions were assayed by exchange techniques. Unlike a 91% recovery of the depleted cytosol E2R in the nuclear fraction of ovariectomized rats, only 39.2 and 27.5% were recovered in the adult and ovariectomized estrogen prestimulated rat uterus respectively. Moreover, depending on the temperature and duration of nuclear suspension incubation, from 18 up to 80% of the recovered nuclear E2R were solubilized in the incubation medium and nuclear post-incubation washes and could be measured by hydroxylapatite treatment (HAP). Saturation assays showed a plateau from 12 nM E2 3H onwards up to 80 nM. The Kd values computed for the receptors in the nucleus and HAP in all the three groups were of the order of 2 X 10(-9) M. In conclusion, after E2 administration to adult or ovariectomized estrogen prestimulated rats, a stoichiometric recovery of the depleted cytosol E2R in the nuclear fraction was not observed, even when leakage of nuclear receptor into the medium in course of exchange was taken into account. Chronic estrogenization appeared to modify the dynamics of uterine receptor.     

Development and validation of a quantitative assay for the determination of tamoxifen and its five main phase I metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry

A sensitive bioanalytical assay for the quantitative determination of tamoxifen and five of its phase I metabolites (N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen and 4'-hydroxytamoxifen) in serum is described. The method has been fully validated at ranges covering steady-state serum concentrations in patients receiving therapeutic dosages of tamoxifen. The bioanalytical assay is based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using multiple reaction monitoring for drug (-metabolite) quantification. The sample pretreatment consists of protein precipitation with acetonitrile using only 50 μL of serum. In the past, numerous assays have been developed by other groups for the quantification of tamoxifen and its phase I metabolites. However, the number of metabolites included in these studies is very limited and only very few of these assays have been fully validated. A liquid chromatography tandem mass spectrometry assay for the quantification of tamoxifen and four phase I metabolites in human serum that was previously developed by our group is now explicitly improved and described herein. Time of analysis has been reduced by 50% and sensitivity was increased by a reduction of the lower limit of quantification from 1.0 to 0.2 ng/mL for 4-hydroxytamoxifen and 4'-hydroxytamoxifen. Additionally, two phase I metabolites that have never been quantified in human serum hitherto, namely 4'-hydroxytamoxifen and N-desmethyl-4'-hydroxytamoxifen, were included in this assay. Validation results demonstrate an accurate and precise quantification of tamoxifen, N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen and 4'-hydroxytamoxifen in human serum. The applicability of the assay was demonstrated and it is now successfully used to support clinical studies in which patient-specific dose optimization is performed based on serum concentrations of tamoxifen metabolites.     

Characterization of an antiestrogen-binding protein in high salt extracts of human breast cancer tissue

An antiestrogen binding protein which binds [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)-phenyl]1,2-diphenylbut-1(Z)-ene) with high affinity (Kd = 1.1 X 10(-9) M) is present in high salt (0.6 M KCl) extracts of washed breast cancer tissue pellets. Its concentration in high salt extract is higher than its concentration in cytosol. The characteristics of the antiestrogen binding protein from cytosol and salt extract of breast cancer tissue are indistinguishable. It specifically binds triphenylethylene and other nonsteroidal antiestrogens and displays little or no binding affinity for estrogens, progesterone, dihydrotestosterone and cortisol. The antiestrogen binding protein is of unusually large size as judged by gel filtration on agarose 0.5 m and sedimentation analysis on 5-20% sucrose density gradients. Differential centrifugation studies indicate that it is not principally microsomal in origin. This protein is more thermostable than the estrogen receptor from which it can also be distinguished by ion exchange chromatography. The antiestrogen binding protein was eluted from DEAE-Sephacel by 0.05 M KCl indicating that it is less negatively charged than the estrogen receptor which was eluted by 0.1 M KCl. Lipoprotein fractionation of breast cancer cytosol using potassium bromide density gradients did not reveal specific antiestrogen binding activity associated with any recognized class of lipoprotein. Specific [3H]tamoxifen binding sites were pelleted in potassium bromide gradients consistent with the apparent large size of this protein. The physical characteristics of the antiestrogen binding protein in normal human tissue (myometrium) and neoplastic tissue (breast cancer) are remarkably similar, possibly reflecting a highly conserved structure.     

Regulation of cytosol and nuclear progesterone receptors in rabbit uterus by estrogen, antiestrogen and progesterone administration.

A synthetic progestin, 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione (ORG 2058), was utilized to measure progesterone receptors from the rabbit uterus. This steroid has a high affinity for both cytosol and nuclear receptors, with KD values of 1.2 nM (at 0--4 degrees C) and 2.3 nM (at 15 degrees C), respectively. Administration of estradiol-17 beta or a non-steroidal antiestrogen, tamoxifen, for 5 days to estrous rabbits led to a progressive rise in the cytosol receptor levels: from 34,000 to 120,000 (estradiol-17 beta) and 80,000 (tamoxifen) receptors/cell, without any major influence on the nuclear receptor content. A single intravenous injection of progesterone (5 mg/kg) elicited a 3-fold increase in the mean nuclear receptor content at 30 min after injection (from 18,000 to 48,000 receptors/nucleus). Nuclear receptor accumulation was short-lived and returned to control levels within 4 h after treatment. A second dose of progesterone given 24 h later doubled the nuclear receptor level (from 18,000 to 35,000 receptors/nucleus). The concomitant decline in the cytosol receptor content was twice that accounted for by the nuclear receptor accumulation (70,000 vs. 30,000, and 40,000 vs. 17,000 receptors/cell, after the first and second progesterone injection, respectively). Following progesterone administration, the cytosol receptor level reached a nadir by 30 min, exhibited minimal replenishment within the ensuing 24 h, and remained at approx. 50% of the pretreatment values. After a single dose or two consecutive doses of progesterone, total uterine progesterone receptor content declined to about 60% of the level prior to each dose, a nadir being reached at 2 h after treatment.     

Dynamics of oestrogen-receptor distribution between the cytosol and nuclear fractions of immature rat uterus after oestradiol administration.

1. The nuclear-myofibrilar (800g pellet) fraction of the uterus from immature (22-23 days old) rats not exposed to oestrogen exhibits saturable binding of oestradiol. The nuclear binding capacity represents approximately 10% of that of the cytosol fraction (approx. 3.5 fmol/mug of DNA). The predominant part (0.3.5 fmol/mug of DNA) of the nuclear binind sites are present in the residual pellet after extraction with 0.5 M-KC1. 2. By using an exchange technique in vitro, determinations of the nuclear binding sites have been carried out after administration of 1 mug of oestradiol in vivo. Within 0.5h after the hormone injection, the concentration of nuclear bindng sites increased to approx. 0.4 fmol/mug of DNA in the 0.5 M-KC1-extractable fraction, and to approx. 1.2 fmol/mug of DNA in the residual fraction. Meanwhile the cytosol oestrogen-receptor concentration decreased to approx. 10% of its initial value. In the following period from 0.5 h after the oestradiol injection onwards, the concentration of nuclear oestrogen receptors decreased with halflife values of approx. 140 and 200 min for the KC1(0.5 M)-extractable and residual form respectively. At the same time, the cytosol receptor concentration increased to reach approx. 50% of the initial value by the 6h. This increase could not be blocked by cycloheximide. The initial concentration of cytosol receptor was restored approx. 11h after the injection and the increase during the 6-11h period was sensitive to cycloheximide inhibition, suggesting protein-synthesis-dependence of the process. 3. With the (more) physiological dose of oestradiol (0.1 mug), the decrease the cytosol receptor was only 50% by 4h and this was followed by a period (up to 12h after injection) during which the initial concentration was restored. During this period the increase of the receptor can be blocked by cycloheximide.