Glucocorticoid receptors in cytosol and nuclear extract of human leukocytes

Cortisol binding by cytosol and 0.4 M KCl extract of the nuclear fraction of human leukocytes were studied by gel chromatography and ion exchange filtration on DEAE cellulose. The cytoplasmic cortisol binding protein has a molecular weight 95 000 and the soluble nuclear binding protein 50 000. The absence of the uptake of radioactive cortisol by isolated nuclei and the apparent requirement of the cytosol for glucocorticoid specific binding in nuclear receptor sites was observed. The association constant characterising the binding of cortisol to cytosol was KA = 3.5 . 10(9) l/mol.     

Differentiation of estradiol receptors in rat uterine cytosol by sensitivity to tamoxifen.

Analysis of rat uterine cytosol by means of immobilized antibody discloses the presence of two distinct high affinity, low capacity estradiol binding components. One of these is readily saturable by the antiestrogen tamoxifen (TS = tamoxifen sensitive), the other is tamoxifen insensitive (TI). Only TS-estradiol binding shows positive cooperativity at low ligand concentration. TS but not TI is lost when frozen tissue is thawed at 4°C then refrozen and stored for an additional 12 hours. Experiments with ovariectomized rats show that TI is formed only in the presence of estradiol. In the estrus cycle TS increases in the order: metestrus, diestrus, proestrus and estrus. The quantity of TI is the same in metestrus and diestrus and also the same but fractionally higher in proestrus and estrus.     

Chemical differentiation of type I and type II receptors for adrenal steroids in brain cytosol

Studies outlined here compare the properties of mineralocorticoid (Type I) and glucocorticoid (Type II) receptors in cytosol from adrenalectomized mouse brain. Pretreating cytosol with dextran-coated charcoal (DCC) produced a 4.7-fold increase in the subsequent macromolecular binding of the mineralocorticoid, [3H]aldosterone (20 nM ALDO, in the presence of a 50-fold molar excess of the highly specific synthetic glucocorticoid, RU 26988), whereas it produced a 55% decrease in the binding of the glucocorticoid, [3H]triamcinolone acetonide (20 nM TA). Scatchard analyses revealed that DCC pretreatment had no effect on the affinity or maximal binding of Type I receptors for [3H]ALDO (in the presence of a 0-, 50- or 500-fold excess of RU 26988), whereas it produced a 3- to 6-fold increase in the Kd, and an 8-43% decrease in the maximal binding, of Type II receptors for [3H]TA and [3H]dexamethasone. Optimal stability of unoccupied Type I receptors at 0 degree C was found to be achieved in buffers containing glycerol, but lacking molybdate. Although the addition of molybdate was found to reduce the loss in Type I receptor binding observed after incubating unlabelled cytosol at 12 or 22 degrees C, this stabilization was accompanied by a concentration-dependent reduction in the binding of [3H]ALDO at 0 degree C. Scatchard analyses showed that this reduction was due to a shift in the maximal binding, and not the affinity, of the Type I receptors for [3H]ALDO. The presence or absence of dithiothreitol in cytosol appeared to have little effect on the stability of Type I receptors. In contrast to our finding for Type I receptors, it was possible to stabilize the binding capacity of unoccupied Type II receptors, even after 2-4 h at 12 or 22 degrees C, if the glycerol containing buffers were supplemented with both molybdate and dithiothreitol. In summary, these results indicate distinct chemical differences between Type I and Type II receptors for adrenal steroids.     

Distribution of progesterone receptors between the cytosol and nuclear fraction in normal and neoplastic human endometrium

The progesterone receptor (PR) content was determined in the cytosol and in the nuclear fraction in 28 cases of endometrial adenocarcinoma after in vitro incubation of tissue slices in the presence of progesterone. In 17 instances besides the malignant, nontransformed tissue from the same uterus was taken for analysis. The level of PR was estimated by Scatchard plot analysis or by one-point assay, whenever not enough tissue was available. The distribution of PR between the cytosol and the nuclear fraction was studied. Positive correlation between the concentrations of total and of nuclear progesterone binding sites was found in all samples of nontransformed tissue whereas in cancerous tissue this was true only in about 40% of cases. In about 60% of neoplastic tissue samples the PR were namely found predominantly in the cytosol. Since we observe that in many malignant endometria the PR are poorly transferred from the cytoplasm to the nucleus even under optimal conditions for in vitro activation/translocation, we conclude that in these cases the action of progesterone is for some reason altered either at the level of receptor activation or at the level of translocation of the hormone-receptor complex from the cytoplasm to the nucleus.     

Characterization of ecdysteroid receptors in cytosol and naive nuclear preparations of Drosophila Kc cells

Significant ecdysteroid binding activity can be demonstrated in nuclear extracts obtained from hormonally naive Drosophila Kc cells. The kinetic and physical characteristics of this nuclear binding are presented and compared with those exhibited by a high speed cytosol preparation of Kc cells. Examination of the effect of in vivo ecdysteroid exposure on the number of nuclear binding sites revealed that the quantity of nuclear receptors was not detectably altered. In addition, an effective synthesis of the biologically active ecdysteroid radioligand, [3H]ponasterone A, is described.     

Solubilization and characterization of adrenal and uterine angiotensin II receptors after photoaffinity labeling

The physical characteristic of the receptors for angiotensin II in dog adrenal cortex and uterus were determined after affinity labeling. 125I-nitro-5-azido-benzoyl-angiotensin II, a photosensitive angiotensin II analogue which retained aldosterone-stimulating activity, was used to couple the octapeptide specifically and irreversibly to its membrane receptors. After solubilization with Triton X-100, the covalent hormone . receptor complex was analyzed by gel filtration and sucrose density gradient centrifugation. Two radioactive species were consistently observed, with calculated Mr values of 126,000 +/- 10,000 and 64,500 +/- 11,000. the elution profiles of solubilized adrenal and uterine particles were almost identical. When the solubilized complexes were subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, a single radioactive band was detected upon autoradiography, with Mr - 65,000 +/- 6,000 for adrenal cortex and 68,000 +/- 7,000 for myometrium. These results indicate that the receptors for angiotensin II in adrenal cortex and uterus are composed of two subunits of similar molecular weight, and that the common functional properties of the receptors from both tissues are probably related to their similar physicochemical characteristics.     

Characterization of protein kinases from adrenal medulla. A study of cytosol and nuclear enzymes.

Since phosphorylation of chromosomal proteins by cyclic AMP-dependent protein kinases (EC 2.7.1.37) enhances template activity of adrenal medulla chromatin (9), we have studied the properties and regulation of protein kinases isolated from chromaffin cell cytosol and nuclei. DEAE-cellulose chromatography revealed three peaks of kinase activity in the nucleus (nPKI, nPKII, nPKIII) and two in the cytosol (cPKI, cPKII). The three nuclear enzymes, as well as cPKII, did not require cyclic AMP to express their catalytic activity. nPKI and nPKIII preferred acidic substrates as PO3-4 acceptors, while nPKII and the cytosol enzymes preferred basic PO3-4 acceptors. Enzyme recombination experiments using protein kinase regulatory subunits from cytosol suggested that cPKII was the catalytic subunit of cPKI. In contrast, the nuclear enzymes were not catalytic subunits of the cyclic AMP-dependent protein kinase in the cytosol (cPKI). Only the cytosol protein kinases could be inhibited by endogenous heat-stable protein kinase inhibitors. The nuclear and cytosol cyclic AMP-independent protein kinases were distinguishable on the basis of their sedimentation constants as well as Mc2+ and Mn2+ requirements.     

Effect of estrogens on follicular development and ovarian and uterine estrogen receptors in the immature rabbit, guinea pig and mouse

Immature rabbits, guinea pigs and mice were injected with estradiol cyclopentylpropionate (ECP) or diethylstilbestrol (DES) for 3 days to evaluate whether estrogen enhances follicular maturation. Also, estrogen receptors in the ovary and uterus from these animals were measured. Uterine weight increased in all animals treated with ECP or DES, whereas actual ovarian weight increased only in the guinea pig. This correlated with the ability of estrogens to significantly increase the number of antral follicles in the guinea pig ovary. In the rabbit and mouse, estrogen increased only the number of small or large preantral follicles. However, the number of estrogen binding sites in the ovarian cytosol and nucleus was greater in the rabbit and the mouse than in the guinea pig. The affinity of ovarian cytosol receptors was the lowest for the guinea pig among the 3 species. Thus it is seen that estrogen does not enhance follicular maturation in all animal species. The ovarian response to estrogen is not only dependent upon estrogen receptors but also unknown mechanism(s) that may be related to paracrine or autocrine functions.     

Neonatal castration of male and female rats affects hypothalamic and pituitary estrogen nuclear and progestin cytosol receptors

We compared the effects of neonatal or adult castration (7 days) and 2 or 7 days of estrogen treatment on the concentrations of estradiol cystolic (ERc) and nuclear (ERn), and progestin cytosolic receptors (PRc) in the hypothalamus, amygdala and pituitaries of adult rats. Two days of estradiol (E2) treatment greatly increased ERn levels, but no further concentration changes occurred by Day 7 in any of the tissues. Long- and short-term castrated males and females had comparable ERn concentrations on Day 2 versus Day 7. Tissue ERn levels were significantly lower in short-term males compared to short-term females or neonatally castrated males and females. In a second study, ERn levels were compared in E2-treated short-term castrated males and females on Day 2. A sex difference was observed, with females having greater ERn levels in most areas. Estrogen significantly increased PRc levels in pituitary (PIT) and hypothalamus, and these levels were comparable in Day 2 and Day 7 animals. Thus, the ability of estrogen to induce PRc synthesis is somewhat refractory in long-term castrated rats.     

Effect of phospholipids on estrogen receptor of rat uterine cytosol

Phospholipids such as phosphatidyl inositol and phosphatidyl serine inhibit the binding of R5020 and progestin receptors. The effect of phospholipids on the binding of estrogen and estrogen receptors of rat uterine cytosol was studied. Phosphatidyl choline, sphingomyelin, phosphatidyl inositol, phosphatidyl ethanolamine, cardiolipin, and phosphatidic acid inhibited the binding of estradiol and estrogen receptors. This inhibitory effect of phosphatidyl inositol and cardiolipin was dose dependent.     

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

A synthetic progestin, 16α-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 K_D values of 1.2 nM (at 0–4°C) and 2.3 nM (at 15°C), respectively. Administration of estradiol-17β 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-17β) 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 receptor/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.