Characterization of estrogen and progesterone receptors in monkey endometrium: methodology and effects of estradiol and/or progesterone on endometrium of castrate monkeys.

Appropriate methods for repeated surgical collection of endometrial tissue from rhesus monkeys, and characterization of cytosol and nuclear estrogen (E₂) and progesterone (P) receptors (R) are described. Tissue collection was made in the mid-luteal phase at abdominal fundal hysterotomy. Functional status of the ovaries was determined by visual inspection and RIA of E₂ and P in serum. Receptor assay procedures were devised permitting the measurement of total cytosol and nuclear receptor concentration. Sucrose density gradients of labelled cytosol were made and a 4S saturable binding component for ³H P and for ³H E₂ were found. Equilibrium dissociation constants of ³H E₂ and ³H R5020 were 2.1×10^?10M and 3.6 × 10^?9M, respectively. These binding characteristics are similar to those found in human endometrium and suggest that these surrogate primates have extensive utility in investigation of factors influencing E₂R and PR concentrations in endometrial tissue during the menstrual cycle and implantation. Simulated menstrual cycle were produced in 20 castrate monkeys by sequential treatment with estradiol and progesterone in silastic capsules. RIA of E₂ and P, and gonadotropins in peripheral serum provided assuredness of the hormonal status of each monkey under treatment. Cytosol and nuclear receptors for E₂ and P were measured in the endometrium after different intervals of the treatment. E₂ receptor (E₂R) levels were not changed during the estrogen sequence, but were lowered by progesterone therapy in both cytosol and nuclear components. Progesterone receptor (PR) synthesis in cytosol was induced by exogenous estrogen. The total concentration of PR decreased with the uptake of P by the cell; meanwhile, the ratio of cytosol to nuclear P receptors declined. These data suggest that this sequential estrogen-progesterone regimen induces the changes in E₂R and PR patterns in the endometrium of ovariectomized monkeys which occurs due to ovarian cyclicity in the normal menstrual cycle.     

Absence of correlation between antiestrogenic activity and binding affinity for the estrogen receptor.

We have compared the binding to the estrogen receptor (R) of different androgens and antiestrogens with their antiestrogenic activities on uterine growth. We found that estradiol (E₂)¹ and hydroxytamoxifen, a potent antiestrogen, displayed the same affinity for R. Conversely, androgens which have a much lower affinity for R and a much higher dissociation rate than E₂, behave at high doses as full estrogens, with no significant antiestrogenic activity. We conclude that there is no correlation between the dissociation rate from R and the antiestrogenic activity of R ligands and that one cannot discriminate between estrogen and antiestrogen ligands by simply evaluating their $\text{in vitro}$ binding to the cytosol R.     

Antiestrogens: studies using an in vitro estrogen-responsive uterine system

Three antiestrogens have been tested $\text{in vitro}$ for their capacity to affect the cytosol binding and nuclear uptake of 17β-estradiol (E₂) and induction of the synthesis of a specific uterine protein (IP). CI-628 competes with E₂ for binding sites, inhibits IP induction, and is itself much less effective than E₂ in promoting the IP response; U-11, 100A is a binding competitor and a weak inducer of IP synthesis, but does not antagonize the E₂-induced IP response. Dimethylstilbestrol is an effective inhibitor of E₂ binding, elicits a high IP response, but does not antagonize E₂-induced IP synthesis. Higher concentrations of antiestrogens are required to inhibit nuclear binding of E₂ than expected from their relative binding ability to cytosol.     

Characterization and localization of prostaglandin E1 receptors in rat liver plasma membranes

Methodology for measurement and characterization of prostaglandin binding to membranes has been developed. The binding assay was used to study the presence of prostaglandin receptors in high purified cell fractions derived from rat liver. High affinity binding receptors which have a saturation value of 1.0 pmole/mg protein and a dissociation constant of 1.2 nM were found exclusively in the plasma membrane. High affinity receptors were not found in cell fractions containing nuclei, rough microsomes. Golgi complex or mitochondria. The binding by other prostaglandins was competitive with prostaglandin E₁. Competitive binding studies were used to obtain dissociation constants for prostaglandins F_1α, F_2α, B₁, B₂, A₁, A₂, and 15-keto prostaglandin E₂ which were 1100, 100, 300, 180, 16. 16 and 700 nM, respectively. Eicosa-5.8.11.19-tetraynoic acid, an inhibitor of prostaglandin synthesis did not bind appreciably to the prostaglandin E receptor, whereas two prostaglandin analogues, which have high physiological activity compete effectively with prostaglandin E₁ for the receptor. Thus, the binding receptor for the E-type prostaglandins is highly specific both with respect to cell localization as well as the type of substrate. Numerical routines for the fitting of the data and a procedure for the determination of the specific activity of the labelled prostaglandin are provided.     

Characterization of a cytosolic estrogen receptor and its up-regulation by 17β-estradiol and the xenoestrogen 4-tert-octylphenol in the liver of eelpout (Zoarces viviparus)

Estrogen binding activity was revealed in the cytosolic fraction of hepatic extracts from adult male and female eelpout (Zoarces viviparus). The binding moiety was characterized by a single class of high affinity binding sites (K_d=0.59±0.05 nM in males and 1.06±0.10 nM in females). The affinity was significantly higher in males. Binding sites were satiable and binding capacity was significantly elevated in vitellogenic females (2.92±0.28 pmol/g) compared to males (1.67±0.11 pmol/g). The binding was specific to known estrogens but not to other tested steroids. The binding moiety was able to bind to DNA–cellulose and was extractable by high salt concentrations. A time-course study of estrogen binding activity in liver cytosol and of vitellogenin (Vtg) in plasma, after intraperitoneal (i.p.) injections of 17β-estradiol (E₂) in male eelpout, was carried out. It was shown that both are inducible by E₂. Estrogen binding activity was significantly elevated 48 h and Vtg 72 h after E₂ treatment. The binding moiety was hereafter designated as a cytosolic estrogen receptor (ER). The estrogenicity of 4-tert-octylphenol (OP) was evaluated by measuring ER and Vtg after i.p. treatment. OP-treatment increased both receptor levels and Vtg concentrations in male fish, indicating that OP acts as an estrogen in male eelpout.     

The effects of long-term estrogen exposure on the induction of sexual behavior and measurements of brain estrogen and progestin receptors in the female rat

The purpose of this study was to investigate long-term effects of estradiol-17β (E₂) on sexual receptivity and to study the molecular basis for estrogen potentiated changes in receptivity. We therefore examined the long-term effects of E₂ on E₂ and progestin receptors in the hypothalamus-preoptic area-septum (HPS) and pituitary (PIT) of the female rat. Twenty-one days following ovariectomy, females received a 5-mm Silastic capsule of E₂ or cholesterol (C) for 1 week (pretreatment). Some animals were sacrificed for chemical analyses (i). The remainder had their capsules removed and 5 days later these animals either were sacrificed for chemical analyses (ii), or received E₂ (reimplantation). Forty-six hours after reimplantation, females either were sacrificed for chemical analyses (iii), or were tested for receptivity. When tested with sexually active males or by a manual stimulation method, animals pretreated with E₂ showed significantly better lordosis scores than animals pretreated with C. The tests for lordosis were carried out after administration of E₂ or E₂ + P to all subjects tested. During E₂ pretreatment, HPS and PIT cytosol progestin receptors increased significantly, while available estrogen receptor levels decreased significantly, as compared with C controls. Five days after E₂ pretreatment, HPS progestin receptor levels had decreased to the level observed in C controls, while PIT progestin receptors were slightly elevated. In HPS and PIT, levels of available E₂ receptor in E₂ and C pretreated animals were indistinguishable from each other. Neither the saturation capacity of the estrogen receptor nor the dissociation constant for binding [³H]E₂ was altered by E₂ pretreatment, as shown by Scatchard equilibrium analysis. Forty-six hours following E₂ reimplantation, progestin HPS and PIT receptor levels in E₂ and C pretreated animals were identical. Long-term potentiation of lordosis by E₂ does not result from a change in estrogen or progestin receptor dynamics in HPS of female rats.     

Autoradiographic Localization of 5-HT1E and 5-HT1F Binding Sites in Rat Brain: Effect of Serotonergic Lesioning

Abstract

5-carboxamidotryptamine (5-CT)-insensitive binding sites labelled by [³H]5-hydroxytryptamine (5-HT) in the presence of 100 nM 5-CT and 100 nM mesulergine, were examined by semi-quantitative autoradiography in rat brain. Under these conditions most of the labelled sites correspond to 5-HT_1E and 5-HT_1F sites. The 5-CT-insensitive binding is located mainly in cortical layer V, caudate-putamen, interpeduncular nucleus and claustrum. In cortex and caudate-putamen, a large proportion of 5-CT-insensitive sites is displaced by 250 nM sumatriptan and can be attributed to the presence of 5-HT_1F receptors. A low, but significant, level of displacement by sumatriptan was observed in the choroid plexus. Lesions of serotonergic neurones by intracerebroventricular 5,7-dihydroxytryptamine injection does not significantly modify the densities of 5-HT_1E or 5-HT_1F binding sites. Our findings suggest that the 5-HT_1F receptor has a limited distribution in rat brain, mainly located on non-serotonergic neurones.     

Simultaneous Measurements of Estrogen Nuclear (E2Rn) and Progestin Cytosol (PRc) Receptors in the Hypothalamus and Pituitary Gland of Rats

Abstract

The present methods used to measure estrogen nuclear (E₂Rn) and progestin cytosol (PRc) receptors in the hypothalamus and pituitary gland require that separate assays be performed to determine the concentrations of each receptor. In the present studies we describe a method which simultaneously measures both E₂Rn and PRc in hypothalamic and pituitary tissue. Tissue samples were homogenized in tris-EDTA-glycerol-dithiothreitol buffer and centrifuged at 1500 × g for 5 min. The supernatant was purified for the PRc assay while the nuclear pellet was extracted for the E₂Rn exchange assay.

For the PRc assay, the supernatant was centrifuged at 106,500 × g for 30 min and aliquots from the resultant supernatant then were incubated with ³H-R5020. For the E₂Rn exchange assay, the original pellet was purified further by successively resuspending and centrifuging it through several sucrose solutions. Estrogen-receptor complexes were extracted from the chromatin pellet with a 0.4M KC1 solution and aliquots of the final supernatant were incubated with ³H-estradiol.

In both assays, the samples were placed onto Sephadex LH20 columns and the receptor bound ³H-steroid was eluted directly into scintillation vials. Scatchard analyses revealed that these assays measure a single class of binding sites for E₂Rn and PRc with dissociation constants (K_D) and maximal number of binding sites (Bmax) similar to those previously reported using a separate assay for each receptor.     

Modulation of Progestin Binding Activity in Cultured Human Breast Carcinoma Cells: The Effect of Serum Type and Concentration

Abstract

Progesterone receptor levels in MCF-7 human breast cancer cells increase as a specific response to estrogen and to some nonsteroidal antiestrogens. In the present study we demonstrate that the type and quantity of serum present during culture of these cells modifies the level of progestin binding activity, but not the level of estradiol binding activity.

MCF-7 cells maintained in media supplemented with 5% charcoal-dextran treated calf serum (CDCS) contain 0.3 - 0.4 pmol of cytosol progesterone receptor (PR_c) per mg DNA. When cells previously maintained in 5% CDCS-media are shifted to media containing 5% charcoal-dextran treated fetal calf serum (CDFCS), the level of progestin binding increases after day 16, and stabilizes at 2 - 3 pmol/mg DNA at days 30 to 40. Shifting these cells back to 5% CDCS-media, reduces PR_c to 0.2 - 0.4 pmol/mg DNA within 3 days. This reduction is dose dependent with a half-optimal decrease at 1% CDCS, and a full decrease at 2% CDCS (4d incubation). Nuclear progestin binding was uniformly low (0.2 - 0.4 pmol/mg DNA) and unaffected by type or concentration of serum, and no consistent change in cytosol or nuclear estrogen receptor levels was observed. These cytoplasmic progestin binding sites are translocated to the nucleus by progesterone, and are similar to estradiol (E₂) induced sites by Scatchard binding and sucrose gradient analysis. Similar serum-dependent changes are also observed in the T₄₇D human breast cancer cell line where growth in CDFCS-media results in 4-fold higher progestin binidng levels than observed in CDCS-media. Our findings suggest the presence of non-dialyzable stimulatory factor(s) in CDFCS that influence the progestin receptor level the highlight the fact that serum components can alter dramatically the cellular progestin binding activity.     

Binding of [H]methyltrienolone to androgen receptor in rat liver

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [³H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the K_d value was 3.3 · 10⁻⁸ M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the K_d value was 2.5 · 10⁻⁸ M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to of that in the untreated cytosol. The profile of glycerol gradient centrifiguration indicated that [³H]methyltriemolone-bound receptor migrated in the 8–9 S region in both untreated and triamcinolone-blocked cytosols, but the 8–9 S peak in triamcinolone-blocked cytosol was reduced to about of that of untreated cytosol.     

Kinetic Analysis of Estrogen and Antiestrogen Competition for Hypothalamic Cytosol Estrogen Receptors. Evidence for Noncompetitive Ligand-Receptor Interactions

Abstract

The binding of (³H)-estradiol (E₂) to cytoplasmic estrogen receptors isolated from female rat hypothalamus-preoptic area by unlabeled estrogen and antiestrogens (nafoxidine and tamoxifen) was examined when the concentrations of both the agonist (³H)-E₂) and unlabeled competitors were varied over a wide range. Concentrations of unlabeled E₂ up to 10^-10M decreased the affinity of the labeled steroid for its receptor; at higher E₂ concentrations, the apparent number of binding sites (B_max) began to decline. Thus at some concentrations, E₂ may act as a mixed competitive and noncompetitive inhibitor of its own binding to hypothalamic cytosol receptors. The antiestrogens differed markedly from E2 in their interactions with hypothalamic estrogen receptors. Only at relatively high competitor concentrations (e.g., > 10^-9M) did the antagonists appear to competitively inhibit (³H)-E₂-receptor binding. The most striking observation was that tamoxifen and nafoxidine significantly inhibited (³H)-E₂-receptor binding at very low competitor concentrations (e.g., 1 pM), but only slightly inhibited estrogen binding at intermediate concentrations (e.g., 10^-10M). It was proposed that the non-linear, concentration-dependent effects of antiestrogens on the neural estrogen receptors might be due to complex interactions of the antagonists with a non-estrogen binding site.     

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.     

Binding of [3H]methyltrienolone to androgen receptor in rat liver

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [³H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the K_d value was 3.3 · 10^?8 M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the K_d value was 2.5 · 10^?8 M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to $\text{3}\text{4}$ of that in the untreated cytosol. The profile of glycerol gradient centrifiguration indicated that [³H]methyltriemolone-bound receptor migrated in the 8–9 S region in both untreated and triamcinolone-blocked cytosols, but the 8–9 S peak in triamcinolone-blocked cytosol was reduced to about $\text{3}\text{4}$ of that of untreated cytosol.     

Oligodeoxynucleotide base recognition by steroid hormone receptors

Oligodeoxynucleotides covalently linked to cellulose were used as probes of the DNA-binding domains of mouse steroid holoreceptors. With uterine cytosol estrogen receptor (E₂R) the relative binding order, in prior studies, was oligo(dG) > oligo(dT) ≧ oligo(dC) > > oligo(dA) > oligo(dI). The binding reactions were salt-sensitive with an optimal KCl concentration of 0.1–0.2 M. There was no enhancement of binding by activation, either temperature- or salt-induced. In the present study, using the oligomer ligands at a lower concentration, oligo(dT) binding was greater than that to oligo(dC). Quantitative differences in oligodeoxynucleotide binding were elicited by a number of inhibitors. These differences are again seen by exposure of E₂R to chaotropic salts such as SCN^?, ClO₄^? and NO₃^? as well as to putative modifiers of receptor amino acids, ie, iodoacetamide, 1,2 cyclohexanedione, and Rose Bengal. These results, and the quantitative differences following heat and purification, led to a designation of two types of subsites within the DNA-binding domain of uterine E₂R. These are stable G sites, which interact with oligo(dG); and labile N sites, which bind to oligo(dT), oligo(dC) and oligo(dA). Stimulation of binding to N sites and stabilization of the holoreceptor was effected by histones H2A and H2B. However, the differential response to incubation at 37°C was not altered by addition of H2B. Treatment of uterine E₂R by limited proteolysis also eliminated the stimulatory response to H2B. The above data, as well as prior studies, indicate that steroid holoreceptors can discriminate between the structural features of deoxynucleotide bases and this recognition process can be modulated by accessory proteins.     

A novel effect of molybdate on the binding of [3H]aldosterone to gel-filtered type I receptors in brain cytosol

Recently we reported that adding molybdate to crude steroid-free cytosol at 0°C results in a dose-dependent reduction in the binding of [³H]aldosterone ([³H]ALDO), to Type I adrenocorticosteroid receptors. In the experiments outlined here, we found that addition of molybdate to steroid-free brain cytosol produces a 30–50% increase in the subsequently measured maximal specific binding capacity (B _MAX) of [³H]ALDO-Type I receptors if the cytosol is subjected to Sephadex G-25 gel filtration prior to steroid addition. These manipulations were found to have no effect on the equilibrium dissociation constant (K _d) of the receptors. In contrast, when gel filtration of steroid-free cytosol was performed in the absence of molybdate, there was a 2-fold increase in the K_d and over a 50% reduction in the subsequently measuredB _MAX of [³H]ALDO-Type I receptors. When molybdate was added to this steroid-free cytosol immediately following gel filtration, there was no reduction (or increase) in Type I receptor [³H]ALDO binding capacity compared with nongel-filtered controls. The addition of as little as 2 mM molybdate to crude steroid-free cytosol was found to stabilize the binding capacity of Type I receptors during exposure to 22°C incubations; however, when gel-filtered steroid-free cytosol was exposed to these conditions at least 10 mM molybdate was required to stabilize Type I receptor binding capacity. Adding the sulfhydryl reducing reagent, dithiothreitol, to the various steroid-free cytosols had little effect on [³H]ALDO-Type I receptor binding. The effects of molybdate, revealed in this study, on Type I receptors in brain cytosol subjected to gel filtration are clearly different from those seen with receptors in crude cytosol preparations, as well as from those reported in the literature for other steroid receptors. Possible mechanisms of action of molybdate on unoccupied Type I receptors in crude and gel-filtered cytosol are discussed.     

Prostaglandin E1 binding and adenylate cyclase activation in normal and transformed fibroblasts

The binding of [³H]prostaglandin E₁ to membranes of clones of normal rat kidney fibroblasts (NRK cells) has been measured. Cell lines that responded to prostaglandin E₁, such as NRK and NRK transformed with Schmitt-Ruppin strain of Rous sarcoma virus (SR-NRK cells), have a high affinity prostaglandin E₁ binding site. Murine-sarcoma-virus-transformed lines of NRK cells are unresponsive to prostaglandin E₁ and have reduced prostaglandin E₁ binding. Exposure of cells to prostaglandin E₁ results both in decreases prostaglandin E₁ responsiveness and reduced prostaglandin E₁ binding.Activation of adenylate cyclase is correlated to binding of prostaglandin E₁ to receptors in both NRK and SR-NRK cell membranes. Mathematical models suggest that GTP decreases the affinity of hormone for its receptor while increasing the catalytic efficiency of adenylate cyclase, and that aggregates of occupied receptors may play an important role in the activation of adenylate cyclase.     

Further characterization of the estrogen binding macromolecule in human pancreas

The estrogen binding protein in human pancreas has been purified from pancreatic cytosol by chromatography on Concanavalin-A-Sepharose and hydroxyl-apatite followed by ion-exchange chromatography carried out using a fast-protein liquid chromatography apparatus (FPLC). The purified protein, still able to bind labelled [3H]estradiol, appeared as one single band corresponding to 31 K in SDS-gel electrophoresis. Total amino acid analysis revealed high levels of histidine, glutamic acid and leucine. The capacity of the purified protein to bind estrogens could be increased more than 4-fold by addition of a cytosolic factor, probably being a small peptide, that is present in crude cytosol, but lost during the purification procedure. The iodinated protein does not bind to DNA-cellulose or phosphocellulose, and shows no similarities to estrogen receptor proteins.     

Prostaglandin E2, prostaglandin E1 and thromboxane B2 release from human monocytes treated with C3b or bacterial lipopolysaccharide

C3b or lipopolysaccharide treatment of human peripheral blood monocytes in culture stimulates an early release of thromboxane B₂ and a delayed release of prostaglandin E into culture supernatants. Immunoreactive thromboxane B₂ release is maximal from 2–8 h, whereas prostaglandin E release is maximal from 16–24 h after stimulation of monocytes in culture. We further examined this process by comparing the time course of labelled prostaglandin E₂, prostaglandin E₁ and thromboxane B₂ release from human monocytes which were pulse or continuously labelled with [³H]arachidonic acid and [¹⁴C]eicosatrienoic acid. The release of labelled eicosanoids was compared with the release of immunoreactive prostaglandin E and thromboxane B₂. The time course of prostaglandin E₂ release was virtually identical to the release of prostaglandin E₁ in all culture supernatants regardless of labelling conditions. However, release of immunoreactive prostaglandin E paralleled the release of labelled prostaglandin E₁ and E₂ only for continuously labelled cultures. The release of labelled prostaglandin E₁ and E₂ from pulse labelled cultures paralleled the release of thromboxane B₂ and not immunoreactive prostaglandin. In contrast, labelled and immunoreactive thromboxane B₂, quantitated in the same culture supernatants, demonstrated similar release patterns regardless of labelling conditions. These findings indicate that the differential pattern of prostaglandin E and thromboxane B₂ release from human monocytes is not related to a time-dependent shift in the release of prostaglandin E₁ relative to prostaglandin E₂. Because thromboxane B₂ and prostaglandin E₂ are produced through cyclooxygenase mediated conversion of arachidonic acid, these results further suggest that prostaglandin E₂ and thromboxane B₂ are independently metabolized in human monocyte populations.     

Heterogeneity of steroid hormone receptor in adult rat lung cytosol

Chromatography on cellulose DEAE-52 columns revealed that the glucocorticoid receptor from rat lung cytosol consisted of a component in the 0.001 M prewash, revealed with synthetic steroids and natural mineralocorticoids, a second component eluted with 0.04 M PO₄, labelled with triamcinolone, dexamethasone, and a third moiety in the 0.06 M PO₄ region, evident with natural glucocorticoids (corticosterone, cortisol) as well as mineralocorticoids (aldosterone, deoxycorticosterone). The thrid component coelutedf with rat blood serum transcortin in double labelled experiments. Rat lung was devoid of another component in the 0.02 M PO₄ found in rat liver supernate and of the mineralocorticoid receptor evident only in rat kidney. Chromatography on Sephadex G-200 columns revealed a shift of radioactivity from a higher to a lower molecular weight region in the presence of 0.4 M KCl. Collectively, these studies indicate the subunit nature of the lung receptor as evidenced in most tissues hitherto tested. Moreover, polymorphism within a given subunit component can not be revealed by competition alone, as attempted by others, but can be revealed under selected conditions of physical separation.