Interaction of rat liver glucocorticoid receptor with lectins: is the glucocorticoid receptor a glycoprotein?

Although glucocorticoid receptors have been extensively studied in a variety of tissues, the precise nature of the receptor protein still remains unknown. To further characterize this protein we assessed the effects of various lectins on [3H]dexamethasone binding to prepurified preparations of rat liver glucocorticoid receptor. Among the lectins tested only Ulex europeus and Lens culinaris induced a concentration-related decrease in [3H]dexamethasone binding. Following Ulex europeus or Lens culinaris exposure Scatchard analysis showed that these lectins led to a 3-fold reduction in receptor affinity without influencing the concentration of binding sites. These results provide new experimental evidence that rat liver glucocorticoid receptor would possess alpha-L-fucosyl and alpha-D-mannopyranoside residues in close proximity to the glucocorticoid receptor binding domain.     

The glucocorticoid antagonist 17 alpha-methyltestosterone binds to the 10 S glucocorticoid receptor and blocks agonist-mediated dissociation of the 10 S oligomer to the 4 S deoxyribonucleic acid-binding subunit

The glucocorticoid antagonist 17 alpha-methyltestosterone inhibits binding of the agonist [3H]triamcinolone acetonide ot the glocucorticoid receptor in cytosol prepared from rat pituitary tumor GH1 cells. Competitive binding studies indicate that the dissociation constant for 17 alpha-methyltestosterone is about 1 microM. After incubation of intact GH1 cells with 10 nM [3H]triamcinolone acetonide at 37 C and subsequent cell fractionation at 4 C, three glucocorticoid receptor forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent incubation with 17 alpha-methyltestosterone reduces the amount of [3H]triamcinolone acetonide bound to each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with cytosolic 10 S receptor. Immunochemical analysis using monoclonal antibody BuGR2 indicates that 17 alpha-methyltestosterone does not cause substantial accumulation of glucocorticoid receptors in GH1 cell nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17 alpha-methyltestosterone does not reduce the total amount of cellular glucocorticoid receptor and does not reduce receptor half-life. These results are consistent with a model for glucocorticoid receptor transformation in which binding of agonist promotes the dissociation of an oligomeric 10 S cytosolic receptor protein to its DNA-binding 4 S subunit. The antagonist 17 alpha-methyltestosterone competes with agonist for binding to the 10 S cytosolic receptor but does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[3H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90,000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [3H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [3H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37 degrees C. SDS-polyacrylamide gel electrophoresis of [3H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37 degrees C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei as well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Characterization of glucocorticoid receptor in HeLa-S3 cells

Glucocorticoid receptor of the human cell line HeLa-S3 has been characterized and has been compared to rat and to mouse glucocorticoid receptors. If HeLa cells were lysed in the absence of glucocorticoid, glucocorticoid receptor was isolated in a nonactivated form, which did not bind to DNA-cellulose. If HeLa cells were preincubated with glucocorticoid, glucocorticoid receptor was isolated in an activated, DNA-binding form. HeLa cell glucocorticoid receptor bound [3H]triamcinolone acetonide with a dissociation constant (KD = 1.3 nM at 0 degrees C) that was similar to those of mouse and rat glucocorticoid receptors. Similarly, the relative binding affinities for steroid hormones decreased in the order of triamcinolone acetonide greater than dexamethasone greater than promegestone greater than methyltrienolone greater than aldosterone greater than or equal to moxestrol. Nonactivated and activated receptors were characterized by high-resolution anion-exchange chromatography (FPLC), DNA-cellulose chromatography, and sucrose gradient centrifugation. Human, mouse, and rat nonactivated glucocorticoid receptors had very similar ionic and sedimentation properties. Activated glucocorticoid receptors were eluted at similar salt concentrations from DNA-cellulose columns but at different salt concentrations from the FPLC column. A monoclonal mouse anti-rat liver glucocorticoid receptor antibody [Westphal, H.M., Mugele, K., Beato, M., & Gehring, U. (1984) EMBO J. 3, 1493-1498] did not cross-react with HeLa cell glucocorticoid receptor. Glucocorticoid receptors of HeLa, HTC, and S49.1 cells were affinity labeled with [3H]dexamethasone and with [3H]dexamethasone 21-mesylate. The molecular weights of [3H]dexamethasone 21-mesylate labeled glucocorticoid receptors (MT 96 000 +/- 1000) were undistinguishable by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of corticosterone, 5alpha-dihydrocorticosterone and dexamethasone with proteins in rat-liver cytosol.

The intracellular binding of [3H]corticosterone and [3H]dexamethasone and their metabolites to macromolecules in rat liver cytosol was studied in vivo and in vitro. The macromolecules binding corticosterone and its metabolites were characterized as (a) a steroid conjugate-binding (Stokes radius 2.5 nm and sedimentation coefficient 4.1 S in high ionic strength; pI 8.7, (b) transcortin and (c) a glucocorticoid "receptor". Competition experiments indicate that corticosterone and dexamethasone bind to the same site of the glucocorticoid receptor molecule. Different Stokes radii between the corticosterone-receptor and the dexamethasone-receptor complexes (6.9 and 6.3 nm, respectively, in high ionic strength) indicate that the two ligands induce different conformations of the receptor protein. This may be of importance when explaining the qualitative differences between the cellular effects of natural and synthetic glucocorticoids. 5alpha-Dihydrocorticosterone, on the other hand, competed to a very limited extent with dexamethasone for binding sites on the receptor. An assay of the inductive effect on liver tyrosine aminotransferase and tryptophan oxygenase indicated that 5alpha-dihydrocorticosterone was practically devoid of glucocorticoid activity. It is concluded that 5alpha-dihydrocorticosterone probably does not act as the mediator of corticosterone action in rat liver.     

Binding of dexamethasone to rat liver nuclei in vivo and in vitro: evidence for two distinct binding sites

The binding of [3H]dexamethasone (DEX) to rat liver nuclei in vitro and in vivo have been compared. In vitro, purified nuclei displayed a single class of specific glucocorticoid binding sites with a dissociation constant (Kd) of approximately 10(-7) M for [3H]DEX at 4 degrees C. The glucocorticoid agonists prednisolone, cortisol, and corticosterone and the antagonists progesterone and cortexolone competed avidly for this site, but the potent glucocorticoid triamcinolone acetonide (TA) competed poorly in vitro. Nuclei isolated from the livers of intact rats contained 1-2 X 10(4) [3H]DEX binding sites/nucleus. Up to 85% of the binding sites were recovered in the nuclear envelope (NE) fraction when NE were prepared either before or after labeling with [3H]DEX in vitro. After adrenalectomy, the specific [3H]DEX binding capacity of both nuclei and NE decreased to 15-20% of control values, indicating sensitivity of the binding sites to hormonal status of the animals. Efforts to restore the binding capacity by administration of exogenous glucocorticoids, however, were unsuccessful. After labeling of rat liver nuclei in vivo by intraperitoneal injection of [3H]DEX or [3H]TA into living animals, the steroid specificity and subnuclear localization of radiolabel were different. Both [3H]TA (which did not bind in vitro) and [3H]DEX became localized to nuclei in a saturable fashion in vivo. With either of these ligands, approximately 20% of the total nuclear radiolabel was recovered in the NE fraction. These results suggest the presence of two separate and distinct binding sites in rat liver nuclei, one which is localized to the NE and binds [3H]DEX (but not [3H]TA) in vitro, and another which is not localized to the NE but binds [3H]DEX and [3H]TA in vivo.     

Characterization of an antiserum against the glucocorticoid receptor

An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor from rat liver cytosol contained specific antibodies to glucocorticoid receptor. This was shown following incubation of the [³H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [³H]TA-GR-antibody complex to protein A linked to Sepharose, (II) an increased sedimentation rate of the [³H]TA-GR-antibody complex compared to that of the [³H]TA-GR complex, and (III) an increased molecular size of the [³H]TA-GR-antibody complex when compared to that of the [³H]TA-GR complex as judged from gel filtration. The antibody fraction was characterized with regard to titer, cross-reactivity and specificity. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus), as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. In the rat liver, the antibody bound to both the nuclear and the cytosolic glucocorticoid receptor (Stokes radius 6.1 nm). It did not cross-react with the proteolytic fragments of the glucocorticoid receptor, the 3.6 nm complex or the 1.9 nm complex. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat to rat serum transcortin. With an indirect competitive ELISA (enzyme-linked immunosorbent assay) combined with various separation techniques, based on different physiocochemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. These findings also indicate an immunochemical similarity between glucocorticoid receptors in different tissues as well as in different species, but not between glucocorticoid receptors and other steroid hormone receptor proteins. The cytosolic and nuclear glucocorticoid receptors in rat liver were shown to be immunochemically similar.     

Inhibition by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, of enzyme induction by glucocorticoid and of nuclear translocation of glucocorticoid-receptor complexes

Induction of tyrosine aminotransferase by glucocorticoid in rat hepatocytes was inhibited concentration-dependently by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, but not by N- [2-(methyl-amino)-ethyl]-5-isoquinolinesulfonamide dihydrochloride, an inhibitor of cyclic nucleotide dependent protein kinases. H-7 also inhibited the accumulation of glucocorticoid-receptor complexes in the nuclear fraction with associated accumulation of these complexes in the cytoplasmic fraction, but did not affect incorporation of glucocorticoid into hepatocytes. These results indicate that protein kinase C may be essential in translocation of glucocorticoid-receptor complexes to the nuclei.     

Phosphorylation of immunopurified rat liver glucocorticoid receptor by the catalytic subunit of cAMP-dependent protein kinase

We have examined phosphorylation of the rat liver glucocorticoid receptor (GR) and GR-associated protein kinase (PK) activity in the immunopurified receptor preparations. Affinity labeling of hepatic cytosol with [³H]dexamethasone 21-mesylate showed a covalent association of the steroid with a 94 kDa protein. GR was immunopurified with antireceptor monoclonal antibody BuGR2 (Gametchu & Harrison, Endocrinology 114: 274–279, 1984) to near homogeneity. A 23° C incubation of the immunoprecipitated protein A-Sepharose adsorbed GR with [-³²P]ATP, Mg²⁺ and the catalytic subunit of cAMP-dependent PK (cAMP-PK) from bovine heart, led to an incorporation of radioactivity in the 94 kDa protein. Phosphorylation of GR was not evident in the absence of the added kinase. Of the radioinert nucleotides (ATP, GTP, UTP or CTP) tested, only ATP successfully competed with [-³²P]ATP demonstrating a nucleotide specific requirement for the phosphorylation of GR. Other divalent cations, such as Mn²⁺ or Ca²⁺, could not be substituted for Mg²⁺ during the phosphorylation reaction. Phosphorylation of GR was sensitive to the presence of the protein kinase inhibitor, H-8, an isoquinoline sulfonamide derivative. In addition, the incorporation of radioactivity into GR was both time- and temperature-dependent. The phosphorylation of GR by cAMP-PK was independent of the presence of hsp-90 and transformation state of the receptor. The results of this study demonstrate that GR is an effective substrate for action of cAMP-PK and that the immunopurified protein A-Sepharose adsorbed GR lacks intrinsic kinase activity but can be conveniently used for the characterization of the phosphorylation reaction in the presence of an exogenous kinase.Abbreviations BUGR2 anti-GR monoclonal antibody - cAMP-PK cAMP-dependent protein kinase - DMSO dimethyl sulfoxide - EDTA ethylenediamine tetra acetic acid - GR glucocorticoid receptor - H-8 Isoquinoline sulfonamide derivative - hsp-90 90 kDa heat-shock protein - PMSF phenylmethylsulfonyl fluoride - PR progesterone receptor - NaF sodium fluoride - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - SR steroid receptor - TA triamcinolone acetonide     

Aflatoxin inhibition of glucocorticoid finding capacity of rat liver nuclei.

The effect of aflatoxin B1 on the binding capacity of rat liver cytoplasmic glucocorticoid receptors and the nuclear binding of the activated receptor complex was investigated. No alterations in the kinetics of [3H]desamethasone-cytosol receptor complex formation were noted 2 h after treatment with 1 mg/kg aflatoxin B1. However, a 33% decrease in the concentration of nuclear acceptor sites and a 24% decrease in the glucocorticoid receptor-nuclear binding equilibrium constant of dissociation was observed. This response was near maximal at 2 h and persisted for at least 26 h. Inhibition of nuclear binding capacity was directly related to aflatoxin B1 dose, with a correlation coefficient of 0.99. Actinomycin D treatment (0.1 mg/kg) resulted in a slight reduction (16%) in the concentration of nuclear acceptor sites but had no effect on the nuclear binding dissociation constant. Administration of [3H]dexamethasone to alfatoxin B1 -treated rats produced a similar pattern of glucocorticoid binding distribution in vivo to that observed in vitro. No differences in [3H]dexamethasone-cytoplasmic receptor binding between control and alfatoxin B1 -treated rats were found, whereas nuclear [3H]dexanthasone binding was reduced 34% by alfatoxin B1 -treatment.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[³H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90 000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [³H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [³H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37°C. SDS-polyacrylamide gel electrophoresis of [³H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37°C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei aas well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Glucocorticoid--receptor interactions. Studies of the negative co-operativity induced by steroid interactions with a secondary, hydrophobic, binding site.

The effects of steroids on the binding of [1,2-3H]dexamethasone and [1,2-3H]progesterone to the glucocorticoid receptor of rat thymus cytosol were studied. Although both glucocorticoid agonists and antagonists competed with [1,2-3H]dexamethasone for binding to the receptor under equilibrium conditions, only glucocorticoid antagonists of partial agonists, at micromolar concentrations, were capable of accelerating the rate of dissociation of previously bound [1,2-3H]dexamethasone from the receptor. Antagonists or partial agonists also enhanced the rate of dissociation of [1,2-3H]progesterone from the glucocorticoid receptor, with identical specificity and concentration--response characteristics. These effects are attributed to the presence on the receptor of a secondary, low-affinity, binding site for glucocorticoid antagonists, the occupancy of which produces negatively co-operative interactions with the primary glucocorticoid-binding site. In contrast with the interactions with the primary site, the interactions of steroids with the negatively co-operative site appear to be primarily hydrophobic in nature, and the site resembles the steroid-binding site of progestin-binding proteins in its specificity, though not its affinity. The results also suggest that the initial interactions of both glucocorticoid agonists and antagonists with the receptor under equilibrium conditions are with one primary site on a receptor existing in one conformation only.     

Characterization of cortisol binding sites in chicken liver plasma membrane

1. The presence of sites specifically binding [3H]cortisol in plasma membrane isolated from chicken liver has been determined. The kinetic parameters of this binding are: Kd = 4.5 nM and Bmax = 2225 fmol/mg protein in presence of 10(-6) M progesterone. 2. The affinities of several natural and synthetic steroids for the membrane binding site respect to the binding of 4 nM [3H]cortisol without competitor increased in the following order: Testosterone less than pregnenone less than dexamethasone less than progesterone less than prednisolone less than corticosterone less than deoxycorticosterone. 3. Other steroids such as estradiol, ouabain and triamcinolone acetonide does not bind to the plasma membrane. 4. Metal ions such as Ca2+ and Mg2+ did not modify the binding of [3H]cortisol. 5. Neither propranolol nor phentolamine, beta- and alpha-adrenergic antagonists affected [3H]cortisol binding to the plasma membranes. 6. The result suggest that the binding site detected is more specific for glucocorticoids and it is different of nuclear glucocorticoid receptor and progesterone receptor.     

Propranolol stimulates histone phosphorylation by a putative PK-C in partially purified homogenate of rat testicular interstitial cells. A possible mechanism for increased testosterone secretion by propranolol.

The beta-adrenoceptor blocker propranolol stimulated testosterone secretion by rat testicular interstitial cells (Leydig cell-enriched preparation) in vitro at concentrations ranging from 10(-5) M to 10(-4) M. Treatment of these cells with H7 (20 microM), an inhibitor of protein kinase C, reduced the stimulatory effect of L-propranolol on testosterone secretion by about 5-fold. At concentrations ranging from 31.25 microM to 1000 microM, L-propranolol reduced [3H]phorbol 12,13-dibutyrate binding (IC50 = 75 microM) to rat testicular interstitial cells. At similar concentrations, L-propranolol displaced the binding of [3H]phorbol 12,13-dibutyrate to the homogenate of these cells by only 5%. These findings suggest that the effect of L-propranolol on [3H]phorbol 12,13-dibutyrate binding could be indirect, possibly by increasing the concentration of a chemical mediator interacting with the regulatory domain of protein kinase C. At even lower concentrations (10(-9) M to 10(-7) M), propranolol added directly to the reaction mixture with protein kinase C partially purified from rat testicular interstitial cells increases the phosphorylation of histone. This phosphorylation was comparable to that obtained with (25 microg/ml) phosphatidylserine. The D- and L-stereoisomers of propranolol were equally active. A complete reversal of this propranolol effect on histone phosphorylation was achieved with (20 microM) H-7. In the absence of Ca2+, propranolol was not able to phosphorylate the histone. Taken together, these results suggest that protein kinase C could be the putative kinase involved in this reaction and that its activation by propranolol may be due to interaction of the drug with the regulatory domain of the enzyme at a site differing from the site of interaction with phorbol 12,13-dibutyrate. The ability of propranolol to activate the putative protein kinase C could be related to its stimulatory effect on testosterone secretion by Leydig cells.     

Preparation of an anti-idiotypic antibody directed against anti-dexamethasone antibody and demonstration of its ability to acknowledge the receptor of glucocorticoids

By using a 3-carboxymethyloxime dexamethasone derivative coupled to bovine serum albumin we have prepared specific anti-dexamethasone antibodies in rabbits. These antibodies were then purified by affinity chromatography and administered to a second set of rabbits. One of them produced anti-idiotypic antibodies able to impede the [3H] dexamethasone binding of the initial anti-dexamethasone antibodies and to displace the [3H] dexamethasone-antibodies complexes towards high molecular weight species in gel filtration experiments. Moreover these antibodies we were also able to impede the binding of [3H] dexamethasone to the rat liver glucocorticoid receptor and to recognize the highly purified receptor using Western blot analysis.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

A study of the cytoplasmic receptors for glucocorticoids in intestine of pre- and postweanling rats.

Glucocorticoids cause both enzymic and morphologic changes in the rat intestine during the time of weaning. To obtain information regarding the mechanism of these actions, we examined the cytoplasmic fraction of intestines from 18-day-old rats for the presence of specific glucocorticoid-binding proteins which are characteristics of target tissues. Incubation of slices of intestine with [3H]dexamethasone in a physiological medium at 2 degrees showed the presence of a cytoplasmic binding macromolecule with high specificity for steroids having glucocorticoid activity. The binding reaction was saturable (concentration of binding sites equals 0.24 pmol per mg of protein) and of high affinity (dissociation constant equals 9.3 nM). Binding was reversible on addition of nonlabeled dexamethasone (t 1/2 equals 5.2 hours), indicating that the usual assay procedure measured both corticosterone-filled and unoccupied binding sites. Sucrose density gradient centrifugation showed that the receptor-dexamethasone complex from intestinal cytosol sedimented at the same rate as that from liver (8.2 S). The receptor-dexamethasone complex was stable at 2 degrees for at least 24 hours in intestinal slices, but in isolated cytosol fractions there was considerable loss of binding even in the presence of high concentrations of [3H]dexamethasone. Furthermore, mixing experiments showed that the presence of cytosol from intestinal mucosa (but not from the muscle layers) caused a dissociation of dexamethasone from receptors of liver cytosol. This suggested the presence of some interfering factor in isolated mucosal cytosol and meant that quantitative studies had to be confined to intact slices. Although the reasons for the instability of steroid-receptor complexes in the presence of isolated intestinal cytosol are not understood, the instability is believed to be associated with homogenization and, therefore, is believed to have no physiological significance. Finally, the ontogenesis of cytoplasmic glucocorticoid receptors in intestinal slices was examined and the pattern compared with that in liver and lung. Receptor activity was present in intestine from late fetal life through adulthood, but concentrations were significantly higher during the first two postnatal weeks than at all other times. By contrast, receptor activity detected in cytosol prepared from rat lung was high around the time of birth, while that in liver rose steadily during the first postnatal week and remained at high levels. Thus specific receptors for glucocorticoids are present in the rat intestine during periods of both responsiveness and unresponsiveness. This suggests that although corticosteroids exert their effects through the cytoplasmic receptors, this early event in glucocorticoid action may not be a controlling step for changes in responsiveness during development.     

Arachidonic acid release by basophilic leukemia cells and macrophages stimulated by Ca2+ ionophores, antigen and diacylglycerol: essential role for protein kinase C and prevention by glucocorticosteroids

The role of protein kinase C in phospholipase A2 (PLA2) activation in rat basophilic leukemia cells (RBL-2H3) and macrophages was investigated. 12-O-Tetradecanoyl phorbol 13-acetate (TPA) doubled ionomycin-induced PLA2 activity, assessed by [3H]arachidonate release. Protein kinase C inhibitors, staurosporine and K252a (100 nM) or H-7 (15 micrograms/ml) inhibited ionomycin-stimulation of PLA2 activity by 62, 75 and 80%, respectively. Down-regulation of protein kinase C by prolonged treatment with TPA inhibited Ca2(+)-ionophore A23187 or antigen-stimulation of [3H]arachidonate release by 80%. We examined whether the inhibitory effect of dexamethasone (DEX) on PLA2 activity is related to modulation of protein kinase C activity. The 50% inhibition by DEX of ionomycin elevation of [3H]arachidonate release was almost overcome by addition of TPA. The Ca2+ ionophore and antigen-induced increase in [3H]TPA binding to intact RBL cells was not impaired by DEX. However, DEX markedly reduced phosphorylation of several proteins. 1-Oleoyl-2-acetyl-glycerol (OAG) had a sustained stimulatory effect on PLA2 activity in isolated plasma membranes derived from treated bone-marrow intact mouse macrophages, while both DEX and staurosporine reduced elevated PLA2 activity by 68 and 84%, respectively. The results support an essential role for protein kinase C in regulation of PLA2 activity.     

Aflatoxin inhibition of glucocorticoid binding capacity of rat liver nuclei

The effect of aflatoxin B₁ on the binding capacity of rat liver cytoplasmic glucocorticoid receptors and the nuclear binding of the activated receptor complex was investigated. No alterations in the kinetics of [³H]dexamethasonccytosol receptor complex formation were noted 2 h after treatment with 1 mg/kg aflatoxin B₁. However, a 33% decrease in the concentration of nuclear acceptor sites and a 24% decrease in the glucocorticoid receptor-nuclear binding equilibrium constant of dissociation was observed. This response was near maximal at 2 h and persisted for at least 36 h. Inhibition of nuclear binding capacity was directly related to aflatoxin B₁ dose, with a correlation coefficient of 0.99. Actinomycin D treatment (0.1 mg/kg) resulted in a slight reduction (16%) in the concentration of nuclear acceptor sites but had no effect on the nuclear binding dissociation constant.Administration of [³H]dexamethasone to aflatoxin B₁-treated rats produced a similar pattern of glucocortocoid binding distribution in vivo to that observed in vitro. No differences in [³H]dexamethasone-cytoplasmic receptor binding between control and aflatoxin B₁-treated rats were found, whereas nuclear [³H]dexamethasone binding was reduced 34% by aflatoxin B₁ treatment.     

Occurrence of glucocorticoid binding sites in solubilized microsomes from rat liver

Recent studies suggested the presence of specific glucocorticoid binding sites on rat liver microsomal membranes. We report here a new solubilization procedure which allows the physicochemical characterization of the microsomal glucocorticoid binding sites. Solubilization was achieved with 2 mM CHAPS in the presence of 5 mM benzamidine. Binding of [3H]cortisol showed a high affinity (Kd = 5.1 x 10(-9) M) and a limited capacity (0.72 pmol/mg of protein). The binding activity was abolished by elevated temperature and pronase. Competition experiments revealed that natural glucocorticoids and progesterone were highly potent competitors whereas dexamethasone and triamcinolone acetonide did not compete. Chromatography on DEAE Trisacryl and heparin Ultrogel confirmed that the solubilized protein is different from corticosteroid binding globulin and the cytosolic glucocorticoid receptor. Treatment of microsomal fractions with phosphatidyl inositol phospholipase C promoted the release of specific binding activity suggesting a putative glycosylphosphatidyl anchor for this protein. This finding may have interesting implications concerning the mechanism of glucocorticoid hormone action.