Steroid induction of low-affinity glucocorticoid binding sites in rat liver microsomes

Rat liver contains two glucocorticoid binding sites: the high-affinity or glucocorticoid receptor (GR) and the low-affinity glucocorticoid binding sites, or LAGS. The Kd of LAGS predicts that they can be half-saturated by plasma corticosteroids in some physiological circumstances and, therefore, that they can play relevant roles in the rat liver. [3H]dexamethasone was used as a ligand in exchange assays, to study the relative abundance of GR and LAGS in cell fractions of rat liver. GR were found in the cytosol, but not in the purified nuclei, the mitochondria, or the microsomes. LAGS were found in all the particulate fractions, being more abundant in the smooth-surfaced microsomes, but they were not found in the cytosol. The LAGS of microsomes and purified nuclei showed the same Kd and also the same broad range of steroid competition with [3H]dexamethasone (cortisol = progesterone greater than dexamethasone greater than or equal to corticosterone greater than R5020 greater than DHEA greater than testosterone = estradiol). LAGS were found in liver, placenta and kidney, but not in other GR-containing organs. This suggests that the LAGS could be involved in physiological functions related to the metabolism of steroid hormones. The liver microsome LAGS were undetectable at rat birth, and became present in the 25-day-old rat. The level of LAGS then increased progressively, reaching its maximum level in the 2-3-month-old rats (10 pmol/mg protein), and declining afterwards to reach the adulthood level (5 pmol/mg protein) in 6-month-old rats. LAGS are mainly controlled by the corticoadrenal steroids, which is shown by their dramatic decrease after adrenalectomy, and especially after hypophysectomy. Many steroid hormones, like estradiol, testosterone, and corticosterone (but not progesterone) induce LAGS, estradiol being the most effective. A combination of T4 and corticosterone was more effective in inducing LAGS than when the two hormones were injected separately. It is possible to conclude that rat liver LAGS are mainly microsomal proteins, whose concentration is regulated by a multihormone system under pituitary control.     

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.     

Specific nuclear uptake of intracellularly-produced estrogen by rat granulosa cells

Granulosa cells of the ovarian follicle are unique in that they both synthesize steroid hormones and respond to exogenously-administered steroids. Isolated granulosa cells from ovaries of gonadotropin-primed rats were incubated in the presence of [3H]testosterone, which the cells convert to [3H]estradiol. Nuclear extracts of these cells were analyzed by high-performance liquid chromatography in a system of 40% acetonitrile. When cells were incubated in the presence of [3H]testosterone alone, a significant portion of the radioactivity present in nuclei co-eluted with authentic estradiol. The nuclear binding was considered to be specific, since 50-75% of total binding was suppressed when the incubation medium contained excess unlabeled estrogen. Moreover, when an antibody to estradiol was included in the medium, specific nuclear uptake of [3H]estradiol was not abolished, but rather was increased. Granulosa cells may, therefore, directly utilize endogenously-produced estradiol, a mechanism which may play a role in the regulation of ovarian cells.     

Characterization of a unique corticosterone-binding protein in Candida albicans

This paper further characterizes a protein we have demonstrated in Candida albicans which has the ability to bind corticosterone and related steroid hormones. Fungal cells are disrupted and cytosol is incubated with [3H]corticosterone for 3 h at which time peak steady state binding is achieved. Bound hormone is separated from free using Sephadex G-50 minicolumns or dextran-coated charcoal. Binding was found to be a linear function of protein concentration. The bound hormone co-migrates with authentic corticosterone in thin layer chromatographic systems indicating no metabolism of the radioprobe. Scatchard analysis of the binding in the pseudohyphal form of C. albicans yielded values of 6.3 nM for the Kd and a binding capacity of about 650 fmol/mg of cytosol protein; both determinations are comparable to our findings in the yeast form of this organism. A series of sterols were tested for their ability to displace [3H]corticosterone from the yeast binder, and the results show that the binder is remarkably selective and stereo specific. Physical-chemical studies show the binder to be degraded at high temperatures and that binding is destroyed by trypsin and sulfhydryl blockers. The protein sediments at 4 S on sucrose gradients and does not exhibit ionic dependent aggregation. The molecular weight is estimated to be approximately 43,000 daltons by gel chromatography. We hypothesize that this intracellular protein may represent a primitive form of either the mammalian glucocorticoid receptor or the plasma corticosteroid-binding globulin.     

Corticosterone binding by rat brain cytosol

Significant quantities of corticosterone were associated with macromolecules of the brain cytosol following intrathecal administration of [³H]corticosterone to adrenalectomized rats. Fifteen times more steroid was found associated with proteins from adrenalectomized rats than from control animals or adrenalectomized animals pretreated with corticosterone. Pretreatment of adrenalectomized rats with 11-dehydrocorticosterone, Cortisol and cortisone decreased the amount of [³H]corticosterone found associated with protein, whereas progesterone, oestradiol and testosterone did not interfere with the association of [³H]corticosterone with macromolecules of the cytosol. Further evidence for protein-steroid interaction was obtained by incubating [³H]corticosterone (B), [³H]cortisol (F), or 11-[³H]deoxycortisol (S) with brain cytosols. The degree of binding was in the order B > F > S.     

Distribution of a corticosteroid-binding protein in Candida and other fungal genera

Using [3H]corticosterone as a probe, corticosteroid-binding protein (CBP) was detected in eight out of eight isolates of Candida albicans, of both A and B serotypes. The apparent dissociation constant (Kd) in the various isolates ranged between 8 and 19 nM; the binding capacity varied from 122 to over 2400 fmol (mg cytosol protein)-1. There was no correlation between the amount or affinity of CBP and isolate virulence for murine hosts. Further analysis revealed demonstrable CBP in six out of six Candida species other than C. albicans. One isolate of C. tropicalis has been identified which fails to bind [3H]corticosterone. Saccharomyces cerevisiae, Neurospora crassa and Paracoccidioides brasiliensis also failed to bind [3H]corticosterone. Preliminary attempts were made to determine functions mediated by CBP in Candida, but in vitro growth, phase conversion and glucose oxidation by Candida were unaffected by the addition of a variety of steroid hormones. These data indicate that the presence of CBP in Candida does not correlate with either virulence or serotype. The physiological significance of CBP remains to be determined.     

Dehydroepiandrosterone activates endothelial cell nitric-oxide synthase by a specific plasma membrane receptor coupled to Galpha(i2,3)

The adrenal steroid dehydroepiandrosterone (DHEA) has no known cellular receptor or unifying mechanism of action, despite evidence suggesting beneficial vascular effects in humans. Based on previous data from our laboratory, we hypothesized that DHEA binds to specific cell-surface receptors to activate intracellular G-proteins and endothelial nitric-oxide synthase (eNOS). We now pharmacologically characterize a putative plasma membrane DHEA receptor and define its associated G-proteins. The [3H]DHEA binding to isolated plasma membranes from bovine aortic endothelial cells was of high affinity (K(d) = 48.7 pm) and saturable (B(max) = 500 fmol/mg protein). Structurally related steroids failed to compete with DHEA for binding. The putative DHEA receptor was functionally coupled to G-proteins, because guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) inhibited [3H]DHEA binding to plasma membranes by 69%, and DHEA increased [35S]GTPgammaS binding by 157%. DHEA stimulated [35S]GTPgammaS binding to Galpha(i2) and Galpha(i3), but not to Galpha(i1) or Galpha(o). Pretreatment of plasma membranes with antibody to Galpha(i2) or Galpha(i3), but not to Galpha(i1), inhibited the DHEA activation of eNOS. Thus, DHEA receptors are expressed on endothelial cell plasma membranes and are coupled to eNOS activity through Galpha(i2) and Galpha(i3). These novel findings should allow us to isolate the putative receptor and reevaluate the physiological role of DHEA activity.     

The steroid ligands of estrogen binding proteins in Xenopus laevis liver cytosol are primarily metabolites of 17β-estradiol

The interactions in vitro between [³H]estradiol and liver proteins from Xenopus laevis have been examined to determine if the binding reaction meets criteria of steroid-receptors which may function in the induction of vitellogenesis. Estrogenic hormones associated with proteins in serum and liver cytosol from Xenopus laevis. However, the interactions between soluble liver proteins and estrogens apparently do not result from serum contamination of liver as specific binding was distinguishable by ligand affinity and by differential mobility on polyacrylamide gels. Steroid ligands bound by liver proteins during incubation in vitro were examined by solubility and by thin-layer chromatography. Only a small percentage (13%) of the bound radioactive ligand was recovered as the original tritium-labeled steroid, 17β-estradiol. The major ligand was recovered as a water-soluble metabolite of estradiol which was identified tentatively as an estradiol-glucoside. To investigate whether the protein-bound estradiol metabolite(s) merely masks a small amount of authentic estradiol-receptor complexes or if the metabolite could be an intermediate in estrogen function, isolated liver nuclei were incubated with liver cytosol containing ³H-labeled steroid-protein complexes or with serum protein-bound [³H]estradiol. Nuclei preferentially accumulated ³H-labelea steroids from liver cytosol protein-steroid complexes relative to [³H]estradiol from serum proteins. However, analysis of the steroids recovered in the nuclei after incubation with liver cytosol revealed that both 17β-[³H]estradiol and the ³H-labeled water-soluble metabolite were retained in vitro by nuclei.     

Novel nuclear corticosteroid binding in rat small intestinal epithelia

When small intestinal epithelial cells are incubated with [(3)H]corticosterone, nuclear binding is displaced neither by aldosterone nor RU-28362, suggesting that [(3)H]corticosterone is binding to a site distinct from mineralocorticoid receptor and glucocorticoid receptor. Saturation and Scatchard analysis of nuclear [(3)H]corticosterone binding demonstrate a single saturable binding site with a relatively low affinity (49 nM) and high capacity (5 fmol/microg DNA). Competitive binding assays indicate that this site has a unique steroid binding specificity, which distinguishes it from other steroid receptors. Steroid specificity of nuclear binding mirrors inhibition of the low 11beta-dehydrogenase activity, suggesting that binding may be to an 11beta-hydroxysteroid dehydrogenase (11betaHSD) isoform, although 11betaHSD1 is not present in small intestinal epithelia and 11betaHSD2 does not colocalize intracellularly with the binding site. In summary, a nuclear [(3)H]corticosterone binding site is present in small intestinal epithelia that is distinct from other steroid receptors and shares steroid specificity characteristics with 11betaHSD2 but is distinguishable from the latter by its distinct intracellular localization.     

Effect of sex and prior exposure to a cafeteria diet on the distribution of sex hormones between plasma and blood cells

It is generally assumed that steroid hormones are carried in the blood free and/or bound to plasma proteins. We investigated whether blood cells were also able to bind/carry sex-related hormones: estrone, estradiol, DHEA and testosterone. Wistar male and female rats were fed a cafeteria diet for 30 days, which induced overweight. The rats were fed the standard rat diet for 15 additional days to minimize the immediate effects of excess ingested energy. Controls were always kept on standard diet. After the rats were killed, their blood was used for 1) measuring plasma hormone levels, 2) determining the binding of labeled hormones to washed red blood cells (RBC), 3) incubating whole blood with labeled hormones and determining the distribution of label between plasma and packed cells, discounting the trapped plasma volume, 4) determining free plasma hormone using labeled hormones, both through membrane ultrafiltration and dextran-charcoal removal. The results were computed individually for each rat. Cells retained up to 32% estrone, and down to 10% of testosterone, with marked differences due to sex and diet (the latter only for estrogens, not for DHEA and testosterone). Sex and diet also affected the concentrations of all hormones, with no significant diet effects for estradiol and DHEA, but with considerable interaction between both factors. Binding to RBC was non-specific for all hormones. Estrogen distribution in plasma compartments was affected by sex and diet. In conclusion: a) there is a large non-specific RBC-carried compartment for estrone, estradiol, DHEA and testosterone deeply affected by sex; b) Prior exposure to a cafeteria (hyperlipidic) diet induced hormone distribution changes, affected by sex, which hint at sex-related structural differences in RBC membranes; c) We postulate that the RBC compartment may contribute to maintain free (i.e., fully active) sex hormone levels in a way similar to plasma proteins non-specific binding.     

Characterization of Corticosterone-Induced Protein Synthesis in Hippocampal Slices

: Corticosterone significantly increases the incorporation of [³H]leucine into specific cytosol protein(s) isolated from in vitro hippocampal slices prepared from adult male albino rats. The present study showed that in slices coincubated with glucocorticoid plus a protein synthesis inhibitor (1 mm -cycloheximide), no such enhancement of amino acid incorporation was observed, suggesting that the hormone acts in the hippocampus to increase de novo protein synthesis. Further experiments demonstrated that the steroid-induced protein synthesis was first detectable (+ 5.7%) following a 30-min exposure of slices to corticosterone; slices incubated for 1 or 2 h both showed a 12% increase in synthesis of the affected protein(s) when compared with controls. In an attempt to determine whether the glucocorticoid alteration of protein metabolism was receptor-mediated, hippocampal slices were also incubated with 10 nm -progesterone, a steroid known to compete for corticosterone binding to its cytosol receptor. Progesterone alone, which does not translocate cytoplasmic receptors to the nucleus, did not alter hippocampal protein metabolism and effectively blocked the induction by corticosterone of the 54K protein(s). These studies provide evidence that in the rat hippocampus corticosterone interacts with high-affinity steroid receptors to regulate the synthesis of specific protein(s).     

Production of sex steroid hormones from DHEA in articular chondrocyte of rats

Dehydroepiandrosterone (DHEA), a precursor of sex steroid hormones, is synthesized by cholesterol side-chain cleavage cytochrome P-450 and 17alpha-hydroxylase cytochrome P-450 mainly from cholesterol and converted to testosterone and estrogen by 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17beta-HSD, and aromatase cytochrome P-450. Although sex steroid hormones have important effects in the protection of articular cartilage, it is unclear whether articular cartilage has a local steroidogenic enzymatic machinery capable of metabolizing DHEA. This study was aimed to clarify whether steroidogenesis-related enzymes are expressed in articular chondrocytes, whether expression levels are changed by DHEA, and whether articular chondrocytes are capable of synthesizing sex steroid hormones from DHEA. Articular chondrocytes isolated from adult rats were cultured with DHEA for 3 days. All of the mRNA expressions of steroidogenesis-related enzymes were detected in cultured articular chondrocytes of rats, but the mRNA expression levels of testosterone and estradiol in cultured media increased after the addition of DHEA. These findings provided the first evidence that articular chondrocytes expressed steroidogenesis-related enzyme genes and that they are capable of locally synthesizing sex steroid hormones locally from DHEA.     

Ontogeny of steroid metabolizing enzymes in rat oligodendrocytes

Rat oligodendroglial cells were isolated from newborn and developing brains and used immediately after, for quantification of steroid metabolizing activities. Oligodendrocytes (Ol) and their progenitor cells were incubated with [(14)C] testosterone, [(14)C] progesterone, [(14)C] pregnenolone or [(14)C] dehydroepiandrosterone (DHEA). Oligodendrocytes and their progenitor cells expressed different steroid metabolizing enzymes. The main activities were 5 alpha reduction of testosterone and progesterone and 3 beta hydroxy steroid dehydrogenase-isomerase which transformed pregnenolone into progesterone and DHEA into Delta 4 androstenedione. 5 alpha reductase activity increased in male and female rats in parallel with testosterone or progesterone. Contrary to this, 3 beta hydroxysteroid dehydrogenase-isomerase activity was found to be high in the young rat and to decrease when testosterone and progesterone plasma concentration increased.     

Interrelationship between nuclear histone binding and cell proliferation

1. Binding of non-enzymatically [methyl-14C]-labeled histone H3 to nuclei isolated from young and old rat livers, regenerating rat liver, and tumor cells has been investigated. 2. Scatchard plot analysis indicated that various cell types had different binding capacity and different dissociation constant (Kd). 3. Nuclei isolated from younger rats had fewer binding sites and lower Kd (or higher Ka) values for [methyl-14C]H3 than those from older rats. 4. Fewer binding sites and lower Kd values were also observed with nuclei isolated from the maximally regenerating liver (24 hr after partial hepatectomy) and the fast-growing ascites tumor and Novikoff hepatomas. 5. These results strongly suggest that the number of binding sites and affinity of histone H3 for nuclei appears to be correlated with the degree of cell proliferation. 6. Fractionation of the [methyl-14C]H3 bound nuclei into nuclear membrane and nucleoplasm demonstrates that approx. 94% of radioactivity is associated with the former in which less than 6% of DNA is found, whereas 94% of total DNA is found in nucleoplasm. 7. This suggests that the binding of [methyl-14C]H3 to nuclei is independent of DNA present in each fraction.     

Steroid metabolism in the corpus luteum of the ferret

Implantation in the ferret is believed to be induced by a luteal substance which acts in concert with progesterone (P4) and which is secreted sometime between Days 6 and 8 of pregnancy. This experiment was designed to identify the steroid products synthesized by ferret corpora lutea (CL) on these 2 days of pregnancy. CL were dissected from ferrets on Day 6 or 8 of pregnancy and incubated with [3H] pregnenolone (P3), [3H] P4, or [3H] dehydroepiandrosterone (DHEA). Controls with no tissue or with 50 microliters packed blood cells were incubated at the same time. After incubation of Day 6 CL with [3H] P3 for 180 min, 39% of the added label was found incorporated into P4, 3% into 17 alpha-hydroxyprogesterone (17 alpha-OHP4) and 1% into androstenedione (A). Incubation of Day 8 CL with the same precursor resulted in 35%, 1% and 0.65% of the label being incorporated into the previously mentioned products, respectively. Incubations of Days 6 and 8 ferret CL with [3H] P4 or [3H] DHEA confirmed these results, demonstrating activity of C21-steroid, 17 alpha-hydroxylase and delta 5-isomerase, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). These results suggest that ferret CL primarily accumulate steroids of the delta4 pathway on both Days 6 and 8 of pregnancy, with P4, 17 alpha-OHP4, A and testosterone (T) being the most abundant products after in vitro incubation. Thus, ferret CL appear to metabolize steroids in a manner similar to that observed in rats, sows and mares.     

Steroid metabolism by avian ovarian cells during follicular maturation

The profiles of steroid hormones produced by ovarian cells from the domestic hen were examined. Theca cells from the immature, small white follicles (SWFT), the third largest (T3), and largest (T1) preovulatory follicles, and the ruptured, postovulatory follicle (POFT) were incubated for 3 h at 37 degrees with [3H] progesterone (Prog) or [3H] pregnenolone (Preg). Granulosa cells from the largest preovulatory follicle were incubated with [3H] Preg or were coincubated with theca cells and [3H] Preg. The production of specific steroid metabolites was determined on the basis of coelution of radioactivity with known standard compounds, using an isocratic high-pressure liquid chromatography (HPLC) technique. Granulosa cells converted 93% of [3H] Preg substrate to Prog. More Prog was utilized by T3 cells than by T1 and SWFT cells, either when [3H] Prog was the substrate or when coincubated with granulosa cells and [3H] Preg. The major metabolites of Prog were androstenedione, 17-hydroxyprogesterone, and an unidentified compound with an elution time of 53 min. The POFT cells metabolized [3H] Prog to the same extent as T3 cells did, but their profile of steroidogenesis favored production of the unidentified 53 min metabolite. SWFT cells utilized the least amount of [3H] Preg substrate. The results point to marked changes in enzyme activities in theca cells during maturation and following ovulation.     

Differences between cytosol receptor complexes with corticosterone and dexamethasone in hippocampal tissue from rat brain.

The binding of [3H]corticosterone and [3H]dexamethasone to soluble macromolecules in cytosol of the hippocampal region of the brain has been studied in adrenalectomized male rats. Unlabeled dexamethasone appears to be a less effective competitor than corticosterone in the binding of [3H]corticosterone, while both unlabeled steroids compete equally well for the binding or [3H]dexamethasone. Further investigation of macromolecular complexes with [3H]dexamethasone and [3H]corticosterone revealed that they differ from each other in their behavior during ammonium sulfate precipitation, BioRad A-5M gel permeation chromatography, DE-52 anion exchange chromatography and DNA-cellulose chromatography. (1) After exposure to a 33% ammonium sulfate solution relatively more [3H]dexamethasone complex than [3H]corticosterone complex is precipitated. (2) Treatment of the cytosol with 0.3 M KCl gives disaggregation of the supramolecular 3H-labeled corticoid complexes which are seen eluting with the void volume during gel permeation chromatography on Biorad A-5M at low ionic strength. In 0.3 M KCl, the [3H]dexamethasone complex has an elution volume somewhat smaller than that of bovine serum albumin, while the [3H]-corticosterone complex in 0.3 M KCl is too unstable to survive chromatography with A-5M. (3) Chromatography on DE-52 resolved the 3H-labeled corticoid complexes into three binding components. The complex with [3H]dexamethasone contains a higher percentage (85%) of a component less firmly attached (i.e. eluted by 0.15 M KCl) to the anion exchange resin than is observed for the complex with [3H]corticosterone (49%). (4) The complexes with 3H-labeled corticoids display an enhanced affinity for calf thymus DNA adsorbed to cellulose following "activation", warming to 25 degrees C for 15 min. Concurrently, a fraction of the [3H]dexamethasone complex becomes able to more firmly attach to the DE-52 anion exchange resin. These results with the binding of the cytosol hormone-receptor complexes to DNA-cellulose do not explain the marked in vivo preference of hippocampus for the cell nuclear uptake of [3H] corticosterone. However, the other differences in the properties of the complexes formed with the two labeled glucocorticoids support our previous inference that there may be more than one population of adrenal steroid "receptors" in brain tissue.     

Evidence for the biosynthesis of DHEA from cholesterol by first-trimester human placental tissue: source of androgens.

With a view to establishing whether first-trimester human placentas possess the ability to synthesize DHEA from cholesterol, homogenates of this tissue obtained from two groups of women undergoing elective termination of normally progressing pregnancy between 10 - 12 weeks gestation (n = 5, age 23 - 29 years and n = 5, age 21 - 27 years) were incubated separately with [26-(14)C]cholesterol for the generation of [14C]isocaproic acid + pregnenolone and [7n-3H]pregnenolone for the biosynthesis of [3H]DHEA. Controls consisted of homogenates heated in a boiling water bath for 10 min. Using the reverse-isotope dilution analysis, desmolase efficiency expressed as mean specific activity of [14C]isocaproic acid varied from 282 to 725 dpm/mmol, while that of 17 alpha-hydroxylase and steroid C-17,20-lyase, catalyzed conversion of [7n-3H]pregnenolone to [3H]DHEA varied from 3498 to 26 258 dpm/mmol. The corresponding efficiencies of enzymicconversion varied between 5.8 x 10( -2) and 1.5 x 10( -1) % for [14C]isocaproic acid, but between 5.5 x 10( -2) and 4.1 x 10( -1) % for [3H]DHEA. No such metabolite was evident in the controls of heat-denatured homogenates. These are the first study results to demonstrate that early placentas are capable of converting cholesterol to pregnenolone to DHEA, contrary to the widely held concept of DHEA production by fetal and maternal adrenal glands. This finding has important physiological implications and could provide a new dimension to the concept of fetoplacental steroidogenesis.     

Transformation and nuclear translocation of brain type L corticosteroid receptors complexed with the mineralocorticoid antagonist ZK 91587, aldosterone or dexamethasone.

Type I corticosteroid receptors were determined in cytosol from hippocampus (HIPPO) and amygdala (AMYG), using [3H]aldosterone (ALDO), [3H]dexamethasone (DEX) or the mineralocorticoid antagonist [3H]ZK 91587 as ligands. Incubations with the first two compounds also contained the pure glucocorticoid RU 28362 to block type II receptors. Binding of the three ligands was comparable in cytosol from HIPPO and it was slightly higher for [3H]DEX in AMYG. However, after heat-induced receptor transformation, binding to DNA-cellulose was observed for [3H]ALDO-receptor complex obtained from HIPPO or AMYG, whereas it was negligible for [3H]ZK 91587. Receptors charged with [3H]DEX or [3H]ALDO showed similar retention on DNA-cellulose columns in the case of the AMYG, while binding to the polynucleotide was higher for [3H]ALDO in the HIPPO. Finally, only [3H]ALDO was taken up to a significant extent in purified cell nuclei prepared from slices of HIPPO and AMYG previously incubated with the three ligands. It is concluded that binding of a natural agonist steroid may be a prerequisite for type I receptor transformation and translocation from the cytoplasm into the nuclear fraction. DEX binding to type I receptors resembles a partial agonist with antagonist properties, whereas antagonists such as ZK 91587 are bound and retained in cytoplasm, without further translocation.     

Nucleic acid synthesis in proliferating peroxisomes of rat liver as revealed by electron microscopical radioautography

Summary Thirty albino rats were fed with a diet containing 1, 2 or 4% di-(2-ethylhexyl)-phthalate (DEHP), a peroxisome-proliferating agent. Others were fed with normal diet as controls. Both groups were sacrificed at varying intervals from 3 days to 4 weeks. The livers were either removed and fixed in glutaraldehyde and osmium tetroxide or fixed in glutaraldehyde, incubated in a diaminobenzidine (DAB) medium, postfixed, embedded in Epon, and sectioned. Other tissues were incubated in Eaglés MEM containing either [³H]thymidine or [³H]uridine, fixed, embedded in Epon, sectioned, and radioautographed. Specimens were observed in a Hitachi H-700 electron microscope.The number of peroxisomes showing DAB reactivity increased in DEHP-fed animals as compared with normal controls In radioautograms of normal rats labelled with [³H]thymidine, no silver grains were, observed, whereas grains were observed over some nuclei, mitochondria and peroxisomes of DEHP-fed animals. In contrast, radioautograms of tissue labelled with [³H]uridine revealed a few grains in nuclei and mitochondria or endoplasmic reticulum of normal rats, although grains appeared in nuclei, mitochondria, endoplasmic reticulum and peroxisomes of DEHP-fed animals more frequently.From these results, it is concluded that [³H]thymidine and [³H]uridine were incorporated in the proliferating peroxisomes, suggesting that nucleic acid synthesis had taken place.