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P Robel

Publications and source records attributed to P Robel.

At least 19 recordsLinked to original sources
PubMed1992

Neurosteroid metabolism. 7 alpha-Hydroxylation of dehydroepiandrosterone and pregnenolone by rat brain microsomes.

Two 'neurosteroids', dehydroepiandrosterone (DHEA) and pregnenolone (PREG), are converted by rat brain microsomes into polar metabolites, identified as the respective 7 alpha-hydroxylated (7 alpha-OH) derivatives by the 'twin ion' technique of g.l.c.-m.s. with deuterated substrates. The enzymic reaction requires NADPH and is stimulated 2-4-fold by EDTA. Under optimal conditions (pH 7.4, 0.5 mM-NADPH, 1 mM-EDTA), the Km values for DHEA and PREG are 13.8 and 4.4 microM respectively, and the Vmax. values are 322 and 38.8 pmol/min per mg of microsomal protein respectively. Trace amounts of putative 7 beta-OH derivatives of DHEA and PREG are detected. Oestradiol, at a pharmacological concentration of 5 microM, inhibits DHEA and PREG 7 alpha-hydroxylation. Formation of 7 alpha-hydroxylated metabolites is low in prepubertal rats and increases 5-fold in adults. Derivatives of PREG and DHEA, such as PREG sulphate, DHEA sulphate, progesterone and 3 alpha-hydroxy-5 alpha-pregnan-20-one, are known to be neuroactive. Therefore the quantitatively important metabolism to 7 alpha-OH compounds may contribute to the control of neurosteroid activity in brain.

17-alpha-Hydroxypregnenolone↗
PubMed1992

Delta 5-3 beta-hydroxysteroid acyl transferase activity in the rat brain.

Pregnenolone and dehydroepiandrosterone accumulate in brain as sulfate and fatty acid esters and unconjugated steroids. The steroid fatty acid ester-synthesizing activity was investigated in rat brain microsomes. Endogenous fatty acids in the microsomal fraction were used for the esterification of steroids. The enzyme system had a pH optimum of 4.5 in acetate buffer with [3H]dehydroepiandrosterone as substrate. The apparent Km was 9.2 +/- 3.1 x 10(-5) M and Vmax was 18.6 +/- 3.4 nmol/h/mg protein (mean +/- SEM). The inhibition constants of pregnenolone and testosterone were 123 and 64 microM, respectively. Results were compatible with a competitive type of inhibition. A high level of synthetic activity was found in the brain of 1- to 3-week-old male rats, which rapidly decreased with aging. Saponification of purified [3H]pregnenolone esters yielded pregnenolone and a mixture of palmitate, oleate, linoleate, stearate, and myristate as the predominant fatty acids. Contrasting with the high rates of esterification of several radioactive delta 5-3 beta-hydroxysteroids or 17 beta-hydroxysteroids, no fatty acid esters of either cholesterol, epitestosterone (with a hydroxyl group at position C-17 alpha), or corticosterone (with hydroxyl groups at C-21 and C-11 beta) were formed in the same incubation conditions.

Acyltransferases↗
PubMed1992

Low density lipoprotein-receptors in primary cultures of rat glial cells.

Newborn rat glial cells in primary culture contain an active cholesterol side chain cleavage cytochrome P450. Cholesterol can be supplied either by biosynthesis or derive from low density lipoproteins (LDL), which bind apolipoprotein Band E (apoB,E) (LDL)-receptors and undergo receptor-mediated endocytosis. Using antibodies to purified human plasma LDL and antibodies to bovine adreno-cortical LDL-receptor, the presence of LDL-receptors was demonstrated on rat glial cells after 3-4 weeks of primary culture, by ligand blotting, immunoblotting, and indirect immunofluorescence staining. The latter approach indicated that oligodendrocytes express higher levels of LDL-receptors than astrocytes present in the same culture. The immunofluorescence staining was observed not only at the cell surface, but also within the cytoplasm, suggesting that the LDL-receptor complexes had been internalized. Western blotting of LDL-receptors extracted from glial cells indicated a band of approximately 130 kDa, the size expected for intact receptors. Their functionality was shown by the conversion of [3H]cholesterol linoleate, incorporated into reconstituted LDL and added to the cell cultures, to [3H]pregnenolone and/or its 20 alpha-hydroxy-metabolite. This is the first characterization of functional LDL-receptors on isolated, well characterized, normal brain cells.

Animals↗
PubMed1991

Suppressive effects of dehydroepiandrosterone and 3 beta-methyl-androst-5-en-17-one on attack towards lactating female intruders by castrated male mice. II. Brain neurosteroids.

Dehydroepiandrosterone (DHA) and 3 beta-methyl-androst-5-en-17-one (CH3-DHA) suppress attacks by castrated male mice towards lactating female intruders (Haug et al. Physiol. Behav. 46:955, 1989). Their effects on the concentrations of DHA, pregnenolone (delta 5P), and their sulfate ester (S) have been investigated in the brain of control and treated mice. A more than 2 fold, significant, decrease of delta 5P-S was the only change common to both steroids. DHA and CH3-DHA effects might be related to the antagonistic action of delta 5P-S on GABAA receptors.

Aggression↗
PubMed1991

Immunoreactive cytochrome P-450(17 alpha) in rat and guinea-pig gonads, adrenal glands and brain.

The cytochrome P-450(17 alpha)-hydroxylase, 17----20 lyase (P-450(17 alpha)) is the key enzyme responsible for the biosynthesis of androgens in steroidogenic organs. Its cellular localization has been examined with an immunohistochemical technique. In immature rat ovary, P-450(17 alpha) was first detected in sparse interstitial cells on postnatal Day 8. The number of immunoreactive interstitial cells increased thereafter and the intensity of P-450(17 alpha) staining in these cells was highest at 3 weeks of age. The intensity of staining then started to decline and was very faint at Day 35. From 6 weeks on, the distribution of immunoreactive P-450(17 alpha) was of the adult type: it was detected exclusively in the thecal cells of the large antral, preovulatory, follicles. P-450(17 alpha) was not detectable during pregnancy except on the day of parturition, when thecal cells were transiently immunoreactive. The staining had vanished 24 h after delivery. Human chorionic gonadotrophin (hCG), injected into immature females on Days 24 to 26, induced P-450(17 alpha) prematurely in thecal cells. When injected on Days 12 to 14 of pregnancy, hCG also induced P-450(17 alpha) in the thecal cells surrounding the largest follicles, whereas the interstitial and luteal cells were not immunostained. The antiprogestin RU486, injected on Day 16 of pregnancy, reinstated P-450(17 alpha) (and P-450scc) immunoreactivity in the thecal cells. Oestradiol selectively suppressed P-450(17 alpha) expression in the thecal cells of RU486-treated females. In immature guinea-pig ovary, P-450(17 alpha) was immunostained in thecal cells, not in interstitial cells, although the interstitial cells expressed the delta 5-3 beta-hydroxysteroid dehydrogenase. P-450(17 alpha) was also immunolocalized in the Leydig cells of rat and guinea-pig testes, and in the guinea-pig adrenal cortex (zonae fasciculata and reticularis), but not in the rat adrenal cortex. P-450(17 alpha) was not detectable in the brain of either rat or guinea-pig.

Adrenal Cortex↗
PubMed1991

[Inhibition by dehydroepiandrosterone of aggression responses of spayed female mice towards lactating intruders is potentiated by neonatal androgenization].

Female mice of an inbred Swiss strain were injected subcutaneously (s.c.) with either testosterone propionate or oil-vehicle on the day of birth. Once adult (8 week-old), they were spayed, and then treated either with dehydroepiandrosterone (80 micrograms/day s.c. for 15 days) or with solvent. The decrease of biting attacks by dehydroepiandrosterone was definitely more prominent in neonatally imprinted females than in controls.

Animals↗
PubMed1979

Glucocorticoid receptor in human embryo fibroblasts. I. Kinetic and physicochemical properties.

The kinetics of dexamethasone binding to L 809 E cell line cytosol have been investigated by means of the protamine sulfate precipitation assay. The KDeq for dexamethasone was 1.1--3.3 nM. Binding was specific for glucocorticoids. The mean association rate constant (k+1) was 8.5 x 10(5) M-1 x min-1 and the dissociation rate constant was 4.6 x 10(-5) min-1 at 0 degrees C. The concentration of binding sites was 0.3 pmol/mg of cytosol protein. Binding kinetics were compatible with a model of positive cooperativity. The receptor sedimented at 7.5--9 S in glycerol gradients. By a combination of calibrated ultracentrifugation and polyacrylamide gel electrophoresis, a Stokes radius of 8.5 nm, a molecular weight of 268 000 daltons and a frictional ratio of 1.8 were determined in low ionic strength conditions. When the cells were incubated with 10 nM [3H]dexamethasone for 1 h, a more than 90% depletion of cytosol receptor and an equivalent accumulation of nuclear dexamethasone--receptor complexes was observed.

Cell Line↗
PubMed1979

Relationships among placental, uterine, and circulating concentrations of progesterone and fetal survival in the ovariectomized pregnant rat.

Pregnant rats were ovariectomized or sham operated on day 15 postcoitum. Four days later, progesterone was measured by RIA in peripheral and uterine vein plasma, in uteri, and in placentae. Maintenance of pregnancy was not critically affected by ovariectomy, since fetal survival was 65.7 +/- 5.1% (mean +/- SEM) despite a large decrease of peripheral plasma progesterone from 115.7 +/- 3.4 to 9.3 +/- 0.5 ng/ml. Peripheral and uterine vein plasma progesterone (8.3 +/- 0.9 ng/ml) were identical. In contrast, placental progesterone decreased only slightly, although significantly, from 27.3 +/- 1.3 to 20.3 +/- 1.0 ng/mg. The concentrations of uterine progesterone were variable and positively correlated with the concentrations of peripheral plasma progesterone. It was concluded that uterine progesterone originates from peripheral blood but not from placentae and that fetal survival is positively correlated with residual progesterone concentrations in peripheral plasma and in uterus but not in placentae.

Animals↗
PubMed1979

[Luteal insufficiency. Histoclinical and biological correlations].

Luteal insufficiency, an entity accepted with difficulty, is responsible for 3.5 p. 100 of cases of infertility and 35 p. 100 of early abortions. This study, carried out in 328 women with an average age of 29 years, involved clinical and histological features, as well as the estimation of plasma levels and endometrial receptors of ovarian steroids. Three broad histological patterns emerged:--luteal insufficiency associated with oestrogenic insufficiency indicative of overall ovarian deficiency;--luteal insufficiency with functional dysmaturation indicative of deficient or delayed luteal function, with or without persistence of excessively high oestrogen activity,--luteal insufficiency related to the short life of the corpus luteum which stops on the 20th day. Relationships between histological findings, the measurement of receptors and plasma estimations are discussed and precise analysis of endometrial biopsies must be considered as one of the best test of luteal insufficiency.

Adult↗
PubMed1978

[Response to an antiestrogen as a criterion for hormonal sensitivity of endometrial cancer].

A test is described for the evaluation of hormone sensitivity of endometrial cancer in vivo. The concentrations of progesterone and estradiol receptors, and the activities of ornithine-decarboxylase and 17 beta-hydroxysteroid oxido-reductase enzymes have been measured in the tumor, before and after administration of the anti-estrogen tamoxifen. The responses observed, in particular the increase of progesterone receptor, could allow a more rational approach to hormonal therapy of endometrial cancer.

17-Hydroxysteroid Dehydrogenases↗
PubMed1978

Competitive inhibition of specific steroid-protein binding: practical use of relative competition ratios for the derivation of equilibrium inhibition constants.

The relative competition ratio (RCR) is widely used to express the relative affinities of inhibitor(s) and agonist for a binding protein. The RCR is not a constant; it depends on the concentrations of binding sites and of radioactive hormone, and on the presence of nonsaturable binding component(s). According to the assay conditions used, equating the RCR value to the ratio Ka/Ki of the equilibrium association constants of agonist and inhibitor can lead to large errors. In the case of homogeneous non-interacting binding sites, simple correction factors permit one to calculate the ratio Ka/Ki from the measured RCR value. Calculations are given for the eventual contribution of nonsaturable binding components. Corrections can be unnecessary under well defined experimental conditions, where the bound fraction of hormone in absence of competitor is reduced by using a large dilution of binding protein and/or an increased concentration of radioactive hormone.

Binding, Competitive↗
PubMed1978

Cytoplasmic and nuclear estradiol and progesterone receptors in human endometrium.

Estradiol and progesterone receptors have been characterized in normal human endometrial biopsy samples. The cytosol and nuclei were prepared from 150-250-mg samples, either processed immediately or kept in liquid nitrogen. The total concentration of estradiol-and progesterone-binding sites (available or occupied with endogenous hormone) were measured in both fractions. Results were best expressed in femto-moles per mg DNA, or in sites per cell, assuming an even distribution of receptor throughout the endometrial samples. The contribution to total binding of non-saturable binding components and of plasma proteins (transcortin or sex steroid-binding protein) was taken into account. Measurements were obtained in more than 300 patients, among whom 54 had completely normal menstrual cycles on the basis of clinical, hormonal, and histological features. Total estradiol and progesterone receptors were highest in the late proliferative phase (about 8,000 and 12,000 sites/cell, respectively) and were very significantly lower in the late secretory phase. During the proliferative phase, estradiol receptors were increased only in the nuclear fraction, whereas progesterone receptors were increased mainly in the cytoplasm. In the early luteal phase, estradiol and progesterone receptors decreased in the cytosol, whereas they remained high in the nuclei. Both receptors were at their lowest level in cytosol and nuclei in the late secretory phase. The changes of total estradiol and progesterone receptor sites and of their respective subcellular distributions seem to depend upon the plasma levels of both hormones and to follow the same cause and effect relationships as those demonstrated experimentally in laboratory animals.

Cell Nucleus↗
PubMed1977

[Combined effects of testosterone and estradiol on the ventral lobe of the rat postate in organ culture].

After organ culture without hormone, the epithelial gland cells of Rat veantral prostate undergo atrophic changes, whereas the interstitial stroma components tend to increase. Estradiol (1-1,000 nM),added to the culture medium, is ineffective. On the contrary, testosterone (1-100 nM) maintains epithelial cells and prevents the increase of interstitial stroma. When estradiol (1-1,000 nM) is combined with a physiological concentration of testosterone (1-4 nM), the epithelial cells are well maintained, but the inhibitory action of testosterone on the stroma is counteracted so that the glandular epithelium and the interstitial stroma are both stimulated. However, when testosterone is used at supraphysiological (10-100 nM) concentrations, estradiol is completely ineffective and the structure of the prostate is identical to the one given by the androgen alone.

Animals↗
PubMed1977

Simultaneous radioimmunoassay of tesosterone, dihydrotestosterone, 5 alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol in the plasms of adult male rats.

A single thin layer chromatography and three antibodies were used for the specific radioimmunoassay of four androgens in pooled rat plasma (Sprague-Dawley adult males). The following values were found (pg/ml +/- SD). Testosterone: 3, 138 +/-173; dihydrotestosterone: 374 +/-20; 5alpha-androstane-3alpha, 17beta-diol: 284+/-24; 5alpha-androstane-3beta, 17beta-diol: 223+/-11.

Androstane-3,17-diol↗
PubMed1976

Influence of purified plasma proteins on testosterone uptake and metabolism by normal and hyperplastic human prostate in "constant-flow organ culture".

Surgical samples of human prostate were explanted and submitted to constant-flow organ culture. The medium contained 3H-testosterone 50 nM, and except for controls, increasing concentrations of human serum albumin (HSA) or human sex-steroid-binding plasma protein (SBP). At steady state, the explants were washed and homogenized, and the total radioactivity, radioactive testosterone, androstanolone (17 beta-hydroxyandrostan-3-one), androstane-3 alpha, 17 beta-diol, and androstane-3 beta, 17 beta-diol were determined after the addition of the corresponding internal 14C standards. From these data, testosterone uptake and metabolism were quantitated. The concentration of unbound testosterone in protein-supplemented culture media was measured separately by equilibrium dialysis. In control superfusions without protein, the tissue concentration of total radioactive steroids was equivalent to 182 +/- 18 (mean +/- SEM) pmoles/g of prostate. Androstanolone represented about 2/3, testosterone 1/10, and the two androstanediols together 1/10 of the total radioactivity. No difference was found between "normal" and hyperplastic prostate explants. In experiments with HSA (15-176 muM), is was observed that the uptake of radioactive testosterone in the prostate explants was decreased in direct proportion to the unbound testosterone fraction of the superfusion medium, but the proportions of testosterone metabolities in the superfused explants remained the same. In experiments with SBP (6-135 nM), the concentrations of unbound testosterone in the superfusion medium were reduced to the same levels as in the experiments with HSA. The reduction of tissue radioactivity was somewhat larger than that expected from the reduction of unbound testosterone in the superfusion medium for the concentrations of SBP less than 50 nM, and then remained approximately constant. In addition, SBP altered the metabolism of testosterone: the androstanolone/testosterone ratio in the prostate explants was critically dependent upon the SBP concentration in the superfusion medium. It is therefore suggested that, independent of its effect on the binding of testosterone, SBP has a direct effect on testosterone uptake and metabolism by the human prostate. The underlying mechanism is unknown.

Androstane-3,17-diol↗