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P Robel

Publications and source records attributed to P Robel.

At least 37 records · Page 2Linked to original sources
PubMed1996

Development and regeneration of the nervous system: a role for neurosteroids.

Several steroids, termed 'neurosteroids', are synthesized from cholesterol within both the central and peripheral nervous systems. These include pregnenolone and its sulfate ester, progesterone and its 5 alpha-reduced metabolites. Dehydroepiandrosterone, mainly in its sulfated form, also remains present in the brain long after removal of the steroidogenic endocrine glands. Its biosynthesis in brain remains an open possibility, but the pathways involved are unknown. Little information is available concerning the role of neurosteroids during the maturation of the nervous system, although they are already synthesized by glial cells and by some populations of neurons during embryonic life. Cell culture experiments suggest that neurosteroids may increase the survival and differentiation of both neurons and glial cells. In the adult nervous system, neurosteroids modulate neurotransmission by acting directly on the neuronal membrane and also produce structural changes in neurons and in astrocytes. Studies of neurosteroid levels are currently conducted to examine their possible role during aging. We have recently reported that progesterone, synthesized by Schwann cells, promotes the formation of new myelin sheaths after lesion of the mouse sciatic nerve. Thus, neurosteroids may also play an important role during regeneration of the nervous system.

Animals↗
PubMed1995

Progesterone synthesis and myelin formation by Schwann cells.

Progesterone is shown here to be produced from pregnenolone by Schwann cells in peripheral nerves. After cryolesion of the sciatic nerve in male mice, axons regenerate and become myelinated. Blocking either the local synthesis or the receptor-mediated action of progesterone impaired remyelination. Administration of progesterone or its precursor, pregnenolone, to the lesion site increased the extent of myelin sheath formation. Myelination of axons was also increased when progesterone was added to cultures of rat dorsal root ganglia. These observations indicate a role for locally produced progesterone in myelination, demonstrate that progesterone is not simply a sex steroid, and suggest a new therapeutic approach to promote myelin repair.

Animals↗
PubMed1995

Modulation of recombinant alpha 6 beta 2 gamma 2 GABAA receptors by neuroactive steroids.

The sensitivity of the recombinant alpha 6 beta 2 gamma 2 GABAA receptor expressed in HEK 293 cells to neuroactive steroids was studied. The steroids 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP), pregnenolone sulfate and 3 alpha-OH-DHP sulfate have different modulatory effects on [3H]muscimol or [3H]Ro15-4513 binding to the alpha 6 beta 2 gamma 2 than to the alpha 1 beta 2 gamma 2 receptor. Binding of both radioactive ligands to the alpha 6 beta 2 gamma 2 receptor was maximally potentiated with each steroid used (10 nM) and decreased with further increases in steroid concentration. Using whole-cell recording, the GABA response of clusters of transfected HEK 293 cells was strongly potentiated by 3 or 10 nM 3 alpha-OH-DHP. In contrast, this response was reduced by 100 nM 3 alpha-OH-DHP. This latter effect appears to be related to the acceleration of the GABA response desensitization, observed in isolated cells. 3 alpha-OH-DHP (10 or 100 nM) was able to activate a response in the absence of GABA. It is proposed that the interaction of neuroactive steroids with the alpha 6 beta 2 gamma 2 receptor involves at least two distinct binding sites. One of them might be located close to the GABA binding site.

Azides↗
PubMed1995

A progesterone metabolite stimulates the release of gonadotropin-releasing hormone from GT1-1 hypothalamic neurons via the gamma-aminobutyric acid type A receptor.

The reduced progesterone metabolite tetrahydroprogesterone (3 alpha-hydroxy-5 alpha-pregnan-20-one; 3 alpha,5 alpha-THP) is a positive modulator of the gamma-aminobutyric acid type A (GABAA) receptor. Experiments performed in vitro with hypothalamic fragments have previously shown that GABA could modulate the release of gonadotropin-releasing hormone (GnRH). Using GT1-1 immortalized GnRH neurons, we investigated the role of GABAA receptor ligands, including 3 alpha,5 alpha-THP, on the release of GnRH. We first characterized the GABAA receptors expressed by these neurons. [3H]Muscimol, but not [3H]flunitrazepam, bound with high affinity to GT1-1 cell membranes (Kd = 10.9 +/- 0.3 nM; Bmax = 979 +/- 12 fmol/mg of protein), and [3H]muscimol binding was enhanced by 3 alpha,5 alpha-THP. mRNAs encoding the alpha 1 and beta 3 subunits of the GABAA receptor were detected by the reverse transcriptase polymerase chain reaction. In agreement with binding data, the benzodiazepine-binding gamma subunit mRNA was absent. GnRH release studies showed a dose-related stimulating action of muscimol. 3 alpha,5 alpha-THP not only modulated muscimol-induced secretion but also stimulated GnRH release when administered alone. Bicuculline and picrotoxin blocked the effects of 3 alpha,5 alpha-THP and muscimol. Finally, we observed that GT1-1 neurons convert progesterone to 3 alpha,5 alpha-THP. We propose that progesterone may increase the release of GnRH by a membrane mechanism, via its reduced metabolite 3 alpha,5 alpha-THP acting at the GABAA receptor.

Animals↗
PubMed1995

Neurosteroids, via sigma receptors, modulate the [3H]norepinephrine release evoked by N-methyl-D-aspartate in the rat hippocampus.

N-Methyl-D-aspartate (NMDA, 200 microM) evokes the release of [3H]norepinephrine ([3H]NE) from preloaded hippocampal slices. This effect is potentiated by dehydroepiandrosterone sulfate (DHEA S), whereas it is inhibited by pregnenolone sulfate (PREG S) and the high-affinity sigma inverse agonist 1,3-di(2-tolyl)guanidine, at concentrations of > or = 100 nM. Neither 3 alpha-hydroxy-5 alpha-pregnan-20-one nor its sulfate ester modified NMDA-evoked [3H]NE overflow. The sigma antagonists haloperidol and 1-[2-(3,4-dichlorophenyl)-ethyl]-4-methylpiperazine, although inactive by themselves, completely prevented the effects of DHEA S, PREG S, and 1,3-di(2-tolyl)guanidine on NMDA-evoked [3H]NE release. Progesterone (100 nM) mimicked the antagonistic effect of haloperidol and 1-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-piperazine. These results indicate that the tested steroid sulfate esters differentially affected the NMDA response in vitro and suggest that DHEA S acts as a sigma agonist, that PREG S acts as a sigma inverse agonist, and that progesterone may act as a sigma antagonist. Pertussis toxin, which inactivates the Gi/o types of guanine nucleotide-binding protein (Gi/o protein) function, suppresses both effects of DHEA S and PREG S. Since sigma 1 but not sigma 2 receptors are coupled to Gi/o proteins, the present results suggest that DHEA S and PREG S control the NMDA response via sigma 1 receptors.

Animals↗
PubMed1995

Non-genomic mechanisms of action of steroid hormones.

Sex steroid hormones are known to act through intracellular receptors and their cognate hormone response elements, located in the promoters of hormone-regulated genes. However, this classical mechanism of action cannot account for a variety of rapid effects of steroids (within seconds or minutes). In this review, non-genomic modes of target cell responses to sex steroids are described. The prototypical example is the resumption of meiosis in amphibian oocytes, triggered by progesterone at the plasma membrane level. Membrane effects of progesterone may also account for sperm maturation. Other membrane-mediated effects of steroids are reviewed. Whether a steroid hormone might elicit responses from a single cell through both genomic and membrane mechanisms remains an open question.

Animals↗
PubMed1995

Biosynthesis and assay of neurosteroids in rats and mice: functional correlates.

Pregnenolone (PREG), synthesized de novo in rodent brain, is the precursor of PREG sulfate (S) and progesterone (PROG). PROG is further converted to 5 alpha-pregnane 3, 20-dione (DH PROG) and to 3 alpha-hydroxy-5 alpha-pregnan-20-one (TH PROG). PROG, DH PROG and TH PROG have been measured in the brain of male and female rats. Neither PROG nor DH PROG disappeared from brain, contrary to plasma, after combined adrenalectomy (ADX) and gonadectomy (CX). Trilostane decreased PROG and increased PREG in the brain of CX+ADX rats and mice, in accordance with a precursor to product relationship. As previously described in CX male mice, the neurosteroid DHEA and its analog 3 beta-methyl-androst-5-en-17-one (CH3-DHEA) inhibited the aggressive behavior of female mice towards lactating female intruders. The decrease of biting attacks by DHEA was definitely more prominent in females neonatally imprinted with testosterone. The degree of inhibition of aggressive behavior was related to the decrease of PREG S concentrations in brain. The memory-enhancing effects of DHEA S and PREG S in male mice have been previously documented. Infusion of PREG S (12 fmol) into the nucleus basalis magnocellularis (NBM) of the rat after the acquisition trial enhanced memory performance in a two-trial recognition task (TTRT). Conversely, TH PROG (6 fmol), which potentiates GABAergic neurotransmission, disrupted performance when injected before the acquisition trial. Accordingly, we have found a positive correlation between the performances of 2-year-old rats in the TTRT and the concentrations of PREG S in the hippocampus, namely animals which performed best had the highest steroid levels.

Adrenalectomy↗
PubMed1995

Androgen biosynthesis in the stomach: expression of cytochrome P450 17 alpha-hydroxylase/17,20-lyase messenger ribonucleic acid and protein, and metabolism of pregnenolone and progesterone by parietal cells of the rat gastric mucosa.

Dehydroepiandrosterone (DHEA) and its conjugates persist in the rat brain, for up to 1 month after ablation of both adrenals and gonads. Since DHEA synthesis in brain from pregnenolone (PREG) was excluded, we have considered other tissular sources including the digestive tract. In situ hybridization with specific oligonucleotide probes showed that the parietal cells of the gastric mucosa, contrary to other cell types, strongly expressed P450(17) alpha messenger RNA. Expression of the enzyme in the parietal cells was confirmed by immunocytochemistry with specific antibodies. An intense reaction was observed in the stomach of adult males and of cyclic or pregnant females. Access to food did not influence the intensity of immunostaining. It appeared at postnatal days 16-21, then the number of positive cells increased rapidly and leveled off at adult age. Parietal cells were released by pronase digestion of everted stomachs from adult male and female rats and were purified by density gradient centrifugation on Nycodenz. 5 x 10(4) to 1.6 x 10(6) cells were incubated with either 1 microM 14C-PREG or 14C-progesterone (14C-PROG) at 37 C under 95% O2-5% CO2, for 10-180 min. PREG was converted to 17-OH PREG and to androstenediol, whereas PROG was converted to 17-OH PROG and to testosterone. Only minute amounts of either DHEA or androstenedione, respectively, were detected at any incubation time, indicating their fast conversion to the corresponding 17 beta-hydroxysteroids. 3H-25-OH cholesterol was not metabolized to 3H-PREG, and 14C-PREG was not converted to 14C-PROG, in accordance with negative immunocytochemical results with antibodies to cytochrome P450scc and 3 beta-hydroxysteroid dehydrogenase delta 5-->4-isomerase (3 beta-HSD). In conclusion, the parietal cells, which are known as the source of gastric acid secretion, can synthesize testosterone from PROG and androstenediol from PREG. The physiological relevance of such conversions remains to be established.

Androgens↗
PubMed1995

Neurosteroids: biosynthesis and function.

The term "neurosteroids" applies to those steroids that are both synthesized in the nervous system, either de novo from cholesterol or from steroid hormone precursors, and that accumulate in the nervous system to levels at least in part independent of steroidogenic gland secretion rates. Glial cells play a major role in neurosteroid formation and metabolism. Several neurosteroids are involved in either auto- or paracrine mechanisms implicating both regulation of target gene expression and effects on membrane receptors (including those of neurotransmitters). The neuromodulatory role of neurosteroids in regulating the estrus cycle and pregnancy, stress, memory, developmental and aging processes awaits further investigation.

Animals↗
PubMed1994

Progesterone and RU486: opposing effects on human sperm.

Progesterone induced a rapid influx of calcium in capacitated human sperm, followed by a long-lasting, dose-dependent increase of intracellular free calcium. Thereafter, progesterone increased the fraction of hyperactivated sperm and the acrosome reaction. On the contrary, the progesterone antagonist RU486 (mifepristone) induced an immediate and transient, dose-dependent decrease of intracellular free calcium and a drop in the values of sperm movement parameters related to hyperactivation. Moreover, RU486 counteracted the effects of progesterone on calcium influx, lateral sperm head displacement, and the acrosome reaction. Therefore, RU486 effects were opposite to those of progesterone. The nature of the membrane receptor(s) involved is unknown. Several steroids bearing 11 beta-phenyl substitutions, with different pharmacological profiles, were also investigated. It was concluded that the steroid structure and chemical groups added to the 11 beta-phenyl influence effects on calcium influx.

Acrosome↗
PubMed1994

Actions of steroid hormones- and growth factors on glial cells of the central and peripheral nervous system.

Primary cultures of oligodendrocytes and astrocytes and purified cultures of Schwann cells were prepared respectively from forebrain and sciatic nerves of newborn rats. The effects of steroid hormones and growth factors on glial cell growth and on the production of myelin-specific proteins and lipids were investigated. Progesterone (P, 100 nM) decreased the proliferation of glial cells of the central nervous system. This inhibitory effect of P was abolished by the simultaneous administration of the antagonist RU486, thus suggesting a receptor-mediated action of the hormone. The expression of myelin-specific proteins, including the myelin basic protein (MBP) and the 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase), and of a myelin-specific lipid, galactocerebroside (Gal C), was also measured during cell differentiation under different hormonal conditions. The expression of MBP in oligodendrocytes was increased by P, and this effect was not blocked by RU486. The combined application of P and insulin promoted a synergistic stimulation of MBP expression. Insulin, by itself, also increased the number of MBP-positive oligodendrocytes in culture. The effects of P and insulin appeared to be selective as dexamethasone, dehydroepiandrosterone, pregnanolone and epidermal growth factor (EGF) had no effect. Only estradiol (E2, 500 nM) increased the number of MBP-immunoreactive cells, but in contrast to P, only a small synergism between E2 and insulin on MBP expression was observed. The expression of CNPase, another myelin-specific protein, was also increased by P and, here again, a synergy between P and insulin could be observed. In contrast, the expression of Gal C, a myelin-specific lipid, was not modified by P or other steroid hormones. Moreover, the increase in Gal C-positive cells observed in response to insulin alone was not further potentiated by P. Glial cells of the peripheral nervous system, namely Schwann cells, are also sensitive to steroid hormones. Schwann cells contain estrogen receptors, and E2 stimulates their proliferation in the presence of forskolin or dibutyryl cyclic AMP (dbcAMP). The mitogenic effect of E2 was abolished by the pure antiestrogen ICI-164,384. Insulin, at micromolar concentration, also stimulated Schwann cell growth when forskolin or dbcAMP were present in the culture medium. The mitogenic effect of insulin was mediated by insulin-like growth factor I (IGF-I) receptors. Indeed, at a physiological nanomolar concentration, IGF-I but not insulin or IGF-II, increased the proliferation of Schwann cells in synergy with forskolin. In addition, Schwann cells express receptors for IGF-I.

Animals↗
PubMed1993

Infusion of neurosteroids into the nucleus basalis magnocellularis affects cognitive processes in the rat.

The neurosteroids, pregnenolone sulfate (PREG-S) and tetrahydroprogesterone (TH-PROG), act on the GABAA receptor with antagonist or agonist-like properties, respectively. In this study the effect of the infusion of PREG-S and TH-PROG into the nucleus basalis magnocellularis (NBM) of the rat was examined in a two-trial memory task. The results show that PREG-S (5 ng in 0.5 microliter) enhances memory performance when injected after an acquisition trial; conversely TH-PROG (2 ng in 0.5 microliter) disrupts performance when injected before an acquisition trial. A role for neurosteroids in memory processes subserved by the nucleus basalis magnocellularis is of interest in view of the implication of this structure and these substances in neurodegenerative processes.

Animals↗
PubMed1993

Pregnenolone metabolism in rodent embryonic neurons and astrocytes.

The rat CNS has been previously shown to synthesize pregnenolone (PREG) and to convert it into progesterone (PROG) and some of its 5 alpha-reduced metabolites. However, the brain cell types involved in the metabolic conversions of PREG are poorly known. Selective conditions were used to obtain purified cultures of neurons and astrocytes from mouse or rat fetal striatum and cerebral cortex. Neurons converted PREG to only one identified metabolite, 20 alpha-dihydro PREG, whereas astrocytes converted PREG also to PROG, 5 alpha-dihydro PROG, and 3 alpha (3 beta)-5 alpha-tetrahydro PROG. Therefore, astrocytes can convert the neurosteroid PREG into the steroid hormone PROG and the neuromodulatory steroid 3 alpha, 5 alpha-tetrahydro PROG, whereas neurons lack the delta 5-3 beta-hydroxysteroid dehydrogenase isomerase activity (and cholesterol side-chain cleavage activity), necessary for the biosynthesis of PROG. Provision of steroid substrates is another example of cross-talk between glial cells and neurons.

Animals↗
PubMed1993

Insulin-like growth factor I: a mitogen for rat Schwann cells in the presence of elevated levels of cyclic AMP.

To develop effective procedures for improving the regeneration of peripheral nerves and for preventing the formation of neurofibromas, it is necessary to identify the different mitogens that stimulate the proliferation of Schwann cells. Insulin-like growth factor I (IGF-I), which is a potent autocrine growth factor in many tissues, is synthesized by proliferating Schwann cells. However, the role of IGF-I in stimulating their division is still uncertain. Here we show that nanomolar concentrations of IGF-I stimulate the growth of Schwann cells in primary culture. IGF-I alone was uneffective, but in the presence of forskolin (5 microM) or dibutyryl cyclic AMP (dbcAMP, 10 microM), it became a potent mitogen. Neither IGF-II nor epidermal growth factor (EGF) were effective even in the presence of forskolin. Insulin also stimulated Schwann cell proliferation in the presence of forskolin, but only at micromolar concentration. Receptors for IGF-I were visualized on the Schwann cell surface by indirect immunofluorescence staining using anti-human IGF-I receptor antibodies. Their presence was also assessed by binding assays using [125I]-IGF-I as a ligand. Scatchard analysis showed a single class of high-affinity receptors (Kd = 1.5 nM). Competitions studies with unlabeled IGF-I or insulin indicated a half-maximal displacement of [125I]-IGF-I by IGF-I at about 5 nM, while insulin was about 500-fold less effective. The number of binding sites for IGF-I was increased by exposing cells for 3 days to forskolin (-forskolin: about 5,100; + forskolin: about 12,200 binding sites/cell).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Astrocytes and neurosteroids: metabolism of pregnenolone and dehydroepiandrosterone. Regulation by cell density.

The rat central nervous system (CNS) has previously been shown to synthesize pregnenolone (PREG) and convert it to progesterone (PROG) and 7 alpha-hydroxy-PREG (7 alpha-OH PREG). Astrocytes, which participate to the regulation of the CNS function, might be involved in the metabolism of neurosteroids. Purified type 1 astrocytes were obtained from fetal rat forebrain with the use of selective culture conditions and were identified by immunostaining with specific antibodies (GFAP+, A2B5-). They were plated at low, intermediate, or high densities (2.5-5 x 10(5), 1-2 x 10(6), or 4-8 x 10(6) cells/dish, respectively) and maintained for 21 d. They were then incubated with 14C-PREG and 14C-DHEA for 24 h and the steroids extracted from cells and media were analyzed. Most radioactive derivatives were released into incubation media. Two metabolic pathways were mainly observed. PREG and DHEA were oxidized to PROG and androstenedione (ADIONE), respectively, [3 beta-hydroxysteroid-dehydrogenase, delta 5-->4 3-ketosteroid-isomerase (3 beta-HSD) activity], and converted to 7 alpha-OH PREG and 7 alpha-OH DHEA, respectively (7 alpha-hydroxylase activity). After low density plating, the formation of PROG and ADIONE was approximately 10% of incubated radioactivity, tenfold larger than that of 7 alpha-hydroxylated metabolites. In contrast, after high density plating, low levels of PROG and ADIONE were formed, whereas the conversion to either 7 alpha-OH PREG or 7 alpha-OH DHEA was > or = 50%. The results expressed per cell indicated that the 3 beta-HSD activity was almost completely inhibited at high cell density, in contrast to the 7 alpha-hydroxylation which was maintained or increased. The pattern of steroid metabolism was related to cell density at the time of measurement and not to an early commitment of cells: when primary cultures were plated at high density (8 x 10(6) cells/dish), then subcultured after several dilutions (3-, 9-, or 27-fold), the 3 beta-HSD activity was recovered only at low density. Furthermore, when 5 x 10(5) cells were centrifuged and the resulting clusters were plated, 3 beta-HSD activity was decreased, whereas steroid 7 alpha-hydroxylation was enhanced. This implies that cell density per se, but neither cell number nor a diffusible factor(s) is involved in the regulation of steroid metabolism. We conclude that astrocytes in culture metabolize PREG and DHEA, and that the metabolic conversions and, therefore, the related enzymatic activities depend on cell-to-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗