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Showing papers on "Pregnenolone published in 2001"


Journal ArticleDOI
TL;DR: This cell line is considered to be a very useful model for understanding the regulation of steroidogenesis, cell growth, and apoptosis in human granulosa cells.
Abstract: We established a steroidogenic human ovarian granulosa-like tumor cell line, designated KGN, from a patient with invasive ovarian granulosa cell carcinoma. KGN had a relatively long population doubling time of about 46.4 h and had an abnormal karyotype of 45,XX, 7q-, -22. A steroid analysis of the cultured medium by RIA performed 5 yr after the initiation of culture showed that KGN was able to secrete pregnenolone and progesterone, and both dramatically increased after stimulation with (Bu)(2)cAMP. However, little or no secretion of 17alpha-hydroxylated steroids, dehydroepiandrosterone, androstenedione, or estradiol was observed. The aromatase activity of KGN was relatively high and was further stimulated by (Bu)(2)cAMP or FSH. These findings showed a pattern similar to that of steroidogenesis in human granulosa cells, thus allowing analysis of naturally occurring steroidogenesis in human granulosa cells. Fas-mediated apoptosis of KGN was also observed, which mimicked the physiological regulation of apoptosis in normal human granulosa cells. Based on these findings, this cell line is considered to be a very useful model for understanding the regulation of steroidogenesis, cell growth, and apoptosis in human granulosa cells.

454 citations


Book ChapterDOI
TL;DR: A physiological function of neurosteroids in the central nervous system is strongly suggested by the role of hippocampal PREGS with respect to memory performance, observed in aging rats, and a new mechanism of PREG action discovered in the brain involves specific steroid binding to microtubule associated protein and increased tubulin polymerization for assembling microtubules.
Abstract: Neurosteroids are synthetisized in the central and the peripheral nervous system, in glial cells, and also in neurons, from cholesterol or steroidal precursors imported from peripheral sources. They include 3 beta-hydroxy-delta 5-compounds, such as pregnenolone (PREG) and dehydroepiandrosterone, their sulfate esters, and compounds known as reduced metabolites of steroid hormones, such as the tetrahydroderivative of progesterone 3 alpha-hydroxy-5 alpha-pregnan-20-one. These neurosteroids can act as modulators of neurotransmitter receptors, such as GABAA, NMDA, and sigma 1 receptors. Progesterone itself is also a neurosteroid, and a progesterone receptor has been detected in peripheral and central glial cells. At different sites in the brain, neurosteroid concentrations vary according to environmental and behavioral circumstances, such as stress, sex recognition, or aggressiveness. A physiological function of neurosteroids in the central nervous system is strongly suggested by the role of hippocampal PREGS with respect to memory performance, observed in aging rats. In the peripheral nervous system, a role for PROG synthesized in Schwann cells has been demonstrated in remyelination after cryolesion of the sciatic nerve in vivo and in cultures of dorsal root ganglia. A new mechanism of PREG action discovered in the brain involves specific steroid binding to microtubule associated protein and increased tubulin polymerization for assembling microtubules. It may be important to study the effects of abnormal neurosteroid concentration/metabolism in view of the possible treatment of functional and trophic disturbances of the nervous system.

326 citations


Journal ArticleDOI
TL;DR: Observations imply that hippocampal neurons synthesize neurosteroids, which may be stimulated and regulated by glutamate-mediated synaptic communication.
Abstract: Neurosteroidogenesis has not been well elucidated due to the very low level of steroidogenic proteins in the brain. Here we report the first demonstration of the neuronal localization of neurosteroidogenic systems as well as the regulation of neurosteroidogenic activity in the adult rat hippocampus. Significant localization of cytochrome P450scc was observed in pyramidal neurons and granule neurons by means of immunohistochemical staining of slices. We also observed the colocalization, in hippocampal neurons, of P450scc with redox partners, hydroxysteroid sulfotransferase and steroidogenic acute regulatory protein. The distributions of astroglial cells and oligodendroglial cells showed very different patterns from that of the P450scc-containing cells. The expression of P450scc, redox partners, the sulfotransferase, and steroidogenic acute regulatory protein was also confirmed by Western blot analysis. The process of active neurosteroidogenesis was stimulated by exposing neurons to N-methyl-D-aspartate. Upon stimulation with N-methyl-D-aspartate, Ca(2+) influx through the N-methyl-D-aspartate subtype of glutamate receptors occurred, and significant net production of pregnenolone and pregnenolone sulfate was observed in the hippocampus. This neurosteroid production was considerably suppressed by the addition of antagonists of N-methyl-D-aspartate receptors, by Ca(2+) depletion, or by the addition of an inhibitor of P450scc. Upon stimulation with N-methyl-D-aspartate, the processing of full-length steroidogenic acute regulatory protein (37-kDa) to the truncated 30-kDa steroidogenic acute regulatory protein was observed. Taken together, these observations imply that hippocampal neurons synthesize neurosteroids. This synthesis may be stimulated and regulated by glutamate-mediated synaptic communication.

242 citations


Journal ArticleDOI
TL;DR: The therapeutical interest of selective sigma1 receptor agonists in alleviating aging-related cognitive deficits will be discussed.

223 citations


Journal ArticleDOI
TL;DR: This review summarizes data in animals and humans in favor of a role of neurosteroids in cognitive aging and proposes the use of new methods of quantification of Neurosteroids as a useful tool for understanding their respective role in improving learning and memory impairments associated with normal aging and/or with pathological aging, such as Alzheimer's disease.

198 citations


Journal ArticleDOI
TL;DR: The demonstration that the neurosteroid pregnenolone sulfate (PREGS) is active on memory function at both the physiological and pharmacological levels led to examining in detail the effects of the steroid on spatial working memory by using a two-trial recognition task in a Y-maze, a paradigm based on the natural drive in rodents to explore a novel environment.
Abstract: The demonstration that the neurosteroid pregnenolone sulfate (PREGS) is active on memory function at both the physiological and pharmacological levels led to us examining in detail the effects of the steroid on spatial working memory by using a two-trial recognition task in a Y-maze, a paradigm based on the natural drive in rodents to explore a novel environment. Dose–response studies in young male adult Sprague–Dawley rats and Swiss mice, after the postacquisition intracerebroventricular injection of steroid, showed an U-inverted curve for memory performance and indicated a greater responsiveness in rats compared with mice. Remarkably, the synthetic (−) enantiomer of PREGS not only also displayed promnesiant activity, but its potency was 10 times higher than that of the natural steroid. Intracerebroventricular coadministration experiments with dl-2-amino-5-phosphonovaleric acid, a competitive selective antagonist of the N-methyl-d-aspartate receptor, abolished the memory-enhancing effect of PREGS, but not that of the PREGS enantiomer, evoking enantiomeric selectivity at the N-methyl-d-aspartate receptor and/or different mechanisms for the promnestic function of the two enantiomers.

142 citations


Journal ArticleDOI
TL;DR: An additional unexpected mechanism of steroid action is reported here: pregnenolone binds to neural microtubule-associated protein of type 2 (MAP2) and increases both the rate and extent of tubulin polymerization, forming microtubules of normal electron microscopic appearance, which may play a role in regulatingmicrotubule formation and dynamics and thus neuronal plasticity and function.

127 citations


Journal ArticleDOI
TL;DR: The results indicate that human brain makes steroids in a cell‐specific manner and suggest that dehydroepiandrosterone synthesis can be regulated by intracellular free radicals.
Abstract: Neurosteroids in rodents can originate from peripheral tissues or be locally synthesized in specific brain areas There is, as yet, no information about the synthesis and regulation of neurosteroids in human brain We examined the ability of human brain cells to synthesize steroids from a radiolabeled precursor and the mRNA and protein expression of key components of peripheral steroidogenic machinery Oligodendrocytes are the source of pregnenolone in human brain Human astrocytes do not synthesize radiolabeled pregnenolone, nor do human neurons There is potential for all three cell types to metabolize pregnenolone to other neurosteroids, including dehydroepiandrosterone mRNA and protein for cytochrome P450 17α-hydroxylase were found in all cell types, although no activity could be demonstrated We examined the ability of the cells to make dehydroepiandrosterone via an alternative pathway induced by treatment with Fe2+ Oligodendrocytes and astrocytes make dehydroepiandrosterone via this pathway, but neurons do not In searching for a natural regulator of dehydroepiandrosterone formation, we observed that treating oligodendrocytes with β-amyloid, which increases reactive oxygen species, also increased dehydroepiandrosterone formation These effects of β-amyloid were blocked by vitamin E These results indicate that human brain makes steroids in a cell-specific manner and suggest that dehydroepiandrosterone synthesis can be regulated by intracellular free radicals

115 citations


Journal ArticleDOI
TL;DR: A dual response mechanism of T(3) on steroidogenesis and StAR expression, and on LH receptor (LHR) expression and binding in mLTC-1 cells is reported, which further characterize the thyroid-gonadal connection and provide insights into the mechanisms for a dual regulatory role of thyroid hormone in Leydig cell functions.
Abstract: Recently, we demonstrated that triiodothyronine (T3) stimulated steroid hormone biosynthesis and steroidogenic acute regulatory (StAR) protein expression in mLTC-1 mouse Leydig tumor cells through the mediation of steroidogenic factor 1 (SF-1). We now report a dual response mechanism of T3 on steroidogenesis and StAR expression, and on LH receptor (LHR) expression and binding in mLTC-1 cells. T3 acutely (8 h), induced a 260% increase in StAR messenger RNA (mRNA) expression over the basal level which was coincident with an increase in progesterone (P) production. In contrast, chronic stimulation with T3 (beyond 8 h), resulted in an attenuation of StAR expression and P production. This attenuation was most likely caused by a decrease in cholesterol delivery to the inner mitochondrial membrane as demonstrated by incubations with the hydrophilic steroid precursors, 22R hydroxycholesterol and pregnenolone, which restored P synthesis. In similar studies, chronic treatment with T3 increased the levels of cytochr...

112 citations


Journal ArticleDOI
TL;DR: Results indicate that in different endocrine steroid-secreting cells P450 (scc), 3 beta-HSD and P450(c17) have the same association with cytoplasmic organelles, suggesting similar intracellular pathways for biosynthesis of steroid hormones.
Abstract: The biosynthesis of steroid hormones in endocrine steroidsecreting glands results from a series of successive steps involving both cytochrome P450 enzymes, which are mixed-function oxidases, and steroid dehydrogenases. So far, the subcellular distribution of steroidogenic enzymes has been mostly studied following subcellular fractionation, performed in placenta and adrenal cortex. In order to determine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural localization of the three key enzymes: P450 side chain cleavage (scc) which converts cholesterol to pregnenolone; 3-hydroxysteroid dehydrogenase (3HSD) which catalyzes the conversion of 3-hydroxy-5ene steroids to 3-oxo-4-ene steroids (progesterone and androstenedione); and P450c17 which is responsible for the transformation of C21 into C19 steroids (dehydroepiandrosterone and androstenedione). Immunogold labeling was used to localize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P450scc and 3-HSD immunoreactivities were detected in the reticular, fascicular and glomerular zones. P450scc was exclusively found in large mitochondria. In contrast, 3-HSD antigenic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P450 c17 could not be detected in adrenocortical cells. In the testis, the three enzymes were only found in Leydig cells. Immunolabeling for P450 scc and 3-HSD was restricted to mitochondria, while P450 c17 immunoreactivity was exclusively observed in ER. In the ovary, P450 scc and 3-HSD immunoreactivities were found in granulosa, theca interna and corpus luteum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P450c17 could also be detected in theca interna cells of large developing and mature follicles. As observed in Leydig cells, P450c17 immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P450scc ,3 -HSD and P450c17 have the same association with cytoplasmic organelles (with the exception of 3-HSD in Leydig cells), suggesting similar intracellular pathways for biosynthesis of steroid hormones.

111 citations


Journal ArticleDOI
TL;DR: Data suggest that PXR may play certain roles in perinatal period, possibly in the protection of the feto-maternal system from the toxic effect of endogenous steroids and foreign substrates.

Journal ArticleDOI
TL;DR: Characterization of the newly described human hydroxysteroid sulfotransferase SULT2B1 isoforms shows that these enzymes are selective for the sulfation of 3 beta-hydroxysteroids, such as pregnenolone, epiandrosterone, DHEA, and androstenediol.

Book ChapterDOI
TL;DR: Activation of 7 alpha-hydroxylation by increased close contacts of astrocytes and after glucocorticoid treatment suggested that the regulated production of 7alpha-hydroxysteroids was a key event for the neuroprotection conferred by neurosteroids.
Abstract: Robert Morfin Laboratoire de Biotechnologie Conservatoire National des Arts et M~tiers 75003, Paris, France Luboslav St6rka Institute of Endocrinology 116 94 Prague 1, Czech Republic I. Neurosteroid Metabolism in the Brain II. 7~-Hydroxylation Studies in the Brain III. 7/3-Hydroxylation Studies in the Brain 1V. 7~-Hydroxy-DHEA and 7/3-Hydroxy-DHEA as Native Anti-glucocorticoids V. The Brain and Other Organs VI. Sex Steroid Metabolism in the Brain VII. Concluding Remarks References The neurosteroids pregnenolone (PREG) and dehydroepiandrosterone (DHEA) are precursors for both oxidized and hydroxylated metabolites in the brain. Thus, brain production of 7-hydroxylated derivatives is sec- ond to that in the liver, and P4507Bl-containing hippocampus is the major site for 7c~-hydroxylation. Other P450s and/or oxido-reductive mechanisms may be responsible for 7/3-hydroxylation. In addition to regulating neuros- teroid brain levels, when produced, the 7-hydroxylated derivatives of PREG and DHEA were investigated for antiglucocorticoid-mediated neuropro- tective potencies, and both 7c~- and 7/3-hydroxy-DHEA were efficient in preventing the nuclear uptake of [3H]dexamethasone-activated glucocor- ticoid receptor in brain cells. Activation of 7c~-hydroxylation by increased close contacts of astrocytes and after glucocorticoid treatment suggested that the regulated production of 7c~-hydroxysteroids was a key event for the neuroprotection conferred by neurosteroids. ~.~2001 Academic Press.

Journal ArticleDOI
TL;DR: An E. coli expression system is developed that permits the efficient production of biochemically homogeneous ApoD via secretion into the bacterial periplasm and it appears that ApiD discriminates well in its binding function between closely related compounds.
Abstract: Apolipoprotein D (ApoD) constitutes an atypical lipoprotein in so far as it is predominantly found associated with HDL particles but belongs to the lipocalin structural family. Apart from its involvement in serum lipid transport it is abundant in various tissues, and differing physiological functions have been ascribed to it. We have now developed an E. coli expression system that permits the efficient production of biochemically homogeneous ApoD via secretion into the bacterial periplasm. Detailed ligand binding studies by fluorescence titration revealed that progesterone and arachidonic acid are complexed with dissociation constants both in the 1 microM range, whereas the presumed ligands pregnenolone, bilirubin and E-3M2H are not recognized by the recombinant protein. In contrast with previous reports it thus appears that ApoD discriminates well in its binding function between closely related compounds.

Journal ArticleDOI
TL;DR: Results are consistent with the conclusion that compromise of StAR-mediated cholesterol transport may play a key role in age-related reductions in Leydig cell steroidogenesis.
Abstract: Primary points of control in steroidogenesis are the transport of cholesterol from intracellular stores to the inner mitochondrial membrane, and the subsequent conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage enzyme (P450 scc ). Testosterone production has been shown to decline in Brown Norway rat Leydig cells as the rats age. To better understand the mechanism by which aging Leydig cells lose steroidogenic function, we examined the effect of aging on steroidogenic acute regulatory protein (StAR), an important Leydig cell cholesterol transfer protein, and on P450 scc . Leydig cells isolated from middle-aged (14 months) and old (24 months) rats produced significantly less testosterone than cells from young (4 months) rats. StAR mRNA (1.7 kilobase [kb]) was significantly reduced in Leydig cells from middle-aged and old rats, by 26% and 52%, respectively. Significant reductions also were seen in the steady-state levels of mRNA for P450 scc , of 29% and 50%, respectively. Western blots revealed significant reductions in StAR protein, by 47% and 74%, respectively, and in P450 scc protein, by 38% and 54%, respectively. In response to LH stimulation in vitro, testosterone production by Leydig cells in young, middle-aged, and old rats increased by 30-, 40-, and 33-fold, respectively, although the amounts of testosterone produced by the young cells significantly exceeded that produced by the middle-aged and old cells. StAR protein also increased in response to LH by 1.4-, 3-, and 11-fold, respectively, whereas P450 scc protein remained unchanged. These results are consistent with the conclusion that compromise of StAR-mediated cholesterol transport may play a key role in age-related reductions in Leydig cell steroidogenesis. However, because P450 scc is reduced in old Leydig cells, the reaction catalyzed by this enzyme would be rate-limiting under circumstances in which saturating amounts of cholesterol entered the mitochondria.

Journal ArticleDOI
TL;DR: Using median blood levels as a cut-off indicator, higher blood levels of DHEA and DHEAS were associated with fewer ADHD symptoms, in particular hyperactivity symptomatology, suggesting a possible protective effect of various neurosteroids on the expression of ADHD symptom atology.
Abstract: Neurosteroids are important neuroactive substrates with demonstrated involvement in several neurophysiological and disease processes. Attention deficit hyperactivity disorder (ADHD) has been associated with dysregulation of the catecholaminergic and serotonergic systems, however its relationship to irregularities or changes in neurosteroid levels remains unknown. We examined the relationship between blood levels of dehydroepiandrosterone (DHEA), its principal precursor pregnenolone and its principal metabolite dehydroepiandrosterone sulphate (DHEAS) in 29 young male subjects aged 7-15 years with DSM-IV criteria of ADHD. Subjects were evaluated by a specially designed scale, following which patients were divided into two groups according to severity of symptomatology. Results indicated significant inverse correlations between clinical symptomatology and levels of DHEA and pregnenolone in the total group. These inverse correlations were particularly evident in the less severe group of subjects. Levels of DHEA and DHEAS were inversely correlated with the hyperactivity subscale. Furthermore, using median blood levels as a cut-off indicator, higher blood levels of DHEA and DHEAS were associated with fewer ADHD symptoms, in particular hyperactivity symptomatology. Our findings suggest a possible protective effect of various neurosteroids on the expression of ADHD symptomatology.

Journal ArticleDOI
TL;DR: The expression of messenger RNA (mRNA) encoding for the enzyme cytochrome P450 17alpha-hydroxylase/c17,20-lyase (P450(17alpha,lyase), which converts pregnenolone to dehydroepiandrosterone via 17 alpha-hydroxyprogesterone, suggests that the avian brain possesses P450 (17alpha) as well as P450scc and 3beta-HSD in both sexes.

Journal ArticleDOI
TL;DR: Results show that all five of these metabolites rise markedly during pregnancy and suggest that alterations in progesterone metabolites may be involved in the mood changes of pregnancy and the puerperium.
Abstract: Progesterone and its 5 alpha reduced metabolite, 5 alpha-dihydroprogesterone, rise greatly in pregnancy. Both are known to have anesthetic properties, as do a number of other ring A-reduced progesterone metabolites. The possible significance of these steroids with respect to the mood changes that are common in pregnancy and in the puerperium has not been explored. In this study, pregnenolone, progesterone, and five neuroactive progesterone metabolites: the 5 alpha and 5 beta dihydroprogesterones (DHP), and three tetrahydroprogesterones (THP)-3 alpha,5 alpha-THP, 3 beta,5 beta-THP, and 3 beta,5 alpha-THP-were studied at various stages of pregnancy and in the early postpartum period. Levels of all of the steroids rose greatly during pregnancy (P < 0.001), being highest for progesterone (562-fold the follicular level), 5 alpha-DHP (161-fold), 3 beta,5 alpha-THP (56-fold), 3 alpha,5 alpha-THP (37-fold), pregnenolone (30-fold), 5 beta-DHP (16-fold) and 3 beta,5 beta-THP (16-fold) at 37 wk of gestation. During the period 2-7 d postpartum, the level of progesterone fell precipitously, whereas those of pregnenolone and the metabolites fell more slowly and mean levels were still elevated compared with follicular levels 2 wk after delivery. By 7 wk postpartum, only 3 alpha,5 alpha-tetrahydroprogesterone and 3 beta,5 beta-tetrahydroprogesterone remained slightly elevated (P < or = 0.012 and 0.007, respectively). Mean levels of the progesterone metabolites tended to be higher in depressed patients compared with controls, and this difference reached significance for 5 alpha-dihydroprogesterone both at 27 wk (P = 0.04) and at 37 wk (P = 0.02) of gestation (combined, P = 0.003). These results show that all five of these metabolites rise markedly during pregnancy and suggest that alterations in progesterone metabolites may be involved in the mood changes of pregnancy and the puerperium.

Journal ArticleDOI
TL;DR: The results suggested that the physiological level of lactate stimulated testosterone production in rat Leydig cells through a mechanism involving the increased activities of adenylyl cyclase, cytochrome P450scc, and L‐type Ca2+ channel.
Abstract: Previously we found that the increased plasma testosterone levels in male rats during exercise partially resulted from a direct and luteinizing hormone (LH)-independent stimulatory effect of lactate on the secretion of testosterone. In the present study, the acute and direct effects of lactate on testosterone production by rat Leydig cells were investigated. Leydig cells from rats were purified by Percoll density gradient centrifugation subsequent to enzymatic isolation of testicular interstitial cells. Purified rat Leydig cells (1 x 10(5) cells/ml) were in vitro incubated with human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (an adenylyl cyclase activator, 10(-5) M), or 8-bromo-adenosine-3':5'-cyclic monophosphate (8-Br-cAMP, 10(-4) M), SQ22536 (an adenylyl cyclase inhibitor, 10(-6)-10(-5) M), steroidogenic precursors (25-hydroxy-cholesterol, pregnenolone, progesterone, and androstenedione, 10(-5) M each), nifedipine (a L-type Ca(2+) channel blocker, 10(-5)-10(-4) M), or nimodipine (a potent L-type Ca(2+) channel antagonist, 10(-5)-10(-4) M) in the presence or absence of lactate at 34 degrees C for 1 h. The concentration of medium testosterone was measured by radioimmunoassay. Administration of lactate at 5-20 mM dose-dependently increased the basal testosterone production by 63-187% but did not alter forskolin- and 8-Br-cAMP-stimulated testosterone release in rat Leydig cells. Lactate at 10 mM enhanced the stimulation of testosterone production induced by 25-hydroxy-cholesterol in rat Leydig cells but not other steroidogenic precursors. Lactate (10 mM) affected neither 30- nor 60-min expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein. The lactate-stimulated testosterone production was decreased by administration of nifedipine or nimodipine. These results suggested that the physiological level of lactate stimulated testosterone production in rat Leydig cells through a mechanism involving the increased activities of adenylyl cyclase, cytochrome P450scc, and L-type Ca(2+) channel.

Journal ArticleDOI
TL;DR: Serum levels of allopregnanolone, pregnenolone sulfate, and dehydroepiandroster one sulfate were measured in 8 male patients with generalized anxiety disorder (GAD) and 8 healthy control subjects and suggest that patients with GAD have significantly lower levels of pregnenoline sulfate.
Abstract: Serum levels of allopregnanolone, pregnenolone sulfate, and dehydroepiandrosterone sulfate were measured in 8 male patients with generalized anxiety disorder (GAD) and 8 healthy control subjects Results suggest that patients with GAD have significantly lower levels of pregnenolone sulfate than control subjects

Journal ArticleDOI
TL;DR: Results show that pregnenolone has neuroprotective effects against both glutamate and amyloid beta protein neuropathology and prevention of glucocorticoid receptor (GR) localization to the nucleus may be involved in the observed neuroProtective effects of pregnenolsone against glutamate neurotoxicity.
Abstract: In the present work we have examined whether the neurosteroid pregnenolone has any neuroprotective effects against glutamate and amyloid beta protein neurotoxicity using immortalized clonal mouse hippocampal cell line (HT-22). The neurosteroid pregnenolone protects HT-22 cells against both 5 mM glutamate and 2 μM amyloid beta protein induced cell death in a concentration dependent manner. Optimum protection was attained at 500 nM pregnenolone, against both 5 mM glutamate as well as 2 μM amyloid beta protein induced HT-22 cell death. Furthermore, using confocal immunoflourescence microscopy we observed that 20 hours of treatment with 5 mM glutamate resulted in intense nuclear localization of the glucocorticoid receptor (GR) in HT-22 cells as compared to control untreated cells. Interestingly, 500 nM pregnenolone treatment for 24 hours, followed by 20 hours treatment with 5 mM glutamate resulted in dramatic reduction in GR nuclear localization. These results show that (i) pregnenolone has neuroprotective effects against both glutamate and amyloid beta protein neuropathology and (ii) prevention of glucocorticoid receptor (GR) localization to the nucleus may be involved in the observed neuroprotective effects of pregnenolone against glutamate neurotoxicity.

Journal ArticleDOI
TL;DR: The influence of membrane cholesterol on some pharmacological properties of the GABAA receptor was investigated in acutely dissociated rat hippocampal neurones with whole cell patch clamp recording and effects on the non‐steroidal potentiators of GABA of cholesterol‐enrichment or addition of epicholesterol to the neurones are suggested to be due to changes in membrane fluidity.
Abstract: 1. The influence of membrane cholesterol on some pharmacological properties of the GABA(A) receptor was investigated in acutely dissociated rat hippocampal neurones with whole cell patch clamp recording. The cholesterol levels were varied between 56% and 235% control using methyl-beta-cyclodextrin as the cholesterol carrier. 2. Enrichment of neurones with cholesterol increased the effects of the non-steroidal GABA potentiators propofol, flunitrazepam and pentobarbitone. A similar result was obtained after pre-incubation of neurones with epicholesterol, the 3alpha-hydroxy isomer of cholesterol. 3. In contrast, the effects of the steroidal GABA potentiators pregnanolone and alfaxalone were reduced by cholesterol enrichment, but not by epicholesterol. Depletion of membrane cholesterol increased the potentiation of GABA by pregnanolone and alfaxalone but did not affect the non-steroidal potentiators. 4. The steroidal antagonist of GABA, pregnenolone sulphate, reduced the maximum response to GABA. This effect, also, was diminished in cholesterol-enriched neurones and enhanced in cholesterol-depleted neurones. 5. The effects of the cholesterol manipulations that were selective for the steroidal modulators of GABA are suggested to arise from direct interactions between membrane cholesterol and the GABA(A) receptor. The separate effects on the non-steroidal potentiators of GABA of cholesterol-enrichment or addition of epicholesterol to the neurones are suggested to be due to changes in membrane fluidity. 6. In view of the likely physiological modulation of GABA(A) receptors by endogenous neuroactive steroids and evidence of the in vivo lability of membrane cholesterol, the present observations may have physiological as well as pharmacological relevance.

Journal ArticleDOI
TL;DR: A repetitive increase in the brain concentrations of neuroactive steroids may contribute to the therapeutic action of fluoxetine, as shown in rats chronically treated with this drug.
Abstract: Rationale: Recent preclinical and clinical studies have shown that selective serotonin re-uptake inhibitors modulate neurosteroid synthesis in an opposite manner. Objectives: The action of long-term administration of fluoxetine was investigated on the peripheral and central concentrations of 3α,5α-tetrahydroprogesterone (3α,5α-TH PROG) and 3α,5α-tetrahydrodeoxycorticosterone (of 3α,5α-TH DOC), progesterone, and pregnenolone in rats. We also investigated the effect of chronic treatment with fluoxetine on the foot-shock stress-induced increase in the plasma and brain concentrations of these steroids. Methods: Fluoxetine was administered acutely (20 mg/kg) or chronically (10 mg/kg, once daily for 15 days). Steroids were extracted from plasma and brain, separated and purified by means of high-performance liquid chromatography, and quantified by means of radioimmunoassay. Results: A single dose of fluoxetine (20 mg/kg, i.p.) induced in 20 min significant increases in the cerebral cortical and plasma concentrations of 3α,5α-TH PROG (+96% and +13%, respectively), 3α,5α-TH DOC (+129 and +31%, respectively), progesterone (+111 and +58%, respectively), and pregnenolone (+151 and +59%, respectively). In addition, the plasma concentration of corticosterone was also significantly increased (+24%) after acute administration of fluoxetine. In contrast, long-term administration of fluoxetine reduced the basal concentrations of these various steroids (ranging from –22 to –43%), measured 48 h after the last drug injection, in both brain and plasma. A challenge injection of fluoxetine (20 mg/kg, i.p.), however, was still able to increase the concentrations of steroids in both the brain and plasma of rats chronically treated with this drug. Acute foot-shock stress increased the cortical and plasma concentrations of steroids in rats chronically treated with fluoxetine to extents similar to those apparent in control rats. Conclusions: A repetitive increase in the brain concentrations of neuroactive steroids may contribute to the therapeutic action of fluoxetine.

Journal ArticleDOI
TL;DR: The observations bring a molecular basis to the modulatory role of σ1 receptors in memory, and reveal that the anti‐amnesic action of neuroactive steroids may not similarly involve an interaction with ρ1 receptors.
Abstract: 1. The sigma(1) (sigma(1)) receptor cDNA was cloned in several animal species. Molecular tools are now available to identify its endogenous effectors, such as neuroactive steroids, and to establish its precise physiological role. In particular, the sigma(1) receptor is involved in memory processes, as observed in pharmacological and pathological rodent models of amnesia. 2. In order to establish the involvement of sigma(1) receptors in memory, a 16-mer oligodeoxynucleotide antisense to the sigma(1) receptor cDNA (aODN), and its mismatched control (mODN) were prepared and centrally administered into the mouse brain. The anti-amnesic effects induced by the selective sigma(1) agonist PRE-084 and the steroid dehydroepiandrosterone (DHEA) sulphate or pregnenolone sulphate were examined in ODN-treated animals. 3. The aODN treatment failed to affect the dissociation constant (K(d)) but significantly decreased the number of sigma(1) sites (B(max)) labelled with [(3)H]-(+)-SKF-10,047 in the hippocampus and cortex. In these structures, the in vivo binding levels were also diminished, according to the dose and number of injections, as compared with control animals injected with saline or mODN. 4. Cannulation and injections failed to affect the open-field behaviour of the animals. However, the anti-amnesic effects of PRE-084 and DHEA sulphate against the dizocilpine-induced impairments were blocked after aODN treatment in the short- and long-term memory tests. The anti-amnesic effects of pregnenolone sulphate remained unchanged. 5. These observations bring a molecular basis to the modulatory role of sigma(1) receptors in memory, and reveal that the anti-amnesic action of neuroactive steroids may not similarly involve an interaction with sigma(1) receptors.

Journal ArticleDOI
TL;DR: It is reported that progestins alter cholesterol synthesis in HepG2 cells and rat testes to increase levels of major MAS (FF‐MAS and T‐MAS), which provides a plausible explanation for the elevated concentration of MAS in gonads and suggests a new role for progestin in fertility.
Abstract: The resumption of meiosis is regulated by meiosis-preventing and meiosis-activating substances in testes and ovaries. Certain C29 precursors of cholesterol are present at elevated levels in gonadal tissue, but the mechanism by which these meiosis-activating sterols (MAS) accumulate has remained an unresolved question. Here we report that progestins alter cholesterol synthesis in HepG2 cells and rat testes to increase levels of major MAS (FF-MAS and T-MAS). These C29 sterols accumulated as a result of inhibition of Delta24-reduction and 4alpha-demethylation. Progesterone, pregnenolone, and 17alpha-OH-pregnenolone were potent inhibitors of Delta24-reduction in an in vitro cell assay and led to the accumulation of desmosterol, a Delta5,24 sterol precursor of cholesterol. A markedly different effect was observed for 17alpha-OH-progesterone, which caused the accumulation of sterols associated with inhibition of 4alpha-demethylation. The flux of 13C-acetate into lathosterol and cholesterol was decreased by progestins as measured by isotopomer spectral analysis, whereas newly synthesized MAS accumulated. The combined evidence that MAS concentrations can be regulated by physiological levels of progestins and their specific combination provides a plausible explanation for the elevated concentration of MAS in gonads and suggests a new role for progestins in fertility.

Journal ArticleDOI
TL;DR: It is suggested that 24-h exposure to OP + hCG has no effect on 17beta-hydroxysteroid dehydrogenase, but that it inhibits the 17alpha-hydroxylase/C17-20 lyase step, which converts progesterone to androstenedione, and potentially OP could inhibit cholesterol side/chain cleavage activity.

Journal ArticleDOI
TL;DR: Data suggest that PREG-S can influence cognitive processes, particularly in senescent subjects, through a modulation of ACh neurotransmission associated with paradoxical sleep modifications; furthermore the recent data suggest a role for neurosteroids in the modulation of hippocampal neurogenesis.

Journal ArticleDOI
TL;DR: Investigating the immunohistochemical localization and activity of 3beta-hydroxysteroid dehydrogenase, 17beta-HSD and hydroxysteroid sulfotransferase in the frog brain demonstrates that the process of neurosteroid biosynthesis occurs in amphibians as previously seen in mammals.

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TL;DR: The possibility that DHEA, among other neurosteroids, could modulate directly the age-associated impairment of PKC signal transduction is addressed and experimental evidence that D HEA can revert the alteration of RACK-1 anchoring protein expression is provided.

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TL;DR: Progesterone production may be constant during embryonic and post-hatched development and in adulthood, whereas 5beta-dihydroprogesterone may be produced actively only in embryonic life due to 5 beta-reductase.