Showing papers on "Pregnenolone published in 2004"

Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017α and P450 aromatase localized in neurons

Abstract: In adult mammalian brain, occurrence of the synthesis of estradiol from endogenous cholesterol has been doubted because of the inability to detect dehydroepiandrosterone synthase, P45017α. In adult male rat hippocampal formation, significant localization was demonstrated for both cytochromes P45017α and P450 aromatase, in pyramidal neurons in the CA1–CA3 regions, as well as in the granule cells in the dentate gyrus, by means of immunohistochemical staining of slices. Only a weak immunoreaction of these P450s was observed in astrocytes and oligodendrocytes. ImmunoGold electron microscopy revealed that P45017α and P450 aromatase were localized in pre- and postsynaptic compartments as well as in the endoplasmic reticulum in principal neurons. The expression of these cytochromes was further verified by using Western blot analysis and RT-PCR. Stimulation of hippocampal neurons with N-methyl-d-aspartate induced a significant net production of estradiol. Analysis of radioactive metabolites demonstrated the conversion from [3H]pregnenolone to [3H]estradiol through dehydroepiandrosterone and testosterone. This activity was abolished by the application of specific inhibitors of cytochrome P450s. Interestingly, estradiol was not significantly converted to other steroid metabolites. Taken together with our previous finding of a P450scc-containing neuronal system for pregnenolone synthesis, these results imply that 17β-estradiol is synthesized by P45017α and P450 aromatase localized in hippocampal neurons from endogenous cholesterol. This synthesis may be regulated by a glutamate-mediated synaptic communication that evokes Ca2+ signals.

A novel pathway for sequential transformation of 7‐dehydrocholesterol and expression of the P450scc system in mammalian skin

Abstract: Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side-chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7-dehydrocholesterol (7-DHC; precursor to cholesterol and vitamin D3) to 7-dehydropregnenolone (7-DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7-DHC into 7-DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7-DHC can form 7-DHP through P450scc side-chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7-steroidal dienes (7-DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB-induced intramolecular rearrangements to vitamin D3-like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB.

Local synthesis and dual actions of progesterone in the nervous system: neuroprotection and myelination

Abstract: Progesterone (PROG) is synthesized in the brain, spinal cord and peripheral nerves. Its direct precursor pregnenolone is either derived from the circulation or from local de novo synthesis as cytochrome P450scc, which converts cholesterol to pregnenolone, is expressed in the nervous system. Pregnenolone is converted to PROG by 3beta-hydroxysteroid dehydrogenase (3beta-HSD). In situ hybridization studies have shown that this enzyme is expressed throughout the rat brain, spinal cord and dorsal root ganglia (DRG) mainly by neurons. Macroglial cells, including astrocytes, oligodendroglial cells and Schwann cells, also have the capacity to synthesize PROG, but expression and activity of 3beta-HSD in these cells are regulated by cellular interactions. Thus, Schwann cells convert pregnenolone to PROG in response to a neuronal signal. There is now strong evidence that P450scc and 3beta-HSD are expressed in the human nervous system, where PROG synthesis also takes place. Although there are only a few studies addressing the biological significance of PROG synthesis in the brain, the autocrine/paracrine actions of locally synthesized PROG are likely to play an important role in the viability of neurons and in the formation of myelin sheaths. The neuroprotective effects of PROG have recently been documented in a murine model of spinal cord motoneuron degeneration, the Wobbler mouse. The treatment of symptomatic Wobbler mice with PROG for 15 days attenuated the neuropathological changes in spinal motoneurons and had beneficial effects on muscle strength and the survival rate of the animals. PROG may exert its neuroprotective effects by regulating expression of specific genes in neurons and glial cells, which may become hormone-sensitive after injury. The promyelinating effects of PROG were first documented in the mouse sciatic nerve and in co-cultures of sensory neurons and Schwann cells. PROG also promotes myelination in the brain, as shown in vitro in explant cultures of cerebellar slices and in vivo in the cerebellar peduncle of aged rats after toxin-induced demyelination. Local synthesis of PROG in the brain and the neuroprotective and promyelinating effects of this neurosteroid offer interesting therapeutic possibilities for the prevention and treatment of neurodegenerative diseases, for accelerating regenerative processes and for preserving cognitive functions during aging.

Mitotic and neurogenic effects of dehydroepiandrosterone (DHEA) on human neural stem cell cultures derived from the fetal cortex

Abstract: Dehydroepiandrosterone (DHEA) is a neurosteroid with potential effects on neurogenesis and neuronal survival in humans. However, most studies on DHEA have been performed in rodents, and there is little direct evidence for biological effects on the human nervous system. Furthermore, the mechanism of its action is unknown. Here, we show that DHEA significantly increased the growth rates of human neural stem cells derived from the fetal cortex and grown with both epidermal growth factor (EGF) and leukemia inhibitory factor (LIF). However, it had no effect on cultures grown in either factor alone, suggesting a specific action on the EGF/LIF-responsive cell. Precursors of DHEA such as pregnenolone or six of its major metabolites, had no significant effect on proliferation rates. DHEA did not alter the small number ( 95%) of nestin-positive precursors. However, the number of glial fibrillary acidic protein-positive cells, its mRNA, and protein were significantly increased by DHEA. We found both N-methyl-d-aspartate and sigma 1 antagonists, but not GABA antagonists, could completely eliminate the effects of DHEA on stem cell proliferation. Finally we asked whether the EGF/LIF/DHEA-responsive stem cells had an increased potential for neurogenesis and found a 29% increase in neuronal production when compared to cultures grown in EGF/LIF alone. Together these data suggest that DHEA is involved in the maintenance and division of human neural stem cells. Given the wide availability of this neurosteroid, this finding has important implications for future use.

A steroid modulatory domain on NR2B controls N-methyl-d-aspartate receptor proton sensitivity

Abstract: N-methyl-d-aspartate (NMDA) receptor function is modulated by several endogenous molecules, including zinc, polyamines, protons, and sulfated neurosteroids. Zinc, polyamines, and phenylethanolamines exert their respective modulatory effects by exacerbating or relieving tonic proton inhibition. Here, we report that pregnenolone sulfate (PS) uses a unique mechanism for enhancement of NMDA receptor function that is independent of the proton sensor. We identify a steroid modulatory domain, SMD1, on the NMDA receptor NR2B subunit that is critical for both PS enhancement and proton sensitivity. This domain includes the J/K helices in the S2 region of the glutamate recognition site and the fourth membrane transmembrane region (M4). A molecular model based on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor structure suggests that steroid modulatory domain 1 contributes residues to a hydrophobic pocket that is capable of accommodating PS. The results demonstrate that the J/K helices and the fourth membrane transmembrane region participate in transducing allosteric interactions induced by steroid and proton binding to their respective sites.

Steroid sulfatase (STS) expression in the human temporal lobe: enzyme activity, mRNA expression and immunohistochemistry study.

Abstract: Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are suggested to be important neurosteroids. We investigated steroid sulfatase (STS) in human temporal lobe biopsies in the context of possible cerebral DHEA(S) de novo biosynthesis. Formation of DHEA(S) in mature human brain tissue has not yet been studied. 17α-Hydroxylase/C17-20-lyase and hydroxysteroid sulfotransferase catalyze the formation of DHEA from pregnenolone and the subsequent sulfoconjugation, respectively. Neither their mRNA nor activity were detected, indicating that DHEA(S) are not produced within the human temporal lobe. Conversely, strong activity and mRNA expression of DHEAS desulfating STS was found, twice as high in cerebral neocortex than in subcortical white matter. Cerebral STS resembled the characteristics of the known placental enzyme. Immunohistochemistry revealed STS in adult cortical neurons as well as in fetal and adult Cajal-Retzius cells. Organic anion transporting proteins OATP-A, -B, -D, and -E showed high mRNA expression levels with distinct patterns in cerebral neocortex and subcortical white matter. Although it is not clear whether they are expressed at the blood–brain barrier and facilitate an influx rather than an efflux, they might well be involved in the transport of steroid sulfates from the blood. Therefore, we hypothesize that DHEAS and/or other sulfated 3β-hydroxysteroids might enter the human temporal lobe from the circulation where they would be readily converted via neuronal STS activity.

Overview of dehydroepiandrosterone biosynthesis

Abstract: The biosynthesis of dehydroepiandrosterone (DHEA) from cholesterol involves only two enzymes, both cytochrome P450s. The conversion of cholesterol to pregnenolone is mediated by cholesterol side-chain cleavage enzyme (CYP11A1), which is found in the mitochondria. The cleavage of pregnenolone to DHEA requires both the 17alpha-hydroxylase and 17,20-lyase activities of CYP17, which is found in the endoplasmic reticulum. These conversions require pairs of electron transfer proteins or redox partners, which are adrenodoxin and adrenodoxin reductase for CYP11A1 and cytochrome P450-oxidoreductase and cytochrome b5 for CYP17. In addition, the steroidogenic acute regulatory (StAR) protein regulates the flux of cholesterol into the biosynthetic pathway and represents the mechanism of acute regulation. Finally, in addition to possessing CYP11A1 and CYP17, it is equally important that a steroidogenic cell not contain other enzymes that drain the flux of pregnenolone to DHEA. These characteristics are illustrated by the fetal adrenal cortex and the zona reticularis, which are dedicated to the synthesis of DHEA and DHEA-sulfate.

Impact of neuropathic pain on the gene expression and activity of cytochrome P450side-chain-cleavage in sensory neural networks

Abstract: Development of efficient therapy against chronic and stubborn pains requires fundamental identification of adequate cellular and molecular targets. This study combined cellular, molecular and biochemical approaches to investigate the gene expression and enzymatic activity of cytochrome P450side-chain-cleavage (P450scc) in spinal neural networks under normal and neuropathic pain states. P450scc is the key onset enzyme for steroidogenesis in endocrine glands and for neurosteroid biosynthesis in nerve cells. The P450scc gene was over-expressed in spinal and supra-spinal networks during neuropathic pain provoked by sciatic nerve ligature. Plasticity was observed in P450scc cellular distribution in pain circuits and its activity also increased inducing in vivo, hyper-secretion of pregnenolone and allopregnanolone which strongly stimulates type A receptors for g-aminobutyric acid, a pivotal neurotransmitter involved in pain modulation. These results, by establishing a direct link between neuropathic pain and neuroactive steroid formation in the nervous system, open new perspectives for chronic-pain modulation by endogenous neurosteroids.

Neurosteroids and sigma1 receptors, biochemical and behavioral relevance.

Abstract: The sigma(1) receptor is a 223 amino acid protein sharing no homology with other mammalian protein. It is an intracellular protein present on the endoplasmic reticulum membrane, which can translocates to other organelles and plasma membranes after activation. Activation of the sigma(1) receptor results in modulation of calcium mobilization from inositol trisphosphate receptor-gated intracellular pools and, at the plasma membrane, in modulation of several neurotransmitter responses. Behaviorally, sigma(1) receptors are involved in learning and memory, response to stress and depression, psychostimulant-induced sensitization, vulnerability to addiction and pain perception. Numerous synthetic compounds bind to sigma(1) receptor, playing the role of activator/agonist or blocker/antagonist, and these include benzomorphans, neuroleptics, antidepressants, cocaine, peptides related to neuropeptide Y or calcitonin gene-related peptide. It is also the case of neuro(active)steroids, i. e., circulating neuroactive steroids and neurosteroids synthesized de novo by the brain, which appear as the most important endogenous modulators of sigma(1) receptor. Pregnenolone and dehydroepiandrosterone act as sigma(1) receptor agonists and progesterone is a potent antagonist. The present paper will review the molecular and biochemical features concerning the sigma(1) receptor and focus on the recent studies examining the impact of the neuro(active)steroid/sigma(1) receptor interaction on the antidepressant activity of sigma(1) receptor agonists in the context of neurodegenerative diseases.

Feedback Inhibition of Steroidogenic Acute Regulatory Protein Expression in Vitro and in Vivo by Androgens

Abstract: Mullerian-inhibiting substance (MIS) reduces testosterone synthesis in Leydig cells by inhibiting cytochrome P450C17 hydroxylase/C17–20 lyase expression However, in mouse Leydig MA-10 cells, MIS also enhances the cAMP-induced expression of mRNA for steroidogenic acute regulatory protein (StAR), which transports cholesterol to the inner mitochondrial membrane for conversion to pregnenolone We hypothesized that the MIS-induced StAR expression is the indirect result of reduced testosterone synthesis in Leydig cells caused by MIS We show that, in addition to MIS, flutamide, an androgen receptor antagonist, enhanced StAR mRNA expression when added to cAMP-treated MA-10 cells, whereas dihydrotestosterone, a potent androgen receptor agonist, attenuated these responses Progesterone, dexamethasone, and estradiol also inhibited StAR mRNA expression Addition of MIS to cAMP-treated MA-10 cells transfected with a StAR-promoter luciferase reporter resulted in increased StAR promoter activity over cAMP alone; this

Molten Globule Structure and Steroidogenic Activity of N-218 MLN64 in Human Placental Mitochondria

Abstract: Progesterone synthesis by the human placenta requires the conversion of mitochondrial cholesterol to pregnenolone by cytochrome P450scc. Most steroidogenic tissues use the steroidogenic acute regulatory protein (StAR) to deliver cholesterol to the inner mitochondrial membrane where P450scc is located, but StAR is not expressed in the human placenta. However, the human placenta does express MLN64, which has a C-terminal domain homologous to StAR that can also transport cholesterol. We investigated the ability of bacterially expressed N-218 MLN64 and N-62 StAR to transport cholesterol between artificial membranes and to its inner membrane site of use in placental mitochondria. Urea denaturation experiments show that N-218 MLN64 undergoes a pH-dependent and denaturant-dependent structural transition to a molten globule state, as reported previously for N-62 StAR. N-218 MLN64 stimulated cholesterol transfer between artificial phospholipid vesicles with an initial rate of 6.5 mol/min·mol N-218 MLN64. Both N-21...

Increased allopregnanolone levels in the fetal sheep brain following umbilical cord occlusion.

Abstract: Allopregnanolone (AP) is a potent modulator of the GABAA receptor Brain AP concentrations increase in response to stress, which is thought to provide neuroprotection by reducing excitation in the adult brain Umbilical cord occlusion (UCO) causes hypoxia and asphyxia in the fetus, which are major risk factors associated with poor neurological outcome for the neonate, and may lead to adverse sequelae such as cerebral palsy The aims of this study were as follows: (i) to determine the effect of 10 min UCO on AP concentrations in the extracellular fluid of the fetal brain using microdialysis, and (ii) to compare the content of the steroidogenic enzymes P450scc and 5α-reductase type II (5αRII) with brain and CSF neurosteroid concentrations UCO caused fetal asphyxia, hypertension, bradycardia and respiratory acidosis, which returned to normal levels after 1–2 h AP concentrations in dialysate samples from probes implanted in grey and white matter of the parietal cortex were significantly increased 1 h after UCO from control levels of 104 ± 04 and 124 ± 03 to 260 ± 51 and 276 ± 64 nmol l−1, respectively (P < 005), before returning to pre-occlusion levels by 3–4 h after UCO When fetal brains were collected 1 h after a 10 min UCO, the relative increases of AP and pregnenolone content in the parietal cortex were similar to the increase observed in the extracellular (dialysate) fluid AP, but not pregnenolone, was increased in CSF at this time P450scc and 5αRII enzyme expression was significantly increased in the cerebral cortex in the UCO fetuses compared to control fetuses These results suggest that the fetal brain is capable of transiently increasing neurosteroid production in response to asphyxia The action of the increased neurosteroid content at GABAA receptors may serve to diminish the increased excitation due to excitotoxic amino acid release, and provide short-term protection to brain cells during such stress

Deletion of the mouse P450c17 gene causes early embryonic lethality.

Abstract: Dehydroepiandrosterone (DHEA), a 19-carbon precursor of sex steroids, is abundantly produced in the human but not the mouse adrenal. However, mice produce DHEA and DHEA-sulfate (DHEAS) in the fetal brain. DHEA stimulates axonal growth from specific populations of mouse neocortical neurons in vitro, while DHEAS stimulates dendritic growth from those cells. The synthesis of DHEA and sex steroids, but not mouse glucocorticoids and mineralocorticoids, requires P450c17, which catalyzes both 17-hydroxylase and 17,20lyase activities. We hypothesized that P450c17-knockout mice would have disordered sex steroid synthesis and disordered brain DHEA production and thus provide phenotypic clues about the functions of DHEA in mouse brain development. We deleted the mouse P450c17 gene in 127/SvJ mice and obtained several lines of mice from two lines of targeted embryonic stem cells. Heterozygotes were phenotypically and reproductively normal, but in all mouse lines, P450c17 / zygotes died by embryonic day 7, prior to gastrulation. The cause of this early lethality is unknown, as there is no known function of fetal steroids at embryonic day 7. Immunocytochemistry identified P450c17 in embryonic endoderm in E7 wild-type and heterozygous embryos, but its function in these cells is unknown. Enzyme assays of wild-type embryos showed a rapid rise in 17-hydroxylase activity between E6 and E7 and the presence of C17,20-lyase activity at E7. Treatment of pregnant females with subcutaneous pellets releasing DHEA or 17-OH pregnenolone at a constant rate failed to rescue P450c17 / fetuses. Treatment of normal pregnant females with pellets releasing pregnenolone or progesterone did not cause fetal demise. These data suggest that steroid products of P450c17 have heretofore-unknown essential functions in early embryonic mouse development. The synthesis of steroid hormones in the adrenals, gonads, placenta, and brain requires the expression of several steroidogenic enzymes. In all tissues, steroidogenesis is initiated by conversion of cholesterol to pregnenolone by the mitochondrial cholesterol side chain cleavage enzyme, P450scc. Thereafter, the specific steroid that is synthesized by a particular tissue depends upon the differential expression of additional steroidogenic enzymes. The conversion of pregnenolone and progesterone to their 17-hydroxylated products and then to either dehydroepiandrosterone (DHEA) or androstenedione, respectively, is mediated by a single microsomal enzyme, P450c17 (33, 34, 48), encoded by a single gene (22, 39). The pattern of P450c17 expression in steroidogenic tissues is species specific: it is expressed in the human adrenal and gonad but not placenta (9, 11, 15, 44), and it is expressed in the rodent gonad and placenta but not adrenal (19, 23, 25). P450c17 is also expressed in the fetal mouse brain beginning at embryonic day 9.5 (E9.5) (12). At this time, P450c17 is found in cells migrating from the neural crest, and subsequently, P450c17 is found in many cells derived from the neural crest. P450c17 is also expressed in the neocortical subplate, a region that receives thalamic projections, produces signals for cortical projections, and may produce signals for efferent thalamic projections from the cortex (32, 41). We hypothesized that DHEA, a steroid product of P450c17, may be an endogenous signal in the subplate to target axons coming from this region to specific sites in the developing cortex and showed that DHEA increased axonal outgrowth while DHEA-sulfate (DHEAS) increased dendritic growth (13). DHEA, but not DHEAS, induced other morphological indices of synaptic contacts, increased mRNAs for Tau-1 and type 1 and 2 dopamine receptors (14), mediated increases in intracellular calcium via N-methyl-D-aspartic acid receptors (13), protected hippocampal neurons from glutamate toxicity (27), and stimulated neurogenesis in the hippocampus (26). Thus, DHEA may serve as an endogenous neurotrophic agent. However, all these studies have been performed with cultured neuronal cells derived from specific regions of the nervous system at particular times in development. Thus, we sought to determine the function of DHEA and DHEAS in vivo during the development of the nervous system by deleting the gene encoding P450c17 in genetargeted mice.

Modulation of the firing activity of female dorsal raphe nucleus serotonergic neurons by neuroactive steroids.

Abstract: Important gender differences in mood disorders result in a greater susceptibility for women. Accumulating evidence suggests a reciprocal modulation between the 5hydroxytryptamine (5-HT) system and neuroactive steroids. Previous data from our laboratory have shown that during pregnancy, the firing activity of 5-HT neurons increases in parallel with progesterone levels. This study was undertaken to evaluate the putative modulation of the 5-HT neuronal firing activity by different neurosteroids. Female rats received i.c.v. for 7 days a dose of 50 µg/kg per day of one of the following steroids: progesterone, pregnenolone, 5-pregnane-3,20-dione (5-DHP), 5pregnan-3-ol,20-one, 5-pregnan-3-ol,20-one, 5pregnane-3,20-dione, 5-pregnan-3-ol,20-one (allopregnanolone, 3,5-THP), 5-pregnane-3-ol,20-one and dehydroepiandrosterone (DHEA). 5-DHP and DHEA were also administered for 14 and 21 days (50 µg/ kg per day, i.c.v.) as well as concomitantly with the selective sigma 1 (1) receptor antagonist NE-100. In vivo, extracellular unitary recording of 5-HT neurons performed in the dorsal raphe nucleus of these rats revealed that DHEA, 5-DHP and 3,5-THP significantly increased the firing activity of the 5-HT neurons. Interestingly, 5-DHP and DHEA showed different timeframes for their effects with 5-DHP having its greatest effect after 7 days to return to control values after 21 days, whereas DHEA demonstrated a sustained effect over the 21 day period. NE-100 prevented the effect of DHEA but not of 5-DHP, thus indicating that its 1 receptors mediate the effect of DHEA but not that of 5-DHP. In conclusion, our results offer a cellular basis for potential antidepressant effects of neurosteroids, which may prove important particularly for women with affective disorders.

Endocrine disrupters as disrupters of brain function: a neurosteroid viewpoint.

Abstract: The mechanisms of neurosteroid synthesis in the rat hippocampus were investigated. Metabolism assay demonstrated the pathway of "cholesterol alpha pregnenolone --> dehydroepiandrosterone --> androstenedione --> testosterone --> estradiol." Upon exposure of pups to bisphenol A (BPA) from the embryonic stage until 3 week-old stage, a significant facilitation of the synthesis of estradiol was observed in the hippocampus. The localization of cytochrome P450s (P450scc, P45017alpha, and P450arom) as well as estrogen receptor alpha (ER(alpha)) was observed in pyramidal and granule neurons, using immunohistochemical staining. Furthermore, the synaptic localization of P45017alpha, P450arom and ER(alpha) was demonstrated with immuno-electron microscopic analysis. The acute action of estradiol and endocrine disrupters were then analyzed with an electrophysiological measurement of hippocampal pyramidal neurons. A 30 min preperfusion of diethylstylbesterol (DES) enhanced the induction of long-term potentiation (LTP) by almost an identical magnitude to that obtained by estradiol perfusion. On the other hand, although the application of BPA alone did not affect LTP-induction, the co-perfusion of BPA with estradiol completely suppressed the enhancement effect of LTP by estradiol. The current investigations demonstrate in the hippocampus (1) that locally synthesized estrogen rapidly enhances the synaptic plasticity of neurons, and (2) that BPA and DES modulate the synaptic plasticity as well as the synthesis of estradiol. The probable targets of BPA and DES are ER(alpha) and steroidogenic proteins.

Fetal alcohol exposure alters neurosteroid levels in the developing rat brain

Abstract: Neurosteroids are modulators of neuronal function that may play important roles in brain maturation. We determined whether chronic prenatal ethanol exposure altered neurosteroid levels in the developing brain. Rat dams were exposed to: (i) a 5% ethanol-containing liquid diet that produces peak maternal blood alcohol levels near the legal intoxication limit (approximately 0.08 g/dL); (ii) an isocaloric liquid diet containing maltose-dextrin instead of ethanol with pair-feeding; (iii) rat chow ad libitum. Neurosteroid levels were assessed in offspring brains using radioimmunoassay or gas chromatography-mass spectrometry techniques. A prenatal ethanol exposure-induced increase in pregnenolone sulfate levels, but not dehydroepiandrosterone sulfate levels, was evident at the earliest time point studied (embryonic day 14). This effect lasted until post-natal day 5. Levels of other neurosteroids were assessed at embryonic day 20; pregnenolone levels, but not allopregnanolone levels, were elevated. Pregnenolone sulfate levels were not altered in the maternal brain. Neither pregnenolone nor pregnenolone sulfate levels were significantly altered in the fetal liver, placenta and maternal blood, indicating that the effect of ethanol is not secondary to accumulation of peripherally-produced steroids. Fetal ethanol exposure has been shown to decrease both cellular and behavioral responsiveness to neurosteroids, and our findings provide a plausible explanation for this effect.

Modulation of bradykinin-induced calcium changes in SH-SY5Y cells by neurosteroids and sigma receptor ligands via a shared mechanism

Abstract: In this study we investigated the effects of sigma receptor ligands and neurosteroids on bradykinin-induced intracellular calcium concentration ([Ca2+]i) changes in SH-SY5Y neuroblastoma cells. [Ca2+]i levels in cells loaded with fura-2 were monitored with dual-wavelength ratiometric fluorescence measurement. Submicromolar concentrations of bradykinin elicited [Ca2+]i responses with a fast rise followed by a slow decline in these cells. Preincubation of low micromolar concentrations of the neurosteroids pregnenolone, dehydroepiandrosterone (DHEA), or the prototypic sigma (sigma) receptor agonist (+)pentazocine potentiated bradykinin-induced [Ca2+]i changes in SH-SY5Y cells. The sigma receptor antagonist haloperidol blocked the enhancing effects on [Ca2+]i by (+)pentazocine or pregnenolone. Progesterone did not significantly affect the basal [Ca2+]i level or bradykinin-induced [Ca2+]i changes in these cells. However, coincubation of progesterone with (+)pentazocine, pregnenolone, or DHEA reversed their potentiating effects. The antagonistic effects of haloperidol and progesterone on the potentiating effects of (+)pentazocine and pregnenolone suggested that these ligands might act through a common mechanism. We further showed that progesterone, pregnenolone, and DHEA competed for [3H]+pentazocine binding in SH-SY5Y cells with Ki values of 0.13 +/- 0.03 microM, 0.98 +/- 0.34 microM, and 5.2 +/- 1.4 microM, respectively. Thus, the modulation of bradykinin-induced [Ca2+]i changes by neurosteroids in these cells is likely due to their actions on sigma receptors.

The role of progesterone metabolism and androgen synthesis in renal blood pressure regulation.

Abstract: 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a crucial role in converting hormonally active cortisol into inactive cortisone, conferring specificity onto the human mineralocorticoid receptor (MR). Progesterone binds with even higher affinity to the MR, but acts as an MR antagonist. How aldosterone is able to keep its function as predominant MR ligand in clinical situations with high progesterone concentrations, such as pregnancy, is not clear. We have shown in vitro that the human kidney possesses an effective enzyme system that metabolizes progesterone to inactive metabolites in a process similar to the inactivation of cortisol by 11beta-HSD2. In studies on patients with adrenal insufficiency, we have shown that the in vivo anti-mineralocorticoid activity of progesterone is diminished by inactivating metabolism of progesterone, local formation of the deoxycorticosterone mineralocorticoid from progesterone, and inhibition of 11beta-HSD2 by progesterone and its metabolites resulting in decreased inactivation of cortisol and hence increased MR binding by cortisol. The enzymes involved in progesterone metabolism are also responsible for the capability of the human kidney to convert pregnenolone to DHEA and androstenedione leading to the formation of active androgens, testosterone and 5alpha-DH-testosterone. Locally produced androgens might be responsible for the observed difference in blood pressure between men and women and higher susceptibility to hypertension in men.

The potential function of steroid sulphatase activity in steroid production and steroidogenic acute regulatory protein expression

Abstract: The first step in the biosynthesis of steroid hormones is conversion of cholesterol into pregnenolone. StAR (steroidogenic acute regulatory) protein plays a crucial role in the intra-mitochondrial movement of cholesterol. STS (steroid sulphatase), which is present ubiquitously in mammalian tissues, including the placenta, adrenal gland, testis and ovary, desulphates a number of 3beta-hydroxysteroid sulphates, including cholesterol sulphate. The present study was designed to examine the effect of STS on StAR protein synthesis and steroidogenesis in cells. Steroidogenic activities of COS-1 cells that had been co-transfected with a vector for the cholesterol P450scc (cytochrome P450 side-chain-cleavage enzyme) system, named F2, a StAR expression vector (pStAR), and an STS expression vector (pSTS) were assayed. Whole-cell extracts were subjected to SDS/PAGE and then to Western blot analysis. pSTS co-expressed in COS-1 cells with F2 and pStAR increased pregnenolone synthesis 2-fold compared with that of co-expression with F2 and pStAR. Western blot analysis using COS-1 cells that had been co-transfected with pSTS, F2 and pStAR revealed that StAR protein levels increased, whereas STS and P450scc protein levels did not change. The amount of StAR protein translation products increased when pSTS was added to an in vitro transcription-translation reaction mixture. Pulse-chase experiments demonstrated that the 37 kDa StAR pre-protein disappeared significantly ( P <0.01) more slowly in COS-1 cells that had been transfected with pSTS than in COS-1 cells that had not been transfected with pSTS. The increase in StAR protein level is not a result of an increase in StAR gene expression, but is a result of both an increase in translation and a longer half-life of the 37 kDa pre-StAR protein. In conclusion, STS increases StAR protein expression level and stimulates steroid production.