Showing papers on "Pregnenolone published in 1996"

Relationship between symptom severity and steroid variation in women with premenstrual syndrome: study on serum pregnenolone, pregnenolone sulfate, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one.

Abstract: The relationship between symptoms of premenstrual syndrome (PMS) and serum levels of pregnenolone (Pe), pregnenolone sulfate (PS), 5 alpha-pregnane-3,20-dione (5 alpha-DHP), 3 alpha-hydroxy-5 alpha- pregnan-20-one (5 alpha-THP), LH, 17 beta-estradiol (E2), and progesterone (P) was investigated during 2 consecutive menstrual cycles in 12 patients using daily measurements. Corresponding hormones were also measured during 1 cycle in 8 control women. Pe, PS, 5 alpha-DHP, and 5 alpha-THP showed a significant cyclicity within menstrual cycles and a high rate of correlation with P variation in both PMS patients and controls. No significant difference was found between PMS patients and controls in average serum concentrations of Pe, PS, 5 alpha-DHP, 5 alpha-THP, and LH during the luteal phase, whereas a significantly higher level of E2 and a lower level of P were observed in PMS patients. The variation in symptom scores was compared with that in hormone levels within each woman. The symptom peak showed a delay of...

Time-dependent changes in rat brain neuroactive steroid concentrations and GABAA receptor function after acute stress.

Abstract: The time courses of changes in rat brain neuroactive steroid concentrations and gamma-aminobutyric acid type A (GABAA) receptor function elicited by acute stress were investigated in animals exposed to CO2 for 1 min, a treatment known to induce stress in rats and panic attacks in humans. Inhalation of CO2 induced increases in cerebral cortical steroid concentrations, the time dependence of which varied with the steroid examined. Thus, progesterone and deoxycorticosterone showed maximal increases (10- and 4-fold, respectively) 10 min after CO2 inhalation and had returned to basal values by 30 and 60 min, respectively. In contrast, pregnenolone and 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) concentrations showed maximal increases (+174 and + 200%, respectively) at 30 min, were still higher than control at 60 min and returned to control values 120 min after stress. Inhalation of CO2 also resulted in increases in plasma steroid concentrations, most of which peaked at 30 min and had returned to control values by 60 min. A parallel analysis of the stress-induced changes in GABAA receptor function, assessed either biochemically by t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to cerebral cortical membranes or behaviorally by the punished responding score in Vogel's test, showed that the effects of CO2 inhalation on both parameters were maximal (+51 and -40%, respectively) after 10 min; the behavioral reaction returned to normal after 60 min, whereas [35S]TBPS binding had returned to control values 120 min after stress. The results show that: (a) the maximal increase in the brain concentrations of allopregnanolone, a potent and efficacious positive modulator of GABAA receptors, occurred at a time (30 min) when both conflict behavior and [35S]TBPS binding begun to decrease, and (b) both allopregnanolone concentrations and [35S]TBPS binding had returned to control values 120 min after CO2 inhalation. The data are thus consistent with a physiological role of neuroactive steroids in restoring GABAergic tone after stress.

Potentiation of neuronal NMDA response induced by dehydroepiandrosterone and its suppression by progesterone: effects mediated via sigma receptors.

Abstract: We have shown previously that low doses of selective sigma (sigma)- receptor ligands potentiate the excitatory response of pyramidal neurons to NMDA in the CA3 region of the dorsal hippocampus in the rat Because progesterone competitively displaces the binding of the ligand N-[3H]allyl-normetazocine (SKF-10,047), the present studies were undertaken to determine in vivo the effect of neuroactive steroids on NMDA-induced excitation of rat CA3 pyramidal neurons Low doses of dehydroepiandrosterone (DHEA) potentiated the NMDA response selectively and dose-dependently The effect of DHEA was reversed by the selective sigma antagonist N-dipropyl-2-(4-methoxy-3- (2-phenylethoxy)phenyl)- ethylamine monohydrochloride (NE-100) and by haloperidol, but not by spiperone Progesterone had no effect by itself but reversed, at low doses, the potentiation of the NMDA response induced by DHEA as well as those induced by nonsteroidal sigma ligands Neither pregnenolone nor pregnenolone sulfate had any effect on the NMDA response--nor did they antagonize the potentiation of the NMDA response induced by DHEA and by nonsteroidal sigma ligands A pertussis toxin pretreatment, which inactivates Gi/o-proteins, abolished the potentiating effects of DHEA Ovariectomy enhanced the potentiation of the NMDA response by the nonsteroidal sigma ligand di(2-tolyl)guanidine (DTG) There was a reciprocal occlusion of the effects of DHEA and DTG; DTG did not potentiate the NMDA response further after DHEA, and DHEA did not do so after DTG These results suggest that some neuroactive steroids modulate the NMDA response via sigma receptors

The zona reticularis is the site of biosynthesis of dehydroepiandrosterone and dehydroepiandrosterone sulfate in the adult human adrenal cortex resulting from its low expression of 3 beta-hydroxysteroid dehydrogenase

Abstract: Based on indirect evidence, it has often been assumed that the zona reticularis of the adult human adrenal cortex is the source of the adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), but direct tests of this concept have been few. Using the techniques of cell culture, Northern blotting, and RIA, we compared the properties of separated adult zonal cells to those of fetal zone cells, a cell type well known to secrete large amounts of DHEA(S) due to its low expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). In nine glands from donors of a wide age range, the zona fasciculata and zona reticularis were separated and dissociated, and the cells were placed in culture. After 5 days, serum was removed by a 24-h period in serum-free defined medium followed by a 24-h exposure to cAMP analogs, with the optional addition of insulin, also in serum-free medium. The separated fasciculata and reticularis cells showed large differences in the DHEA(S)/cortisol (F) production ratios from added pregnenolone precursor, consistent with the synthesis of only F and essentially no DHEA(S) by fasciculata cells and with the synthesis of mostly DHEA(S) with little or no F by both reticularis cells and fetal zone cells. The different patterns of steroidogenesis were accompanied by a much lower level of expression of type II 3 beta HSD in reticularis cells, similar to that in fetal zone cells. In contrast, other genes were similarly regulated in the two adult zones and in the fetal zone by both cAMP and insulin. The levels of messenger ribonucleic acids for 17 alpha-hydroxylase, cholesterol side-chain cleavage enzyme, 21-hydroxylase, and 11 beta-hydroxylase responded to cAMP and insulin in both reticularis cells and fetal zone cells in the same pattern as that previously established in fasciculata cells. The central role of the limited expression of 3 beta HSD in the DHEA(S)-synthesizing property of reticularis cells was established by inhibition of 3 beta HSD in fasciculata cells with trilostane, which caused them to increase their DHEA/F production ratio to a level exceeding even that in fetal zone cells. There did not appear to any age-related changes in gene expression that could account for the large age-related decline in DHEA(S) biosynthesis in humans in either reticularis or fasciculata cells. Thus, the most likely cause of the age-related decline in adrenal androgen biosynthesis is an age-related decline in the number of functional reticularis cells, without a major change in the differentiated properties of the zonal cells as a function of age.

Localization of 17beta-hydroxysteroid dehydrogenase and characterization of testosterone in the brain of the male frog

Abstract: Several enzymes involved in the formation of steroids of the pregnene and pregnane series have been identified in the brain, but the biosynthesis of testosterone has never been reported in the central nervous system. In the present study, we have investigated the distribution and bioactivity of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) (EC 1.1.1.62; a key enzyme that is required for the formation of testosterone and estradiol) in the brain of the male frog Rana ridibunda. By using an antiserum against human type I placental 17beta-HSD, immunoreactivity was localized in a discrete group of ependymal glial cells bordering the telencephalic ventricles. HPLC analysis of telencephalon and hypothalamus extracts combined with testosterone radioimmunoassay revealed the existence of two peaks coeluting with testosterone and 5alpha-dihydrotestosterone. After HPLC purification, testosterone was identified by gas chromatography/mass spectrometry. Incubation of telencephalon slices with [3H]pregnenolone resulted in the formation of metabolites which coeluted with progesterone, 17alpha-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, and 5alpha-dihydrotestosterone. The newly synthesized steroid comigrating with testosterone was selectively immunodetected by using testosterone antibodies. These data indicate that 17beta-HSD is expressed in a subpopulation of gliocytes in the frog telencephalon and that telencephalic cells are capable of synthesizing various androgens, including dehydroepiandrosterone, androstenedione, testosterone, and 5alpha-dihydrotestosterone.

Potentiation by dehydroepiandrosterone of the neuronal response to N-methyl-D-aspartate in the CA3 region of the rat dorsal hippocampus: an effect mediated via sigma receptors.

Abstract: We have previously shown in vivo that low doses of selective sigma (sigma) receptor ligands potentiate selectively and dose-dependently the excitatory response of pyramidal neurons to microiontophoretic applications of N-methyl-D-aspartate (NMDA) in the CA3 region of the rat dorsal hippocampus. As several neuroactive steroids such as progesterone and testosterone have a high affinity for sigma receptors, the effects of some neuroactive steroids on the NMDA-induced neuronal response were assessed using extracellular unitary recordings of CA3 dorsal hippocampus pyramidal neurons obtained in anesthetized Sprague-Dawley rats. Low doses of dehydroepiandrosterone (DHEA) potentiated selectively and dose-dependently the NMDA response without affecting those to acetylcholine or quisqualate. This potentiating effect of DHEA was suppressed by the selective sigma 1 antagonist NE-100 and by the non-selective sigma antagonist haloperidol. Low doses of progesterone and of testosterone did not modify the NMDA response, but reversed the potentiating effects of DHEA as well as those of the non-steroidal sigma ligands di-tolylguanidine (DTG), (+)pentazocine and JO-1784. The two neuroactive steroids with a low affinity for sigma receptors, pregnenolone and pregnenolone sulfate, had no effect on the NMDA response, and did not modify the potentiation of the NMDA response induced by DHEA and by non-steroidal sigma ligands. The potentiation of the NMDA response by DTG (1 microgram/kg i.v.) was significantly greater in ovariectomized rats than in males and non-ovariectomized females on either day one or three of the estrous cycle. These results suggest that some neuroactive steroids such as DHEA, progesterone and testosterone modulate the NMDA response via sigma receptors. Furthermore, they also indicate that endogenous progesterone and testosterone, by acting as non-selective sigma antagonists, may produce a tonic dampening of the function of sigma receptors and consequently a decrease in the NMDA receptor function.

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

Abstract: 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 it

Stress-induced increase in brain neuroactive steroids: Antagonism by abecarnil

Abstract: Acute foot shock stress elicits a selective and time-dependent increase of neuroactive steroid (pregnenolone, progesterone, allotetrahydrodeoxycorticosterone) concentrations in rat brain cortex, accompanied by a marked increase of plasma corticosterone. The brain cortical neuroactive steroid levels peaked between 10 and 30 min poststress and returned to control values by 2 h. Abecarnil (0.3 mg/kg), i.p.), a beta-carboline derivative with anxiolytic properties, completely antagonized the effect of foot shock on brain cortical neuroactive steroids. A single administration of the anxiogenic beta-carboline FG 7142 (15 mg/kg, i.p.), in contrast, mimicked the effect of foot shock. These data support the hypothesis for the existence of a functional relationship between brain neuroactive steroid concentrations and GABAA receptor function/emotional state of the animal.

Neurosteroids block the memory-impairing effects of ethanol in mice

Abstract: Using a win-shift foraging paradigm to assess working memory in C57BL/6 mice, the memory-enhancing effect of low doses of the neurosteroids 5-pregnen-3β-ol-20-one [pregnenolone (PE)], 5-pregnen-3β-ol-20-one sulfate [pregnenolone sulfate (PS)], 5-androsten-3β-ol-17-one [dehydroepiandrosterone (DHEA)], and 5-androsten-3β-ol-17-one sulfate [dehydroepiandrosterone sulfate (DHEAS)] were demonstrated. The neurosteroids 5β-pregnan-3α-ol-20-one [pregnanolone (PA)] and 5β-pregnan-3β-ol-20-one [epipregnanolone (EPI)] disrupted memory in this paradigm. PE, PS, DHEA, DHEAS, and PA were also capable of blocking the memory-impairing effect of 0.5 g/kg ethanol. EPI prevented PA from blocking the effect of ethanol. The influence of these compounds on memory and their interactions on this behavior are consistent with their actions on the GABA A system.

The neurosteroid progesterone increases the expression of myelin proteins (mbp and cnpase) in rat oligodendrocytes in primary culture

Abstract: Steroid hormones are known to act in the central nervous system (CNS), affecting brain development and behavior. They have profound influences on the growth, maturation, differentiation and functioning of brain cells. The biological effects of these steroids are mediated by specific high-affinity intracellular receptors, and their presence in the brain has been described by several groups (for review see McEwen et al., 1982). In earlier studies, we have shown that newborn rat glial cells in primary culture can synthesize pregnenolone and progesterone (P) and that these brain cells contain 4 classes of steroid hormone receptors, progesterone, glucocorticoid, estrogen and androgen receptors (PR, GR, ER and AR). After treating glial cells with estrogen, only the PR was induced, and this induction was most significant in cultures established from female rat pups (Jung-Testas et al., 1991). In agreement with the presence of intracellular steroid hormone receptors, we have shown direct effects of steroids on cell multiplication, morphology and differentiation. For instance, after treatment of cells with P, an increased synthesis of myelin proteins was observed in oligodendrocytes (Jung-Testas et al., 1992). In the CNS, oligodendrocytes are responsible for myelin formation and maintenance. In rodents, central myelin is composed of about 70% lipids, among which galactocerebroside (Gal C) is a specific marker for oligodendrocytes (Raft et al., 1987), and of about 30% proteins, mainly proteolipid protein, myelin

Development and Regeneration of the Nervous System: A Role for Neurosteroids (Paart 2 of 2)

Abstract: 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 it

Regulation of adrenal steroidogenesis during chronic stress

Abstract: The mechanism of the altered adrenal responsiveness during chronic stress was studied by analysis of ACTH and Ang II responses and the expression and activity of steroidogenic enzymes in the adrenal cortex of rats subjected to repeated immobilization (2 hr/day for 14 days), or repeated i.p. injection of 1.5 M NaC1. Concomitant with increased pregnenolone production and reduced aldosterone secretion by isolated adrenal glomerulosa cells of chronically stressed rats, P-450scc mRNA were increased and P-450aldo mRNA levels were decreased in adrenal zona glomerulosa. Consistent with elevated plasma corticosterone levels, isolated adrenal fasciculata cells from stressed rats showed higher cAMP, pregnenolone and corticosterone responses to ACTH. Adrenal fasciculata area and levels of P-450scc, but not those of P-45011β hydroxylase were significantly increased. The effects of repeated stress on adrenal steroidogenesis were mimicked by repeated ACTH injections. The half life of corticosterone in plasma measured wi...

Possible Role for Mitochondrial Calcium in Angiotensin II- and Potassium-Stimulated Steroidogenesis in Bovine Adrenal Glomerulosa Cells*

Abstract: In adrenal zona glomerulosa cells, the action of angiotensin II (Ang II) and of potassium (K’) on aldosterone synthesis is mediated by the Ca2+ messenger system. The major part of the steroidogenic pathway takes place inside the mitochondria, and Ca2+ must enter the mitochondrial matrix to stimulate the steroidogenic cascade. To examine how changes in the cytosolic free calcium concentration ([Ca”],) induced by Ang II and K+ are relayed into the mitochondrial matrix, we transfected bovine adrenal zona glomerulosa cells in primary culture with a chimeric complementary DNA encoding for the signal presequence targeting human cytochrome c oxidase subunit VIII to the matrix, linked to a complementary DNA coding for the Ca’+sensitive photoprotein aequoiin. Resting mitochondrial free calcium concentration ([Ca2+],) amounted to 0.41 2 0.18 FM (n = 40). Ang II induced a concentration-dependent (EC,, = 11.3 t 6.0 nM), biphasic rise of [Ca’+],. After a large transient &tial peak (5.13 + O.&l FM, n = 28), [Ca”], decreased to a plateau that remained higher than basal [Ca’+], for several minutes in the presence of the hormone. By contrast, studies in cells transfected withcytosolic aequorin indicate2 that the rise of [Ca2’], triggered by Ang II was confined to 1.34 2 0.26 ~.LM (n = 17). In Ca’+-free medium, a reduced peak [Ca2’], response to Ang II occurred without a secondary plateau. On readdition of extracellular Ca”‘, in the presence ofthe hormone, the resulting Ca2+ influx was accompanied by small rise of [Ca’+],. The mitochondrial uncoupler, carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone, prevented the Ang II-induced [Ca”], rise but not the [Ca2+lc response, thus demonstrating the mitochondrial location of transfected aequorin. In contrast to Ang II, K+ (13 InM) induced a sustained [Ca’+], response, which was relayed without amplification into the mitochondrial matrix as a plateau of [Ca2’],. This plateau of [Ca”], was suppressed by the addition of the dihydropyridine, nifedipine (200 nM). The inhibitor of the mitochondrial Na+/Ca’+ exchanger, CGP37157, reduced significantly the rate of decrease of [Ca’+], following the peak induced by Ang II. In cells whose [Ca’+], was clamped at various levels (0.05-0.860 PM) with ionomycin, a concentrationdependent stimulation of pregnenolone output was induced by Ca2+. Under these conditions, the output of pregnenolone - the early product of steroidogenesis - was markedly potentiated by CGP37157. These results suggest the existence of microdomains ofhigh [Ca”], elicited by Ang II in the proximity of mitochondria. Moreover, our observations are consistent with a mitochondrial site of action for calcium in the activation of the steroidogenic cascade. 137: 55445551, 1996)

StAR-A tissue specific acute mediator of steroidogenesis.

Abstract: The rate-limiting and acutely regulated step in steroid hormone biosynthesis is the translocation of cholesterol, the precursor of all steroid hormones, from the mitochondrial outer membrane to the inner membrane, where it is converted to pregnenolone by the cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc). This step has long been known to be dependent upon the de novo synthesis of a labile protein factor, which is required for the intramitochondrial translocation of cholesterol. Recently, the Steroidogenic Acute Regulatory (StAR) protein has been shown to have an indispensable role in acute steroid production and is proposed to be this labile protein factor. Given the fundamental importance of StAR as a key regulator of steroid hormone biosynthesis, the next frontier for researchers is elucidating the molecular mechanisms that control StAR expression and function.

Prenatal alcohol exposure influences the effects of neuroactive steroids on separation-induced ultrasonic vocalizations in rat pups

Abstract: Fetal alcohol exposure has been reported to be associated with hyper-responsiveness to stress. Using a maternal separation paradigm, this study examined whether prenatal alcohol exposure affected sensitivity to neurosteroid modulation of stress. We have shown that the neuroactive steroid allopregnanolone reduces ultrasonic vocalizations (USVs) after brief maternal separation in week-old rat pups. Prenatal alcohol exposure, however, resulted in reduced sensitivity to this neurosteroid. In this study's first experiment, the behavioral effects of pregnenolone sulfate, a neurosteroid with reportedly opposite modulatory effects on the GABAA receptor, were characterized. Pregnenolone sulfate had a triphasic effect on the production of ultrasonic vocalizations and on open field activity. Blockade of conversion of pregnenolone sulfate to allopregnanolone via the 5 alpha-reductase inhibitor 4-MA also blocked the drug-related reduction in USVs, but not the higher-dose augmentation. The enzyme inhibitor alone had no significant effects on USV production, nor did progesterone. These results suggest that the neuroactive steroid pregnenolone sulfate may play an independent role in the stress response after maternal separation as well as being a precursor for the anxiolytic neurosteroid allopregnanolone. In the second experiment, prenatal alcohol exposure was found to eliminate both the low dose USV-reducing effect and the higher dose USV-increasing effect. These results support previous results demonstrating that prenatal alcohol exposure may cause an altered sensitivity to the neuromodulatory effects of neurosteroids.

Steroidogenesis in the Ovarian Follicles of the Medaka (Oryzias latipes) during Vitellogenesis and Oocyte Maturation

Abstract: Changes in the steroidogenic pathway in medaka (Oryzias latipes) ovarian follicles during vitellogenesis and oocyte maturation were investigated in vitro by incubation of follicles with several radiolabeled steroid precursors, followed by thin layer chromatography (TLC) fractionation and recrystallization. When vitellogenic follicles collected at 18 hr before the expected time of spawning were incubated with 3H-labeled pregnenolone, the major metabolites were 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, and androstenedione. Incubations of vitellogenic follicles with androstenedione produced testosterone and estradiol-17β By contrast, when maturing follicles (postvitellogenic follicles undergoing maturation) collected at 10 hr before spawning were incubated with 3H-labeled pregnenolone, the major metabolites were 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, and 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-DP, maturation-inducing hormone of medaka); androstenedione was not detected. Neith...

Glucocorticoid receptor-independent transcriptional induction of cytochrome P450 3A1 by metyrapone and its potentiation by glucocorticoid.

Abstract: Metyrapone administration to 21- and 90-day-old male rats causes a transcriptional induction of the hepatic glucocorticoid-inducible CYP3A1 gene within an hour as determined by nuclear run-on experiments. Analyses performed 24 hr after metyrapone administration in both ages of rat demonstrate that the transcriptional induction of CYP3A1 gene expression is followed by significant increases in CYP3A1 mRNA, CYP3A-immunoreactive microsomal protein and total microsomal cytochrome P450 (CYP). In 21-day-old rats, there is a significant increase in microsomal CYP3A dependent steroid 6 beta-hydroxylase activity but not in 90-day-old rats, possibly because of a slower clearance of this drug, which inhibits CYP activities. In hepatocytes cultured in serum- and glucocorticoid hormone-free medium, metyrapone alone induces CYP3A1 mRNA expression, which demonstrates that metyrapone transcriptionally induces hepatic CYP3A1 by a direct interaction with the liver. Metyrapone does not compete with the binding of the synthetic glucocorticoid and potent transcriptional CYP3A1 inducer dexamethasone to the glucocorticoid receptor (GR) in soluble fractions from liver. This suggests that metyrapone is not a ligand for the GR and induces CYP3A1 by a mechanism independent of the GR. Addition of glucocorticoid to cultured hepatocytes at levels that induce GR-dependent genes potentiate CYP3A1 mRNA induction by metyrapone without inducing CYP3A1 mRNA alone. A GR-dependent mechanism may therefore mediate the potentiation of CYP3A1 transcriptional induction by metyrapone. The CYP3A1 transcriptional inducer and glucocorticoid antagonist pregnenolone 16 alpha-carbonitrile at 100 microM blocks dexamethasone binding to the GR in 21-day-old rat liver soluble fractions but is less effective in 90-day-old rat liver soluble fractions in contrast with 10 microM glucocorticoid antagonist RU486, which is equally effective at blocking dexamethasone binding to the GR. The inability of pregnenolone 16 alpha-carbonitrile to fully compete with dexamethasone for cytosolic binding in adult animals suggests that there may exist variant receptors with different affinities for dexamethasone and pregnenolone 16 alpha-carbonitrile and may explain the mechanism by which low concentrations of dexamethasone potentiate the transcriptional induction of CYP3A1 mediated by high concentrations of pregnenolone 16 alpha-carbonitrile [J. Biol, Chem. 270:28917-28923 (1995)]. Examination of membrane-bound dexamethasone binding activity, with which other steroidal and nonsteroidal CYP3A inducers have been shown to compete, indicates that binding activity is detectable in 90- but not 21-day-old rat liver microsomes. The absence of membrane-bound glucocorticoid binding site activity and the presence of a functional CYP3A1 transcriptional response in 21-day-old rats suggest that membrane-bound glucocorticoid binding site activity is not involved in the transcriptional activation of CYP3A1 expression. These data suggest that both glucocorticoids and nonsteroidal compounds may trigger the transcriptional induction of CYP3A1 by a GR-independent mechanism that may be potentiated by a GR-dependent mechanism.

Identification of Intermediates in the Conversion of Cholesterol to Pregnenolone with a Reconstituted Cytochrome P-450scc System: Accumulation of the Intermediate Modulated by the Adrenodoxin Level

Abstract: Dihydroxycholesterol and pregnenolone were clearly detected on HPLC when 22R-hydroxycholesterol was incubated with a reconstituted P450scc system containing equimolar amounts of P450scc and adrenodoxin. The dihydroxycholesterol, which has been accepted to be an intermediate in the conversion of 22R-hydroxycholesterol to pregnenolone, accumulated when adrenodoxin was at a subsaturating level with respect to P450scc. The formation of the intermediate increased with increasing pH in the range of 7.2 to 8.1, and the ratio of the intermediate to the product, pregnenolone, increased with increasing pH. When the binding of P450scc to adrenodoxin was weakened by elevation of the ionic strength, the formation of the intermediate relative to the product increased. The apparent Km for dihydroxycholesterol at a subsaturating level of adrenodoxin was about 7 microM, in contrast to 4 microM at a saturating level of adrenodoxin, implying that the affinity of dihydroxycholesterol is lower at a subsaturating level of adrenodoxin than at a saturating one. These results suggest that a subsaturating level of adrenodoxin weakened the binding of dihydroxycholesterol to P450scc and thus the intermediate, dihydroxycholesterol, was released. An intermediate other than dihydroxycholesterol, obtained when cholesterol was used as the substrate, was identified as 22R-hydroxycholesterol by HPLC and mass spectroscopic analysis. The intermediate obtained when 22R-hydroxycholesterol was used as the substrate was identified as 20R,22R-dihydroxycholesterol by HPLC, mass, and 1H-NMR spectroscopic analyses. These results provide direct evidence that cholesterol is metabolized to pregnenolone by way of 22R-hydroxycholesterol and 20R,22R-dihydroxycholesterol by P45 scc.

Derivatives of estra 1,3,5(10)triene-17-one, 3-amino compounds and their use

Abstract: This invention discloses compounds useful as steroid sulfatase inhibitors. The compounds comprise the formula (1) ##STR1## wherein (a) R is selected from the group consisting of hydrogen, a lower alkyl group, an alkoxy group, halogen, NH2, NO2, C.tbd.N and N═C═S; and (b) the ring system ABCD is a steroid nucleus selected from the group consisting of estrone, dehydroepiandrosterone, estradiols, estradiolesters, pregnenolone, substituted estrones, substituted dehydroepiandrosterones, substituted estradiols, substituted estradiolesters and substituted pregnenolone. The compounds also comprise the formula (5) ##STR2## wherein (a) R1 is hydrogen and R2 is selected from the group consisting of SO2 CF3, SO2 NH2, SO2 (C1 -C6 -alkyl), COCF3, CONH2, CO(C1 -C6 -alkyl); and (b) the ring system ABCD is a steroid nucleus selected from the group consisting of estrone, dehydroepiandrosterone, estradiol, estradiolester, pregnenolone, substituted estrones, substituted dehydroepiandrosterone, substituted estradiols, substituted estradiolesters and substituted pregnenolone. The invention also discloses methods of treating a patient therapeutically and prophylactically for estrogen dependent diseases with the compounds of this invention.