Topic
Pregnenolone
About: Pregnenolone is a research topic. Over the lifetime, 3539 publications have been published within this topic receiving 126444 citations. The topic is also known as: (3b)-3-hydroxy-Pregn-5-en-20-one & 3-Hydroxypregn-5-en-20-one.
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TL;DR: The molecular cloning and functional characterization of a full-length complementary DNA for estrogen sulfotransferase from mouse testis are reported and suggest complex regulatory mechanisms in its expression under normal and pathophysiological conditions.
Abstract: Sulfation represents a major pathway for the inactivation of steroid hormones such as estrogens and is catalyzed by a group of enzymes called sulfotransferases. Aberrant regulation of an estrogen sulfotransferase has been demonstrated previously in the livers of obese and diabetogenic C57BL/KsJ-db/db strain mice. In this paper, we report the molecular cloning and functional characterization of a full-length complementary DNA for estrogen sulfotransferase from mouse testis. The mouse estrogen sulfotransferase complementary DNA encodes 295 amino acids. It shares 88%, 77%, 75%, and 68% identity in amino acid sequence with the rat liver, human liver, guinea pig adrenal, and bovine placental estrogen sulfotransferase, respectively. The mouse enzyme was expressed as a glutathione-S-transferase fusion protein in Escherichia coli. The fusion protein was affinity purified, and milligram quantities of pure enzyme were obtained after cleavage of the fusion protein with thrombin. The expressed enzyme exhibits a high substrate specificity toward estrogens, including estradiol and estrone. Neither dehydroepiandrosterone, pregnenolone, testosterone, nor a simple phenolic compound, 4-nitrophenol appears to be a substrate. Northern hybridization indicates that messenger RNA (1.3 kilobases) for the estrogen sulfotransferase is expressed exclusively in the testes in control C57BL/KsJ mice. However, both the messenger RNA and protein are dramatically induced in the livers of obese and diabetogenic C57BL/KsJ-db/db mice. In contrast to the liver, the constitutive expression of the enzyme in the testis is not affected by the db/db genotype. These results recapitulate the species-specific nature in the tissue distribution of estrogen sulfotransferase and suggest complex regulatory mechanisms in its expression under normal and pathophysiological conditions.
86 citations
TL;DR: Results support the view that steroid synthesis occurs in a glioma cell line and provide evidence for a rapid steroid synthesis in C6-2Bglioma cells, which in turn appears to be accelerated by 1-100 nM 4'CD, a MDR ligand
Abstract: An experimental model to study synthesis of cholesterol and pregnenolone from the precursor mevalonolactone (MVA) was developed in C6-2B glioma cells. The steroidogenic capability of this cell line and the regulation of pregnenolone production by 4'-chlorodiazepam (4'CD), a specific ligand for the mitochondrial diazepam binding inhibitor (DBI) receptor (MDR), were investigated. Cells maintained in serum-free media were incubated with lovastatin (20 microM) and two inhibitors of pregnenolone metabolism, trilostane (25 microM) and 1,2,3,4-tetrahydro-4-oxo-7-chloro-2-naphthylpyridine (10 microM). Under these conditions the incorporation of [3H]MVA into cholesterol and pregnenolone formation was biphasic, with an initial rapid phase (within 1 min) followed by a slower phase. Cholesterol and pregnenolone were identified by coelution with authentic steroids from a Si 60 Lichrosorb column and gas chromatography/mass spectrometry. Pregnenolone synthesis in intact C6-2B glioma cells was stimulated by nanomolar concentrations of 4'CD after 5 min of incubation with MVA. The stimulatory effect was dependent on drug concentration and the maximal effect was achieved at 10 nM. The time course showed that the incorporation of MVA into pregnenolone is accelerated by the MDR ligand. Cholesterol synthesis is only slightly and not significantly affected by 4'CD. These results support the view that steroid synthesis occurs in a glioma cell line. Moreover, we provide evidence for a rapid steroid synthesis in C6-2B glioma cells, which in turn appears to be accelerated by 1-100 nM 4'CD, a MDR ligand.
85 citations
TL;DR: 7DHP, its photoderivative 20-oxopregnacalciferol, and pregnenolone exhibited biological activity in skin cells including inhibition of proliferation of epidermal keratinocytes and melanocytes, and melanoma cells.
Abstract: Since P450scc transforms 7-dehydrocholesterol (7DHC) to 7-dehydropregnenolone (7DHP) in vitro, we investigated sequential 7DHC metabolism by adrenal glands ex vivo. There was a rapid, time- and dose-dependent metabolism of 7DHC by adrenals from rats, pigs, rabbits and dogs with production of more polar 5,7-dienes as detected by RP-HPLC. Based on retention time (RT), UV spectra and mass spectrometry, we identified the major products common to all tested species as 7DHP, 22-hydroxy-7DHC and 20,22-dihydroxy-7DHC. The involvement of P450scc in adrenal metabolic transformation was confirmed by the inhibition of this process by DL-aminoglutethimide. The metabolism of 7DHC with subsequent production of 7DHP was stimulated by forscolin indicating involvement of cAMP dependent pathways. Additional minor products of 7DHC metabolism that were more polar than 7DHP were identified as 17-hydroxy-7DHP (in pig adrenals but not those of rats) and as pregna-4,7-diene-3,20-dione (7-dehydroprogesterone). Both products represented the major identifiable products of 7DHP metabolism in adrenal glands. Studies with purified enzymes show that StAR protein likely transports 7DHC to the inner mitochondrial membrane, that 7DHC can compete effectively with cholesterol for the substrate binding site on P450scc and that the catalytic efficiency of 3βHSD for 7DHP (Vm/Km) is 40% of that for pregnenolone. Skin mitochondria are capable of transforming 7DHC to 7DHP and the 7DHP is metabolized further by skin extracts. Finally, 7DHP, its photoderivative 20-oxopregnacalciferol, and pregnenolone exhibited biological activity in skin cells including inhibition of proliferation of epidermal keratinocytes and melanocytes, and melanoma cells. These findings define a novel steroidogenic pathway: 7DHC→22(OH)7DHC→20,22(OH)27DHC→7DHP, with potential further metabolism of 7DHP mediated by 3βHSD or CYP17, depending on mammalian species. The 5–7 dienal intermediates of the pathway can be a source of biologically active vitamin D3 derivatives after delivery to or production in the skin, an organ intermittently exposed to solar radiation.
85 citations
TL;DR: Combining a simple chromatographic system on celite microcolumns with specific antisera as binding reagents, simultaneous radioimmunoassay of pregnenolone, 17-5Δ-P, and dehydroepiandrosterone (DHEA) could be performed on the same aliquot of plasma, showing no significant difference between these two phases of the cycle.
Abstract: Combining a simple chromatographic system on celite microcolumns with specific antisera as binding reagents, simultaneous radioimmunoassay of pregnenolone (5Δ-P), 17-hydroxypregnenolone (17-5Δ-P), and dehydroepiandrosterone (DHEA) could be performed on the same aliquot of plasma. These steroids have been measured in plasma samples obtained from subjects of both sexes under various conditions. The plasma levels of 5Δ-P, 17-5Δ-P, and DHEA were found to be within the same range when 12 prepubertal boys and 15 men were studied. The mean levels of these steroids were respectively: 1.57, 0.95, and 1.2 ng/ml in prepubertal boys and 0.88, 0.73, and 1.82 in adult men. Six premenopausal women were studied during a complete menstrual cycle. Comparing the levels of 5Δ-P, 17-5Δ-P, and DHEA during the follicular and luteal phases showed no significant difference between these two phases of the cycle. However, plasma 5Δ-P was found to be significantly elevated in the luteal phase when the mean follicular and lu...
85 citations
TL;DR: Results support the suggestion that these neurosteroids could play a role in the initial response to stress and indicate that further work needs to be done to determine the mechanism for the interaction with ethanol.
Abstract: The neurosteroids pregnenolone and pregnenolone sulfate were tested for anxiogenic/anxiolytic effects in mice on the elevated plus-maze Pregnenolone in a dose of 001 micrograms/kg increased motor activity and caused an anxiogenic response, ie, a decreased number of entries onto the open arms of the plus-maze Pregnenolone sulfate had no effect on motor activity in the doses tested but showed a biphasic response on the plus-maze: at 100 and 10 micrograms/kg pregnenolone sulfate caused an anxiogenic response but at 01 microgram/kg it produced an anxiolytic response When administered with 15 g/kg ethanol, neither neurosteroid altered the depression in motor activity caused by ethanol However, all doses of pregnenolone tested blocked the anxiolytic effect of ethanol on the plus-maze while one dose of pregnenolone sulfate, 10 microgram/kg, attenuated the response to ethanol These results support the suggestion that these neurosteroids could play a role in the initial response to stress and indicate that further work needs to be done to determine the mechanism for the interaction with ethanol
85 citations