Pregnenolone and its sulfate ester in the rat brain

Corpéchot, C.; Synguélakis, Monique; Talha, Saliha; Axelson, Magnus; Sjövall, Jan; Vihko, R.; Baulieu, Étienne-Émile; Röbel, P

doi:10.1016/0006-8993(83)90797-7

Cited by 359 publications

(185 citation statements)

References 16 publications

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“…The values for P, D and their sulfate esters are much higher than those of the sex steroids in all areas of the brain and in the same range as those reported in the rat (1,2). The values for unconjugated P and D are much higher than their respective values in plasma.…”

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confidence: 55%

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The Cholesterol Side‐Chain Cleavage Complex in Human Brain White Matter

Goascogne

Gouézou

Röbel

et al. 1989

J Neuroendocrinology

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Specific antibodies obtained in rabbits after injection of bovine cholesterol side-chain cleavage enzymes cytochrome P -~~O S C C , adrenodoxin and adrenodoxin-reductase were used for immunohistochemical studies in human brain. The three enzymes were co-localized in the white matter of the cerebellum. This observation strongly suggests the existence of steroidogenic activity in human oligodendrocytes, as previously reported in the rat, and suggests that the concept of 'neurosteroids' can be applied to A5-38-hydroxysteroid metabolites of cholesterol that accumulate in human brain.Two A5-3P-hydroxysteroid metabolites of cholesterol, pregnenolone (P) and deliydroepiandrosterone (D), have been detected in the brain of several mammalian species (rat, mouse, monkey and occasionally pig and man) (1-3). Definitive identification of the steroid moiety was made in rat brain extracts by gas/liquid chromatography-mass spectrometry (1, 2). These A5-38-hydroxysteroids persisted in brain after removal of steroidogenic organs, and we therefore proposed that their formation and/or accumulation in the brain depends on in situ mechanisms unrelated to the peripheral endocrine gland system. P is the key steroid synthesized from cholesterol in steroidogenic glands. The oxidative side-chain cleavage of cholesterol is operated by a specific enzyme complex, which includes cytochrome P -~~O S C C , adrenodoxin-reductase (Red) and adrenodoxin (Adx) (4). We have used specific antibodies to the bovine adrenal cytochrome P-45Oscc for the immunohistochemical localization of the enzyme in the adult rat brain, and we have found that the white matter was selectively immunostained throughout the brain ( 5 ) . Since the myelin of the white matter is made by a particular type of glial cell, the oligodendrocyte, we have isolated oligodendrocyte mitochondria, incubated them with The concentrations of P and D have been measured in human brains removed at autopsy 5 to 24 h post-mortem (7, 8). The values for P, D and their sulfate esters are much higher than those of the sex steroids in all areas of the brain and in the same range as those reported in the rat (1,2). The values for unconjugated P and D are much higher than their respective values in plasma. Hence, it was tempting to speculate that human brain is capable of de novo biosynthesis of steroids, as reported for the rat brain. Thus, we have attempted to localize, with the immunoperoxidase technique, cytochrome P-45Oscc, Adx and Red in human brain. We used specific antibodies to the adrenal enzymes of cattle origin. Though there are marked structural homologies between bovine and human side-chain cleavage enzymes, we first checked, using a testis sample, that available antibodies could be used for immunohistochemical staining of human tissues. Resu Its The human testisThe Leydig cells were strongly immunostained with the anticytochrome P-45Oscc IgGs, in contrast to the seminiferous tubules which served as control of tissue specificity (Fig. IA). A similar immunoperoxidase staining was ob...

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confidence: 55%

“…Two A5-3P-hydroxysteroid metabolites of cholesterol, pregnenolone (P) and deliydroepiandrosterone (D), have been detected in the brain of several mammalian species (rat, mouse, monkey and occasionally pig and man) (1)(2)(3). Definitive identification of the steroid moiety was made in rat brain extracts by gas/liquid chromatography-mass spectrometry (1, 2).…”

mentioning

confidence: 99%

The Cholesterol Side‐Chain Cleavage Complex in Human Brain White Matter

Goascogne

Gouézou

Röbel

et al. 1989

J Neuroendocrinology

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“…6) However, these methods have some shortcomings, such as requirement of complicated pretreatment and solvolysis steps and a decline in the recovery rate of the steroid. To overcome these problems, we employed liquid chromatography (LC)-electrospray ionization (ESI)-MS-MS to determine PREGS in rat brains without deconjugation, 7) but contrary to our expectations, its levels were less than one tenth of those previously reported (more than 5 ng/g tissue).…”

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confidence: 99%

Studies on Neurosteroids XVI. Levels of Pregnenolone Sulfate in Rat Brains Determined by Enzyme-linked Immunosorbent Assay Not Requiring Solvolysis

Higashi

Sugitani

Yagi

et al. 2003

Biological & Pharmaceutical Bulletin

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In the 1980s, Baulieu and co-workers demonstrated that some steroids, such as pregnenolone (PREG), dehydroepiandrosterone and their sulfates, are present in higher concentrations in the brain than in blood and are synthesized de novo in the central nervous system. 1) Such steroids are now universally referred to as neurosteroids. They act as modulators of several neurotransmitter receptors (g-aminobutyric acid A , N-methyl-D-aspartate and s 1 receptors) either as stimulators or inhibitors 1,2) and are involved in learning and memory performance.3) For these reasons, there is a considerable interest in determining the location and levels of different neurosteroids in the brain.The levels of PREG sulfate (PREGS) have conventionally been evaluated by measuring its genin formed after solvolysis by 5) or radioimmunoassay (RIA). 6) However, these methods have some shortcomings, such as requirement of complicated pretreatment and solvolysis steps and a decline in the recovery rate of the steroid. To overcome these problems, we employed liquid chromatography (LC)-electrospray ionization (ESI)-MS-MS to determine PREGS in rat brains without deconjugation, 7) but contrary to our expectations, its levels were less than one tenth of those previously reported (more than 5 ng/g tissue).4-6) One potential explanation for this discrepancy is that the LC-MS-MS method is more specific for brain samples than the GC-MS or RIA assay combined with solvolysis; the GC-MS may be detecting PREG derived from PREGS as well as that from other conjugated forms, such as sulfolipid conjugate, by solvolysis, and the RIA employs an antibody that reacts with other steroids having structures closely related to that of PREG. Although our LC-MS-MS method was specific and did not need a solvolysis procedure, it did not have sufficient sensitivity for the analysis of PREGS in the brain (limit of quantitation: ca. 160 pg/injection), and the obtained data using ca. 400 mg of tissue were not so reliable.7) Based on the results, we developed an enzyme-linked immunosorbent assay (ELISA) for PREGS using an antibody raised against 11a-hemiglutaryloxy-PREGS-bovine serum albumin conjugate. 8) This ELISA is sensitive (limit of quantitation: 1 pg/well) and does not require solvolysis procedure. The present paper describes the application of the ELISA to the determination of PREGS in rat brains and some data concerning the source of the brain PREGS. MATERIALS AND METHODS Materials and ReagentsStandard PREGS, PREG and all other reagents were those used in the previous study. 8)Oasis HLB cartridges (60 mg: Waters Assoc., Milford, MA, U.S.A.) were successively washed with AcOEt (1 ml), EtOH (2 ml) and H 2 O (2 ml) prior to use.Rats and Treatment Wistar strain rats (7 weeks old, male, 190-200 g) obtained from Japan S.L.C. (Hamamatsu, Japan) were used and euthanized by decapitation. Brain samples were collected from the rats assigned either to a control group (without injection, nϭ10), a group of animals receiving an intrapertioneal (i.p.) vehicle injection (5% EtOH/ s...

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“…Intracellular receptors that are involved in the regulation of specific gene transcription have been identified in neuroendocrine structures, and they account for the many molecular events that are involved in steroid hormone action. However, the characterization of pregnenolone (PREG) in the rat brain (at higher concentrations than in blood) and its persistence after removal of steroidogenic endocrine glands (adrenals and gonads) (2) led to the discovery of a steroid biosynthetic pathway in the nervous system (3). PREG, which retains the carbon skeleton of cholesterol, is the precursor of steroid hormones, but does not bind to any nuclear receptor of steroid hormones (4).…”

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confidence: 99%

Microtubule-associated protein 2 (MAP2) is a neurosteroid receptor

Fontaine-Lenoir

Chambraud

Fellous

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The neurosteroid pregnenolone (PREG) and its chemically synthesized analog 3β-methoxypregnenolone (MePREG) bind to microtubule-associated protein 2 (MAP2) and stimulate the polymerization of microtubules. PREG, MePREG, and progesterone (PROG; the physiological immediate metabolite of PREG) significantly enhance neurite outgrowth of nerve growth factor-pretreated PC12 cells. However, PROG, although it binds to MAP2, does not increase the immunostaining of MAP2, contrary to PREG and MePREG. Nocodazole, a microtubule-disrupting agent, induces a major retraction of neurites in control cultures, but pretreatment with PREG/MePREG is protective. Decreasing MAP2 expression by RNA interference does not modify PROG action, but it prevents the stimulatory effects of PREG and MePREG on neurite extension, showing that MAP2 is their specific receptor.

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Pregnenolone and its sulfate ester in the rat brain

Cited by 359 publications

References 16 publications

The Cholesterol Side‐Chain Cleavage Complex in Human Brain White Matter

The Cholesterol Side‐Chain Cleavage Complex in Human Brain White Matter

Studies on Neurosteroids XVI. Levels of Pregnenolone Sulfate in Rat Brains Determined by Enzyme-linked Immunosorbent Assay Not Requiring Solvolysis

Microtubule-associated protein 2 (MAP2) is a neurosteroid receptor

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