Neurosteroids: pregnenolone in human sciatic nerves.

The characterization and quantification of pregnenolone in human sciatic nerves were undertaken, following previous demonstration of the synthesis of this steroid in rat brain oligodendrocytes, to explore the hypothesis that Schwann cells may demonstrate the same biosynthetic activity. Pregnenolone was definitively identified by mass spectrometry and quantified by specific radioimmunoassay. Its concentration (mean +/- SD, 63.9 +/- 45.9 ng/g of wet tissue, n = 12) was greater than or equal to 100 times the plasma level and concentration found in tendons and muscle. No correlation was found with sex or age. Free dehydroepiandrosterone as well as sulfate and fatty acid esters of pregnenolone and dehydroepiandrosterone were also measured. Results are discussed in terms of the concept that these "neurosteroids" may be synthesized in the peripheral nervous system.     

Neurosteroids: biosynthesis of pregnenolone and progesterone in primary cultures of rat glial cells

Cells dissociated from newborn rat forebrains were established in long term primary cultures. The cultures were made up almost exclusively of oligodendrocytes and astrocytes, as confirmed by indirect immunofluorescence staining with monoclonal antibodies to galactocerebroside and glial fibrillary acidic protein, respectively. After 3 weeks of culture, the oligodendrocytes were also highly immunoreactive to monospecific polyclonal antibodies against cytochrome P450scc, an enzyme involved in the conversion of cholesterol to pregnenolone (P). Biosynthesis of [3H]cholesterol, [3H]P, and [3H]Pregn-5-ene-3 beta, 20 alpha-diol was demonstrated in these primary cultures by incubating cells with [3H]mevalonolactone in the presence of mevinolin and trilostane. The activity of the 2',3'-cyclic nucleotide 3'-phosphodiesterase enzyme, a documented indicator of oligodendrocyte differentiation, increased rapidly after day 10 of culture, together with the onset of steroid biosynthetic activity. Both reached a maximum at 3 weeks of culture and remained stable up to 6.5 weeks. In the absence of trilostane, [3H]P was converted by glial cell cultures to [3H]progesterone, [3H]5 alpha-pregnane-3,20-dione, and [3H]3 alpha-hydroxy-5 alpha-pregnan-20-one. The demonstration of P, pregn-5-ene-3 beta,20 alpha-diol, and progesterone synthesis by normal rat glial cells, once oligodendrocytes have undergone their differentiation process, brings additional support to the concept of "neurosteroids."     

Neurosteroids: biochemistry and clinical significance.

The brain, like the adrenals, gonads and the placenta, is a steroidogenic tissue. However, unlike classic steroidogenic tissues, the synthesis of steroids in the nervous system requires coordinated expression and regulation of genes encoding the steroidogenic enzymes in several different cell types (neurons and glia) at different locations in the nervous system, often at some distance from the cell bodies. Furthermore, the synthesis of these steroids might be developmentally regulated and related to their functions in the developing brain. The steroids synthesized by the brain and nervous system, given the name 'neurosteroids', have a wide variety of diverse functions. In general, they mediate their actions not through classic steroid hormone nuclear receptors, but through other mechanisms, such as ion-gated neurotransmitter receptors or direct/indirect modulation of other neurotransmitter receptors. We summarize the biochemistry of the enzymes involved in the biosynthesis of neurosteroids, their pharmacological properties and modes of action. The physiological relevance and potential uses of neurosteroids in certain human diseases are discussed.     

Effects of luteinizing hormone withdrawal on Leydig cell smooth endoplasmic reticulum and steroidogenic reactions which convert pregnenolone to testosterone

Depletion of endogenous LH with sc implants of testosterone-17 beta-estradiol (T-E) caused a reduction in the Leydig cell smooth endoplasmic reticulum (SER) over a 10-day treatment period. Decreases also occurred in some, but not all, of the testicular steroidogenic reactions responsible for the conversion of pregnenolone (PREG) to testosterone. The conversions of progesterone (PROG) to 17 alpha-hydroxyprogesterone, 17 alpha-hydroxyprogesterone to androstenedione, and androstenedione to testosterone were significantly correlated (P less than 0.05) with the loss of Leydig cell SER. In contrast, the testicular conversion of PREG to PROG in rats deprived of endogenous LH for up to 10 days was identical to that in intact controls. Similar results were obtained when rats were hypophysectomized for 10 days. These results indicate that the Leydig cell enzyme activities responsible for converting PREG to PROG are distributed in the Leydig cell SER fraction which remains in Leydig cell cytoplasm 10 days after LH withdrawal, and thus, the bulk of these enzyme activities are sequested in a SER compartment that is resistant to LH withdrawal.     

Neurosteroids Biosynthesis and function.

The term neurosteroids applies to those steroids that are both synthesized in the nervous system, either de novo from cholesterol or from steroid hormone precursors, and that accumulate in the nervous system to levels that are at least in part independent of steroidogenic gland secretion rates. Glial cells play a major role in neurosteroid formation and metabolism. Several neurosteroids are involved in either auto- or paracrine mechanisms involving both regulation of target gene expression and effects on membrane receptors (including those of neurotransmitters). The neuromodulatory role of neurosteroids in regulating the estrous cycle and pregnancy, stress, memory, and developmental as well as aging processes awaits further investigation.     

Regulation of cholesterol metabolism in rat adrenal mitochondria.

Addition of cholesterol to rat adrenal mitochondria resulted in a stimulation of pregnenolone synthesis. The slow step of the mitochondrial cholesterol side-chain cleavage reaction could be the interaction of the sterol with cytochrome P-450. The rate of cholesterol binding to this enzyme as observed spectroscopically correlated with the equilibration period (20 min) of the mitochondria and exogenous cholesterol required for maximal rates of pregnenolone synthesis. It is suggested that translocation of cholesterol between different sterol pools occurs within the mitochondria. Potential intracellular effectors that could be of importance in the movement or regulation of mitochondrial cholesterol include bivalent metallic ions, prostaglandins, cyclic nucleotides, polyamines and polylysine. Of the effectors studied, only calcium ions and polylysine markedly stimulated pregnenolone synthesis. These effectors might stimulate steroidogenesis by lateral displacement of cholesterol in the mitochondrial membrane into a compartment easily accessible to the cholesterol side-chain cleavage enzyme complex.     

Cholesterol pools in rat adrenal mitochondria: use of cholesterol oxidase to infer a complex pool structure.

ACTH stimulates the side-chain cleavage of cholesterol in the adrenal cortex in a cycloheximide-inhibitable manner. Its mechanism involves mobilizing cholesterol to a "steroidogenic pool" where the sterol can be metabolized to pregnenolone. This pool has been proposed to be in the inner mitochondrial membrane where cytochrome P-450scc resides, and regulation may involve transport of cholesterol from the outer to the inner membrane. To investigate the structure of the mitochondrial cholesterol pools, cholesterol oxidase has been used as a membrane-impermeant probe which should have selective access to outer membrane cholesterol. At 37 C, almost all the cholesterol in mitochondria from ether-stressed rats was metabolized by cholesterol oxidase. Depletion of an intermembrane space but not a matrix marker enzyme indicated partial disruption of the outer membrane. However, at 16 C, mitochondria remained largely intact, and cholesterol oxidase identified a unique pool of cholesterol, which was about two-thirds of the total. In experiments using mitochondria from ether-stressed rats, the size of the 16 C cholesterol oxidase accessible and inaccessible pools was compared with that of the steroidogenic pool. The steroidogenic pool was enhanced by pretreatment of some animals with aminoglutethimide (a P-450scc inhibitor) or eliminated with cycloheximide, both of which increased the total mitochondrial cholesterol. This approach reveals that the steroidogenic pool is not equivalent to the cholesterol oxidase-inaccessible pool. Rather, it overlaps both the cholesterol oxidase accessible and inaccessible pools. These results are not consistent with a simple two pool model, but can be explained by assuming a minimum of three cholesterol pools.     

Antibodies to cholesterol.

Cholesterol-dependent complement activation has been proposed as a factor that might influence the pathogenesis of atherosclerosis. Although antibodies to cholesterol conjugates have been reported, cholesterol is widely regarded as a poorly immunogenic substance. Monoclonal IgM complement-fixing antibodies to cholesterol were obtained in the present study after immunizing mice with liposomes containing high amounts of cholesterol (71 mol % relative to phosphatidylcholine) and lipid A as an adjuvant. Clones were selected for the ability of secreted antibodies to react with liposomes containing 71% cholesterol but not with liposomes containing 43% cholesterol. The antibodies also reacted with crystalline cholesterol in a solid-phase enzyme-linked immunosorbent assay. Binding of monoclonal antibodies to the surface of crystalline cholesterol was demonstrated by electron microscopy by utilizing a second antibody (anti-IgM) labeled with colloidal gold. The immunization period required to induce monoclonal antibodies was very short (3 days) and a high fraction of the hybrid cells (at least 70%) were secreting detectable antibodies to cholesterol. The results demonstrate that cholesterol can be a highly immunogenic molecule and that complement-fixing antibodies to cholesterol can be readily obtained.     

Mice convert melatonin to 6-sulphatoxymelatonin

The principal objective of this study was to establish whether mice can convert melatonin to 6-sulphatoxymelatonin (aMT6s). Precision-cut liver slices from C3H/He, C57BL/6, and BALB/c mice were incubated with melatonin, and the concentration of aMT6s in the culture media was determined using a sensitive and specific radioimmunoassay procedure. All three strains of mice generated aMT6s in a time-dependent manner; no significant strain differences were observed. When samples of the media were treated with sulphatase prior to analysis, aMT6s was not detectable. In contrast, similar treatment with beta-glucuronidase had no effect. 6-Sulphatoxymelatonin was present in the urine of both control and melatonin-treated C3H/He and C57BL6 mice. Treatment with melatonin led to a dramatic rise in the urinary levels of aMT6s in both mouse strains. Pre-treatment of the urines with sulphatase, but not beta-glucuronidase, markedly decreased the levels of aMT6s. Finally, in both strains urinary excretion of aMT6s displayed diurnal rhythmicity, peak excretion occurring during the dark hours. It may be inferred that: (a) mice can convert melatonin to aMT6s, both in vivo and in vitro, and (b) mice generate aMT6s in a rhythmic manner. Finally, the present studies confirm that determination of aMT6s rhythms in mice could provide an alternative, non-invasive, approach for assessing circadian clock function.     

Studies of the human testis. VII. Conversion of pregnenolone to progesterone.

Delta5-3beta-Hydroxysteroid dehydrogenase (EC.1.1.1.145) and steroid delta-isomerase (EC.5.3.3.1) were extracted from frozen human testicular tissue and co-precipitated by addition of ammonium sulfate. The activities of both enzymes were localized in the 0-40% (NH4)2SO4 fraction. The enzyme preparation catalyzed conversion of pregnenolone, 17alpha-hydroxypregnenolone, dehydroepiandrosterone, and androstenediol to the corresponding delta4-3-oxosteroid. Since isomerization appeared not to be the rate-limiting step of the overall reaction, measurement of activity of delta5-3beta-hydroxysteroid dehydrogenase was related to the amount of delta4-3-oxosteroid produced from the corresponding delta5-3beta-hydroxysteroid. Delta5-3beta-Hydroxysteroid dehydrogenase required NAD for maximal activity. The Michaelis constants (Km) for NAD were 50 muM, 33 muM and 14 muM, respectively for the dehydrogenation of pregnenolone, 17alpha-hydroxypregnenolone, androstenediol and dehydroepiandrosterone. Km values for each substrate were: pregnenolone 10 muM, 17alpha-hydroxypregnenolone and dehydroepiandrosterone 2.5 muM and androstenediol 3.0 muM. Human testicular delta5-3beta-hydroxysteroid dehydrogenase was inhibited by most of the steroids procued by the testis. The following steroids acted as competitive inhibitors with pregnenolone: 17alpha-hydroxypregenolone (Ki = 1.3 muM), androstenediol (Ki = 2.4 muM), dehydroepiandrosterone (Ki = 0.74 muM), 20alpha-dihydroprogesterone (Ki = 1.1 muM) estrone (Ki = 0.33 muM) and estradiol-17beta (Ki = 0.87 muM). 17alpha-Hydroxyprogesterone, testosterone and androstenedione showed mixed-type inhibition of the enzyme for pregnenolone. Progesterone and NADH were noncompetitive inhibitors of the enzyme for pregnenolone. Ki values, with respect to prenenolone, were 7.4 muM for progesterone and 150 muM for NADH. NADH, however, acted competitively with NAD and Ki value was 30 muM.     

The mechanism of the prolonged stimulatory effect of corticotrophin on pregnenolone production by guinea-pig adrenocortical mitochondria

The postulated prolonged stimulatory influence of ACTH on the adrenocortical mitochondrial synthesis of pregnenolone in response to ACTH was studied in adrenal mitochondria isolated from control guinea-pigs and from animals treated s.c. with 100 micrograms ACTH(1-24) twice daily on the day before the animals were killed. The animals from both groups were injected with 100 micrograms ACTH s.c. 30 min before killing. The mitochondrial production of pregnenolone (expressed in nmol per mg mitochondrial protein after 10-min incubation) increased from 1.52 +/- 0.46 (S.E.M.) in the control group to 4.50 +/- 0.59 for mitochondria from ACTH-treated animals, despite a similar free cholesterol content in the mitochondria, even when determined after a previous in-vivo treatment with aminoglutethimide to block further metabolism of cholesterol into pregnenolone. In addition, in the presence of an excess of exogenous cholesterol (100 mumol/l), the production of pregnenolone remained higher for mitochondria from ACTH-treated animals. In contrast, when the calcium concentration in the incubation medium was raised to 1 mmol/l, with subsequent enhancement in pregnenolone synthesis, the mitochondrial pregnenolone production became similar for both groups (8.28 +/- 1.11 nmol in the ACTH-treated group and 9.55 +/- 1.90 nmol in the control group), even in the presence of 100 mumol cholesterol/l (13.5 +/- 1.80 nmol in ACTH-treated animals and 14.8 +/- 1.93 nmol in controls). Cycloheximide treatment administered on the day before the animals were killed was without any effect on pregnenolone production in control animals (3.51 +/- 0.43 nmol before and 3.65 +/- 0.63 nmol after cycloheximide treatment).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transplantation of an oligodendrocyte cell line leading to extensive myelination.

Oligodendrocytes, the myelin-forming cells of the central nervous system, can be generated from progenitor cell lines and assayed for their myelinating properties after transplantation. A growth-factor-dependent cell line of rat oligodendrocyte progenitors (CG4) was carried through 31-48 passages before being transplanted into normal newborn rat brain or the spinal cord of newborn myelin-deficient (md) rats. In md rat spinal cord, CG4 oligodendrocyte progenitors migrated up to 7 mm along the dorsal columns, where they divided and myelinated numerous axons 2 weeks after grafting. CG4 cells were transfected with the bacterial lacZ gene and selected for high beta-galactosidase expression. The cell migration and fate of these LacZ+ cells were analyzed after transplantation. In normal newborn brain, LacZ+ oligodendrocyte progenitors migrated along axonal tracts from the site of injection and integrated in the forming white matter. In md rats, extensive migration (up to 12 mm) was revealed by staining for beta-galactosidase activity of the intact spinal cord where many grafted cells had moved into the posterior columns. Similar migration and integration of grafted cells occurred in the spinal cord of normal myelinated rats and after a noninvasive grafting procedure. Thus, oligodendrocyte progenitors can maintain their ability to migrate and myelinate axons in vivo after multiple passages in vitro. Such progenitor cell lines can be used to study the molecular mechanisms underlying oligodendrocyte development and the repair of myelin in dysmyelinating diseases.     

Heterogeneous pools of cholesterol side-chain cleavage activity in adrenal mitochondria from ACTH-treated rats: differential responses to different reducing precursors

Side-chain cleavage (SCC) of endogenous cholesterol in adrenal mitochondria isolated from ACTH-treated rats indicates that the size of the reactive cholesterol pool depends on the reducing precursor. At optimal concentrations of reductant, this pool was typically at least 2 times greater for isocitrate than for succinate. Succinate-supported reactions were rapidly completed, were highly sensitive to a 2-min preincubation, and failed to deplete spectrally detected P-450SCC-cholesterol complexes. Cholesterol SCC with 1 mM isocitrate exhibited 2-3 times more fast-phase metabolism, a pronounced slow phase, insensitivity to preincubation, and 60% depletion of spectrally detected cholesterol-P-450SCC complexes. Addition of bovine serum albumin (BSA) and EDTA, either during homogenization or directly to the incubation, prevented preincubation losses in response to succinate and removed most of the difference between succinate and isocitrate activities. This effect of BSA/EDTA was reversed within 5 min by octanoate by a mechanism that was enhanced by Ca2+. These distinct reductant characteristics suggest that only a subpopulation of mitochondria or of pools of activity within individual mitochondria can support cholesterol SCC with succinate while isocitrate is necessary for the remainder. The rapid responses of succinate-supported metabolism to preincubation or to octanoate suggest depletion of a critical factor for cholesterol metabolism. Metabolism of added 20 alpha-hydroxycholesterol or deoxycorticosterone established that NADPH remained fully available after succinate-supported cholesterol metabolism had stopped or after preincubation. Cessation of pregnenolone formation, therefore, results from a failure to supply cholesterol, not inadequate NADPH. The preincubation effect suggests loss of an energy-dependent component that enhances this supply of cholesterol. One possibility tested was that GTP, an activator of intermembrane cholesterol transfer (Xu et al. (1989) J. Biol. Chem. 264, 17674-17680), was being lost. Added GTP slightly activated succinate-supported pregnenolone production but did not prevent preincubation-induced losses. alpha-Ketoglutarate, which can generate matrix GTP, is an effective reductant that, in combination with succinate, prevents preincubation-induced losses.     

Estrogen increases precursor for pregnenolone synthesis with temperature-sensitive occupancy of P-450scc in mitochondria of rabbit corpus luteum

To examine the mechanism of estrogen's direct stimulation of steroidogenesis in the rabbit corpus luteum, we tested the hypothesis that the effect of estrogen on progestin production occurs at the site of processing of the precursor for pregnenolone (i.e. cholesterol) in the mitochondrion. For this purpose, we manipulated a model of estrogen stimulation by 1) removing sc estradiol-filled polydimethylsiloxane capsules from superovulated rabbits on day 9 of pseudopregnancy or 2) leaving the capsules in place to preserve a chronic estrogen stimulus. In the estrogen-deprived rabbits, the serum progesterone level fell precipitously in vivo within 24 h, but in rabbits with chronic estrogen stimulation, serum progesterone levels remained high. Our results show that the loss in progestin production caused by estrogen deprivation could not be attributed to loss of the mitochondrial cytochrome P-450 side-chain cleavage enzyme (P-450scc), a common rate-limiting step in progestin synthesis in many steroidogenic tissues. In addition, we confirmed that there was no loss in the catalytic activity of this enzyme. Treatment with aminoglutethimide in vivo followed by electron paramagnetic resonance spectroscopic analysis of mitochondria (prepared in aminoglutethimide-free buffers) showed that incubation of isolated mitochondria at 37 C and pH 6.2 caused an increased high spin state (g = 8.2 signal) and a concomitant decreased low spin state. This shift from low to high spin states, which is indicative of cholesterol-P-450scc complex formation, occurred in the luteal mitochondria from both estrogen-deprived and estrogen-stimulated rabbits. In further studies to localize estrogen's regulatory point, we determined that the initial (first minute) rate of production of pregnenolone (per mg protein or per U P-450scc) from endogenous precursor proceeded equally fast in mitochondria from estrogen-deprived and those from estrogen-stimulated rabbits. However, the rapid pregnenolone production in the estrogen-deprived group lasted for a shorter time and, after 30 min, yielded less pregnenolone per mg protein or per U P-450scc than did mitochondria from estrogen-stimulated rabbits. Addition of 25-hydroxycholesterol did not increase the initial rate of pregnenolone formation, indicating that precursor availability is not limiting during the initial period. In aggregate, these observations suggest that the effect of estrogen on progestin production in the rabbit corpus luteum is not regulation of the movement of cholesterol to the catalytic site on the inner mitochondrial membrane, even though this is a step in the regulation of protein hormone-stimulated steroidogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)