Metabolism Of Pregnenolone Research Articles

Stromal Cells of the Human Postmenopausal Ovary Display a Distinctive Biochemical and Molecular Phenotype

It has been postulated that the postmenopausal ovary, which consists chiefly of stromal cells, produces androgens, but studies of steroidogenic activity in these cells have given mixed results. To learn whether key steroidogenic enzymes involved in androgen synthesis are expressed in the postmenopausal ovarian stroma, stromal cells were isolated so that gene expression could be compared with that in cultured human theca cells and dermal fibroblasts. Genes involved in steroidogenesis include steroidogenic acute regulatory protein (StAR), P450scc (side-chain cleaving enzyme), 3β-hydroxysteroid dehydrogenase (3β-HSD), P450c17 (17-hydoxj1ase, 17,20 lyase), and P450c27. Several growth factor-binding proteins were also studied, including gremlin, IGF-binding protein-4, follistatin, and secreted frizzled-related proteins (sFRP)-1 and -4. In addition, the production of pregnenolone, progesterone, and hydroxysterol metabolites and the metabolism of 3 H-pregnenolone in ovarian stromal cells were examined. Isolated postmenopausal stromal cells had a stellate morphology distinct from the appearance of ovarian surface epithelial cells in culture. The stromal cells co-expressed both vimentin and cytokeratin. Transcript levels of StAR, P450scc, and 3β-HSD were 25- to 45-fold greater in theca cells than in stromal cells. Transcript levels of P450scc and 3β-HSD in stromal cells were similar to those in dermal fibroblasts, but StAR transcripts were much more abundant in stromal cells. Neither cell type expressed significant amounts of P450c17. The cholesterol-metabolizing enzyme P450c27 was expressed in both stromal and theca cells. There was no appreciable metabolism of pregnenolone in ovarian stromal cells, and no progesterone secretion was detected. Gremlin transcripts were 10-fold higher in ovarian stromal and fibroblast cells than in theca cells, but levels of IGF-binding protein-4 did not differ significantly. Follistatin transcripts were 24 times higher in stromal than in theca cells, and levels of sFRP-1 were 40-fold higher in stromal cells. Levels of sFRP-4 were 15 times higher than in theca cells and 80 times higher than in fibroblasts. Postmenopausal ovarian stromal cells have a biochemical/molecular phenotype distinct from that of fibroblasts. They do not appear to have substantial steroidogenic potential despite their ability to metabolize cholesterol to hydroxysterols. Expression of growth factor-binding proteins in stromal cells suggest that the stroma may help to limit the action of regulatory molecules in the premenopausal ovary.

11-hydroxylation in the biosynthesis of endogenous ouabain: multiple implications.

Accumulating evidence indicates that mammals use steroidal glycosides with "digitalis-like" activity. An endogenous ouabain (EO) has been described and is linked with long-term changes in sodium balance and cardiovascular structure and function. In the adrenal gland, the biosynthesis of EO and similar compounds appears to involve cholesterol side-chain cleavage with sequential metabolism of pregnenolone and progesterone. The more distal events in the biosynthesis have not been elucidated. Preliminary work using primary cell cultures from the bovine adrenal cortex suggests that the biosynthesis of EO is affected by inhibitors of 11beta-hydroxylase. Direct participation of 11-hydoxylase in EO synthesis would lead to an 11beta isomer of ouabain in mammals and, in vivo, an 11beta-oriented hydroxyl group would spontaneously form a mixture of two 11-19 hemiketal isomers. The latter isomers would likely be converted back to a single 11beta isomer of ouabain during isolation. The existence of an additional ring in the hemiketals, along with reduced flexion of the steroidal A, B, and C rings, raises the possibility that their in vivo physiological targets and actions differ from the isolated form of EO.

In vitro metabolism of pregnenolone to 7α-hydroxypregnenolone by rainbow trout embryos

Tissues taken from rainbow trout embryos at several developmental stages, were incubated in the presence of radioactively-labelled pregnenolone in order to determine the capability of salmonid embryos to metabolize steroids, such as pregnenolone, that are incorporated into the oocyte during gonadal growth and maturation. High performance liquid chromatography was used to separate the steroid products, and gas chromatography–mass spectrometry was applied for the chemical identification of the product. 7α-Hydroxypregnenolone, previously known to be produced only by ovarian tissues, was found to be the sole metabolite of pregnenolone metabolism by rainbow trout embryos. Sulfate and glucuronide conjugated forms of 7α-hydroxypregnenolone were also produced. We hypothesize that this metabolite provides a pathway for excretion of pregnenolone, enabling the embryo to maintain its own steroid milieu, although the possibility of 7α-hydroxypregnenolone also playing a physiological role cannot be excluded.

Cyp7b1 catalyses the 7alpha-hydroxylation of dehydroepiandrosterone and 25-hydroxycholesterol in rat prostate.

Dehydroepiandrosterone (DHEA) is the most prominent circulating steroid in humans, and it is a precursor for sex-steroid synthesis in peripheral tissues, including the prostate. Recently, enzyme-mediated pre-receptor metabolism has been recognized as a key step in determining steroid action in vivo. Hydroxylation of 3beta-steroids at the 7alpha-position has been reported in rat and human prostate to be a major inhibitory pathway to sex-steroid synthesis/action. However, the molecular identity of the enzyme responsible is so far unknown. We recently described a novel cytochrome P450 enzyme, cyp7b1, strongly expressed in the hippocampus of rodent brain, which catalyses the metabolism of DHEA, pregnenolone and 25-hydroxycholesterol to 7alpha-hydroxy products. In the light of this new enzyme, we have examined its possible role in 7alpha-hydroxylation conversion in rat prostate. NADPH-dependent 7alpha-hydroxylation was confirmed for 3beta-hydroxysteroids including DHEA and androstenediol, as well as 25-hydroxycholesterol. Kinetic analysis yielded an apparent K(m) of 14+/-1 microM for 7alpha-hydroxylation of DHEA in the prostate gland, a value similar to that recorded for recombinant cyp7b1 enzyme [13.6 microM; Rose, Stapleton, Dott, Kieny, Best, Schwarz, Russell, Bjoorkheim, Seckl and Lathe (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4925-4930]. The V(max) value of the prostate was 46+/-2 pmol/min per mg, and this activity was inhibited by clotrimazole, a P450-enzyme blocker. Moreover, RNA analysis (reverse-transcription PCR, Northern blotting and in situ hybridization) revealed a high expression of cyp7b1 mRNA in the rat prostate, restricted to the epithelium, suggesting that cyp7b1 catalyses oxysterol 7alpha-hydroxylation in the prostate gland.

Cyp7b1 catalyses the 7α-hydroxylation of dehydroepiandrosterone and25-hydroxycholesterol in rat prostate

Dehydroepiandrosterone (DHEA) is the most prominent circulating steroid in humans, and it is a precursor for sex-steroid synthesis in peripheral tissues, including the prostate. Recently, enzyme-mediated pre-receptor metabolism has been recognized as a key step in determining steroid action in vivo. Hydroxylation of 3β-steroids at the 7α-position has been reported in rat and human prostate to be a major inhibitory pathway to sex-steroid synthesis/action. However, the molecular identity of the enzyme responsible is so far unknown. We recently described a novel cytochrome P450 enzyme, cyp7b1, strongly expressed in the hippocampus of rodent brain, which catalyses the metabolism of DHEA, pregnenolone and 25-hydroxycholesterol to 7α-hydroxy products. In the light of this new enzyme, we have examined its possible role in 7α-hydroxylation conversion in rat prostate. NADPH-dependent 7α-hydroxylation was confirmed for 3β-hydroxysteroids including DHEA and androstenediol, as well as 25-hydroxycholesterol. Kinetic analysis yielded an apparent Km of 14±1µM for 7α-hydroxylation of DHEA in the prostate gland, a value similar to that recorded for recombinant cyp7b1 enzyme [13.6µM; Rose, Stapleton, Dott, Kieny, Best, Schwarz, Russell, Bjoorkheim, Seckl and Lathe (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4925-4930]. The Vmax value of the prostate was 46±2pmol/min per mg, and this activity was inhibited by clotrimazole, a P450-enzyme blocker. Moreover, RNA analysis (reverse-transcription PCR, Northern blotting and in situ hybridization) revealed a high expression of cyp7b1 mRNA in the rat prostate, restricted to the epithelium, suggesting that cyp7b1 catalyses oxysterol 7α-hydroxylation in the prostate gland.

Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera: Dytiscidae): II. Metabolism of 3β-hydroxypregn-5-en-20-one (pregnenolone)

The in vitro metabolism of pregnenolone by two Bacillus strains (HA-V6-3 and HA-V6-11) isolated from the foregut of the water beetle Agabus affinis (Payk.) was examined in the course of our studies about a possible participation of gut micro-organisms in the biosynthesis of prothoracic defensive steroids of dytiscids. The transformation products were identified by EI GC–MS of culture extracts after derivatization. The dominating reactions were hydroxylations, with 7α-hydroxypregnenolone as the major product. With considerably lower yields, 7β- and 15ξ-hydroxypregenolone were formed by both strains, while 11, 17 and 16α-hydroxypregnenolone were produced only by HA-V6-3. The occurrence of 7, 11α- and 7β, 11α-dihydroxypregnenolone as well as several minor products containing a 17α-OH group proved the capability of HA-V6-11 to hydroxylate pregenenolone at C 11 and C 17 as well. The monohydroxylated 7-OH-pregnenolones were partly oxidized to 7-oxopregnenolone by both strains. In trace amounts, HA-V6-3 performed 3β-acetylation of pregnenolone.

Stimulation of pregnenolone metabolism and aromatase activity by luteinizing hormone in mouse uterus.

In vitro metabolism of pregnenolone (P5) as well as production of 17beta-estradiol (E2) were studied in uteri of untreated and luteinizing hormone (LH)-treated mice that had been ovariectomized (OVX) at late-diestrus stage. In the uteri of untreated mice, [H]pregnenolone was shown to be metabolized to Delta-components such as 17alpha-hydroxypregnenolone (17alpha-P5) and dehydroepiandrosterone (DHEA), whereas LH treatment resulted in significant increases in the formation of progesterone (P4), 17alpha-hydroxyprogesterone (17alpha-P4), androstenedione (AD) and testosterone (T). This was assessed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The content and release of P4 was shown to be stimulated by LH. Trilostane, an inhibitor of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), inhibited LH-induced P4 synthesis and its release in a dose-dependent manner. A considerable increase in [H]estradiol formation from [H]testosterone was recorded in LH-stimulated uterine tissue as compared with the control, indicating the stimulatory effect of LH on aromatase activity. LH-stimulation in the synthesis of P4 and E2 in OVX mouse uteri was mimicked by dbcAMP (cell-permeable cAMP). Incubation with LH was shown to augment the conversion of P4 to various delta-3-oxosteroids. In vitro effects of LH on the synthesis and metabolism of P4, as well as on the stimulation of aromatase activity, were more pronounced in the uterine tissue of LH-primed OVX mice. Thus the results of the present study indicate that, under specific conditions, the uterus of the mouse behaves like steroidogenic tissue. Its prompt response to LH reveals the probable physiological relevance of the existence of LH receptors of high binding affinity in the uterine tissue of the mouse, as reported earlier.

3beta-hydroxysteroid dehydrogenase/delta5-->4-isomerase activity associated with the human 17beta-hydroxysteroid dehydrogenase type 2 isoform.

The type 2 isoform of human 17beta-hydroxysteroid dehydrogenase (17betaHSD2) efficiently catalyzes the oxidative metabolism of androgens and estrogens, and it is expressed in a large series of human peripheral tissues. To obtain a better understanding of the regulation of local steroid biosynthesis and metabolism in human tissues, we have established a dual steroidogenic activity of the 17betaHSD2 enzyme after transfection of human 17betaHSD2-transfected human embryonic kidney (293) cells. After transient transfection, the metabolism of testosterone, pregnenolone, and dehydroepiandrosterone (DHEA) in intact transfected 293 cells was evaluated by TLC-based radiometric assays. 17betaHSD2-transfected cells converted 91% of testosterone (1 micromol/L) into androstenedione in a 2-h incubation period. In addition, pregnenolone (1 micromol/L) was converted to progesterone (18.5%), whereas DHEA (1 micromol/L) was metabolized to androstenedione (8.3% conversion) in a 15-h incubation period. The kinetics of the 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17betaHSD2 activities using cell homogenate protein of stably transfected 293 cells indicated that the catalytic efficiency (apparent catalytic efficiency = maximum velocity/Km) of this 3betaHSD activity is approximately 2000-fold (pregnenolone as substrate) or 3000-fold (DHEA as substrate) weaker than that of 17betaHSD2 activity. It is noteworthy, however, that the apparent catalytic efficiency of the HSD3B2 gene product is only approximately 50-fold higher than that of the 3betaHSD aspect of the 17betaHSD2 gene product. Pregnenolone or DHEA effectively inhibited 17betaHSD2 activity in a noncompetitive fashion. Furthermore, the potent 5alpha-reductase/3betaHSD inhibitor, 17beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5alpha-androstane-3-one , inhibited neither 3betaHSD nor 17betaHSD2 activities. We conclude that human 17betaHSD2 enzyme exhibits 3betaHSD activity. Notwithstanding that this 3betaHSD activity is reduced compared to 17betaHSD oxidative activity, it may account for at least some of the reports of 3betaHSD activity found in human peripheral tissues that express notable amounts of the 17betaHSD2 isozyme as well as in individuals with severe classic 3betaHSD deficiency.

Identification of maturation-inducing steroid in a freshwater perchAnabas testudineus and differential responses of intact follicles and denuded oocytes to cyclic AMP in oocyte maturation

Postvitellogenic follicles of freshwater perch Anabas testudineus incubated with [(3)H]pregnenolone as exogenous precursor produced several metabolites, including 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) and 5 beta-pregnane-3 alpha, 17 alpha,20 beta-triol (5 beta-3 alpha,17 alpha,20 beta-P). These were identified by chromatography, microchemical reactions, and crystallization to constant specific activity. Following stimulation with fish (perch) pituitary extract (FPE) there was significant high production of DHP and 5 beta-3 alpha,17 alpha,20 beta-P, concomitant with a high percentage of germinal vesicle breakdown (GVBD). Inhibitor of steroidogenesis (trilostane) and inhibitors of protein synthesis (cycloheximide and actinomycin-D) completely blocked FPE-induced pregnenolone metabolism and oocyte maturation. The effectiveness of various C(21) steroids in inducing GVBD was examined. Results indicate that DHP was the most potent inducer of GVBD than other structurally related C(21) steroids. In intact follicles, FPE-stimulated production of DHP was shown to be mediated through the adenylate cyclase-cAMP pathway. Addition of IBMX or forskolin, which increases the endogenous cAMP level, as well as directly supplementing dbcAMP to the incubation medium, had no inhibitory effect on DHP-induced GVBD in the intact follicles. But all these agents were shown to inhibit GVBD in fully denuded oocytes. This study provides evidence that DHP, produced by postvitellogenic follicles through the adenylate cyclase-cAMP pathway, is the maturation-inducing steroid in freshwater perch and that the role played by cAMP in the induction of GVBD in intact follicles is different from that in the denuded oocytes. J. Exp. Zool. 287:294-303, 2000.

Pregnenolone metabolized to 17α-hydroxyprogesterone in yeast: biochemical analysis of a metabolic pathway

The cDNA coding for the human 3β-hydroxy-5-ene steroid dehydrogenase/5-ene-4-ene steroid isomerase (3β-HSD) has been expressed in yeast. When expressed from identical vectors except for the coding sequence, the specific activity of the type I is lower than that of the type II enzyme. A mutant of the human 3β-HSD type II lacking the putative membrane spanning domain 1 was generated by site directed mutagenesis: its apparent K m for pregnenolone (PREG) is significantly increased and its V reduced to the level of the type I enzyme. The influence of the kinetic properties of 3β-HSD in the accumulation of 17α-hydroxyprogesterone was probed by co-expression of the bovine 17α-hydroxylase cytochrome P450 (P45017α) cDNA. The metabolism of PREG was followed with time using the membrane fraction. Kinetic properties of the 3β-HSD were modulated such that its activity was in excess, limiting or balanced with respect to the activity of the P45017α and the accumulation of intermediates and products recorded. Conditions for the generation of the by-products resulting from the 17,20-Lyase activity of the P45017α were found. The potential applications of the system are discussed.

The inhibitor of phospholipase-A2, AACOCF3, stimulates steroid secretion by dispersed human and rat adrenocortical cells

AACOF3 is a trifluomethylketone analog of arachidonic acid, which inhibits phospholipase-A2 (PLA2). AACOCF3 was found to concentration-dependently increase basal aldosterone and corticosterone secretion by dispersed rat zona glomerulosa and zona fasciculata/reticularis cells, respectively, as well as aldosterone and cortisol production by dispersed human adrenocortical cells Maximal effective concentration was 10 −5M, and elicited about 2.5–3.0-fold rises in steroid output. 10 −5 M AACOCF3 also enhanced submaximally ( 10 −15 10 −12M ), but not maximally (10 −9 M) ACTH-stimulated hormonal secretion. Quantitative HPLC showed that 10 −5 M AACOCF3 evokes similar increases (from 2.0- to 3.0-fold) in the basal release of the entire spectrum of adrenocortical steroids ( i.e. both intermediate and definitive products of steroid synthesis), thereby suggesting that AACOCF3 acts on the early steps of steroid synthesis. Accordingly, when pregnenolone metabolism is prevented by cyanoketone, 10 −5 M AACOCF3 increased by about 8–10-fold the production of this steroid. In conclusion, we have demonstrated a side-effect of AACOCF3, which may become relevant in studies where this chemical is used to inhibit PLA2 in tissues able to convert cholesterol to pregnenolone.

Pregnenolone and progesterone metabolism by the testes of Bufo arenarum.

Sliced testis tissue from Bufo arenarum was incubated in the presence of [3H]pregnenolone. Testis fragments were also used for double isotope experiments using [3H]pregnenolone and [14C]progesterone. Specific activities were equated with the addition of radioinert pregnenolone. When yields of radiometabolites were analysed, pregnenolone was found to be a good precursor for C19 steroids such as dehydroepiandrosterone, 5-androsten-3 beta, 17 beta diol, testosterone, 5 alpha-dihydrotestosterone and a C21 steroid, 5 alpha-pregnan-3,20 dione. Progesterone mainly converts to 5 alpha-pregnan-3,20 dione, a steroid with unknown function in amphibians. The 5-ene pathway, including 5-androsten-3 beta, 17 beta diol as intermediate, could be predominant for androgen biosynthesis. Testes bypass not only progesterone but also androstenedione for testosterone biosynthesis.

Neurosteroids: expression of functional 3beta-hydroxysteroid dehydrogenase by rat sensory neurons and Schwann cells.

Steroids which are synthesized within the nervous system, such as progesterone, have been termed 'neurosteroids'. Levels of progesterone are much larger in peripheral nerves of rats and mice than in plasma, and persist after removal of the steroidogenic endocrine glands. Schwann cells are a source of progesterone: when isolated from embryonic dorsal root ganglia, they can synthesize progesterone from pregnenolone, the obligate precursor of all steroids. Locally produced progesterone has been shown to play an important role in myelination of peripheral nerve. We show here that sensory neurons from embryonic dorsal root ganglia also express 3beta-hydroxysteroid dehydrogenase and can convert [3H]pregnenolone to [3H]progesterone. Moreover, when cultured under different conditions and incubated for 24 h in the presence of 100 nM [3H]pregnenolone, they produce 5-10 times more [3H]progesterone than Schwann cells. The conversion of pregnenolone to progesterone by neurons is further increased by a diffusible factor produced by Schwann cells. Sensory neurons can also metabolize progesterone to 5alpha-dihydroprogesterone, but unlike Schwann cells, they do not produce 3alpha,5alpha-tetrahydroprogesterone, a potent positive allosteric modulator of gamma-aminobutyric acid type A receptors. We also show that cells isolated from the adult nervous system still have the capacity to convert [3H]pregnenolone to progesterone and its 5alpha-reduced metabolites: neurons and Schwann cells purified from dorsal root ganglia of 6 week old male rats show a similar pattern of pregnenolone metabolism to cells isolated from 18 day old embryos. These findings further support the important role of progesterone in the development and regeneration of the peripheral nervous system.

Effect of age on synthesis of the GABAergic steroids 5-alpha-pregnane-3,20-dione and 5-alpha-pregnane-3-alpha-ol-20-one in rat cortex in vitro.

Progesterone 5-alpha-reductase activity and 3-alpha-hydroxysteroid dehydrogenase activity were determined in the cortex of male and female rats in vitro. Age effects were investigated. The age of the male rats was 3-23 months, and that of the female rats 4-23 months. On addition, we investigated the enzyme 3 beta-hydroxysteroid oxidoreductase, 5-ene-isomerase in rat cortex in order to estimate the local synthesis of progesterone from pregnenolone. We found age-related increases in progesterone 5-alpha-reductase activity in the female rats (r = 0.64, p < 0.01, n = 6) and in the male rats (r = 0.5, p < 0.05, n = 18). 3-alpha-HSDH activity remained constant with age in female and male rats. The ratio of 3-alpha-hydroxysteroid dehydrogenase activity to 5-alpha-reductase activity tended to decrease with age (not significantly) in both male rats (r = -0.45, p = 0.06, n = 19) and the female rats (r = -0.36, p = 0.17, n = 16). We could not detect significant metabolism of pregnenolone to progesterone in rat cortex in vitro. The sensitivity of the assays of 3 beta-hydroxysteroid oxidoreductase, 5-ene-isomerase was calculated from the mean of the blank values + 3SD; the sensitivity of the assay was calculated as 0.103 fmol/mg protein/min. No significant metabolism of pregnenolone could be detected in cortex pooled from several male rats. The mean metabolism of progesterone was 1,200 times higher than the detection threshold of the assay for 3 beta-hydroxysteroid oxidoreductase, 5-ene-isomerase. We conclude that modifications of the inhibitory effects of the GABAergic steroids 5-alpha-pregnane-3,20-dione and 5-alpha-pregnane-3-alpha-ol-20-one via altered progesterone metabolism in rat cortex are possible with aging. A connection with the age-related increase in incidence of epileptic attacks, and with age-related changes in the effects of anticonvulsant and GABAA-active drugs, appears possible.

Differential effect of trilostane on the progestin milieu in the pregnant mare.

Trilostane, a competitive inhibitor of 3 beta-hydroxysteroid dehydrogenase, was administered intravenously to pregnant mares (n = 3) between day 277 and day 282 of gestation to determine its effect on the progestin milieu. In addition, placental tissue from mares at mid-gestation (150-300 days) (n = 4) were exposed to either deuterium-labelled pregnenolone alone or deuterium-labelled pregnenolone and trilostane to examine the effect of trilostane on placental metabolism of pregnenolone. Blood samples were collected from indwelling jugular catheters at frequent intervals for 48 h after infusion. Both plasma samples and incubation media were quantitatively analysed, after solid phase extraction and steroid derivitization, for concentrations of eight different progestin metabolites using gas chromatography and mass spectrometry. Trilostane infusion differentially affected the progestin milieu in vivo without inducing abortion. Forty-five minutes after infusion, maternal plasma concentrations of pregnenolone, 5-pregnene-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta,20 beta-diol and 3 beta-hydroxy-5 alpha-pregnan-20-one increased (P < 0.05) and remained high for 37 h. Concentrations of 5 alpha-pregnane-3,20-dione, 20 alpha-hydroxy-5 alpha-pregnan-3-one and 5 alpha-pregnane-3 beta,20 alpha-diol decreased 15 min after infusion (P < 0.05), yet 1.5 h after infusion, concentrations had increased and remained high until 37 h after infusion. Trilostane inhibited the conversion of pregnenolone to progesterone in vitro (P < 0.001) while mediating an increase (P < 0.05) in concentrations of 5 alpha-pregnane-3,20-dione and 3 beta-hydroxy-5 alpha-pregnan-20-one. These observations demonstrate that the pregnant mare possesses unique steroid hormone metabolic activity and suggests that another steroid, perhaps 5 alpha-pregnane-3,20-dione and not progesterone, is the important steroid precursor for the other progestin metabolites found in circulating plasma.

VASCULITIS ASSOCIATED WITH CONNECTIVE TISSUE DISORDERS

This article focuses on the important association of vasculitis with the connective tissue diseases. The immunopathologic mechanisms in these secondary vasculitides, with complement and immune-complex involvement, differ from those involved in primary systemic necrotizing vasculitis even though histologically the lesions may appear identical. Any size of vessel may be involved, so biopsy evidence of small vessel disease does not exclude arterial lesions elsewhere. The clinical manifestations are equally wide ranging and vary from one connective tissue disease to another. Treatment of vasculitis is generally with immunosuppressive agents but is dependent on the degree and severity of the vasculitis. There is an important need to make a distinction between vasculopathy, where there is no evidence of underlying inflammation, and true vasculitis because the former requires anticlotting therapy rather than immunosuppression.

Androgen biosynthesis in the stomach: expression of cytochrome P450 17 alpha-hydroxylase/17,20-lyase messenger ribonucleic acid and protein, and metabolism of pregnenolone and progesterone by parietal cells of the rat gastric mucosa.

Dehydroepiandrosterone (DHEA) and its conjugates persist in the rat brain, for up to 1 month after ablation of both adrenals and gonads. Since DHEA synthesis in brain from pregnenolone (PREG) was excluded, we have considered other tissular sources including the digestive tract. In situ hybridization with specific oligonucleotide probes showed that the parietal cells of the gastric mucosa, contrary to other cell types, strongly expressed P450(17) alpha messenger RNA. Expression of the enzyme in the parietal cells was confirmed by immunocytochemistry with specific antibodies. An intense reaction was observed in the stomach of adult males and of cyclic or pregnant females. Access to food did not influence the intensity of immunostaining. It appeared at postnatal days 16-21, then the number of positive cells increased rapidly and leveled off at adult age. Parietal cells were released by pronase digestion of everted stomachs from adult male and female rats and were purified by density gradient centrifugation on Nycodenz. 5 x 10(4) to 1.6 x 10(6) cells were incubated with either 1 microM 14C-PREG or 14C-progesterone (14C-PROG) at 37 C under 95% O2-5% CO2, for 10-180 min. PREG was converted to 17-OH PREG and to androstenediol, whereas PROG was converted to 17-OH PROG and to testosterone. Only minute amounts of either DHEA or androstenedione, respectively, were detected at any incubation time, indicating their fast conversion to the corresponding 17 beta-hydroxysteroids. 3H-25-OH cholesterol was not metabolized to 3H-PREG, and 14C-PREG was not converted to 14C-PROG, in accordance with negative immunocytochemical results with antibodies to cytochrome P450scc and 3 beta-hydroxysteroid dehydrogenase delta 5-->4-isomerase (3 beta-HSD). In conclusion, the parietal cells, which are known as the source of gastric acid secretion, can synthesize testosterone from PROG and androstenediol from PREG. The physiological relevance of such conversions remains to be established.

Androgen biosynthesis in the stomach: expression of cytochrome P450 17 alpha-hydroxylase/17,20-lyase messenger ribonucleic acid and protein, and metabolism of pregnenolone and progesterone by parietal cells of the rat gastric mucosa

Androgen biosynthesis in the stomach: expression of cytochrome P450 17 alpha-hydroxylase/17,20-lyase messenger ribonucleic acid and protein, and metabolism of pregnenolone and progesterone by parietal cells of the rat gastric mucosa

Carbamazepine increased pregnenolone synthesis blocked by peripheral type benzodiazepine receptor antagonist

AbstractDespite the widespread use of carbamazepine (CBZ) in the treatment of a number of neuropsychiatric disorders, its mechanism(s) of action remain to be elucidated. In view of the postulated interaction of CBZ with peripheral type benzodiazepine (BDZ) receptors and the role of these receptors in regulating neuroactive steroid biosynthesis, we investigated the effects of CBZ on pregnenolone accumulation in cultured rat C6 glioma cells. We found that CBZ elevates pregnenolone accumulation over a period of 60 min, and that this effect was not due to inhibition of pregnenolone metabolism. Preincubation of C6 cells with the selective peripheral type benzodiazepine receptor antagonist PK11195 blocked the CBZ‐induced elevation in pregnenolone‐induced elevation in pregnenolone accumulation. The results suggest that CBZ stimulated pregnenolone biosynthesis in cultured C6 cells involves a direct or indirect interaction with peripheral type benzodiazepine receptors. These effects may provide additional insights into the mechanisms by which CBZ regulates neuronal excitability. Depression 3:267‐y (1995/1996). © 1996 Wiley‐Liss, IncThis article is a US Government work and, as such, is in the public domain in the United States of America. .

Insulin as an Amplifier of Gonadotropin Action on Steroid Production: Mechanisms and Sites of Action in Goldfish Prematurational Full-Grown Ovarian Follicles

The present study examines the effects of mammalian insulin and insulin-like growth factor-1 (IGF-I) on steroid production by goldfish vitellogenic and prematurational full-grown ovarian follicles in vitro. Insulin (0.4-40 μg/ml) had no effect on its own, but potentiated human chorionic gonadotropin (hCG) and 25-hydroxycholesterol-stimulated testosterone production by prematurational full-grown follicles in both a dose- and time-dependent manner. By comparison, in vitellogenic follicles insulin (4 μg/ml) had no effect on hCG- or 25-hydroxycholesterol-stimulated testosterone and 17β-estradiol production. IGF-I (11-100 ng/ml) neither affected basal nor hCG- and 25-hydroxycholesterol-stimulated testosterone production by vitellogenic or prematurational full-grown follicles. Studies to determine the site(s) of action of insulin showed that insulin had no effect on basal cyclic adenosine 3′.5′-monophosphate (cAMP) generation, but it enhanced hCG-stimulated cAMP and testosterone production by prematurational full-grown follicles. Insulin also enhanced conversion of 25-hydroxycholesterol to testosterone, but had no effect on the metabolism of pregnenolone, suggesting that insulin either enhances mobilization of cholesterol to the mitochondria or increases the activity of the cytochrome P450 side-chain cleavage enzyme. The ability of insulin to enhance hCG and/or 25-hydroxycholesterol-stimulated testosterone production was blocked by the addition of the protein kinase A (PKA) inhibitor H89, which confirmed that these actions of insulin are exerted, at least in part, by the cAMP/PKA pathway. These findings suggest that insulin, but not IGF-I, is capable of participating in the regulation of ovarian steroid biosynthesis through effects at multiple steps within the steroid cascade and that ovarian responsiveness to insulin changes during the course of follicular development in the goldfish. Together, these results provide new insights into the biological action of insulin in the fish ovary.