Biosynthesis and metabolism of steroid hormones by human adrenal carcinomas.
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Over a 15-year period, our university-based laboratory obtained 125 adrenal tumors, of which 15 (12%) were adrenal cortical carcinomas. Of these, 6 (40% of the carcinomas) occurred in patients with clear clinical manifestations of steroid hormone excess. Adrenal cortical carcinoma cells derived from the surgically resected tumors in 4 of these patients were isolated and established in primary culture. Radiotracer steroid interconversion studies were carried out with these cultures and also on mitochondria isolated from homogenized tissues. Large tumors had the lowest steroidogenic activities per weight, whereas small tumors had more moderately depressed enzyme activities relative to cells from normal glands. In incubations with pregnenolone as substrate, 1 mM metyrapone blocked the synthesis of corticosterone and cortisol and also the formation of aldosterone. Metyrapone inhibition was associated with a concomitant increase in the formation of androgens (androstenedione and testosterone) from pregnenolone. Administration of metyrapone in vivo before surgery in one patient resulted in a similar increase in plasma androstenedione, though plasma testosterone levels were not significantly affected. In cultures of two of four tumors examined, dibutyryl cAMP stimulated 11ss-hydroxylase activity modestly; ACTH also had a significant stimulatory effect in one of these tumors. Unlike results obtained with normal or adenomatous adrenal cortical tissues, mitochondria from carcinomatous cells showed a lack of support of either cholesterol side-chain cleavage enzyme complex or steroid 11ss-hydroxylase activity by Krebs cycle intermediates (10 mM isocitrate, succinate or malate). This finding is consistent with the concept that these carcinomas may tend to function predominantly in an anaerobic manner, rather than through the oxidation of Krebs cycle intermediates. (+info)
Increased progesterone secretion and 3 beta-hydroxysteroid dehydrogenase activity in human cumulus cells by pregnenolone is limited to the high steroidogenic active cumuli.
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PURPOSE: Several reports imply that lower progesterone secretion by cumulus-oocyte complexes (COCs) is associated with lower fertilization in the corresponding oocyte. The possible role of progesterone in oocyte fertilization in humans was studied using two approaches: (a) increasing the total progesterone secretion by culturing more than one COC per dish; and (b) increasing the cumulus cell progesterone secretion by providing pregnenolone as a substrate. METHODS: Mature COCs were cultured individually or cocultured in groups. Oocyte fertilization and progesterone secretion were tested after 20 hr and 3 days in culture, respectively. The cumuli from individually plated COCs were cultured in the absence of oocyte for an additional 3 days in order to test the effects of pregnenolone on progesterone secretion and the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. A comparable study with pregnenolone was performed on the corresponding granulosa-lutein cells. RESULTS: Increasing the number of COC to two instead of one led to a significant increase in both fertilization rate and progesterone secretion. The addition of pregnenolone during days 3-6 increased significantly both progesterone secretion and 3 beta-HSD activity. Comparable results were observed in granulosa-lutein cells subjected to pregnenolone treatment. Following the first 3 days culture, cumulus masses were categorized as secreting high or low progesterone levels. Adding pregnenolone had a greater effect on both progesterone secretion and 3 beta-HSD activity in the high-progesterone-secreting cumuli. CONCLUSIONS: Addition of pregnenolone increased progesterone secretion and 3 beta-HSD more efficiently in the higher-progesterone-secreting cumuli. Coculture of two COCs instead of one led to a higher fertilization rate and greater progesterone secretion. (+info)
Steroidogenic acute regulatory protein (StAR) and the intramitochondrial translocation of cholesterol.
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The steroidogenic acute regulatory (StAR) protein regulates the rate limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. Insight into the structure and function of StAR was attained through molecular genetic studies of congenital lipoid adrenal hyperplasia, a rare disease caused by mutations in the StAR gene. Subsequent functional analysis defined two major domains within the StAR protein, the N-terminal mitochondrial targeting sequence and the C-terminus, which promotes the translocation of cholesterol between the two mitochondrial membranes. Two models of StAR's mechanism of action, (1) stimulation of cholesterol desorption from the outer mitochondrial membrane and (2) an intermembrane shuttle hypothesis, are discussed with respect to the known biochemical and biophysical events associated with the process of steroidogenesis and the structure of StAR. StAR gene expression is regulated primarily at the transcriptional level, and the roles of transcription factors that govern basal and cAMP-dependent StAR expression including SF-1, C/EBP beta, Sp1 and GATA-4 are reviewed. (+info)
Multiple actions of steroid hormones--a focus on rapid, nongenomic effects.
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According to the traditional model, steroid hormones bind to intracellular receptors and subsequently modulate transcription and protein synthesis, thus triggering genomic events finally responsible for delayed effects. Based upon similarities in molecular structure, specific receptors for steroids, vitamin D(3) derivatives, thyroid hormone, retinoids, and a variety of orphan receptors are considered to represent a superfamily of steroid receptors. In addition, very rapid effects of steroids mainly affecting intracellular signaling have been widely recognized that are clearly incompatible with the genomic model. These rapid, nongenomic steroid actions are likely to be transmitted via specific membrane receptors. Evidence for nongenomic steroid effects and distinct receptors involved is presented for all steroid groups including related compounds like vitamin D(3) and thyroid hormones. The physiological and clinical relevance of these rapid effects is still largely unclear, but their existence in vivo has been clearly shown in various settings including human studies. Drugs that specifically affect nongenomic steroid action may find applications in various clinical areas such as cardiovascular and central nervous disorders, electrolyte homeostasis, and infertility. In addition to a short description of genomic steroid action, this review pays particular attention to the current knowledge and important results on the mechanisms of nongenomic steroid action. The modes of action are discussed in relation to their potential physiological or pathophysiological relevance and with regard to a cross-talk between genomic and nongenomic responses. (+info)
Stimulation of pregnenolone metabolism and aromatase activity by luteinizing hormone in mouse uterus.
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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. (+info)
Regulating ankyrin dynamics: Roles of sigma-1 receptors.
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Ankyrin is a cytoskeletal adaptor protein that controls important cellular functions, including Ca(2+) efflux at inositol 1,4,5-trisphosphate receptors (IP(3)R) on the endoplasmic reticulum. The present study found that sigma-1 receptors (Sig-1R), unique endoplasmic reticulum proteins that bind certain steroids, neuroleptics, and psychotropic drugs, form a trimeric complex with ankyrin B and IP(3)R type 3 (IP(3)R-3) in NG-108 cells. The trimeric complex could be coimmunoprecipitated by antibodies against any of the three proteins. Sig-1R agonists such as pregnenolone sulfate and cocaine caused the dissociation of an ankyrin B isoform (ANK 220) from IP(3)R-3. This effect caused by Sig-1R agonists was blocked by a Sig-1R antagonist. The degree of dissociation of ANK 220 from IP(3)R-3 caused by Sig-1R ligands correlates excellently with the ligands' efficacies in potentiating the bradykinin-induced increase in cytosolic free Ca(2+) concentration. Immunocytohistochemistry showed that Sig-1R, ankyrin B, and IP(3)R-3 are colocalized in NG-108 cells in perinuclear areas and in regions of cell-to-cell communication. These results suggest that Sig-1R and associated ligands may play important roles in cells by controlling the function of cytoskeletal proteins and that the Sig-1R/ANK220/IP(3)R-3 complex regulating Ca(2+) signaling may represent a site of action for neurosteroids and cocaine. (+info)
Inhibition of basal and stimulated progesterone synthesis by dichlorodiphenyldichloroethylene and methoxychlor in a stable pig granulosa cell line.
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The effects of the insecticide dichlorodiphenyldichloroethylene (DDE) and methoxychlor in a stable pig granulosa cell line, JC-410, were investigated. The studies of DDE and methoxychlor were conducted in combination with studies of cholera toxin, the protein kinase A activator that stimulates cAMP and progesterone synthesis and gene expression of P450 cholesterol side chain cleavage (P450scc), which converts cholesterol to pregnenolone. Administration of DDE at 3000 and 10 000 ng ml (-1) was found to decrease progesterone synthesis 0.49- and 0.25-fold, respectively, and to block the stimulatory effect of 100 ng cholera toxin ml (-1), after 24 h incubation. At 1-100 ng ml (-1), methoxychlor did not affect progesterone synthesis after 48 h incubation. However, 1000 ng methoxychlor ml (-1) decreased progesterone synthesis 0.32-fold, and both 100 and 1000 ng methoxychlor ml (-1) blocked the stimulatory effect of cholera toxin. At 3000 and 10 000 ng ml(-1), DDE decreased cAMP synthesis 0.66-and 0.36-fold, respectively. At 300, 3000 and 10 000 ng ml (-1), DDE also decreased cholera toxin-stimulated cAMP synthesis 0.84-, 0.68-, and 0.52-fold, respectively. Administration of 1-100 ng methoxychlor ml (-1) did not affect basal or cholera toxin-stimulated cAMP synthesis. Cholera toxin increased P450scc mRNA 1.4-fold after 24 h incubation, while 3000 and 10 000 ng DDE ml (-1) led to 0.39- and 0.18-fold reductions, respectively. The stimulatory effect of cholera toxin on P450scc mRNA was blocked by 3000 and 10 000 ng DDE ml(-1). Cholera toxin increased P450scc mRNA 3.48-fold after 48 h incubation, while 100 and 1000 ng methoxychlor ml (-1) increased P450scc mRNA 1.79- and 3.0-fold, respectively, and further increased the stimulatory effect of cholera toxin 6.47- and 5.44-fold, respectively. The results of the present study indicate that DDE inhibits granulosa cell steroidogenesis by affecting cAMP production and P450scc gene expression. However, methoxychlor appears to inhibit steroidogenesis by a mechanism occurring before the conversion of cholesterol into pregnenolone. (+info)
Regulation and substrate specificity of a steroid sulfate-specific hydroxylase system in female rat liver microsomes.
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The sulfate-specific hydroxylase system in liver microsomes from rats has been investigated with respect to its substrate specificity. Eighteen different C18, C19, C21, and C27 steroid sulfates and the coresponding free steroids have been incubated with microsomal preparations from male and female rats. The sulfate-specific system was only present in preparations from female rats and primarily catalyzed hydroxylation in position 15beta but also in position 7beta. In contrast to this, male liver microsomes were more efficient than female liver microsomes in hydroxylating free steroids; these were hydroxylated in positions 2alpha,2beta,6alpha,6beta,7alpha,7beta,16alpha, and 18. The sulfate-specific hydroxylase system in female liver microsomes was found to have rigid requirements c concerning the structure of ring D in the substrate molecule; only 17beta-sulfates (C18 and C19 steroids) and 21-sulfates (C21 steroids) were hydroxylated. Less rigid criteria, however, exist concerning the structure of ring A. The following K-m values were determined for microsomal 15beta-hydroxylation: 5alpha-androstane-3alpha,17beta-diol disulfate, 17.2 muM; 5beta-androstane-3alpha,17beta-diol disulfate, 16muM;5alpha-androstane-3alpha,17beta-diol 17-sulfate, 26 muM; and estradiol 17-sulfate, 181 muM. Some of the regulatory mechanism controlling the activity of the sex-specific 15beta-hydroxylase system also have been studied and compared to the mechanism controlling the activities of the less specific 2alpha-, 7alpha-, and 18-hydroxylase systems active on 5alpha-[4-14C]androstane-3alpha,17beta-diol. Biliary drainage did not affect the 15beta-hydroxylase activity, whereas the 2alpha- and 7alpha-hydroxylase activities decreased.. (+info)