Structure of the murine constitutive androstane receptor complexed to androstenol: a molecular basis for inverse agonism.
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The nuclear receptor CAR is a xenobiotic responsive transcription factor that plays a central role in the clearance of drugs and bilirubin while promoting cocaine and acetaminophen toxicity. In addition, CAR has established a "reverse" paradigm of nuclear receptor action where the receptor is active in the absence of ligand and inactive when bound to inverse agonists. We now report the crystal structure of murine CAR bound to the inverse agonist androstenol. Androstenol binds within the ligand binding pocket, but unlike many nuclear receptor ligands, it makes no contacts with helix H12/AF2. The transition from constitutive to basal activity (androstenol bound) appears to be associated with a ligand-induced kink between helices H10 and H11. This disrupts the previously predicted salt bridge that locks H12 in the transcriptionally active conformation. This mechanism of inverse agonism is distinct from traditional nuclear receptor antagonists thereby offering a new approach to receptor modulation. (+info)
Epostane and indomethacin actions on ovarian kallikrein and plasminogen activator activities during ovulation in the gonadotropin-primed immature rat.
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Kallikrein and plasminogen activator (PA) are serine proteases that have been implicated in the ovulatory process. Epostane and indomethacin are anti-ovulatory agents that inhibit steroid and eicosanoid synthesis, respectively. This study examines the effects of these two anti-ovulatory agents on ovarian kallikrein and PA activities during ovulation. The proteases were assayed by their actions on chromogenic peptide substrates S-2266 and S-2251, respectively. The ovulatory process was induced in 25-day-old Wistar rats by giving them hCG (10 IU, s.c.) 2 days after the animals had been primed with eCG (10 IU, s.c.). Control animals ovulated approximately 60-70 ova/rat, with the first ova appearing in the oviducts at 10-12 h after hCG administration, and this was the same time ovarian kallikrein and PA activities reached a peak. When doses of epostane ranging from 0.1-5.0 mg/rat or doses of indomethacin ranging from 0.03 to 3.16 mg/rat were administered s.c. at 3 h after hCG, the two drugs inhibited ovulation and ovarian kallikrein and PA activities in a dose-dependent manner. However, the anti-ovulatory action of the two drugs was more closely correlated with suppression of kallikrein activity than with PA activity. Treatment of the animals with exogenous progesterone reversed the inhibitory action of epostane, but not of indomethacin. The results suggest that the increase in ovarian progesterone at the time of ovulation may influence ovarian kallikrein and PA activities. (+info)
Identification of key amino acids responsible for the substantially higher affinities of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) for substrates, coenzymes, and inhibitors relative to human 3beta-HSD2.
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The human type 1 (placenta, breast tumors, and prostate tumors) and type 2 (adrenals and gonads) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1 and 3beta-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern. Our recent studies have shown that His156 contributes to the 14-fold higher affinity that 3beta-HSD1 exhibits for substrate and inhibitor steroids compared with human 3beta-HSD2 containing Tyr156 in the otherwise identical catalytic domain. Our structural model of human 3beta-HSD localizes His156 or Tyr156 in the subunit interface of the enzyme homodimer. The model predicts that Gln105 on one enzyme subunit has a higher probability of interacting with His156 on the other subunit in 3beta-HSD1 than with Tyr156 in 3beta-HSD2. The Q105M mutant of 3beta-HSD1 (Q105M1) shifts the Michaelis-Menten constant (Km) for 3beta-HSD substrate and inhibition constants (Ki) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3beta-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3beta-HSD2. Our model also predicts that Gln240 in 3beta-HSD1 and Arg240 in 3beta-HSD2 may be responsible for the 3-fold higher affinity of the type 1 isomerase activity for substrate steroid and cofactors. The Q240R1 mutation increases the isomerase substrate Km by 2.2-fold to a value similar to that of 3beta-HSD2 isomerase and abolishes the allosteric activation of isomerase by NADH. The R240Q2 mutation converts the isomerase substrate, cofactor, and inhibitor kinetic profiles to the 4-14-fold higher affinity profiles of 3beta-HSD1. Thus, key structural reasons for the substantially higher affinities of 3beta-HSD1 for substrates, coenzymes, and inhibitors have been identified. These structure and function relationships can be used in future docking studies to design better inhibitors of the 3beta-HSD1 that may be useful in the treatment of hormone-sensitive cancers and preterm labor. (+info)
Olfactory responsiveness to two odorous steroids in three species of nonhuman primates.
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Social communication by means of odor signals is widespread among mammals. In pigs, for example, the C19-steroids 5-alpha-androst-16-en-3-one and 5-alpha-androst-16-en-3-ol are secreted by the boar and induce the mating stance in the sow. In humans, the same substances have been shown to be compounds of body odor and are presumed to affect human behavior. Using an instrumental conditioning paradigm, we here show that squirrel monkeys, spider monkeys and pigtail macaques are able to detect androstenone at concentrations in the micromolar range and thus at concentrations at least as low as those reported in pigs and humans. All three species of nonhuman primates were considerably less sensitive to androstenol, which was detected at concentrations in the millimolar range. Additional tests, using a habituation-dishabituation paradigm, showed that none of the 10 animals tested per species was anosmic to the two odorous steroids. These results suggest that androstenone and androstenol may be involved in olfactory communication in the primate species tested and that the specific anosmia to these odorants found in approximately 30% of human subjects may be due to their reduced number of functional olfactory receptor genes compared with nonhuman primates. (+info)
The pheromone androstenol (5 alpha-androst-16-en-3 alpha-ol) is a neurosteroid positive modulator of GABAA receptors.
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Androstenol is a steroidal compound belonging to the group of odorous 16-androstenes, first isolated from boar testes and also found in humans. Androstenol has pheromone-like properties in both animals and humans, but the molecular targets of its pheromonal activity are unknown. Androstenol is structurally similar to endogenous A-ring reduced neurosteroids that act as positive modulators of GABA(A) receptors. Here we show that androstenol has neurosteroid-like activity as a GABA(A) receptor modulator. In whole-cell recordings from cerebellar granule cells, androstenol (but not its 3beta-epimer) caused a concentration-dependent enhancement of GABA-activated currents (EC(50), 0.4 microM in cultures; 1.4 microM in slices) and prolonged the duration of spontaneous and miniature inhibitory postsynaptic currents. Androstenol (0.1-1 microM) also potentiated the amplitude of GABA-activated currents in human embryonic kidney 293 cells transfected with recombinant alpha1beta2gamma2 and alpha2beta2gamma2 GABA(A) receptors and, at high concentrations (10-300 microM), directly activated currents in these cells. Systemic administration of androstenol (30-50 mg/kg) caused anxiolytic-like effects in mice in the open-field test and elevated zero-maze and antidepressant-like effects in the forced swim test (5-10 mg/kg). Androstenol, but not its 3beta-epimer, conferred seizure protection in the 6-Hz electroshock and pentylenetetrazol models (ED(50) values, 21.9 and 48.9 mg/kg, respectively). The various actions of androstenol in the whole-animal models are consistent with its activity as a GABA(A) receptor modulator. GABA(A) receptors could represent a target for androstenol as a pheromone, for which it is well suited because of high volatility and lipophilicity, or as a conventional hormonal neurosteroid. (+info)
Crystallographic analysis of murine constitutive androstane receptor ligand-binding domain complexed with 5alpha-androst-16-en-3alpha-ol.
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The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily. In contrast to classical nuclear receptors, which possess small-molecule ligand-inducible activity, CAR exhibits constitutive transcriptional activity in the apparent absence of ligand. CAR is among the most important transcription factors; it coordinately regulates the expression of microsomal cytochrome P450 genes and other drug-metabolizing enzymes. The murine CAR ligand-binding domain (LBD) was coexpressed with the steroid receptor coactivator protein (SRC-1) receptor-interacting domain (RID) in Escherichia coli. The mCAR LBD subunit was purified away from SRC-1 by affinity, anion-exchange and size-exclusion chromatography, crystallized with androstenol and the structure of the complex determined by molecular replacement. (+info)
Relationship between the expression of hepatic but not testicular 3beta-hydroxysteroid dehydrogenase with androstenone deposition in pig adipose tissue.
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This study investigated the relationship between expression of hepatic and testicular 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and accumulation of androstenone in adipose tissue because of its relation to boar taint. The experiments were performed on 13 Large White (50%) x Landrace (50%) and Meishan (25%) x Large White (25%) x Landrace (50%), pigs, which differed in the level of backfat androstenone. Our previous work showed that the major product of the hepatic androstenone metabolism is 3beta-androstenol. In this study, the formation of 3beta-androstenol was inhibited by the specific 3beta-HSD inhibitor trilostane. These results are the first direct confirmation that 3beta-HSD is the enzyme responsible for androstenone metabolism in the pig. The expression of the hepatic but not testicular 3beta-HSD protein showed a negative relationship with the level of backfat androstenone (r2 = 0.64; P < 0.001) and was accompanied by a reduced rate of the hepatic androstenone clearance. Low expression of 3beta-HSD protein in the liver of high androstenone pigs was also accompanied by a reduced level of 3beta-HSD mRNA (P < 0.001), which suggests a defective regulation of the hepatic 3beta-HSD expression at the level of transcription. In contrast, expression of the testicular 3beta-HSD protein did not differ between animals with high and low androstenone levels (P > 0.05) and was lower compared with the hepatic 3beta-HSD expression. Cloning and sequencing of the 3beta-HSD coding regions established that the hepatic and testicular 3beta-HSD cDNA have identical sequences, which were 98% similar to the human 3beta-HSD isoform I. It is suggested that expression of a single 3beta-HSD gene is regulated by different mechanisms in pig liver and testis. The liver-specific regulation of 3beta-HSD expression contributes to the low rate of hepatic androstenone metabolism and therefore can be considered as one of the factors regulating deposition of androstenone in pig adipose tissue and subsequent development of boar taint. (+info)
Is 3alpha-androstenol pheromone related to menstrual synchrony?
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BACKGROUND AND METHODOLOGY: The ovarian cycles of females living and interacting together may synchronise due to pheromones released from axillary secretary glands, the highest concentration of which is produced in the mid-follicular phase, prior to ovulation. The objective of this study was to find evidence for menstrual synchrony in a group of female students living together and to obtain a correlation between the ability to smell the putative pheromone, 5alpha-androst-16-en-3alpha-ol (3alpha-androstenol), found in apocrine secretions and menstrual synchrony. This cross-sectional study involved 88 students who completed a standard questionnaire and whose sense of smell was measured using ten varying thresholds. The menstrual history, friendship scale and menstrual hygiene score was determined for the participants. RESULTS: A total of 59.1% of the subjects studied were found to have menstrual synchrony. There was no significant association between menstrual synchrony and smelling threshold. However, a significant correlation was found between menstrual synchrony and personal hygiene score (p<0.01). CONCLUSIONS: The phenomenon of menstrual synchrony may be related to various factors. The results failed to demonstrate any significant difference between synchronised and non-synchronised subjects in detecting the steroid by sense of smell. However, the odours associated with menstrual blood or vaginal discharge might have an affect on menstrual synchrony. (+info)