Regulation of acute cortisol synthesis by cAMP-dependent protein kinase and protein kinase C in a teleost species, the rainbow trout (Oncorhynchus mykiss).
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The effects of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) on acute ACTH-stimulated cortisol secretion were assessed using a specific PKA inhibitor (H-89) and a PKC activator (phorbol 12-myristate 13-acetate, PMA) in dispersed head kidney cells of rainbow trout (Oncorhynchus mykiss). To investigate the sites of action of both PKA and PKC, pregnenolone (a cortisol precursor stemmed from the rate limiting step in cortisol synthesis) and 25-OH-cholesterol (an exogenous substrate that bypasses the rate limiting step) were used as substrates, with and without ACTH stimulation. Inhibition of PKA decreased ACTH-stimulated cortisol secretion while activation of PKC had the same effect, demonstrating that PKA stimulates and PKC inhibits cortisol synthesis. Inhibition of PKA and activation of PKC had no significant effect on pregnenolone-stimulated cortisol synthesis, indicating that both PKA and PKC act upstream from the pregnenolone step. Inhibition of PKA and activation of PKC had no significant effect on basal cortisol secretion in the presence of 25-OH-cholesterol, suggesting that PKA and PKC exert their effects on the mitochondrial cholesterol translocation step. This study provided evidence for the stimulatory role of PKA and the inhibitory role of PKC in the signalling pathways leading to cortisol synthesis in teleosts. (+info)
Oxidized adrenodoxin acts as a competitive inhibitor of cytochrome P450scc in mitochondria from the human placenta.
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The conversion of cholesterol to pregnenolone by cytochrome P450scc is the rate-determining step in placental progesterone synthesis. The limiting component for placental cytochrome P450scc activity is the concentration of adrenodoxin reductase in the mitochondria, where it permits cytochrome P450scc to work at only 16% of maximum velocity. Adrenodoxin reductase serves to reduce adrenodoxin as part of the electron transfer from NADPH to cytochrome P450scc. We therefore measured the proportion of adrenodoxin in the reduced form in intact mitochondria from the human placenta during active pregnenolone synthesis, using EPR. We found that the adrenodoxin pool was only 30% reduced, indicating that the adrenodoxin reductase concentration was insufficient to maintain the adrenodoxin in the fully reduced state. As both oxidized and reduced adrenodoxin can bind to cytochrome P450scc we tested the ability of oxidized adrenodoxin to act as a competitive inhibitor of pregnenolone synthesis. This was done in a fully reconstituted system comprising 0.3% Tween 20 and purified proteins, and in a partially reconstituted system comprising submitochondrial particles, purified adrenodoxin and adrenodoxin reductase. We found that oxidized adrenodoxin is an effective competitive inhibitor of placental cytochrome P450scc with a Ki value half that of the Km for reduced adrenodoxin. We conclude that the limiting concentration of adrenodoxin reductase present in placental mitochondria has a two-fold effect on cytochrome P450scc activity. It limits the amount of reduced adrenodoxin that is available to donate electrons to cytochrome P450scc and the oxidized adrenodoxin that remains, competitively inhibits the cytochrome. (+info)
Direct inhibitory effect of digitalis on progesterone release from rat granulosa cells.
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Digoxin (10(-7) - 10(-5) M) or digitoxin (10(-7) - 10(-5) M) decreased the basal and human chorionic gonadotropin (hCG)-stimulated release of progesterone from rat granulosa cells. Digoxin (10(-5) M) or digitoxin (10(-5) M) attenuated the stimulatory effects of forskolin and 8-bromo-cyclic 3' : 5'-adenosine monophosphate (8-Br-cAMP) on progesterone release from rat granulosa cells. Digoxin (10(-5) M) or digitoxin (10(-5) M) inhibited cytochrome P450 side chain cleavage enzyme (cytochrome P450(scc)) activity (conversion of 25-hydroxyl cholesterol to pregnenolone) in rat granulosa cells but did not influence the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Neither progesterone production nor P450scc activity in rat granulosa cells was altered by the administration of ouabain. Digoxin (10(-5) M) or digitoxin (10(-5) M), but not ouabain, decreased the expression of P450scc and steroidogenic acute regulatory (StAR) protein in rat granulosa cells. The present results suggest that digoxin and digitoxin decrease the progesterone release by granulosa cells via a Na(+),K(+)-ATPase-independent mechanism involving the inhibition of post-cyclic AMP pathway, cytochrome P450scc and StAR protein functions. (+info)
Pregnenolone sulfate block of GABA(A) receptors: mechanism and involvement of a residue in the M2 region of the alpha subunit.
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Neurosteroids are produced in the brain, and can have rapid actions on membrane channels of neurons. Pregnenolone sulfate (PS) is a sulfated neurosteroid which reduces the responses of the [gamma]-aminobutyric acid A (GABA(A)) receptor. We analysed the actions of PS on single-channel currents from recombinant GABA(A) receptors formed from [alpha]1, [beta]2 and [gamma]2L subunits. Currents were elicited by a concentration of GABA eliciting a half-maximal response (50 microM) and a saturating concentration (1 mM). PS reduced the duration of clusters of single-channel activity at either concentration of GABA. PS had no discernable effect on rapid processes: no effects were apparent on channel opening and closing, nor on GABA affinity, and a rapidly recovering desensitised state was not affected. Instead, PS produced a slowly developing block which occurred at a similar rate for receptors with open or closed channels and with one or two bound GABA molecules. The rate of block was independent of membrane potential, implying that the charged sulfate moiety does not move through the membrane field. Change in a specific residue near the intracellular end of the channel lining portion of the [alpha]1 subunit had a major effect on the rate of block. Mutation of the residue [alpha]1 V256S reduced the rate of block by 30-fold. A mutation at the homologous position of the [beta]2 subunit ([beta]2 A252S) had no effect, nor did a complementary mutation in the [gamma]2L subunit ([gamma]2L S266A). It seems likely that this residue is involved in a conformational change underlying block by PS, instead of forming part of the binding site for PS. (+info)
Neuroactive steroid-serotonergic interaction: responses to an intravenous L-tryptophan challenge in women with premenstrual syndrome.
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OBJECTIVE: To evaluate the circulating concentrations of the neuroactive steroids in response to an i.v. L-tryptophan (L-TP) challenge across the menstrual cycle in women with premenstrual syndrome (PMS) and in controls. METHOD: An i.v. L-TP challenge was administered eight times during 1 month to five women with prospectively documented PMS and five age- and body mass-matched controls. Progesterone, allopregnanolone pregnenolone and 3alpha-5alpha-tetrahydrocorticosterone were assessed 15 and 0 min before, and at 30, 60 and 90 min after the challenge, across the menstrual cycle. RESULTS: In response to L-TP challenge, only allopregnanolone concentrations were significantly increased across the cycle and this increase was of a greater magnitude in women with PMS. Pregnenolone and 3alpha-5alpha-tetrahydrocorticosterone concentrations were not affected in women with PMS or controls after L-TP challenge. CONCLUSIONS: The data provide evidence for possible interaction between the serotonergic system and the neuroactive steroid, allopregnanolone. Women with PMS demonstrated a more significant increase in allopregnanolone concentrations in response to L-TP challenge, which could be due to an initial low basal serotonergic tone in the luteal phase in the PMS group. (+info)
The interaction of bovine adrenodoxin with CYP11A1 (cytochrome P450scc) and CYP11B1 (cytochrome P45011beta ). Acceleration of reduction and substrate conversion by site-directed mutagenesis of adrenodoxin.
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The kinetics of protein-protein interaction and heme reduction between adrenodoxin wild type as well as eight mutants and the cytochromes P450 CYP11A1 and CYP11B1 was studied in detail. Rate constants for the formation of the reduced CYP11A1.CO and CYP11B1.CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4-108), Adx-(4-114), T54S, T54A, and S112W, and the double mutants Y82F/S112W, Y82L/S112W, and Y82S/S112W (the last four mutants are Delta113-128) are presented. The rate constants observed differ by a factor of up to 10 among the respective adrenodoxin mutants for CYP11A1 but not for CYP11B1. According to their apparent rate constants for CYP11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants). The adrenodoxin mutants forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone conversion. This strong correlation suggests that C-terminal truncation of adrenodoxin in combination with the introduction of a C-terminal tryptophan residue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system. Such a variation of the adrenodoxin structure resulted in a mutant protein (S112W) showing a 100-fold increased efficiency in conversion of cholesterol to pregnenolone. (+info)
The conversion of pregnenolone-7alpha-3H and progesterone-4-14C to estrogens by corpora lutea of menstrual cycles and pregnancy.
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The metabolism of pregnenolone-7alpha-3H and progesterone-4-14C by human corpora lutea tissue of menstrual cycles and pregnancy was studied. In the incubations, equimolar mixtures of pregnenolone-7alpha-3H and progesterone-4-14C were used as substrates. Three corpora lutea of cycles were used as minced tissue. From those corpora lutea progesterone, 17-hydroxyprogesterone and androstenedione were formed, although no estrogens were formed. One corpus luteum of cycle and one corpus luteum of pregnancy were used as homogenated tissue, and those formed estrone and estradiol as well as the same three delta4-metabolites. The corpus luteum of cycle also formed testosterone. All metabolites including estrogens showed the lower 3H to 14C ratio than the starting ratio. 17-hydroxypregnenolone in only one corpus luteum, and no delta5-metabolites in the other four corpus luteum were identified. It is therefore proposed that the major pathway for estrogen formation in human corpus luteum is pregnenolone yields progesterone yields 17-hydroxyprogesterone yields androstenedione (or testosterone) yields estrone and estradiol. (+info)
Development of an enzyme immunoassay for serum 16-dehydropregnenolone.
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We have developed an enzyme immunoassay (EIA) for serum 16-dehydropregnenolone (3beta-hydroxy-5,16-pregnadien-20-one; 16-DHP). The antiserum against 16-DHP-3-hemisuccinate conjugated bovine serum albumin (16-DHP-3HS-BSA) was raised in rabbits. For use as an enzyme labeled antigen, 16-DHP-3HS was conjugated to alkaline phosphatase. The minimal amount of 16-DHP detected was 4 pg (0.013 pmol)/assay and the measurable range was from 0.06-60 ng/ml (0.191-191 nmol/l). The intra-assay coefficient of variation (C.V.) was 4.1% (0.73+/-0.03 ng/ml, mean+/-S.D., n=6), and inter-assay C.V. was 7.7% (0.13+/-0.01 ng/ml, n=6). A liner relation was observed between the serum sample dilution and the 16-DHP concentration. For the recovery study, authentic 16-DHP was added to a serum sample (original concentration: 0.10-0.14 ng/ml), and the recovery was found to be 94.4-96.8% (final 16-DHP concentrations calculated: 0.29-16.3 ng/ml). To investigate the reliability of the present EIA, the values from our EIA were compared with those obtained by GC-MS. The 16-DHP concentration could not be measured in serum by GC-MS because of its sensitivity. Therefore, the conjugated steroid, 16-DHPS, was first enzymatically hydrolysed and then the 16-DHP measured by both methods. There was a good correlation between the levels determined by these methods (Pearson's correlation coefficient: r=0.927, p<0.001, y=0.74x+3.61, n=27). The serum concentrations of 16-DHP in neonates and umbilical vein were 0.53+/-0.09 ng/ml and 0.88+/-0.61 ng/ml, respectively. No 16-DHP was detected in serum from normal healthy adults using the present EIA. These results suggest that 16-DHP originates from the fetus and neonate. (+info)