Biomedical subjects

P F Hall

Publications and source records attributed to P F Hall.

At least 55 records · Page 3Linked to original sources

PubMed1990

Diagnosis of dystocia and management with cesarean section among primiparous women in Ottawa-Carleton.

We carried out a chart review study to determine the rate of diagnosis of dystocia (abnormal progress) and the use of cesarean section to treat dystocia among 3887 primiparous women who gave birth to a single baby in the vertex presentation at four hospitals in Ottawa-Carleton in 1984. Of the 3740 women who had some labour 1127 (30.1%) were given a diagnosis of dystocia. Cesarean section for dystocia was done during all phases of labour (41% of procedures in the latent phase, 38% in the active phase and 21% in the second stage). The cesarean section rate varied among the hospitals from 11.8% to 19.6%. A total of 75% of the cesarean sections were for dystocia, disproportion or failed induction. The findings suggest that cesarean section is being done for disproportion without a trial of labour beyond the latent phase and for dystocia in the absence of fetal distress. If these practices were modified the cesarean section rate could be reduced from 16% to about 8%, the rate found in some other centres and that observed in Canada in the early 1970s.

Adult↗

PubMed1990

The role of calmodulin in the responses to adrenocorticotropin of plasma membranes from adrenal cells.

Inhibitors of calmodulin [trifluoperazine, chlorpromazine, pimozide, and calmidazolium N-(6-aminohexyl)5-chloro-1-napthalenesulphonamide (W7)] and calmodulin antibodies were used to investigate the role of calmodulin in the response of Y-1 mouse adrenal cells to ACTH, with particular reference to events in the plasma membrane. In whole cells it was found that two responses (production of steroids and cAMP) to two stimulating agents (ACTH and forskolin) were inhibited by trifluoperazine at concentrations consistent with those involved in binding of the inhibitor to pure calmodulin (10-25 microM). The steroidogenic responses were also inhibited by the three other inhibitors of calmodulin (chlorpromazine, calmidazolium, and W-7). Trifluoperazine and pimozide (1-500 microM) did not inhibit binding of an [125I]ACTH analog to highly purified plasma membranes of Y-1 cells or to the cells themselves. With Y-1 plasma membranes it was found that trifluoperazine, pimozide, W-7, and calmodulin antibodies inhibited the increase in adenylate cyclase activity in response to ACTH, but not the cyclase responses to cholera toxin or forskolin. Moreover, the effect of cholera toxin on the ADP-ribosylation of specific membrane substrates was independent of the presence or absence of endogenous and/or exogenous Ca2+/calmodulin. The response of adenylate cyclase to ACTH was also decreased in plasma membranes from which calmodulin was removed by washing, and exogenous calmodulin partly reversed this decrease. Anti-calmodulin immunoglobulin inhibited the stimulation of adenylate cyclase produced in plasma membrane by ACTH, but was without effect on the responses to cholera toxin and forskolin. Exogenous calmodulin partly reversed the inhibition of stimulation by ACTH of adenylate cyclase produced by the antibody. It is concluded that calmodulin influences the events taking place in the plasma membrane in response to ACTH, after the binding of the hormone to its receptor and before the action of the G protein (Gs). That is, calmodulin is involved in coupling the occupied receptor to Gs. The effects of inhibitors of calmodulin in whole cells must involve some additional effect(s) requiring the intact cell.

Adenosine Diphosphate Ribose↗

PubMed1990

Voltage-dependent Ca2+ channels are involved in regulation of steroid synthesis by bovine but not rat fasciculata cells.

Bovine but not rat fasciculata cells show a concentration-dependent stimulation of the production of corticosteroids by addition of external Ca2+ to the incubation medium. Both cell types respond to ACTH in a concentration-dependent manner. Increasing concentrations of K+ (0-20 mM) cause increased production of corticosteroids and accelerated influx of Ca2+ in bovine fasciculata cells but no change in either of these two parameters in rat fasciculata cells. Two inhibitors of Ca2+ channels (nifedipine and PY108-068) inhibit both the production of steroids by unstimulated bovine cells and the stimulation produced by three agents (ACTH, (Bu)2 cAMP, and K+). Half-maximal inhibition of these responses was produced in each case by submicromolar or low micromolar concentrations of the inhibitors. These inhibitors are without effect in rat fasciculata cells. One Ca2(+)-channel agonist (BAY K8644) stimulated synthesis of steroids by bovine cells and potentiated the response to ACTH. The agonist acts in the low micromolar range but was without effect on rat cells or on the responses of steroid synthesis by these cells to either ACTH or dibutyryl cAMP. Moreover, bovine fasciculata cells show specific binding sites for [+]PN 200-110, a specific ligand for 1,4-dihydropyridine receptors associated with voltage-dependent Ca2+ channels [dissociation constant (Kd), 14.3 nM; maximum number of binding sites (Bmax), 0.52 pmol/10(6) cells]. Rat cells show no specific binding of PN 200-110. We conclude that bovine fasciculata cells possess voltage-dependent Ca2+ channels which are involved in the regulation of steroid synthesis in these cells and that rat fasciculata cells are without such channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1990

The role of intermediate filaments in adrenal steroidogenesis.

Cholesterol is stored in adrenal cells as ester in lipid droplets, which are transported to mitochondria to provide a substrate for steroid hormone synthesis. Using mouse adrenal tumour cells (Y-1), we show here that approximately 33% of the adrenal cell cholesterol ester is bound tightly to intermediate filaments while the rest is either loosely attached or free in the cytosol. Specific binding of droplets to intermediate filaments was demonstrated by immunofluorescence and electron microscopy. Immunofluorescence was based upon Nile Red to stain lipid and antibodies to vimentin, actin and tubulin. Electron microscopy, including immunoelectron microscopy with protein A conjugated to gold particles (5 nm), was used to examine whole mounts of cytoskeletons and intermediate filaments. Immunofluorescence reveals that bound droplets are surrounded by a capsule containing vimentin and can be removed from the filaments by extraction with ethanol or 6 M urea. Negative staining of the urea extracts revealed isolated droplets. To the extent that cholesterol ester is the storage form of steroidogenic cholesterol, the knowledge that lipid droplets containing such esters are attached to intermediate filaments may prove important in unravelling the complex process of the transport of cholesterol to mitochondria.

Adrenal Gland Neoplasms↗

PubMed1989

Isolation and characterisation of calcineurin from adrenal cell cytoskeleton: identification of substrates for Ca2+-calmodulin-dependent phosphatase activity.

Ca2+-calmodulin-dependent protein phosphatase activity is found in cytoskeletons of Y-1 mouse adrenal and bovine fasciculata cells. The activity is inhibited by three inhibitors of calmodulin (trifluoperazine, W-7 and pimozide) with EC50 in the low micromolar range. Protein phosphatase activity is inhibited by vanadate, fluoride, Zn2+ and pyrophosphate, stimulated by Mn2+ and found to be tightly bound to the cytoskeleton. Substrates for endogenous phosphatase activity were defined by one- and two-dimensional polyacrylamide gels. Phosphatase activity was seen with proteins that are substrates for both cyclic AMP-dependent and cyclic AMP-independent kinase enzymes. One specific Ca2+-calmodulin-dependent phosphatase, namely calcineurin, was purified to near homogeneity from cytoskeletons of Y-1 cells. The enzyme was found to be a heterodimer (MW 61,000 and 16,000) and the smaller subunit was shown to cross-react with antibodies raised against calcineurin from bovine brain. The purified enzyme catalyzes dephosphorylation of proteins (phosphorylase kinase and casein), phosphoamino acids (tyr greater than thre greater than ser) and a synthetic substrate (p-nitrophenyl phosphate). In addition, a new application of membrane transfer was devised by which the purified enzyme was incubated with a Western blot of cytoskeleton following incubation with [32P]ATP. This method defined four specific substrates of the enzyme (MW 150,000, 55,000, 35,000 and 30,000). Anti-calcineurin revealed that only a single Ca2+-calmodulin-dependent phosphatase is found in adrenal cell cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Identification of des-(Gly-Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor.

Delivery of cholesterol to inner mitochondrial membranes is rate-limiting for steroidogenesis in the zona fasciculata of adrenal cortex. A protein that stimulates this process was isolated to homogeneity from bovine adrenal tissue. This protein's primary structure has been determined in its entirety by a combination of automated Edman microsequencing, fast-atom bombardment mass spectrometry (FAB-MS). The sequence was identical to that previously reported for bovine brain endozepine, except that it lacks the last two residues, -Gly-Ile, at the C terminus. To our knowledge, isolation of an endozepine-related protein from a tissue other than brain has not been reported previously. Endozepine competes with benzodiazepines for saturable binding sites in synaptosomes and in mitochondria of specific peripheral tissues. Previous reports have localized the adrenal benzodiazepine receptor to the outer mitochondrial membrane. In this report, we show that the prototypic benzodiazepine, diazepam, effects a stimulation of adrenal mitochondrial cholesterol delivery similar to that observed for endozepine. The effective diazepam concentration was consistent with that previously shown to displace a high-affinity ligand of the mitochondrial benzodiazepine receptor. The action of diazepam in adrenal mitochondria suggests that the mediation of corticotropin-induced steroidogenesis may be the physiological function of the peripheral-type benzodiazepine receptor. These studies provide new insights into the previously unknown function of peripheral benzodiazepine receptors and should allow new investigations into the stimulation of steroidogenesis by endozepines and benzodiazepines in the brain and in certain peripheral tissues.

Adrenocorticotropic Hormone↗

PubMed1989

Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton.

The cytoskeletons of Y-1 mouse adrenal tumor cells contain a calcium and phospholipid-dependent protein kinase (protein kinase C) that is bound sufficiently tight to resist extraction by 0.5% Triton but not by 1.0% Triton. The enzyme has been purified to near homogeneity from cytoskeleton and cytosol. It shows features typical of this type of kinase, namely a requirement for Ca2+ and phospholipid, stimulation by tumor promoters but not by nontumor-promoting phorbol esters, and inhibition by trifluoperazine. The enzyme shows specificity for four substrates found in the cytoskeleton, namely 80, 33, 20, and 18 kD. The first three substrates are phosphorylated by the enzyme; the fourth is dephosphorylated and is therefore affected by the kinase indirectly. The 80-kD protein is the kinase enzyme itself which is autophosphorylated in vitro and in the cytoskeleton. The 20-kD protein is myosin light chain. The 33- and 18-kD proteins are unidentified. The same substrates were phosphorylated when Y-1 cells were permeabilized with digitonin and incubated with [gamma-32P]ATP and phorbol-12-myristate-13-acetate. Partly purified protein kinase C changes the extent of phosphorylation of the same substrates when added to cytoskeletons previously extracted to remove endogenous protein kinase C. Addition of Ca2+, phosphatidylserine, and phorbol-12-myristate-13-acetate to cytoskeletons, and addition of these three agents plus protein kinase C to extracted cytoskeletons, causes these structures to undergo a rapid and extensive rounding. A similar change is induced in intact cells by addition of phorbol ester. It is concluded that protein kinase C is capable of changing the shape of adrenal cells by an action that involves autophosphorylation and phosphorylation of myosin light chain. This response may in turn be related to the steroidogenic responses to ACTH and cyclic AMP.

Adenosine Triphosphate↗

PubMed1989

Cytochromes P-450 in steroidogenesis: are these enzymes more specific than those of drug metabolism?

1. In contrast to drug-metabolizing cytochromes P-450, the corresponding steroidogenic enzymes appear to be highly specific with respect to substrates, reactions catalysed, and the sites in the substrate molecule that are attacked. 2. Recent studies have shown that important exceptions to this generalization are encountered. 3. The conversion of 11-deoxycorticosterone to aldosterone requires three enzymatic reactions, the last of which (aldehyde synthetase), like the first two, requires a P-450 mono-oxygenation. The involvement of P-450 in this third step (from alcohol to aldehyde) was demonstrated by photochemical action spectra and determination of stoichiometry. The three reactions were shown to be catalysed by a single enzyme as demonstrated by immunoprecipitation and the use of a homogenous enzyme. 4. The conversion of pregnenolone and progesterone to the corresponding delta 16-C19 steroids, which are pheromones, is catalysed by a cytochrome P-450 that also catalyses the conversion of progesterone to androstenedione (that is, C21 side-chain cleavage; hydroxylase/lyase). The synthesis of the delta 16 compounds requires cytochrome b5. 5. The conversion of testosterone and epitestosterone to androstenedione is catalysed by P-450b. Studies of kinetic isotope effects using deuterium and 18O2 show that P-450b catalyses this reaction with epitestosterone via the formation of a gem diol, whereas, with testosterone as substrate, one-third of the product is formed via the same mechanism while the remainder results from the mechanism described as dual hydrogen abstraction.

Animals↗

PubMed1989

Forskolin activates voltage-dependent Ca2+ channels in bovine but not in rat fasciculata cells.

The action of forskolin on bovine and rat fasciculata cells was examined in freshly prepared cells. Bovine cells show a close parallelism between production of steroids and production of cAMP as a function of the concentration of ACTH up to 10(-8) M. By contrast, forskolin (10(-7)-10(-5) M) causes a similar increase in steroid synthesis but relatively little effect on the production of cAMP. cAMP-dependent protein kinase shows a similar response to ACTH but no response to forskolin in the same range of concentrations. ACTH and forskolin, at submaximal concentrations, cause greater steroid production when added together than when added separately, but the two agents at high concentrations produce the same response whether added together or separately. The inhibitors of voltage-dependent Ca2+ channels inhibit the steroidogenic response to forskolin (IC50 for nifedipine is 0.1 microM and for Py108-068 is 0.4 microM). A Ca2+ channel agonist (BAY K8644) increases the steroidogenic response of bovine adrenal cells to forskolin, but not that of ACTH. Finally, forskolin causes a concentration-dependent uptake of Ca2+ by these cells; in the concentration range of 0.1-10 microM, forskolin caused an increase in [Ca2+] from 185 nM to 345 nM. By contrast, forskolin caused some stimulation of the production of cAMP, but not that of steroids in rat fasciculata cells. It is concluded that in bovine fasciculata cells forskolin activates voltage-dependent Ca2+ channels with a consequent increase in steroid synthesis. This effect is independent of the well known action of forskolin on adenylate cyclase. Rat fasciculata cells, on the other hand, do not possess such Ca2+ channels and do not show a steroidogenic response to forskolin.

Adenylyl Cyclase Inhibitors↗

PubMed1989

The influence of exogenous free cholesterol on steroid synthesis in cultured adrenal cells.

Previous studies showed that Y-1 cells in which the concentration of cholesterol in plasma membranes (expressed relative to phospholipid as a molar ratio, C/P) was raised by incubation with cholesterol produced more 20 alpha-dihydroprogesterone than cells with normal or low plasma membrane C/P values. This difference was not associated with any difference in the production of cAMP. The cells respond to (Bu)2cAMP in the order high greater than normal greater than low C/P. High C/P is associated with higher levels of cholesterol in the cytosol and higher concentrations of this steroid in the inner mitochondrial membrane. The rate of transport of cholesterol to the inner membrane differed in the three groups of cells: high greater than normal greater than low C/P. Production of pregnenolone by mitoplasts was no different in the three groups, and the same applies to the production of pregnenolone by mitochondria from cells incubated with and those incubated without (Bu)2cAMP. 25-Hydroxycholesterol increased the production of 20 alpha-dihydroprogesterone by cells showing normal or low C/P values, but not those with high values. It is concluded that the transport of cholesterol to the inner mitochondrial membrane is regulated by at least two processes, namely the supply of substrate (cholesterol), which can saturate this transport mechanism, and some mechanism(s) that is stimulated by cAMP and is capable of stimulating transport above maximal levels produced by high concentrations of cholesterol alone. The second mechanism requires the synthesis of a new protein(s).

20-alpha-Dihydroprogesterone↗

PubMed1989

Aromatase cytochrome P450 and cholesterol side-chain cleavage cytochrome P450 in corpora lutea of pregnant rats: diverse regulation by peptide and steroid hormones.

In previous studies we have shown that aromatase cytochrome P450 (P450arom) mRNA and protein increase markedly in luteal tissue between days 10-19 of gestation, whereas cholesterol side-chain cleavage cytochrome P450 (P450scc) appears to be constitutively maintained regardless of hormonal changes occurring during pregnancy. To identify pituitary and placental hormones that regulate these two P450 enzymes in the rat corpus luteum, serum LH activity and pituitary PRL release were selectively inhibited by administration of LH antiserum (LH-Ab) or CB-154, respectively. Placental hormones were removed by hysterectomy. Hormonal activities were replaced by the administration of hCG, PRL, testosterone (T), or estradiol (E), given individually or in combination. Induction of aromatase mRNA transcripts (3.3, 2.6, and 1.9 kilobases) and protein (54,000 mol wt) between days 10-15 of gestation was blocked by either surgical hysterectomy or LH-Ab treatment. Hysterectomy on day 10 combined with CB-154 abolished not only aromatase mRNA, but also markedly reduced P450scc mRNA (2.0 kilobases) by day 12. Induction of aromatase was partially restored in the day 10-15 hysterectomized rats by treatment with PRL plus E (most effective), PRL plus T, or PRL alone, but not by either T or E alone. Similar results were observed 2 days after hysterectomy (day 12), except that hysterectomy alone caused a transient 3.5-fold increase in P450arom mRNA and protein, most likely due to a transient release of pituitary LH. Aromatase mRNA and protein were also increased in intact pregnant rats treated with hCG between days 10-12. However, no effect of hCG was observed before (days 8-10) or after (days 13-19) midgestation. Likewise, LH-Ab had no effect if given after day 13. Despite hormone-specific regulation of the content of aromatase protein, E biosynthesis in vitro was not strictly related to aromatase enzyme content. We conclude that aromatase mRNA and protein are maintained by PRL at a low level of expression in the first half of pregnancy, can be modulated by LH at midgestation, and are subsequently induced to high levels in the second half of gestation by placental factors (rat placental lactogen-1 and T) and the conversion of T to E in the corpus luteum. P450scc appears to be constitutively maintained. Thus, two P450 genes known to be regulated by LH/cAMP in the rat follicle are controlled by diverse peptide and steroid signal transduction mechanisms in the corpus luteum.

Animals↗

PubMed1989

Human aromatase.

Explore the source record for details and available documents.

Aromatase↗

PubMed1988

Mechanism of androstenedione formation from testosterone and epitestosterone catalyzed by purified cytochrome P-450b.

A purified rat hepatic monooxygenase system containing cytochrome P-450b oxidizes testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone at approximately equal rates. The metabolism of epitestosterone by the same system is characterized by a marked stereoselectivity in favor of 16 beta-hydroxylation (4- to 5-fold relative to 16 alpha-hydroxylation), formation of 15 alpha-hydroxyepitestosterone, and a rate of androstenedione formation which is three to five times higher than that observed with testosterone. Apparent Km values for 16 alpha- and 16 beta-hydroxylation and androstenedione formation are 20-30 microM with either substrate. Mass spectral analysis of the androstenedione formed from [16,16-2H2]testosterone and [16,16-2H2] epitestosterone indicates essentially complete retention of deuterium, thereby ruling out a mechanism of androstenedione formation via C-16 hydroxylation followed by loss of water and rearrangement. Mass spectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 18O2 shows essentially complete incorporation of 18O (greater than 95%). Androstenedione formed from testosterone is enriched in 18O only 2-fold (5-8%) over background, while the androstenedione formed from epitestosterone shows 84% enrichment. Kinetic experiments utilizing [17-2H]testosterone and [17-2H]epitestosterone as substrates indicate that cleavage of the C-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both substrates. Taken together, our results indicate that androstenedione formation from epitestosterone proceeds exclusively through the gem-diol pathway, while androstenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen abstraction pathways.

Androstenedione↗

PubMed1988

Conversion of 11-deoxycorticosterone and corticosterone to aldosterone by cytochrome P-450 11 beta-/18-hydroxylase from porcine adrenal.

Highly purified cytochrome P-450 11 beta-/18-hydroxylase and the electron carriers adrenodoxin and adrenodoxin reductase were prepared from porcine adrenal. When the enzyme was incubated with the electron carriers, 11-deoxycorticosterone (DOC) and NADPH, the following products were isolated and measured by HPLC: corticosterone, 18-hydroxy-11-deoxycorticosterone (18-hydroxyDOC), 18-hydroxycorticosterone and aldosterone. All of the DOC consumed by the enzyme can be accounted for by the formation of these four steroids. Aldosterone was identified by mass spectroscopy and by preparing [3H]aldosterone from [3H]corticosterone followed by recrystallization at constant specific activity after addition of authentic aldosterone. Corticosterone and 18-hydroxycorticosterone were also converted to aldosterone. Conversion of corticosterone and 18-hydroxycorticosterone to aldosterone required P-450, both electron carriers, NADPH and substrate. The reaction is inhibited by CO and metyrapone. Moreover, all three activities of the purified enzyme decline at the same rate when the enzyme is kept at room temperature for various periods of time and when the enzyme is treated with increasing concentrations of anti-11 beta-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11 beta-/18-hydroxylase can convert DOC to aldosterone via corticosterone and 18-hydroxycorticosterone. The stoichiometry of this conversion was found to be 3 moles of NADPH, 3 moles of H+ and 3 moles of oxygen per mole of aldosterone produced.

Adrenal Glands↗

PubMed1988

Hormonal regulation, tissue distribution, and content of aromatase cytochrome P450 messenger ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea: relationship to estradiol biosynthesis.

The following study was undertaken to compare the content of aromatase cytochrome P450 (P450arom) mRNA with the content of the enzyme in rat ovarian tissues and to relate these changes with estradiol biosynthesis by follicles and corpora lutea isolated throughout pregnancy. A deoxyoligonucleotide (62 mer) probe derived from an amino acid sequence of purified human placental P450arom was used to screen a rat granulosa cell lambda gt11 cDNA expression library. Seven cDNA clones, ranging in size from 0.6-2.0 kilobases (kb), were identified and plaque purified. In vitro translation using mRNA that had been selected by hybridization to a 1.2-kb rat P450arom cDNA insert yielded an 35S-labeled translation product that bound antihuman aromatase immunoglobulin and comigrated with purified human placental aromatase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thus verifying that the clones do encode for P450arom. Using the 1.2-kb cDNA insert as a radiolabeled probe, the hormonal regulation, tissue distribution, content, and size of mRNA for P450arom were analyzed. Filter hybridization assays demonstrated that P450arom mRNA was low in small antral (SA) follicles, increased 16-fold in preovulatory (PO) follicles, and reached a peak in granulosa cells within 1 h after an ovulatory dose of hCG. In the corpus luteum of pregnancy, P450arom mRNA content was low on day 4, and increased 3-fold on days 7-11 and 10-fold on days 15-19 of gestation. P450arom mRNA then decreased on days 21 and 23, the day of parturition. Northern analyses of RNA from PO follicles and corpora lutea revealed three bands of P450arom mRNA that were 3.3, 2.6, and 1.9 kb in size. Immunoblots of soluble cell extracts of SA, PO, and luteinizing (PO plus hCG) follicles and corpora lutea of pregnancy demonstrated that aromatase enzyme was low in SA follicles, increased 1.5- to 3-fold in PO follicles, and decreased within 3-5 h after an ovulatory dose of hCG. Changes in the content of P450arom enzyme in luteal cells during pregnancy exhibited a pattern similar to that observed for P450arom mRNA. In contrast, changes in estradiol biosynthesis by follicles and corpora lutea were not directly related to the contents of P450arom mRNA and enzyme. For example, although corpora lutea isolated on days 15-21 of gestation contain the highest amount of P450arom mRNA and enzyme, these tissues did not produce the most estradiol when incubated for 5 h at 37 C in the presence of aromatizable androgen substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Testosterone-enhanced oxygen-mediated degradation of P-450(17) alpha in mouse Leydig cell cultures.

The present study investigated the process by which the microsomal cytochrome P-450 17 alpha-hydroxylase enzyme (P-450(17) alpha) activity in Leydig cells is decreased by steroid products in an oxygen-dependent manner. Leydig cells were maintained in primary culture at ambient (19% O2) or reduced (1% O2) oxygen tension and treated with 8-bromo-cAMP or steroids for 48 h. The amount and activity of P-450(17) alpha present in the treated cells were measured to determine whether changes in the activity of the enzyme due to treatment correlated with changes in the amount of the enzyme protein. At ambient oxygen tension the amount of P-450(17) alpha declined in Leydig cells treated with cAMP, and this decrease could be prevented when cultures were maintained at reduced oxygen tension. Treatment of cultures with testosterone caused an oxygen-sensitive decrease in the amount of the enzyme similar in extent to the cAMP-induced decrease. Reductions in the amount of P-450(17) alpha corresponded to reductions in the activity of the enzyme. The decline in the amount of P-450(17) alpha was due to increased decay of the enzyme protein and not to a decrease in the rate of synthesis of P-450. In contrast to testosterone, neither estradiol nor cortisol affected the amount of P-450(17) alpha. The data are consistent with the proposal that steroid products acting as pseudosubstrates cause decreases in P-450(17) alpha activity by enhancing oxygen radical-mediated damage to the enzyme. The damaged enzyme resulting from this process is more prone to degradation than is the intact protein.

Aerobiosis↗

PubMed1988

Isolation and characterization of rat fetal Sertoli cells.

Sertoli cells were prepared from fetal rats, aged 15, 18, and 21 days. Cultures of these cells can be prepared at a purity of 85% by a method that is widely used to prepare the same cells from postnatal rats. The fetal cells are identical in appearance to postnatal Sertoli cells. Fetal Sertoli cells round up in response to (Bu)2cAMP, but not in response to FSH. Protein synthesis in the cells is not stimulated by (Bu)2cAMP or FSH. However, incorporation of [35S]methionine into secreted proteins appearing in the incubation medium is stimulated by the cyclic nucleotide, but not by FSH. Reproducible patterns for the incorporation of [35S]methionine into cellular and secreted proteins are presented as autoradiograms of one- and two-dimensional electrophoretograms. These autoradiograms show that the response of secreted proteins to (Bu)2cAMP is a general one; most or all proteins participate in the response. No clear differences were observed in the nature of the proteins synthesized when cells from fetal rats of 15, 18, and 21 days of age were compared. Fetal Sertoli cells produced approximately 2 nmol lactate/10(6) cells.h, which is approximately one fifth of the amount produced by postnatal cells. Lactate production was increased by the addition of FSH or (Bu)2cAMP to the medium. Fetal Sertoli cells also synthesize androgen-binding protein, and this activity is not increased by either FSH or (Bu)2cAMP.

Androgen-Binding Protein↗

PubMed1988

The regulation of intracellular transport of cholesterol in bovine adrenal cells: purification of a novel protein.

We have purified to homogeneity a protein from bovine adrenal (fasciculata) cells that is capable of: 1) stimulating synthesis of pregnenolone (in a concentration-dependent fashion) by mitochondria when added with exogenous cholesterol; 2) increasing the concentration of cholesterol in outer and inner membrane when added to mitochondria with exogenous cholesterol; 3) increasing the rate of transport of cholesterol from outer membrane to inner membrane when the two membranes are incubated together in an aqueous buffer and subsequently separated by centrifugation; and 4) increasing the proportion of molecules of C27 side-chain cleavage P-450 that are bound to the substrate cholesterol. The protein is homogeneous according to two-dimensional polyacrylamide gels, shows a molecular weight of 8200 and is therefore referred to as 8.2 K. It is proposed that 8.2 K may be involved in the regulation of steroid synthesis in bovine fasciculata cells, possibly by stimulating entry of cholesterol into the outer membrane and by altering the intramitochondrial distribution of this substrate.

Adrenal Cortex↗