Presence Of Aminoglutethimide Research Articles

A Regulatory Mechanism Between Progesterone and Notch Signaling Pathway Maintains the Functionality of the Corpus Luteum from Pregnant Rats.

The Notch signaling pathway includes a family of transmembrane receptors (Notch1-4) that interact with specific ligands (Delta-like family, Jagged1 and Jagged2). This system regulates cell fate decisions, including proliferation, differentiation and apoptosis. The transmembrane receptors are cleaved upon binding of their ligands, leading to the release of the intracellular domain (NICD), which translocates to the nucleus where it functions as a transcriptional coactivator of regulatory genes of cellular fate. This processing requires the activity of two proteases, namely tumour necrosis factor a-converting enzyme and presenilin/gamma-secretase. We have previously shown that Notch1-4 receptors and the ligand Dll4 are expressed in the corpus luteum (CL) of pregnant rats and that the administration of prostaglandin F2 alpha decreases the levels of mRNA and protein expression of these proteins. Furthermore, blocking ovarian Notch system with the gamma-secretase inhibitor DAPT decreases serum levels of progesterone (P4) and increases pro-apoptotic protein expression in the CL. However, the role of P4 in regulating the Notch pathway in the CL of pregnant rats remains unclear. In this study CL′s were isolated from ovaries of rats on day 16 of gestation and cultured for 4 hours in the presence of aminoglutethimide (inhibitor of cytochrome P450scc 0.15 mM), DAPT (gamma secretase inhibitor, 20 uM), both inhibitors and the inhibitors with the addition of P4 (500ng/ml). Luteal function was assessed by measuring P4 levels in the medium by RIA. The CL levels of Notch1-4 and members of the ERK and PI3K/AKT pathways were measured by Western blot. The treatment of CL′s with each inhibitor significantly decreased P4 levels (P < 0.05, n = 5) in the culture medium, effect which was similar in the presence of both compounds. Furthermore, these inhibitors decreased protein levels of Notch1 intracellular fragment (NICD) compared to the control group (P < 0.05, n = 5). However, no significant difference was observed for NICD of Notch4. In addition, ERK and AKT phosphorylation decreased with the addition of aminoglutethimide or/and DAPT in the CL culture medium. These results suggest the existence of a regulatory mechanism between progesterone and the Notch signaling pathway to maintain the functionality of the CL in pregnancy. Supported by: CONICET (PIP1223), Universidad de Buenos Aires (X-218) and ANPCYT (PICT, 2010-0248).

Effects of cycloheximide, aminoglutethimide, indomethacin, cytochalasin B and colchicine on ovulation and the ultrastructure of the ovarian follicle wall in the stellate sturgeon Acipenser stellatus Pall

The inhibitor of protein synthesis cycloheximide, inhibitor of steroidogenesis aminoglutethimide, and inhibitor of prostaglandin synthesis indomethacin, as well as the drugs affecting the cell cytoskeleton, such as cytochalasin B and colchicine, were used for studying the mechanisms of ovulation in the stellate sturgeon Acipenser stellatus Pall. Follicles were isolated from the body cavity within certain time intervals after the injection of pituitary suspension to a female and cultivated in media with the inhibitors. In the case of follicles isolated in the middle of the period from hormonal injection until ovulation, cycloheximide, cytochalasin B, and aminoglutethimide suppressed ovulation most effectively, while in the case of oocytes isolated during the last quarter of this period, aminoglutethimide and cytochalasin B were the most effective. It was shown using TEM and SEM that cycloheximide suppressed all processes related to the preparation for ovulation, except the initial ones: contraction of follicle cells and their processes and secondary flattening of these cells. In the presence of aminoglutethimide, the follicle cells underwent pathological changes. Incubation in the media containing indomethacin and colchicine prevented degradation of the outer theca layer at the follicle apex. In the presence of cytochalasin B affecting the cytoskeleton, the drawing of follicle cell processes from the jelly coat channels was blocked, the outer theca cells were strongly contracted, but the cell layer integrity was affected and it was divided in separate fragments. A relationship is discussed between the metabolic processes and morphological changes that lead to ovulation. It was proposed on the basis of the present and previous data that the preovulatory preparation of the follicle tissues comprises two contractile and two apoptotic processes distinctly coordinated in time and space.

Detection of P450c17-independent pathways for dehydroepiandrosterone (DHEA) biosynthesis in brain glial tumor cells.

Dehydroepiandrosterone (D) is biosynthesized in the brain by a pathway different from that existing in the adrenal cortex. C6 rat glioma tumor cells in culture biosynthesize both pregnenolone (P) and D. They possess the mRNA, protein, and side-chain cleavage activity of P450scc. On the other hand, P450c17 was not detected. Adding FeSO4 to C6 cells increased the synthesis of both P and D. Even in the presence of aminoglutethimide, an inhibitor of P450scc, FeSO4 increased the synthesis of both steroids, indicating that the Fe2+-sensitive process does not involve P450scc. Likewise, the FeSO4-induced formation of D was not blocked by the P450c17 inhibitor, SU-10603. These results suggest that the FeSO4-induced synthesis of D as well as of P in C6 cells may be due to the fragmentation of in situ-formed tertiary hydroperoxides. It is likely, however, that the effect of the Fe2+ is not limited to this one reaction. When exogenous P was added to C6 microsomes, along with FeSO4, the amount of D formed was greater than control values, indicating that Fe2+ facilitated the conversion of P to D. Unlike the constituents that are converted by Fe2+ to P, the precursor of D in C6 cells is not soluble in a 1:1 mixture of ether and ethylacetate. Treatment of C6 cells with KI, NaBH4, or HIO4 resulted in an increase in D synthesis. From this it seems clear that a precursor of the D produced in C6 cells is a steroid where both C-17 and C-20 are oxygenated.

N-cadherin-mediated cell contact inhibits granulosa cell apoptosis in a progesterone-independent manner.

Previous studies have shown that both progesterone and cell contact inhibit granulosa cell (GC) apoptosis in vitro. Since the progesterone concentration associated with aggregated GCs may be higher than that of single GCs, experiments were conducted to differentiate progesterone's action from that of cell contact. For these studies, GCs were isolated from immature rats. Large GCs were collected after Percoll gradient centrifugation and placed in serum-free culture for 24 h. These studies confirmed that the rate of apoptosis was 2-3 times higher for single GCs than for aggregated GCs. This relationship was observed in the presence of aminoglutethimide, where progesterone concentrations were 3 ng/ml or less. A dose-response studied revealed that a minimum of 100 ng/ml progesterone were required to suppress apoptosis of single GCs. In addition, a single cell contact was shown to be sufficient to suppress apoptosis, with a small nonsteroidogenic GC being as effective as a large steroidogenic GC. Taken together, these data support the concept that cell contact blocks apoptosis in a progesterone-independent manner. GC contact is due to the presence of gap and adhesion-type junctions. To assess which, if either, of these junctions is involved in mediating the antiapoptotic action of cell contact, cocultures were set-up between GCs and R2C cells. Contact with R2C cells inhibits GC apoptosis, but does not result in the formation of functional gap junctions. This demonstrates that gap junctions are not essential to maintain GC viability. Adhesion-type junctions result from a homophilic binding of N-cadherin, which is expressed by both GCs and R2C cells. When this binding is inhibited by treatment with either an antibody to N-cadherin or a synthetic N-cadherin peptide, cell aggregation is attenuated. For those cells that form cell contacts in the presence of these N-cadherin-binding inhibitors, the percentage of apoptotic cells is increased compared to that in controls. These observations suggest that homophilic binding of N-cadherin molecules on the surface membranes of adjacent GCs initiates a signal transduction cascade that ultimately inhibits apoptosis.

Regulation of oxytocin production by purified adult rat Leydig cells in vitro: effects of LH, testosterone and lipoproteins.

The aim of the present study was to determine whether LH stimulates oxytocin production by adult rat Leydig cells directly or indirectly via testosterone. Purified adult rat Leydig cells were cultured in the presence or absence of 0.1 ng/ml LH or 1, 10 or 100 ng/ml testosterone for 22 h. Culture medium was collected at 2-hourly intervals and assayed for oxytocin and testosterone. In the presence of LH, Leydig cells produced significantly higher levels of both testosterone (basal production 1.4 +/- 0.13 ng, LH-stimulated 4.1 +/- 0.13 ng/10(6) cells per 2 h) and oxytocin (basal production 8.3 +/- 1.2 pg, LH-stimulated 20.2 +/- 1.3 pg/10(6) cells per 2 h). Testosterone also stimulated oxytocin secretion. However, the increase was smaller compared with that seen with LH and was not found to be dose-dependent. Furthermore, testosterone production was only significantly increased by LH during the first 10 h of the 22-h culture period whereas LH stimulated oxytocin production throughout the whole culture period. To further determine the effect of LH on oxytocin production, cultures were performed in the presence of LH and/or 400 microM aminoglutethimide. In the presence of aminoglutethimide both the basal and LH-stimulated production of testosterone was significantly reduced. However, in the same cultures aminoglutethimide did not alter either the basal or LH-stimulated production of oxytocin. These data show that LH does not act via testosterone to stimulate oxytocin production and therefore acts directly or by some alternative indirect mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of C6 glioma cell steroidogenesis by adenosine 3',5'-cyclic monophosphate.

Incubation of C6 glioma cells in the presence of aminoglutethimide, an inhibitor of cholesterol metabolism, together with either adenosine 3',5'-cyclic monophosphate (cAMP) analogues or agents that increase cAMP synthesis, such as cholera toxin, forskolin, and isoproterenol, stimulated the rate of pregnenolone formation by their isolated mitochondria. This effect of cAMP was blocked by the antagonist (Rp)-cAMPS. The incorporation rate of mevalonolactone into pregnenolone was also increased by the stimulation of adenylyl cyclase activity in intact C6 cells. It is concluded that cAMP stimulates glial cell steroidogenesis by increasing the movement of the substrate, cholesterol, to the mitochondria, where it will be metabolized to pregnenolone by the side chain cleavage cytochrome P450 enzyme.

Localization of prostaglandin F2 alpha inhibition of lipoprotein use by bovine luteal cells.

Prostaglandin F2 alpha (PGF2 alpha) inhibits lipoprotein-stimulated progesterone production by bovine luteal cells in vitro and the objective of this study was to localize the site of action of PGF2 alpha. Cultured bovine luteal cells were treated with PGF2 alpha for seven days, and then with either lipoproteins or 25-hydroxycholesterol in the presence of aminoglutethimide (which inhibits cholesterol side-chain cleavage) for the final 48 h. The effects of PGF2 alpha on progesterone production, cellular cholesterol content, mitochondrial cholesterol content and cholesterol side-chain cleavage activity were determined. As expected, PGF2 alpha inhibited (P less than 0.05) lipoprotein-stimulated progesterone production. However, PGF2 alpha did not inhibit low-density lipoprotein-stimulated, or high density lipoprotein-stimulated, increases in cellular cholesterol (P less than 0.05) or inhibit lipoprotein-induced increases in mitochondrial cholesterol content (P less than 0.05). Additionally, cholesterol content of mitochondria increased (P less than 0.05) in the presence of PGF2 alpha alone. To determine if the PGF2 alpha-induced inhibition of steroidogenesis occurred at, or after, the side-chain cleavage reaction, we treated cells with the readily diffusable sterol, 25-hydroxycholesterol. Prostaglandin F2 alpha did not inhibit 25-hydroxycholesterol-stimulated progesterone production (P less than 0.05). Prostaglandin F2 alpha may therefore exert its luteolytic effect at a site after cholesterol transport to the mitochondria but before cholesterol side-chain cleavage.

Inhibition of estrone sulfatase enzyme in human placenta and human breast carcinoma

Estrone sulfatase is an important mechanism of local synthesis of biologically active estrogens in human breast cancer. The human placental microsome and breast carcinoma mitochondrial/microsomal estrone sulfatase activity were characterized and inhibition studies performed. The K m of the placental tissue enzyme was 6.83 μM, V max 0.015 nmol/min/mg, and for the breast carcinoma tissue K m was 8.91 μM and V max 0.022 nmol/min/mg. Danazol produced a significant inhibition of estrone sulfatase (20% with 50 μM danazol). No significant inhibition was seen in the presence of aminoglutethimide, rogletimide, tamoxifen, 4-hydroxyandrostenedione, stilboestrol, or any metabolites of danazol or tamoxifen. Studies with synthetic and naturally occuring steroids demonstrated that the presence of a sulfate group at the 3 position to be the most important factor in determining inhibition, and the most potent inhibitor was 5α-androstene-3β, 17β-diol-3-sulfate ( K i of 2.0 μM). The naturally occuring 3-sulfated steroids all demonstrated competitive inhibition. These studies could form the basis for the design of a potent estrone sulfatase inhibitor which would have potential therapeutic activity in the management of breast cancer.

Aminoglutethimide augments follicle-stimulating hormone-induced aromatase activity in cultured porcine granulosa cells.

The role of endogenous progestin synthesis in the modulation of FSH-induced aromatase activity was examined. Granulosa cells isolated from nonatretic medium-sized (3-5 mm) follicles of prepubertal pigs were cultured for an initial 48-h period, during which time aromatase activity was induced by FSH in the absence or presence of aminoglutethimide (AG). After induction, the cell monolayers were washed before being cultured for a further 6-h period in the presence of the substrate testosterone (0.5 microM). The aromatase activity was assessed by measuring the accumulation of estradiol during the test period. Basal aromatase activity was negligible and was unaffected by the presence of AG (0.1-100 microM) during the induction period. But when cells were cultured with FSH and AG (0.1-1000 microM) during the induction period, there was a dose-dependent, biphasic increase in the FSH-induced estradiol synthesis during the test period. Maximal enhancement was obtained with 10 microM AG (3.5-fold). Thereafter the aromatase activity declined and, at 1000 microM AG, was significantly (P less than 0.05) inhibited. At the same time, the FSH-stimulated progestin production during the induction period was inhibited in a dose-related fashion by AG. This AG-enhanced aromatase activity was dose and time dependent but was independent of the FSH concentration used. The apparent median effective dose of AG was 2.4 microM and a minimal time of 24 h or less was needed to potentiate the induction of aromatase activity by FSH. If AG was, however, added to the cell cultures during the test period, the FSH-induced aromatase activity was inhibited, showing that AG is an inhibitor of FSH-induced aromatase activity. This action of AG during the test period could be alleviated by the addition of testosterone during the induction period. The viability of the granulosa cells and the total cellular protein were not significantly (P greater than 0.05) altered by AG. These results show that the induction of aromatase activity by FSH could be enhanced by AG, which probably acts by inhibiting progestin production during the induction period, leading us to conclude that endogenous progestins might play an important role in modulating the induction of aromatase activity by FSH.

Regulation of low density lipoprotein receptor gene expression in cultured human granulosa cells: roles of human chorionic gonadotropin, 8-bromo-3',5'-cyclic adenosine monophosphate, and protein synthesis.

Treatment of human granulosa cells with human CG (hCG) or an analog of its second messenger, cAMP, promotes a rapid increase in low density lipoprotein (LDL) receptor mRNA content. After 1 h of treatment with 8-bromo-cAMP, an appreciable increase in hybridizable LDL receptor mRNA was found which increased to apparently maximal levels within 4-6 h. Treatment of the granulosa cells with 25-hydroxycholesterol, in the presence of aminoglutethimide, resulted in a reduction in LDL receptor mRNA content within 6 h of treatment. However, hCG or 8-bromo-cAMP were able to stimulate an increase in LDL receptor mRNA content in the presence of this inhibitory signal. We further investigated the mechanism by which tropic agents increased mRNA content. While inhibition of RNA synthesis with actinomycin D blocked the hCG or cAMP-induced rise in LDL receptor mRNA content, inhibition of protein synthesis with cycloheximide augmented basal or hCG- or cAMP-stimulated LDL receptor mRNA levels. We conclude that human steroidogenic cells possess a cAMP-mediated mechanism for rapid upregulation of LDL receptor mRNA which is distinct from, and supercedes, cholesterol negative feedback of LDL receptor gene expression. The actions of hCG and 8-bromo-cAMP do not require ongoing protein synthesis. Indeed, a cycloheximide-sensitive mechanism modulates receptor mRNA levels in these cells such that the effects of hCG and 8-bromo-cAMP are enhanced when cells are pretreated with this drug.

Inhibition of ACTH action on cultured bovine adrenal cortical cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin through a redistribution of cholesterol.

The conversion of cholesterol to cortisol by cultured bovine adrenal cortical cells is stimulated 6-fold by adrenocorticotropin and is limited by the movement of cholesterol to the mitochondria (DiBartolomeis, M.J., and Jefcoate, C.R. (1984) J. Biol. Chem. 259, 10159-10167). Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10(-8)M), for 24 h prior to adrenocorticotropin (ACTH) addition decreased the rate of ACTH-stimulated steroidogenesis but did not affect the basal rate. TCDD was more effective against stimulation at 10(-11) M ACTH (4-fold) than at 10(-7) M ACTH (10%), consistent with an increase in EC50 for ACTH. Stimulation of bovine adrenal cortical cells by cAMP was similarly decreased by TCDD. In both cases the effectiveness of TCDD increased with time of exposure to the stimulant. The transfer of cholesterol to mitochondria in intact cells was quantitated by means of the 2-h accumulation of mitochondrial cholesterol in the presence of aminoglutethimide, an inhibitor of cholesterol side chain cleavage. Although cholesterol accumulated in the presence of ACTH (13 to 28 micrograms/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13 to 8 micrograms/mg). The effect of TCDD was produced relatively rapidly (t1/2 approximately 4 h). In absence of TCDD, the mitochondria of ACTH-stimulated cells also eventually lose cholesterol (after 2 h). It is concluded that TCDD pretreatment may increase the presence of a protein(s) that cause mitochondrial cholesterol depletion when the cells are stimulated by ACTH or cAMP. TCDD-enhanced cholesterol efflux from mitochondria diminishes cholesterol side chain cleavage when mitochondrial cholesterol is sufficiently depleted (after 2-4 h).

Adenosine 3′,5′-Monophosphate-Mediated Induction of 17α-Hydroxylase and C17–20Lyase Activities in Cultured Mouse Leydig Cells Is Enhanced by Inhibition of Steroid Biosynthesis*

We recently reported that treatment of mouse Leydig cell cultures for 5 days with LH or cAMP caused an induction of the microsomal cytochrome P-450 activities 17 alpha-hydroxylase and C17-20 lyase. We also have shown that the microsomal P-450s are very sensitive to oxygen-mediated loss of these activities and that this decrease is enhanced by steroids produced during acute cAMP stimulation of Leydig cells. In the present study, we investigated whether steroids produced during chronic cAMP treatment of Leydig cells limit the extent of induction of 17 alpha-hydroxylase and C17-20 lyase. Treatment of Leydig cell cultures with 8-bromo-cAMP in the presence of aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, resulted in a 4- to 7-fold enhancement of cAMP-mediated induction of the 17 alpha-hydroxylase and C17-20 lyase activities and, after 11 days of treatment, completely restored the activities to those found in freshly isolated Leydig cells. Treatment with aminoglutethimide in the absence of cAMP had no effect on these enzyme activities. Addition of the steroid products androstenedione and/or testosterone (5 microM) to cAMP-plus amino-glutethimide-treated cultures caused a significant reduction in cAMP-mediated induction of microsomal P-450, while addition of estradiol (50 nM) had no significant effect. 3 beta-Hydroxysteroid dehydrogenase-isomerase, another microsomal enzyme that is not a P-450 enzyme, was not induced by cAMP in either the presence or absence of aminoglutethimide. The data suggest that Leydig cell microsomal P-450 activities are maintained in vivo by a balance between two processes: cAMP-mediated induction and steroid product-induced degradation.

Sterol carrier protein 2 (SCP 2)-mediated transfer of cholesterol to mitochondrial inner membranes

Rats treated with cycloheximide accumulate cholesterol in the adrenal mitochondria. This cholesterol is largely associated with the mitochondrial outer membrane. The addition of homogeneous sterol carrier protein2 (SCP2) to these mitochondria stimulated pregnenolone production to a greater extent than with mitochondria from untreated rats. Adrenal mitochondria from cycloheximide-treated rats were incubated in the presence of aminoglutethimide and cycloheximide to prevent further utilization of cholesterol for pregnenolone synthesis. Outer and inner membrane fractions of these mitochondria incubated with and without 0.75 microM SCP2, were prepared following hypotonic disruption. Incubations of mitochondria in the presence of SCP2 caused a marked shift in cholesterol from outer to inner membrane fractions. The findings provide direct evidence for a role of SCP2 or a similar peptide in modulating transfer of cholesterol to the inner membrane site of cholesterol side chain cleavage.

Intramitochondrial movement of adrenal sterol carrier protein with cholesterol in response to corticotropin.

Sterol carrier protein (SCP) is a highly abundant, ubiquitous, low molecular weight protein that has a rapid turnover and multifunctional roles in lipid metabolism and transport. It is also known as the fatty acid-binding protein. These and other characteristics led to studies on the possible role of SCP in mediation of steroidogenic responses of the adrenal gland to corticotropin. To quantitate the level of SCP in adrenal tissue and subcellular fractions, a specific immunochemical assay was developed using an antibody to homogeneous liver SCP. SCP is a major protein in adrenal cells (greater than 100 micrograms/mg of total protein). The bulk of it is present in the soluble (60%) and mitochondrial (35%) fractions. Nearly all mitochondrial SCP is present in the inner membrane. Adrenal SCP undergoes a dramatic diurnal variation, varying from 2% to 12% of total protein. When corticotropin was administered in vivo in the presence of aminoglutethimide, an inhibitor of steroidogenesis, there was a parallel movement of SCP with cholesterol to the inner mitochondrial membrane. Other work showed mitochondrial SCP levels increase only in situations where there is an increase in cholesterol levels. These findings strongly suggest that one of the functions of adrenal SCP is to participate in teh corticotropin-stimulated movement of cholesterol to the inner mitochondrial membrane for steroidogenesis.

Regulation of intramitochondrial cholesterol transfer to side-chain cleavage cytochrome P-450 in rat adrenal gland.

Rat adrenal mitochondria accumulated cholesterol during ether stress in vivo when side-chain cleavage was inhibited by aminoglutethimide (control = 14.6 vs. aminoglutethimide = 26.5 micrograms of cholesterol per mg of protein). This accumulation was insensitive to simultaneous administration of cycloheximide (24.2 micrograms/mg), but side chain cleavage in the mitochondria was greatly decreased. Outer and inner mitochondrial membrane fractions were separated by discontinuous Ficoll gradient centrifugation. Quantitation of marker enzymes for inner, outer, and microsomal enzymes indicated that outer membranes contained less than 5% inner membranes. The inner membrane fraction contained less than 7% outer membrane and included 90% of mitochondrial cytochrome P-450. Electron microscopy revealed outer membranes as circular intact ghosts, whereas inner membranes were largely intact and retained vesicular structure typical of intact adrenal cortex mitochondria. Administration of aminoglutethimide effected a 2-fold increase in inner membrane cholesterol (9.4 vs. 20.1 micrograms/mg) but simultaneous administration of cycloheximide completely blocked this increase (10.9 micrograms/mg). We conclude that: (i) in the presence of aminoglutethimide, stress stimulates accumulation of cholesterol in the inner membrane of adrenal mitochondria; and (ii) transfer of cholesterol from outer to inner membranes requires a cycloheximide-sensitive agent.

Adenosine 3',5'-cyclic monophosphate and adrenal tumor cell morphology.

Cultured adrenal tumor cells respond to adrenocorticotropin (ACTH), adenosine 3′,5′-cyclic monophosphate (cAMP), cholera enterotoxin (CT), andEscherichia coli enterotoxin with increased steroidogenesis and a reversible alteration of monolayer morphology. The morphological response is not due specifically to the interaction of ACTH or CT with a membrane receptor site, because both cAMP and theophylline, which work intracellularly, also stimulated steroidogenesis and rounding of adrenal cells. The morphological alteration appears to be unrelated to stimulation of steroido-genesis or protein synthesis, inasmuch as CT in the presence of aminoglutethimide or cycloheximide still caused rounding and cAMP accumulation without increased steroid production. These results suggest that increased intracellular cAMP concentration may be related to the observed change of flattened adrenal cells to a spherical shape in monolayer cultures.

The Involvement of Human Placental Microsomal Cytochrome P-450 in Aromatization

The aromatization of androstenedione is inhibited in the presence of aminoglutethimide, diethylaminoethyl-2,2-diphenyl valerate HCl (SKF 525-A), and 10-3m KCN, but not carbon monoxide or metyrapone. The role of cytochrome P-450 in this reaction could not, therefore, be unequivocally established by inhibition studies. However, it was determined that steroid binding to placental microsomal cytochrome P-450 is absolutely specific for aromatase substrates, intermediates, and inhibitors. Moreover, the substrate 19-nortestosterone, aromatization of which is inhibited by CO, is a competitive inhibitor of both the aromatization and cytochrome P-450 binding of androstenedione, and vice versa. These results suggest that a single species of cytochrome P-450 is responsible for the aromatization of both steroids. Furthermore, both 19-hydroxyandrostenedione and 19-oxoandrostenedione also compete with androstenedione and 19-nortestosterone for the same species of cytochrome P-450. This suggests that all three hydroxylation reactions of C19 aromatization as well as the aromatization of 19-nortestosterone are carried out at the same enzyme site. In addition, an antibody raised against porcine hepatic NADPH-cytochome c reductase, which specifically inhibits cytochrome P-450 mediated reactions, is a potent inhibitor of the aromatization of both C19 and C18 estrogen precursors. The extent of inhibition of the CO-sensitive aromatization of C18 substrates by the antibody is similar to that observed for CO-sensitive drug hydroxylations occurring in liver microsomes. On the other hand, inhibition of the aromatization of the non-CO-sensitive substrate, androstenedione, by the antibody was atypical in that a much greater inhibitory effect on aromatization than on cytochrome c reduction was found. The distinct patterns of inhibition of aromatization by the reductase antibody are consistent with the hypothesis that the rate of entry of the first electron into the oxidized P-450 substrate complex may be rate limiting in the aromatization of C19 but not C18 steroids. The difference in the rate of entry of the first electron would be reflected in the extent to which the reduced P-450 substrate complex accumulates during the steady state. Since CO inhibition occurs at the level of the reduced P-450 substrate complex, variation in the steady state concentration of this intermediate could explain the difference in CO sensitivity observed between C19 and C18 substrates.

Effects of ACTH and cyclic-AMP in vitro on incorporation of 3H-leucine and 14C-orotic acid into protein and RNA in the presence of an inhibitor of cholesterol side chain cleavage.

Aminoglutethimide [α- (p-aminophenyl)- α-ethyl-glutarimide], an inhibitor of cholesterol side chain cleavage, largely reverses the inhibitory effects of ACTH and adenosine- 3′,5′-monophosphate (CAMP) on in vitro incorporation of 3H-leucine and 14C-orotate into rat adrenal protein and RNA, respectively. With aminoglutethimide present, small stimulatory effects of ACTH and CAMP on leucine incorporation are apparent, and these effects are not blocked by actinomycin D. Small stimulatory effects of ACTH on orotate incorporation are also apparent in the presence of aminoglutethimide, but CAMP produces a biphasic response with inhibition and reversal of inhibition at lower and higher CAMP concentrations, respectively. The inhibitory effect of CAMP may be due to conversion to 5′-AMP since the latter is purely inhibitory and since caffeine blocks CAMP-induced inhibition of orotate incorporation. (Endocrinology85: 1209, 1969)