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Biomedical subjects

E R Simpson

Publications and source records attributed to E R Simpson.

At least 217 records · Page 12Linked to original sources
PubMed1986

Synthesis of the cholesterol side-chain cleavage enzymes in cultured rat ovarian granulosa cells: induction by follicle-stimulating hormone and dibutyryl adenosine 3',5'-monophosphate.

The effects of FSH and (Bu)2cAMP on synthesis of the components of the cholesterol side-chain cleavage (SCC) enzyme complex, namely SCC cytochrome P-450 (P-450scc), the iron-sulfur protein adrenodoxin (ISP), and NADPH:ISP reductase (Red), were investigated in granulosa cells obtained from ovaries of immature estrogen-primed rats cultured for up to 72 h in defined medium in the presence or absence of FSH and (Bu)2cAMP. The cells were lysed, and proteins were subjected to polyacrylamide gel electrophoresis, followed by immunoblotting using antibodies specific to bovine adrenocortical P-450scc, ISP, and Red. A time-dependent increase was observed in the specific contents of these three components of SCC, but not of the reference mitochondrial protein, F1-ATPase, upon treatment with FSH or (Bu)2cAMP. The increase in the content of these three enzymes was accompanied by a rise in progesterone and 20 alpha-hydroxyprogesterone production. The synthesis of P-450scc, ISP, and Red increased 3- to 4-fold with time upon FSH or (Bu)2cAMP treatment respectively, as evidenced by pulse labeling of the cell proteins with [35S]methionine, followed by immunoprecipitation. Immunoprecipitation of P-450scc and ISP from an in vitro translation system programmed by RNA isolated from cultured cells revealed that treatment with FSH or (Bu)2cAMP resulted in an increase in the levels of translatable mRNA specific for these proteins, and that the initial products of translation were precursor forms of cytochrome P-450scc and ISP, similar to those observed in bovine adrenal and granulosa cells. It is concluded that in cultured rat ovarian granulosa cells, FSH induces the synthesis of cytochrome P-450scc, ISP, and Red by increasing the content of translatable mRNA coding for the precursor forms of these enzymes and that this action is mediated by cAMP. Furthermore, the effects of FSH and (Bu)2cAMP provide an explanation for the action of these compounds to stimulate progestin synthesis in cultured ovarian cells.

Animals↗
PubMed1986

Immunolocalization of cholesterol side-chain-cleavage cytochrome P-450 and 17 alpha-hydroxylase cytochrome P-450 in bovine ovarian follicles.

Follicles were collected from cows and processed for electron microscopy and for immunofluorescent staining at the light microscope level. Key regulatory steroidogenic enzymes cholesterol side-chain-cleavage cytochrome P-450 (P-450scc) and 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha) were immunolocalized using specific IgG fractions raised against these enzymes. In larger follicles in which the theca interna had differentiated, positive staining for cytochromes P-450scc and P-450(17) alpha was observed in the cells of the theca interna. Electron microscopic examination showed that these cells were rich in endoplasmic reticulum, mainly rough, and had moderate numbers of mitochondria with tubular and lamellar cristae. Positive staining was also present in the theca of follicles undergoing atresia. Positive staining for cytochrome P-450(17) alpha was not observed in the membrana granulosa but cytochrome P-450scc was present in the membrana granulosa in some follicles, particularly in the larger antral follicles. By contrast, positive staining for both enzymes was not observed in stroma, surface epithelium or in small preantral follicles in which the theca interna had not differentiated. These results indicate good agreement between the type(s) of steroidogenic enzyme(s) present in tissues and the type(s) of steroid hormone(s) produced. It is concluded that regulation of steroid hormone production involves, at least in part, regulation of the levels of steroidogenic enzymes.

Animals↗
PubMed1986

Immunolocalization of cholesterol side-chain-cleavage cytochrome P-450 and ultrastructural studies of bovine corpora lutea.

Corpora lutea were collected from cows at four stages of the luteal phase and prepared for immunostaining at the light microscope level. Other corpora lutea, which were fully developed, were dispersed by collagenase treatment and freshly isolated and cultured cells were processed for immunostaining. Electron microscopy was carried out on mature corpora lutea and freshly isolated cells. Positive staining for cholesterol side-chain-cleavage cytochrome P-450 (P-450scc), an inner-mitochondrial membrane enzyme considered to catalyse the rate-limiting step in the conversion of cholesterol to progesterone, was observed in all corpora lutea. The intensity of staining was much greater in mature corpora lutea than in young or regressing corpora lutea. Only small and large luteal cells stained positively and cells of the vasculature and other connective tissue elements did not. When cells were cultured and had become flatter, the intensity of immunostaining was observed to be greater in large luteal cells than in small luteal cells which was interpreted to be due, in part, to the greater volume density of mitochondria in these cells. In some cultured small luteal cells the pattern of immunostaining appeared as whorls of strands encircling the nucleus. This pattern was interpreted as a three-dimensional network of mitochondria organized into 'strands', more than one mitochondrion in cross-section, perhaps formed during the process of attachment and elongation of the cells. Further observations made at the electron microscope level, included the presence of close (5-8 nm) contacts with interconnecting septa between small luteal cells in tissue.

Animals↗
PubMed1986

Control of gene expression of adrenal steroid hydroxylases and related enzymes.

Utilization of cDNA probes specific for various components of the bovine adrenocortical steroidogenic pathway have led to the conclusion that there are three levels of regulation of steroid hydroxylase gene expression. In each case it is postulated that specific classes of proteins bind to regulatory regions of these genes and modulate their transcription. Throughout adult life, cAMP-dependent regulation via SHIP protein(s) is the predominant mechanism by which optimal steroidogenic capacity is maintained. A second type of regulation is tissue-specific. One subclass of tissue-specific expression is the "all-or-none" type whereby steroid 21-hydroxylase and 11 beta-hydroxylase gene expression occur only in adrenal cortex and not in other steroidogenic tissues. A second subclass of tissue-specific expression is the "variable" type whereby 17 alpha-hydroxylase and cholesterol side chain cleavage (SCC) activity are both expressed in ovarian thecal cells but only SCC activity is expressed in corpus luteum. The third type of regulation is cAMP-independent and leads to fetal-imprinting (initial expression of steroid hydroxylase genes during fetal life).

Adrenal Cortex↗
PubMed1985

Regulation of cytochrome P-45011 beta gene expression by adrenocorticotropin.

Recombinant DNA clones corresponding to 11 beta-hydroxylase cytochrome P-450 (P-450(11)beta) have been identified in a bovine adrenocortical cDNA library. These clones, pB11 beta-1 and pB11 beta-2, hybridize to at least three RNA species, 7.2, 6.2, and 4.3 kilobases in length, present in the adrenal cortex. All three RNA species directed cytochrome P-450(11)beta synthesis in an in vitro translation system. Expression of the cytochrome P-450(11)beta gene is tissue specific in that these transcripts were not detected in liver, heart, kidney, or corpus luteum. In cultured bovine adrenocortical cells, adrenocorticotropin (ACTH) or dibutyryl cAMP increased the concentration of cytochrome P-450(11)beta transcripts. This increase appears to be at least partially due to de novo transcription as shown by the action of actinomycin D which effectively blocked the ACTH-induced increase in cytochrome P-450(11)beta RNA level. Furthermore, cycloheximide administered prior to or along with ACTH resulted in the blockage of any new transcription of the cytochrome P-450(11)beta gene as evidenced from the level of RNA. Thus, in addition to potential effects on RNA stabilization, a primary action of ACTH in the regulation of the level of cytochrome P-450(11)beta may be to mediate the induction of an unidentified adrenocortical protein which is required for the activation of cytochrome P-450(11)beta gene expression. These results, together with previous studies (Kramer, R.E., Rainey, W.E., Funkenstein, B., Dee, A., Simpson, E. R., and Waterman, M. R. (1984) J. Biol. Chem. 259, 707-713) that showed increased intracellular levels of cAMP upon treatment of bovine adrenocortical cell cultures with ACTH, suggest a role for cAMP-mediated events in the regulation of cytochrome P-450(11)beta gene expression.

Adrenocorticotropic Hormone↗
PubMed1985

Effects of adrenocorticotropin on 17 alpha-hydroxylase activity and cytochrome P-450(17 alpha) synthesis in bovine adrenocortical cells.

The effects of adrenocorticotropin (ACTH) on 17 alpha-hydroxylase activity and cytochrome P-450(17 alpha) synthesis have been studied utilizing bovine adrenocortical cells in primary monolayer culture. A 20-fold stimulation of the conversion of pregnenolone to 17 alpha-hydroxypregnenolone was observed in postmitochondrial supernatant fractions from cells treated with ACTH as compared to controls. This increase in 17 alpha-hydroxylase activity was found to be due to a change in the Vmax and not a change in the Km(app). By immunoisolation of newly synthesized protein from an RNA-directed cell-free translation system we found that the level of P-450(17 alpha) was many-fold greater when RNA from ACTH-stimulated cells was used, as compared to RNA from control cells. A similar pattern was obtained when the rate of P-450(17 alpha) synthesis was analyzed by immunoisolation from radiolabeled cellular protein from ACTH-stimulated cells. When the total amount of P-450(17 alpha) was measured by immunoblotting we found that the levels of enzyme present correlated with the 17 alpha-hydroxylase activity. In addition, we found that these ACTH-mediated effects could be mimicked by treatment of cells with analogs of cyclic AMP. These results indicate that the activity of P-450(17 alpha) is regulated primarily by cyclic AMP-mediated changes in synthesis, probably at the transcriptional level, which in turn has a profound effect on the pattern of steroid secretion. Thus, we believe cytochrome P-450(17 alpha) to be a key regulatory enzyme in the steroidogenic pathway.

17-alpha-Hydroxypregnenolone↗
PubMed1985

Preparation and characterization of polyclonal and monoclonal antibodies against human aromatase cytochrome P-450 (P-450AROM), and their use in its purification.

Aromatase cytochrome P-450 (P-450AROM) was partially purified from human placental microsomes by hydrophobic affinity chromatography using Phenyl-Sepharose and ion-exchange chromatography on DEAE-cellulose. The resulting preparation had a specific activity of 2 nmol/mg protein with respect to cytochrome P-450 content and displayed a type I difference spectrum upon addition of the substrate androstenedione. When the cytochrome P-450-enriched fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue, there was an enrichment of two proteins having apparent molecular weights of 50,000 and 55,000. The bands containing these proteins were removed from unstained polyacrylamide gels and injected separately or together into three rabbits. An aliquot of the serum or an immunoglobulin (IgG) fraction prepared from the serum of the rabbit injected with the 55-kDa band or with both the 50- and 55-kDa bands inhibited aromatase activity of human placental microsomes by 80%; this IgG had no effect on 17 alpha-hydroxylase or 21-hydroxylase activities of human fetal adrenal microsomes. In contrast, the serum of the rabbit injected with the 50-kDa band had little capacity to inhibit placental aromatase activity. By immunoblot analysis, it was found that the IgG from the serum of the rabbit immunized with the 55-kDa protein bound specifically to a protein of 55 kDa in human placental microsomes. Monoclonal antibodies were prepared from a hybridoma cell line derived from the spleen cells of mice immunized against the 55-kDa protein. The monoclonal IgG was covalently linked to a Sepharose 4B column and was used for immunoaffinity chromatography of cytochrome P-450AROM. The finding that cytochrome P-450 and the 55-kDa protein were selectively retained by the affinity column and eluted with NaCl (2 M) and glycine (0.2 M, pH 3.0) and that this fraction contained aromatase activity upon reconstitution with purified NADPH-cytochrome P-450 reductase and phospholipid, is indicative that the 55-kDa protein is indeed cytochrome P-450AROM. These findings are also indicative that both the monoclonal and polyclonal IgGs are specific for human cytochrome P-450AROM.

Animals↗
PubMed1985

Regulation of the biosynthesis of cytochromes P-450 involved in steroid hormone synthesis.

The actions of ACTH to regulate the synthesis of the various enzymes involved in steroid hormone biosynthesis have been studied using bovine adrenocortical cells in monolayer culture. ACTH causes an increase in the synthesis of both the mitochondrial and the microsomal forms of cytochrome P-450 involved in steroid hormone biosynthesis, as well as of the iron-sulfur protein involved in transferring electrons to the mitochondrial forms of cytochrome P-450, namely, adrenodoxin. This increased synthesis is reflective of an increase in translatability of mRNA species specific for these various proteins, and appears in each case to be mediated by cyclic AMP. Whereas the mitochondrial proteins are synthesized as precursors of higher molecular weight which are processed upon insertion into the mitochondria, the microsomal proteins are synthesized as species identical in molecular weight to the mature forms. In order to determine whether the action of ACTH to increase the rate of synthesis of these proteins is the result of an increase in the levels of specific mRNA species, cDNA clones complementary to these mRNA species are being isolated. These probes will also make it possible to characterize the genes encoding the steroidogenic enzymes, as well as to identify regulatory elements which control their transcription.

Adrenal Cortex↗
PubMed1985

Regulation of aromatase activity of rat granulosa cells: induction of synthesis of NADPH-cytochrome P-450 reductase by FSH and dibutyryl cyclic AMP.

Aromatase is an enzyme complex that is composed of a specific form of cytochrome P-450 and a flavoprotein, NADPH-cytochrome P-450 reductase. Aromatase activity of granulosa cells is increased markedly by follicle-stimulating hormone (FSH) and by analogs of cyclic AMP. It was the objective of the present study to investigate the effects of FSH and dibutyryl cyclic AMP (Bt2cAMP) on the synthesis of NADPH-cytochrome P-450 reductase in rat granulosa cells maintained in vitro. Granulosa cells were obtained from the ovaries of diethylstilbestrol (DES)-treated immature rats and were incubated in the presence of DES (10(-7) M), DES + FSH (250 ng/ml), or DES + Bt2cAMP (1 mM) for up to 72 h. After 72 h of incubation, aromatase activity of cells incubated with DES alone was 5 pmoles estrogen formed 2 h-1 mg-1 protein and was increased greater than 60-fold in cells incubated with FSH or Bt2cAMP. NADPH-cytochrome P-450 reductase was immunoisolated from [35S]methionine-labeled lysates of granulosa cells incubated for 72 h in the absence or presence of stimulatory factors. The rate of synthesis of reductase was found to be increased about 3-fold in cells incubated with DES + FSH or DES + Bt2cAMP as compared to cells incubated with DES alone. By immunoblot analysis we found that the cellular content of reductase was increased about 2-fold by FSH and Bt2cAMP treatment. Reductase specific activity was 10 nmoles min-1 mg-1 protein in membrane fractions of DES-treated cells and was increased 1.6-fold by FSH treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Molecular cloning and amino acid sequence of the precursor form of bovine adrenodoxin: evidence for a previously unidentified COOH-terminal peptide.

Several recombinant cDNA clones specific for the mitochondrial iron-sulfur protein adrenodoxin have been identified in a bovine adrenocortical cDNA library. One clone (pBAdx4) contains a 900-base-pair insert that includes the entire amino acid coding region of the adrenodoxin precursor protein. The amino acid sequence of mature adrenodoxin deduced from the nucleotide sequence of pBAdx4 is identical with that determined by protein sequencing except for three amide changes. The previously undetermined sequence of the adrenodoxin NH2-terminal precursor segment (58 amino acids) contains several basic residues, a characteristic feature of the precursor segment of proteins destined for mitochondria. In addition, a 14 amino acid extension is present at the COOH terminus of the mature adrenodoxin sequence. Whether this represents a COOH-terminal precursor segment is not clear. Three different adrenodoxin mRNAs are present [1.75, 1.4, and 0.95 kilobase(s) long] in bovine adrenocortical RNA. RNA from bovine corpus luteum, liver, and kidney contains transcripts that hybridize to adrenodoxin cDNA. Accumulation of adrenodoxin mRNA occurs in cultured bovine adrenocortical cells after treatment with ACTH or dibutyryl-cAMP, similar to that observed for the mitochondrial steroid hydroxylases that it services--namely, the cholesterol side-chain-cleavage cytochrome P-450 and the steroid 11 beta-hydroxylase cytochrome P-450.

Adrenal Cortex↗
PubMed1985

Factors affecting the conversion of androstenedione to estrogens by human fetal hepatocytes in monolayer culture.

The purpose of the present investigation was to characterize and determine what hormones affect the activity of aromatase in human fetal hepatocytes maintained in primary monolayer culture. The major product of aromatization of androstenedione was estrone sulfate. Optimal conditions for assay of aromatase activity in fetal liver cells were determined. The apparent Km for androstenedione was 50 nM. Aromatase activity was stimulated by glucocorticoids in the presence of fetal calf serum. The concentration of dexamethasone required for half-maximal stimulation was 10(-8) M, similar to the concentration required for half-maximal binding to glucocorticoid receptors. This action of dexamethasone was inhibited by cortisol 21-mesylate, a glucocorticoid antagonist. Aromatase activity was also stimulated by (Bu)2cAMP and cholera toxin, and was inhibited by fetal calf serum. This effect of fetal calf serum was mimicked by epidermal growth factor. However, epidermal growth factor did not mimic the permissive action of serum to stimulate aromatase activity by dexamethasone. In these respects, the regulation of aromatase activity of human fetal hepatocytes is similar to that of human adipose stromal cells. A polycyclic hydrocarbon, benzo(a)pyrene, which causes induction of aryl hydrocarbon hydroxylase activity in fetal hepatocytes, inhibited the stimulation of aromatase activity by dexamethasone. Of a number of hormones tested, including glucagon, insulin, angiotensin II, ACTH, hCG, GH, PRL, and T3, only glucocorticoids were effective in stimulating aromatase activity of human fetal hepatocytes. These results emphasize the complex and multiparameter nature of the regulation of aromatase activity in this as in other tissues.

Androstenedione↗
PubMed1985

Effects of aging and obesity on aromatase activity of human adipose cells.

Adipose tissue is the principal site of estrogen formation in postmenopausal women; with advancing age as well as with increased body weight, there is an increase in the fractional conversion of circulating androstenedione to estrone. We have studied the effects of aging as well as body weight on aromatase activity of adipose tissue specimens taken from 50 women of various ages and weights. Since aromatase activity of adipose tissue is detectable primarily in stromal cells, these cells were incubated with [1-3H]androstenedione (150 nM), and estrogen formation was assayed by measuring the incorporation of tritium into [3H]water. The aromatization rate, when normalized on the basis of equal numbers of cells, increased with increasing age (P less than 0.03; r = 0.43). In contrast, when expressed as a function of body weight, no change in aromatase activity of adipose stromal cells were found. Aromatization of androstenedione by cells from young women who had undergone oophorectomy was not increased compared with that of cells from young women with normal ovarian function, indicating that the onset of menopause per se and the accompanying increase in circulating gonadotropin levels were not causative factors in the increased aromatase activity of adipose stromal cells. We conclude, therefore, that increased estrone production associated with aging may result from an increase in the specific activity of the aromatase enzyme in adipose stromal cells and is not affected by changes in gonadotropin concentrations associated with menopause. On the other hand, the increase in estrogen formation as a function of obesity is probably due to increased numbers of adipose cells, rather than to an increase in the specific activity of aromatase in those cells.

Adipose Tissue↗
PubMed1985

Adenylate cyclase activity in neocortex and fetal zone membrane fractions of the human fetal adrenal gland.

Whether peptide hormones other than ACTH may be responsible for the difference in size or rate and pattern of steroidogenesis of the fetal zone (FZ) compared to those of the neocortex (NC) of the human fetal adrenal gland is controversial. In the present investigation, the activity of adenylate cyclase in membrane fractions of separated zones of the human fetal adrenal gland was determined. Basal adenylate cyclase activity was 2- to 3-fold greater in NC than in FZ membrane fractions. The addition of ACTH-(1-24) stimulated adenylate cyclase activity in both zones, but the activity was more sensitive to ACTH (10(-10) M) in NC fractions than in FZ fractions (10(-7) M). In addition to ACTH-(1-24), the effect of other ACTH-related peptides on the activity of adenylate cyclase in the separated zones of the adrenal gland was investigated. 16K fragments 2-36, gamma 3MSH, alpha MSH, beta-endorphin, leu-enkephalin, and met-enkephalin, as well as hCG, FSH, prostaglandin E2, prostaglandin F2 alpha, epinephrine, and norepinephrine did not stimulate adenylate cyclase activity in either zone. It is concluded that basal and ACTH-(1-24)-stimulated adenylate cyclase activities are greater in NC than in FZ membrane fractions. In addition, the results of the present investigation do not support the concept that other ACTH-related peptides or peptide or protein hormones increase steroidogenesis by stimulating adenylate cyclase activity in the human fetal adrenal gland.

Adenylyl Cyclases↗
PubMed1984

Cholesterol synthesis by human fetal hepatocytes: effect of lipoproteins.

The purpose of the present investigation was to determine the effect of various lipoproteins on the rate of cholesterol synthesis of human fetal liver cells maintained in culture. This was accomplished by measuring the rate of incorporation of tritium from tritiated water or carbon 14-labeled acetate into cholesterol in human fetal liver cells. Optimal conditions for each assay were determined. When human fetal liver cells were maintained in the presence of low-density lipoprotein, cholesterol synthesis was inhibited in a concentration-dependent fashion. Intermediate--density lipoprotein and very-low-density lipoprotein also suppressed cholesterol synthesis in human fetal liver cells. In contrast, high-density lipoprotein stimulated cholesterol synthesis in human fetal liver cells. The results of the present as well as our previous investigations suggest that multiple interrelationships exist between fetal liver cholesterol synthesis and lipoprotein-cholesterol utilization by the human fetal adrenal gland and that these processes serve to regulate the lipoprotein-cholesterol levels in fetal plasma.

Acetates↗
PubMed1984

Induction of synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin by follicle-stimulating hormone, 8-bromo-cyclic AMP, and low density lipoprotein in cultured bovine granulosa cells.

The actions of follicle-stimulating hormone (FSH), 8-bromo-cyclic AMP (8-Br-cAMP), and low density lipoprotein (LDL) to stimulate the production of progesterone and the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450ssc) and adrenodoxin were investigated in bovine granulosa cells maintained in primary monolayer culture. Treatment of granulosa cells in culture with FSH resulted in an increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc in a concentration-dependent fashion with a maximal effect being obtained at an FSH concentration of 500 ng/ml. Treatment of granulosa cells with FSH also resulted in the induction of synthesis of adrenodoxin. The cyclic AMP analog, 8-Br-cAMP, induced the synthesis of both cytochrome P-450scc and adrenodoxin to a greater extent than did FSH. LDL also stimulated the synthesis of both cytochrome P-450scc and adrenodoxin, when added to cells maintained in the presence of lipoprotein-poor serum. The presence of FSH or 8-Br-cAMP together with LDL resulted in a higher rate of enzyme synthesis than that observed with each effector alone. FSH, 8-Br-cAMP, and LDL also stimulated progesterone production by cultured granulosa cells. The results of this study offer a possible mechanism whereby granulosa cells undergo cytodifferentiation in vivo into luteal cells. The concentration of LDL in follicular fluid is very low. Following ovulation, vascularization of the follicle occurs and thus the granulosa cells are exposed to high levels of LDL, allowing for provision of substrate cholesterol, as well as stimulation of the synthesis of the enzymes involved in cholesterol side chain cleavage.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Induction of synthesis of mitochondrial steroidogenic enzymes of bovine adrenocortical cells by analogs of cyclic AMP.

The long term action of cyclic AMP analogs to stimulate the synthesis of cytochromes P-450scc, P-45011 beta, and adrenodoxin has been studied utilizing confluent monolayers of adult bovine adrenocortical cells maintained for periods of time up to 72 h in the absence or presence of dibutyryl cyclic AMP (1 mM), 8-bromo cyclic AMP (1 mM), or ACTH (adrenocorticotropin) (10(-6) M). The synthesis of these proteins was examined by radiolabeling cellular proteins with [35S]methionine or else by translating RNA extracted from such cells in a cell-free system in the presence of [35S]methionine. In each case, the protein under study was immunoprecipitated utilizing specific antisera, or IgG fractions prepared from such antisera. ACTH and both analogs of cyclic AMP caused an increase in the synthesis of cytochrome P-450scc which reached a maximum 36-48 h after addition, and then declined. On the other hand, butyric acid (1 mM) had no effect on the synthesis of cytochrome P-450scc. Cytochrome P-450scc activity measured as pregnenolone production by both intact cells or isolated mitochondria from such cells was increased following incubation of cells with either dibutyryl cyclic AMP or ACTH. The binding of rabbit anti-cytochrome P-450scc IgG was also increased in cells incubated with dibutyryl cyclic AMP or ACTH as estimated by immunofluorescence microscopy using fluorescein-tagged anti-rabbit IgG. Furthermore, dibutyryl cyclic AMP and ACTH both increased the synthesis of adrenodoxin and of cytochrome P-45011 beta, as well as the activity of 11 beta-hydroxylase. In addition, ACTH stimulated the secretion of cyclic AMP in a time- and concentration-dependent fashion. Thus, it is concluded that analogs of cyclic AMP can mimic the long term actions of ACTH to induce the synthesis of steroidogenic enzymes, and that this action of ACTH is likely mediated by cyclic AMP.

8-Bromo Cyclic Adenosine Monophosphate↗