Characterization of the lipoidal derivatives of pregnenolone prepared by incubation of the steroid with adrenal mitochondria.

Using mass spectrometric, radioisotopic, chromatographic and chemical techniques, five fatty acid esters of 3 beta-hydroxy-5-pregnen-20-one (pregnenolone) have been identified as components of the lipoidal derivatives biosynthesized in vitro with bovine adrenal mitochondria. The five compounds are: pregnenolone arachidonate, pregnenolone linoleate, pregnenolone oleate, pregnenolone palmitate, and pregnenolone stearate. The distribution of the fatty acids among these five esters is different from the previously reported (Cmelik, S.H.W., and Ley, H. (1977) Comp. Biochem. Physiol. 56B, 267-270) fatty acid composition of these organelles.     

Presence of fatty acid esters of pregnenolone in follicular fluid from women undergoing follicle stimulation

In the following investigation the presence of lipoidal pregnenolone derivatives in the preovulatory follicular fluid obtained from women undergoing in vitro fertilization was established. Concentrations of lipoidal pregnenolone proved to be at least twofold greater than those of the unconjugated counterpart. Indirect identification of these lipoidal pregnenolone derivatives was achieved by comparing the C-18 column, thin-layer silica gel (TLC), high performance liquid chromatography (HPLC), and gas chromatography/mass spectrometry (GC/MS) chromatographic properties of the endogenous lipoidal pregnenolone derivatives in follicular fluid with those of synthetic acyl pregnenolone esters. Lipoidal pregnenolone derivatives recovered after HPLC subfractionation were treated with alkali to hydrolyze the acyl group thus liberating nonconjugated pregnenolone. Concentrations of this steroid were then measured using radioimmunoassay upon which analysis of HPCL and gas chromatograms permitted the calculation of the individual pregnenolone ester contributions within the samples. Five lipoidal pregnenolone derivatives constituted more than 90% of the total lipoidal pregnenolone concentration observed, these derivatives being: pregnenolone oleate (30.7%), linoleate (20.7%), palmitate (20.1%), linolenate (14.8%), and palmitoleate (7.1%).     

Formation of lipoidal steroids in follicular fluid

The presence of high levels of lipoidal pregnenolone in follicular fluid has recently been established although no evidence has been presented concerning its possible origin. The following investigation focuses on the enzymatic conversion of non-conjugated steroids into their lipoidal derivatives in preovulatory follicular fluid obtained from women undergoing in vitro fertilization. Our observations indicated that pregnenolone, an important precursor steroid, was acylated at a similar rate as cholesterol in follicular fluid. Similar studies were subsequently conducted with serum obtained from a pool of normal women and women undergoing follicular stimulation which showed little difference to the results obtained in follicular fluid. Further studies using dehydroepiandrosterone, androst-5-ene-3 beta,17 beta-diol, estradiol and dihydrotestosterone were were also performed to monitor their respective lipoidal conversion percentages in follicular fluid which revealed a marked difference of conversion rates between steroids. The indirect identification of the lipoidal pregnenolone derivatives formed in follicular fluid was also conducted by incubating radiolabelled pregnenolone in follicular fluid. The fatty acid components of the resulting lipoidal pregnenolone derivatives showed a marked resemblance to those of cholesteryl esters formed in plasma by the enzymatic activity of lecithin:cholesterol acyltransferase. The pregnenolone derivatives were comprised predominantly of unsaturated fatty acids such as linoleate, palmitoleate, oleate, linolenate and arachidonate while saturated fatty acids, namely palmitate, constituted 20% of the total lipoidal pregnenolone.     

Administration of pregnenolone and dehydroepiandrosterone to guinea pigs and rats causes the accumulation of fatty acid esters of pregnenolone and dehydroepiandrosterone in plasma lipoproteins.

Steroids were administered continuously to guinea pigs and rats using subcutaneously applied silastic tubing implants, and the effects on circulating steroid and steroid conjugate levels were monitored. Using implants filled with pregnenolone, we observed that pregnenolone had a marked effect on increasing the levels of its fatty acid-esterified derivative, while dehydroepiandrosterone-releasing implants produced a rise in circulating nonconjugated dehydroepiandrosterone, androst-5-ene-3 beta,17 beta-diol, androstenedione, testosterone, and lipoidal derivatives of both dehydroepiandrosterone and androst-5-ene-3 beta,17 beta-diol. Implants filled with androstenedione produced a 20-fold increase in plasma androstenedione levels relative to untreated controls and a corresponding five-fold increase over control testosterone levels. No fatty acid-esterified derivative of testosterone could be detected within the plasma. Lipoproteins were isolated from both rats and guinea pigs treated with implants filled with pregnenolone or dehydroepiandrosterone. The steroid and steroid fatty acid esters present in each fraction were analyzed, revealing that approximately 75% of all the fatty acid esters of pregnenolone recovered in the lipoproteins was localized within the high-density lipoprotein (HDL) fraction of both guinea pig and rat plasma. Similarly, lipoidal dehydroepiandrosterone was found associated predominantly with the low-density lipoprotein and HDL fractions in the guinea pig, while in the rat this steroid conjugate was exclusively within the HDL fraction. High-density lipoprotein-incorporated tritiated pregnenolone fatty acid esters and dehydroepiandrosterone fatty acid esters were injected into castrated male guinea pigs to study the fate of these complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of 3β-hydroxysteroid-dehydrogenase/isomerase from bovine adrenal cortex

The formation of 4-ene-3-ketosteroids from 3β-hydroxy-5-ene precursors is an obligatory step in the biosynthesis of hormonal steroids such as glucocorticoids, mineralocorticoids, estrogens and androgens. In the adrenal cortex, pregnenolone, 17-hydroxy-pregnenolone and dehydroisoandrosterone are converted to progesterone, 17-hydroxy-progesterone and androstenedione, respectively, by the enzymatic system 3β-hydroxy-5-ene steroid dehydrogenase and 3-keto-5-ene steroid isomerase (3β-HSD/I).

The present work reports a two step purification procedure which yields an homogenous preparation of 3β-HSD/I from bovine adrenal cortex. It uses solubilization of the microsomal proteins followed by two chromatographic steps, i.e. DEAE-cellulose and heparine-sepharose columns. The enzyme was obtained as an homogeneous protein exhibiting an apparent molecular size of 45 kDa upon SDS-gel electrophoresis and of 81 kDa upon gel filtration. The purified enzyme exhibits both the 5-ene-3β-ol steroid dehydrogenase and isomerase activities in contrast to previous work using a more complex procedure which yielded a final preparation having lost its isomerase activity [Hiwatashi et al., Biochem. J. 98 (1985) 1519–1525]. N-terminal aminoacid (29 residues) sequence of the purified protein was determined and was found identical to that predicted from the nucleic acid sequence of the recently identified enzyme cDNA [Zhas et al. FEBS Lett. 259 (1989) 153–157].     

Catabolism of cholesterol by bovine adrenal-cortex enzymes: in vitro formation of oxygenated sterols and side-chain cleavage products.

The identification of sterols and steroids formed by incubation of [7alph-3H, 26-14C]cholesterol with mitochondrial enzymes from bovine adrenal cortex is reported. Only 17% of the radioactivity associated with cholesterol metabolites was found in pregnenolone, whereas 15% was reliable to oxygenated sterols and 6% to steroid compounds. The significance of the formation of these compounds is discussed particularly as regards oxygenated cholesterol derivatives.     

Estimation of pregnenolone synthesis in rat adrenal homogenates: some cofactor requirements: effects of stress, hypophysectomy, cortisone and ACTH.

Pregnenolone synthesis was estimated in whole adrenal homogenates incubated in the presence of cyanoketone (2alpha-cyano-4,4,17alpha-trimethyl-androst-5-en-17beta-ol-3-one). The yield of pregnenolone depended on the type of incubation medium employed. Both Ca++ and bovine serum albumin (BSA) markedly stimulated the rate of pregnenolone synthesis as did NADPH or NADPH generating system. Aminoglutethimide added in vitro inhibited cholesterol sidechain cleavage activity. Ether stress in vivo stimulated pregnenolone synthesis in vitro, and hypophysectomy of 24 hours duration resulted in a decrease. Cortisone administration for 8 days reduced the formation of pregnenolone by rat adrenal homogenates, an effect prevented by concomitant treatment with ACTH. Similarly, hypophysectomy of 8 days duration resulted in a marked diminution of pregnenolone synthesis and ACTH replacement reversed this effect. Changes in pregnenolone synthesis were paralleled by changes in corticosterone and total steroid production.     

Wide spectrum of steroids serving as substrates for the formation of lipoidal derivatives in ZR-75-1 human breast cancer cells

Recently, several natural steroids have been found to be esterified to long-chain fatty acids (FAE) in various mammalian tissues. The purpose of the present study was to determine the ability of a series of 3H-labeled steroids to serve as substrates for the formation and accumulation of such non-polar derivatives in intact cells, using the hormone-responsive ZR-75-1 human breast cancer cell line as model. All 14 steroids tested were found to be converted, directly or following further metabolism, to lipoidal ester derivatives. The percentage of intracellular steroids recovered as FAE derivatives was usually substantial (14-90%), especially in the case of C-19 steroids (75-90%). The composition of the lipoidal steroid fractions recovered from the labeled cell extracts was characterized by chromatographic comparison with synthetic steroid FAEs and by saponification of the steroid FAEs and identification of the released steroidal moieties. Following metabolism, most steroid substrates were converted into multiple lipoidal esters. Furthermore, 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane-3 beta, 17 beta-diol, as well as androst-5-ene-3 beta, 17 beta-diol formed lipoidal diesters in addition to the monoester form. The high level of intracellular steroid FAE accumulation reported in this study suggests that these yet poorly known steroid derivatives may play important functions in the regulation of steroid hormone metabolism and action.     

Ovine steroid 17alpha-hydroxylase cytochrome P450: characteristics of the hydroxylase and lyase activities of the adrenal cortex enzyme

The steroid 17-hydroxylase cytochrome P450 (CYP17) found in mammalian adrenal and gonadal tissues typically exhibits not only steroid 17-hydroxylase activity but also C-17,20-lyase activity. These two reactions, catalyzed by CYP17, allow for the biosynthesis of the glucocorticoids in the adrenal cortex, as a result of the 17-hydroxylase activity, and for the biosynthesis of androgenic C(19) steroids in the adrenal cortex and gonads as a result of the additional lyase activity. A major difference between species with regard to adrenal steroidogenesis resides in the lyase activity of CYP17 toward the hydroxylated intermediates and in the fact that the secretion of C(19) steroids takes place, in some species, exclusively in the gonads. Ovine CYP17 expressed in HEK 293 cells converts progesterone to 17-hydroxyprogesterone and pregnenolone to dehydroepiandrosterone via 17-hydroxypregnenolone. In ovine adrenal microsomes, minimal if any lyase activity was observed toward either progesterone or pregnenolone. Others have demonstrated the involvement of cytochrome b(5) in the augmentation of CYP17 lyase activity. Although the presence of cytochrome b(5) in ovine adrenocortical microsomes was established, ovine adrenal microsomes did not convert pregnenolone or 17-hydroxypregnenolone to dehydroepiandrosterone. Furthermore the addition of purified ovine cytochrome b(5) to ovine adrenal microsomes did not promote lyase activity. We conclude that, in the ovine adrenal cortex, factors other than cytochrome b(5) influence the lyase activity of ovine CYP17.     

Amino-steroids as inhibitors and probes of the active site of cytochrome P-450scc. Effects on the enzyme from different sources

A series of analogues of cholesterol, each having a primary amine attached to a shortened side chain, were tested for their effects on cytochrome P-450scc from several different sources. Reconstituted enzyme systems using disrupted mitochondria from bovine adrenal and placenta, adult human adrenal and placenta, neonatal human adrenal, and rat adrenal and testis were used to assay for inhibitory effects on the side chain cleavage of cholesterol to pregnenolone. Two of the derivatives tested, 22-amino-23,24-bisnor-5-cholen-3 beta-ol and 23-amino-24-nor-5-cholen-3 beta-ol, were found to be potent inhibitors of this reaction; the derivatives in which the amine was attached closer to or further from the steroid ring, (20 R and S)-20-amino-5-pregnen-3 beta-ol and 24-amino-5-cholen-3 beta-ol, were much weaker inhibitors. In addition, spectral studies with rat adrenal mitochondria and a soluble preparation of human placental cytochrome P-450scc showed that binding of the 22-amine derivative to the enzyme produces difference spectra characteristic of nitrogen bonding to the heme; this indicates that the heme is positioned close to C-22 in the steroid-enzyme complex. These findings on the relative effectiveness of the amino-steroid inhibitors and the type of complex formed are similar to results obtained with purified bovine adrenocortical cytochrome P-450scc. This establishes that the proximity of the substrate binding site and the heme-iron catalytic site is a feature common to the enzyme from several sources and is therefore likely to be a necessary property of the active site structure.     

Steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities in mouse tissues

The metabolism of estrone sulfate and dehydroisoandrosterone sulfate to the free, unconjugated steroids, estrone and dehydroisoandrosterone, was demonstrated in more than thirty different tissues from male and female BALB/c mice. The activity of steroid sulfatase, when expressed per mg tissue, was greatest in both the pituitary gland and the adrenal glands. The pituitary gland, however, had the lowest capacity for hydrolysis of steroid sulfates while the liver had the greatest capacity. 17 beta-Hydroxysteroid oxidoreductase activity also was demonstrated in all mouse tissues by the formation of estradiol-17 beta when using estrone sulfate as the substrate. The highest apparent activity for 17 beta-hydroxysteroid oxidoreductase was found in lung tissue, and the greatest capacity to form estradiol-17 beta from estrone sulfate was found in liver, lungs, kidneys and testes. This study demonstrates that the majority of mouse tissues have steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities.     

Inhibition of the conversion of cholesterol to pregnenolone in bovine adrenocortical preparations.

The inhibition of the conversion of [4-14C] cholesterol to [4-14C] pregnenolone by a number of steroids has been studied in bovine adrenocortical mitochondrial acetonedried preparations. At equimolar substrate and inhibitor concentrations (3.3 muM) the most potent inhibitors were cholesterol derivatives containing a nitrogen function at c-22, followed by derivatives containing oxygen functions at c-22 or c-20 or both. The presence of a hydroxyl group at c-17 or the replacement of the 3beta-hydroxyl group by fluorine reduced the inhibitory efficacy. In the presence of inhibitors that were also relatively good substrates of the cholesterol side-chain cleavage system, such as some cholesterol derivatives hydroxylated in the side-chain,the rate of [4-14C] pregnenolone formation increased with time as the inhibitor was consumed. (20S)-20,21-Dihydroxycholesterol exerted such an effect on the kinetics of [4-14C]pregnenolone formation, and yielded 21-hydroxypregnenolone which was identified by gas chromatography-mass spectrometry. The synthesis of (20R)-22-ketocholesterol, of (20R,22R)-22hydroxycholesterol, (20R,22S)-hydroxycholesterol, and of (20S)-desmosterol is described.     

Effects of cholesterol side chain cleavage inhibitors on 11β-hydroxylation: linkage of mitochondrial oxygenase systems

Potent inhibitors of cholesterol side chain cleavage were tested for inhibition of 11β-hydroxylation of 11-deoxycortisol by bovine adrenal cortex mitochondria. Compounds which inhibited 11β-hydroxylation were metyrapone, 4-phenylimidazole, 1-benzylimidazole, 17β-ureido-1,4-androstadien-3-one, SU-8000, 4-methylaminoglutethimide, and 20α-hydroxycholestrol. Compounds which did not inhibit 11β-hydroxylation at concentrations of 0.5 mM were d-aminoglutethimide tartrate, 1-aminoglutethimide tartrate, N-methylaminoglutethimide, 16α-methylpregnenolone, 16β-methylpregnenolone, 20-tolylpregnenediol, 16α-chloropregnenolone-3-acetate, 16α-benzyloxypregnenolone-3-acetate and cyanoketone. The results obtained indicate that aminoglutethimide and its congeners, the 16-halogenated and 16-benzoylated derivatives of pregnenolone and cyanoketone are specific inhibitors of cholesterol side chain cleavage enzyme. The two mitochondrial steroid oxyganase systems are linked through their competition for a single electron source.     

Measurement of steroid synthesis in zona glomerulosa cells by liquid chromatography-electrospray ionization-mass spectrometry: inhibition by nitric oxide

A liquid chromatography-electrospray ionization-mass spectrometry method was developed to simultaneously determine the concentrations of aldosterone, corticosterone, cortisol, deoxycorticosterone, pregnenolone, and progesterone in bovine adrenal zona glomerulosa (ZG) cells. Steroids were extracted by liquid-liquid extraction, separated on a reverse-phase C18 column, ionized by electrospray, and detected by single-quadrupole mass spectrometry in a positive ion mode. All steroids formed sodium adducts at high abundance. Factors affecting the formation and signal of sodium adducts were investigated. The limits of detection (S/N=3) using selected ion monitoring are 2 pg for these steroids and 10 pg for pregnenolone. DETA NONOate, a nitric oxide donor, inhibited the basal, angiotensin-II-stimulated, and 25-hydroxycholesterol-stimulated syntheses of these steroids in ZG cells in a concentration-dependent manner. The technique demonstrates the ability to determine the individual steroid in each enzymatic step of aldosterone synthesis and the activity of steroidogenic enzymes in adrenal ZG cells.     

C21 steroid side chain cleavage enzyme from porcine adrenal microsomes. Purification and characterization of the 17 alpha-hydroxylase/C17,20-lyase cytochrome P-450

The properties and the purity of a cytochrome P-450 (17 alpha-hydroxylase) from porcine adrenal microsomes have been examined following a report that the corresponding enzyme from bovine adrenocortical microsomes is inactive as a 17 alpha-hydroxylase and fails to show a high spin spectrum on addition of substrate, once the enzyme has been purified (Bumpus, J. A., and Dus, K. M. (1982) J. Biol. Chem. 257, 12696-12704). The purity of the porcine enzyme was demonstrated by electrophoresis on polyacrylamide with sodium dodecyl sulfate, immunoelectrophoresis, and NH2-terminal amino acid sequence (16 residues). The pure enzyme shows Mr = 54,000, heme content of greater than 0.8 nmol/nmol of protein, and absorption spectra typical of cytochrome P-450. The enzyme is active with both delta 4 (progesterone) and delta 5 (pregnenolone) substrates as a 17 alpha-hydroxylase and with the corresponding 17 alpha-hydroxysteroids as a C17,20-lyase. All four substrates produce typical type I spectra with the enzyme (so-called high spin form). We conclude that: 1) porcine adrenal microsomes contain a 17 alpha-hydroxylase/C17,20-lyase which is a single protein molecule readily purified to an enzymatically active form; 2) the C17,20-lyase activity is largely suppressed in the microsomes; and 3) the enzyme closely resembles that found in testicular microsomes. We propose that this enzyme be referred to as the adrenal C21 steroid side chain cleavage enzyme.     

Range of substrates and steroid bioconversion reactions performed by recombinant microorganisms Saccharomyces cerevisiae and Yarrowia lipolytica expressing cytochrome P450c17

The relationship between 17α-hydroxylation and 20-oxidation-reduction of progesterone and some of its derivatives was studied in yeast strains Saccharomyces cerevisiae YEp51α, Yarrowia lipolytica E129A15, and expressing cytochrome P450c17. The key metabolites were found to be 17α-hydroxyprogester-one and 17α,20(α,β)-dihydroxypregn-4-ene-3-ones. The bioconversion pathways of pregn-4-ene-20(α,β)-ol-3-ones were determined. They included cycles of 20-oxidation, 17α-hydroxylation, and stereospecific 20-reduction. The efficiency and kinetic parameters of steroid bioconversion by the recombinant strains were determined. The role of yeast analogs of mammalian steroid dehydrogenases is discussed. It was found that any of the desired derivatives, 17α-hydroxyprogesterone or progesterone 17α,20(α,β)-diols, could be obtained from progesterone. Cholesterol bioconversion yields important metabolites: steroid hormones, the vitamin-D group, and bile acids [1, 2]. Attention to various cytochrome-P450 species participating in the biosynthesis of mammalian steroid hormones is caused by two circumstances: (1) the necessity of detecting structural-function abnorm alities of some of the enzymes of steroid-synthesis that cause human diseases, and (2) the potential of regio-and stereospecific cytochrome P450 species of mammals in chemoenzymatic synthesis of pharmacologically valuable steroids. Concerning the second line of inquiry, the development of transgenic Saccharomyces cerevisiae yeast for the complete synthesis of cortisol by additional expression and elimination of a total of 13 genes was reported [3]. To increase the yield of the target compound, the genes for enzymes performing undesirable steroid modifications were inactivated. These modifications included esterification of pregnenolone [4] and 20α-reduction of 17α-hydroxyprogesterone [5]. A search for analogs of mammalian 20α-hydroxysteroid dehydrogenase (20α-HSD) in the Saccharomyces cerevisiae genome revealed two candidate proteins: Ypr1p (yeast aldo-keto reductase) and Gcy1p (yeast galactose-inducible crystallin-like protein) [3]. Indeed, it was formerly shown that expression of cytochrome P450 from bovine adrenal cortex, performing 17α-hydroxylation and the C17,20-lyase reaction (P450c17) in S. cerevisiae under the control of the GAL10-promoter with the presence of D-galactose as an inducer, was accompanied by the sequential conversion of progesterone to 17α-hydroxyprogesterone and 17α,20(α,β)-dihydroxypregn-4-ene-3-one with a high yield [5].     

O-(Carboxymethyl)oxime steroidal haptens linked in the C3 position: study of the characteristics of antibodies against 17alpha-hydroxyprogesterone and testosterone and of their evolution with time.

The production of highly sensitive and specific antisera against 17alpha-hydroxyprogesterone (17 OHP) is reported. 17 OHP was rendered antigenic by covalent linkage to bovine serum albumin through position 3 of the steroid. An unambiguous method to prepare the mono-3-0-(carboxy-methyl) oxime derivative (CMO) of 17 OHP is described starting from 17 OHP-acetate. Antisera raised in rabbits were of high affinity for 17 OHP (Ka = 1 to 2 x 10(10) L/mole) and showed very little (less than 0.7%) or no cross reaction with a variety of steroids. Cross-reaction with 17alpha-hydroxy pregnenolone was however significant. When studied in relation to time, cross-reaction of the latter steroid decreased significantly (18 to 2%) while Ka remained at the same level. The same study of the evolution with time (up to 600 days) of the characteristics of antisera raised against the 3 CMO derivative of testosterone is also reported.     

Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone and absence of their sulfated counterparts in rodent brain

A new sample preparation method coupled to GC-MS analysis was developed and validated for quantification of sulfate esters of pregnenolone (PREG-S) and dehydroepiandrosterone (DHEA-S) in rat brain. Using a solid-phase extraction recycling protocol, the results show that little or no PREG-S and DHEA-S (<1 pmol/g) is present in rat and mouse brain. These data are in agreement with studies in which steroid sulfates were analyzed without deconjugation. We suggest that the discrepancies between analyses with and without deconjugation are caused by internal contamination of brain extract fractions, supposed to contain steroid sulfates, by lipoidal forms of PREG and DHEA (L-PREG and L-DHEA, respectively). These derivatives can be acylated very efficiently with heptafluorobutyric anhydride and triethylamine, and their levels in rodent brain (approximately 1 nmol/g) are much higher than those of their unconjugated counterparts. They are distinct from fatty acid esters, and preliminary data do not favor structures such as sulfolipids or sterol peroxides. Noncovalent interactions between steroids and proteolipidic elements, such as lipoproteins, could account for some experimental data. Given their abundance in rodent brain, the structural characterization and biological functions of L-PREG and L-DHEA in the central nervous system merit considerable attention.     

Cytochrome P-450(17 alpha,lyase)-mediating pathway of androgen synthesis in bovine adrenocortical cultured cells.

Cytochrome P-450(17 alpha,lyase) mediating pathway of dehydroepiandrosterone (DHA) formation from pregnenolone was investigated in primary cultures of bovine adrenocortical fasciculata-reticularis cells. To determine whether DHA formation proceeds predominantly by successive monooxygenase reactions without 17 alpha-hydroxypregnenolone leaving P-450(17 alpha,lyase) the cells were incubated with [14C]pregnenolone and 17 alpha-[3H]hydroxypregnenolone in the presence of Trilostane. Results of the double-substrate double-label experiments indicate that in the presence of high concentration of pregnenolone most of DHA was formed, directly from pregnenolone by the successive reactions. Since the concentration of pregnenolone usually exceeds that of 17 alpha-hydroxypregnenolone in the adrenal glands, DHA is concluded to be formed predominantly by successive reactions from pregnenolone without 17 alpha-hydroxypregnenolone leaving P-450(17 alpha,lyase) in vivo. By chronic ACTH treatment, the activities of 17 alpha-hydroxylation and DHA formation in adrenocortical cultured cells became higher concomitantly with the increase of P-450(17 alpha,lyase) content. Most of DHA was found to be formed by successive reactions from pregnenolone even under such conditions.     

ACTH induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase, cholesterol biosynthesis, and steroidogenesis in primary cultures of bovine adrenocortical cells

The current studies demonstrate that corticosteroidogenesis can be maintained by primary cultures of bovine adrenocortical cells under lipoprotein-depleted conditions. The cholesterol necessary as substrate for steroid synthesis was found to arise from de novo synthesis within these cells. Adrenocorticotropin (ACTH) increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity 5-fold within 12 h after addition to the medium. The increase in activity apparently represented accumulation of enzyme as determined by protein blotting and immunodetection. The predominant immunodetectable species of HMG-CoA reductase from bovine adrenal cells was 97,000 daltons; no higher molecular mass species was detectable. The ACTH induction of HMG-CoA reductase activity could be prevented after inhibition of cholesterol conversion to pregnenolone with clotrimazole. These results are suggestive that ACTH increases adrenocortical cholesterol biosynthesis and HMG-CoA reductase activity after conversion of a cellular pool of cholesterol and/or oxysterol into steroid. The increased rate of cholesterol biosynthesis is then capable of maintaining ACTH-promoted steroid production. This is the first study, in vitro, to demonstrate an ACTH-promoted accumulation of HMG-CoA reductase of adrenocortical cells.