Corticotropin-induced changes in a soluble desmolase preparation

Following simple homogenization, substantial desmolase activity is recovered in rat adrenal 105 000 × g supernatant. The desmolase complex sediments at 3–4 S on sucrose gradients, is found in the clear cytosol, requires NADPH, is derived from mitochondria and is inhibited by aminoglutethimide and pregnenolone. The lipid fraction contains little or no desmolase activity but greatly enhances pregnenolone synthesis in soluble desmolase preparations, presumably by supplying free cholesterol substrate. Prior adrenocorticotropin (ACTH) administration enhances pregnenolone synthesis in the 105 000 × g supernatant, and cycloheximide, an inhibitor of adrenal protein synthesis, does not block this effect of ACTH (but rather potentiates it). The ACTH effect may be largely explained by an increase in free cholesterol, which enhances the activity of both the lipid fraction and clear cytosol, since: free cholesterol levels are increased by ACTH, particularly with cycloheximide pretreatment; type I and inverted type I difference spectrum changes, indicating greater cholesterol availability for binding to cytochrome P-450, are enhanced by ACTH with or without cycloheximide treatment; cholesterol-rich lipid fraction enhances such spectral changes and obliterates the differences in spectral and pregnenolone-synthesizing activities betwen control and ACTH-stimulated soluble desmolase preparations; and desmolase stimulatory properties of clear cytosol co-chromatographs with [¹⁴C]cholesterol. Since cycloheximide blocks ACTH-induced effects in intact mitochondria but not in the soluble desmolase preparation, it is postulated that the labile protein required during ACTH action functions to overcome a ?restraining influence’ which is present in intact mitochondria but not in the soluble desmolase system. The ‘restraining influence’ may be due to limited cholesterol-desmolase interaction.     

The enhancement of pregnenolone production as the main mechanism of the prolonged stimulatory effect of ACTH on cortisol production by guinea-pig adrenocortical cells

The prolonged stimulatory influence of corticotropin (ACTH) on the adrenocortical steroidogenic response to ACTH was studied in guinea-pig adrenocortical cells harvested from control and ACTH-treated animals (ACTH1-24, 50 micrograms s.c. twice daily on the day preceding the in vitro experiment). The maximal capacity to produce cortisol in response to ACTH (by 10(5) cells and 2 h incubation) was increased from 341.8 +/- 36.3 ng (control group) to 663.3 +/- 37.6 ng for cells obtained from guinea-pigs treated in vivo with ACTH. In the presence of trilostane, added to the cells in order to block the conversion of pregnenolone to cortisol, the net maximal output of pregnenolone and 17-hydroxypregnenolone in response to ACTH was significantly increased in adrenocortical cells from ACTH-treated animals (449.5 +/- 35.8 ng pregnenolone and 85.7 +/- 10.5 ng 17-hydroxypregnenolone vs 269.1 +/- 36.3 ng pregnenolone and 43.7 +/- 8.51 ng 17-hydroxypregnenolone for cells from control guinea-pigs). It appeared therefore that the total production of pregnenolone (as estimated by the sum of pregnenolone and 17-hydroxypregnenolone produced by the cells incubated with trilostane) nearly reached the level of the maximal production of cortisol in response to ACTH and was also significantly enhanced for cells from ACTH-treated animals (532.2 +/- 38.4 ng vs 312.8 +/- 40.0 ng for cells from control group). By contrast, no effect was documented on 17 alpha-hydroxylase activity since 17 alpha-hydroxylation index was similar for both types of adrenocortical cells (16.3 +/- 2.05% for ACTH-treated animals and 14.2 +/- 2.83% for control group). It was concluded therefore that the prolonged stimulatory influence of ACTH on pregnenolone production is the main mechanism of the enhancement of cortisol synthesis by guinea-pig adrenocortical cells previously stimulated by ACTH.     

Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization

Addition of homogeneous rat liver sterol carrier protein2 (SCP2) or an adrenal cytosolic fraction enhanced pregnenolone production by adrenal mitochondria. Pretreatment of SCP2 or adrenal cytosol with anti-SCP2 IgG abolished the stimulatory effect of both preparations on mitochondrial pregnenolone output. Incubation of mitochondria with aminoglutethimide, which blocks interaction of cholesterol with inner membrane cytochrome P-450scc, resulted in decreased pregnenolone production and a decreased level of mitoplast cholesterol. Addition of SCP2 to the incubation media caused an almost 2-fold increase in cholesterol associated with the mitoplast, but did not enhance mitochondrial pregnenolone production. Studies with reconstituted cytochrome P-450scc in phospholipid vesicles also suggested that SCP2 did not affect interaction of cholesterol with the hemoprotein. Treatment of rats with cycloheximide alone or with adrenocorticotropic hormone resulted in a dramatic increase in mitochondrial cholesterol. However, these mitochondria did not exhibit increased levels of pregnenolone output under control incubation conditions. When SCP2 was included in the mitochondrial incubation media, pregnenolone production was significantly increased over that observed with adrenal mitochondria from untreated or adrenocorticotropic hormone-treated rats. The results imply that SCP2 enhances mitochondrial pregnenolone production by improving transfer of mitochondrial cholesterol to cytochrome P-450scc on the inner membrane, but does not directly influence the interaction of substrate with the hemoprotein.     

Enhanced 17 alpha-hydroxylation of pregnenolone and increased androgen production by rabbit adrenocortical cells stimulated chronically with corticotropin

The postulated chronic stimulatory effect of corticotropin (ACTH) on pregnenolone production and on 17 alpha-hydroxylase activity was evaluated on adrenocortical cells obtained from control and chronically ACTH-treated rabbits. The cells were incubated with various concentrations of ACTH added alone or together with trilostane, so as to inhibit further conversion of pregnenolone and dehydroepiandrosterone. The maximal steroidogenic effect of ACTH (determined in the absence of trilostane) was increased 2-fold in adrenocortical cells from ACTH-treated animals; furthermore, cortisol production was increased whereas that of corticosterone decreased. While the generation of pregnenolone was of comparable magnitude for cells from both experimental groups, chronic in vivo treatment with ACTH was followed by a 40-fold enhancement in 17-hydroxypregnenolone production. Concomitantly, maximal DHEA production documented in the presence of ACTH and trilostane was enhanced more than 200-fold, from 0.45 +/- 0.20 pmol in control rabbits to 147 +/- 67 pmol in cells from ACTH-treated animals. The corresponding values of DHEA-sulphate production were 0.86 +/- 0.12 and 432 +/- 334 pmol, respectively. Thus, a prolonged stimulatory effect of ACTH on rabbit adrenocortical cells consists in an enhancement of the capacity to generate pregnenolone, and to convert this compound into 17-hydroxylated steroids.     

Increase in the amount of glutathione transferase 4-4 in the rat adrenal gland after hypophysectomy and down-regulation by subsequent treatment with adrenocorticotrophic hormone.

The effect of hypophysectomy and subsequent treatment with adrenocorticotropic hormone (adrenocorticotropin, ACTH) on the isoenzymes of glutathione transferase in the rat adrenal gland was investigated. A large increase (approx. 11-fold) in the level of transferase subunit 4 was observed in hypophysectomized animals by immunoblotting. When the activity of glutathione transferase 4-4 was measured in adrenal cytosol using trans-stilbene oxide as a selective substrate, a 15-fold increase was noted. Lack of the pituitary hormone ACTH is apparently related to this increase, since treatment of hypophysectomized animals with ACTH for 2 weeks partially down-regulated subunit 4. Glutathione transferase subunits 3 and 8 in the adrenal were also increased in amount by hypophysectomy, but not at all to the same extent. The activity of glutathione transferase 4-4 was elevated also in the liver and ovary (5 and 1.5 times respectively) after hypophysectomy. These elevated enzyme levels were, however, not affected by ACTH treatment. This down-regulation of glutathione transferases in the rat adrenal by ACTH may be related to the fact that, under normal conditions, this organ is highly susceptible to the toxic effects of various polycyclic hydrocarbons, whereas under circumstances where there is no ACTH production, as in hypophysectomized rats, the adrenal is resistant to these same hydrocarbons.     

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. III. ACTH-induced temporal subcellular redistributions of steroid precursors to corticosterone

Cholesterol, pregnenolone, progesterone, 11-deoxycorticosterone (11-DOC) and corticosterone were quantitated in subcellular fractions isolated from in vivo adrenocorticotropin (ACTH)-stimulated rat adrenal zona fasciculata/reticularis. Six adrenal subcellular fractions separated by discontinuous sucrose gradient centrifugation (lipid, 0.125 M sucrose, cytosolic, microsomal, mitochondrial and nuclear) were extracted with alkaline ether/ethanol and assayed by high pressure liquid chromatography (HPLC). Lipid fractions contained the major cholesterol stores, while most pregnenolone and progesterone was found in lipid, microsomal and mitochondrial fractions. The 0.125 M sucrose and cytosol fractions together contained approximately 75% of the total 11-DOC and corticosterone. The five steroids were only present in small amounts in organelle fractions containing steroidogenic enzymes. Homogenate and lipid fraction cholesterol decreased between 10 and 15 min and again 30 min after ACTH injection. In the homogenate, lipid, microsomal and mitochondrial fractions, pregnenolone and progesterone were increased after ACTH injection; peak pregnenolone and progesterone concentrations were often measured in adrenal gland sucrose, cytosolic, microsomal and mitochondrial fractions 15 to 20 min after rats were injected with ACTH. Although ACTH increased 11-DOC and corticosterone in all but the mitochondrial and nuclear fractions, the sucrose, cytosolic and microsomal 11-DOC, and cytosolic corticosterone increased most dramatically. In many fractions, peak 11-DOC and corticosterone concentrations were most often observed between the 10 and 15 min periods and again at 30 min.     

Chronic treatment with corticotropin increases the capacity of rabbit adrenocortical cells to convert pregnenolone into androgens

The present study was conducted to evaluate whether the previously demonstrated enhancement in adrenocortical androgen secretion in rabbits chronically treated with ACTH results, in addition to an increased pregnenolone production, from a more efficient conversion of this precursor of steroidogenesis into androgens. To this end, the adrenocortical cells from 14 control and 14 ACTH-treated rabbits (ACTH 1-24,200 micrograms s.c. daily for 12 days) were incubated either in the presence of different concentration of ACTH or with pregnenolone added in amounts from 0.5 to 250 micrograms. The total steroidogenic potency (maximal response to ACTH) was significantly enhanced for cells from ACTH-treated animals, as was the ACTH-induced production of dehydroepiandrosterone (DHEA), DHEA-sulfate, androstenedione and testosterone. In addition the production of these androgens from given amounts of exogenous pregnenolone was also significantly increased. The maximal capacity of adrenocortical cells to convert pregnenolone into androgens averaged (for ACTH-treated vs control group) 130 +/- 34 vs 43 +/- 10 pmol for DHEA, 138 +/- 43 vs 46 +/- 14 pmol for DHEA-sulfate, 99 +/- 31 vs 10 +/- 2 pmol for androstenedione and 8.0 +/- 2.6 vs 2.4 +/- 0.3 pmol for testosterone (P less than 0.001 for all androgens). The addition of ACTH to adrenocortical cells incubated with pregnenolone did not modify the maximal capacity of conversion of pregnenolone into androgens, which was in both experimental groups similar to that documented in the absence of ACTH. Thus, while an acute stimulatory effect of ACTH on adrenocortical steroidogenesis is devoid of any influence on the activity of the post-pregnenolone pathway of androgen synthesis, the chronic exposure of adrenocortical cells to ACTH lead to increased activity of steroidogenic pathway involved in the conversion of pregnenolone into androgens.     

The stimulation by ACTH of 17 alpha-hydroxylation in cultures or rabbit adrenal cells

Adrenal cells from control rabbits (control-cells) and from rabbits that had been injected twice daily for 3 days prior to sacrifice with 25 IU ACTH (ACTH-cells) were cultured both in the absence and presence of 100 mIU ACTH. Culture durations varied from 24 to 120 h in 24 h increments. The culture medium was changed daily and fresh ACTH added to appropriate vessels. At the time of the final media change 0.1 muCi [4-14C]pregnenolone was added. Twenty-four hours later the cultures were terminated and the products formed from the pregnenolone were isolated, quantified and identified by solvent extraction, chromatography and crystallization to constant specific activity. After 72 h ACTH-cells cultured in the presence of ACTH converted 18.5% of the pregnenolone substrate to 17-hydroxycorticosteroids (cortisol plus 11-deoxycortisol) while ACTH-cells cultured in the absence of ACTH converted a maximum of 1.6%. A similar but smaller difference, 10.9 vs 2.1%, was recorded with control cells cultured in the presence and absence of ACTH. Corticosterone production from [4-14C]pregnenolone in the 72-h cultures was increased to a lesser degree by ACTH exposure. In ACTH-cells the conversion climbed from 5.9 to 10.5% and from 9.6 to 12.0% in control cells. Microscopic examination of parallel cultures showed no significant differences in cell density between cells cultured in the presence or absence of ACTH. 17 alpha-Hydroxylase activity arising from the in vivo stimulation did not survive cell division in culture, but required the continual presence of ACTH. In conclusion, the data show that ACTH is capable of stimulating 17-hydroxycorticoid formation in rabbit adrenal cell cultures.     

Factors affecting the adrenocorticotropic hormone stimulation of rabbit adrenal 17α-hydroxylase activity

Adrenocorticotropic hormone (ACTH)-stimulated 17α-hydroxylase activity of rabbit adrenal tissue has been shown to be associated with the subcellular fractions sedimented from 0.25 M sucrose at 33 000 × g for 60 min and at 105 000 × g for 60 min. The fraction sedimenting at 9000 × g for 20 min (mitochondria) contained the majority of the 11β-hydroxylase activity but also had a significant amount of 17α-hydroxylase activity. All subcellular 17α-hydroxylase activity showed an apparent preference for pregnenolone over progesterone. A 1 : 1 mixture of wholehomogenates of adrenal tissue from control and ACTH-stimulated rabbits incubated with[4-¹⁴C]pregnenolone synthesized as much 17α-hydroxylated corticosteroids as homogenate from the ACTH-stimulated tissue alone. However, the mixed homogenate synthesized only 1/4th–1/5th as much 17-deoxycorticosteroids as control, non-stimulated tissue, suggesting that the control tissue contained no inhibitor of 17α-hydroxylation, whereas ACTH-stimulated tissue may contain an inhibitor of 17-deoxycorticoid formation. 24-h dialysis of whole homogenates and subcellular fractions of adrenal tissue from control and ACTH-stimulated animals showed that 17α-hydroxylation was not activated in control tissue and somewhat inactivated in ACTH-stimulated tissue by this treatment. On the other hand, dialysis activated 17-deoxycorticoid formation by whole homogenates, but not in subcellular fractions, of both ACTH-stimulated and control adrenal tissue. Injection of 5 mg/kg cycloheximide prior to the first of 2 daily ACTH injections caused an average of 270 g body weight loss while not affecting the increase in adrenal weight effected by the ACTH. Adrenal tissue homogenates from cycloheximide injected animals produced only 50% as much 17α-hydroxycorticosteroids as homogenates of tissue from animals injected with ACTH alone and produced an amount of17-deoxycorticoids intermediate between homogenates of control and ACTH-stimulated tissue, suggesting the requirement of protein synthesis for 17α-hydroxylation stimulating activity of ACTH.     

Effect of hypophysectomy on responsiveness of rat adrenal slices to ACTH--pregnenolone formation from endogenous cholesterol.

Responsiveness of rat adrenal slices to ACTH was studied by measuring pregnenolone formation from endogenous cholesterol. The yield of pregnenolone by adrenal slices of hypophysectomized for 48 hrs rats was markedly higher as compared to intact animals.     

Adrenocorticotropic hormone does not induce desensitization in human adrenal cells during fetal life

We investigated whether human fetal adrenal cells pretreated with or continuously exposed to adrenocorticotropic hormone (ACTH) would develop refractoriness of the steroidogenic response. Fetal adrenal glands from fetuses of 18-24 wk gestation, were studied. Fetal zone cells were pretreated with increasing doses of ACTH (0-10(-6) M) for 24 h and then restimulated with a single dose of ACTH (10(-6) M) for an additional 24 h. Regardless of the dose of ACTH in the first incubation, the cells responded to the second stimulation with a 2- to 6-fold increase in dehydroepiandrosterone sulfate (DHAS) production. When human fetal adrenal cells were incubated in the continuous presence of 10(-8) M ACTH for 72 h, DHAS production was increased compared to that of the untreated cultures (5-fold at 24 h and 50-fold at 72 h), and the cells remained responsive during the entire experimental period. In contrast, human adult adrenal cells showed a significant decrease of the steroidogenic response after 48 h of ACTH treatment. Twenty-four hours of incubation with increasing doses of ACTH also increased the basal steroidogenic capacity of the fetal adrenal cells. One of the steroidogenic enzymatic steps stimulated by ACTH pretreatment was that of 17 alpha-hydroxylase/17, 20-lyase, since conversion of pregnenolone and 17 alpha-hydroxypregnenolone to DHAS was increased in a dose-dependent manner. These results demonstrate that human fetal adrenal cells, in contrast to those of the adult, do not become desensitized to ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adrenal adenylate cyclase and steroidogenic activities of 63 day old ovine fetuses: in vitro effects of ACTH1-24 and forskolin

This study examines the activity of the adenylate cyclase system and that of some enzymes of the steroidogenic pathway of adrenal cells from 62-63 day old ovine fetuses. Synthetic corticotropin (ACTH1-24), cholera toxin and forskolin stimulated both cAMP and corticoid productions by freshly isolated adrenal cells. The cAMP response to ACTH1-24 was lower than that to forskolin. However, forskolin-induced steroidogenesis was significantly lower than the ACTH1-24-induced steroid output. Freshly isolated cells metabolized quickly [14C]-labeled pregnenolone mainly through the 17-deoxy pathway. The amounts of cortisol and of corticosterone formed, in the presence of exogenous pregnenolone, were roughly 15-fold higher than under maximal stimulation by ACTH1-24. When the cells were cultured for 6 days in the absence or presence of ACTH1-24 (10(-8) M) or forskolin (10(-5) M), a small development of the cAMP response to these factors was observed in the course of the experiment. However, the mechanism of this development appeared different, according to the conditions of culture. The amounts of corticosterone secreted on day 6 by ACTH1-24- or forskolin-treated cells were 2- to 4-fold higher than on day 1, whereas cortisol outputs were much lower on day 6 than on day 1. The response to ACTH1-24 of cells maintained in ACTH-free media decreased dramatically during the culture in terms of both cortisol and of corticosterone. On day 6 of the experiment, the metabolism of [14C]pregnenolone was lower than on day 1 under all 3 conditions of culture. Only the 3 beta-hydroxysteroid dehydrogenase/isomerase activity could be maintained by continuous treatment with forskolin. However, both ACTH1-24 and forskolin enhanced the production of pregnenolone from an endogenous substrate. In conclusion, these results present evidence that: 1) the adenylate cyclase system is not a bottleneck in the steroidogenic response to ACTH1-24 of freshly isolated adrenal cells from 62-63 day old ovine fetuses; 2) the main rate-limiting step for steroidogenesis by these cells is the availability of pregnenolone; 3) neither ACTH1-24 nor forskolin is able to maintain the activity of most enzymes involved in the metabolization of pregnenolone by cultured cells while increasing pregnenolone availability; 4) some inhibiting factors are involved in the loss of adrenal cells responsiveness to ACTH between days 50 and 100 of gestation, and they probably act mainly on the adenylate cyclase system.     

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.     

Some characteristics of adrenal steroidogenesis and their possible relationships to the action of the adrenocorticotropic hormone.

An attempt to define in quantitative terms the characteristics of the biphasic rate curve for pregnenolone synthesis in cell-free systems from the adrenal using male Sprague-Dawley rats is reported. When adrenocorticotropic hormone (ACTH) was used 2 units of .2 ml of .9% saline were injected ip 15 minutes before killing the rats. The effect of ACTH on adrenal steroidogenesis is in the stimulation of the rate of conversion of cholesterol to pregnenolone. This reaction sequence is thought to occur in the mitochondria. Methods of preparing subcellular fractions are described. Incubation of pregnenolone with mitochondria for 20 minutes at 20 degree C resulted in a 70% disappearance of the pregnenolone. This loss does not occur if the mitochondria are boiled, indicating an enzymatic process. The rate of pregnenolone synthesis characteristically shows a biphasic curve with a rapid primary rate and a slower secondary rate. ACTH administration in vivo increased both rates but the percentage increase was greater for the secondary rate. In addition an increase in the duration of the primary rate resulted. Different explanations are offered for these characteristics. Pregnenolone may act as an inhibitor of its own synthesis from cholesterol but not from 20alpha-hydroxycholesterol. Substances that cause mitochondria to swell may stimulate pregnenolone synthesis. Another theory proposes that the limiting ACTH-sensitive step is the rate at which mitochondrial cholesterol is transported to or binds to the cholesterol side-chain cleavage enzyme. The possible role of an inhibitor in the regulation of steroidogenesis is indicated. Data are consistent with the observation that the transition from the primary rate to the slower secondary rate shows the accumulation of an inhibitory substance. The action of ACTH would then be to modify the structure of the cholesterol side-chain cleavage enzyme so that there is a decreased susceptibility of the enzyme to the inhibitor.     

The stimulatory effect of corticotropin on cortisol biosynthetic pathway in guinea-pig adrenocortical cells

The mechanism of the prolonged stimulatory effect of corticotropin (ACTH) on adrenocortical synthesis of cortisol was studied in guinea-pig adrenocortical cells harvested from control animals and from guinea-pigs submitted 24 h before the sacrifice to a prolonged ether anesthesia in an attempt to induce a release of endogenous ACTH. As a result of this in vivo exposure to endogenous ACTH, the maximal capacity to produce glucocorticoids (by 1 X 10(5) cells incubated during 2 h) in response to ACTH increased from 579 +/- 111 ng (control group) to 915 +/- 143 ng for cells from treated animals, whereas the apparent affinity of the steroidogenic response to ACTH remained unchanged. This hyper-reactivity of cells from anesthetized animals was also evident in the presence of dibutyryl cyclic AMP. Moreover, there was increased conversion of exogenous pregnenolone into cortisol by cells from previously anesthetized animals. It was therefore concluded that ACTH increases in a lasting way the activity of steroidogenic pathway leading to cortisol synthesis by adrenocortical cells at sites distal to cyclic AMP generation. Besides an obvious increase of formation of pregnenolone in response to ACTH, it seems that this ACTH-induced enhancement in the capacity of the steroidogenic response to ACTH also implies a prolonged stimulatory influence of the peptide on the post-pregnenolone steroidogenic pathway leading to cortisol synthesis.     

The role of sterol carrier protein 2 in stimulation of steroidogenesis in rat adrenal mitochondria by adrenal cytosol

Cholesterol side-chain cleavage (CSCC) in isolated rat adrenal mitochondria is enhanced by prior corticotropin (ACTH) stimulation in vivo (8-fold). Part of this stimulation is retained in vitro by addition of cytosol from ACTH-stimulated adrenals to mitochondria from unstimulated rats (2.5- to 6-fold). In vivo cycloheximide (CX) treatment fully inhibits the in vivo response and resolves the in vitro cytosolic stimulation into components: (i) ACTH-sensitive, CX-sensitive; (ii) ACTH-sensitive, CX-insensitive; and (iii) ACTH-insensitive, CX-insensitive. These components contribute approximately equally to stimulation by ACTH cytosol. Components (i) and (iii) most probably correspond to previously identified cytosolic constituents steroidogenesis activator peptide and sterol carrier protein 2 (SCP2). SCP2, as assayed by radioimmunoassay or ability to stimulate 7-dehydrocholesterol reductase, was not elevated in adrenal cytosol or other subcellular fractions by ACTH treatment. Complete removal of SCP2 from cytosol by treatment with anti-SCP2 IgG decreased cytosolic stimulatory activity by an increment that was independent of ACTH or CX treatment. Addition of an amount of SCP2, equivalent to that present in cytosol, restored activity to SCP2-depleted cytosol but had no effect alone or when added with intact cytosol, suggesting the presence of a factor in cytosol that potentiates SCP2 action. Pure hepatic SCP2 stimulated CX mitochondrial CSCC 1.5- to 2-fold (EC50 0.7 microM) but was five times less potent than SCP2 in adrenal cytosol. Two pools of reactive cholesterol were distinguished in these preparations characterized, respectively, by succinate-supported activity and by additional isocitrate-supported activity. ACTH cytosol and SCP2 each stimulated cholesterol availability to a fraction of mitochondrial P450scc that was reduced by succinate but failed to stimulate availability to additional P450scc reduced only by isocitrate.     

Forms and activity of high molecular weight adrenocorticotropin in guinea pig

High molecular weight forms of adrenocorticotropin (ACTH) and endorphin were identified in extracts of guinea pig anterior and intermediate/posterior pituitary. Extracts of anterior pituitary contained ACTH immunoactive material with apparent molecular weights of 36,000, 24,000 and 4,500 daltons. The highest molecular weight form the ACTH co-migrated with a peak of endorphin immunoactive material. No material the size of glycosylated ACTH(1--39) was detected. Separated forms of high molecular weight ACTH prepared from mouse tumor cell culture medium stimulated the same maximal production of steroid as ACTH(1--39) in the guinea pig adrenal cell bioassay. Pro-ACTH/endorphin and ACTH biosynthetic intermediate were two orders of magnitude less potent than synthetic human ACTH(1--39); glycosylated ACTH(1--39) was equipotent to ACTH(1--39) although no similar material was detected in guinea pig pituitary extracts. Isolated guinea pig adrenal cortical cells were incubated with the various separated form of mouse tumor cell ACTH and products synthesized from (3H)pregnenolone were analyzed by two-dimensional thin-layer chromatography. The ratio of cortisol-related to corticosterone-related products was the same in response in glycosylated and nonglycosylated ACTH.     

Mechanism of glucocorticoid enhancement of the responsiveness of ovine adrenocortical cells to adrenocorticotropin

The present studies were undertaken to precise the mechanism through which glucocorticoids enhance the responsiveness of ovine adrenocortical cells to ACTH. Experiments using intact cells and crude adrenal membranes have shown that, at the level of the adenylate cyclase system, dexamethasone increases the number of ACTH receptors without modification of the catalytic subunit or of the GTP binding regulatory components Gs and Gi. Cells cultured with dexamethasone secreted more pregnenolone and more corticosteroids in response to 8-BrcAMP than did control cells. By contrast, dexamethasone did not increase corticosterone secretion by cells incubated in the presence of 22-(R)-hydroxycholesterol or of exogenous pregnenolone. Dexamethasone neither affected the incorporation of [14C] acetate into cellular cholesterol nor the amount of cholesterol present in mitochondria of unstimulated cells. However, dexamethasone-treated cells incubated in the presence of both 8-BrcAMP and aminoglutethimide exhibited higher amounts of mitochondrial cholesterol than control cells. These data indicate that dexamethasone enhances the number of cellular ACTH receptors together with increasing the cAMP-induced translocation of cholesterol from the cytoplasm into mitochondria and/or mitochondrial storage of cholesterol.     

Ontogenesis of cholesterol side-chain cleavage activity in the ovine adrenal during late gestation.

The present study examined the activity of the cholesterol side-chain cleavage system, and the amount of cytochrome P450scc in adrenal glands of sheep fetuses and newborn lambs as well as the in vitro regulation of these parameters. Freshly isolated fetal adrenal cells incubated in the presence of 1 mM 8Br-cAMP or 25 microM 22R-OH cholesterol, produced 4- to 5-fold less pregnenolone than neonatal cells under similar conditions. Likewise, pregnenolone production by isolated fetal adrenal mitochondria was lower than that of neonatal mitochondria when endogenous cholesterol was used as a substrate or when 22R-OH cholesterol was added to the incubation medium. Also, the amount of P450scc, determined by immunoblot, was lower in fetal mitochondria than in neonatal mitochondria. In culture, ACTH, despite enhancing both the production of pregnenolone and the incorporation of [14C]acetate in cholesterol and its end-products by fetal adrenal cells, neither increased the amount of pregnenolone formed from 22R-OH cholesterol nor the amount of immunoreactive P450scc. By contrast, during the first 48 h of culture under standard conditions, there was a "spontaneous" increase in the activity of P450scc which reached values observed in neonatal adrenal cells. Such a development was inhibited when 5% ovine fetal serum was added to the culture medium. These results reinforce the view that in the ovine fetal adrenal gland, the development of P450scc is not ACTH-dependent but involves most probably a decrease in inhibitory factors present in fetal blood.     

The influence of cytochalasin B on the response of adrenal tumor cells to ACTH and cyclic AMP.

Cytochalasin B inhibits increase in steroid synthesis by mouse adrenal tumor cells (Y-1), produced either by ACTH or cyclic AMP. Basal levels of steroid synthesis are not decreased and the inhibitor acts by decreasing the response of the side-chain cleavage step (cholesterol → pregnenolone) to ACTH. Inhibition is reversible and is seen in medium without glucose. These observations suggest that microfilaments may play a role in the response of adrenal cells to ACTH.