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.     

Co-induction of 17 alpha-hydroxylase and C-17,20-lyase activities in primary cultures of bovine adrenocortical cells in response to ACTH treatment

Bovine adrenocortical cells in primary culture were used to examine the trophic effect of ACTH on the induction of the 17 alpha-hydroxylase and C-17,20-lyase activities. The addition of exogenous pregnenolone to bovine adrenal microsomes showed the appearance of 17 alpha-hydroxy-pregnenolone before the formation of dehydroepiandrosterone. The same sequence of activities was evident in postmitochondrial supernate from bovine adrenocortical cells cultured 36 h in the presence of 1 microM ACTH but not in postmitochondrial supernate from control cells. In another study, bovine adrenocortical cells were cultured for 36 h after which 30 microM 17 alpha-hydroxypregnenolone was added to the medium and the incubation continued 1 h; there was a 4-fold increase in androgen content in the media from ACTH-treated cells over controls. Measurement of the 17 alpha-hydroxylase and C-17,20-lyase reactions in postmitochondrial supernate from cells cultured 0-72 h in the presence of ACTH or 1 mM dibutyryl cAMP showed concomitant increases in the two activities and both activities were inhibited by the same compounds known to inhibit 17 alpha-hydroxylase activity. These observations support the concept of the co-induction of 17 alpha-hydroxylase and C-17,20-lyase activities in response to ACTH; results in keeping with previous studies indicating that the two activities are catalyzed by a single gene product, the polypeptide chain P-45017a.     

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.     

Glucocorticoid enhancement of glucocorticoid production by cultured ovine adrenocortical cells

The present study examines the effect of chronic treatment with glucocorticoids on the steroidogenic activity of ovine adrenocortical cells in vitro. Cells cultured in the presence of 10(-9) to 10(-5) M dexamethasone produced more glucocorticosteroids in response to ACTH1-24, forskolin or 8 BrcAMP than did control cells. Such an enhancing effect required more than 5 h of treatment and was maximal at 30 h; it was both concentration-dependent and steroid-specific. The maximal secretion of corticosteroids was observed when cells were exposed to 10(-7) M dexamethasone; with higher concentrations the response to ACTH1-24 decreased steadily; the ED50 was 2.8 +/- 0.8 nM. Cortisol and corticosterone enhanced ACTH1-24-induced steroidogenesis to the same extent as dexamethasone, but at concentrations roughly 100-fold higher than for dexamethasone. Testosterone and 17 beta-oestradiol had no enhancing effect. Dexamethasone not only enhanced the maximal steroidogenic response to ACTH1-24 but also decreased its ED50 3-fold. Treatment of cultures with the antiglucocorticoid RU 38486 resulted in a dose-dependent, time-dependent, decrease in ACTH1-24-induced corticosteroid output. Moreover, RU 38486 antagonized the enhancing effect of dexamethasone. The production of corticosteroids by dexamethasone-treated cells incubated in the presence of 22(R)-hydroxycholesterol or of exogenous pregnenolone was similar to that of control cells. The enhancing effect of dexamethasone was also observed when cultures were performed in the absence of insulin and/or in serum-free media. These data suggest that chronic exposure to glucocorticoids is necessary for the full steroidogenic activity of ovine adrenocortical cells. Moreover, they indicate that glucocorticoids exert their effect at least at two different levels in the cell: (i) on the adenylate cyclase system and (ii) at step(s) beyond cAMP but before pregnenolone formation.     

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.     

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.     

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)     

Concerted metabolism of steroid hormones produced by cocultured ovarian cell types

Ovaries of immature, intact rats were dispersed by collagenase-DNase treatment and cultured in serum-free medium (ovarian cell culture). The hormonal responsiveness of the ovarian cell was compared to that exhibited by pure granulosa cells isolated via needle puncturing. Surprisingly, despite the fact that the majority of the cultured cells should have been comprised of granulosa type, no follicle-stimulating hormone-inducible progesterone or 20 alpha-OH-progesterone (20 alpha-OH-P) could be detected by radioimmunoassay, as typically occurs in cultures of pure granulosa cells. Therefore, in order to unravel the cause for the different responsiveness between the granulosa and the ovarian cell, we applied thin-layer chromatography analysis to follow the metabolic fate of added radioactive pregnenolone to intact monolayers in culture. Such TLC analysis revealed that, after priming with follicle-stimulating hormone, added [3H]pregnenolone was converted to progesterone which was rapidly reduced and finally accumulated as 5 alpha-pregnane-3 alpha,20 alpha-diol(pregnanediol). In addition to pregnanediol, a second class of steroid hormones accumulated in the coculture medium, namely androsterone and 5 alpha-pregnane-3 alpha,17 alpha,20 alpha-triol (pregnanetriol). These latter two were specific products of the ovarian coculture, indicating the presence of theca-interstitial cells bearing 17 alpha-hydroxylase and 17,20-lyase activities. Pregnanediol, rather than progesterone, was found to be the progestin precursor for androgen formation. We thus conclude that due to exchange of steroid metabolites between the cocultured cell types, the final steroid products are different by far from the expected contributions of each individually cultured cell type. Moreover, these findings reveal an additional aspect of the "two-cell theory," suggesting a granulosa-thecal concerted metabolism of progestin steroids, rather than exchange of aromatizable androgens.     

Corticotropin-induced changes in enzymatic activities of the post-pregnenolone steroidogenic pathway in rabbit adrenocortical cells

In an attempt to delineate the effect of corticotropin (ACTH) on post-pregnenolone steroidogenesis, the activity of enzymatic systems operative in conversion of pregnenolone into glucocorticoids and androgens was studied in adrenocortical cells from control rabbits and from animals treated with ACTH for 12 days (ACTH 1-24, 200 micrograms s.c. daily). The cells from ACTH-treated rabbits exhibited an increased overall steroidogenic capacity and produced much more cortisol (P less than 0.0005) as well as other 17-hydroxylated steroids as a result of increased activity of 17 alpha-hydroxylase; corticosterone generation was concomitantly reduced. The increased conversion of pregnenolone or progesterone into androgens, as a result of previous treatment with ACTH, provides additional evidence for an effect of ACTH on 17 alpha-hydroxylase activity. A stimulatory effect of ACTH on 11 beta-hydroxylase was also evidenced by these cells, since conversion of 11-deoxycortisol into cortisol was enhanced (P less than 0.005). The increased production of androgens from 17-hydroxylated precursors by cells from ACTH-treated rabbits suggests that ACTH also exerts a prolonged stimulatory effect on 17,20-lyase. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was apparently not influenced by chronic treatment with ACTH, judged from unchanged conversion of dehydroepiandrosterone into androstenedione. The activity of 11 beta-dehydrogenase was likewise unchanged in these conditions.     

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.     

Effects of insulin-like growth factor I (IGF-I) on enzymatic activity in human adrenocortical cells. Interactions with ACTH

Cells obtained from 6 adult human adrenals or adrenal fragments were cultured in serum-free synthetic medium (McCoy's) in order to study the isolated effects of IGF-I on steroidogenesis and its interactions with ACTH. After addition of peptide, changes in the activities of steroidogenic enzymes were assessed by measuring certain steroids in the spent medium. These included pregnenolone, 17-hydroxypregnenolone (17-OH-Preg), dehydroepiandrosterone (DHA), 17-hydroxyprogesterone (17-OH-P), androstenedione (AD), 11-deoxycortisol and glucocorticoids (chiefly cortisol and its immediate precursors, 11-deoxycortisol and 17-OH-P) and cortisol itself.

The steroid responses obtained with repeated doses of IGF-I (40 ng/ml ≈ 10⁻⁹ M), added at 0, 48 and 72 h, over 4 days' culture were quite different from those obtained with repeated doses of ACTH (0.25 ng/ml ≈ 10⁻¹⁰ M). All the steroids measured increased with time of culture under the influence of ACTH and, apart from pregnenolone which peaked, tended to reach a plateau. With IGF-I, by contrast, DHA, AD, 11-deoxycortisol and glucocorticoid production increased initially, then decreased progressively, whereas pregnenolone, 17-OH-Preg and 17-OH-P production was either absent or negative.

Cumulative steroid production over 4 days reached similar levels in response to a single dose of IGF-I and/or ACTH, with two major exceptions: pregnenolone dropped significantly with IGF-I [46% ± 6 (SEM) as opposed to 93% ± 11 with ACTH, P < 0.005, N = 5], as did 17-OH-P (48% ± 11 vs 113% ± 8 with ACTH, P < 0.001, N = 6). Increased formation of down-stream metabolites (DHA, AD, 11-deoxycortisol and glucocorticoids) would suggest that IGF-I induced stimulation of the 17-, 21- and 11β-hydroxylases.

The responses to ACTH stimulation of cells which 4 days previously had been pre-treated with an initial and single dose of IGF-I and/or ACTH emphasized the impact of IGF-I on the 3-hydroxylation steps in cortisol biosynthesis. Compared with ACTH pre-treatment, the effects of which faded in the long term, pre-treatment with IGF-I resulted in a significantly increased steroidogenic response (P between < 0.05 and < 0.01). With the single exception of pregnenolone (43% ± 4.7), production of all the metabolites was amplified: 17-OH-Preg: 348% ± 88; DHA: 643% ± 127; 17-OH-P: 193% ± 36; AD: 725% ± 200; 11-deoxycortisol: 573% ± 110; cortisol: 1000%.

Our findings strongly suggest that IGF-I plays a major rôle in the regulation of steroidogenesis by promoting and maintaining enzymatic activity (17, 21- and 11β-hydroxylases) via which the function of ACTH is achieved, viz., biosynthesis of cortisol.     

Sensitivity of the fetal rat pituitary-adrenal system to corticotropin-releasing factor in organ culture.

Six groups of adrenal glands from 17-day fetal rats were explanted to organ culture for 2 days. In one group, adrenal gland was cultured alone, and in the remaining five groups adrenal gland was cultured with pituitaries from fetuses ranging in age from 14 to 18 days. In each of the groups, half of the cultures had corticotropin-releasing factor (CRF) added to the medium. A histometric parameter utilized the size of adrenocortical cells as an indicator of sensitivity of the pituitary-adrenal system. When 17-day adrenal gland was cultured alone, addition of CRF did not cause any enlargement of cortical cells. When the adrenal gland was cultured with two 14-day pituitaries, cortical cells were enlarged. Addition of CRF to this culture induced no further change. With two 15-day pituitaries in the presence of CRF, cortical cells were slightly larger than those in the absence of CRF. With 16- to 18-day pituitaries, a marked hypertrophy of cortical cells was induced, and the addition of CRF caused further acceleration in their enlargement. These results suggest that, in organ culture, 14-day pituitary can release some adrenocorticotropic hormone (ACTH) with or without additional CRF. Older pituitaries (16- to 18-day) can apparently release an amount of ACTH in the presence of CRF that is greater than their own spontaneous ACTH secretion.     

Effect of cortisol on testosterone production by immature pig Leydig cells.

The direct effects of hydrocortisone (HS) and adrenocorticotropin (ACTH) on testicular testosterone production were studied in purified immature pig Leydig cells in vitro. Leydig cells were obtained from 3- to 4-week-old piglet testes by enzymatical dispersion followed by discontinuous Percoll gradient centrifugation. Leydig cells were treated with HS and ACTH in the absence or presence of luteinizing hormone (LH) after 12 h of incubation. Media were collected 48 h later for testosterone and cyclic adenosine 3',5'-monophosphate (cAMP) measurement. Treatment of Leydig cells with increasing concentrations (0.001-10.0 micrograms/ml) of HS for 48 h resulted in a dose-dependent increase in basal and LH-stimulated testosterone production. Increasing duration (6-72 h) of treatment with HS (100 ng/ml) led to a time-dependent increase in basal and LH-stimulated testosterone production, achieving statistical significance by 48 and 24 h, respectively. HS increased LH-stimulated cAMP production. HS also increased testosterone production induced by (Bu)2 cAMP. Forskolin stimulated testosterone production to an extent comparable to that attained with LH, and HS augmented forskolin-stimulated testosterone production. HS enhanced the conversion of exogenous 17 alpha-hydroxyprogesterone to testosterone, but did not affect the conversion of pregnenolone and progesterone to testosterone, suggesting a specific stimulation of 17,20-desmolase. Porcine ACTH had no influence on basal and LH-stimulated testosterone production. These results suggest that HS directly stimulates immature pig Leydig cell steroidogenesis, at least in part via an enhancement of the generation of cAMP, leading to an increase in the activity of 17,20-desmolase.     

Adrenodoxin biosynthesis by bovine adrenal cells in monolayer culture. Induction by adrenocorticotropin

The long term effect of adrenocorticotropin (ACTH) on the synthesis of adrenodoxin in bovine adrenocortical cells was investigated. Primary, confluent monolayer cultures of adult bovine adrenocortical cells were incubated in the presence or absence of ACTH (10(-6) M) for periods up to 72 h. The amount of adrenodoxin precursor synthesized in a cell-free translation system programmed with RNA isolated from ACTH-treated cells increased to approximately 3 times the control level by 36 h. Similarly, ACTH increased the rate of incorporation of [35S]methionine into mature adrenodoxin in radiolabeled adrenocortical cells, an effect that was maximal 36 h after initiation of ACTH treatment. At longer times (48-72 h), the stimulatory effect of ACTH was not maintained, and adrenodoxin synthesis in both radiolabeled cells and cell-free translation systems declined to control levels. The content of adrenodoxin in cells treated with ACTH for 36 h, as measured by electron paramagnetic resonance spectroscopy, was approximately twice that in control cells. The results indicate that ACTH induces the synthesis of adrenodoxin in bovine adrenocortical cells. Based on the present results as well as those previously reported with respect to the induction of cholesterol side chain cleavage cytochrome P-450 by ACTH (DuBois, R. N., Simpson, E. R., Kramer, R. E., and Waterman, M. R. (1981) J. Biol. Chem. 256, 7000-7005), it is proposed that the synthesis of the mitochondrial components of the adrenocortical steroid hydroxylase system is controlled by ACTH in a coordinate fashion.     

Steroid metabolism by purified adult rat leydig cells in primary culture

To characterize Leydig cell steroidogensis, we examined the metabolism of (³H)pregnenolone (3β-hydroxy-5-pregnen-20-one) to androgens in the presence and absence of human chorionic gonadotropin (hCG) as a function of culture duration. Approximately 20–30% of the (³H)pregnenolone was converted to testosterone (17_β-hydroxy-4-androsten-3-one) by purified Leydig cells at 0, 3 and 5 days (d) of culture. Androstenedione (4-androstene-3,17-dione) and dihydrotestosterone (17_β-hydroxy-5_α-androstan-3-one) were also produced while on day 5 of culture, significant amounts of progesterone (4-pregnene-3, 20-dione) were isolated. The Δ⁵ intermediates, 17-hydroxypregnenolone (3β, 17-dihydroxy-5-pregnen-20-one) and dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one), accounted for less than 1% of substrate conversion, indicating a clear preference for Leydig cells to metabolize (³H)pregnenolone via the Δ⁴ pathway. On day 0 of culture, unidentified metabolites consisted of predominately polar steroids while on day 5 of culture, the unidentified metabolites consisted of predominately nonpolar steroids. In the presence of hCG, (³H)pregnenolone metabolism did not differ from basal on day 0 or 3 of culture. HCG increased the conversion of pregnenolone to progesterone and 17-hydroxyprogesterone (17-hydroxy-4-pregnene-3, 20-dione) on 5d. This suggests that Leydig cells cultured for 5d have decreased C_17–20 desmolase activity or that hCG acutely stimulates 3β-hydroxysteroid dehydrogenase and Δ⁴-Δ⁵ isomerase activities.     

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.     

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.     

A granulosa cell differentiation-stage-specific cytotoxic activity in bovine and rat sera

Heat-inactivated serum is cytotoxic to granulosa cells from preantral follicles but not to cells from preovulatory follicles. A dominant feature of the granulosa cells of preovulatory follicles is the presence of luteinizing hormone (LH) receptors on the surface of the cells. In the present study, we have examined the relationship between the process of LH receptor induction and the acquisition of serum tolerance in granulosa cells in vitro. Granulosa cells from the ovaries of immature rats primed with diethylstilbestrol (DES) were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium containing 30 ng of ovine follicle-stimulating hormone (oFSH; NIH-15). At either 0, 24, or 48 h of culture, heat-inactivated fetal bovine serum (FBS) was added (10% by volume) to separate groups of culture tubes. All cells were cultured for a total of 72 h, at which time the cultures were assessed for LH receptor (specific 125I-human chorionic gonadotropin [hCG] binding) and DNA content. LH receptors were induced in all FSH-containing serum-free cultures by 48 h. Receptors were not induced, however, when serum was added after either 0 or 24 h of culture. Furthermore, serum addition at these times resulted in a cell loss (assessed by DNA) of 40-60%. Serum addition at 48 h to FSH-containing cultures resulted in an inability to detect LH receptors at 72 h and with no significant effect on the culture DNA content. Addition of a protein extract of FBS at the initiation of cell culture prevented FSH-stimulated LH receptor induction and was cytotoxic. A lipid extract of FSH did not interfere with receptor induction and was not cytotoxic.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A potential role for adrenocorticotropin in regulating human B lymphocyte functions

Adrenocorticotropin (ACTH) was found to enhance the growth and differentiation of human B lymphocytes. By using highly purified preparations of human tonsillar B cells, the effects of ACTH on the growth and differentiation of in vitro activated B cells were examined. Optimal concentrations of ACTH were found to increase the proliferation of activated B cells by twofold to threefold when ACTH was present in culture with either a B cell growth factor or recombinant interleukin 2 (IL 2). ACTH had essentially no effects when added to cultures of activated B cells in the absence of the growth factor. Additionally, when ACTH was added in conjunction with an optimal concentration of either a B cell differentiation factor or IL 2 to cultures of activated B cells, the combination of ACTH and factor enhanced Ig secretion by twofold compared with the factor alone. In the absence of the differentiative signal, ACTH had minimal effects on Ig production. Only the first 24 amino acid fragments of ACTH were required to enhance B cell growth and differentiation when combined with the appropriate, more classical signals. Thus, ACTH may have a physiologic role in regulating human B cell function.