Steroidogenesis in dispersed cells of human fetal adrenal

Steroidogenesis in dispersed fetal zone cells of midtrimester human fetal adrenal was stimulated acutely by ACTH. Polypeptide hormones such as hCG, alpha MSH, ovine PRL, and LH did not produce a similar stimulation of steroidogenesis. The principal steroid products of ACTH-stimulated fetal adrenal cells were dehydroisoandrosterone sulfate, pregnenolone, pregnenolone sulfate, and 17 alpha-hydroxypregnenolone. Only minimal production of the delta 4-3-ketosteroids, cortisol, corticosterone, and progesterone, was observed. Cyanoketone (2 alpha-cyano-4,4,17 alpha-trimethyl-17 beta-hydroxyandrost-5-en-3-one; an inhibitor of 3 beta-hydroxysteroid dehydrogenase activity) treatment of the cells caused only a minor increase in 3 beta-hydroxysteroid formation, confirming that 3 beta-hydroxysteroid formation is the principal steroidogenic fate of cholesterol in these cells. SU-10603 [7-chloro-3,4-dihydro-2-(3-pyridyl)naphthalen-1-(2H)one; a steroid 17 alpha-hydroxylase inhibitor] treatment of the cells caused a marked accumulation of pregnenolone sulfate, indicating that the C-19 steroids are produced from C-21 steroids in this tissue and possibly that dehydroisoandrosterone sulfate is synthesized directly from pregnenolone sulfate. ACTH-stimulated pregnenolone synthesis was inhibited by AY-9944 [trans-1,4-bis-(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride; an inhibitor of cholesterol biosynthesis]. Thus, cholesterol synthesized de novo was the likely steroidogenic precursor in the acute hormonally stimulated fetal adrenal cells.     

The control of steroidogenesis by human fetal adrenal cells in tissue culture. IV. The effect of exposure to placental steroids

The effect upon steroidogenesis of adding various steroids produced by the placenta was studied in short term cultures of human fetal adrenal cells. The addition of high concentrations (10(3) ng/ml) of estrone or estriol inhibited the production of cortisol, but only the former elicited a parallel increase in dehydroepiandrosterone (DHA) production. Estradiol was effective in inhibiting delta-4-3-ketosteroid production at concentrations of 10-100 ng/ml, levels which approach those found in the fetal circulation, while DHA production was increased at concentrations of 1 microgram/ml. The addition of progesterone (4 microgram/ml) to the medium caused increased production of cortisol and corticosterone, but had no effect on DHA production. Pregnenolone (4 microgram/ml) increased the basal production of DHA and slightly impaired both basal and ACTH-stimulated aldosterone production, but had no effect on cortisol production. The data demonstrate that the many fetal and placental factors which have been studied to date, only ACTH and estrogens can interact to produce the characteristic fetal pattern of steroidogenesis. Preliminary studies indicate that this effect-stimulated aldosterone production, but had no effect on cortisol production. The data demonstrate that the many fetal and placental factors which have been studied to date, only ACTH and estrogens can interact to produce the characteristic fetal pattern of steroidogenesis. Preliminary studies indicate that this effect-stimulated aldosterone production, but had no effect on cortisol production. The data demonstrate that the many fetal and placental factors which have been studied to date, only ACTH and estrogens can interact to produce the characteristic fetal pattern of steroidogenesis. Preliminary studies indicate that this effect of estrogen is not influenced by other peptide hormones such as hCG, human prl, beta-lipotropin, corticotropin-like intermediate lobe peptide, or beta-endorphin. A revised model of the fetoplacental steroidogenic unit is presented which may explain both normal and fetal hyperplasia and postnatal involution of the adrenal cortex and the variations from this pattern seen in apituitary children.     

In vitro spontaneous and adrenocorticotropin-dependent maturation of the steroidogenic pathway of ovine fetal adrenal cells

Adrenal cells from intact or hypophysectomized fetuses and newborn lambs cultured for 6 days under several conditions were tested for their ability to metabolize [14C]pregnenolone (P5) (5 X 10(-5) M) during a 2-h period. Also, their ability to produce P5 was investigated. At the beginning of the culture, the metabolism of P5 by adrenal cells from hypophysectomized fetuses was lower than that of cells from intact fetuses which in turn was lower than that of cells from newborns. After 6 days in culture in the absence of ACTH-(1-24), the metabolism of P5 by cells from both intact and hypophysectomized fetuses increase, whereas that of cells from newborns slightly decreases. Continuous ACTH-(1-24) (10(-8) M) treatment produced a striking increase in the capacity to metabolize P5 by cells from both intact and hypophysectomized fetuses and the maintainance of this capacity of cells from newborns was demonstrated. At the beginning of the culture the main product of [14C]P5 was corticosterone with cells from intact and hypophysectomized fetuses, and cortisol with newborn cells. After 6 days in culture in ACTH-free medium more than 50% of P5 added appeared as progesterone in all cases. Addition of ACTH-(1-24) to the culture medium induced within 5 days, a striking increase in the activation of 3 beta-hydroxysteroid dehydrogenase-isomerase, 17 alpha-, 21, and 11 beta-hydroxylases of adrenal cells from fetuses. However, the activity of 17 alpha-hydroxylase in adrenal cells from hypophysectomized fetuses was lower than that of cells from intact fetuses. ACTH-(1-24) treatment maintained the enzymatic activity of cultured newborn adrenal cells, except for 11 beta-hydroxylase which declined to about 50% of its activity on day 0. The capacity of fetal adrenal cells to produce P5 after acute ACTH-(1-24) stimulation increased 30-fold when cells were cultured in the absence of ACTH and up to 110-fold when cells were treated with ACTH-(1-24) from day 1 on. These results, together with those reported previously, show that the spontaneous in vitro maturation of fetal adrenal cell steroidogenic pathway is related to an increased activity of the 3 beta-hydroxylase dehydrogenase-isomerase and an enhanced capacity to produce P5. Both steps were stimulated further by ACTH. In addition, the hormone is an absolute requirement for the development and/or the maintenance of 17 alpha-hydroxylase activity.     

Biogenesis of corticosteroids in monolayer cultures of human foetal adrenal cells.

Human foetal adrenal cells were grown in monolayer culture and their steroidogenic capacity observed for up to a month. The cells produced a complex array of steroids and some of their ester sulphates from endogenous as well as from [14C] and [3H] precursors. ACTH stimulated corticoidogenesis, particularly cortisol secretion, and markedly enhanced the incorporation of progesterone and pregnenolone into cortisol. Following incubation with the same precursors, large amounts of radioactivity remained water soluble. From the butanol extractable material of this fraction, dehydroepiandrosterone sulphate was characterized as the main metabolite of pregnenolone and corticosterone and 11-deoxycorticosterone sulphates as the main metabolites of progesterone. With time in culture there was a decrease in steroidogenesis as well as a steady decline in responsiveness to ACTH, mainly manifested by cortisol secretion. The medium from homologous foetal pituitary cultures stimulated cortisol production by the human adrenal cell monolayer.     

The role of calcium in steroidogenesis in fetal zone cells of the human fetal adrenal gland

The human fetal adrenal gland is composed primarily of fetal zone (FZ) cells, which have a high rate of steroidogenesis. The purpose of this study was to examine the role of calcium in the regulation of steroidogenesis by FZ cells. Dispersed FZ cells were incubated in Krebs-Ringers medium at 37 C for 3 h in the presence of ACTH, (Bu)2cAMP, or forskolin in addition to various drugs. The medium contents of dehydroepiandrosterone sulfate (DS), cortisol (F), and cAMP were quantified by RIA. After the addition of ACTH (10(-10)-10(-5) M), DS and cAMP secretion increased. The addition of EGTA to the medium inhibited ACTH- and forskolin-stimulated DS, F, and cAMP secretion by 50% as well as (Bu)2cAMP-stimulated steroidogenesis. The addition of calcium (10(-5)-10(-2) M) had only a slight effect on the secretion of DS or F in the absence of ACTH or (Bu)2cAMP. In the presence of ACTH and (Bu)2cAMP, however, increasing amounts of calcium resulted in a 2- to 3-fold increase in the rates of DS and F secretion. The addition of either A23187, a calcium ionophore, or verapamil, a calcium channel blocker, inhibited ACTH-stimulated DS and F secretion by 90%. The rate of cAMP formation was greater after ACTH plus verapamil treatment than after ACTH treatment alone, whereas A23187 inhibited ACTH-stimulated cAMP secretion to basal levels. Both A23187 and verapamil inhibited ACTH- and cAMP-stimulated pregnenolone secretion. The metabolism of 22R-hydroxycholesterol to pregnenolone was inhibited by A23187 and verapamil. In conclusion, our results suggest that extracellular calcium is important for activation of the human adrenal FZ cell adenylate cyclase system, while intracellular calcium plays a multifaceted role in controlling steroid production.     

Regulation of steroid secretion by adrenal tissue of a human anencephalic fetus

ACTH-stimulated adrenal tissue of a human anencephalic fetus, when maintained in organ culture, secreted appreciable quantities of cortisol but little dehydroisoandrosterone sulfate or pregnenolone sulfate. In the absence of ACTH, cortisol secretion was severely attenuated. Arginine vasopressin or alpha MSH, when added to the culture medium, did not stimulate steroid secretion. When whole human serum was present in the culture medium bathing the adrenal tissue of the anencephalic fetus, the rate of cortisol secretion was similar to that attained when lipoprotein-poor serum was in the medium. Based on these findings, it is concluded that in the presence of ACTH, the adrenals of the anencephalic fetus secrete principally cortisol, and the failure of dehydroisoandrosterone sulfate and pregnenolone sulfate secretion is due to the absence of the fetal zone. The lack of stimulation of cortisol secretion by lipoprotein is probably due to a reduced number of low density lipoprotein receptors resulting from diminished ACTH stimulation before birth.     

A virilizing adrenocortical tumor in a female infant: in vivo and in vitro biochemical characteristics.

Normal adrenal and adrenal tumor cells from a female infant with a virilizing adrenal tumor were grown in tissue culture as monolayers for a period of 7 weeks. Half of the cultures were exposed to ACTH (0.1 U/ml). The cells grew well and continued to produce steroid hormones over the entire period in culture. Production of steroid hormones was measured by RIA of individual steroids in the culture medium. Unstimulated normal and tumor cells produced equivalent amounts of cortisol, 11 beta, 18, 21-trihydroxy-4-pregnene-3,20-dione, and 18,21-dihydroxy-4-pregnene-3,20-dione, but tumor cells produced lesser amounts of dehydroepiandrosterone (DHA), androstenedione, testosterone, and progesterone. Normal cells exposed to ACTH showed an increase in all steroids measured, whereas ACTH-exposed tumor cells showed an increase principally in DHA consistent with a deficiency in 3 beta-hydroxysteroid dehydrogenase activity. Circulating levels of DHA, androstenedione, and testosterone were elevated in the patient before removal of the adrenal tumor. The production of androgens by tumor cells in vitro resembled the pattern of circulating steroids in vivo. These studies demonstrate that tissue culture of human adrenal cells provides a means both to determine their biochemical characteristics and to investigate their responses to exogenous hormones.     

The role of serum lipoproteins in steroidogenesis by the human fetal adrenal cortex.

In the present investigation it was found that human fetal adrenal tissue maintained in organ culture secreted appreciable quantities of dehydroisoandrosterone sulfate (DS) and cortisol. Pregnenolone was also secreted in significant amounts, principally as the sulfate ester. The highest rate of secretion of these steroids by fetal adrenal tissue occurred when both ACTH and whole human serum were present in the culture medium. In the absence of ACTH, steroid secretion was low. When whole serum was replaced by lipoprotein-poor serum, the steroidogenic response to ACTH was markedly attenuated but not abolished. On the basis of these findings, it is concluded (1) that the human fetal adrenal can synthesize steroid hormones de novo from cholesterol, (2) that ACTH is an important stimulant of steroidogenesis by the human fetal adrenal, and (3) that plasma lipoproteins are a major source of the cholesterol utilized by the human fetal adrenal for steroidogenesis. Hence, it is likely that factors which regulate the production of fetal plasma lipoproteins are important determinants of fetal adrenal steroidogenic activity.     

Ovine fetal adrenal synthesis of cortisol: regulation by adrenocorticotropin, angiotensin II and transforming growth factor-beta.

An increase in cortisol production by the fetal adrenal cortex is an important prepartum event. The increase in ovine fetal adrenal synthesis of cortisol appears to rely in part on the ACTH induction of 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha) which occurs before parturition. In the present study we examined the effect of ACTH treatment on cortisol production and P-450(17) alpha expression using primary cultures of ovine fetal adrenal cells. In addition, we examined the effects of angiotensin II (A-II) and transforming growth factor-beta (TGF beta) on ACTH-treated cells. We have demonstrated previously that these factors modulate P-450(17 alpha) levels in adult ovine and bovine adrenal cells. Fetal ovine adrenal cells were isolated from 126- to 130-day fetuses (term = 144 +/- 3 days) and placed in monolayer cell culture. After 1 day in culture the cells were treated with ACTH (10 nM) with or without A-II (0.1-100 nM) or TGF beta (1-100 pM). Medium content of cortisol was low under basal conditions, whereas ACTH-stimulated cortisol production by 10- to 100-fold. A-II and TGF beta inhibited ACTH-induced cortisol production by 70-90%. In addition, 3 days of treatment with ACTH caused a greater than 10-fold induction of P-45017 alpha enzyme activity in fetal adrenal cells. A-II and TGF beta inhibited the ability of ACTH to induce P-45017 alpha activity by at least 75%. Using an antibody to P-45017 and immunoblotting techniques the effects of ACTH, A-II, and TGF beta on enzyme activity were observed to correspond to cellular levels of P-45017 alpha protein. The inhibitory effects of TGF beta and A-II could not be overcome by the cAMP analog (Bu)2cAMP. Interestingly, the expression of the enzyme 3 beta-hydroxysteroid dehydrogenase was much less sensitive to inhibition by A-II or TGF beta. The ability of A-II and TGF beta to suppress P-45017 alpha expression could play a role in determining the pathway of steroidogenesis and specifically the amount of cortisol produced by fetal adrenocortical cells in vivo.     

The control of steroidogenesis by human fetal adrenal cells in tissue culture. III. The effects of various hormonal peptides

The effects upon production of cortisol and dehydroepiandrosterone (DHA) by human fetal adrenal cells in tissue culture were studied using commercial hCG (0.5 and 5 IU/ml), purified hCG (0.7-6.7 IU/ml), the alpha-subunit of hCG (200 and 1000 ng/ml), human GH (50 and 200 ng/ml), human PRL (0.1-100 ng/ml), alpha-MSH (0.1-10 ng/ml), corticotropin-like intermediate lobe peptide (200 ng/ml), human beta-lipotropin (0.1 and 0.2 ng/ml), and beta-endorphin (100 ng/ml). Although each peptide was added to the culture medium in a concentration either similar to that observed in the fetal circulation or (where such information was not available) in amounts several times greater than those effective for ACTH in this system, none demonstrated any significant stimulation of steroid production. In particular, repeated studies with hCG showed that this hormone had no stimulating effect upon DHA production, neither in cultures of whole adrenals nor in cultures of separated fetal zone and definitive zone cells. Furthermore, none of these peptides showed a synergistic effect upon DHA production when they were added to cultures together with concentrations of alpha-ACTH-(1-24) (10(2)-10(3) pg/ml) previously demonstrated to represent the middle of the dose-response curve. Indeed, the only significant interactions with alpha-ACTH-(1-24) observed in these studies were a slight reduction in cortisol production produced by corticotropin-like intermediate lobe peptide and apparent inhibition of DHA production by beta-lipotropin and GH. The data do not lend credence to the suggestion that any of these peptides plays an important role in vivo in stimulating fetal adrenal steroidogenesis.     

The control of steroidogenesis by human fetal adrenal cells in tissue culture. II. Comparison of morphology and steroid production in cells of the fetal and definitive zones

Preparations of dispersed human fetal adrenal cells from the inner third of the gland and from the subcapsular area were maintained in culture, and their ultrastructure and steroid production were studied. The former type of preparation contained only fetal zone cells, while the latter contained definitive zone cells together with varying numbers of fetal zone cells. Both types could be cultured with equal ease, but during short term culture, fetal and definitive zone cells became morphologically indistinguishable. The patterns of steroid production and, in particular, the relative production of delta 4,3-ketosteroids and delta 5,3 beta-hydroxysteroids were similar in both preparations, as were their dose-response relationships during incubation with alpha ACTH-(1-24). Although considerable variability in total steroid production was observed between cells from different adrenal glands, in no specimen was any evidence for functional zonation of the fetal adrenal cortex observed in vitro. The results suggest that the apparently unique histological appearance and function of the fetal adrenal cortex may only reflect intense stimulation by ACTH secondary to the combined influences of a rapid cortisol MCR and of some inhibitor of fetal adrenal 3 beta-hydroxysteroid dehydrogenase activity.     

Steroid formation and differentiation of cortical cells in tissue culture of human fetal adrenals in the presence and absence of ACTH.

Steroid secretion and ultrastructural differentiation of human fetal adrenal cortical cells were analyzed in tissue culture with and without ACTH. The unconjugated and sulfated endogenous neutral steroids were analyzed by gas-liquid chromatography and gas chromatography-mass spectrometry. A fetal pattern of neutral steroids, including high concentrations of sulfate conjugates, was found during the first five days of the cultivation. At 6 to 11 days of cultivation, a decrease was seen in concentrations of these steroids. However, when stimulated with ACTH, an increasing amount of steroids was secreted during days 6 to 11 and their pattern was transformed into the adult type with a 30-200 times higher secretion rate of cortisol. Cortical cells capable of proliferation in the culture had the ultrastructure of the permanent zone cells of the fetal adrenal or adult zona glomerulosa type. ACTH stimulation induced a differentiation of these cells into zona fasciculata type. The results suggest that ACTH is the main hormonal regulator in the genesis of the adult human adrenal cortex and that there is a factor during fetal life which inhibits the synthesis of the 3beta-hydroxysteroid dehydrogenase system.     

Basic fibroblast growth factor expression is regulated by corticotropin in the human fetal adrenal: a model for adrenal growth regulation.

Human fetal adrenal growth after midgestation is very rapid and appears to be dependent upon pituitary adrenocorticotropin (ACTH) in vivo. We hypothesized that the regulation of fetal adrenal growth by ACTH is mediated by ACTH-stimulated local growth factor production. As we have found basic fibroblast growth factor (bFGF) to be a potent mitogen for human fetal adrenal cells in culture, we conducted studies to determine whether bFGF is synthesized by the human fetal adrenal gland and whether bFGF gene expression in primary cultures of human fetal adrenal cells is regulated by ACTH. Bioassayable bFGF-like activity was detected in extracts of whole human fetal adrenal glands and primary cultures of human fetal adrenal cells. Northern blot analysis of total RNA from whole human fetal adrenal glands revealed a characteristic 7-kilobase bFGF mRNA, indicating that the fetal adrenal bFGF bioactivity was most likely due to local synthesis. Slot blot and ribonuclease protection analysis showed that bFGF mRNA was present in very low amounts in total RNA from primary cultures of unstimulated human fetal adrenal cells but was increased 2- to 3-fold in cells exposed to 10 nM ACTH-(1-24) or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate for 24 hr. bFGF mRNA was localized to adrenocortical cells and not fibroblasts by in situ hybridization. bFGF mRNA was barely detectable in unstimulated cells, whereas it was markedly increased in cells exposed to either ACTH or 8-bromoadenosine 3',5'-cyclic monophosphate. These data support our hypothesis that the regulation of human fetal adrenal growth by ACTH at midgestation may be mediated by the stimulation of local growth factor production, and we suggest that bFGF may play a major role in this process.     

Regulation of the fetal human adrenal cortex: effects of adrenocorticotropin on growth and function of monolayer cultures of fetal and definitive zone cells

Monolayer cultures have been prepared from both definitive and fetal zones of the human fetal adrenal cortex. Cultures from each zone consist predominately of adrenocortical cells, as determined by a specific morphological retraction response to ACTH, and by ACTH-induced inhibition of DNA synthesis and cell proliferation. Cell growth was stimulated by fibroblast growth factor. ACTH stimulated steroidogenesis in cells from each zone with an ED50 of 0.4--1.0 nM and at a maximal concentration of 5 nM. Short term stimulation of less than 24 h with ACTH produced a pattern of steroid secretion that was characteristic of the zone of origin. Definitive zone cultures produced both cortisol and dehydroepiandrosterone plus its sulfate (DHA/S), with cortisol production exceeding DHA/S production. Fetal zone cultures produced more DHA/S than cortisol. 3 beta-Hydroxysteroid dehydrogenase, delta 4,5-isomerase enzyme activity was 3-fold less in fetal than in definitive zone cultures. Long term stimulation of 1--4 days with ACTH, 8-bromo-cAMP, or cholera toxin increased steroidogenic capacity in cultures from both zones. The pattern of steroid production by definitive zone cells remained constant, but cortisol production was preferentially increased in fetal zone cells. Forty-eight-hour treatment of fetal zone cells with ACTH increased 3 beta-hydroxysteroid dehydrogenase, delta 4,5-isomerase activity 5-fold. alpha-, beta-, gamma 1-, gamma 2-, and gamma 3-MSH were not effective steroidogenic agents for either zone. These studies indicate that steroidogenic agents induce in fetal zone cells steroid production characteristic of definitive and adult adrenocortical cells.     

Corticotropin-releasing hormone stimulates steroidogenesis in cultured human adrenal cells

The effects of corticotropin releasing hormone (CRH) on steroid production by cultures of human fetal adrenal cells was investigated. We found that CRH, at concentrations that have been reported to exist in human fetal serum, stimulated dehydroepiandrosterone sulfate (DS) and cortisol production by cultured fetal zone and neocortical zone cells. A dose-dependent increase in secretion of both steroids was noted, with the cortisol pathway being preferentially enhanced by CRH at high concentrations. Pretreatment of adrenal cells for 3 days made them more responsive to ACTH stimulation and such effects were dose-dependent also. Inclusion of the antagonist, alpha-helical CRH (9-41) blocked CRH-induced stimulation of DS and cortisol over a broad dose range and also interfered with the augmentation of cortisol secretion noted after ACTH in CRH treated cells. CRH had no effects on adrenal cell proliferation or total cell protein. These studies are suggestive that CRH, either of systemic origin or else produced within the adrenal itself, has the potential to be a modulator of adrenal steroid production in the human.     

Effect of short dexamethasone suppression on plasma steroids in prepubertal and pubertal girls

In 40 girls aged from 2 to 14 years, subdivided into groups according to age and pubertal development, and in 6 adult female volunteers, plasma cortisol (F), pregnenolone (delta 5), dehydroepiandrosterone (DHA) progesterone (P), 17-hydroxyprogesterone (17P), androstenedione (A), testosterone (T) and estradiol (E2) were measured before and after short dexamethasone (DXM) suppression. The results confirmed the capacity of DXM to inhibit plasma steroids in all age groups, except T in 2-9 year old and P1 Tanner's stage girls. The percentage suppression of each given steroid was constant over the age groups from 6-9 years to P4-5 Tanner's stage, while lower suppression was found in 17P, P and DHA in 2-5 year old girls and in 17P, DHA and E2 in adult women. These results emphasize the fundamental role of ACTH as the overall stimulating factor of adrenal steroidogenesis but do not negate the possibility of another factor responsible for the development of the adrenal androgen secreting cells throughout prepuberty and puberty.     

Low expression of 3 beta-hydroxy-5-ene steroid dehydrogenase gene in human fetal adrenals in vivo; adrenocorticotropin and protein kinase C-dependent regulation in adrenocortical cultures.

Human fetal adrenals are very active in steroid production. They make large amounts of dehydroepiandrosterone sulfate which is further converted to estrogens in placenta. Fetal adrenals cannot make cortisol efficiently from cholesterol or pregnenolone, but they can convert progesterone to cortisol. To clarify the molecular basis of the very low activity of 3 beta-hydroxy-5-ene steroid dehydrogenase (3 beta HSD) in human fetal adrenals we studied the expression of 3 beta HSD gene in fetal adrenals in vivo and in culture conditions. Human adult adrenals, placenta and a testicular Leydig cell tumor clearly expressed 3 beta HSD gene when studied by Northern blotting, but fetal adrenals and ovaries had no detectable 3 beta HSD mRNA by this method. Polymerase chain reaction analysis of cDNA samples derived from different human tissues revealed 3 beta HSD gene expression in placenta, adult adrenal and adult ovarian granulosa cells after 25 cycles of amplification. Fetal adrenal samples became positive only after additional amplification cycles, which verifies the very low expression of 3 beta HSD gene in fetal adrenals. In cell culture conditions both ACTH and a protein kinase C regulator 12-O-tetradecanoyl phorbol-13-acetate induced 3 beta HSD gene expression. We conclude: 1) the very low activity of 3 beta HSD in human fetal adrenals is due to the low expression of this gene; 2) both cAMP and protein kinase C-dependent mechanisms regulate 3 beta HSD gene expression in adrenocortical cells.     

Intranasal administration of adrenocorticotropin-(1-24) stimulates adrenocortical hormone secretion

To determine the efficiency of transmucosal absorption of ACTH, we measured serum cortisol, aldosterone, dehydroepiandrosterone (DHEA), and DHEA sulfate (DHEA-S) levels after intranasal (in) vs. iv administration of ACTH-(1-24) (250 microg) in 12 healthy adult men (mean age, 24.3 +/- 3.2 yr; range, 21-31 yr), who had received no prior medication and had no symptoms of rhinitis. Blood was collected at 0, 30, 60, 120, and 180 min after administration of ACTH-(1-24), and the levels of adrenocortical steroids were measured by specific RIAs. There were no side-effects associated with in or iv ACTH administration. After in administration, serum cortisol and aldosterone increased rapidly by 224.7 +/- 39.2% and 147.2 +/- 50.5%, respectively, peaking 30 min after ACTH-(1-24) administration, and decreasing to basal levels within 120 min. These increases in serum cortisol and aldosterone were lower than those obtained after iv administration. Thirty minutes after in or iv administration of ACTH-(1-24), DHEA increased by 49.1 +/- 27.2% and 81.6 +/- 17.1%, respectively, and remained elevated for 180 min. Serum DHEA-S levels did not change after in administration of ACTH-(1-24) and increased only slightly after iv injection. Adrenocortical steroid levels did not increase after in administration of saline. These data demonstrate that adrenocortical steroids are stimulated by in administration of ACTH-(1-24). We suggest that intranasal administration of ACTH offers both a diagnostic approach as an adrenal function test and a therapeutic approach as ACTH replacement therapy in patients with ACTH deficiency. The latter may be more physiological than glucocorticoid replacement.     

Hormonal regulation of messenger ribonucleic acids for P450scc (cholesterol side-chain cleavage enzyme) and P450c17 (17 alpha-hydroxylase/17,20-lyase) in cultured human fetal adrenal cells

ACTH has acute and long term effects on adrenal steroidogenesis by week 14 of fetal life. We used human fetal adrenal cells to investigate the long term effect of physiological doses of ACTH on mRNAs for P450scc (the cholesterol side-chain cleavage enzyme) and P450c17 (17 alpha-hydroxylase/17,20-lyase). Monolayer cultures of 18- to 24-week gestation fetal zone adrenal cells were maintained in the presence and absence of 10(-9) or 10(-8) M ACTH for up to 12 days. As assessed by RNA dot blots probed with cloned homologous human cDNAs, ACTH increased P450scc and P450c17 mRNAs 4- and 9-fold, respectively, over control values on day 7 of culture. ACTH-mediated stimulation was slightly less on day 12 of culture. The ACTH-mediated accumulation of those mRNAs were time dependent. When cells were exposed to a single 10(-8)-M dose of ACTH, the amount of P450scc and P450c17 mRNA was increased by 24 h, reaching a maximum at 48 h and diminishing by 72 h. When cells were maintained in 10(-8) M ACTH continuously, mRNA for both enzymes accumulated in a similar pattern, reaching a peak at 48 h but remaining at nearly maximal values thereafter, up to 96 h. Dibutyryl cAMP (10(-3) M) mimicked these stimulatory actions of ACTH, although its effect was greater at 24 h and more stable up to 96 h. Angiotensin II (1-100 ng/mL) and hCG (1-100 ng/mL) had no effect on accumulation of P450scc and P450c17 mRNAs. The production of both dehydroepiandrosterone sulfate and cortisol also was stimulated by ACTH, suggesting that the increased mRNAs were translated into active enzymes. These results indicate that ACTH induces human fetal adrenal cells to accumulate mRNAs for both P450scc and P450c17; this effect of ACTH is probably mediated by cAMP. Chronic 96-h stimulation of human fetal adrenal cells did not diminish their responsiveness to ACTH. Together with our earlier studies of the human fetal adrenal, these data indicate that fetal adrenal tissue does not exhibit the desensitization to trophic hormone stimulation characteristic of adult tissue.     

Dissociation of cortisol and adrenal androgen secretion in patients with secondary adrenal insufficiency.

Plasma cortisol, dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), and androstenedione (delta4-A) were measured by RIA during ACTH infusion in preadrenarchal children with constitutional short stature, normal adults, and patients with secondary adrenal insufficiency resulting from hypothalamic-pituitary disease or corticosteroid therapy. The plasma levels of all four steroids were decreased in patients with secondary adrenal insufficiency compared to normal adults, but the decrease in DHA and DHAS was considerably greater than that in cortisol and delta4-A, resulting in significant decreases in the plasma ratios of DHA to cortisol, DHAS to cortisol, DHA to delta4-A, and DHAS to delta4-A (P less than 0.00001). The decreased DHA and DHAS responses to ACTH persisted in one glucocorticoid-treated patient after glucocorticoid therapy was terminated and the cortisol response to ACTH had normalized. The data suggest that adrenal atrophy due to hypothalamic-pituitary disease or corticosteroid therapy is associated with a greater impairment in the secretion of the delta5 adrenal androgens DHA and DHAS than in the secretion of cortisol and delta4-A, and that the capacity to secrete cortisol and delta4-A recovers more rapidly than the capacity to secrete the delta5 adrenal androgens when corticosteroid therapy is withdrawn.