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.     

Glucocorticoid induction of the maturation of ovine fetal adrenocortical cells

The aim of the present study was to assess whether glucocorticoids could be directly involved in the maturation of adrenocortical cells from 120-138 days old ovine fetuses. The cAMP response to ACTH1-24 of cells cultured for 24 hours in the presence of ACTH1-24 was 2 fold higher than that of control cells. However, the response of cells cultured in the presence of ACTH1-24 plus metyrapone or aminoglutethimide was lower than that of cells cultured in the presence of ACTH1-24 alone. Cells cultured for 48 hours in the presence of dexamethasone or cortisol released more cAMP than control cells when stimulated by ACTH1-24, but not in response to forskolin. However corticosteroid production stimulated by ACTH1-24, forskolin or dibutyryl cAMP was enhanced by dexamethasone treatment. These results suggest that glucocorticoids can affect the maturation of ovine fetal adrenocortical cells by an auto and/or a paracrine process, and that this effect is exerted, at least, at two different levels in the cell.     

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.     

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.     

Loss of sensitivity to ACTH of adrenocortical cells isolated from maturing domestic fowl

Maturation of domestic fowl corticosteroidogenesis was evaluated using purified adrenocortical cells. Basal corticosterone production decreased steadily from 2 days to 26 weeks after hatching. However, maximally stimulated corticosterone production was not changed. In contrast, the half-maximal steroidogenic concentrations (ED50 values or effective doses for 50% maximal effect) of ACTH analogs increased approximately 40 times by 26 weeks, but the ED50 values of 8-bromo-cyclic AMP and pregnenolone were not changed. This suggests that adrenocortical cell sensitivity to ACTH decreases with maturation of the domestic fowl.     

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.     

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.     

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.     

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.     

Interrelationship between RU38486 and the P450 activities in rat liver

Microsomal P450 monooxygenases contribute actively to the biotransformation of the antiglucocorticoid RU38486, an 11 beta-substituted nor-steroid. Pretreatment of adult rats by inducers of specific forms, belonging to different P450 subfamilies, affects the ability of liver microsomes to metabolize RU38486. Phenobarbital and pregnenolone 16 alpha-carbonitrile increase the metabolic activity of liver microsomes whereas methylcholanthrene decreases their capacity to oxidize the steroid. Thus P450 forms IIIA, IIB1,2 and IIC7 are good candidates to be involved in the degradation of this peculiar molecule. Our study has been completed by investigating whether RU38486 would influence the P450 spectrum. Whereas the treatment of rats with either a glucocorticoid (cortisol, dexamethasone) or an antiglucocorticoid (pregnenolone 16 alpha-carbonitrile) has been shown to induce the P450 activity by increasing the hepatic concentration of form IIIA, we observed a slight decrease of the P450 activity by treating the animals with RU38486. Moreover RU38486 was able to antagonize the P450 induction by the other steroids as well as it inhibits the synthesis of various liver enzymes induced by glucocorticoids (for instance tyrosine aminotransferase). These findings may be important for the therapeutic use of RU38486 since its inhibitory effect on P450 activity may be at the origin of drug interactions by modifying the endogenous hormonal status.     

Genetic-dependent alterations in adrenal stress response and adrenocortical cell function of the domestic fowl (Gallus domesticus)

Strain-dependent differences in adrenocortical function were investigated in male White Leghorn domestic fowl. Adrenocortical function of Cornell K strain (K) (genetic control), autosomal dwarf strain (ADW), and sex-linked recessive dwarf strain (SLD) was evaluated in vivo by measuring plasma corticosterone and in vitro by measuring acute (2 hr) corticosterone production by enriched adrenocortical cell populations. Regardless of strain, there was an age-dependent decrease (27-57%) in plasma corticosterone from 1 to 12 weeks of age. However, there was a tendency for plasma corticosterone values of ADW and SLD to be, respectively, greater and less than that of K. In addition, at 12 weeks of age, plasma corticosterone responses of ADW and SLD to transient heat stress (50 degrees C, 30 min) were, respectively, 22.8% greater and 15.9% less than that of K. Strain differences in adrenal weight and relative adrenal weight (mg% body wt) were also apparent. At 12 weeks of age, adrenal weights of ADW and SLD were, respectively, 33 and 42% less than that of K, whereas relative adrenal weights were, respectively, 27.6% greater and 22.4% less than that of K. In addition there were strain-dependent differences in adrenocortical function at the cellular level. Although there were no consistent strain differences in basal and maximal corticosterone production by cells, there were strain differences in cellular sensitivity to ACTH and pregnenolone. On an equal cell concentration basis, the half-maximal steroidogenic concentrations (ED50 values or effective doses for 50% maximal effect) of ACTH for ADW and SLD adrenocortical cells were, respectively, 0.23 and 2.07 times the ED50 value for K cells. In addition, the ED50 value of pregnenolone for ADW cells was 0.46 times that for K and SLD cells. Since ED50 values are a measure of cellular sensitivity (the greater the ED50 value the lesser the cellular sensitivity), the order of sensitivity to ACTH was ADW greater than K greater than SLD and the order of sensitivity to pregnenolone was ADW greater than K = SLD. However, there were no strain differences in ED50 values of 8-bromo-cyclic AMP. These data suggest that strain differences in plasma corticosterone response to stress are, in part, due to differences in relative adrenal weight and differences in adrenocortical cell function.     

Effect of glucocorticoids pretreatment on steroidogenic capacity of adrenocortical cells isolated from Meishan piglets

The present in vitro experiment was designed to test whether 48 h of pretreatment with glucocorticoids, cortisol, or dexamethasone (DEX), would affect basal and corticotrophin (ACTH) stimulated (24 h) cortisol secretion from primary cultures of pig adrenocortical cells. Cells were divided into six groups: control pretreatment with or without ACTH challenge, cortisol pretreatment with or without ACTH challenge, and DEX pretreatment with or without ACTH. The culture medium and cells were collected at the end of treatment. Cortisol concentration in medium was measured by radioimmunoassay, and protein content of glucocorticoid receptor (GR) and key regulatory factors for steroidogenesis, including melanocortin type 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450scc), were detected by Western blot analysis. The results showed that glucocorticoid pretreatment did not affect cortisol secretion under basal condition without ACTH challenge, but significantly enhanced ACTH-stimulated cortisol secretion. Furthermore, the protein content of GR, MC2R, StAR, and P450scc was all increased in groups pretreated with glucocorticoids. These results indicate that adrenocortical cells pretreated with glucocorticoids display higher steroidogenic capacity under ACTH challenge, through the upregulation of GR and other steroidogenic regulatory factors.     

Ontogenic corticosteroidogenesis of the domestic fowl: response of isolated adrenocortical cells

Ontogenic adrenocortical function of the domestic was investigated using adrenocortical cells isolated from embryonic chicks (18, 19, 20, and 21 days old) and male and female posthatch birds (1 day, 1 week, and 3 weeks old). Production of the predominant corticosteroids secreted by the chicken adrenal gland, corticosterone, cortisol, and aldosterone, was measured by radioimmunoassay after 2-hr incubation of cells with or without steroidogenic agents. Approaching hatch, basal and maximal ACTH-(1-24) (ACTH)-induced corticosteroid production increased steadily and peaked around 1 day posthatch (5-18 times and 3-9 times, respectively, the production values at 18 days embryonic life). Thereafter, corticosteroid production values decreased steadily to 3 weeks posthatch. Corticosterone predominated over the ages studied: Maximal ACTH-induced corticosterone production averaged 52 and 115 times the production values of aldosterone and cortisol, respectively. In addition, maximal ACTH-induced aldosterone production was roughly 2.2 times greater than cortisol production over the ages studied except for a short-lived, disproportionately greater aldosterone production at 1 day posthatch. In addition to perihatch and age-related differences in cellular corticosteroid production, there were also differences in cellular sensitivity to steroidogenic agents as indicated by the differences in half-maximal steroidogenic concentration values (ED50 values) of the steroidogenic agents. Sensitivity to ACTH increased 2.7 times from Day 18 of embryonic life to 1 day posthatch and then decreased steadily to 3 weeks posthatch. In addition, sensitivity to 8-bromo-cAMP (8-Br-cAMP) increased abruptly at 1 day posthatch (nearly 3 times) but then remained constant thereafter. However, a consistent change in cellular sensitivity to 25-hydroxycholesterol was not observed until 3 weeks posthatch (an increase in sensitivity of 3 times that at Day 18 of embryonic life). These data of cellular sensitivity suggest that there were distinct development and maturational alterations in the cellular loci at which ACTH, 8-Br-cAMP, and 25-hydroxycholesterol acted. Thus, during the transition from embryonic to postembryonic life of the domestic fowl, there are alterations in adrenocortical cell steroidogenic capacity and in the function of some cellular loci comprising the corticosteroidogenic pathway.     

In vitro effect of insulin and insulin-like growth factor-I on cell multiplication and adrenocorticotropin responsiveness of fetal adrenal cells

The present study examined the effects of both insulin and insulin-like growth factor-I (IGF-I) on cell division and specific functions of cultured adrenocortical cells from 100- to 122-day-old ovine fetuses. When culture was performed in a serum-free medium containing transferrin and ascorbic acid, the number of cells increased only slightly (1.2-fold) over a 4-day period. Addition of insulin or IGF-I in the culture medium enhanced the number of cells counted on Day 5. The effect of both peptides was dose-dependent, but 10 ng/ml IGF-I was as potent as 10 micrograms/ml insulin. The acute cyclic adenosine 3',5'-monophosphate (cAMP) and steroidogenic responses to adrenocorticotropin (ACTH1-24) decreased in fetal cells cultured in the absence of insulin or ACTH. Insulin at micromolar concentrations not only prevented this decrease but enhanced the acute ACTH1-24-induced cAMP output on Day 5 over that observed on Day 2. Treatment of fetal cells for 4 days with increasing concentrations of insulin or IGF-I enhanced the acute cAMP and steroidogenic responses (3- to 4-fold) to ACTH1-24 over that of control cells. The ED50 of IGF-I was about 3 ng/ml (congruent to 0.4 nM) whereas that of insulin was about 10 ng/ml (1.7 nM). However, a second plateau was apparent at concentrations of insulin above 1 microgram/ml. The acute cholera toxin stimulation of cAMP production of cells cultured in the absence of insulin or ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucocorticoids Enhance Histamine-Evoked Catecholamine Secretion from Bovine Chromaffin Cells

Abstract: The synthetic glucocorticoid dexamethasone enhanced histamine-evoked catecholamine secretion from cultured bovine chromaffin cells. Dexamethasone enhanced the effects of histamine on both adrenergic (epinephrine-rich) and noradrenergic (norepinephrine-rich) chromaffin cells but had a more dramatic effect on noradrenergic cells. Histamine-evoked secretion in noradrenergic cells appeared to become rapidly inactivated, whereas the rate of secretion in adrenergic cells was nearly constant for up to 2 h; dexamethasone treatment attenuated the inactivation seen in noradrenergic cells. The effect of dexamethasone appeared after a lag of several hours and was maximal by 24 h. The EC₅₀ for dexamethasone was ∼1 n M . The effect of dexamethasone was mimicked by the glucocorticoid agonist RU 28362 and was blocked by the antagonist RU 38486, indicating that the effects of these steroids were mediated by the glucocorticoid or type II corticosteroid receptor. Histamine-evoked catecholamine secretion in both dexamethasone-treated and untreated cells was blocked by the H₁ histamine receptor antagonist mepyramine but was not affected by the H₂ antagonist cimetidine; thus, dexamethasone appeared to enhance an H₁ receptor-mediated process. In the absence of glucocorticoids, H₁ receptor mRNA levels were higher in adrenergic than in noradrenergic cells. Dexamethasone increased H₁ receptor mRNA levels in both cell types. The increased expression of H₁ receptors presumably contributes to the enhancement of histamine-evoked catecholamine secretion by glucocorticoids. Glucocorticoids may play a physiological role in modulating the responsiveness of chromaffin cells to histamine and other stimuli.     

Binding studies of the antiglucocorticoid RU38486 in Daudi and Raji lymphoma cells

The activity of RU38486 has been studied in Burkitt's lymphoma cells which are Epstein-Barr virus (EBV) positive. The early antigens (EA) of the virus are induced by dexamethasone (DXM) in Daudi but not in Raji cells, whereas a growth factor (transforming growth factor-beta, TGF-beta) induces the EA in both cell lines. RU38486 blocks the EA induction obtained by DXM or by TGF-beta in either cell line. In order to understand the interaction of RU38486, we considered its binding to specific receptors. We first investigated the binding of the antagonist in whole cells at 22 degrees C. A number of specific binding sites higher for RU38486 than for DXM was found, suggesting that RU38486 may bind to the glucocorticoid receptor and also to other cellular structures which we called the antiglucocorticoid binding sites ("AGBS"). To support this hypothesis, competition experiments have been conducted between RU38486 and other steroid hormones (progesterone and testosterone) since it is known that RU38486 is also able to interact with their cognate receptors. Binding studies of RU38486 in vitro at 4 degrees C in the presence of cytosolic extracts from Daudi and Raji cells led to conclusions similar to those drawn from the whole cell experiments: more complexes were formed with RU38486 than with DXM. Finally, the steroid-receptor complexes were incubated with DNA-cellulose. Since the binding measured for RU38486 was higher than for DXM, we suspect that sites different from the classical glucocorticoid receptor sites are also able to interact with DNA. The blockage exerted by RU38486 on the EA induced by glucocorticoids or by non-steroidal molecules and the lack of responsiveness to glucocorticoids in Raji cells are discussed in the light of the present findings.     

Permissive action of glucocorticoids on catecholamine-induced lipolysis: direct "in vitro" effects on the fat cell beta-adrenoreceptor-coupled-adenylate cyclase system

Exposure of adipose tissue fragments to dexamethasone leads to enhanced lipolytic and cyclic AMP responses of isolated fat cells to isoproterenol. This permissive effect of the steroid is dose-dependent, prevented by the glucocorticoid receptor antagonist RU 38486, maximum after 48 h exposure to 10 nM dexamethasone and affects only the amplitude of the maximal response (+50%). Exposure to dexamethasone also induces an increase in both the number of beta-adrenergic receptors (+30%), and the adenylate cyclase-catalytic activity (+64%) and - responses to GTP (+114%) and isoproterenol (+55%). These data strongly suggest that the permissive effect of glucocorticoids towards lipolysis "in vivo" results at least in part from a glucocorticoid-receptor mediated action of these hormones on the fat cell membranous components involved in the beta-adrenergic control of lipolysis.     

A possible role for endogenous glucocorticoids in orchiectomy-induced atrophy of the rat levator ani muscle: studies with RU 38486, a potent and selective antiglucocorticoid

We employed RU 38486, a potent and selective antiglucocorticoid, to study a possible role for endogenous glucocorticoids in atrophy of the levator ani muscle secondary to castration of male rats. RU 38486 was shown to block [3H]triamcinolone acetonide binding to cytosol from levator ani muscle. Daily oral administration of RU 38486 to castrated rats partially prevented atrophy of the levator ani muscle, as well as a decrease in RNA concentration. In a control group receiving RU 38486 alone, the levator ani underwent significant (20%) hypertrophy. Administration of exogenous dexamethasone also caused pronounced atrophy of the levator ani muscle. This atrophy was prevented, to a significant degree, by simultaneous oral administration of RU 38486. It is concluded that endogenous glucocorticoids, the actions of which are blocked by RU 38486, may be involved in regulation of the mass of the levator ani muscle in intact rats.     

Structure-activity relationships of monomeric and dimeric synthetic ACTH fragments in perifused frog adrenal slices

The effect of synthetic monomeric and dimeric ACTH fragments on spontaneous and ACTH(1-39)-evoked steroidogenesis in frog interrenal tissue was studied in vitro. Infusion of ACTH fragment 11-24 (10(-6) M) or its dimeric conjugates, attached either by their N-terminal, Glu(11-24)2, or their C-terminal amino acid, (11-24)2Lys, had no effect on the spontaneous release of corticosteroids. The monomer ACTH(11-24) and the dimer Glu(11-24)2 were also totally devoid of effect on the steroidogenic response to ACTH(1-39) (10(-9)M). In contrast, the (11-24)2Lys conjugate (10(-6)M) significantly decreased ACTH-induced stimulation of corticosterone and aldosterone (-63 and -62%, respectively). The dimeric conjugate of the fragment ACTH(7-24), linked through the C-terminal ends, (7-24)2Lys (10(-6)M), was also completely devoid of effect on basal steroidogenesis but caused a marked decrease of ACTH-evoked corticosterone and aldosterone release (-72 and -80%, respectively). Conversely, infusion of the dimer (1-24)2Lys gave rise to a dose-related stimulation of corticosterone and aldosterone release. The time-course of the steroidogenic response to the dimer was similar to that of ACTH(1-24). The 1-24 conjugate was 70 times less potent than the monomers ACTH(1-24) and ACTH(1-39). These results suggest that amphibian adrenocortical cells contain only one class of ACTH receptor which recognizes the 11-24 domain of ACTH with an affinity which depends on the presence of a strong potentiator segment, located at the N-terminus end of ACTH(1-39). Since the ACTH-dimers are thought to induce cross-linking of the receptors, our results suggest that aggregation of ACTH receptors causes a down-regulation of the receptors.