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.     

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.     

Clonal variation in response to adrenocorticotropin in cultured bovine adrenocortical cells: relationship to senescence

During cellular senescence, non-clonal cultures of bovine adrenocortical cells show a continuous decline in the rate of production of cyclic AMP (cAMP) stimulated by adrenocorticotropin (ACTH), without changes in the rate of forskolin- or prostaglandin E1-stimulated cAMP production. We investigated the possible mechanisms for loss of response to ACTH by examining the properties of clones of bovine adrenocortical cells. ACTH-stimulated cAMP production rates were measured in clones immediately after isolation, during long-term growth following isolation, and after subcloning. ACTH-stimulated rates were compared with cAMP production in response to forskolin, which acts directly on the catalytic subunit of adenylate cyclase. The results show that cloning is not necessarily associated with a loss of response to ACTH, but that clones with high ACTH response can give rise to subclones with low response. Clones of adrenocortical cells, at the same approximate population doubling level (PDL), showed ACTH response levels that ranged from 12 to 135 pmol cAMP/10(6) cells/min, whereas mass cultures at this PDL showed approximately 50 pmol/10(6) cells/min. Forskolin-stimulated cAMP production rates in clones varied only over the range of 59-119 pmol/10(6) cells/min and showed no correlation with the ACTH-stimulated rates. All clones were adrenocortical cells, as shown by mitogenic response to angiotensin II and cAMP-inducible 17 alpha-hydroxylase activity. The replicative potential of clones varied widely, and there was no apparent correlation between ACTH response levels and growth potential. The level of ACTH response in each clone was stable during proliferation through at least 25 PD beyond the stage at which the clone was isolated. When clones were subcloned, a clone with a high ACTH response level produced sister subclones that had ACTH response levels ranging from 3% of that of the parent clone to a level slightly greater than that of the parent clone. The growth potential of sister subclones varied widely, as for the parent clones, and there was no obvious correlation between growth potential and ACTH response. Two subclones were cloned; in sub-subclones, levels of ACTH response were again different from each other and also from the parent subclone; in one case, the level of ACTH response was approximately eight-fold higher than that of the parent subclone. These experiments show that clonal variation in the extent of expression of a differentiated property may occur in a normal differentiated cell in culture. The loss of ACTH response and the loss of proliferative potential appear to be independent stochastic events.     

Luteotropic and luteolytic responsiveness of ovine luteal cells in long-term culture

Ovine luteal cells were collected and plated 36 h (Day 2) after injection of human chorionic gonadotropin (Day 0) to induce ovulation. Cells were maintained (Days 2-12) in Medium 199 containing 5% calf serum, which was replaced daily. Progesterone secretion was not stimulated (p greater than 0.05) by luteinizing hormone (LH, 10 ng/ml or 100 ng/ml) at any time during culture. However, it was enhanced (p less than 0.05) with a 24-h pulse of dibutyryl adenosine 3', 5'-monophosphate (dbcAMP) during early (2.2-fold stimulation over basal; Days 5,6) or mid- (1.7-fold stimulation over basal: Days 8,9) culture if the pulsing medium contained serum, but not if serum had been withdrawn for 24 h. Continuous exposure of cultures to dbcAMP (2 mM, Days 3-12) resulted in continuously stimulated (p less than 0.05) progesterone secretion (range 1.8- to 4.1-fold stimulation). An increased (p less than 0.05) percentage of cells staining positive for 3 beta-hydroxy-delta 5-steroid dehydrogenase-delta 5, delta 4-isomerase (3 beta HSD) activity were recovered on Day 12 in cultures incubated (Days 3-12) with dbcAMP. Incubation of cultures continuously with prostaglandin F2 alpha (PGF2 alpha) produced dose-dependent inhibition (p less than 0.05) of progesterone secretion. Reduced numbers of 3 beta HSD-positive cells were recovered from these incubations. These experiments demonstrate luteotropic (dbcAMP) as well as luteolytic (PGF2 alpha) effects on ovine luteal cells in long-term culture. This study provides evidence that these cultures will be useful for investigating the development of hormonal regulation of luteal function.     

Transforming growth factor beta treatment decreases ACTH receptors on ovine adrenocortical cells

Transforming growth factor beta (TGF beta) is a potent inhibitor of adrenocortical cell differentiated functions, whereas corticotropin (ACTH) is the main physiological hormone which acts positively on these functions. We have studied the effects of both TGF beta and ACTH on ovine adrenocortical cell ACTH receptors. Ovine adrenocortical cells contained specific high affinity (Kd = 2.7 +/- 1.6 x 10(-10) M) and low capacity (1190 +/- 120 sites/cell) ACTH receptors. Pretreatment of cells with TGF beta resulted in a time- and dose-dependent (ED50 = 50 pg/ml) decrease of 125I-ACTH1-39 binding. The observed decrease in ACTH binding was due to a 2-3-fold decrease in the number of binding sites without modification of the binding affinity. On the contrary, pretreatment of cells with ACTH caused a 4-4.5-fold increase in the number of ACTH binding sites without an effect on the Kd. When cells were pretreated with both ACTH and TGF beta, TGF beta blocked completely the positive trophic effect of ACTH on its own receptors. The variations in ACTH receptor number were associated with parallel changes on acute ACTH-induced cyclic AMP production. Thus, the effects of TGF beta on ACTH receptor content are likely another important negative action of this peptide on adrenocortical cell differentiation. Moreover, these results suggest that regulation of ACTH receptor number may be one mechanism by which hormones and growth factors control adrenocortical differentiation.     

Ontogeny of adrenal VIP content and release from adrenocortical cells in the ovine fetus

The present study was designed to investigate the presence of VIP in fetal adrenals, to determine the changes in adrenal VIP content associated with maturation, and to explore the factors which regulate fetal adrenal VIP release. Adrenal glands from ovine fetuses at 70 to 140 days gestation were used. Adrenal VIP content, as measured by radioimmunoassay, were low at 70 and 80 days of gestation. This was followed by a rapid increase in VIP content from 80 to 110 days reaching a plateau between 110 and 130 days at levels comparable to that in the adult. A significant fall in adrenal VIP content occurred at 140 days, immediately prior to term. Release of VIP from fetal adrenocortical cells in vitro was significantly elevated by angiotensin II at 10(-5) M, while ACTH had no effect. Acetylcholine at 50 microM and high potassium stimulated fetal adrenal VIP release while norepinephrine did not. These results suggest that the VIP neuronal system in the ovine fetal adrenal matures between 80 and 110 days of gestation. Furthermore, the release of VIP from the fetal adrenocortical cells may be regulated by angiotensin II and cholinergic neurotransmitters.     

Mechanism of colchicine-induced steroidogenesis in rat adrenocortical cells

Conflicting data for the effects of colchicine on cholesterol transport and steroidogenesis raise the question of the role of microtubules in cholesterol transport from the lipid droplet to mitochondria in steroidogenic cells. In this study, using corticosterone radioimmunoassay and immunofluorescence microscopy, we re-evaluated the effects of colchicine on hormone production and morphological changes of lipid droplets' and studied the signaling pathway involved in colchicine-induced steroidogenesis. Colchicine stimulated steroid production in a dose- and time-dependent manner. The structural integrity of both the microtubules and the lipid droplet capsule was destroyed by colchicine treatment. Disruption of the lipid droplet capsule occurred later than microtubule depolymerization. After cessation of colchicine treatment and a 3 h recovery in fresh medium, capsular protein relocated to the droplet surface before the cytoplasmic microtubule network was re-established. beta-lumicolchicine, an inactive analogue of colchicine, disrupted the capsule and increased hormone production without affecting microtubular structure. Thus, microtubule depolymerization is not required for the increase in steroid production and capsular disruption. To explore the signaling pathway involved in colchicine-induced steroidogenesis, we measured intracellular cAMP levels. Unlike ACTH, colchicine did not increase cAMP levels, suggesting that the cAMP-PKA system is not involved. Colchicine and ACTH had additive effects on corticosterone production, whereas colchicine and PMA did not, implying that part of the PKC signaling mechanism may be involved in colchicine-induced steroidogenesis. Cycloheximide, a protein synthesis inhibitor, completely inhibited colchicine-induced steroidogenesis and capsular disruption. These results demonstrate that the steroid production and lipid droplet capsule detachment induced by colchicine are both protein neosynthesis-dependent and microtubule-independent.     

Bovine adrenocortical cells exhibit high affinity transforming growth factor-beta receptors which are regulated by adrenocorticotropin

Transforming growth factor beta (TGF-beta) at picomolar concentrations has been previously shown to induce striking alterations of bovine adrenocortical cell differentiated functions, without detectable effect on growth activity (Feige, J.J., Cochet, C., and Chambaz, E. M. (1986) Biochem. Biophys. Res. Commun. 139, 693-700; Hotta, M., and Baird, A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7795-7799). Adrenocortical cells in culture could bind 125I-labeled TGF-beta through at least two different binding systems. The highest affinity TGF-beta binding exhibited a Kd value of 5.7 X 10(-10) M and a calculated capacity of about 100,000 sites/cell, while the low affinity system yielded values of 4.3 X 10(-8) M and 2 X 10(6) sites/cell, respectively. The 125I-labeled TGF-beta bound to adrenocortical cells could be cross-linked using disuccinimidyl suberate and subsequent electrophoretic analysis revealed that TGF-beta was associated with two major cell components of about 280 kDa and 70-75 kDa, respectively, the latter one being resolved as a labeled doublet. Thus bovine adrenocortical cells exhibit a TGF-beta receptor similar to that defined by Massagué and co-workers (Cheifetz, S., Like, B., and Massagué, J. (1986) J. Biol. Chem. 261,9972-9978) in other cell types. Various growth factors, including fibroblast growth factor, as well as established hormonal activators of adrenocortical cell differentiated functions, such as angiotensin II and adrenocorticotropin, were examined as to their effect on TGF-beta receptor activity. A striking increase in the number of high affinity TGF-beta receptors was selectively elicited by ACTH in the nanomolar concentration range. This effect was time- and dose-dependent and was mimicked by cell treatment with dibutyryl cyclic AMP or forskolin. However, the ACTH-induced increase in receptor number was not impaired when protein synthesis was blocked. It is concluded that bovine adrenocortical cells are typical target cells for TGF-beta. This endocrine system represents a model in which, for the first time, the level of TGF-beta receptor is shown to be under hormonal regulation through a cyclic AMP-dependent pathway.     

Immunological selection of variant mouse lymphoid cells with altered glucocorticoid responsiveness.

We have devised an immunological procedure to separate cells on the basis of expression of mouse mammary tumor virus (MMTV) gene products. Plastic petri dishes coated with specific antibodies against MMTV proteins bind cells with an efficiency that correlates with the level of MMTV gene expression. Glucocorticoid-sensitive mouse thymoma cell line W7 was infected with MMTV. Clones from the infected population retain the relatively slow cytolytic glucocorticoid response and, in addition, exhibit a rapid induction of MMTV-specific RNA and proteins. By combining our immunological selection with the selection for resistance to hormone-mediated cytolysis, we have isolated variant cells which are resistant to the cytotoxic effect of glucocorticoids but which retain the induction of viral gene products and must therefore have a functional glucocorticoid receptor protein.     

Mechanism of enhancement of polynucleotide binding to cells by mutagens.

The binding of polyuridylate to cells is substantially increased by proflavine. This enhanced binding is saturable with respect to time and to the concentration of both proflavine and polyuridylate. Enhancement is observed only when cells are exposed to both proflavine and polyuridylate together and depends cooperatively on the proflavine concentration. The resulting complex formed between the cell, proflavine, and polyuridylate can be dissociated with salt but not with sucrose solutions. An increase in the binding of polyuridylate to cells similar to that observed with proflavine was also obtained with cationic dyes such as acridine orange, 9-aminoacridine, and Hoechst 33258, while the introduction of a bulky polysaccharide residue, dextran, into the dyes cancels these effects. Similarly, cationic aromatic compounds such as primaquine and quinacrine which carry bulky nonplanar substituents or aliphatic cationic compounds like ethylenediamine do not enhance binding. Proflavine is unable to augment the binding of a basic macromolecule, diethylaminoethylaminoethyldextran, to cells. The model proposed for the enhanced binding of polyuridylate is based on the cooperative formation of stacked complexes of cationic dye located between the cell surface and the bound polyuridylate.     

Testicular responsiveness to hCG of the ovine fetus in the last third of gestation

The in vivo and in vitro testicular responsiveness to hCG of hemicastrated lamb fetuses 95-99, 110-118 and 130-141 days of gestational age was studied. Basal plasma testosterone (T) levels were similar at all ages (less than 0.25 ng/ml), while the mean testicular concentrations of dehydroepiandrosterone sulfate (DHA-S), 17 alpha-hydroxyprogesterone (17-OHP) and T were higher in 95- to 99-day-fold fetuses. Plasma T levels and the concentration of T, DHA-S, 17-OHP, androstenedione (A) and cyclic adenosine 3'5'-monophosphate (cAMP) were increased by hCG in the hemicastrated animal at all ages. cAMP and T production by enriched preparations of dispersed interstitial cells from control testes was increased by hCG in all groups. In fetuses pretreated with hCG in vivo the addition of hCG in vitro failed to modify cAMP and T production. 100 micrograms of LHRH to a 130-day-old fetus increased plasma LH and T levels. From these experiments, it is suggested that the low plasma LH and T levels found throughout the last trimester of fetal life reflect a relative lack of endogenous LHRH synthesis and/or release, rather than reduced testicular steroidogenic capacity.     

Expression of glucocorticoid receptor isoforms in human adrenocortical adenomas

Introduction

Glucocorticoid receptor (GR) is expressed in the normal human adrenal gland, however, no study has been performed to evaluate the separate expression of α- and β-isoforms (GRα and GRβ) in normal human adrenals and in adrenocortical adenomas.

Experimental

GRα and GRβ mRNA expression was examined by quantitative real-time PCR in 31 adrenal tissues including 19 non-functioning adenomas (NFA), 6 cortisol-producing adenomas (CPA) and 6 normal adrenocortical tissues. In addition, the presence and cellular localization of GRα and GRβ proteins in adrenal tissues were studied by immunohistochemistry.

Results

Compared to normal adrenocortical tissues, both GRα and GRβ mRNAs were significantly increased in CPA but not in NFA. Using anti-GRα antibody a strong nuclear staining was observed in NFA and CPA, and a less remarkable immunoreactivity was detected in some nuclei of normal adrenocortical cells. GRβ immunostaining was absent in normal adrenal tissues and NFA, while a strong cytoplasmic and nuclear immunoreaction was found in CPA.

Conclusions

Altered expression of GRα and GRβ in CPA raises their possible role in the pathophysiology of these adrenal tumors, although further studies are needed to elucidate the potential significance of these findings.     

The role of ether-a-go-go-related gene K(+) channels in glucocorticoid inhibition of adrenocorticotropin release by rat pituitary cells

The present study investigated the role of K(+) channels in the inhibitory effect of glucocorticoid on adrenocorticotropin (ACTH) release induced by corticotropin-releasing hormone (CRH) using cultured rat anterior pituitary cells. Apamin and charybdotoxin (CTX) did not have a significant effect on ACTH release induced by CRH (1 nM). Tetraethylammonium (TEA), a broad spectrum K(+) channel blocker, increased the ACTH response to CRH only at the highest concentration (10 mM). The exposure to 100 nM corticosterone for 60 min inhibited the CRH-induced ACTH release. Neither TEA, apamin, nor CTX affected the inhibitory effect of corticosterone. In contrast, astemizole (Ast) and E-4031, ether-a-go-go-related gene (erg) K(+) channel blockers, abolished the inhibitory effect of corticosterone on CRH-induced ACTH release (1.25+/-0.10 vs. 1.45+/-0.11 ng/well at 10 microM Ast, p>0.05, 1.71+/-0.16 vs. 1.91+/-0.32 ng/well at 10 microM E-4031, p>0.05, vehicle vs. corticosterone). RT-PCR demonstrated all three subtypes of rat-erg mRNAs in the pituitary and corticosterone increased only erg1 mRNA up to 2.47+/-0.54 fold. In conclusion, erg K(+) channels were up-regulated by glucocorticoid, and have indispensable roles in delayed glucocorticoid inhibition of CRH-induced ACTH release by rat pituitary cells.     

Effects of glucagon and glucagon-like peptide-1 on glucocorticoid secretion of dispersed rat adrenocortical cells.

The effects of glucagon and glucagon-like peptide-1 (GLP-1) on the secretory activity of rat adrenocortical cells have been investigated in vitro. Neither hormones affected basal or agonist-stimulated aldosterone secretion of dispersed rat zona glomerulosa cells or basal corticosterone production of zona fasciculata-reticularis (inner) cells. In contrast, glucagon and GLP-1 partially (40%) inhibited ACTH (10(-9) M)-enhanced corticosterone secretion of inner cells, maximal effective concentration being 10(-7) M. The effect of 10(-7) M glucagon or GPL-1 was suppressed by 10(-6) M Des-His1-[Glu9]-glucagon amide (glucagon-A) and exendin-4(3-39) (GPL-1-A), which are selective antagonists of glucagon and GLP-1 receptors, respectively. Glucagon and GLP-1 (10(-7) M) decreased by about 45-50% cyclic-AMP production by dispersed inner adrenocortical cells in response to ACTH (10(-9) M), but not to the adenylate cyclase activator forskolin (10(-5) M). Again this effect was blocked by 10(-6) M glucagon-A or GLP-1-A. The exposure of dispersed inner cells to 10(-7) M glucagon plus GLP-1 completely suppressed corticosterone response to ACTH (10(-9) M). However, they only partially inhibited (by about 65-70%) both corticosterone response to forskolin (10(-5) M) or dibutyryl-cyclic-AMP (10(-5) M) and ACTH (10(-9) M)-enhanced cyclic-AMP production. Quantitative HPLC showed that 10(-7) M glucagon or GLP-1 did not affect ACTH-stimulated pregnenolone production, evoked a slight rise in progesterone and 11-deoxycorticosterone release, and markedly reduced (by about 55%) corticosterone secretion of dispersed inner adrenocortical cells. In light of these findings the following conclusion are drawn: (i) glucagon and GLP-1, via the activation of specific receptors, inhibit glucocorticoid response of rat adrenal cortex to ACTH; and (ii) the mechanism underlying the effect of glucagon and GLP-1 is probably two-fold, and involves both the inhibition of the ACTH-induced activation of adenylate cyclase and the impairment of the late steps of glucocorticoid synthesis.     

Mechanism involved in synergistic adrenocorticotropin response to activating protein kinase-A and -C in rat anterior pituitary cells

Activation of protein kinase C (PKC) stimulates adrenocorticotropin (ACTH) release synergistically in the presence of corticotropin releasing factor (CRF). We examined the effect of a cyclic nucleotide-specific phosphodiesterase inhibitor, 1-isoamyl-3-isobutylxanthine (IIX), on arginine vasopressin (AVP)-induced ACTH release and intracellular cAMP accumulation in normal rat anterior pituitary cells. IIX alone elevated intracellular cAMP accumulation. IIX potentiated AVP-induced ACTH release synergistically without further increase in cAMP accumulation, suggesting that synergistic ACTH release has an alternative mechanism other than the synergistic elevation of intracellular cAMP accumulation which has been reported. Phorbol 12-myristate-13-acetate (PMA) also induced synergistic ACTH release when incubated with IIX. IIX had no additional effect on ACTH response when incubated with maximal dose of CRF, forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Moreover, the combination of PMA and 8-Br-cAMP produced synergistic ACTH response. In conclusion, the synergistic ACTH release from rat pituitary corticotrophs occurs at least in the presence of directly activating events of PKC and PKA as well as PKC-induced inhibition of phosphodiesterase activity.