RESUMO
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.
Assuntos
Corticosteroides/biossíntese , Córtex Suprarrenal/metabolismo , Corticosterona/farmacologia , Dexametasona/farmacologia , Hidrocortisona/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Córtex Suprarrenal/efeitos dos fármacos , Animais , Células Cultivadas , Cosintropina/farmacologia , Estrenos/farmacologia , Glucocorticoides/antagonistas & inibidores , Cinética , Mifepristona , Valores de Referência , OvinosRESUMO
The present study examines the effect of chronic treatment of glucocorticoids on ACTH1-24- or forskolin-induced cAMP output of cultured sheep adrenocortical cells. Cells cultured for 2 days in the presence of 1 microM dexamethasone released more cAMP in response to ACTH1-24 than did untreated cells, both in the absence and presence of 0.5 mM 1-methyl-3-isobutylxanthine. Such an enhancing effect required greater than or equal to 21 h of treatment and was both concentration-dependent and steroid specific. The ED50 of dexamethasone was about 10 nM, while that of cortisol and corticosterone was about 1 microM; testosterone at concentrations less than or equal to 10(-5) M had no enhancing effect. Glucocorticoids enhanced the cAMP response to ACTH1-24 without altering its ED50. Treatment of cultures with aminoglutethimide or the antiglucocorticoid RU 38486 for 48 h resulted in a dose-dependent decrease in ACTH1-24-induced cAMP output. Moreover, RU 38486 antagonized the enhancing effect of dexamethasone. Glucocorticoids did not increase the cAMP response to forskolin. These results suggest that chronic exposure to glucocorticoids is necessary for the full expression of the cAMP response to ACTH1-24 of adrenocortical cells from adult sheep.
Assuntos
Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Cosintropina/farmacologia , AMP Cíclico/biossíntese , Glucocorticoides/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Córtex Suprarrenal/efeitos dos fármacos , Aminoglutetimida/farmacologia , Animais , Células Cultivadas , Colforsina/farmacologia , Corticosterona/farmacologia , Dexametasona/farmacologia , Estrenos/farmacologia , Hidrocortisona/farmacologia , Mifepristona , OvinosRESUMO
The effects of inhibitors of pregnenolone metabolism, WIN-24540 and spironolactone, on adrenocorticotropic hormone (ACTH)- and human chorionic gonadotropin (hCG)-induced cAMP and steroid production by bovine (BAC) and ovine (OAC) adrenal cells and pig Leydig cells (PLC) were investigated. The inhibitors reduced cAMP production by adrenal and Leydig cells by about 75% and 60%, respectively (P less than 0.001). Further, the inhibitors also reduced the cholera toxin- and forskolin-induced cAMP production by pig Leydig cells. In the presence of the inhibitors, corticosterone and testosterone production by BAC and PLC, respectively, following hormonal stimulation was reduced by more than 90%. However, pregnenolone production by BAC and PLC under these conditions represented only 12% and 42% of the corticosterone and testosterone production, respectively, in the absence of inhibitors. Moreover, the inhibitors also reduced the steroidogenic response of PLC to 8-Br-cAMP and the conversion of 22(R)-hydroxycholesterol to pregnenolone by BAC and PLC. The reduced production of pregnenolone in the presence of inhibitors was in part due to the weak inhibition of 17 alpha-hydroxylase by spironolactone. However, when OAC cells were incubated in the presence of WIN-24540 and SU-10603, a potent 17 alpha-hydroxylase inhibitor, the amount of pregnenolone produced in response to ACTH or 22(R)-hydroxycholesterol was only 10% and 19%, respectively, of the steroids (corticosterone plus cortisol) secreted in the absence of inhibitors. The results show that the inhibitors of pregnenolone metabolism reduced, in both adrenal and Leydig cells, the response of adenylate cyclase to several effectors and the activity of the cholesterol side-chain cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glândulas Suprarrenais/metabolismo , Proteínas de Transporte , AMP Cíclico/biossíntese , Endorribonucleases , Peptídeos e Proteínas de Sinalização Intracelular , Células Intersticiais do Testículo/metabolismo , Pregnenolona/metabolismo , Proteínas/metabolismo , Proteínas de Ligação a RNA , Espironolactona/farmacologia , Esteroides/biossíntese , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/efeitos dos fármacos , Animais , Bovinos , Células Cultivadas , Colesterol/metabolismo , Colforsina/farmacologia , Di-Hidrotestosterona/análogos & derivados , Di-Hidrotestosterona/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Fosfoproteínas Fosfatases , Pregnenolona/antagonistas & inibidores , Proteínas/análise , Ovinos , Suínos , Tetra-Hidronaftalenos/farmacologiaRESUMO
We have shown previously that a chronic treatment with glucocorticoids enhances cAMP- or ACTH-induced steroidogenesis of cultured ovine adrenocortical cells. This effect appears to involve a greater amount of cholesterol in mitochondria. Hence, the present study aimed to define the role of glucocorticoids in cholesterol metabolism by these cells. 2-day-old cultures were exposed to different hormones or inhibitors (10(-6) M ACTH, 10(-5) M metyrapone) for 28-48 h. At the end of the treatment period, the cells were stimulated for 2 h with 10(-3) M 8Br-cAMP, in the presence of 10(-3) M aminoglutethimide (in order to load mitochondria with cholesterol). Mitochondria were then isolated and incubated without or with 100 microM cholesterol either in the presence or absence of 10(-3) M CaCl2, or with 25 microM 22R-hydroxycholesterol. Mitochondria isolated from dexamethasone-treated cells produced consistently more pregnenolone than mitochondria from control cells, suggesting that at least part of the additional cholesterol present in these mitochondria was available for steroidogenesis. However, similar differences were obtained when mitochondria were incubated in the presence of exogenous cholesterol, both with or without calcium, or in the presence of 22R-hydroxycholesterol. Pregnenolone production under these latter conditions was much higher than when endogenous cholesterol was the only substrate. Conversely, metyrapone treatment of the cells resulted in lower production of pregnenolone from 22R-hydroxycholesterol by their mitochondria. Likewise ACTH treatment enhanced pregnenolone production by isolated mitochondria irrespective of the incubation conditions. These effects of dexamethasone and ACTH were not related to higher amounts of adrenodoxin, adrenodoxin reductase or cytochrome P450scc. These results indicate that exposure of ovine adrenocortical cells to glucocorticoids or ACTH enhances their steroidogenic potency not only by increasing the amount of cholesterol available for steroidogenesis but also by enhancing some step(s) involved in the transformation of cholesterol into pregnenolone.
Assuntos
Córtex Suprarrenal/metabolismo , Colesterol/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Mitocôndrias/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Córtex Suprarrenal/citologia , Corticosteroides/biossíntese , Hormônio Adrenocorticotrópico/farmacologia , Adrenodoxina/biossíntese , Aminoglutetimida/farmacologia , Animais , Cloreto de Cálcio/farmacologia , Células Cultivadas , Colesterol/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/farmacologia , Ferredoxina-NADP Redutase/biossíntese , Glucocorticoides/antagonistas & inibidores , Hidroxicolesteróis/metabolismo , Hidroxicolesteróis/farmacologia , Metirapona/farmacologia , Mitocôndrias/efeitos dos fármacos , Pregnenolona/biossíntese , RNA Mensageiro/biossíntese , Ovinos , Esteroides/farmacologiaRESUMO
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.
Assuntos
Adenilil Ciclases/metabolismo , Córtex Suprarrenal/metabolismo , Colesterol/biossíntese , Corticosterona/biossíntese , Cosintropina/farmacologia , Dexametasona/metabolismo , Pregnenolona/biossíntese , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Acetatos/metabolismo , Córtex Suprarrenal/efeitos dos fármacos , Animais , Células Cultivadas , Toxina da Cólera/farmacologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Sinergismo Farmacológico , Guanilil Imidodifosfato/farmacologia , Cinética , Pregnenolona/farmacologia , Ovinos , Fluoreto de Sódio/farmacologiaRESUMO
Plasma concentrations of testosterone, measured weekly in 16 Ouled-Djellal rams in Algeria, were lowest in autumn and early winter months, increased in February-March and reached highest levels (about 5 ng/ml) in early summer, before decreasing in autumn. Plasma dihydrotestosterone levels were very low and paralled those of testosterone until July (50 pg/ml); there was then an unexplained rise in October-November (50 pg/ml). The metabolic clearance rate of testosterone was low in November and January, and significantly increased in March and June (about 2000 1/24 h). Consequently, the production rate of testosterone was approximately 5-fold higher in June than in January. These data suggest that the short-day photoperiodic theory is not applicable to all breeds of sheep or to all environmental conditions.
Assuntos
Di-Hidrotestosterona/sangue , Estações do Ano , Testosterona/sangue , Argélia , Animais , Masculino , Taxa de Depuração Metabólica , Ovinos , Testosterona/metabolismoRESUMO
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.
Assuntos
Corticosteroides/biossíntese , Córtex Suprarrenal/embriologia , Hormônio Adrenocorticotrópico/farmacologia , Cosintropina/farmacologia , AMP Cíclico/metabolismo , Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/metabolismo , Aminoglutetimida/farmacologia , Animais , Bucladesina/farmacologia , Células Cultivadas , Colforsina/farmacologia , Feto , Cinética , Metirapona/farmacologia , OvinosRESUMO
Plasma concentrations of prolactin and testosterone were measured weekly, from November 1976 to December 1977, respectively in 29 and 10 Ouled-Djellal rams penned in the Algeria area. Plasma prolactin and testosterone levels exhibited concomitant seasonal variations: they were low in November-December, then they increased from January to maximal values in summer through two steps, the first in February-March, the second in June-July. Taken all around, the hormones changes paralleled those of photoperiod. The synchronism in annual variations of prolactin and testosterone secretory patterns is a feature of the Ouled-Djellal ram. Results are discussed in relation to effects of prolactin on testicular activity.
Assuntos
Periodicidade , Prolactina/sangue , Estações do Ano , Ovinos/fisiologia , Testosterona/sangue , Argélia , Animais , Clima , Masculino , TemperaturaRESUMO
In the ovine fetus, plasma levels of corticosteroids are very low between 60 and 130 days of gestation, then increase dramatically before birth. ACTH appears to be an important regulating hormone for the fetal adrenal cortex, the sensitivity of which to this hormone increases during late gestation. However, the relationship between immunoreactive ACTH and corticosteroids in the fetus is unclear. We review herein recent work performed in our laboratory on the regulation of ACTH secretion by ovine fetal pituitary cells and on the biochemical modifications responsible for the enhancement of the steroidogenic response to ACTH of fetal adrenal cells. It is suggested that qualitative together with quantitative changes in the pituitary drive to the fetal adrenal has to be taken in account to explain the rise of corticosteroids in prepartum animals. Also extra pituitary hormones may be operating during intrauterine life to regulate fetal adrenal function.