RESUMO
This study assessed the role of the extracellular signal-regulated kinase (ERK) signaling pathway on the previously observed enhanced cortisol secretion in response to adrenocorticotropic hormone (ACTH) treatment in fetal adrenocortical cells (FACs) from long-term hypoxic (LTH) ovine fetuses. Ewes were maintained at high altitude (3,820 m) from ~40 to 138-141 days gestation when FACs were collected and challenged with either ACTH (10 nM) or 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP, 10 mM) in the presence or absence of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MEK)/ERK inhibitor UO126 (10 µM). FACs from age-matched normoxic fetuses served as controls. Media and FACs were collected at selected time intervals after ACTH or 8-bromo-cAMP stimulation for cortisol measurement and Western analysis of ERK1/2 and phospho-ERK1 and -2 (pERK1/2). After ACTH or 8-bromo-cAMP treatment, cortisol production was greater in the LTH group compared with control (P < 0.05). UO126 reduced ACTH and 8-bromo-cAMP-mediated cortisol output in both groups (P < 0.01 vs. ACTH or 8-bromo-cAMP alone). Under basal conditions, ERK1/2 and pERK1/2 were not different between LTH and normoxic fetuses. In response to ACTH or 8-bromo-cAMP treatment, ERK1/2 were not different between groups; however, pERK1/2 were elevated in the LTH FACs compared with normoxic control FACs. ERK1/2 phosphorylation declined following ACTH treatment in the control group, but UO126 had no effect on ERK1/2 compared with untreated levels. Both ACTH and 8-bromo-cAMP treatment resulted in a decline of protein levels. UO126 pretreatment virtually eliminated pERK1/2 expression. We conclude that basal ERK signaling in FACs is necessary for normal cortisol production and sustained pERK in LTH adrenals enhances cortisol production.
Assuntos
Glândulas Suprarrenais/embriologia , Glândulas Suprarrenais/metabolismo , Hipóxia Fetal/metabolismo , Hipóxia Fetal/fisiopatologia , Hidrocortisona/biossíntese , Sistema de Sinalização das MAP Quinases/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Córtex Suprarrenal/citologia , Córtex Suprarrenal/embriologia , Córtex Suprarrenal/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Hormônio Adrenocorticotrópico/farmacologia , Animais , Western Blotting , Butadienos/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Gravidez , OvinosRESUMO
We previously reported that in the sheep fetus, long-term hypoxia (LTH) resulted in elevated basal plasma adrenocorticotropic hormone (ACTH(1- 39)) whereas the cortisol levels were not different from normoxic controls. We also showed that LTH enhances endothelial nitric oxide synthase (eNOS) expression in the fetal adrenal. This study was designed to determine the effect of NO on cortisol production in adrenocortical cells from LTH fetal sheep. Ewes were maintained at high altitude (3820 m) from â¼40 days' gestation (dG) to near term. Between 138 and 141 dG, fetal adrenal glands were collected from LTH and age-matched normoxic control fetuses. Adrenal cortical cells were pretreated with sodium nitroprusside (SNP), nitro-L-arginine methyl ester (L-NAME), L-arginine, or diethyleneamine NO (DETA-NO) and then challenged with 10 nmol/L ACTH. Cortisol responses were compared after 1 hour. Adrenocorticotropic hormone -induced cortisol secretion was significantly higher in LTH versus control (P < .01). Enhancement of NO with L-arginine resulted in a significant reduction of ACTH-mediated cortisol production in the LTH group. DETA-NO also caused a significant decrease in ACTH-mediated cortisol production (P < .05). Inhibition of NOS with L-NAME significantly increased cortisol production in the LTH group (P < .05 compared to ACTH alone), whereas the effect on the control group was not significant. Nitric oxide synthase activity was significantly higher in the LTH group compared to control, but this difference was eliminated following ACTH treatment. These data indicate that LTH enhances adrenal cortical sensitivity to the inhibitory effects of NO on cortisol production. Nitric oxide may, therefore, play an important role in regulating ACTH-induced cortisol production in the LTH fetal adrenal.
Assuntos
Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Hipóxia Fetal/metabolismo , Hidrocortisona/metabolismo , Óxido Nítrico/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Feto , Hidrocortisona/análise , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Nitroprussiato/farmacologia , Gravidez , Ovinos , Triazenos/farmacologiaRESUMO
This study was designed to test the hypothesis that fetal adrenal nitric oxide synthase (NOS) is elevated in response to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for approximately the last 100 days of gestation. Between days 138 and 141 of gestation, adrenal glands were collected from LTH fetuses and age-matched normoxic controls. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western analysis were used to quantify NOS expression, and NOS distribution was examined by immunohistochemistry and double-staining immunofluorescence for endothelial NOS (eNOS) and 17alpha-hydroxylase (CYP17). Neuronal NOS (nNOS) was expressed at very low levels and with no differences between groups. Expression of eNOS was significantly greater in the LTH group compared with control. Neuronal NOS was distributed throughout the cortex while the greatest density of eNOS was observed in the zona fasciculata/reticularis area and eNOS co-localized with CYP17. We conclude that LTH enhances eNOS expression in the inner adrenal cortex which may play a role in regulation of cortisol biosynthesis in the LTH fetus.