Adrenocorticotropic Hormone and PI3K/Akt Inhibition Reduce eNOS Phosphorylation and Increase Cortisol Biosynthesis in Long-Term Hypoxic Ovine Fetal Adrenal Cortical Cells

Abstract

This study was designed to determine the role of the MEK/ERK1/2 and PI3K/Akt pathways in cortisol production and endothelial nitric oxide synthase (eNOS) phosphorylation (peNOS) in the ovine fetal adrenal in response to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for the last 100 days of gestation (dGa). At 138 to 142 dGa, fetal adrenal cortical cells (FACs) were collected from LTH and age-matched normoxic fetuses. Cortisol production and peNOS were measured in response to pretreatment with the MEK/ERK1/2 pathway inhibitor UO126 (UO) and adrenocorticotropic hormone (ACTH) stimulation. UO126 reduced ACTH-stimulated cortisol in both normoxic and LTH FACs. UO126 alone or in combination with ACTH reduced peNOS in the normoxic group, while ACTH alone or ACTH + UO inhibited peNOS in LTH FACs. Additionally, cortisol was measured in response to pretreatment with UO and treatment with 22R-hydroxycholesterol (22R-OHC) or water-soluble cholesterol (WSC) with and without ACTH stimulation. UO126 had no effect on 22R-OHC-treated cells, but reduced cortisol in cells treated with WSC and/or ACTH. Cortisol and peNOS were also measured in response to pretreatment with PI3K/Akt pathway inhibitor Wortmannin (WT) and ACTH stimulation. Wortmannin further increased cortisol under ACTH-stimulated conditions and, like ACTH, reduced peNOS in LTH but not normoxic FACs. Together, these data suggest that in LTH FACs MEK/ERK1/2 does not regulate peNOS but that UO acts downstream from eNOS, possibly at cholesterol transport, to affect cortisol production in LTH FACs, while the PI3K/Akt pathway, along with ACTH, regulates peNOS and plays a role in the fetal adaptation to LTH in FACs.

Keywords: adrenocorticotropic hormone; hypoxia; sheep.

Figure 1.

Time course of cortisol production in normoxic and LTH FACs with MEK/ERK1/2 inhibition and ACTH stimulation. Stimulation with ACTH (100 pmol/L) increased cortisol production in both normoxic and LTH FACs. UO126 (10 µmol/L) pretreatment had no effect on basal cortisol but prevented increased cortisol biosynthesis in response to ACTH stimulation in both normoxic and LTH FACs (Normoxic n = 7, LTH n = 5). Values represent mean values ± standard error of the means (SEMs). *P < .05 compared to time 0, ^# P < .05 compared to UO + ACTH. FACs indicates fetal adrenocortical cells; LTH, long-term hypoxia; UO, UO126; ACTH, adrenocorticotropic hormone.

Figure 2.

Protein expression (A) and phosphorylation (B) of eNOS in response to MEK/ERK1/2 inhibition and ACTH stimulation in normoxic and LTH FACs as determined by Western analysis. Pretreatment with UO (10 µmol/L) with and without ACTH (100 pmol/L) stimulation had no effect on eNOS in either normoxic or LTH FACs. Pretreatment with UO and treatment with ACTH reduced on peNOS in normoxic FACs. Pretreatment with UO had no effect on peNOS in LTH FACs. Treatment with ACTH with and without UO pretreatment reduced peNOS in LTH FACs (Normoxic n = 7, LTH n = 5). Values represent mean values ± standard error of the means (SEMs). *P < .05 compared to control. FACs indicates fetal adrenocortical cells; LTH, long-term hypoxia; eNOS, endothelial nitric oxide synthase; peNOS, phosphorylated endothelial nitric oxide synthase; ROD, relative optical density; UO, UO126; ACTH, adrenocorticotropic hormone.

Figure 3.

Cortisol production in response to UO126 pretreatment and 22R-OHC or WSC stimulation with or without ACTH in normoxic and LTH FACs. ACTH (100 pmol/L), 22R-OHC (10 µmol/L), and WSC (10 µmol/L) stimulation increased cortisol production in both normoxic and LTH FACs, with a greater increase in dual stimulated cells. Pretreatment with UO blocked cortisol increase in cells stimulated with ACTH and WSC but had no effect on cells stimulated with 22R-OHC in both normoxic and LTH FACs (normoxic n = 6, LTH n = 7). Values represent mean values ± standard error of the means (SEMs). *P < .05 compared to untreated control, ^# P < .05 compared to normoxic. FACs indicates fetal adrenocortical cells; LTH, long-term hypoxia; C, Control; UO, UO126; ACTH, adrenocorticotropic hormone; 22R-OHC, 22R-hydroxycholesterol; WSC, water-soluble cholesterol.

Figure 4.

Time course of cortisol production in normoxic and LTH FACs with PI3K/Akt inhibition and ACTH stimulation. Stimulation with ACTH (100 pmol/L) increased cortisol production in both normoxic and LTH FACs. WT (10 nmol/L) pretreatment had no effect on basal cortisol but enhanced cortisol biosynthesis in response to ACTH stimulation in both normoxic and LTH (normoxic n = 7, LTH n = 9). Values represent mean values ± standard error of the means (SEMs). *P < .05 compared to time 0, ^# P < .05 compared to ACTH alone. FACs indicates fetal adrenocortical cells; LTH, long-term hypoxia; WT, Wortmannin; ACTH, adrenocorticotropic hormone.

Figure 5.

Protein expression (A) and phosphorylation (B) of eNOS in response to PI3K/Akt inhibition and ACTH stimulation in normoxic and LTH FACs as determined by Western analysis. Pretreatment with WT (10 nmol/L) with and without ACTH (100 pmol/L) stimulation had no effect on eNOS in both normoxic and LTH FACs. Pretreatment with WT (10 nmol/L) with and without ACTH (100 pmol/L) stimulation had no effect on peNOS in normoxic FACs but reduced peNOS in LTH FACs (normoxic n = 7, LTH n = 9). Values represent mean values ± standard error of the means (SEMs). *P < .05 compared to time 0. FACs indicates fetal adrenocortical cells; LTH, long-term hypoxia; eNOS, endothelial nitric oxide synthase; peNOS, phosphorylated endothelial nitric oxide synthase; ROD, relative optical density; WT, Wortmannin; ACTH, adrenocorticotropic hormone.