Long-Term Hypoxia Enhances Cortisol Biosynthesis in Near-Term Ovine Fetal Adrenal Cortical Cells

Abstract

This study was designed to determine the potential mechanism(s) of previously observed enhanced fetal cortisol secretion following exposure to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3,820 m) for approximately the last 100 days of gestation. Between days 138-141 of gestation, adrenal glands were collected from LTH and age-matched normoxic control fetuses. cAMP, cortisol and steroidogenic acute regulatory protein (StAR) were measured in response to ACTH stimulation. Cortisol responses to ACTH were also measured in the presence of either the PKA inhibitor H-89, POMC or 22kDa pro-ACTH. Cortisol output was higher in the LTH group compared to the control, (p< 0.05) following ACTH treatment while the cAMP response was similar in both groups. Although PKA inhibition decreased cortisol production in both groups, however no differences were observed between groups. Western analysis revealed a significant increase in protein expression for StAR in the LTH group (p<0.05, compared to control). POMC and 22 kDa proACTH did not alter the cortisol response to ACTH treatment. Results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in the LTH group is not the result of differences in cAMP generation or PKA. We conclude that enhanced cortisol production in LTH adrenals is the result of enhanced protein expression of StAR and potential downstream signaling pathways.

Extracellular signal-regulated kinases (ERK1/2) signaling pathway plays a role in cortisol secretion in the long-term hypoxic ovine fetal adrenal near term

“…In an effort to elucidate the mechanism(s) responsible for the enhanced cortisol biosynthesis in response to an acute stress (or ACTH) in the LTH fetus, we recently investigated the effect of LTH on key elements of the ACTH/cAMP signaling pathway that regulate cortisol biosynthesis in fetal FACs (40). We found no apparent differences in cAMP production or subsequent PKA activation that could explain the observed difference in cortisol production between control and LTH FACs.…”

“…Absorbance was measured at 562 nm on a Synergy HT MultiDetection Microplate reader (BioTek, Winooski, VT) using Gen5 software. Levels of ERK1/2 protein were determined by Western blotting using methods, which we have previously described and validated (28,29,40). Briefly, protein samples were denatured by boiling for 5 min, and 3 g (ERK1/2) or 4 g (pERK1/2) of protein were loaded per lane.…”

“…On days 138 -141 of gestation, both LTH and normoxic control ewes were sedated with pentobarbital sodium, intubated, and maintained under general anesthesia with 1.5-2% halothane in oxygen. Fetuses were then delivered through a midline laparotomy, and the fetal adrenal glands were collected in ice-cold media M-199 (Sigma-Aldrich, St. Louis, MO), containing 2.2 g sodium bicarbonate, 2.0 g bovine serum albumin, and 0.1 g L-glutamine as we have previously described (40).…”

“…All experiments were conducted on FACs at 37°C. All procedures were performed by methods that we have previously described and validated (40).…”

“…While the LTH fetus maintains seemingly normal basal plasma cortisol concentrations, paradoxically, basal ACTH and acute secondary stress-induced cortisol biosynthesis is enhanced (2,21,28). We recently demonstrated that the enhanced cortisol production observed in the LTH fetal adrenocortical cells (FACs) compared with normoxic FACs in response to ACTH was not the result of enhanced cAMP production and/or protein kinase A (PKA) stimulation (40).…”