Long-term hypoxia enhances cortisol biosynthesis in near-term ovine fetal adrenal cortical cells

Abstract

This study was designed to determine the potential mechanism/mechanisms of previously observed enhanced fetal cortisol secretion following exposure to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for approximately the last 100 days of gestation. Between the gestation days of 138 and 141, adrenal glands were collected from LTH and age-matched normoxic control fetuses. Cyclic adenosine monophosphate (cAMP), cortisol, and steroidogenic acute regulatory (StAR) protein were measured in response to adrenocorticotropic hormone (ACTH) stimulation. Cortisol responses to ACTH were also measured in the presence of the protein kinase (PKA) inhibitor H-89, proopiomelanocortin (POMC), or 22-kDa pro-ACTH. Cortisol output was higher in the LTH group compared to the control (P < .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 < .05, compared to control). Proopiomelanocortin and 22-kDa pro-ACTH 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.

Figure 1.

Time course of basal (A) and ACTH-stimulated (B) cortisol production in control and LTH FACs. Under basal conditions, cortisol output was significantly greater in the LTH group compared with control (*P < .05). Although ACTH (10 nmol/L) increased cortisol biosynthesis in both groups (n = 6 for each group), the response was significantly greater in the LTH group compared to control (P < .05). LTH indicates long-term hypoxia; FACs, fetal adrenal cortical cells; ACTH, adrenocorticotropic hormone.

Figure 2.

Time course of basal (A) and ACTH-stimulated (B) cAMP production in control and LTH FACs. There was no difference in either basal or ACTH-stimulated cAMP generation between control and LTH FACs (n = 6 for each group). LTH indicates long-term hypoxia; FACs, fetal adrenal cortical cells; ACTH, adrenocorticotropic hormone; cAMP, cyclic adenosine monophosphate.

Figure 3.

Effects of ACTH-stimulated cortisol production in response to PKA inhibition with H-89. In response to ACTH alone, cortisol secretion increased significantly in both groups compared to basal cortisol secretion (*P < .05). Pretreatment with H-89 returned cortisol secretion to basal levels. ACTH, adrenocorticotropic hormone; PKA, protein kinase. Control n = 6, LTH n = 7

Figure 4.

Steroidogenic acute regulatory (StAR) protein expression in control (n = 6) and LTH (n = 6). FACs before and after ACTH treatment. Protein expression was significantly greater in LTH versus control FACs under both basal conditions and ACTH stimulation (*P < .05 compared to control). LTH indicates long-term hypoxia; FACs, fetal adrenal cortical cells; ACTH, adrenocorticotropic hormone.

Figure 5.

Adrenocorticotropic hormone (ACTH)-stimulated cortisol production in control (n = 5) and LTH (n = 5) FACs in response to ACTH alone or simultaneous treatment with ACTH and either POMC or 22-kDa pro-ACTH. ACTH treatment significantly increased cortisol production in both groups, which was unaffected by POMC or 22-kDa pro-ACTH (P < .05 compared to basal. LTH indicates long-term hypoxia; FACs, fetal adrenal cortical cells; POMC, proopiomelanocortin.