Rapid Glucocorticoid Feedback Inhibition of ACTH Secretion Involves Ligand-Dependent Membrane Association of Glucocorticoid Receptors

Abstract

The hypothesis that rapid glucocorticoid inhibition of pituitary ACTH secretion mediates a feedforward/feedback mechanism responsible for the hourly glucocorticoid pulsatility was tested in cultured pituitary cells. Perifusion with 30 pM CRH caused sustained the elevation of ACTH secretion. Superimposed corticosterone pulses inhibited CRH-stimulated ACTH release, depending on prior glucocorticoid clearance. When CRH perifusion started after 2 hours of glucocorticoid-free medium, corticosterone levels in the stress range (1 μM) caused a delayed (25 min) and prolonged inhibition of CRH-stimulated ACTH secretion, up to 60 minutes after corticosterone withdrawal. In contrast, after 6 hours of glucocorticoid-free medium, basal corticosterone levels inhibited CRH-stimulated ACTH within 5 minutes, after rapid recovery 5 minutes after corticosterone withdrawal. The latter effect was insensitive to actinomycin D but was prevented by the glucocorticoid receptor antagonist, RU486, suggesting nongenomic effects of the classical glucocorticoid receptor. In hypothalamic-derived 4B cells, 10 nM corticosterone increased immunoreactive glucocorticoid receptor content in membrane fractions, with association and clearance rates paralleling the effects on ACTH secretion from corticotrophs. Corticosterone did not affect CRH-stimulated calcium influx, but in AtT-20 cells, it had biphasic effects on CRH-stimulated Src phosphorylation, with early inhibition and late stimulation, suggesting a role for Src phosphorylation on the rapid glucocorticoid feedback. The data suggest that the nongenomic/membrane effects of classical GR mediate rapid and reversible glucocorticoid feedback inhibition at the pituitary corticotrophs downstream of calcium influx. The sensitivity and kinetics of these effects is consistent with the hypothesis that pituitary glucocorticoid feedback is part of the mechanism for adrenocortical ultradian pulse generation.

The Pulsatility of ACTH Secretion in the Rat Anterior Pituitary Cell Perifusion System

“…Preliminary experiments in a previous study indicated that 5-minuteincreasing pulses of CRH resulted in concentration-dependent ACTH secretion with a threshold of 30 pM [20]. In this study, the lowest physiological levels of CRH, 10pM was not able to produce a marked increase in ACTH secretion (Fig.…”

Non-Genomic Action of Androgens is Mediated by Rapid Phosphorylation and Regulation of Androgen Receptor Trafficking

“…After cells were harvested, cytoplasmic, membrane, and nuclear protein fractions were separated using the subcellular protein fractionation kit for cultured cells (Pierce, Life Technologies) as previously described [22]. Protein concentrations were determined by the bicinchoninic acid protein assay.…”

Accelerated Episodic Luteinizing Hormone Release Accompanies Blunted Progesterone Regulation in PCOS-like Female Rhesus Monkeys (Macaca Mulatta) Exposed to Testosterone during Early-to-Mid Gestation

“…In addition, mifepristone is inconsistent in elevating LH levels when administered to healthy women during the early follicular phase [112][113][114] and fails to reverse exogenous E 2 and progesterone-induced slowing of GnRH pulse frequency in healthy women when it replaces 10 days of prior exogenous progesterone therapy [69]. Such unexpectedly mixed effects of mifepristone may stem from its suppression of Ca ++ -mediated membrane depolarization [115] crucial for exocytosis of GnRH and LH, and from its antagonism at the glucocorticoid receptor [116], potentially inducing compensatory elevation in hypothalamic CRH, and potentially, GnRH suppression [117].…”