CRH and AVP-induced changes in synthesis and release of ACTH from the ovine fetal pituitary in vitro: negative influences of cortisol.

Abstract

During late gestation in sheep, fetal plasma adreno-corticotrophin (ACTH) and cortisol levels increase, and these are associated with increased pro-opiomelanocortin (POMC) mRNA levels in the anterior pituitary. Corticotrophin-releasing hormone (CRH) and vasopressin (AVP) are the primary hypophysiotrophic factors regulating ACTH secretion from the fetal sheep pituitary corticotroph, but previous reports with term fetal tissue have failed to show effects on levels of POMC mRNA. The objectives of the present study were to establish the effects of CRH and AVP on both synthesis and secretion of ACTH before term, and to determine how cortisol affects these responses. Fetal pituitaries were removed at d 138 of gestation (term approximately d 147), the anterior pituitary was separated, and the cells dispersed and placed in monolayer tissue culture. After 4 d, cells were treated for 18 h with several different concentrations (10(-6)-10(-9) M) and combinations of CRH, AVP, and cortisol. Following incubation, the medium was removed for ACTH analysis, and the cells fixed for POMC mRNA measurement and immunoreactive (ir)-ACTH localization. Separately, CRH and AVP significantly (p < 0.05) stimulated ACTH secretion in a dose-dependent manner. Simultaneous treatment of maximally stimulating levels of CRH and AVP augmented (p < 0.05) the output of ACTH. Cortisol did not affect basal (nonstimulated) ACTH output, but attenuated the neuropeptide-induced increases in ACTH secretion. This effect of cortisol was more pronounced in cells treated with CRH than in cells treated with AVP. POMC mRNA levels were increased by both CRH and AVP treatments in a dose-dependent manner, though there was no further increase in POMC mRNA when CRH and AVP were added together. Cortisol attenuated (p < 0.05) the neuropeptide-induced increases in POMC mRNA, though AVP-stimulated POMC mRNA levels were significantly higher than in cells treated with cortisol alone. Cortisol failed to alter non-stimulated POMC mRNA levels. We conclude that in late gestation: 1) Fetal pituitary corticotrophs respond to CRH and AVP by increasing POMC mRNA levels and ACTH secretion 2) AVP is more potent than CRH at the level of ACTH secretion, but not POMC transcription 3) Cortisol attenuates the synthetic and secretory responses to CRH and AVP, but has little effect in the non-stimulated state.