Lipocortin 1 mediates an early inhibitory action of glucocorticoids on the secretion of ACTH by the rat anterior pituitary gland in vitro.

Abstract

Lipocortin 1 (LC1), a mediator of anti-inflammatory steroid action in some peripheral tissues, may contribute to the acute inhibitory effects of these steroids on hypothalamo-pituitary-adrenal (HPA) function. Accordingly, in the present study we have used an in vitro model to examine the potential role of this protein in the regulation of the release of corticotrophin (ACTH) from the anterior pituitary gland. Hypothalamic extracts (0.05-0.4 HE/ml), the 41 amino acid corticotrophin-releasing factor (CRF-41, 1-100 nM), the adenyl cyclase stimulator, forskolin (0.1 microM-10 mM), and the L-Ca2+ channel opener, BAY K8644 (0.01-10 mM), all caused concentration-dependent increases in the release in vitro of immunoreactive (ir)-ACTH from segments of rat anterior pituitary tissue. The secretory responses to submaximal concentrations of these secretagogues were overcome by preincubation of the tissue with dexamethasone (0.1 and 1 microM). LC1 was readily detectable by Western blotting in protein extracts of freshly excised pituitary tissue; a small proportion of the protein was found to be attached to the outer surface of the cell membranes where it was retained by a Ca(2+)-dependent mechanism. Exposure to dexamethasone (0.1 microM) in vitro did not affect the total LC1 content of the pituitary tissue but, over a 2-hour period, it caused a progressive two- to fivefold increase in the amount of LC1 attached to the outer surface of the cell; this response developed in parallel with the inhibitory effects of the steroid on ir-ACTH release. Both the dexamethasone-induced 'externalization' of LC1 by the pituitary tissue and the concomitant steroid-induced inhibition of peptide release were blocked by cycloheximide (1.0 microgram/ml) but not by actinomycin D (0.5 microgram/ml). A stable N-terminal lipocortin 1 fragment, LC1(1-188) (10 pg-10 ng/ml), attenuated (p < 0.01) the release of ir-ACTH evoked by HE (0.1 HE/ml), CRF-41 (1 nM), forskolin (1 mM) and BAY K8644 (1 nM). Conversely, inclusion of the anti-LC1 antibody in the medium substantially overcame the inhibitory effects of dexamethasone (0.1 microM) on the release of ir-ACTH evoked by the secretagogues whilst a control isotype matched antibody was without effect. The results suggest that LC1 plays a key role in effecting the acute inhibitory actions of glucocorticoids on the secretion of ir-ACTH by the rat anterior pituitary gland.