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The role of glucocorticoids in the regulation of lipocortin 1 (LC1) mRNA/protein expression in the brain is uncertain. This study has examined the influence of dexamethasone on the disposition of LC1 protein in various central and peripheral tissues in the rat. LC1 was readily detectable in all tissues studied by Western blot analysis. Exposure to dexamethasone in vitro (0.1 microM, 3 h) or in vivo (20 micrograms/100 g i.p., 24 h before autopsy) had no discernible effects on intracellular LC1 levels but increased the amount of LC1 attached to the outer surface of cells (pericellular LC1) in cortex, hippocampus, hypothalamus, pituitary gland and peritoneal macrophages (in vitro only). The results suggest that in central tissues, as in the periphery, glucocorticoids promote the translocation of LC1 from intracellular to pericellular sites.

Our previous studies have identified a role for annexin 1 (also called lipocortin 1) in the regulatory actions of glucocorticoids (GCs) on the release of PRL from the rat anterior pituitary gland. In the present study we used antisense and immunoneutralization strategies to extend this work. Exposure of rat anterior pituitary tissue to corticosterone (1 nM) or dexamethasone (100 nM) in vitro induced 1) de novo annexin 1 synthesis and 2) translocation of the protein from intracellular to pericellular sites. Both responses were prevented by the inclusion in the medium of an annexin 1 antisense oligodeoxynucleotide (ODN; 50 nM), but not by the corresponding sense and scrambled ODN sequences. Unlike the GCs, 17beta-estradiol, testosterone, and aldosterone (1 nM) had no effect on either the synthesis or the cellular disposition of annexin 1; moreover, none of the steroids or ODNs tested influenced the expression of annexin 5, a protein closely related to annexin 1. The increases in PRL release induced in vitro by drugs that signal via cAMP/protein kinase A [vasoactive intestinal polypeptide (10 nM), forskolin (100 microM), 8-bromo-cAMP (0.1 microM)] or phospholipase C (TRH, 10 nM) were attenuated by preincubation of the pituitary tissue with either corticosterone (1 nM) or dexamethasone (100 nM). The inhibitory actions of the steroids on the secretory responses to vasoactive intestinal polypeptide, forskolin, and 8-bromo-cAMP were specifically quenched by inclusion in the medium of the annexin 1 antisense ODN (50 nM) or a neutralizing antiannexin 1 monoclonal antibody (antiannexin 1 mAb, diluted 1:15,000). By contrast, the ability of the GCs to suppress the TRH-induced increase in PRL release was unaffected by both the annexin 1 antisense ODN and the antiannexin 1 mAb. In vivo, interleukin-1beta (10 ng, intracerebroventricularly) produced a significant increase in the serum PRL concentration (P < 0.01), which was prevented by pretreatment of the rats with corticosterone (100 microg/100 g BW, sc). The inhibitory actions of the steroid were specifically abrogated by peripheral administration of an antiannexin 1 antiserum (200 microl, sc); by contrast, when the antiserum was given centrally (3 microl, intracerebroventricularly), it was without effect. These results support our premise that annexin contributes to the regulatory actions of GCs on PRL secretion and suggest that it acts at point distal to the formation of cAMP.

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This study exploited established immunoneutralization protocols and an N‐terminal annexin 1 peptide (annexin 1Ac2 – 26) to advance our knowledge of the role of annexin 1 as a mediator of acute glucocorticoid action in the rat neuroendocrine system in vivo. Rats were treated with corticosterone (500 μg kg−1, i.p.) or annexin 1Ac2 – 26 (0.1 – 10 ng rat−1, i.c.v.) and 75 min later with interleukin 1β (IL‐1β, 10 ng rat−1, i.c.v. or 500 μg kg−1, i.p). Blood was collected 1 h later for hormone immunoassay. Where appropriate, anti‐annexin 1 polyclonal antiserum (pAb) was administered subcutaneously or centrally prior to the steroid challenge. Corticosterone did not affect the resting plasma corticotrophin (ACTH) concentration but suppressed the hypersecretion of ACTH induced by IL‐1β (i.p. or i.c.v.). Its actions were quenched by anti‐annexin 1 pAb (s.c. or i.c.v) and mimicked by annexin 1Ac2 – 26. By contrast, corticosterone provoked an increase in serum growth hormone (GH) which was ablated by central but not peripheral administration of anti‐annexin 1 pAb. IL‐1β (i.c.v. or i.p.) did not affect basal GH but, when given centrally but not peripherally, it abolished the corticosterone‐induced hypersecretion of GH. Annexin 1Ac2 – 26 (i.c.v.) also produced an increase in serum GH which was prevented by central injection of IL‐1β. The results support the hypothesis that the acute regulatory actions of glucocorticoids on hypothalamo‐pituitary‐adrenocortical function require annexin 1. They also provide novel evidence that the positive influence of the steroids on GH secretion evident within this timeframe is effected centrally via an annexin 1‐dependent mechanism which is antagonized by IL‐1β. British Journal of Pharmacology (2001) 134, 887–895; doi:10.1038/sj.bjp.0704324

The hypothalamic-pituitary-adrenocortical (HPA) responses to bacterial infection are mediated, in part, by the actions of lipopolysaccharide (LPS) on pituitary folliculostellate (FS) cells that release pro-inflammatory cytokines [e.g. interleukin (IL)-6] and thereby facilitate adrenocorticotrophic hormone (ACTH) release from neighbouring corticotrophs. In the present study, two murine pituitary cell lines [TtT/GF (FS cells) and AtT20 D16:16 (corticotrophs)], alone and in co-culture, and an in vivo model of endotoxaemia were used to examine the potential role of nuclear factor-kappa B (NF-κB) in mediating LPS-induced ACTH secretion. Both cell lines expressed mRNAs for the key components of the LPS signalling system. LPS stimulated IL-6 release from TtT/GF cells via a glucocorticoid-sensitive, NF-κB-dependent mechanism; it also activated NF-κB in AtT20 cells, as did corticotrophin-releasing hormone (CRH). IL-6 potentiated (but LPS reduced) the stimulatory effects of CRH on ACTH release from AtT20 cells, whereas blockade of NF-κB (SC-514) increased the ACTH release induced by CRH in the presence or absence of LPS. In co-cultures, CRH and LPS acted synergistically to induce release of both IL-6 and ACTH. However, although SC-514 suppressed the release of IL-6 evoked by CRH and LPS, it potentiated the concomitant increase in ACTH release. In vivo both immunological (LPS) and psychological (restraint) stress increased intrapituitary NF-κB, whereas an NF-κB inhibitor (PHA781535E) attenuated the LPS-induced release of ACTH and abolished the HPA response to restraint stress. The results obtained in the present study support the premise that NF-κB plays an important role in mediating LPS signalling in the anterior pituitary gland, particularly in relation to IL-6 and ACTH secretion, and provide novel evidence that NF-κB blockade in vivo compromises stress-induced ACTH release.

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