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 µM–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 µM). 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 Ca²⁺-dependent mechanism. Exposure to dexamethasone (0.1 µM) 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 µg/ml) but not by actinomycin D (0.5 µg/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 µM) 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.

To examine the influence of the putative satiety factor (GLP-1) on the hypothalamo-pituitary-gonadal axis, we used GT1-7 cells as a model of neuronal luteinizing hormone- releasing hormone (LHRH) release. GLP-1 caused a concentration-dependent increase in LHRH release from GT1-7 cells. Specific, saturable GLP-1 binding sites were demonstrated on these cells. The binding of [125I]GLP-1 was time-dependent and consistent with a single binding site (Kd = 0.07+/-0.016 nM; binding capacity = 160+/-11 fmol/mg protein). The specific GLP-1 receptor agonists, exendin-3 and exendin-4, also showed high affinity (Ki = 0.3+/-0.05 and 0.32+/-0.06 nM, respectively) as did the antagonist exendin-(9-39) (Ki = 0.98+/-0.24 nM). At concentrations that increased LHRH release, GLP-1 (0.5-10 nM) also caused an increase in intracellular cAMP in GT1-7 cells (10 nM GLP-1: 7.66+/-0.4 vs. control: 0.23+/-0.02 nmol/mg protein; P < 0.001). Intracerebroventricular injection of GLP-1 at a single concentration (10 microg) produced a prompt increase in the plasma luteinizing hormone concentration in male rats (GLP-1: 1.09+/-0.11 vs. saline: 0.69+/-0.06 ng/ml; P < 0.005). GLP-1 levels in the hypothalami of 48-h-fasted male rats showed a decrease, indicating a possible association of the satiety factor with the low luteinizing hormone levels in animals with a negative energy balance.

Glucocorticoids have been shown repeatedly to inhibit the secretion of TSH in experimental animals and in man but their site and mode of action are unknown. In the present study, we have used an in vitro model to examine the effects of dexamethasone on the resting and pharmacologically evoked secretion of TSH by the rat anterior pituitary gland, and to show how they are influenced by inhibitors of RNA/protein synthesis. In addition, we have investigated the potential role of lipocortin 1 (LC1), a protein shown previously to contribute to glucocorticoid action in several systems, as a mediator of the glucocorticoid-induced suppression of TSH release in our in vitro preparation. The significant (P < 0.01) increases in the release of immunoreactive (ir)TSH from rat anterior pituitary tissue initiated by submaximal concentrations of TRH (10 nmol/l), vasoactive intestinal polypeptide (VIP, 10 nmol/l) or the adenyl cyclase activator, forskolin (100 mumol/l) were reduced significantly (P < 0.05) by preincubation of the tissue with dexamethasone (0.1 mumol/l). In contrast, irTSH secretion evoked by a submaximal concentration of the L-Ca2+ channel opener BAY K8644 (10 mumol/l) was unaffected by the steroid, although readily antagonised (P < 0.01) by nifedipine (1-100 mumol/l). Inclusion of actinomycin D (1.78 mumol/l) or cycloheximide (0.8 mumol/l), inhibitors of RNA and protein synthesis respectively, in the medium effectively abrogated the inhibitory effects of dexamethasone (0.1 mumol/l) on the secretory responses to TRH (10 nmol/l), VIP (10 nmol/l) and forskolin (100 mumol/l). LC1 was readily detectable by Western blotting in protein extracts of freshly excised anterior pituitary tissue. A small proportion of the protein was found to be attached to the outer surface of the cells where it was retained by a Ca(2+)-dependent mechanism. Exposure of the tissue to dexamethasone (0.1 mumol/l) caused a pronounced increase in the amount of cellular LC1 attached to the outer surface of the cells and a concomitant decrease in the intracellular LC1 pool. Progesterone (0.1 mumol/l) and aldosterone (0.1 mumol/l) were also weakly active in this regard, but thyroxine and tri-iodothyronine (0.1 mumol/l) were not. Addition of an N-terminal LC1 fragment, LC1(1-188) (0.05-0.53 pmol/l) to the incubation medium reduced significantly (P < 0.01) the increases in irTSH release induced by TRH (10 nmol/l), VIP (10 nmol/l) and forskolin (100 mumol/l), but failed to influence (P < 0.05) those initiated by BAY K8644 (10 mumol/l). Furthermore, the inhibitory actions of dexamethasone (0.1 mumol/l) on the release of irTSH provoked by TRH (10 nmol/l), VIP (10 nmol/l) and forskolin (100 mumol/l) were substantially reversed (P < 0.01) by a specific monoclonal anti-LC1 antibody, while an isotype-matched control antibody was without effect. The results show clearly that dexamethasone, a semi-synthetic glucocorticoid, acts at the pituitary level to inhibit the neurochemically evoked release of irTSH. They also provide novel evidence that the inhibitory act...

Glucocorticoids have been shown repeatedly to inhibit the release of prolactin (PRL) in the rat but their site and mode of action is unknown. In the present study, we used an in vitro model to examine the requirement for protein synthesis for dexamethasone to suppress the release of immunoreactive (ir)-PRL release from the rat pituitary gland. In addition we have performed a series of in vitro and in vivo experiments to investigate the potential role in this regard of lipocortin 1 (LC1), a protein shown previously not only to mediate aspects of the anti-inflammatory and anti-proliferative actions of the glucocorticoids but also to contribute to the regulatory actions of the steroids in the brain-neuroendocrine system. In vitro, the release of ir-PRL from rat anterior pituitary tissue initiated by submaximal concentrations of VIP (10 nM), TRH (10 nM) or the adenyl cyclase activator forskolin (100 µM) was reduced significantly (p < 0.01) by preincubation (2 h) of the tissue with dexamethasone (0.1 µM). By contrast, ir-PRL release evoked by a submaximal concentration of the L-Ca²⁺ channel opener BAY K8644 (10 µM) was unaffected by the steroid although readily antagonised (p < 0.01) by nifedipine (1–100 µM). Exposure of the pituitary tissue to dexamethasone (0.1 µM) also caused a pronounced and highly significant increase in de novo protein synthesis, as assessed by the incorporation of ¹⁴C-lysine into the tissue (p < 0.001). This response was reduced markedly by the inclusion of the RNA and protein synthesis inhibitors, actinomycin-D (0.5 µg/ml) or cycloheximide (1.0 µg/ml), in the incubation medium (p < 0.001), both of which also effectively abrogated (p < 0.01) the dexamethasone-induced inhibition of the release of ir-PRL evoked by TRH, VIP and forskolin. Lipocortin 1 was readily detectable by Western blotting in protein extracts of freshly excised anterior pituitary tissue; a small proportion of the protein was found to be attached to the outer surface of the cells where it was retained by a Ca²⁺-dependent mechanism. Exposure of the tissue in vitro to dexamethasone (0.1 µM) or corticosterone (0.1 µM) but not 17β-oestradiol (0.1 µM) caused a pronounced increase in the amount of LC1 attached to the outer surface of the cells and concomitant decrease in the LC1 content of the intracellular LC1 pool. Addition of an N-terminal LC1 fragment, LC1_1–188 (10 pg-10 ng/ml), to the incubation medium reduced significantly (p < 0.01) the increases in ir-PRL release induced in vitro by VIP (10 nM) and forskolin (100 µM). By contrast, at all concentrations tested, LC1_1–188 (10 pg-10 ng/ml) failed to influence (p < 0.05) the highly significant (p < 0.01) ir-PRL response to TRH (10 nM). Similarly, the inhibitory actions of dexamethasone (0.1 µM) on the release of ir-PRL induced by VIP (10 nM) or forskolin (100 µM) but not by TRH (10 nM) were substantially reversed (p < 0.01) by a specific monoclonal anti-LC 1 antibody while an isotype-matched control antibody was without effect...

Our recent studies suggest that lipocortin 1 (LC1), a potential mediator of the anti-inflammatory, antiproliferative and anti-fever actions of glucocorticoids in peripheral tissues, may also contribute to the powerful negative feedback actions of the steroids on the hypothalamo-pituitary-adrenal (HPA) axis. In the present study we have used (1) an in vitro model to examine the influence of a specific neutralizing monoclonal anti-LC1 antibody (anti-LC1 mAb) on the capacity of dexamethasone to suppress the cytokine-induced release of the 41-amino acid corticotropin-releasing factor (CRF-41) and arginine vasopressin (AVP) from the rat hypothalamus and (2) a passive immunization protocol to assess the contribution of LCI to the inhibitory actions of dexamethasone on the HPA responses to immunological (i.p. injection of interleukin 1β, IL-1β) and surgical (laparotomy under ether anaesthesia) stress. In vitro, Il-1α (0.2 ng/ml), IL-1β (0.5 ng/ml), IL-6 (10ng/ml) and IL-8 (1 ng/ml) each caused significant increases in the release of immunoreactive (ir)-CRF-41 and ir-AVP from hypo-thalami removed from rats adrenalectomized 10-12 days before autopsy; these responses were readily inhibited by preincubation of the tissue with dexamethasone (10-⁷M). The inhibitory actions of the steroid were attenuated and, in many instances, abolished by inclusion in the medium of a monoclonal anti-LCI antibody (LCI mAb, diluted 1:15,000); an isotype-matched control antibody (antispectrin α+β, diluted 1:15,000) was ineffective in this regard. IL-1α (0.2 ng/ml), IL-1β (0.5 ng/ml) and IL-6 (10 ng/ml) also initiated similar increases in the release of CRF-41 and AVP from hypothalami from intact rats which were effectively blocked by dexamethasone (10^–7M). However, although the inhibitory actions of the steroid on the pharmacologically evoked release of CRF-41 were specifically overcome by anti-LC1 mAb (diluted 1:15,000), the steroid blockade of AVP release was not. In vivo, rats pretreated with either a polyclonal anti-LC 1 antibody (anti-LCI pAb, 1 ml/day s.c. for 2 days) or a corresponding volume of a nonimmune sheep serum (NSS) responded to immunological (IL-1β, 3 µg/kg i.p.) or surgical (laparotomy under ether anaesthesia) trauma with significant increases in the serum ACTH and corticosterone concentrations. In the NSS-treated groups, dexamethasone (100 µg/kg), which had no effect on the prestress concentrations of ACTH and corticosterone in the blood, completely prevented the HPA responses to both IL-1β and laparotomy. The steroid treatment also abolished the HPA responses to laparotomy in rats pretreated with anti-LC1 pAb. By contrast, passive immunization against LC1 largely overcame the ability of dexamethasone to inhibit the hypersecretions of ACTH and corticosterone provoked by IL-1β (3 µg/kg i.p.). The results suggest that LC1 plays an important role in mediating the inhibitory actions of dexamethasone on the HPA responses to cytokines in vitro and in vivo.