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Annexin 1 (Lipocortin 1) Mediates the Glucocorticoid Inhibition of Cyclic Adenosine 3',5'-Monophosphate-Stimulated Prolactin Secretion¹

Department of Neuroendocrinology, Division of Neuroscience and Psychological Medicine, Imperial College School of Medicine, Charing Cross Hospital, London, United Kingdom W6 8RF; Department of Human Anatomy and Genetics, University of Oxford (J.F.M.), Oxford, United Kingdom OX1 3QX; and Department of Biochemical Pharmacology, The William Harvey Research Institute, St. Bartholomew’s and the Royal London School of Medicine at Queen Mary and Westfield College (R.J.F.), London, United Kingdom EC1M 6BQ

Address all correspondence and requests for reprints to: Prof. Julia Buckingham, Department of Neuroendocrinology, Division of Neuroscience and Psychological Medicine, Imperial College School of Medicine, Charing Cross Hospital, Fulham Palace Road, London, United Kingdom W6 8RF. E-mail: j.buckingham{at}ic.ac.uk

	Abstract

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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, 17ß-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 µM), 8-bromo-cAMP (0.1 µM)] 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-1ß (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 µg/100 g BW, sc). The inhibitory actions of the steroid were specifically abrogated by peripheral administration of an antiannexin 1 antiserum (200 µl, sc); by contrast, when the antiserum was given centrally (3 µl, 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.

	Introduction

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STRESS INITIATES a number of changes in the activity of the neuroendocrine system. The most obvious of these is activation of the hypothalamo-pituitary-adrenocortical axis, but in many instances the release of PRL is also increased markedly (1, 2, 3). The stress-induced release of corticotropin (ACTH) is tempered by the glucocorticoids (GCs), which exert powerful negative feedback effects at the levels of the anterior pituitary gland, the hypothalamus and elsewhere in the central nervous system (4). No such obvious feedback loop exists for PRL, which lacks a specific endocrine target gland, but a substantial body of evidence suggests that the GCs may also contribute to the regulation of this pituitary hormone. For example, in the rat exogenous GCs suppress the hypersecretion of PRL provoked by various physical (5, 6, 7, 8) and psychological (3) stresses. Endogenous GCs are also effective in this regard, and pharmacological blockade of GC receptors with, for example, mifepristone potentiates PRL release (9, 10), whereas adrenalectomy produces GC-reversible increases in the basal and stress-induced release of PRL (11). The inhibitory effects of the steroids on PRL release have been attributed to direct actions on the pituitary gland and the brain, in particular the hypothalamus (12, 13, 14, 15, 16). At the pituitary level, GCs inhibit PRL synthesis by repressing transcription of the PRL gene (12, 15). In addition, they exert a significant inhibitory influence on PRL release (13, 14) through a mechanism that is blocked by messenger RNA (mRNA) and protein synthesis inhibitors and must therefore require de novo generation of a protein messenger(s) (16). The nature of these proteins is as yet unknown. One potential candidate is annexin 1, a GC-inducible protein that has been implicated in the control of PRL release by decidual tissue (17, 18).

Annexin 1 (also known as lipocortin 1) is a well characterized member of the annexin family of Ca²⁺- and phospholipid-binding proteins. It was first identified as a potential mediator of the therapeutically important antiinflammatory actions of the GCs (19) and has since also been shown to contribute to the signaling mechanisms effecting the regulatory actions of the steroids in the neuroendocrine system (19). Annexin 1 is found in abundance in the anterior pituitary gland, particularly in the S100-positive folliculo-stellate cells (20), and in specific loci in the hypothalamus, where its expression and cellular disposition are regulated by GCs (19, 21, 22, 23, 24, 25). Our functional studies, which have involved the use in vitro and in vivo of immunoneutralization and antisense strategies and examination of the actions of recombinant annexin 1 and related peptides, have identified a key role for annexin 1 in effecting the acute inhibitory actions of GCs on the release of CRH and ACTH from the hypothalamus and pituitary gland, respectively (19, 24, 25, 26, 27). They have also provided novel evidence for a role for annexin 1 in the control of PRL secretion. They have thus demonstrated that the inhibitory effects of dexamethasone on the release of PRL induced in vitro by vasoactive intestinal polypeptide (VIP) or forskolin are mimicked by an N-terminal annexin 1 peptide [annexin-1(1–188)] and specifically ablated by an antiannexin 1 monoclonal antibody (mAb). In addition, they have shown that the suppressive effect of dexamethasone on the rise in serum PRL provoked in vivo by a surgical stress is quenched by passive immunization against annexin 1 (16). The present study, which exploits our established antisense and immunoneutralization strategies, was designed to examine further the role of annexin 1 in effecting the inhibitory actions of GCs on PRL release in vitro and in vivo.

	Materials and Methods

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Animals
Adult male Sprague Dawley (200 g) rats were housed in a quiet room with controlled lighting (lights on, 0800–2000 h), temperature (21–23 C), and humidity (50%). Food and water were available ad libitum. Animals used for in vitro experiments were bred in-house from a closed colony (coded CFY) and housed after weaning in groups of five per cage. Those used for the in vivo studies were purchased from Harlan Olac (Banbury, UK) and caged in pairs; they were handled daily by the person responsible for the subsequent experimental procedures for 2 weeks before use. All experiments were started between 0800–0900 h to avoid any circadian influences.

Oligodeoxynucleotide preparations
In line with our previous studies (27), the annexin 1 antisense oligodeoxynucleotide (ODN) sequence was targeted to bases 83–98 inclusive (3'-G GTC CTG GTG GAA ACA-5'), which code for amino acids 29–33. This 16-base sequence, which comprises approximately 60% GC residues, is unique and specific to annexin 1 (27). From this sequence the complementary antisense ODN (3'-TCT TTC CAC CAG GAC C-5') and a scrambled ODN sequence (3'-TTC CTC TAC GAC CGA G-5') were constructed together with the annexin 1 sense sequence (3'-G GTC CTG GTG GAA ACA-5'). The ODNs were protected from degradation by the addition of two phosphorothioate groups at both the 3'- and the 5'-end (100% efficiency at 10 µM; Oswel, University of Southampton, Southampton, UK),

Antiannexin 1 antisera
Neutralizing antiannexin 1 monoclonal and polyclonal antisera of proven efficacy and specificity were employed (24, 25, 26). The monoclonal antibody (antiannexin 1 mAb, Zymed Laboratories, Inc., Cambridge Biosciences, Cambridge, UK; clone Z013, raised against bovine lung annexin 1) was used for the in vitro studies together with an isotype-matched (IgG1) control mAb (antispectrin and ß mAb, Sigma, Poole, UK). A polyclonal antiannexin 1 antiserum was used in the in vivo studies (antiannexin 1 pAb, a gift from Dr Jamie Croxtall, William Harvey Research Institute, London, UK). It was raised in sheep against human recombinant annexin 1 and purified (26) with an Immunopure (A/G) IgG purification kit (Pierce Chemical Co., Rockford, IL) before use; a similarly purified nonimmune sheep serum (NSS) was used a control. Antiannexin 1 pAb was also used for immunoprecipitation of annexin 1. Where appropriate, annexin 5 was precipitated using a polyclonal antiserum raised in sheep against human placental annexin 5 (antiannexin 5 pAb, gift from Dr. Jeff Browning, Cambridge, MA).

Incubation of pituitary tissue
Preparation and incubation of dispersed anterior pituitary cells. Suspensions of dissociated anterior pituitary cells were prepared as described previously (27). Briefly, the cells, obtained postmortem from decapitated rats, were dissociated by incubation (1 h, 37 C) with collagenase (0.2%, wt/vol; Roche Molecular Biochemicals, Sussex, UK) and deoxyribonuclease (0.05%, wt/vol; Sigma) in Earle’s Balanced Salt Solution (EBSS; Sigma, pH 7.4, phenol red free) enriched with BSA (0.4%; Sigma); the dispersion was aided by gentle trituration (30 sec every 10 min). The resulting cell suspension was centrifuged (300 x g, 10 min), the pellet was resuspended in 5 ml BSA-enriched EBSS, and the suspension was filtered through a 20-µm pore size nylon mesh to remove any large clumps of debris. The filtrate was then centrifuged (300 x g, 10 min), and the pellet was resuspended in 5 ml incubation medium [1% aprotinin (vol/vol; Bayer Corp. Ltd., Saffron Walden, UK) and 1% penicillin/streptomycin (vol/vol; Sigma) in EBSS, pH 7.4]. The cells were examined at the light microscope level to verify the effectiveness of the dispersion and were counted using a hemocytometer. Cell viability was assessed by the trypan blue exclusion test and was always greater than 95%.

The cells were plated out at a density of 2.5 x 10⁵ cells/ml·well in 24-well cell culture plates (Costar, Cambridge, MA) and incubated for 2.5 h at 37 C in a humidified atmosphere saturated with 95% O₂-5% CO₂ gas. They were then challenged for 1 h with VIP (0.1 nM to 1 µM), forskolin (0.1 nM to 1 mM), 8-bromo-cAMP (8-Br-cAMP; 10 pM to 10 µM) or TRH (1–100 nM); controls were incubated with an equal volume of medium/vehicle alone. After centrifugation (600 x g, -4 C, 10 min), the supernatant fluid was harvested and either assayed immediately for immunoreactive (ir-) PRL or stored in aliquots (300 µl) at -20 C for subsequent peptide measurement. In some experiments the pituitary cells were retained for annexin 1 and annexin 5 measurements. Where appropriate steroids [corticosterone, (10 nM), dexamethasone (100 nM), and 17ß-estradiol, testosterone, and aldosterone (1 nM)] were included in the medium throughout the experiment. Annexin 1 antisense, sense, or scrambled ODNs (50 nM) were also added to the medium at the beginning of the experiment as required and replenished at 1.5 and 2.5 h. For studies involving the measurement of newly synthesized annexin 1 or annexin 5, the pituitary cells were preincubated with ³⁵S-labeled cysteine/methionine (SA, >1000 Ci/nmol; Pro-mix, Amersham International, Aylesbury, UK) for 10 min before the addition of steroids and/or the ODNs.

Static incubation of anterior pituitary segments. The method used was described detail by Taylor et al. (16). Briefly, anterior pituitary glands were removed from rats immediately after decapitation and divided into 4 pieces of approximately equal size. The segments were distributed randomly (1 segment/well) in the wells of 24-well tissue culture plates (Costar, Cambridge, MA) and incubated in 1 ml EBSS (pH 7.4; phenol red free) enriched with aprotinin (1%; Bayer Corp. Ltd.) for 2 h at 37 C in a humidified atmosphere saturated with 95% O₂-5% CO₂ gas; the medium was changed after 1 and 1.5 h. The segments were then incubated for an additional 1 h in medium containing VIP (0.1 nM to 1 µM), forskolin (0.1 nM to 1 mM), 8-Br-cAMP (10 pM to 10 µM), or TRH (1–100 nM); controls were exposed to an equal volume (1 ml) of medium/vehicle alone. Where appropriate, corticosterone was included in the medium throughout both the preincubation and final incubation periods. Antiannexin 1 mAb or antispectrin and ß mAb (both diluted 1:15,000) were added to the final incubation medium as required. The medium from the final incubation was collected and either assayed immediately for ir-PRL or stored in aliquots (300 µl) at -20 C for subsequent peptide measurement. The pituitary segments were weighed on a torsion balance and discarded.

In vivo experiments
In a preliminary operation the rats were anaesthetized with sodium pentobarbitone (Sagital; 0.6 mg/100 g BW in a volume of 2 ml/100 g BW), and guide cannulas with stoppers were implanted stereotaxically into the third ventricle (0.0 mm anterior/posterior; 0.0 mm lateral; -8.0 mm dorsoventral from bregma). The rats were allowed to recover for 7–10 days. Antiannexin 1 mAb or NSS (control) was administered peripherally (200 µl, sc) or centrally [3 µl/rat, intracerebroventricularly (icv)] 24 h or 15 min, respectively, before corticosterone challenge [100 µg/kg, ip; controls received a corresponding volume (2 ml/kg) of the saline vehicle]. After an additional 75 min, the rats were treated with either rat IL-1ß (10 ng/rat, icv) or a corresponding volume of its saline vehicle (3 µl, icv). The rats were decapitated 1 h later, and trunk blood was collected. The plasma was separated and stored (-80 C) for assay of PRL and TSH. The position of the cannula was verified postmortem.

Detection of [³⁵S]annexin 1 and [³⁵S]annexin 5
[³⁵S]Annexin 1 and [³⁵S]annexin 5 in pituitary cells preloaded in vitro with [³⁵S]cysteine-methionine were separated by immunoprecipitation and SDS-PAGE and visualized by autoradiography (27). For measurement of total annexin 1/annexin 5, the proteins were extracted initially by resuspending and homogenizing the pituitary cells in 1 ml PBS (Oxoid Chemicals Ltd., Basingstoke, UK), containing EDTA (10 mM; Sigma), Triton (1%, vol/vol; BDH Chemicals Ltd., Poole, UK), phenylmethylsulfonylfluoride (1 mM; Sigma) and aprotinin (1%, vol/vol). In some instances, proteins bound to the outer surface of the cell membranes by Ca²⁺-dependent mechanisms (pericellular annexin 1/annexin 5) were removed before the tissue extraction by washing the cells for 1 min in a cocktail (250 µl) of EDTA (1 mM; a Ca²⁺ chelator) and protease inhibitors [1 mM phenylmethylsulfonylfluoride and 1% (vol/vol) aprotinin]. Annexin 1 and annexin 5 in the remaining tissue (intracellular annexin 1/annexin 5) were then extracted as described above.

Annexin 1 and annexin 5 were precipitated by a double antibody method either immediately or after storage of the extracted samples at -80 C (27). Briefly, antiannexin 1 pAb or antiannexin 5 pAb (10 µl; diluted 1:200 in 1 ml PBS) was added to each tube. The tubes were incubated for 24 h at 4 C, after which an immunoprecipitating antibody [50 µl; donkey antisheep (IDS Ltd., Bolton, UK) diluted 1:10 in PBS] was added, and the resultant suspension was vortexed and incubated for an additional 2 h. The suspension was centrifuged (4000 rpm, 1 h, 4 C), and the supernatant fluid was aspirated and discarded. The pellets from the pericellular and total/intracellular samples were resuspended in 50 or 250 µl PBS, respectively, and their protein contents were determined. The proteins [4 µg/channel (washes) and 40 µg/channel (extracts) in a volume of 20 µl] were separated by electrophoresis on SDS-polyacrylamide gels (midget gel electrophoresis system and power pack, LKB, Milton Keynes, UK) as described previously (24). The gels were then wrapped in cling film to prevent them from drying out and exposed to x-ray film (Kodak, Clywd, UK) for at least 2 days for detection of ³⁵S-labeled cysteine/methionine-labeled annexin 1 and annexin 5 (27). The film was developed using conventional techniques and reagents (all from Kodak). The OD of the bands was measured semiquantitatively, using a Fujix Bas 1500 imaging system (Fuji, Tokyo, Japan) with a low level light-sensitive camera and TINA software (Raytek, Sheffield, UK). Specificity studies confirmed that none of the drugs or ODNs employed in the experiments interfered with either the running of the gels or the binding of the antibody.

Hormone assays
PRL was measured in duplicate by a direct RIA (16) using reagents supplied by the National Hormone and Pituitary Program (Ogden Bioservices, Rockville, MD). The reference preparation, antiserum, and tracer (labeled with ¹²⁵I) were coded rat PRL RP-3, antirat PRL S-9, and PRL 1–5, respectively. The sensitivity of the assay was 0.2 ng/ml, with inter- and intraassay coefficients of variation of 9.4% and 10.1%, respectively (n = 6). Dilution curves of test samples were parallel with those of the standard PRL preparation. The antiserum showed negligible cross-reactivity with rat LH, TSH, GH, FSH, and ACTH; VIP, forskolin, 8-Br-cAMP, TRH, antiannexin 1 mAb, antispectrin and ß mAb, antiannexin 1 pAb, annexin 1 ODNs (antisense, sense and scrambled sequences), corticosterone, dexamethasone, 17ß-estradiol, testosterone, and aldosterone were also inactive in the assay in concentrations likely to be present in the samples. Serum TSH was measured by RIA using reagents supplied by the National Hormone and Pituitary Program (Ogden Bioservices) and established protocols (28).

Drugs
The following were used: VIP (Bachem, Saffron Walden, UK), forskolin (Sigma), 8-Br-cAMP (Sigma), TRH (Roche, Herts, UK), dexamethasone sodium phosphate (David Bull Laboratories, Inc., Warwick, UK), and corticosterone, 17ß-estradiol, testosterone, and aldosterone (all from Sigma). Forskolin, corticosterone, 17ß-estradiol, testosterone, and aldosterone were each dissolved initially in small amounts of ethanol and diluted subsequently in incubation medium; the final concentration of ethanol never exceeded 0.01%, and appropriate controls were included in all experiments. The remaining drugs were dissolved directly in incubation medium immediately before use.

Data analysis
Comparisons of band densities on the autoradiographs were made within gels only. Responses to the steroids and ODNs were calculated as a percentage of the corresponding drug-free (i.e. basal) control and expressed as the mean ± SD (n = 3 gels); statistical comparisons between groups were made using the Mann-Whitney U test.

Data from the release studies were normally distributed (Shapiro and Wilks test) and were analyzed by standard parametric tests, ANOVA with post-hoc comparisons by Duncan’s multiple range test (in vitro experiments) and Scheffe’s test (in vivo experiments). Statistical comparisons were made within experiments only, and differences were considered significant if P < 0.05. Each of the studies was repeated several times (for specific details, see figure legends), and in all instances the data profiles were similar.

	Results

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Preliminary studies
Initial studies identified concentrations of VIP (10 nM), forskolin (100 µM), 8-Br-cAMP (1 µM), and TRH (10 nM) that initiated significant (P < 0.01), but submaximal (80–90%), increases in ir-PRL release that were almost completely abolished (90–100%) by corticosterone (1 nM) or dexamethasone (100 nM; data not shown). These concentrations were used in subsequent experiments.

Inhibition of GC-induced annexin 1 synthesis by antisense ODN
Figure 1 shows the effects of corticosterone (1 nM) and dexamethasone (100 nM) on the synthesis of total annexin 1 and total annexin 5 by pituitary cells (as [³⁵S]annexin 1 and [³⁵S]annexin 5, expressed as a percentage of the control) in the presence and absence of annexin 1 antisense, sense, and scrambled ODNs. [³⁵S]Annexin 1 (37 kDa) was detected in all samples (Fig. 1a). Exposure of the cells to corticosterone or dexamethasone caused a marked increase in [³⁵S]annexin 1 expression (P < 0.01 vs. basal). In the absence of the steroids, none of the ODN sequences tested had any obvious effect on the expression of [³⁵S]annexin 1. However, the annexin 1 antisense ODN prevented the rise in [³⁵S]annexin 1 induced by corticosterone and dexamethasone. By contrast, the annexin 1 sense and scrambled ODN sequences were inert in this regard. Parallel measurements of [³⁵S]annexin 5 (36 kDa) demonstrated that the synthesis of this closely related protein was unaffected by corticosterone, dexamethasone, and/or the antisense, scrambled, or sense ODNs (Fig. 1b).

View larger version (37K): [in this window] [in a new window]   Figure 1. Effects of corticosterone (1 nM) and dexamethasone (100 nM) on the expression of newly synthesized annexin 1 (a; i.e. [35S]annexin 1) and annexin 5 (b; [35S]annexin 5) in anterior pituitary cells in the presence and absence of annexin 1 antisense, sense or scrambled ODNs (50 nM). , Control; , corticosterone; , dexamethasone. Values were derived by scanning autoradiographs (see Materials and Methods) and represent the mean ± SD (n = 3) density of bands of [35S]annexin 1 (37 kDa) and [35S]annexin 5 (36 kDa) immunoreactivity, expressed as a percentage of the control. **, P < 0.01 vs. corresponding steroid-free control; , P < 0.01 vs. corresponding ODN-free control (by Mann-Whitney U test).

View larger version (72K): [in this window] [in a new window]   Figure 2. Autoradiographs demonstrating the effects of corticosterone (1 nM) in the presence and absence of LC1 antisense or control ODNs (50 nM) on the distribution of newly synthesized annexin 1 (as [35S]annexin 1) between the pericellular (a) and intracellular (b) pools in anterior pituitary cells. Parallel measurements of intracellular annexin 5 (as [35S]annexin 5) are shown in c. Lane 1, Basal; lane 2, corticosterone; lane 3, annexin 1 antisense ODN; lane 4, annexin 1 antisense ODN and corticosterone; lane 5, scrambled ODN sequence; lane 6, scrambled ODN sequence and corticosterone; lane 7, annexin 1 sense ODN; lane 8, annexin 1 sense ODN and corticosterone. For quantification of the labeled bands, see Table 2.

View larger version (120K): [in this window] [in a new window]   Figure 3. Autoradiographs comparing effects of corticosterone (1 nM) with those of dexamethasone (100 nM) and other steroids (17ß-estradiol, testosterone, and aldosterone; 1 nM) on the distribution of newly synthesized annexin 1 (as [35S]LC1) between the pericellular (a) and intracellular (b) pools in anterior pituitary cells. Parallel measurements of intracellular annexin 5 (as [35S]annexin 5) are shown in c. Lane 1, Basal; lane 2, corticosterone; lane 3, dexamethasone; lane 4, 17ß-estradiol; lane 5, testosterone; lane 6, aldosterone. For quantification of the labeled bands, see Table 3.

In vitro studies
Antisense studies. Figures 4 and 5 illustrate the inhibitory effects of GCs on the secretagogue-driven release of ir-PRL from enzymatically dispersed pituitary cells in vitro and show how they are influenced by the annexin 1 antisense and control ODNs. VIP (10 nM; Fig. 4a), forskolin (100 µM; Fig. 4b), 8-Br-cAMP (1 µM; Fig. 4c), and TRH (10 nM; Fig. 4d) all produced significant increases in ir-PRL release, which were inhibited by preincubation of the tissues with corticosterone (1 nM). In the absence of corticosterone, none of the ODNs tested (annexin 1 antisense, annexin 1 sense or the scrambled sequence; 50 nM) influenced either the basal release of ir-PRL (P > 0.05) or the rises in ir-PRL release induced by any of the four secretagogues; they also failed to influence resting ir-PRL release in the presence of corticosterone (P > 0.05). However, the annexin 1 antisense ODN (but not the sense or scrambled sequences) effectively reversed (P < 0.01) the inhibitory effects of the steroid on the release of ir-PRL evoked by VIP, forskolin, and 8-Br-cAMP; by contrast, it failed to quench the corticosterone-induced blockade of TRH-stimulated ir-PRL release (Fig. 4d). Further experiments (Fig. 5) revealed that the annexin 1 antisense ODN also specifically quelled the capacity of dexamethasone (100 nM) to suppress the significant increases (P < 0.01) in ir-PRL release induced by VIP (10 nM, Fig. 5a) or forskolin (100 nM, Fig. 5b); however, it failed to modify the suppressive effect of dexamethasone on the ir-PRL response to TRH (10 nM; Fig. 5c).

View larger version (46K): [in this window] [in a new window]   Figure 4. Effects of an annexin 1 antisense ODN and control ODN sequences on the inhibitory effects of corticosterone (1 nM) on the release of ir-PRL from freshly dispersed rat anterior pituitary cells induced in vitro by VIP (a; 10 nM), forskolin (b; 100 µM), 8-Br-cAMP (c; 1 µM), and TRH (d; 10 nM). , ODN free; , annexin 1 antisense ODN (50 nM); , scrambled ODN sequence (50 nM); , annexin 1 sense ODN (50 nM). The open areas at the base of each column represent peptide release in the absence of secretagogues in corresponding groups. Each column represents the mean ± SEM (n = 6). **, P < 0.01 vs. corresponding secretagogue-free control; , P < 0.01 vs. corresponding corticosterone-free control (by ANOVA and Duncan’s multiple range test). Typical data from three or four replicate experiments are shown.

View larger version (19K): [in this window] [in a new window]   Figure 5. Effects of an annexin 1 antisense ODN and control ODN sequences on the inhibitory effects of dexamethasone (100 nM) on the release of ir-PRL from freshly dispersed rat anterior pituitary cells induced in vitro by VIP (a; 10 nM), forskolin (b; 100 µM) and TRH (c; 10 nM). , ODN free; , annexin 1 antisense ODN (50 nM); , scrambled ODN sequence (50 nM); , annexin 1 sense ODN sequence (50 nM). The open areas at the base of each column represent peptide release in the absence of secretagogues in corresponding groups. Each column represents the mean ± SEM (n = 6). **, P < 0.01 vs. corresponding secretagogue-free control; , P < 0.01 vs. corresponding dexamethasone-free control (by ANOVA and Duncan’s multiple range test). Typical data from three or four replicate experiments are shown.

View larger version (39K): [in this window] [in a new window]   Figure 6. Effects of a neutralizing antiannexin 1 monoclonal antibody (antiannexin 1 mAb) and an isotype-matched control mAb (antispectrin and ß) on the inhibitory effects of corticosterone (1 nM) on the release of ir-PRL from rat anterior pituitary segments induced in vitro by VIP (a; 10 nM), forskolin (b; 100 µM), 8-Br-cAMP (c; 1 µM), and TRH (d; 10 nM). , Antibody free; , anti-LC1 mAb (diluted 1:15,000); , antispectrin and ß (diluted 1:15,000). The open areas at the base of each column represent peptide release in the absence of secretagogues in corresponding groups. Each column represents the mean ± SEM (n = 6). **, P < 0.01 vs. corresponding secretagogue-free control; , P < 0.01 vs. corresponding corticosterone-free control (by ANOVA and Duncan’s multiple range test). Typical data from three or four replicate experiments are shown.

View larger version (20K): [in this window] [in a new window]   Figure 7. Effects of a neutralizing antiannexin 1 polyclonal antibody (annexin 1 pAb) and control NSS administered centrally (a; 3 µl/rat, icv) or peripherally (b; 200 µl, sc) on the inhibitory effects of corticosterone (100 µg/kg, ip) on the IL-1 (50 µg/rat, icv)-induced increases in serum PRL concentrations. , NSS; , annexin 1 pAb. Each column represents the mean ± SEM (n = 6–8). **, P < 0.01 vs. corresponding secretagogue-free control; , P < 0.01 vs. corresponding corticosterone-free control (by ANOVA and Scheffe’s test). Typical data from two replicate experiments are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our experimental design exploited established immunoneutralization (16, 24, 26) and antisense (27) strategies to examine the role of annexin 1 in the regulation of PRL secretion in vivo and in vitro. The in vivo studies showed clearly that the ability of exogenous corticosterone to block the hypersecretion of PRL induced by a central injection of rIL-1ß is blocked by peripheral administration of an antiannexin 1 antiserum; they thus suggest that annexin 1 plays an obligatory role in effecting the regulatory actions of the steroid in this experimental paradigm and accord with our previous findings that indicated that the inhibitory actions of dexamethasone on the PRL response to surgical stress require annexin 1 (16). These arguments are supported by evidence that the passive immunization protocol we used has been shown to generate antibody titers sufficient to neutralize endogenous annexin 1 (26) and to quench the acute regulatory actions of GCs on the hypothalamo-pituitary-adrenocortical axis (26) and in several models of inflammation and cell growth (19, 29, 30); moreover, the actions of the antibody appear to be specific, as in the experiments reported here and previously (16, 24) a control (nonimmune) serum was consistently without effect. Our previous studies suggest that when given sc, the antiannexin 1 antiserum gains access to the brain and that substantial antibody titers are maintained in the hypothalamus and elsewhere in the central nervous system as well as in the periphery for 4–5 days after the injection (26). Our finding that central administration of the antibody in a dose sufficient to block specifically the acute regulatory actions of corticosterone on the release of ACTH and GH (31) failed to quench the inhibitory influence of corticosterone on IL-1ß-driven PRL secretion is thus interesting and suggests that the principal site of action of the antibody, and hence of the annexin 1-dependent regulatory actions of corticosterone on PRL release, is within the periphery, probably at the pituitary level. Our in vitro studies, which involved both analysis of annexin 1 expression and examination of the effects of ablating annexin 1 by means of antisense probes and neutralizing antibodies on PRL release, provided firm evidence to support this view.

The in vitro studies were based on two well established models (viz. pituitary segments or enzymatically dispersed pituitary cells, both maintained in static incubation) which respond reproducibly to VIP, forskolin, 8-Br-cAMP, and TRH with concentration-dependent increases in PRL release that are readily prevented by preincubation of the tissue/cells with corticosterone or dexamethasone (16). The advantages and disadvantages of these systems have been reviewed recently (32). The segment system, which we have validated for annexin 1 immunoneutralization studies (16, 24, 28), has a key advantage in that it retains the three-dimensional structure of the heterogeneous cell population and thus sustains the autocrine and paracrine communication inherent to the tissue as a whole. Diffusion is, however, inevitably restricted, and concerns about the ability of nucleotide probes to penetrate the tissue pieces and gain access to their intracellular targets prompted us to undertake the antisense studies in a dispersed cell preparation. In this system, as in the segment system, the ultrastructure of the cells is well maintained throughout the incubation period (data not shown); however, although the improved diffusion characteristics permit rapid entry of small nucleotides probes into the cells (27), the loss of the three-dimensional structure and the consequent realignment of the cells could be disadvantageous.

The expression studies confirm reports that annexin 1 (37 kDa) is expressed in abundance by rat anterior pituitary tissue in vitro (22, 23, 24, 27) as it is in vivo (21, 22). They also show for the first time that corticosterone mimics the well defined effects of dexamethasone (22, 23, 24, 27) on the synthesis and cellular disposition of annexin 1 in the pituitary cells; thus, corticosterone induces de novo annexin 1 synthesis (indexed by the incorporation of ³⁵S-labeled amino acids into immunoprecipitated protein) and subsequent translocation of the newly synthesized protein from an intracellular compartment to a pericellular site, where it adheres to the surface of cells by a Ca²⁺-dependent mechanism (24). By contrast, aldosterone, testosterone, and 17ß-estradiol were without effect, suggesting that the effects of dexamethasone and corticosterone are GC specific. Moreover, none of the steroids tested influenced the expression annexin 5, a closely related protein that is abundant in the anterior pituitary gland (21, 27, 33) but does not appear to be exported from the cells in a manner analogous to annexin 1 and is thus undetectable in the EDTA cell surface wash. Interestingly, two recent reports describe estrogen-reversible increases in the expression of both annexin 1 and annexin 5 in the rat pituitary gland after ovariectomy (33, 34). These effects were, however, slow to emerge and would therefore have been unlikely to be evident within the time frame of the present study (3.5 h); additional studies using a longer contact times and tissue taken from female animals after ovariectomy are required to investigate this further.

Although there has been criticism in the literature of antisense technology and further controversy regarding the molecular basis of antisense action, there are now many examples where the exploitation of antisense strategies has helped to define the functional role of gene products (35, 36, 37, 38, 39). In the present study we sought to overcome these criticisms by supporting our antisense studies with complementary experiments in which annexin 1 activity was ablated by immunoneutralization and also by using antiannexin 1 probes (antisense ODN and neutralizing antiserum) of proven efficacy and specificity (16, 24, 28). The antisense probe was directed against a nucleotide sequence specific to rat annexin 1 mRNA and designed according to the criteria of Wahlstadt (34); it thus comprised 16 bases with a guanine/cytosine content of 55% and was protected from degradation by the addition of 2 phosphorothioate groups to both the 3'- and 5'-ends of the molecule; complementary sense and scrambled ODN sequences were used as controls. As the pharmacokinetics and potency of the ODN probes may vary according to the tissue preparation used, and phosphorothioate derivatives are potentially toxic, preliminary concentration/time-response studies were performed to determine the minimum concentration (50 nM) and contact time (3.5 h) of antisense required to ablate GC-induced de novo annexin 1 expression. Using these parameters, the antisense ODN effectively prevented the increase in annexin 1 synthesis (as shown by [³⁵S]annexin 1) induced by corticosterone and, thus, the downstream appearance of newly synthesized protein on the cell surface. These findings, which correspond with those reported here and previously (27) with dexamethasone, appear to be specific, as the sense and scrambled ODN sequences were inert in this regard; moreover, none of the ODNs tested altered the expression of annexin 5 in the presence or absence of corticosterone. Interestingly, in the steroid-free controls, a low level of annexin 1 synthesis persisted regardless of the presence of the antisense probe. The reasons for this are obscure. In an earlier study using confocal microscopy and fluorescein tagging, we showed that our antisense ODN concentrates mainly in the nucleus of pituitary cells; it may therefore target the primary transcript rather than the mature mRNA already in the cytoplasm (27) or inhibit gene transcription by forming DNA/DNA hybrids or triplex DNA structures (35, 36).

Our functional studies showed clearly that exposure of the dispersed pituitary cells to the antiannexin 1 antisense ODN effectively and specifically reversed the marked inhibitory effects of corticosterone and dexamethasone on the release of PRL induced by VIP, forskolin, and 8-Br-cAMP. By contrast, the antisense probe failed to modify the inhibitory effects of the steroids on TRH-induced PRL secretion. The complementary experiments in which annexin 1 activity in corticosterone-treated tissue was specifically quenched with a well characterized (16, 24, 28) neutralizing monoclonal antibody yielded a similar profile of data. Importantly, the sense and scrambled ODN sequences failed to modify the resting or evoked release of PRL in the presence or absence of steroid as also did the control monoclonal antibody (antispectrin and ß), suggesting that the responses were specific. These findings are in full accord with the data from our previous study that demonstrated 1) that an N-terminal annexin 1 peptide [annexin-1(1–188)] inhibits the release of PRL induced by VIP and forskolin without affecting the secretory response to TRH, and 2) that immunoneutralization of annexin 1 substantially reverses the inhibitory actions of dexamethasone on VIP- and forskolin-stimulated PRL release in vitro, but fails to suppress the blockade of TRH-evoked PRL secretion induced by the steroid (16). As VIP and forskolin signal via cAMP, whereas TRH acts via phospholipase C (40), these findings together suggest that annexin 1 inhibits cAMP-dependent stimulus secretion coupling in the lactotrophs, acting at a point distal to the formation of cAMP in the signaling cascade; annexin 1 may thus augment the powerful inhibitory influence of dopamine (which depresses cAMP generation) on PRL release. Disruption of cAMP-dependent signaling may also explain the data from our in vivo experiments that identified a role for annexin 1 in the pituitary gland in effecting the inhibitory actions of corticosterone on the PRL response to IL-1ß. Although there are conflicting reports in the literature on the effects of exogenous IL-1 on PRL secretion in the rat (41, 42), possibly because the vast majority of workers have employed human rather than rat recombinant proteins, in our hands rIL-1ß reproducibly produces a marked increase in serum PRL when given centrally (via an indwelling icv cannula), but not peripherally. The mechanism involved is unclear, but complementary studies using human IL-1 suggest that the cytokine triggers a nitric oxide-dependent reduction in dopaminergic tone to the anterior pituitary gland (41); the consequent cAMP-driven increase in PRL release would thus represent a potential target for annexin 1.

The molecular mechanism by which annexin 1 disrupts cAMP-dependent signaling in the lactotrophs remains to be determined. We have previously suggested that the cellular exportation of annexin 1 is critical to its action, as it provides a means by which the protein may gain access to receptors on the outer surface of the cells and thereby initiate a biological response; annexin 1 may thus act as a paracrine or autocrine agent. This concept is supported by several lines of evidence. Firstly, treatments that block the exportation of the protein (annexin 1 antisense ODNs or protein synthesis inhibitors) also inhibit the regulatory actions of the steroids on cAMP-driven PRL release (16). Secondly, the antiannexin antisera that specifically reverse the inhibitory actions of GCs on cAMP-dependent PRL release would not be expected to penetrate cell membranes readily, but could effectively sequester annexin 1 at a pericellular site (24); similarly, annexin-1(1–188), which blocks cAMP-stimulated PRL secretion (16), may not enter cells easily. Thirdly, we have demonstrated the presence of high affinity (K_d, 13 nM), saturable, proteinaceous annexin 1-binding sites on the surface of several pituitary cell types, including lactotrophs (43); these sites resemble those on human peripheral leukocytes that have been deemed essential for annexin 1 activity (29). Finally, although we detected annexin 1 in both secretory and nonsecretory adenohypophyseal cells by fluorescence-activated cell sorting analysis, our immunohistochemical studies at the light and electron microscope levels suggest that the principal source of annexin 1 in the anterior pituitary gland is the S100-positive folliculostellate cells (20); these cells are well positioned to exert paracrine influences on hormone secretion, as their stellate projections lie in close apposition with the secretory cells (20).

In conclusion, our results provide new evidence that the well defined regulatory effects of GCs on PRL secretion are effected predominantly at the pituitary level through induction of a protein, annexin 1, that suppresses cAMP-driven PRL secretion. In addition, they confirm and extend our previous finding that these steroids also suppress TRH-stimulated PRL secretion, acting via an annexin 1-independent mechanism that requires de novo protein synthesis (16). The nature and molecular mode of action of the protein(s) generated are obscure, but reports (44) that dexamethasone increases both the synthesis and the activity of ATP-sensitive K⁺ channels in two PRL-secreting pituitary cell lines, GH₃ and GH₄C₁, together with evidence that the secretory response to TRH is associated with a decrease in membrane K⁺ flux (44, 45, 46, 47), raise the possibility that K⁺ channels are important in this regard. A further interesting and consistent observation in the present study is that, contrary to expectation, corticosterone proved consistently to be more potent in our in vitro preparations than dexamethasone. The reason for this is unclear. Dexamethasone was administered as the sodium phosphate salt, and it may be, therefore, that its access to its receptors is limited by the rate at which the free base is liberated. Alternatively, delivery of corticosterone to its receptors may be enhanced by type 1 11ß-hydroxysteroid dehydrogenase, which is present in the anterior pituitary gland (48) and would reactivate any corticosterone converted to the inactive 11-dehydro species.

	Acknowledgments

 
We are grateful to Dr. Jamie Croxtall (William Harvey Research Institute, London, UK) for the sheep antiannexin 1 antiserum, to Dr. Jeff Browning (Biogen Co., Inc., Cambridge, MA) for antiannexin 5 antiserum, and to the National Hormone and Pituitary Program for reagents for the PRL and TSH assays.

	Footnotes

 
¹ This work was supported by the Wellcome Trust (051887/B/97/Z) and the Charing Cross Hospital Trustees.

Received December 28, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Black PH 1994 Central nervous system-immune interactions: psychoneuroendocrinology of stress and its immune consequences. Antimicrobial Agents Chemother 38:1–6[Abstract/Free Full Text]
2. Weiner RI, Findell PR, Kordon C 1988 Role of classic and peptide neuromediators in the neuroendocrine regulation of LH and prolactin. In: Knobil E, Neill JD (ed) The Physiology of Reproduction. Raven Press, New York, pp 1253–1283
3. Yelvington DB, Weiss GK, Ratner A 1984 Effect of corticosterone on the prolactin response to psychological and physical stress in rats. Life Sci 35:1705–1711[CrossRef][Medline]
4. Buckingham JC, Cowell A-M, Gillies GE 1997 The neuroendocrine system: anatomy, physiology and responses to stress. In: Buckingham JC, Cowell A-M, Gillies GE (ed) Stress, Stress Hormones and the Immune System. Wiley & Sons, pp 9–47
5. Copinschi G, L’Hermite M, Léclerq R, Goldstein J, Vanhaelst L, Virasoro E, Robyn C 1975 Effects of glucocorticoids on pituitary hormonal responses to hypoglycaemia. Inhibition of prolactin release. J Clin Endocrinol Metab 40:442–449[Abstract]
6. Rossier J, French E, Rivier C, Shibasaki T, Guillemen R, Bloom FE 1980 Stress-induced release of prolactin: blockade by dexamethasone and naloxone may indicate ß-endorphin mediation. Proc Natl Acad Sci USA 77:666–669[Abstract/Free Full Text]
7. Piroli G, Grillo C, Ferrini M, Diaz Torga G, Rey E, Libertun C, De N 1993 Restoration by bromocriptine of glucocorticoid receptors and glucocorticoid negative feedback on prolactin secretion in estrogen-induced pituitary tumours. Neuroendocrinology 58:273–279[CrossRef][Medline]
8. Lopez-Calderon A, Ariznavarreta C, Calderon MD, Tresguerres JAF, Gonzalez Quijano MI 1989 Role of adrenal cortex in chronic stress-induced inhibition of prolactin secretion in male rats. J Endocrinol 120:269–273[Abstract]
9. Nishino Y, Michna H, Hasan SH, Schneider MR 1992 Involvement of the adrenal glands in the prolactin rise induced in the female rat by an anti-progestin, onapristone. J Steroid Biochem Mol Biol 41:841–845[CrossRef][Medline]
10. Caron RW, Salicioni AM, Deis RP 1994 Mifepristone treatment demonstrates the participation of adrenal glucocorticoids in the regulation of oestrogen-induced prolactin secretion in ovariectomized rats. J Steroid Biochem Mol Biol 48:385–389[CrossRef][Medline]
11. Watanobe H 1990 The immunostaining for the hypothalamic vasoactive intestinal peptide, but not for ß-endorphin, dynorphin-A or methionine-enkephalin, is affected by the glucocorticoid milieu in the rat: correlation with the prolactin secretion. Regul Pept 28:310–311
12. Evans, RH, Birnberg, NC, Rosenfeld MG 1992 Glucocorticoid and thyroid hormones transcriptionally regulate growth hormone gene expression. Proc Natl Acad Sci USA 79:7659–7663
13. Houben H, Denef C 1992 Negative regulation by dexamethasone of the potentiation of neuromedin C-induced growth hormone and prolactin release by estradiol in anterior pituitary cell aggregates. Life Sci USA 50:775–780
14. Robberecht W, Andries M, Denef C 1992 Stimulation of prolactin secretion from rat pituitary by luteinizing hormone releasing hormone: evidence against mediation by angiotensin II acting through a (Sar¹-Ala⁸)-angiotensin II-sensitive receptor. Neuroendocrinology 56:185–194[Medline]
15. Stephanou A, Sarlis NJ, Knight RA, Lightman SL, Chowdrey HS 1992 Glucocorticoid-mediated responses of plasma ACTH and anterior pituitary pro-opiomelanocortin, growth hormone and prolactin mRNAs during adjuvant-induced arthritis in the rat. J Mol Endocrinol 9:273–278[Abstract]
16. Taylor AD, Cowell A, Flower RJ, Buckingham JC 1995 Dexamethasone suppresses the release of prolactin from the rat anterior pituitary gland by lipocortin 1 dependent and independent mechanisms. Neuroendocrinology 62:530–542[Medline]
17. Handwerger S, Markoff E, Richards R 1991 Regulation of the synthesis and release of decidual prolactin by placental and autocrine/paracrine factors. Placenta 12:121–130[Medline]
18. Pihoker C, Feeney RJ, Su J-L, Handwerger S 1991 Lipocortin-1 inhibits the synthesis and release of prolactin from human decidual cells: evidence for autocrine/paracrine regulation by lipocortin 1. Endocrinology 128:1123–1128[Abstract]
19. Buckingham JC, Flower RJ 1997 Lipocortin 1:a second mesenger of glucocorticoid action in the hypothalamo-pituitary adrenal axis. Mol Med Today 93:296–302
20. Traverso VT, Christian HC, Morris JF, Buckingham JC 1999 Lipocortin 1 (annexin 1) is localised in pituitary folliculostellate cells. Endocrinology 140:4311–4319[Abstract/Free Full Text]
21. Smith T, Flower RJ, Buckingham JC 1993 Lipocortins 1, 2 and 5 in the central nervous system and pituitary gland ofthe rat: selective induction by dexamethasone of lipocortin 1 in the anterior pituitary gland. Mol Neuropharmacol 3:45–55
22. Philip JG, Flower, RJ, Buckingham JC 1997 Glucocorticoids modulate the cellular disposition of lipocortin 1 in the rat brain in vivo and in vitro. NeuroReport 8:1871–1876[Medline]
23. Christian HC, Flower RJ, Morris JF, Buckingham JC 1999 Localisation and semi-quantitative measure of lipocortin 1 in rat anterior cells by fluorescence activated cell analysis/sorting and electron microscopy. J Neuroendocrinol 11:707–714[CrossRef][Medline]
24. Taylor AD, Cowell AM, Flower RJ, Buckingham JC 1993 Lipocortin 1 mediates an early inhibitory action of dexamethasone on the secretion of ACTH by the rat anterior pituitary gland in vitro. Neuroendocrinology 58:430–439[Medline]
25. Loxley HD, Cowell A-M, Flower RJ, Buckingham JC 1993 Modulation of the hypothalamo-pituitary-adrenocortical response to cytokines in the rat by lipocortin 1 and glucocorticoids: a role for lipocortin 1 in the feedback inhibition of CRF-41 release? Neuroendocrinology 57:801–814[Medline]
26. Taylor AD, Loxley HD, Flower RJ, Buckingham JC 1995 Immunoneutralisation of lipocortin 1 reverses the acute inhibitory effects of dexamethasone on the hypothalamo-pituitary adrenocortical responses to cytokines in the rat in vitro and in vivo. Neuroendocrinology 62:19–31[Medline]
27. Taylor AD, Christian HC, Morris JF, Flower RJ, Buckingham JC 1997 An antisense oligonucleotide to lipocortin 1 reverses the inhibitory actions of dexamethasone on the release of adrenocorticotropin from rat pituitary tissue in vitro. Endocrinology 138:2909–2918[Abstract/Free Full Text]
28. Taylor AD, Flower RJ, Buckingham JC 1995 Dexamethasone inhibits the release of thyrotrophin from the rat anterior pituitary gland in vitro by mechanisms dependent on de novo protein synthesis and lipocortin 1. J Endocrinol 147:533–544[Abstract]
29. Perretti M, Croxtall JD, Wheller S, Goulding NJ, Hannon R, Flower RJ 1996 Mobilising lipocortin 1 in adherent leukocytes down regulates their transmission. Nature Med 2:1259–1262[CrossRef][Medline]
30. Croxtall JD, Flower RJ 1992 Lipocortin 1 mediates dexamethasone-induced growth arrest of the A549 lung adenocarcinoma cell line. Proc Natl Acad Sci USA 89:3571–3575[Abstract/Free Full Text]
31. Philip JG, Cover PO, Morris JF, Flower RJ, Croxtall JD, Buckingham JC 1998 Regulatory actions of glucocorticoids on pituitary function in rats treated centrally or peripherally with anti-lipocortin 1 antisera. J Endocrinol 159:OC29
32. Gillies GE, Buckingham JC 1997 The application of in vitro models pituitary function for toxicity testing. In: Hare SO, Atterwill C (eds) In Vitro Toxicity Testing Protocols. Humana, Towton, vol 43:81–93
33. Kawaminami M, Yamaguschi K, Miyagawa S, Numazawa S, Ioka H, Kurusu S, Hashimoto I 1998 Ovariectomy enhances the expression and nuclear translocation of annexin 5 in rat anterior pituitary gonadotrophs. Mol Cell Endocrinol 141:73–78[CrossRef][Medline]
34. Christian HC, Buckingham JC, Flower RJ, Morris JF 1997 Regulation of anterior pituitary lipocortin 1 during the oestrous cycle: suppression by oestradiol. J Endocrinol 155:OC8
35. Wahlstadt C 1995 Antisense oligonucleotide strategies in neuropharmacology. Trends Physiol Sci 15:42–46
36. Murray JAM (ed) 1992 Antisense RNA and DNA. In: Modern Cell Biology. Wiley Liss, New York, vol 11:1–401
37. Matteucci MD, Wagner RW 1996 In pursuit of antisense. Nature 384:20–22[Medline]
38. Stein CA, Cheng YC 1993 Antisense oligonucleotides as therapeutic agents-is the bullet really magic? Science 261:1004–1012[Abstract/Free Full Text]
39. Wagner RW 1995 The state of art of antisense research. Nat Med 1:1116–1118[CrossRef][Medline]
40. Lamberts SWJ, Macleod RM 1990 Regulation of prolactin secretion at the level of the lactotroph. Physiol Rev 70:279–318[Free Full Text]
41. Rettori V, Jurovivcova J, McCann SM 1987 Central action of interleukin-1 in altering the release of TSH, growth hormone and prolactin in the male rat. J Neurosci Res 18:179–183[CrossRef][Medline]
42. Rivier C, Erikson G 1993 The chronic intracerebroventricular infusion of interleukin1ß alters the activity of the hypothalamo-pituitary-gonadal axis of cycling rats. II. Induction of pseudopregnant-like corpora lutea. Endocrinology 133:2431–2436[Abstract]
43. Christian HC, Taylor AD, Morris JF, Goulding NJ, Flower RJ, Buckingham JC 1997 Characterisation and localisation of lipocortin 1 binding sites in the anterior pituitary gland by fluorescence activated cell analysis/sorting. Endocrinology 138:5341–5351[Abstract/Free Full Text]
44. Levitan ES, Hemmick LH, Birnberg WC, Kackmar LK 1991 Dexamethasone increases potassium channel messenger RNA and activity in clonal pitu