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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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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demonstrates the steroid
specificity of the corticosterone-induced increase in
[35S]annexin 1 expression in the pituitary
cells. Like corticosterone (1 nM), dexamethasone (100
nM) produced an overt increase in the tissue content of
[35S]annexin 1 (P < 0.01). By
contrast, 17ß-estradiol, testosterone, and aldosterone (1
nM) were ineffective in this regard. None of the
steroids tested significantly influenced the expression of
[35S]annexin 5.
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illustrate [35S]annexin 1 in the pericellular
(Fig. 2a) and intracellular (Fig. 2b) compartments of pituitary
cells exposed to corticosterone (1 nM) and/or annexin
1 antisense, sense, and scrambled ODNs; corresponding measures of band
density are shown in Table 2. Exposure of
the cells to corticosterone (1 nM) caused a marked increase
in the amount of [35S]annexin 1 (37 kDa)
detected in the pericellular pool (Fig. 2a, lane 2 vs. lane
1, and Table 2; P < 0.01) and a concomitant decrease
in the amount of newly synthesized annexin 1 contained within the cells
(Fig. 2b, lane 2 vs. lane 1, and Table 2; P
< 0.01). Annexin 1 antisense ODN alone did not influence the
distribution of [35S]annexin 1 between the
pericellular (Fig. 2a, lane 3 vs. lane 1, and Table 2) and
intracellular (Fig. 2b, lane 3 vs. lane 1, and Table 2)
pools. However, it prevented the appearance of newly synthesized
annexin 1 on the outer cell surface normally induced by corticosterone;
thus, in the presence of the antisense ODN, corticosterone had no
obvious effect on the amounts of 35S-labeled
annexin 1 contained within either the pericellular (Fig. 2a, lane 3
vs. lane 4, and Table 2; P < 0.01) or the
intracellular (Fig. 2b, lane 3 vs. lane 4, and Table 2;
P < 0.01) pools. By contrast, the responses to
corticosterone were unaffected by either the scrambled or sense ODN
sequences; thus, in these groups corticosterone induced increases in
[35S]annexin 1 in the pericellular pool and
concomitant reductions in the [35S]annexin 1
content of the intracellular pool analogous to those observed in the
nucleotide-free control groups (Fig. 2, a and b, lanes 5 and 6 and
lanes 7 and 8 vs. lanes 1 and 2, and Table 2;
P < 0.01).
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and Table 3 compare the effects
of corticosterone (1 nM) on the distribution of
[35S]annexin 1 between the pericellular (Fig. 3a) and intracellular (Fig. 3b) compartments with those of
dexamethasone (100 nM), 17ß-estradiol, testosterone, and
aldosterone (1 nM). Like corticosterone, dexamethasone
caused an increase in the amount of
[35S]annexin 1 contained within the
pericellular pool (Fig. 3a, lane 3 vs. lane 1, and Table 3; P < 0.01) and a
concomitant decrease in intracellular
[35S]annexin 1(Fig. 3a, lane 3 vs.
lane 2, and Table 3; P < 0.01). In contrast, the other
steroids tested had no apparent effect on the cellular disposition of
[35S]annexin 1 (Fig. 3a, lanes 4–6
vs. lane 1, and Table 3).
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and Table 2); and 2) dexamethasone (100
nM), 17ß-estradiol, testosterone, or aldosterone (1
nM; Fig. 3c and 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).
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demonstrates the ability of
corticosterone to inhibit the release of ir-PRL from rat anterior
pituitary segments induced in vitro by submaximal
concentrations of VIP (Fig. 6a), forskolin (Fig. 6b), 8-Br-cAMP
(Fig. 6c), and TRH (Fig. 6d) and shows how the responses are modified
by a neutralizing antiannexin 1 mAb. VIP (10 nM),
forskolin (100 µM), 8-Br-cAMP (1
µM), and TRH (10 nM) all
caused significant (P < 0.01) increases in ir-PRL
release, which were inhibited by corticosterone (1
nM). Addition of either antiannexin 1 mAb
(diluted 1:15000) or an equivalent dilution of an isotype-matched
control antibody (antispectrin and ß mAb) to the medium had no
significant effect on either the basal release of ir-PRL in the
presence or absence of corticosterone or the release induced by each of
the four secretagogues alone. However, antiannexin 1 mAb quenched
(P < 0.01) the inhibitory actions of corticosterone on
the release of ir-PRL evoked by VIP (Fig. 6a), forskolin (Fig. 6b), or
8-Br-cAMP (Fig. 6c), whereas antispectrin and ß mAb was inert in
this regard. By contrast, like the antisense ODN (Figs. 4d and 5c),
antiannexin 1 mAb failed to reverse the corticosterone-induced blockade
of TRH-stimulated ir-PRL release (Fig. 6d).
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demonstrates the effects of
a neutralizing antiannexin 1 polyclonal antibody (antiannexin 1 pAb)
administered either peripherally (200 µl, sc) or centrally via an
indwelling cannula to the third ventricle (3 µl, icv) on the ability
of corticosterone (100 µg/kg, ip) to suppress the IL-1ß (10 ng,
icv)-induced release of PRL. IL-1ß produced a marked increase in
serum ir-PRL (P < 0.001) in rats treated centrally (3
µl/rat, icv) or peripherally (200 µl, sc) with antiannexin 1 pAb or
the control serum (NSS). The responses to the cytokine were inhibited
(P < 0.01) in NSS-treated rats by pretreatment with
corticosterone (100 µg/kg, ip), which alone had no effect on the
serum PRL concentration. Corticosterone also ablated the response to
IL-1ß in rats treated centrally with antiannexin 1 pAb (3 µl, icv;
P < 0.01; Fig. 7a). However, when the antibody was
given peripherally (200 µl, sc), it completely reversed the
inhibitory effect of the steroid on the IL-1ß-induced changes in
serum ir-PRL (Fig. 7b). None of the treatments influenced the serum TSH
concentration (Table 4). Further studies
revealed that when given peripherally, IL-1ß (50 µg/100 g BW, ip)
had no effect on serum ir-PRL (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (Kd, 
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.
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Received December 28, 1999.
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, 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; 
, 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; 
, annexin 1 antisense
ODN (50 nM);
