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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; the Department of Biochemical Pharmacology, The William Harvey Research Institute, St. Bartholomew’s and the Royal London School of Medicine and Dentistry at Queen Mary and Westfield College (R.J.F.), London, United Kingdom EC1M 6BQ; and the Department of Human Anatomy, University of Oxford (J.F.M.), Oxford, United Kingdom OX1 3QX
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}cxwms.ac.uk
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Introduction |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of dispersed anterior pituitary cells
Suspensions of dissociated anterior pituitary cells were
prepared according to established protocols (16, 20). Briefly, the
cells of anterior pituitary glands removed postmortem from 24 rats were
dissociated with collagenase (0.2%, wt/vol) and deoxyribonuclease
(0.05%, wt/vol; both from Sigma Chemical Co., Poole, UK) in buffer A
[Earle’s balanced salts solution (EBSS) enriched with BSA (0.4%,
wt/vol; Sigma Chemical Co., St. Louis, MO) and
Na2HCO3 (28.5 mM)]; the resulting
cell suspension was centrifuged twice through a solution of 4% BSA
(200 x g, 10 min) to remove small debris and
erythrocytes and filtered through a 20-µm nylon mesh to remove any
remaining tissue clumps. In some experiments (Fig. 2
, B and C, and Fig. 4, A–D), the cells were incubated (37 C) for an additional 10 min with
trypsin (Sigma Chemical Co., St. Louis, MO; 0.05%, wt/vol, in buffer
A); the protease action was halted by adding a 10-fold volume of EBSS
containing BSA (2%), after which the cells were washed three times in
buffer A and finally resuspended in buffer A. Samples of collagenase-
or collagenase/trypsin-dispersed cells were examined by light
microscopy to verify the effectiveness of the dispersion (>80%) and
counted using a hemocytometer. The viability of the cells (normally
>95%) was assessed by the trypan blue exclusion test. Additional
samples of the cells were retained for examination at the electron
microscope level.
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and ß mAb; Sigma Chemical Co., St. Louis, MO; diluted 1:15,000)
was added to the final incubation medium (8, 20); if required,
dexamethasone (10 nM) was included in the medium during
both the preincubation and the final incubation periods. The
specificity of the anti-LC1 mAb was previously described (8); when used
for Western blot analysis, it reveals a single band of LC1
immunoreactivity in pituitary extracts with a molecular mass (37 kDa)
corresponding to that of the LC1 standard preparation.
Detection of cell surface LC1-binding sites by FAC analysis
Peripheral blood leukocytes. Preliminary experiments
investigated binding of LC1 to rat peripheral blood leukocytes by
methods analogous to those reported previously for detection of murine
and human leukocyte LC1-binding sites (17, 18). Cellular fractions were
isolated from trunk blood collected immediately after decapitation and
diluted with sodium citrate (0.36%, wt/vol, in distilled water; 1–6
ml blood), using a single step Ficoll-Hypaque M-85 gradient (Pharmacia,
Uppsala, Sweden; Winthrop Pharmaceuticals, Rennsaler, NY) (21). The
leukocyte fractions were pooled, washed twice in PBS (10 ml, 400
x g, 10 min, 22 C), resuspended in buffer A (5 ml), and
examined at the light microscope level with the aid of a hemocytometer;
a population comprising lymphocytes (70%), monocytes (15%), and
polymorphonuclear neutrophils (PMNs; 15%) was observed. The cells were
then washed with PBS containing EDTA (1 mM), which chelates
Ca2+ and thereby removes any LC1 that may be associated
with the cell membrane (18, 22), and diluted in buffer A to a
concentration of 2.5–5 x 106 cells/ml. Aliquots (20
µl) of the cell suspension were placed in the wells of a 96-well
microtiter plate (Falcon, Becton Dickinson, Oxford, UK) and incubated
in the presence of graded concentrations of hu-r-LC1 (0.5–500
nM) for 60 min at 4 C. The cells were washed in PBS (200
µl) containing BSA (0.2%, wt/vol) and CaCl2 (1.3
mM; PBC) to remove any free hu-r-LC1. Hu-r-LC1 bound to the
cell surface was detected by a double antibody method. Briefly, the
cells were incubated in buffer A at 4 C for 60 min in the absence
(controls) or the presence of a specific antihuman LC1 mAb (coded
anti-LC1 mAb 1B; 20 µg/ml) together with rat IgG (5 mg/ml; Sigma
Chemical Co., St. Louis, MO) to block nonspecific antibody binding. The
cells were then washed three times with cold PBC (200 µl), incubated
at 4 C for an additional 30 min with goat antimouse IgG-fluorescein
isothiocyanate (FITC) conjugate (diluted 1:100; Caltag, San Francisco,
CA), and washed in cold PBC; they were then resuspended in PBC (200
µl), fixed in an equal volume of paraformaldehyde (2%, wt/vol, in
PBS) and stored (4 C) for FAC analysis (see below), which was performed
within 4 days.
Anterior pituitary cells. Pituitary cell suspensions (0.6
ml; 3–5 x 105 cells/ml) were prepared and
preincubated for 2 h as described above. The cells were then
washed twice with PBS (2 ml; pH 7.4) and once with PBS containing EDTA
(1 mM) and resuspended in buffer A (1–2 x
107 cells/ml). Aliquots (20 µl) of the cell suspension
were transferred to the wells of a flat-bottomed 96-well microtiter
plate and incubated with hu-r-LC1 (0.5–500 nM) at 4 C (or
37 C for experiments shown in Fig. 3D) for 1 h. LC1 bound to the
cell surface was determined by FAC analysis using the protocol
described above for leukocytes. As an additional control, an equal
concentration of an isotype-matched (IgG2a) control mAb
(antitropomyosin; 20 µg/ml; Sigma Chemical Co.) was substituted for
anti-LC1 mAb. The specificity of the LC1 binding was further determined
by coincubating the pituitary cells with hu-r-LC1 (2–20
nM) and other polypeptides [CRH-41 (2–200
nM), GH (100–200 nM), or annexin 5 (100–200
nM); all diluted in PBC] and by spiking the cell samples
with leukocytes. In the latter case leukocytes (5 x
105 in 20 µl, prepared as described above) were mixed
with anterior pituitary cells (5 x 105 in 20 µl) in
96-well plates and washed in PBS-EDTA (200 µl) before the LC1 binding
protocol was performed. In further experiments (Fig. 4
, B–D),
pituitary cells separated by collagenase-trypsin treatment were
cultured in the presence or absence of inhibitors of RNA (actinomycin
D, 0.1 µg/ml) or protein [cycloheximide (0.5 µg/ml) and puromycin
(2 µg/ml)] synthesis for 24 h before incubation with graded
concentrations of hu-r-LC1 (0.5–500 nM); the
concentrations of RNA/protein synthesis inhibitors were selected on the
basis of previous experiments in which de novo protein
synthesis in pituitary tissue in vitro was measured directly
(9).
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In some experiments, the fluorescence-labeled cells (i.e. those expressing cell surface LC1-binding sites) were separated by FAC sorting (FACStar Plus, Becton Dickinson). The positive cells were then characterized on a morphological basis and by immunogold cytochemistry at the electron microscope level (see below). Cell surface fluorescence was also visualized by conventional fluorescence (BH2-RFL reflected fluorescence microscope, Olympus, Lake Purchase, NY) and confocal (MRC-500 laser scanning device, Bio-Rad, Richmond, CA) microscopy after smearing the labeled cells onto gelatin-coated slides.
Electron microscopy
Anterior pituitary cells (freshly dispersed and after separation
by FAC sorting) were prepared for electron microscopy using standard
methods. Briefly, the cells were fixed with glutaraldehyde (Sigma
Chemical Co., St. Louis, MO; 2.5%, vol/vol, in PBS), postfixed in
osmium tetroxide (1%, wt/vol, in 0.1 M phosphate buffer),
stained with uranyl acetate (2%, wt/vol, in distilled water),
dehydrated through a series of increasingly concentrated ethanol
(70–100%), and embedded in Spurr’s resin (Agar Scientific, Stansted,
UK). Ultrathin sections (50–80 nm) were viewed with a JEOL
transmission microscope (JEM-1010, JEOL, Peabody, MA). Cells in
sections taken systematically from different depths of the embedded
cell pellet were identified on the basis of their secretory granule
populations (shape, electron density, size, and distribution),
organelle structures, nucleus size, and chromatin characteristics (24)
and by immunogold labeling (25). Cells from individual samples were
always identified and counted on four to eight randomized grids in a
systematic manner.
Determination of ACTH
ACTH was determined in duplicate by RIA (16, 26) using a primary
antibody of defined specificity raised in rabbits against human
ACTH-(1–39) (National Hormone and Pituitary Program, Bethesda, MD),
synthetic human ACTH-(1–39) as a reference preparation (National
Institute for Biological Standards and Control, South Mimms, UK), and
[125I]ACTH-(1–39) as the tracer. The assay sensitivity
was 10 pg/ml, and the inter- and intraassay coefficients of variation
were 10.0% and 5.2%, respectively. Dilution curves of the test
samples were parallel those of the standard ACTH preparation.
Drugs
The following were used: dexamethasone sodium sulfate (David
Bull Laboratories, Slough, UK); CRH-41 (Bachem, Torrance, CA); anti-LC1
mAb (for in vitro immunoneutralization studies, Zymed, clone
Z013); hu-r-LC1 and anti-LC1 mAb 1B (for FAC analysis/sorting, both
from Biogen Research Corp., Cambridge, MA); FITC-conjugated goat
antimouse IgG antibody and FITC-conjugated goat antirabbit IgG antibody
(both from Caltag); human placental annexin 5 (gift from Dr. F.
Russo-Marie, Paris, France); GH (National Hormone and Pituitary
Program, Bethesda, MD); and actinomycin D, puromycin, cycloheximide,
antispectrin and ß mAb, and antitropomyosin mAb (all from Sigma
Chemical Co., St. Louis, MO). The drugs were dissolved and/or diluted
as appropriate in incubation medium immediately before use.
Statistical analysis
Preliminary analysis by the Shapiro and Wilkes test showed that
the data were normally distributed. Subsequent analysis was performed
by ANOVA with post-hoc comparisons by Duncan’s multiple
range test. Differences were considered significant if
P < 0.05. Statistical analyses were made within
experiments only. Each of the studies shown was repeated at least three
times (for specific details, see legends), and in all instances a
similar profile of data were seen.
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demonstrates the binding
of hu-r-LC1 to rat peripheral blood leukocytes as detected by anti-LC1
mAb 1B with an antimouse IgG FITC-conjugated second antibody and
quantified by computerized FAC analysis. Concentration-dependent
binding of hu-r-LC1 (0.5–500 nM) to PMNs and monocytes was
observed, with saturation at 50 nM, giving a total of
33,000 ± 4,000 binding sites/cell (mean ± SEM)
for the monocytes and 27,000 ± 4,000 for the PMNs (n = 4).
Approximately 95% monocytes and PMNs displayed a fluorescence shift on
LC1 binding. In contrast, the lymphocyte population did not bind
hu-r-LC1. Scatchard analysis of the data (by the approximation for flow
cytometry) yielded straight line plots, with Kd
and Bmax of 3 ± 1 nM and
50 ± 13 pM, respectively, for PMNs and 4 ± 2
nM and 42 ± 18 pM for monocytes (n =
3; Fig. 1B). Removal of Ca2+ from the medium completely
prevented the binding of hu-r-LC1 (2.6–260 nM;
P < 0.01, Ca2+ free vs.
control; n = 4; Fig. 1C). Similarly, the binding of hu-r-LC1
(2.6–260 nM) was almost completely abolished by
pretreatment of the monocytes with trypsin (0.05%; 10 min; 37 C;
P < 0.01, trypsin-treated vs. control;
n = 4; Fig. 1D).
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); a small proportion of
nonsecretory cells (
5%) was also observed, but the bulk of the
folliculostellate and macrophage populations was lost during the
dispersion process.
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illustrates the effects of an
anti-LC1 mAb (diluted 1:15,000) on the ability of dexamethasone (10
nM) to suppress CRH-41-stimulated release of ir-ACTH
in vitro from anterior pituitary cells preincubated either
for 2 h after dispersion with collagenase (Fig. 2A) or
collagenase/trypsin (Fig. 2B) or for 24 h after dispersion with
collagenase/trypsin (Fig. 2C). In the presence of the control antibody
(antispectrin and ß mAb, diluted 1:15,000), all three cell
preparations responded to CRH-41 (1 nM) with significant
increases in ir-ACTH release; however, the responses of cells separated
with collagenase/trypsin and preincubated for only 2 h before
stimulation were generally smaller and showed a greater variance (Fig. 2B; P < 0.05) than those of either
collagenase-dispersed cells (Fig. 3A;
P < 0.01) or cells allowed to recover for 24 h in
culture after separation with collagenase/trypsin (Fig. 2C;
P < 0.01). Nonetheless, the responses to CRH-41 in the
three preparations were blocked by dexamethasone (P <
0.05), which alone did not influence the spontaneous release of the
pituitary hormone. In the absence of dexamethasone, inclusion of
anti-LC1 mAb in the medium had no significant effect (vs.
the control mAb) on either the basal release of ir-ACTH or the
secretory responses to CRH-41, nor did the antibody influence basal
peptide release in the presence of dexamethasone (Fig. 2, A–C).
However, in the collagenase-dispersed rat anterior pituitary cells
(Fig. 2A), anti-LC1 mAb effectively reversed the inhibitory effects of
dexamethasone on the increases in ir-ACTH evoked by CRH-41. By
contrast, anti-LC1 mAb failed to influence the modest inhibitory
actions of dexamethasone on CRH-41-stimulated ir-ACTH release in cells
preincubated for 2 h after dispersion with collagenase/trypsin
(Fig. 2B). However, when the collagenase/trypsin-dispersed cells were
permitted to recover in culture for 24 h, responsivity to the
antibody was reinstated; thus, treatment of these cells with anti-LC1
mAb fully reversed the inhibitory actions of dexamethasone on
CRH-41-stimulated ir-ACTH release (Fig. 2C).
Detection of LC1-binding sites on anterior pituitary cells
Figure 3 demonstrates the ability of hu-r-LC1 to bind to the
surface of collagenase-dispersed pituitary cells as determined by FAC
analysis using anti-LC1 mAb 1B as a probe. Concentration-dependent
binding of hu-r LC1 (0.5–500 nM) was observed with
saturation at 250 nM, giving a total of 139,000 ±
15,000 binding sites/cell (Fig. 3A). By contrast, when a control mAb
(antitropomyosin) was substituted for anti-LC1 mAb, no change in
fluorescence above the background level was observed (data not shown).
Scatchard analysis of the data (using the approximation for flow
cytometry) yielded a straight line plot, an apparent Kd of
14 ± 3 nM, and a Bmax of
32 ± 13 pM (Fig. 3B). Removal of Ca2+
from buffer A during the incubation with hu-r-LC1 (0.5–500
nM) and from PBC during the subsequent washes completely
abolished the ability of hu-r-LC1 to bind to the cells
(P < 0.01, Ca2+ vs.
Ca2+-free at all concentrations of hu-r-LC1; Fig. 3C).
Performing the incubation with hu-r-LC1 (0.5–500 nM) at 37
C also reduced LC1 binding significantly (P < 0.01
vs. binding at 4 C; Fig. 3D).
Figure 4 illustrates the effects of
exposing collagenase-dispersed anterior pituitary cells to trypsin
(0.05%; 10 min at 37 C) on the expression of cell surface
hu-r-LC1-binding sites. The binding of hu-r-LC1 (1–500 nM)
to the cells was promptly abolished (P < 0.01; n
= 3) by trypsin treatment, but was fully reinstated when the
trypsinized cells were allowed to recover for 24 h in culture
before study (binding sites per cell at saturation: trypsin plus
24 h in culture, 139,000 ± 15,000; nontrypsinized cells,
132,000 ± 17,000; Kd: trypsin plus 24 h in
culture, 15 ± 3 nM; vehicle, 14 ± 2
nM; n = 3; Fig. 4A). Regeneration of the binding sites
was prevented by inclusion in the medium of the protein synthesis
inhibitors cycloheximide (0.5 µg/ml; Fig. 4C) or puromycin (2
µg/ml; Fig. 4D), but not by the RNA synthesis inhibitor actinomycin D
(0.1 µg/ml; Fig. 4B).
The binding of hu-r-LC1 (2 nM) to anterior pituitary cells
was unaffected by the inclusion of CRH-41 (20–200 nM) in
the medium (Fig. 5A); similarly, hu-r-LC1
(20 nM) binding was unaffected by GH (100–200
nM; Fig. 5B). Moreover, when the pituitary cells were mixed
with leukocytes before incubation with hu-r LC1 and FAC
analysis/sorting, the lymphocytes were confined to the population
negative for LC1-binding sites, whereas the monocytes and neutrophils
were selectively contained within the population displaying
fluorescence for LC1 binding (Table 2).
However, annexin 5 (100 and 200 nM) blocked the binding of
hu-r-LC1 (20 nM) to the cells by approximately 60% and
95%, respectively, suggesting competition for a single site (Fig. 5C).
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, A–D; n = 8). Visualization of
pituitary cells separated by FAC sorting (on the basis of hu-r-LC1
binding) at the electron microscope level confirmed that the
ultrastructural integrity of the cells was well maintained during
passage through the cytometer (Fig. 7).
It also showed that the proportion of pituitary cells expressing
hu-r-LC1-binding sites represents approximately 80% of the cell
population tested. All of the main secretory (i.e.
endocrine) cells were identified on the basis of morphological criteria
in this fraction (i.e. somatotrophs, gonadotrophs,
corticotrophs, thyrotrophs, and lactotrophs; Fig. 7) in proportions
comparable to those observed in normal pituitary tissue or in
collagenase-dispersed cells before sorting (Table 1). Identification
and quantification of cell types on the basis of morphology correlated
well with the data obtained when identification was assisted by
immunogold labeling for the respective stored pituitary peptides.
Further examination of cells showing strong fluorescence for hu-r-LC1
binding by conventional confocal microscopy confirmed that this
population comprised cells of differing sizes that appeared intact.
Cells not expressing hu-r-LC1-binding sites separated from a mixture of
anterior pituitary cells were found to comprise mainly lymphocytes, but
also somatotrophs and gonadotrophs (Table 2).
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FAC analysis has been used effectively to detect and characterize high affinity LC1-binding sites on the surface of human and murine peripheral blood leukocytes, and indeed, these sites are deemed necessary for the biological actions of the protein (3, 17, 18, 27). In line with these findings, we report for the first time the presence of similar high affinity, saturable, LC1-binding sites on the surface of rat peripheral blood monocytes and PMNs, but not lymphocytes. In addition, we have revealed by fluorescence and confocal microscopy that the distribution of the binding sites on the cell surface is punctate and variable between cells; this pattern is consistent with the widespread phenomenon of ligand-induced receptor clustering, a process that may be essential for internalization (28). There was no significant difference between the monocytes and PMNs with regard to either the estimated Kd or the Bmax; moreover, these values closely resemble those reported for LC1 binding to human and murine leukocytes (17, 18, 27). It is evident from our study that LC1 binding is Ca2+ dependent, which supports the known Ca2+ requirements for LC1 biological activity (1). The fact that LC1 does not bind to lymphocytes argues against the possibility that the protein is merely binding nonspecifically to surface membrane phospholipids as does the Kd, which is indicative of a putative receptor. Indeed, our finding that the LC1 Bmax of the cells is destroyed by trypsin suggests that the binding site(s) is proteinaceous in nature; similar conclusions have been drawn from a recent study on human monocytes in which LC1 binding was ablated by pretreatment of the cells with proteases (18, 29). In accord with these findings, two LC1-binding proteins (with molecular masses of 15 and 18 kDa) have been identified on the surface of human monocytes (18), but their exact nature is not yet known.
Although flow cytometry is used routinely to type immune cells on the
basis of specific cell surface marker expression, little work has been
published on the use of this technology to detect and quantify surface
antigens or receptors on anterior pituitary cells. Inevitably, with a
solid tissue such as the pituitary gland, there are methodological
problems in balancing the need to preserve surface proteins with the
conflicting requirement for FAC analysis of a preparation of single
cells, the production of which entails digestion of the connective
tissue between the cells with proteases that are themselves likely to
damage the surface proteins. We thus investigated three methods of
anterior pituitary cell preparation (viz. collagenase,
collagenase/trypsin, and collagenase/trypsin plus 24 h in culture)
and complemented our study of the capacity of the dispersed cells to
bind LC1 with measures of the morphological and functional integrity of
the cells. Our data indicate that both the ultrastructure and the
viability of the pituitary cells were well maintained
regardless of the method of separation. Moreover, the
three preparations each responded readily to CRH-41 in vitro
with significant rises in ir-ACTH release that were readily attenuated
by preincubation of the cells with dexamethasone; however, the
responses of the collagenase/trypsin-treated cells to both CRH-41 and
dexamethasone (Fig. 2B) were generally smaller and more variable than
those of the other groups (Fig. 2, A and C). High affinity, saturable
LC1-binding sites were readily detected by FAC analysis on the surface
of approximately 80% of anterior pituitary cells separated by
collagenase; in contrast, cells exposed to trypsin failed to bind
hu-r-LC1, although their binding capacity was restored after 24 h
in culture. There was an excellent correlation between the presence of
the cell surface binding site and the capacity of our anti-LC1 antibody
to quench the powerful inhibitory effects of dexamethasone on ir-ACTH
release. Thus, in accord with our previous observations on pituitary
segments (8), anti-LC1 mAb effectively suppressed the inhibitory
actions of dexamethasone on CRH-41-stimulated ir-ACTH release from the
collagenase-dispersed cells; the action of the antibody appeared to be
specific, because an isotype-matched control antibody was ineffective
in this regard. However, in cells in which the surface hu-r-LC1-binding
sites were destroyed by trypsin, the LC1-neutralizing antibody was
without effect. In contrast, when the trypsinized cells were maintained
in culture for 24 h to reinstate LC1 binding, the ability of the
antibody to quench the inhibitory actions of the steroid was restored.
These data add further support to our hypothesis (2, 5, 7, 8, 9, 10, 11) that LC1
acts via membrane-bound proteins to suppress peptide release. The fact
that cells subjected to collagenase/trypsin treatment still showed weak
responses to dexamethasone despite their loss of sensitivity to
anti-LC1 mAb is interesting and supports our premise (2, 5) that the
steroid may exploit more than one mechanism of action in a given target
cell.
Our data also show clearly that the binding of hu-r-LC1 to collagenase-dispersed pituitary cells is concentration, temperature, and Ca2+ dependent and that the binding sites assume a punctate pattern of distribution across the cell surface that appeared to vary between cells. The overt reduction in Bmax evident at 37 C vs. that at 4 C is difficult to explain, but may reflect more rapid internalization of the ligand-binding site complex. Two lines of evidence indicate that, as in leukocytes, LC1-binding sites on anterior pituitary cells are protein in nature. Firstly, the capacity of the pituitary cells to bind hu-r-LC1 is destroyed by trypsin; secondly, the subsequent regeneration of the binding sites in culture is prevented by the inclusion in the medium of the protein synthesis inhibitors cycloheximide and puromycin, although, surprisingly, not by the RNA synthesis inhibitor actinomycin D. These findings indicate that the reinstatement of the binding site in culture is dependent on the translation, but not the transcription, of new protein; they thus suggest that a readily translatable pool of RNA (primary transcript or mature messenger RNA) for the LC1-binding protein RNA exists within anterior pituitary cells.
The specificity of the binding measured was an important consideration. In all of our experiments, fluorescence in the absence of ligand and/or primary antibody was minimal; the specificity of the primary antibody was further assured by the fact that our isotype-matched control antibody (antitropomyosin) was repeatedly inert. Three additional questions were addressed to gain some insight into the specificity of hu-r-LC1 binding to collagenase-dispersed anterior pituitary cells; namely, was the binding of profile hu-r-LC1 altered by the addition to the cell suspension of 1) a cell known not to express LC1 binding sites (i.e. lymphocytes), 2) high physiological concentrations of other hormones, and 3) other annexins? Spiking of the pituitary cell suspension with leukocytes had no discernible effect on the binding profile; moreover, morphological examination of samples harvested by FAC sorting confirmed that the lymphocytes were confined to the population negative for LC1 binding. The binding of hu-r-LC1 to the pituitary cells was also unaffected by CRH-41 and GH, which suggests that the interaction of LC1 with the cell membranes was not via a nonspecific protein association. An effect of CRH-41 might have been expected, as the N-terminal of LC1 exhibits some degree of sequence homology with members of the CRH peptide family, notably sauvagine (30). Our finding that annexin 5 causes an apparent concentration-dependent inhibition of hu-r-LC1 binding to anterior pituitary cells raises the possibility that annexin family members may bind to a common site on the cell membrane. This view, however, is not supported by our recent experiments showing that hu-r-LC1 does not affect the binding of annexin 5 to pituitary cells. As annexins readily form multimers (31, 32), one possible explanation of our data is that hu-r-LC1 combined with annexin 5 in solution to form a heteromultimer in which the epitope required for association of hu-r-LC1 either with its cell surface binding site or with the antibody probe (i.e. anti-LC1 mAb 1B) was masked.
FAC sorting permitted ready separation of the 80% of collagenase-dispersed anterior pituitary cells that displayed fluorescence for hu-r-LC1-binding sites. Identification of the cells harvested on the basis of morphological criteria at the electron microscope level indicated that all principal secretory (i.e. endocrine) cells emerged within this fraction, with no obvious enrichment of any particular secretory cell type; thus, somatotrophs, lactotrophs, corticotrophs, gonadotrophs, and thyrotrophs were all shown to express hu-r-LC1-binding sites. Parallel studies in which the pituitary cells were identified and quantified on the basis of immunogold labeling of the respective stored pituitary peptides led to similar conclusions. Unfortunately, the number of cells contained within the population not expressing LC1-binding sites was not sufficient for quantitative evaluation. However, in experiments in which the pituitary cells were mixed with leukocytes before FAC analysis/sorting, the population negative for LC1 binding was found to comprise not only the predicted abundance of lymphocytes, but also a surprisingly high proportion of somatotrophs and gonadotrophs. The reason why some cells of one secretory type should exhibit differences in hu-r-LC1 Bmax is unclear. It is possible that some cells are more prone to damage by protease treatment, but the ultrastructural morphology of the pituitary cells not expressing LC1-binding sites appeared intact. A more plausible explanation is that cells negative for LC1 binding are either at a different stage of a particular cycle (22), or they belong to specific subgroups of the principal pituitary cell types that may be identified more readily on a chemical, rather than a morphological, basis.
An important question emerges as to whether the LC1-binding sites
expressed on the pituitary cells are identical with those observed on
leukocytes. There were no obvious differences between the two
populations with regard to the LC1 Bmax; moreover
in both cases, LC1 binding was Ca2+ dependent and destroyed
by pretreatment of the cells with trypsin. However, comparisons of the
binding affinity point to possible differences between the pituitary
(Kd = 14 nM) and monocyte/PMN
(Kd = 2–4 nM) sites. Moreover, despite the
similar Bmax values for leukocytes and anterior
pituitary cells, the maximum number of binding sites per cell was
approximately 3-fold greater on anterior pituitary cells. This may
reflect the relatively large surface area of the cells (vs.
leukocytes); alternatively, it may be explained by a low affinity, high
capacity site. Unfortunately, direct statistical comparisons of the
measures of affinity and capacity were not possible, as parallel
measurements of LC1 binding to leukocytes and anterior pituitary cells
were not made in a sufficient number of experiments.
In conclusion, this study describes for the first time the presence of saturable, high affinity binding sites for hu-r-LC1 on the surface of multiple anterior pituitary cell types. These findings accord with data from our functional studies that have identified a role for LC1 in the regulation of secretion of several pituitary hormones, and they thus lend further support to our hypothesis that the actions of this protein are effected via cell surface, membrane-bound, binding sites. It remains to be determined whether these sites are true receptors, but both their high affinity and their punctate distribution accord with this view. As LC1 is expressed mainly by the agranular folliculostellate cells in the adenohypophysis, but also by the endocrine cells in the adenohypophysis (34), it is not yet clear whether LC1 binds to and acts on the cells from which it is released (i.e. as an autocrine agent) or whether it modulates the activity of adjacent cells (i.e. as a paracrine agent). Our finding that anti-LC1 mAb ablates the inhibitory actions of dexamethasone on ACTH release from collagenase-dispersed anterior pituitary cells in vitro, as it does in preparations in which the three-dimensional structure of the tissue is retained (8), suggests that the actions of the protein are not dependent on cell-cell contacts. Nonetheless, we cannot exclude the possibility that there may be a requirement for cell juxtaposition in vivo for the LC1-producing cells to generate an effective concentration of the protein at the surface of the LC1-responsive cells. The folliculostellate cells would be well suited to participate in such mechanism because they are particularly rich in LC1, and their elongated processes make close contact with secretory endocrine cells. Further studies are now underway to identify the binding protein, the factors that modulate its expression, and the signal transduction pathways it uses in the pituitary gland to regulate hormone release.
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Acknowledgments |
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Footnotes |
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Received April 22, 1997.
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V. Traverso, H. C. Christian, J. F. Morris, and J. C. Buckingham Lipocortin 1 (Annexin 1): A Candidate Paracrine Agent Localized in Pituitary Folliculo-Stellate Cells Endocrinology, September 1, 1999; 140(9): 4311 - 4319. [Abstract] [Full Text]
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H C Christian and J F Morris Rapid actions of 17{beta}-oestradiol on a subset of lactotrophs in the rat pituitary J. Physiol., March 1, 2002; 539(2): 557 - 566. [Abstract] [Full Text] [PDF]
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, Control mAb alone; 
, CRH-41 and
control mAb; 
, anti-LC1 mAb alone; 
, CRH-41 and anti-LC1 mAb.
Each column represents the mean ± SEM (n = 5).
*, P < 0.05; **, P < 0.01
(vs. corresponding CRH-41-free control). 
,
P < 0.05; 
, trypsin treatment without subsequent
culture; 
, trypsin treatment and 24-h culture. B–D show the effects
of inclusion in the culture medium of the RNA synthesis inhibitor
actinomycin D (B; 0.1 µg/ml) and the protein synthesis inhibitors
cycloheximide (C; 0.5 µg/ml) and puromycin (D; 2 µg/ml) on the
ability of collagenase/trypsin-dispersed cells maintained in culture
for 24 h to regenerate hu-r-LC1 binding sites. •, Controls; 
