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Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, Kentucky 40536
Address all correspondence and requests for reprints to: James F. Hyde, Ph.D., Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, 800 Rose Street (MN224), Lexington, Kentucky 40536-0084. E-mail: jfhyde00{at}pop.uky.edu
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Introduction |
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Galanin in the anterior pituitary is regulated in a manner similar to that of the hormones of the specific cell types in which it is colocalized (10, 11). Estrogen is a potent stimulator of galanin secretion and gene expression (12, 13), with pituitary levels of galanin peptide exhibiting fluctuations coincident with changing circulating estrogen concentrations (14, 15). These observations suggested that galanin of pituitary origin may serve as an endocrine hormone. However, estimates of circulating levels of galanin peptide indicate that only 30% of galanin in the general circulation originates from the pituitary, with the remaining source being the gastrointestinal system (16, 17). Because the majority of galanin in plasma originates from sources other than the hypothalamic-pituitary axis, it seems redundant to have galanin produced within pituitary cells unless it serves some role within the local environment of the anterior pituitary gland. The recent report of a novel galanin receptor subtype (GALR2) to be localized within the anterior pituitary (18) adds credence to this hypothesis.
Previous investigations designed to determine the extent of the influence of estrogen on pituitary galanin expression demonstrated an association between the levels of galanin peptide and the growth of lactotroph tumors (prolactinomas) in the estrogen-sensitive Fischer 344 rat (11, 19, 20, 21). Galanin expression has also been correlated to the expansive growth of two lactotroph tumor cell lines, 235–1 and MtTW-10 pituitary cells (22, 23). The results of these studies suggest that galanin may have a role in mediating the growth and maintenance of prolactinomas. Recently, our laboratory reported that estrogen up-regulates galanin gene expression in the anterior pituitaries of Fischer 344 rats by increasing both the number of galanin-positive cells and the levels of gene expression (24). Furthermore, it was demonstrated that the majority of these galanin-positive cells were lactotrophs.
Concurrently, we have been studying the role of galanin in a model of estrogen-independent pituitary somatotroph hyperplasia, the human GH-releasing hormone (hGHRH) transgenic mouse. Using this animal model, we showed that galanin messenger RNA (mRNA) levels and peptide contents were increased in the male hGHRH transgenic mice compared with those in their nontransgenic siblings (25). Linear regression analysis revealed a positive correlation between pituitary concentrations of galanin peptide and the sizes of the anterior pituitary glands of control and hGHRH transgenic mice. The increased pituitary concentrations of galanin were peptide specific, as vasoactive intestinal peptide concentrations were not increased within the anterior pituitaries of the hGHRH transgenic mice (26). Because galanin is predominately localized within somatotrophs in the male rat anterior pituitary (9), the increased production, and presumed hypersecretion, of galanin in the male hGHRH transgenic mice may reflect the participation of galanin in the augmentation of GH secretion. However, the specific pituitary cell types producing galanin in the mouse anterior pituitary are unknown, and galanin secretion from individual pituitary cells has not been studied in either normal or hGHRH transgenic mice.
Thus, to further investigate the role(s) of galanin peptide within estrogen-independent anterior pituitary tumors, we 1) evaluated the changes in pituitary cell populations in hGHRH transgenic mice compared with those in control animals; 2) performed cell counts to determine whether the increased levels of galanin within the anterior pituitaries of hGHRH transgenic mice are the result of increased production by the same number of galanin-producing cells or an increase in the number of galanin-producing cells per se; 3) identified the phenotypes and percentages of galanin-producing pituitary cells in hGHRH transgenic mice compared with nontransgenic siblings; 4) ascertained whether GH and galanin were hypersecreted from individual pituitary cells of hGHRH transgenic mice; 5) determined whether any relationship exists between the colocalization of galanin and the secretion of GH from somatotrophs in hGHRH transgenic and nontransgenic mice; 6) examined the effects of immunoneutralizing galanin on GH secretion in vitro; and 7) evaluated the expression of the galanin receptors (GALR2) within the anterior pituitaries of control and hGHRH transgenic mice.
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Materials and Methods |
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Identification of transgenic mice
The hGHRH transgenic mice were identified by PCR. After death, a
small piece of skin from the ear or tail of each animal was removed and
immediately placed on dry ice. The DNA from the skin tissue was
extracted with 0.5 mg/ml proteinase K in 50 mM Tris, 20
mM NaCl, and 0.1% SDS at 55 C overnight. An aliquot of the
skin digest was then used for PCR. Oligonucleotide primers were
synthesized at the Oligonucleotide Core Facility at the University of
Kentucky. One primer was designed to anneal to sequences within the
mouse metallothionein-1 gene promoter (5'-CCA GTC GTG CCA AAG GGG
CGG-3'), and a second primer was designed to anneal to a portion of the
hGHRH sequence within the transgene (5'-ACA GGC GGG CGT TCG ACG
AGG-3'). Aliquots of the PCR reactions were analyzed by electrophoresis
through a 1% agarose gel and visualized with ethidium bromide. A
single PCR product of approximately 642 bp identified the heterozygous
transgenic mice. Age-matched nontransgenic siblings were used as
control animals for all experiments.
Tissue preparation and immunocytochemistry of paraffin-embedded
pituitary glands
After decapitation, whole pituitaries from hGHRH transgenic and
control mice were immediately dissected and immersed in Bouin’s
fixative at 4 C for 6 h. The pituitaries were then transferred
into 10 mM phosphate buffer at 4 C for 18 h. The
pituitaries were dehydrated (70–100% ethanol and xylene) and embedded
in paraffin. The paraffin-embedded tissue was sectioned with a
microtome at a thickness of 10 µm and mounted on SuperFrost
Plus slides (Erie Scientific, Portsmouth, NH).
The pituitary sections were deparaffinized at 55 C and rehydrated (xylene-70% ethanol). After a 15-min incubation in buffer (10 mM Tris-buffered saline-1% Tween-20, pH 7.4), the slides were immersed in a methanol-3% H2O2 solution to block endogenous peroxidase activity. After three 5-min washes in buffer, the tissue was covered with 1.5% normal goat serum for 20 min at room temperature to block nonspecific immunostaining. A rabbit-generated rat galanin antiserum (1:2000; see below) was applied to the tissue sections for 1 h at room temperature. The tissue was then washed three times for 5 min each time with Tris-buffered saline-Tween buffer and incubated at room temperature for 30 min with a biotinylated goat antirabbit IgG serum. After three 5-min washes in buffer, the tissue was covered with freshly prepared avidin-biotin-peroxidase complex reagent (Vector Laboratories, Inc., Burlingame, CA) for 1 h. The tissue was washed three times for 5 min each time with buffer and covered with a metal-enhanced diminobenzidene substrate solution (Pierce Chemical Co., Rockford, IL) for 2.5 min. The tissue was then washed three times with buffer, dehydrated (70–100% ethanol and xylene), and coverslipped using Entellan (Electron Microscopy, Fort Washington, PA). Negative controls included 1) preabsorption of the galanin antiserum with synthetic rat galanin, 2) omission of galanin antiserum, and 3) omission of secondary antibody.
The galanin-specific antiserum used in all experiments was prepared in our laboratory as previously described (28). The antiserum used in this study was obtained from the fifth bleeding of rabbit JFH2319. No significant cross-reactivity with rat (r) GH, rPRL, rLH, ACTH, rGHRH, TRH, LHRH, somatostatin-14, rCRH, arginine vasopressin, oxytocin, bradykinin, vasoactive intestinal peptide, angiotensin II, or human galanin was observed. The antiserum appears to recognize the carboxyl-terminus of the rat galanin peptide due to the fact that the first 15 amino acids of rat and human galanin are identical, and this antiserum failed to recognize human galanin. The predicted amino acid sequence of mouse galanin peptide (GenBank accession no. Z23069) is identical to that of rat galanin.
Anterior pituitary cell preparation
Monodispersed anterior pituitary cells from hGHRH transgenic and
control animals were used to 1) identify the phenotypes of the
pituitary cell types by immunofluorescence, 2) evaluate the secretion
of GH and galanin from individual pituitary cells by using the cell
immunoblot assay (CIBA), and 3) determine the effects of passive
immunoneutralization of galanin on GH secretion. Pituitary cells were
prepared as previously described (11). After removal of the
neurointermediate lobe, the anterior pituitaries were cut into small
pieces and incubated with 0.2% trypsin (Worthington Biochemical Corp., Freehold, NJ) for 33 min at 37 C. After treatment with
deoxyribonuclease I (Sigma Chemical Co., St. Louis, MO; or
Worthington Biochemical Corp.) and lima bean trypsin
inhibitor (Worthington Biochemical Corp.), the cells were
dispersed by gentle trituration with a siliconized Pasteur pipette.
Pituitary cells (2.5 x 104) were applied to
poly-L-lysine (0.2 mg/ml)-treated SuperFrost Plus
microscope slides in 0.05 ml of a Krebs-Hensleit-BSA-glucose-amino acid
solution [125 mM NaCl, 5 mM KCl, 14
mM glucose, 1 x MEM amino acids (Life Technologies, Grand Island, NY), 1.25 mM
KH2PO4, 1 x MEM vitamins (Life Technologies), 13.5 mM NaHCO3, and 1%
BSA], or the cells were used for the CIBA or static incubation as
described below. The slides were left at room temperature for 30 min to
allow for cell attachment and then submerged in phosphate-buffered 4%
paraformaldehyde at 4 C overnight. The following morning the cells were
sequentially washed in 0.1 M phosphate buffer,
diethylpyrocarbonate-treated water, acetic anhydride (0.25%) diluted
in 80 mM (pH 8.0) triethanolamine buffer, and 2 x SSC
(1 x SSC = 0.15 M NaCl and 0.015 MM
sodium citrate). The slides were then dehydrated through an ethanol
gradient (70–100%) and stored at -20 C until immunofluorescent
procedures were performed.
Immunofluorescence
Single or dual immunofluorescent staining was performed to
determine the percentage of each pituitary cell phenotype and the
percentage of each cell type colocalized with galanin within the
anterior pituitaries of five hGHRH transgenic mice and five
nontransgenic littermates. The pituitary cell slide preparations were
warmed to room temperature and washed with 1 mM PBS-0.3%
Triton X-100 buffer. The slides were then placed in 3% normal serum of
the species of the secondary antibodies for 30 min to block nonspecific
binding of the fluorescent antibody. The slides were then placed in the
single or paired primary antisera in 3% normal serum overnight at room
temperature. The primary antibodies used for dual immunofluorescence
were: rabbit (Rb) anti-rGAL (JFH2319; 1:500), guinea pig (Gp)
anti-rLHß (1:1000), Gp anithuman ACTH (anti-hACTH; 1:250), Gp
anti-rPRL (1:1000), Gp anti-mTSH (1:500), and monkey anti-rGH (1:500).
With the exception of the galanin antisera, all primary antibodies were
obtained from Dr. A. F. Parlow at the National Hormone and
Pituitary Program, University of California-Los Angeles-Harbor Medical
Center. The antibodies used to identify each of the pituitary hormone
cell types were tested for cross-reactivity with all of the pituitary
hormones used in this study and displayed no detectable
cross-reactivity. In addition, the secondary antibodies were tested for
cross-reactivity with each other, and none of the secondary antibodies
displayed cross-reactivity.
The following day, the slides were washed with buffer and then incubated with the single or paired secondary fluorescent-conjugated antibodies for 1 h [Dk anti-Rb CY3 (1:350), Dk anti-Gp fluorescein isothiocyanate (FITC; 1:350; Jackson ImmunoResearch Laboratories, Inc. West Grove, PA), and goat antimonkey FITC (1:350; ICN Pharmaceuticals, Inc., Costa Mesa, CA)]. The slides were next washed with buffer and coverslipped with Vectashield mounting medium (Vector Laboratories, Inc.). Immunofluorescent cells were visualized using a Nikon fluorescence Diaphot microscope system (Nikon, Melville, NY) and a x40 objective. FITC immunofluorescence was visualized using an FITC filter cube (Omega Optical Imaging, Inc., Brattleboro, VT) that has an excitation wavelength of 485 nm and allows for a 515-nm FITC emission wavelength. Cy3 immunofluorescence was visualized using a rhodamine (TRITC) filter cube (Nikon) that has an excitation wavelength of 554 nm and allows for a 570-nm Cy3 emission wavelength. The percentage of each pituitary cell type was determined by examining at least 1000 total cells from each animal for each paired hormone preparation and counting the number of single labeled, dual labeled, as well as unlabeled cells.
CIBA/immunocytochemistry
The CIBA procedure (29) relies on the protein-adsorbing
properties of the Immobilon polyvinylidene difluoride transfer membrane
(Millipore Corp., Bedford, MA). The Immobilon membrane is
hydrophobic and has a high capacity for protein binding. This membrane
will adsorb proteins secreted from cells in apposition to its surface.
The proteins can then be identified by immunocytochemical methods.
Before applying living pituitary cells to the Immobilon membranes, the
membranes were prewetted with methanol to open the pores and allow
protein adsorption. The membranes were washed with sterile water and
kept moist with phenol red-free DMEM (Life Technologies)
containing nonessential amino acids, antibiotics, and 20 mM
HEPES until the pituitary cells were applied. One thousand pituitary
cells in 0.2 ml medium were pipetted onto a pretreated piece of
Immobilon membrane (
0.752 in.). The cells were incubated
in a water-saturated atmosphere of 5% CO2-95% air at 37
C. After the specified number of hours of incubation, secretion was
terminated by fixing the membranes with phosphate-buffered 4%
paraformaldehyde for 15 min. The membranes were stored in 0.1
M Sorenson’s buffer at 4 C until immunocytochemical
techniques were performed. After incubation and fixation, a 0.05
M Tris-0.05% Tween-20 buffer was applied to the membranes
for 15 min. After washing in buffer, 10% normal goat serum was applied
to the membranes to block nonspecific binding of antibody. After 1
h, antirat GH (1:10,000; A. F. Parlow) or rat galanin antibody
(1:2,000) in 10% normal goat serum was applied to the membranes, and
incubation was continued overnight at 4 C. The membranes were washed in
buffer (3 times, 5 min each), and a biotinylated IgG solution [goat
antirabbit (Vector Laboratories, Inc.) for galanin and
goat antimonkey (E-Y Laboratories, Inc., San Mateo, CA) for GH] was
applied for 1 h. After another wash in buffer, an
avidin-conjugated horseradish peroxidase solution was applied for 60
min, and then a solution of hydrogen peroxide-activated,
3,3'-diaminobenzidine tetrahydrochloride (DAB; 0.5 mg/ml; Sigma Chemical Co.) was applied for 4 min. The membranes were washed
in buffer, air-dried, and examined under a Nikon
microscope equipped with a Pulnix CCD camera (Pulnix America,
Sunnyvale, CA). Negative controls included 1) preabsorption of the
galanin antibody with synthetic rat galanin, 2) omission of the primary
antibodies for each of the hormones analyzed, and 3) omission of the
secondary antibodies. Secretion areas were analyzed using a Macintosh
IIci (Apple Computer, Inc., Cupertino, CA) and the NIH Image program.
The mean hormone secretion area for each animal was determined by
averaging the areas obtained by image analysis from at least 50
hormone-secreting cells, resulting in 1 value for each animal per
hormone.
Validation of the CIBA
To validate the CIBA, a series of endocrine manipulations was
performed on pituitary cells from hGHRH transgenic mice to determine
whether the expected physiological secretory responses could be
elicited. To evaluate the effect of estradiol on galanin secretion,
three hGHRH transgenic mice were anesthetized with ether and 2-mm
SILASTIC brand capsules (id, 0.020 in.; od, 0.037 in.; Dow Corning Corp., Midland, WI) filled with crystalline
17ß-estradiol (6 mg crystalline powder) were implanted sc for 1
week. The pituitaries of the hGHRH mice that received 17ß-estradiol
capsules were harvested and processed as described above. Three male
hGHRH transgenic mice without prior estradiol treatment were used as
controls. During the in vitro incubation of the anterior
pituitary cells from estradiol-treated and control mice, some of the
cells were challenged with rat GHRH (10 nM; Peninsula Laboratories, Inc., Belmont, CA), dopamine (500 nM,
in 0.1 mM ascorbic acid; Sigma Chemical Co.),
or 0.1 mM ascorbic acid alone. After each treatment, the
membranes were immunostained for galanin or GH, as described above, and
data for hormone secretion areas were obtained by image analysis.
CIBA/dual immunocytochemistry
For dual immunocytochemistry with the CIBA membranes, the
immunostaining procedures were modified as follows. The pituitary cells
were incubated and fixed as described above for the single labeling
protocol. The dual labeling procedure used the combination of two
primary antibodies (rabbit antigalanin, 1:2,000; monkey anti-GH,
1:10,000). After overnight incubation with the primary antiserum, the
membranes were washed and then incubated with a 1:500 dilution of
Cy3-conjugated goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 60 min. The membranes
were subsequently washed in buffer (three times, 5 min each time), and
a biotinylated goat anti-monkey IgG solution (500 ng/ml; E-Y
Laboratories Inc.) was applied for 1 h. After another wash in
buffer, an avidin-conjugated horseradish peroxidase was applied for 60
min, and a DAB chromagen solution was applied for 4 min. The membranes
were then washed, air-dried, and examined under a Nikon
microscope as described above. Galanin-immunoreactive (galanin-IR)
cells were identified using fluorescent imaging as described above.
Galanin-IR cells showed greatly intensified CY3 fluorescence compared
with the low, yet visible, level of background label displayed by all
cells on the membrane. The mean GH secretion areas of galanin-positive
and galanin-negative cells for each animal were determined by averaging
the areas of immunostaining, obtained by using NIH Image software, from
50 GH-secreting cells of each subtype (i.e. galanin-positive
and galanin-negative), resulting in 1 value for each animal per cell
type.
Image analysis
The relative amount of hormone secreted by single pituitary
cells was determined using a Nikon light microscope
equipped with a Pulnix black and white CCD video camera linked with a
Macintosh IIci with NIH Image software. The membranes were viewed with
a x20 objective, and one pixel of the digitized images was determined
to equal 1 µm2. The digitized images were scaled by gray
levels (1–256). A value of 1 indicates the greatest amount of light
allowed to pass through the membranes. Therefore, each membrane was
calibrated identically by setting the pixels representing the center of
the pores present on the Immobilon membranes to the lowest gray
scale value. Background levels were determined by measuring the mean
optical density of an area of membrane void of immunostained secreted
product. The Image analysis density slice tool was used to
highlight any pixels with gray levels 10 or more density units darker
than membrane background. By using the Wand Auto Measure tool of
NIH Image analysis, the area of highlighted immunostaining
(i.e. cell secretion area) was determined. Overlapping
secretion profiles were not analyzed. Fifty cell secretion areas were
measured for each animal and averaged to determine one mean hormone
secretion area per animal.
Pituitary cell culture: static incubation
Monodispersed anterior pituitary cells from normal (n = 8)
and hGHRH transgenic (n = 6) mice were cultured (30,000
cells/well) in 96-well tissue culture plates (Nunclon, Thomas Scientific, Swedesboro, NJ) coated with
poly-D-lysine (0.5 mg/ml; Sigma Chemical Co.).
The cells were cultured in phenol red-free DMEM containing 10% gelded
horse serum and antibiotics and maintained in a humidified atmosphere
of 5% CO2-95% air at 37 C. After 24 h in culture,
the cells were washed three times (15 min each time) with serum-free
medium 199 (Life Technologies) containing 0.1% BSA. The
cells were then incubated for 3 h in medium 199-BSA alone or
medium 199-BSA containing normal rabbit serum (1:100), galanin
antiserum (1:100), or synthetic galanin (1 µM;
Peninsula Laboratories, Inc. Belmont, CA). After
incubation, the medium was collected and stored at -20 C until assayed
for GH content by RIA as previously described (11).
Ribonuclease protection assay (RPA)
RPA was performed to quantify GALR2 mRNA levels (RPA II kit,
Ambion, Inc., Austin, TX). A mouse complementary DNA
(cDNA) fragment of GALR2 was obtained by RT-PCR using total RNA
isolated from mouse anterior pituitary gland. Rat GALR2 primers (sense,
5'-GAC GTC GAG CCA TGG ACC TCT GCA CC-3'; antisense, 5'-GAC CAG AGC GTA
AAC GAT GGG GTT GAC AC-3'; Integrated DNA Technologies, Inc.,
Coralville, IA) were used for PCR. The single PCR fragment was
subcloned into pGEM-T (Promega Corp., Madison, WI) and
verified by dideoxy chain termination sequencing (Sequenase, version
2.0, U.S. Biochemical Corp., Cleveland, OH). The mouse GALR2 cDNA
sequence was more than 90% identical to that of rat GALR2. The GALR2
cDNA template was linearized with SacII and transcribed with
SP6 RNA polymerase to produce a 32P-labeled complementary
RNA (cRNA) probe. A ß-actin cRNA probe was generated as previously
described (30). Twenty micrograms of total RNA were then hybridized
with the cRNA probes overnight at 45 C, followed by ribonuclease A/T1
digestion. The protected fragments were separated on a 6% denaturing
polyacrylamide gel. The gel was then dried, and the protected bands
were quantified using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). The ratios of the integrated densities
from the GALR2 and ß-actin bands were used for statistical
analysis.
Statistical analyses
All data are expressed as the mean ± SEM.
Statistical analyzes for all comparisons were performed by one- or
two-way ANOVA, followed by Newman-Keuls multiple range test where
appropriate (31). Differences were considered statistically significant
at P < 0.05.
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Results |
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). To quantify the increase
in galanin-IR cells in hGHRH transgenic and control pituitaries,
immunocytochemistry and cell counts were performed using dispersed
anterior pituitary cells, rather than tissue sections, to avoid
possible variations in the regional distribution of specific pituitary
cell phenotypes.
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Quantification of specific pituitary cell types
Table 1A shows the percentages of
specific anterior pituitary cell phenotypes from male hGHRH transgenic
and nontransgenic mice determined by fluorescent
immunocytochemistry. For each cell phenotype identified, the
percentages compared between groups were significantly different. When
these percentages were converted to total cell numbers (Table 1B), it
was apparent that the decreased percentages of corticotrophs,
thyrotrophs, and gonadotrophs were largely due to a dilutional effect
as a result of the somatotroph proliferation. Conversely, the total
numbers of every cell phenotype were actually increased significantly
within the anterior pituitary glands of hGHRH transgenic mice.
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). Immunofluorescent cytochemistry revealed that the dispersed
anterior pituitary of the hGHRH transgenic mouse contained a
significantly (P = 0.038) higher percentage of
galanin-IR cells compared with pituitaries from nontransgenic siblings.
This increased percentage of galanin-IR cells translates to a 4-fold
increase in the total number of galanin-IR cells in the entire anterior
pituitary of hGHRH transgenic mice (Table 1B).
The photomicrographs in Fig. 2 illustrate
representative examples of dual immunofluorescent staining for GH, TSH,
and PRL colocalization with galanin. Table 1C shows that most (90%) of
the galanin-IR cells were also immunoreactive for GH, whereas the
remaining few percent were immunoreactive for either PRL or TSH. Table 1D shows the percentages of somatotrophs, lactotrophs, and thyrotrophs
that colocalize with galanin. In hGHRH transgenic mice, each pituitary
cell phenotype had an increased percentage of galanin-containing cells,
but only the percentages of galanin-containing lactotrophs
(P = 0.01) and thyrotrophs (P = 0.027)
were significantly greater than those in control animals.
Galanin-IR was not detected in cells immunopositive for LH or
ACTH.
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depicts an Immobilon membrane immunostained for galanin released from
pituitary cells of an hGHRH transgenic mouse. GH secretion was readily
detectable after 1 h of incubation, whereas galanin secretion was
not clearly detectable at any time point before 6 h. A time-course
evaluation of hormone secretion revealed an increase in GH and galanin
secretion over time (Fig. 4). These
results were used to determine the earliest time points at which it was
possible to detect pituitary hormone secretion and to ensure that
hormone secretion was monitored within a linear range of the detection
system. In all subsequent experiments, GH secretion was measured at
2 h, and galanin secretion was measured at 6 h.
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, A and B, revealed a
significant (P < 0.0001) effect of time and group on
the secretion of GH and galanin. The pituitary cells of hGHRH
transgenic mice secreted more GH and galanin than those of their
nontransgenic siblings, and the secretion of both hormones increased
with time. Further analysis using Newman-Keuls multiple range test
revealed that both GH and galanin secretion areas were significantly
(P < 0.05) larger in pituitary cells from hGHRH
transgenic mice at each time point examined.
The results of endocrine manipulations of galanin secretion are
depicted in Figs. 5 and 6. One week of in vivo
treatment with estradiol caused a significant increase in the mean
secretion areas of galanin (2-fold; P = 0.009) from
anterior pituitary cells of hGHRH transgenic mice compared with
secretion areas from pituitary cells of untreated hGHRH transgenic mice
(Fig. 5). Furthermore, treatment of pituitary cells from
estradiol-exposed hGHRH transgenic mice with 500 nM
dopamine in vitro significantly decreased the secretion
areas of galanin (55% estradiol alone; P = 0.002) to
areas that were not significantly different from those galanin
secretion areas from untreated pituitary cells of hGHRH transgenic mice
(Fig. 5). Ascorbic acid alone had no effect on galanin secretion from
pituitary cells of hGHRH transgenic mice (data not shown). Finally, the
average galanin (P = 0.0374) and GH (P
= 0.0325) secretion areas from anterior pituitary cells of hGHRH
transgenic mice were both increased 1.5-fold by in vitro
treatment with 10 nM GHRH (Fig. 5).
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depict an Immobilon membrane stained by
the modified dual CIBA method. The left panel shows
pituitary cells that secreted GH-immunoreactive peptide. The
right panel shows the same cells examined for galanin
immunofluorescence. Figure 7 shows that
somatotrophs found to be galanin-IR had significantly greater secretion
areas of GH in pituitary cell preparations from both hGHRH transgenic
(1.7-fold) and nontransgenic (2-fold) mice compared with somatotrophs
void of galanin-IR.
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). Moreover, immunoneutralization
of galanin in vitro significantly (P <
0.05) decreased GH secretion from pituitary cells of hGHRH transgenic
mice by 35%, but had no effect on GH release from pituitary cells of
normal mice (Fig. 8). Synthetic galanin caused a significant
(P < 0.01) 1.8-fold increase in GH secretion from
pituitary cells of normal mice, but failed to alter GH release from
hGHRH transgenic mice. Normal (preimmune) rabbit serum had no effect on
GH secretion in vitro.
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) and hypothalamus (data not shown)
revealed a single protected band of the same size (325 bp) in each
tissue. GALR2 mRNA levels (micrograms of GALR2 mRNA per µg total RNA)
in the anterior pituitaries of the hGHRH transgenic mice were 1.4-fold
(P < 0.05) decreased compared with those in their
nontransgenic siblings (Fig. 9B). However, when the data were expressed
as GALR2 mRNA per pituitary gland, GALR2 mRNA levels were significantly
(P < 0.05) increased more than 2-fold in the
pituitaries of transgenic mice. RPA analysis of hypothalamic levels of
GALR2 mRNA in control and hGHRH transgenic mice revealed no significant
differences in GALR2 expression between hGHRH transgenic and normal
mice (Fig. 9B).
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From these observations, it appears that the early stages of pituitary tumor formation in hGHRH transgenic mice involve a promiscuous increase in all pituitary cell types. One possible explanation for this phenomenon is that the increased concentrations of GHRH may cause an excessive differentiation of pituitary progenitor cells. The adult pituitary gland contains many undifferentiated cells that theoretically possess the potential to mature into functional pituitary cells. The elevated concentrations of GHRH may stimulate the progenitor cells to differentiate into mature cells, thus producing pituitary hyperplasia. In addition, increased regulatory factors may stimulate normal mitosis within the pituitary gland that also could contribute to pituitary hyperplasia of normal pituitary tissue. The focal adenomas observed within the anterior pituitary glands of the hGHRH transgenic mice finally occur due to the extra sensitivity of somatotrophs to GHRH. After rapidly repeated mitotic events, a mutation may occur in one or more somatotrophs that dramatically changes the genotype of the dividing cells, ultimately resulting in adenoma formation.
Exposure of rat pituitary cells to 5 nM GHRH for 18 days
in vitro increases thymidine incorporation and somatotroph
proliferation (34). The pituitary glands in hGHRH transgenic mice are
exposed to circulating concentrations of GHRH approaching 30 ng/ml
(600 nM) throughout their lifetimes (35). The mechanism
by which GHRH stimulates the proliferation of somatotrophs is
uncertain, although a cAMP-dependent process is probably involved (34).
We postulate that galanin may facilitate or mediate in part the
proliferative actions of GHRH.
Previously, we reported that galanin peptide concentrations and mRNA levels were significantly increased within the anterior pituitary glands of hGHRH transgenic mice (25). The increased concentrations of galanin peptide were positively correlated to the increased size of the pituitary glands, whereas the plasma concentrations of GH remained constant and were not directly correlated to the absolute size of the pituitary. We now report that the elevated levels of pituitary galanin peptide and mRNA in hGHRH transgenic mice are due to a 4-fold increase in the number of galanin-producing pituitary cells, rather than merely to an increased production of galanin by the same number of galanin-producing pituitary cells. Even though the anterior pituitaries of the hGHRH transgenic mice contain 3 times as many total cells, the percentage of galanin-containing cells is also significantly increased compared with that in the nontransgenic pituitaries.
The finding that 90% of galanin-positive cells are somatotrophs in the control male mice is not surprising, as somatotrophs are also the primary cell type containing galanin in male rats (9). The observations that the same percentage of galanin-containing, GH-IR cells is maintained in the hyperplastic anterior pituitary of the hGHRH transgenic mouse and that the percentage of somatotrophs containing galanin remains the same suggest that galanin plays an important role in this subpopulation of somatotrophs. Galanin may be a necessary component in the processes regulating the proliferation and secretory activities of somatotrophs. These data establish that galanin-containing pituitary cells are undergoing a hyperplastic response during the formation of the ensuing pituitary tumors in hGHRH transgenic mice.
The localization of galanin within a small percentage of thyrotrophs is consistent with reports using male rats (9). The observation of galanin localization in a small percentage of lactotrophs in both hGHRH transgenic and control animals was unexpected. Previously, galanin was only localized within somatotrophs and thyrotrophs of the male rat (9). The localization of galanin within lactotrophs of mice may reflect a species difference. It is tempting to speculate that the colocalization of galanin with PRL may, in fact, represent the localization of galanin in mammosomatotrophs. Indeed, a mammosomatotroph hyperplasia has been reported to occur in the 10-month-old hGHRH transgenic mice (33). Triple labeling experiments will need to be performed to address this possibility. Another possible explanation for the localization of galanin within lactotrophs of the male mouse is the method of detection used in this study. By dispersing the pituitary cells before performing immunofluorescent detection techniques, peptides within the cells may be more accessible for recognition by antibodies. Furthermore, we used a different antiserum to galanin than was previously used by Steel and colleagues in their investigation of the male rat pituitary gland (9). Analysis of male rat pituitaries with this galanin antibody using cell dispersion techniques should reveal whether a species difference, regarding the localization of galanin, truly exists.
In addition to the changes observed regarding the production of galanin within the anterior pituitary of the hGHRH transgenic mice, changes were observed concerning the basal secretion of galanin from individual anterior pituitary cells. Using the CIBA, we found that in vitro, individual anterior pituitary cells from hGHRH transgenic mice secrete a significantly greater amount of galanin peptide than cells from nontransgenic pituitaries. Furthermore, in vitro treatments of pituitary cells from hGHRH transgenic mice with dopamine had no significant effect on the secretion of galanin unless the cells were previously exposed to estradiol in vivo. This supports the finding that the majority of galanin cells are somatotrophs. If there were substantial numbers of lactotrophs containing galanin, we would have expected dopamine to have a greater effect on the secretion of galanin peptide in untreated transgenic mice. However, estradiol exposure greatly increases the expression of galanin within lactotrophs (9), where its secretion is inhibited by dopamine (11). These findings demonstrate that in addition to the increased expression of galanin in the anterior pituitaries of hGHRH transgenic mice, galanin is being hypersecreted from the increased number of cells.
Pituitary cells from the hGHRH transgenic mice also secrete greater basal amounts of GH in vitro compared with pituitary cells from nontransgenic mice. The hyperplasia occurring within the anterior pituitary glands of the hGHRH transgenic mice has previously been described as primarily consisting of somatotrophs (33, 35). Therefore, within the pituitaries of these animals there is not only an increase in the number of somatotrophs, but these cells can innately secrete greater amounts of GH. The increased basal secretory rate of GH (and galanin) from cells of the hGHRH transgenic mice probably reflects increased synthesis due to the continual exposure to hGHRH in vivo. Furthermore, our analysis of GH and galanin secretion from pituitary cells from hGHRH transgenic mice in vitro confirm the previous observation of Kovacs et al. (36) that these cells remain responsive to GHRH stimulation despite the lifelong overexposure of extremely elevated circulating levels of hGHRH.
We now present evidence for a possible role of galanin in the normal and hyperplastic anterior pituitary gland. In addition to the proposed mitogenic actions of galanin, earlier studies using rat pituitary cells in vitro implicated a direct role of galanin on GH secretion (37, 38). The increased production and hypersecretion of galanin in the anterior pituitary gland of hGHRH transgenic mice may reflect the participation of galanin in the augmented secretion of endogenous GH. Because galanin is primarily localized within somatotrophs of male mice, we evaluated whether the colocalization of galanin in somatotrophs was correlated with the amount of GH secretion.
Our current studies revealed that those somatotrophs that contain galanin-IR secrete a significantly greater amount of GH than those somatotrophs that are void of detectable galanin peptide. In this context, galanin may be acting in an autocrine manner to increase the secretion of GH. Upon the binding of GHRH molecules to their receptors on somatotroph membranes, the cAMP pathway is activated within the receptive cell, resulting in an influx of calcium (39). The increased levels of intracellular calcium result in the exocytotic release of GH from the secretory granules within the cytoplasm of the somatotrophs. We hypothesize that the corelease of galanin with GH may augment the secretory process. We previously showed that galanin and GH are localized within the same secretory granules of somatotrophs in the male rat (40). Somatotrophs that corelease galanin with GH could conceivably possess galanin receptors that become activated by the high concentrations of galanin that surround the cells during secretory events. Neighboring cells that possess galanin receptors could also be stimulated in a paracrine manner by the local increases in galanin concentrations.
The data obtained from the pituitary cell static cultures support our hypothesis of the autocrine/paracrine role of galanin in the anterior pituitary gland. These results suggest that the effect of endogenous galanin on GH secretion in the hGHRH transgenic mouse has reached its maximum because exogenous galanin could not further stimulate GH secretion as it did in the nontransgenic mice. Furthermore, it appears that the low levels of galanin being secreted from pituitary cells of the control mice are not sufficient to cause a measurable increase in the secretion of GH. However, in situations when the secretion of galanin is increased, as in the hyperplastic pituitary of the hGHRH transgenic mouse, GH secretion is increased, and specifically neutralizing galanin peptide significantly attenuates this increase. Therefore, there appear to be upper and lower thresholds of galanin concentrations that regulate the secretion of GH from somatotrophs.
The recent identification of a novel galanin receptor (GALR2) and the localization of the mRNA of this receptor in somatotrophs also are in support of our hypothesis (18). In the current study, we found that the concentration of GALR2 mRNA was decreased 1.4-fold in the anterior pituitaries of the hGHRH transgenic mouse. However, there was a 4-fold increase in the total amount of RNA in the pituitaries of the transgenic mice. Thus, analyses of mRNA concentrations may be subject to dilution by changes in the larger pool of mRNA. By expressing these values as the amount of GALR2 mRNA per pituitary, we found that the anterior pituitaries of the hGHRH mice, in fact, contained a significantly greater amount of GALR2 mRNA. The increase we observed in total GALR2 mRNA levels in the hGHRH transgenic mouse is probably the result of the increase in the number of pituitary cells. It is tempting to speculate that the majority of the proliferating cells within the anterior pituitary gland of the hGHRH transgenic mouse are those that contain the GALR2 receptor. The increase in the number of somatotrophs accounts for two thirds of the increase in total number of pituitary cells within the hGHRH transgenic mouse. It is possible that the increase in the level of GALR2 mRNA is parallel to the increase in the somatotroph cell population. Further investigations into the cellular distribution of the GALR2 receptor within normal and hGHRH transgenic mouse pituitaries are necessary for the determination of the role of the GALR2 receptor in pituitary cell proliferation.
In summary, we now show that galanin-containing pituitary cells are undergoing a hyperplastic response within the anterior pituitaries of hGHRH transgenic mice. The galanin-containing cells in control and hGHRH transgenic mice are primarily somatotrophs, whereas only a small percentage is either thyrotrophs or lactotrophs. We demonstrated that galanin and GH are hypersecreted by pituitary cells in hGHRH transgenic mice compared with those from control pituitaries. In addition, we established that somatotrophs containing galanin-IR secrete greater amounts of GH than those void of galanin-IR, suggesting an autocrine role for galanin in the regulation of GH secretion. We also demonstrated that when pituitary galanin levels are high (as in the hGHRH transgenic mouse), immunoneutralization of secreted galanin peptide significantly decreased GH secretion from pituitary cells in vitro. Finally, we showed that exogenous galanin can directly increase the secretion of GH from normal mouse pituitary cells in vitro.
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2 Present address: National Institute of Neurological Disorders and
Stroke, National Institutes of Health, Building 36, Room 4d08, MSC
4150, Bethesda, Maryland 20892-4150.
Received September 1, 1998.
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