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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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The regulation of pituitary galanin synthesis and secretion parallels that of PRL in many regards. PRL is one of the best studied anterior pituitary hormones (14), and much is known about the factors regulating its gene expression and secretion. Estrogen dramatically increases PRL synthesis and secretion (15). PRL is secreted in a very heterogeneous pattern, which does not correlate with PRL gene expression at the level of the single cell (16, 17). Fischer 344 rats develop a dramatic pituitary hyperplasia after 2 weeks of estrogen treatment, and more prolonged estrogen treatment can induce hyperprolactinemia and prolactinomas (18). We reported that estrogen stimulates a similar pattern of secretion of galanin and PRL in vitro (10), and that galanin and PRL peptides are located within the same secretory granules in lactotrophs of estrogen-treated rats (19). Moreover, receptor binding studies indicate that there are high affinity galanin receptors in the pituitary (20), and a recently cloned galanin receptor (GALR2) is expressed in the rat anterior pituitary (21). Therefore, we hypothesized that the function of pituitary galanin is to regulate the secretion of PRL in an autocrine and/or paracrine manner.
To test our hypothesis and to better understand the function of galanin in the anterior pituitary, we performed the following experiments. First, we quantified the precise distribution of the population of galanin-positive cells in the anterior pituitary by colocalizing galanin mRNA with PRL and GH mRNAs by using dual in situ hybridization. Second, we compared PRL secretion in both galanin-positive and galanin-negative lactotrophs at the level of the individual cell by using the reverse hemolytic plaque assay combined with in situ hybridization. Finally, we examined whether galanin exerts autocrine and/or paracrine effects on PRL secretion by blocking the access of galanin to galanin-positive lactotrophs with a specific galanin antiserum, and treating galanin-negative lactotrophs with synthetic galanin.
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Materials and Methods |
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Experimental design
Exp 1. Pituitaries from ovariectomized (n = 3) and
ovariectomized estrogen-treated (n = 3) rats were frozen on dry
ice, and then sectioned (7.5 µm) on a cryostat. Three representative
sections from each pituitary were used to perform in situ
hybridization to measure the effects of estrogen on galanin gene
expression.
Exp 2. To quantify the galanin-positive cell population in the anterior pituitary, dual in situ hybridization was performed using dispersed anterior pituitary cells from estrogen-treated rats. Anterior pituitaries (n = 3; 4 slides per animal) were carefully dissected from the neural and intermediate lobes. Cells were dispersed using a standard trypsin treatment as previously described (10). Briefly, pituitary fragments were incubated at 37 C with trypsin (0.2%) for 30 min, DNase I (0.1%) for 2 min, and then rinsed with lima bean trypsin inhibitor (0.075%) twice. Cell counts were performed using a hemocytometer. Approximately 50,000 cells were placed onto microscope slides freshly coated with poly-L-lysine (200 µg/ml; Sigma Chemical Co., St. Louis, MO). Cells were allowed to attach to the microscope slides for 30 min at room temperature, and then fixed in phosphate-buffered 4% paraformaldehyde. Dual in situ hybridization histochemistry was performed on these slides with 35S-UTP (galanin) and digoxigenin-UTP labeled (PRL and GH) complementary RNAs (cRNAs).
Exp 3. The reverse hemolytic plaque assay (22) combined with in situ hybridization was performed in this study to measure PRL secretion and galanin gene expression at the level of the individual cell (n = 3 animals, 3 slides per animal). Conjugation of protein A (Sigma) with ovine red blood cells (oRBC; CO Serum Co., Denver, CO), was performed 24 h before the assay (23). The next day, dispersed anterior pituitary cells were obtained from estrogen-treated rats as described in Exp 2. Protein A-coated oRBCs and monodispersed anterior pituitary cells [20% oRBCs, 4 x 104/ml anterior pituitary cells in DMEM/0.1% BSA (Sigma)] were incubated in a Cunningham chamber with different treatments: medium alone, normal rabbit serum (1:25), galanin antiserum (1:100 and 1:25) (24) or synthetic rat galanin (1 µM; Peninsula Laboratories, Inc., Belmont, CA). The antiserum used in the present study was obtained from the fifth bleeding of rabbit JFH2319. No significant molar cross-reactivity with rat GH, rat PRL, rat LH, ACTH, rat GHRH, TRH, LHRH, somatostain-14, rat CRH, arginine vasopressin, oxytocin, bradykinin, VIP, angiotensin II, or human galanin was observed. The antiserum appears to recognize the carboxy-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 immunoglobulins in the normal rabbit serum and galanin antiserum were purified by ammonium sulfate precipitation, followed by extensive dialysis in borate-buffered saline (25). After a 45-min incubation, all chambers were washed with DMEM/0.1% BSA, and then treated with a rPRL antibody (26) (1:70, provided by Dr. N. Ben-Jonathan) combined with the treatment solution for one additional hour. After washing the cells, a guinea pig complement solution (1:60, Life Technologies, Gaithersburg, MD) was added to the chamber and incubated for 50 min. All incubations were performed at 37 C in a water-saturated CO2 incubator (5%CO2/95% air). The slides were then fixed in 4% paraformaldehyde for in situ hybridization of galanin mRNA.
Complementary DNA (cDNA) templates and cRNA probe
preparation
A 585-bp rat galanin cDNA was generated in our laboratory by
RT-PCR using total RNA from an estrogen-treated rat anterior pituitary.
This cDNA (+110 to +695) corresponds to a part of the coding region of
galanin mRNA (27) and was ligated into pGEM2 (Promega, Madison, WI).
The PCR product was verified by dideoxy chain termination sequencing
(Sequenase, v2.0, USB Corp., Cleveland, OH). The template was
linearized with AvaI and transcribed with T7 RNA polymerase
to obtain a 486-bp antisense galanin cRNA. To obtain a sense galanin
cRNA, this same template was linearized with HindIII and
transcribed with SP6 RNA polymerase. Rat GH and PRL cDNAs were generous
gifts from Drs. J. D. Baxter and R. A. Maurer, respectively.
The original GH cDNA insert was digested with KpnI and
PstI and subcloned into pGEM3Zf(-) (Promega, Madison, WI).
The GH template was then linearized with PstI and
transcribed with T7 RNA polymerase to produce a 380-bp cRNA probe. The
PRL template was linearized with ApalI and transcribed with
SP6 RNA polymerase to produce a 430-bp cRNA. A total of 50
mM UTP and 500 mM ATP, CTP, and GTP were used
in the following transcription reactions. In Exp 1, the galanin cRNA
was transcribed with 7.5 mM 35S-labeled UTP and
42.5 mM unlabeled UTP, and purified through a G-50 Quick
Spin Column (Boehringer Mannheim, Indianapolis, IN), which yielded an
antisense cRNA with a specific activity of 3.4 x 108
dpm/µg RNA. Sense galanin cRNA was transcribed in a similar manner
and yielded a comparable specific activity. In Exp 2, the galanin cRNA
was transcribed with 30 mM 35S-labeled UTP and
20 mM unlabeled UTP to yield a probe with a much higher
specific activity (1.3 x 109 dpm/µg RNA). GH and
PRL cRNAs were transcribed with 30 mM digoxigenin-labeled
UTP and 20 mM unlabeled UTP. In Exp 3, the galanin cRNA was
transcribed as described in Exp 2.
In situ hybridization, dual in situ hybridization, in situ
hybridization combined with the reverse hemolytic plaque assay
In situ hybridization histochemistry was performed
according to the method of Cai and Wise (28). The tissues were fixed in
4% paraformaldehyde for different lengths of time in individual
experiments. In Exp 1, pituitary sections were fixed for only 5 min,
whereas in Exp 2, dispersed pituitary cells were maintained in the
paraformaldehyde solution overnight. In Exp 3, after the reverse
hemolytic plaque assay, slides were immersed in 4% paraformaldehyde
for up to 2 days. The slides were then 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, 0.015 M sodium citrate). Hybridization
buffer (25 µl, Amresco, Solon, OH) containing 0.3 µg/ml cRNA of
galanin was applied to each slide. For dual in situ
hybridization, the radiolabeled galanin cRNA was added together with
either GH or PRL cRNAs. The slides were coverslipped and incubated
overnight (16 h) at 55 C in a humidified incubator. After
hybridization, the coverslips were removed in 4 x SSC. The slides
were then treated with RNase A (20 µg/ml) at 37 C for 30 min, washed
in RNase buffer at 37 C for 30 min, followed by two washes in 0.2
x SSC at room temperature for 15 min each and 0.1 x SSC at 60 C
for 1 h. The slides were briefly dehydrated in 70% alcohol for 10
sec and then air dried. The slides from Exp 1 and 3 were then dipped in
1:1 diluted Kodak NTB-2 emulsion (Eastman Kodak, Rochester, NY) and
exposed for 4 days and 1 week, respectively, to visualize the
hybridized signals.
The detection of the signals from the digoxigenin-labeled PRL and GH cRNAs in Exp 2 was performed according to the method of Eyigor and Jennes with modifications (29). After preincubation (4% lamb serum, 0.2% Triton-X-100 in 2 x SSC), the slides were rinsed in freshly prepared buffer A (100 mM Tris pH 7.5, 150 mM NaCl), and incubated with antidigoxigenin (Boehringer Mannheim, Indianapolis, IN) solution (1:1000 anti-digoxigenin, 5% lamb serum, 0.3% Triton-X-100 in buffer A) overnight. Digoxigenin signals were detected the following morning in a dark environment. Signals for both GH and PRL mRNAs could be detected after a 30-min incubation in chromagen solution, which consisted of 35 µl X-phosphate and 45 µl NBT (Boehringer Mannheim, Indianapolis, IN) in freshly prepared buffer B (100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl2). Eosin counterstain was used to visualize all of the cells. The slides were then briefly dehydrated in 70% alcohol for 10 sec, air dried, and dipped in 1:1 diluted Ilford emulsion (Polysciences, Inc., Warrington, PA), and exposed for 1 week to visualize the signals for galanin mRNA. The percentage of the galanin-positive cells in the dispersed anterior pituitary cells using single-labeled in situ hybridization was identical to that in dual in situ hybridization (data not shown).
Image and data analysis
In Exp 2, the numbers of total cells, lactotrophs,
galanin-positive cells, and cells coexpressing galanin/PRL were counted
on the same slides probed with galanin and PRL cRNAs. Numbers of total
cells, somatotrophs, galanin-positive cells, and cells coexpressing
galanin/GH were counted on the same slides probed with galanin and GH
cRNAs. Cells were counted from at least 20 different representative
areas (totaling approximately 2000 cells) on each slide, the mean
number of the total counts was used to represent the animal (4 slides
per animal). Data are presented as means ± SE (n = 3)
of the percentage of specific cell types in the anterior pituitary cell
population.
In Exp 3, the areas of PRL-secreting plaques were measured using an image analysis system (BioQuant OS/2; R&M Biometrics, Nashville, TN) (28). Plaque areas of PRL-secreting cells were obtained as arbitrary units. Plaques were divided into two groups by the presence and the absence of galanin gene expression. Mean plaque areas of different treatments from each group of animals were calculated and analyzed by two-way ANOVA to compare between the galanin-positive and galanin-negative PRL-secreting cells. When two-way ANOVA revealed a significant interaction between treatment [medium alone, normal rabbit serum (1:25), galanin antiserum (1:100 and 1:25), and galanin (1 µM)] and group (galanin-positive and galanin-negative cells) (P < 0.05), we performed multiple range comparison analyses to further compare the effects of the different treatments within and between the two groups. The video counted areas from the cluster of grains of the galanin-positive cells were also measured simultaneously. Regression analysis was performed on galanin-positive PRL-secreting cells from each animal to analyze the correlation of galanin gene expression (by the video counted area of grains) and PRL secretion (by the plaque areas).
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Results |
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). Estrogen treatment
dramatically increased galanin gene expression in the anterior
pituitary, and no signals were detected in the posterior pituitary
(neural or intermediate lobes; Fig. 1B). In addition, no signal was
observed using a sense galanin cRNA (Fig. 1C), confirming the
specificity of the galanin antisense cRNA probe. As illustrated in Fig. 1, the cells are extremely compacted within the rat anterior pituitary
gland, and it is not possible to reliably or accurately distinguish and
quantify single radiolabeled cells. Therefore, we performed dual
in situ hybridization in Exp 2 using dispersed anterior
pituitary cells. Because we could not detect any signal for galanin
mRNA in ovariectomized rats, we performed the following experiments in
ovariectomized estrogen-treated rats only.
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and 3).
Figure 2 shows representative photomicrographs of the dual in
situ hybridization results using the galanin and PRL cRNAs. The
distribution of the three different cell types (galanin-, PRL-, and
GH-positive cells) in the anterior pituitary and their relationships
are shown in Fig. 3. The percentages of somatotrophs, galanin-positive
cells and lactotrophs in the total population of estrogen-treated
anterior pituitary cells were 13.5%, 16%, and 51%, respectively.
Fifteen percent of the total number of cells coexpressed galanin and
PRL mRNAs, whereas only 0.3% of the total cell population coexpressed
galanin and GH mRNAs. One-third of all lactotrophs expressed galanin
mRNA. More than 93% of the galanin-positive cells were lactotrophs,
and less than 2% of the galanin-positive cells were somatotrophs in
estrogen-treated Fischer 344 female rats.
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and 5).
Figure 4 shows representative photomicrographs of PRL-secreting cells
using the reverse hemolytic plaque assay combined with in
situ hybridization. PRL secretion from single cells was measured
in all treatments. For the purpose of analysis, the plaque areas from
each treatment were divided into two groups based upon the detection of
galanin mRNA: galanin-positive and galanin-negative lactotrophs.
Two-way ANOVA revealed a significant interaction between treatment
[medium alone, normal rabbit serum (1:25), galanin antiserum (1:100
and 1:25) and galanin (1 µM)] and group
(galanin-positive and galanin-negative lactotrophs) (P
< 0.005). The galanin-positive lactotrophs in medium alone, as well as
after normal rabbit serum treatment, had significantly larger plaque
areas as compared with those of galanin-negative lactotrophs
(P < 0.05) (Figs. 4 and 5). When we compared the
plaque areas among different treatments in the group of
galanin-positive lactotrophs, we detected significantly smaller plaque
areas using both concentrations of galanin antiserum, and no
differences using normal rabbit serum and galanin treatment compared
with medium alone (Fig. 5). When we compared the plaque areas among
different treatments in the group of galanin-negative lactotrophs, we
detected significantly larger plaques areas after galanin treatment,
and no differences after normal rabbit serum and different
concentrations of galanin antiserum treatment compared with medium
alone (Fig. 5). Regression analysis revealed no correlation between PRL
secretion (plaque area) and galanin gene expression (video count area
of grains) (r = -0.1776; P < 0.20, n =
82 cells).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our present study provides direct evidence for an autocrine and paracrine function for galanin in regulating PRL secretion. The dramatic increase of the levels of galanin mRNA in the anterior pituitary gland by estrogen is not only due to an increase in the number of galanin-expressing cells, as previously shown by immunocytochemistry (10, 12), but is also the result of increasing the level of galanin gene expression within each cell. No galanin mRNA was detected in either the neural or intermediate lobes of the pituitary, although immunoreactive galanin peptide is present in the axon terminals of the neural lobe (30; unpublished observation). The present study is the first to quantify the subpopulation of galanin-expressing cells. In estrogen-treated female rats, more than 93% of galanin-positive cells are lactotrophs. This large degree of colocalization with PRL strongly supports our hypothesis that one of the functions of pituitary galanin is to regulate the secretion of PRL. The small degree of colocalization of galanin and GH mRNAs suggests that galanin may not exert a major function in regulating GH in the estrogen-treated anterior pituitary. Nevertheless, a small subpopulation of somatotrophs appears to be sensitive to estrogen, as determined by the induction of the galanin gene. It is tempting to speculate that this population of somatotrophs may, in fact, be mammosomatotrophs. However, triple-labeling experiments will be needed to prove this possibility.
The present study is also the first to compare PRL secretion between galanin-positive and galanin-negative lactotrophs at the level of individual cell. Neill and Frawley showed previously that the amount of PRL secreted from a lactotroph correlates with the size of the plaque in the reverse hemolytic plaque assay (23, 31). That is, lactotrophs secreting more PRL have larger plaque areas, and therefore the plaque area and PRL secretion are directly correlated. Galanin-positive lactotrophs secrete significantly greater amounts of PRL as compared with galanin-negative lactotrophs. Therefore, by increasing PRL secretion at the level of individual cell, galanin may mediate, at least in part, the estrogen-induced stimulation of PRL secretion. Because galanin is only contained in one-third of the lactotrophs, we believe that galanin in the anterior pituitary may also contribute to the heterogeneous secretion pattern of PRL.
Data from the plaque assay shed light on the cellular and molecular mechanisms of galanin-mediated PRL secretion. Our previous studies showed that galanin and PRL are located in the same secretory granules and are released simultaneously (19). We hypothesized that galanin stimulated PRL secretion in an autocrine and/or paracrine manner. By placing the pituitary cells far apart from one another in the plaque assay, we severely diminished the opportunity for galanin secreted from one cell to reach adjacent cells. Therefore, we predicted that galanin secreted from a PRL-releasing cell, if acting as a stimulatory autocrine factor, should exert its effects on that cell, i.e. increasing the size of the plaque. Our data support this hypothesis, in that those lactotrophs expressing galanin secrete more PRL than those lactotrophs that do not contain galanin mRNA. To prove that galanin is regulating PRL secretion in an autocrine manner, we blocked the availability of endogenous galanin to the cells by immunoneutralization. Galanin antiserum caused a significant attenuation of PRL secretion from galanin-positive lactotrophs, and the levels of PRL secretion were lowered to levels similar to those in galanin-negative lactotrophs. The failure of the galanin antiserum to alter PRL secretion from galanin-negative lactotrophs argues against any nonspecific effects of the antiserum on hormone secretion. In addition, we did not see a further increase of PRL secretion from galanin-positive lactotrophs after galanin treatment. This may be due to the high extracellular concentrations of endogenous galanin surrounding the galanin-positive lactotrophs after estrogen-treatment, and a further increase in the galanin concentration by the addition of exogenous galanin is not capable of increasing PRL secretion (i.e. the functional galanin receptors are completely occupied).
We also tested the hypothesis that galanin regulates PRL secretion in a paracrine manner by using in vitro treatment with synthetic galanin. Others have shown that the addition of synthetic galanin is capable of stimulating PRL secretion from rat pituitary cells in vitro (20, 32). The paracrine regulation of PRL secretion by galanin was also suggested by Wynick et al. using an immunoblot assay (32). By maintaining a large distance between the pituitary cells in the Cunningham chambers, we are doubtful that galanin secreted from a galanin-positive lactotroph reached neighboring cells. Therefore, if galanin is capable of acting as a paracrine factor to stimulate PRL secretion, exogenous galanin treatment should exert a stimulatory effect on PRL secretion from galanin-negative lactotrophs, i.e. increasing the size of the plaque. Indeed, exogenous galanin increased the PRL plaque area by approximately 50% from galanin-negative cells. It is also important to consider that treatment with the galanin antiserum did not significantly alter the sizes of the plaques from galanin-negative lactotrophs, suggesting that either 1) these cells were not exposed to endogenous galanin; or 2) that these lactotrophs were exposed to endogenous galanin, but that the peptide had no effect on PRL secretion from this subpopulation of PRL-secreting cells. The former possibility is upheld because treatment with galanin significantly increased PRL secretion from galanin-negative lactotrophs. To relate these findings to the whole animal, we infer that PRL secretion from lactotrophs not producing galanin (or producing undetectable levels of galanin mRNA) may be stimulated by endogenous galanin received from either adjacent galanin-positive pituitary cells or from hypothalamic neurons.
In light of previous data, it is not surprising that steady-state galanin gene expression does not correlate with PRL secretion at the level of the individual cell. Scarbrough et al. (16) showed previously that PRL gene expression does not correlate with PRL secretion at the level of the individual cell in ovariectomized and ovariectomized estrogen-treated Sprague-Dawley rats using the reverse hemolytic plaque assay, and Castaño et al. (17) showed that PRL gene transcription failed to correlate with hormone secretion in lactating rats. We and others showed that estrogen induced the synthesis and secretion of both galanin and PRL with similar time courses (10, 11). However, it appears that not only are PRL gene expression and secretion independent events, but that the absolute levels of galanin gene expression are also unrelated to PRL secretion. These data suggest that global gene expression per se in the lactotroph may be tightly regulated during specific times within individual cells but that overall gene expression is not coincident with secretion, nor is it strongly coupled to active secretory function.
Galanin in the anterior pituitary may play an intermediate role, via autocrine/paracrine mechanisms, in the formation of estrogen-induced lactotroph hyperplasia and tumorigenesis. Recent studies indicate that autocrine and paracrine factors play important roles in the genesis of some tumors (33, 34). Galanin stimulates cell proliferation in small cell lung cancer cells (33) and 235–1 cells, a rat pituitary tumor cell line (32). After 2 weeks of estrogen treatment, Fischer 344 rats develop a dramatic pituitary hyperplasia, and more prolonged estrogen treatment can induce hyperprolactinemia and prolactinomas (18). We previously showed that bromocriptine and the somatostatin analog SMS 201–995 inhibit galanin gene expression and galanin secretion, and also inhibit estrogen-induced prolactinoma formation (8, 9). Moreover, pituitary somatotroph adenomas are also associated with increased galanin gene expression and peptide production (35). Therefore, pituitary galanin may mediate the formation of pituitary hyperplasia and adenomas, not only in estrogen-induced models but also in estrogen-independent models. However, galanin may also subserve a variety of other functions in the pituitary such as interacting with endogenous growth factors or membrane receptors and their second messenger signaling cascades. The recent development of mice with a targeted disruption of the galanin gene (36), and transgenic mice overexpressing galanin in somatotrophs (37) and lactotrophs (Cai and Hyde, manuscript in preparation) will undoubtedly assist in our understanding of the diverse roles of galanin in pituitary function.
In summary, estrogen upregulates galanin gene expression by increasing both the number of galanin-positive cells and the levels of gene expression. Galanin-positive cells in the estrogen-treated anterior pituitary are mainly lactotrophs. One of the functions of pituitary galanin is to stimulate PRL secretion by acting as an autocrine and paracrine factor.
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Acknowledgments |
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Footnotes |
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Received November 26, 1997.
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