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Endocrinology, doi:10.1210/en.2007-1396
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Endocrinology Vol. 149, No. 5 2313-2324
Copyright © 2008 by The Endocrine Society

β1-Adrenoceptor Expression in Rat Anterior Pituitary Gonadotrophs and in Mouse {alpha}T3-1 and LβT2 Gonadotrophic Cell Lines

Kristel Janssens, Olga Krylyshkina, Nicole Hersmus, Hugo Vankelecom and Carl Denef

Laboratory of Cell Pharmacology, University of Leuven, Medical School, Gasthuisberg, B-3000 Leuven, Belgium

Address all correspondence and requests for reprints to: Professor Carl Denef, Laboratory of Cell Pharmacology, University of Leuven, Medical School, Campus Gasthuisberg (O & N), B-3000 Leuven, Belgium. E-mail: Carl.Denef{at}med.kuleuven.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The rat anterior pituitary expresses β2-adrenoceptors (ARs) on somatotrophs, lactotrophs, and corticotrophs. The present study investigates whether β1-ARs exist in the anterior pituitary, in which cell type(s) they are found, and whether they are regulated by glucocorticoids. As determined by quantitative RT-PCR and Western immunoblotting, the rat anterior pituitary expressed β1-AR mRNA and protein. Unlike the β2-AR, expression decreased to very low levels after 5-d aggregate cell culture but was strongly up-regulated in a dose- and time-dependent manner by dexamethasone (DEX). Glucocorticoids attenuated isoproterenol-induced down-regulation of β1-AR mRNA levels. As examined by immunofluorescence confocal microscopy, β1-AR immunoreactivity was detected in a subpopulation of gonadotrophs, but not in somatotrophs, lactotrophs, corticotrophs, thyrotrophs, or folliculo-stellate cells. β1-AR-immunoreactivity cells were often surrounded by cup-shaped lactotrophs. Consistent with these findings, β1-AR mRNA was considerably more abundant in the gonadotrophic {alpha}T3-1 and LβT2 cell lines than in the GHFT, GH3, and TtT/GF cell lines. DEX did not affect expression level in the cell lines. DEX also failed to up-regulate β1-AR mRNA levels in aggregates from a subpopulation enriched in large gonadotrophs obtained by gradient sedimentation. In contrast, excessive DEX-dependent up-regulation of β1-AR mRNA was found in a subpopulation enriched in small nonhormonal cells. The present data indicate that β1-AR is expressed in a subpopulation of gonadotrophs with a topographical relationship to lactotrophs. However, the glucocorticoid-induced up-regulation does not seem to occur directly in the gonadotrophs but within (an)other unidentified cell type(s), or is transduced by that cell type on gonadotrophs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CATECHOLAMINES AND glucocorticoids play an essential role in the stress response and contribute to the pathophysiology of cardiovascular diseases, depression, and anxiety disorders (1), and exert a suppressive influence on reproduction (2). All pituitary hormones participate at diverse levels during coping with stress (3), and, therefore, it is not surprising that secretion and expression of these hormones are modulated by catecholamines and glucocorticoids. Catecholamines control the secretion of ACTH (4, 5, 6, 7, 8, 9), GH (4, 5, 10, 11, 12, 13, 14), prolactin (PRL) (4, 15), TSH (4, 16), and LH (4, 17, 18) at the level of the hypothalamus and/or the pituitary. The effects of catecholamines are mediated by {alpha}- and β-adrenoceptors (ARs) that are members of a large superfamily of G protein-coupled receptors (19).

Glucocorticoids are known to modulate β-AR levels, thereby regulating the cellular responsiveness to catecholamines (20). At the pituitary level, glucocorticoids have fast and delayed effects on various hormonal cells. Fast nongenomic effects are mediated by folliculo-stellate (FS) cells via translocation of annexin-1 on the cell surface, and result in inhibition of ACTH, GH, and PRL release by a paracrine action (21). Genomic effects are established at a slower rate via the glucocorticoid nuclear receptor. In rat pituitary cell aggregates, Baes and Denef (13) showed a strong potentiation by dexamethasone (DEX) of isoproterenol (ISO) and epinephrine-stimulated GH release through β2-ARs. In addition, glucocorticoids enhance GH release in response to {alpha}1-AR activation (14). Baes and Denef (13) furthermore showed the importance of intercellular communication in these potentiating effects of DEX. Therefore, the anterior pituitary represents an interesting model to study the impact of local regulation in catecholamine action.

The β2-AR is the predominant β-AR subtype in the pituitary (22), which led research to be mainly focused on this subtype in previous studies (7, 11, 15, 22, 23, 24, 25). The presence of the β1-AR has been demonstrated, but no precise cell type localization was made, although it may participate in the regulation of GH secretion (26). Based on pharmacological experiments, bovine FS cells in culture have expressed both β1- and β2-AR in approximately equal proportions, and responded to β-AR stimulation in terms of cAMP accumulation (27). However, no evidence for β1-AR expression could be found in rat pituitary cell cultures (4, 5, 10, 11, 12, 13, 14).

The increasing evidence that the anterior pituitary is a direct target of peripheral signals related to stress (21), reproduction (28), and energy homeostasis (29) revitalized our interest in the role of catecholamines at the anterior pituitary level. The present study was intended to further investigate β1-AR expression in the anterior pituitary, its responsiveness to glucocorticoids, and its cellular distribution. We used β1- and β2-AR mRNA as a primary read-out system by quantitative RT-PCR (qRT-PCR) analysis that unequivocally detects expression of the receptor, confirmed expression at the protein level, and determined the cellular distribution by immunocytochemistry. Furthermore, we investigated β1-AR mRNA and protein expression in various pituitary cell lines representative for each hormonal cell type (GHFT, GH3, AtT-20, {alpha}T3-1, and LβT2), and FS cells (TtT/GF). We demonstrate that β1-AR is expressed in the anterior pituitary, more precisely in gonadotrophs that are juxtaposed to lactotrophs. Receptor mRNA level is strongly up-regulated by glucocorticoids, but this glucocorticoid action does not occur directly in gonadotrophs but occurs either within another unidentified cell type or is transduced by that cell type on the gonadotrophs. In a companion paper (72), we show that the β1-AR constitutively activates adenylate cyclase via a pertussis toxin-sensitive transduction system, suggesting the hypothesis that its function may be related to the trophic action of gonadotrophs on lactotrophs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult (11–12 wk old) male Wistar rats were purchased from Elevage Janvier (Bio-Services, Uden, The Netherlands). The animals were kept in the animal house facilities of the University of Leuven under constant temperature, humidity, and day-night cycle, and had free access to pellet food and water. The rats were anesthetized with CO2 and killed by decapitation. All experiments were conducted in accordance with the Guidelines for Care and Use of Experimental Animals and were approved by the University Ethical Committee.

Anterior pituitary aggregate cell culture
Single cells were obtained by dispersion of the anterior pituitary lobes using methods described previously (30). Briefly, the pituitaries were cut into small blocks with a razor blade and enzymatically treated with porcine trypsin [2.5% from ICN Biomedicals (Aurora, OH) or 0.7% from Roche Diagnostics (Indianapolis, IN)], deoxyribonuclease (DNase) (Sigma-Aldrich, Steinheim, Germany), soybean trypsin inhibitor (Sigma-Aldrich), and 2 mM EDTA in Ca2+- and Mg2+-free medium. After mechanical dispersion with a polished and narrowed Pasteur pipette, cells were treated again with DNase and centrifuged at 190 g through a 3% BSA layer (fraction V, standard grade; Serva, Heidelberg, Germany) to remove cell debris. The cell pellet was then resuspended in serum-free defined culture medium as previously described (30, 31). This medium consists of DMEM/F12 1/1 with selenite and ethanolamine (prepared as powder by Life Technologies, Inc.-Invitrogen, Grand Island, NY), supplemented with 0.5% BSA (lyophilized, cell culture grade or fraction V receptor grade; Serva), 5 mg/liter insulin-Zn (Sigma-Aldrich), 5 mg/liter transferrin (Invitrogen), 50 mg/liter streptomycin (Sigma-Aldrich), 35 mg/liter penicillin (Sigma-Aldrich), 10 mM ethanol, 1 mg/liter catalase (Roche Diagnostics), and 1 g/liter NaHCO3. Final concentration of iron (Fe2+ and Fe3+) was 0.6 µM and of glucose 1.4 g/liter (7.8 mM). No phenol red was added to the medium.

To establish three-dimensional aggregate cell cultures, anterior pituitary cells were distributed at 2 x 106/2 ml culture medium in 35-ml nontreated culture dishes (Iwaki, Scitech division, Chiba, Japan), and allowed to re-associate on a gyratory shaker at 63 rpm in a humidified 1.5–1.9% CO2 incubator at 37 C. Unless otherwise stated, this medium was supplemented with 50 pM T3 (Serva) diluted from a 0.5 µM stock solution (in 0.9% NaCl) and/or various doses of DEX (Serva) diluted from a 0.8 mM stock solution (in ethanol). Vehicle was added to the controls. After 2 d the aggregates of each dish corresponding to a particular experimental treatment were divided equally over three dishes (each corresponding to ~666,000 cells) with fresh medium added for further analysis with TaqMan real-time qRT-PCR. Analysis was performed on the fifth day of culture, and each experiment was repeated three times.

Velocity sedimentation at unit gravity
Freshly dispersed cells were allowed to sediment through a 0.3–2.4% BSA (Fraction V, standard grade; Serva) gradient to prepare subpopulations enriched for a particular cell type, as previously described (30, 32, 33). Eleven fractions of 270 ml were collected from the gradient and called fractions A, B, and 1–9. In this study cells from fraction 3 and fraction 7–9 (further indicated as fraction 7 cells) were used. The proportion of the different pituitary cell types in the freshly isolated fractions was determined previously (10, 13, 32, 34, 35, 36). Because analysis of β1-AR mRNA was done at d-5 aggregate cell culture, we reanalyzed the proportions of pituitary cell types in fraction 3 and 7 after 5 d in aggregate cell culture by single-cell RT-PCR of mRNA of GH, PRL, proopiomelanocortin (POMC), and the glycoprotein hormone {alpha}-subunit ({alpha}GSU). The method has been described previously in detail (37, 38).

Cell line culture
GHFT cells, LβT2 cells, {alpha}T3-1 cells (all from P. Mellon, University of California, San Diego, CA), and AtT20 cells (ATCC; LGC Promochem, Teddington, UK) were cultured in Advanced DMEM/F12 mixture (Invitrogen) supplemented with GlutaMAX, streptomycin, penicillin (Invitrogen), and 10% fetal calf serum (FCS) (Cambrex Bio Science, Verviers, Belgium). GH3 cells (ATCC; LGC Promochem) and TtT/GF cells (Riken Cell Bank, Tsukuba, Ibaraki, Japan) were cultured in Advanced DMEM/F12 supplemented with GlutaMAX, streptomycin, penicillin, and 12.5% horse serum (Invitrogen), –2.5% FCS and 10% horse serum –2.5% FCS, respectively. All cells were seeded in 75 cm2 culture flasks and trypsinized twice a week using TrypLE (Invitrogen), except TtT/GF cells, which were released using a mixture of TrypLE and trypsin, and were dispersed in a way similar to pituitary cell dispersion. Cultures were maintained in a humidified 5% CO2 incubator at 37 C. According to the experimental design, the medium was supplemented with 80 nM DEX or with vehicle during 3–4 d before experimentation.

Immunofluorescent staining
To avoid interpretation problems of immunostaining signals due to autofluorescent red blood cells, rats were anesthetized with CO2 and perfused subsequently with a PBS solution and a 4% paraformaldehyde (Riedel-deHaën, Seelze, Germany)-PBS solution. Complete pituitaries were then dissected and further fixed using 4% paraformaldehyde (2 h at 4 C), rinsed with Ca2+- and Mg2+-free PBS, and embedded in 2% agarose (ICN Biomedicals) (in PBS). The samples were mounted on a Vibratome (Leica VT 1000 S; Leica Microsystems, Wetzlar, Germany), and sections of 40 µm were made. The slices were transferred to 24-well plates and stored in PBS at 4 C until additional processing. The complete staining procedure was performed at room temperature. The floating sections were rinsed with PBS and permeabilized with 0.5% saponin (Sigma-Aldrich) in PBS (2 x 10 min). To block nonspecific binding sites, sections were incubated with 20% normal goat serum (Sigma-Aldrich). Sections were treated overnight with the primary antibodies for the β1-AR and one of the pituitary hormones. Affinity purified rabbit antibody A272 against rat β1-AR (1:500 final dilution) was obtained from Sigma-Aldrich. The antibody was raised against a synthetic peptide (Gly-Asp-Arg-Pro-Arg-Ala-Ser-Gly-Cys-Leu-Ala-Arg-Ala-Gly) derived from amino acids 394–408 of mouse and rat β1-AR C-terminal domain. Specificity for β1-AR was shown by the manufacturer for both immunoblotting and immunofluorescent staining of sections of mouse kidney distal tubule cells, and further tested in the present study by antibody omission and preadsorption experiments (see Results). We also found positive staining on sections of rat kidney. The guinea pig antisera against rat {alpha}GSU (1:1000), LHβ (1:4000), TSHβ (1:4000), GH (1:1000), and PRL (1:1000) were obtained from Dr. A. F. Parlow through the National Hormone and Pituitary Program (Harbor-University of California Los Angeles Medical Center, Torrance, CA), and monoclonal mouse antibody against human POMC (1–50) (1:100) was obtained from Biogenesis (Poole, UK). As negative controls, primary antibodies were omitted. After excessive rinsing, sections were incubated with secondary antibodies for 90 min while being shielded from light. Secondary goat antirabbit Alexa 488 (1:1000), goat antiguinea pig Alexa 555 (1:2000), and goat antimouse Alexa 555 (1:1000) antibodies were purchased from Invitrogen. TOPRO-3 (1:100) (Invitrogen) was added during 10 min to stain the nuclei. All steps were performed in 0.5% saponin-PBS. After thorough rinsing with PBS, sections were mounted in Vectashield (Vector Laboratories, Burlingame, CA) on glass slides and covered with glass coverslips. Sections were scanned using a confocal laser-scanning microscope (LSM 510; Zeiss, Zaventem, Belgium) and were analyzed using the Zeiss LSM Image Browser as previously described (39).

Western blotting
Membrane proteins were extracted according to the method of Boulanger et al. (40). Treatment of cell lines and anterior pituitary cell aggregates was performed as described previously, except that the aggregates were treated with 80 nM DEX instead of 20 nM. All cells were pooled and spun down at 150 g for 20 sec and rinsed with ice-cold PBS. Subsequently, the pellet was sonicated for 30 sec in 500 µl TAME buffer [50 mM Tris-acetate (pH 7.4), 5 mM MgCl2, 5 mM EDTA, and 250 mM sucrose] to which complete protease inhibitor (Hoffmann-La Roche Ltd., Basel, Switzerland) was added. This mixture was centrifuged for 10 min at 800 g. The supernatant was spun down at 45,000 g for 30 min. The pellet was resuspended in TAME buffer, and quantification of the protein content was done with the Pierce Micro BCA Protein Assay Reagent Kit (Perbio Science, Erembodegem, Belgium). All steps were performed at 4 C. Proteins were denaturated in loading buffer containing Tris-HCl, glycerol, sodium dodecyl sulfate, bromophenol blue, and 2-mercapto-ethanol, and subsequently boiled for 5 min. Thirty to 75 µG protein of the samples was loaded on a sodium dodecyl sulfate/polyacrylamide gel (12%), as well as a broad-range prestained protein marker (New England Biolabs, Ipswich, MA). After electrophoresis, proteins were blotted on an Immobilon-P membrane (Millipore, Brussels, Belgium). The blots were blocked for nonspecific binding using 5% BSA in Tris-buffered saline to which 0.1% Tween-20 was added. A rabbit antirat β1-AR (1:250) primary antibody was obtained from Sigma-Aldrich (see Immunofluorescent staining section above). The antibody was added overnight in Tris-buffered saline to which 0.1% Tween 20 was added (4 C), after which an alkaline-phosphatase-coupled antirabbit secondary antibody was added for 1 h. After incubating the blots with ECF substrate (GE Healthcare, Diegem, Belgium), chemiluminescence was measured using the Storm 840 Phosphor/FluorImager, and analyzed using ImageQuaNT 5.0 software (Molecular Dynamics, Sunnyvale, CA). To check equal loading of samples, the blots were stripped with methanol, and the amount of the peroxisomal membrane protein peroxin 14P (PEX14P) was detected with rabbit anti-PEX14P (41) as the primary antibody.

qRT-PCR
Pituitary cell aggregates were collected on d-5 culture and transferred to 0.5 ml TriPure RNA isolation reagent (Hoffmann-La Roche Ltd.). Cell lines were trypsinized and transferred to 1 ml TriPure. RNA isolation and RT were performed as described before (31), with some minor modifications. In brief, the RNA solution was treated with Rnase-free DNase I (1 U/µl; Invitrogen) to destroy contaminating genomic DNA. The RT reaction was performed on 200 ng RNA in a final volume of 20 µl RT mixture. qRT-PCR was performed as described previously (42) using TaqMan Universal PCR Master Mix (Applied Biosystems, Lennik, Belgium). Primers to amplify rat GH and PRL cDNA, and rat as well as mouse β1- and β2-AR cDNA (Table 1Go) were purchased from Invitrogen, and TaqMan probes were obtained from Eurogentec (Seraing, Belgium). The primers for GH and PRL were designed over an intron. Given the absence of introns in β1- and β2-AR genes, precautions had to be taken to avoid amplification of genomic DNA. First, as mentioned, a DNase treatment was performed on the RNA extract. Second, for each sample to be analyzed, a negative RT control (i.e. RT in the absence of reverse transcriptase) was included. Amplification signal was not different from background in all the negative RT controls. Normalization of the mRNA level was performed against 18S rRNA, measured using the TaqMan Ribosomal RNA Control Reagents Kit (Applied Biosystems).


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  		TABLE 1. Primer and probe oligonucleotide sequences for qRT-PCR and accession numbers in GenBank for rat GH (rGH), rat β1 (rβ1)-, rat (rβ2)-, mouse β1 (mβ1)-, and mouse β2 (mβ2)-ARs

 
To quantify receptor mRNA levels, the equivalent of 17 ng initial RNA was used for qPCR. To quantify receptor expression, too high amounts of pituitary cDNA were needed to set up a standard curve. Therefore, a standard of receptor cDNA was made as follows. β1- and β2-AR fragments were amplified by PCR from pituitary cDNA. The amplified fragment was loaded onto a 2% agarose gel, extracted using GenElute Gel Extraction kit (Sigma-Aldrich), ligated into 2.1 Topo vector (Topo TA Cloning kit; Invitrogen), and the plasmids used to transform TOP10F’ Escherichia coli cells. The cells were plated on LB agar with ampicillin overnight. Colonies were picked and transferred to liquid LB ampicillin. Plasmid DNA was isolated using the boiling-prep-method and checked by enzyme restriction for insertion of the PCR fragment, after which plasmids were purified by phenol/chloroform extraction and sequenced by Lark (Essex, UK). Plasmid DNA (containing the receptor cDNA) was quantified using PicoGreen to calculate copy numbers. This stock was serially diluted (from 105 to one copy) to use as standard curve in qRT-PCR. To analyze GH mRNA levels, the RT product was further diluted in such a way that the equivalent of 170 pg initial RNA was added to the final 25 µl qPCR mixture. For quantification pituitary cDNA was used in a dilution series from 10 ng to 0.1 pg and run together with the samples to be analyzed.

The mRNA values measured were normalized for 18S rRNA content. The validity of 18S rRNA as internal control for qRT-PCR was verified against other housekeeping genes (β-actin and glyceraldehyde-3-phosphate dehydrogenase). Cycle threshold standard curves for all housekeeping gene products were parallel, and relative expression levels obtained for pituitary hormone mRNAs were similar, regardless of the housekeeping gene used for normalization. All samples were analyzed in triplicate.

Statistical analysis
All experiments were independently repeated at least three times. Values were expressed as mean ± SEM. Most data were log or ln transformed because of heterogeneity of variance. Data were compared by one-way, two-way, or three-way generalized linear model (GLM) ANOVA with the Tukey-Kramer comparison test, depending on the experimental design. The statistical package used for all experiments was NCSS (Statistical Solutions Ltd., Cork, Ireland).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
β1- and β2-AR mRNA is differentially expressed in the anterior and neuro-intermediate pituitary ex vivo
Intact anterior pituitary as well as neuro-intermediate lobe of adult male rats expressed both β1- and β2-AR mRNA (Fig. 1Go). mRNA levels of both receptors, normalized against 18S rRNA, were higher in the neuro-intermediate lobe than in the anterior pituitary (β1-AR: 2-fold; β2-AR: 20-fold). In the anterior lobe, β1-AR mRNA was approximately three times lower than the β2-AR mRNA level. As expected, GH mRNA levels in the neuro-intermediate lobe were negligible compared with levels in the anterior pituitary.


Figure 1
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  		FIG. 1. Expression of β1-AR, β2-AR, and GH mRNA in the rat anterior lobe (AL) and neuro-intermediate lobe (NIL). Three samples each consisting of three pituitaries were analyzed, and data are expressed as the percentage of anterior lobe expression levels, which are set at 100%. Measured mRNA values of anterior lobe, normalized for 18S rRNA content, are shown above the respective bars. Statistical analysis by one-way GLM ANOVA: **/***, significantly different from expression in anterior lobe with P = 0.01/0.001.

 
In pituitary aggregate cell culture β1- but not β2-AR expression is highly dependent on glucocorticoids
Pituitary cells were established in aggregate cell culture, and the dependence of β1-AR and β2-AR mRNA expression on DEX was tested. As an internal control for DEX action, GH mRNA was measured.

Both β1- and β2-AR remained expressed upon establishing anterior pituitary cells in aggregate cell culture for 5 d. However, the expression level of β1-AR mRNA was 10 times lower compared with the ex vivo levels (Fig. 2AGo). Supplementing the culture medium with DEX (80 nM) prevented the decrease in the β1-AR mRNA level. In contrast, β2-AR mRNA levels were not affected by culturing, and DEX caused a much smaller increase (Fig. 2BGo). GH mRNA levels (Fig. 2CGo) decreased in culture, but this was less pronounced than observed for β1-AR mRNA levels.


Figure 2
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  		FIG. 2. Influence of establishing anterior pituitaries in aggregate cell culture and of DEX (80 nM)-supplemented medium on β1-AR (A), β2-AR (B), and GH (C) mRNA levels. Ex vivo samples consisted of three pooled anterior pituitaries per experiment, cultured samples consisted of three dishes of aggregates in each experiment, and experiments were repeated three times. Values are expressed as the percentage of mRNA levels detected in the ex vivo pituitaries (set to 100%). Data were log transformed, and two-way GLM ANOVA revealed that *** was significantly different from ex vivo levels with P = 0.001, and ###/## was significantly different from control medium condition with P = 0.001/P = 0.01.

 
Up-regulation of β1-AR mRNA expression by DEX was dose dependent (Fig. 3AGo). A 5-fold increase was seen at 0.4 nM, the lowest dose used, and a maximal effect (20-fold increase) was seen at 20 nM DEX. In contrast, β2-AR mRNA levels increased only from a dose of 1 nM DEX (Fig. 3BGo) and did not further increase at higher doses of DEX. As expected (43, 44), DEX increased GH mRNA levels (Fig. 3CGo) from a dose of 4 nM, although no further dose response was found.


Figure 3
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  		FIG. 3. Dose response of the effect of DEX on β1-AR (A), β2-AR (B), and GH (C) mRNA levels in anterior pituitary cell aggregates. DEX was supplemented to the medium during the 5-d culture period. Two-way GLM ANOVA was performed on log-transformed data: ***/**/*, significantly different from control levels with P = 0.001/P = 0.01/P = 0.05; ###/##/#, significantly different from 0.4 nM DEX condition with P = 0.001/P = 0.01/P = 0.05; and °, significantly different from 1 nM DEX condition with P = 0.05.

 
To have an estimate of the time course of the β1-AR mRNA response to 80 nM DEX, the steroid was added to the cultures for a period of 1, 4, and 16 h before the end of the 5-d culture period, and expression level was compared with that of aggregates cultured in the presence of DEX during the entire 5 d-culture period. β1-AR mRNA levels had already increased within 1-h DEX exposure (6-fold increase) (Fig. 4AGo). The magnitude of this increase was maintained at approximately the same level when DEX was added for 4 and 16 h. However, when present for 5 d, DEX caused a 25-fold difference in β1-AR levels compared with expression found in steroid-free medium. β2-AR mRNA levels (Fig. 4BGo) slightly but significantly increased after 1-h DEX treatment, but this was not maintained after 4 and 16 h. The presence of DEX during the 5-d culture period resulted in a 2-fold increase in β2-AR mRNA levels compared with the steroid-free condition. GH mRNA levels (Fig. 4CGo) were significantly increased after 16-h exposure to DEX to 50% of that after the 5-d DEX treatment.


Figure 4
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  		FIG. 4. Time course of the effect of DEX on β1-AR (A), β2-AR (B), and GH (C) mRNA levels in cultured anterior pituitary cell aggregates. Time points indicated are the intervals during which aggregates were exposed to DEX at the end of the culture period with the 5-d condition corresponding to the complete culture period. Data are expressed as the percentage of mRNA levels detected in corresponding aggregates cultured in control medium with all control values set to 100%. The mRNA values, normalized for 18S rRNA content, of all control groups were comparable to each other. Three-way GLM ANOVA on ln-transformed data revealed that: ***/**/*, significantly different from corresponding control condition with P = 0.001/0.01/0.05; and ###, significantly different from all previous time points with P = 0.001.

 
Down-regulation by ISO confirms DEX dependency of β1-AR mRNA expression
It has been shown that activation of functional β-ARs by agonists results in down-regulation of receptor expression (45). A 6-h treatment of aggregates with the nonselective β-agonist ISO resulted in a decline of β1-AR mRNA level (Fig. 5AGo) in DEX (20 nM)-supplemented culture condition, but not in control culture condition. However, it should be noted that the vehicle itself also caused a decrease in β1-AR mRNA level and only in the steroid-free condition, making interpretation of the findings difficult. In contrast, in both the control and DEX conditions, the β2-AR mRNA level (Fig. 5BGo) was reduced, at least with a dose of 100 nM ISO. Interestingly, ISO slightly, but significantly, depressed GH mRNA levels independently of DEX treatment (Fig. 5CGo).


Figure 5
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  		FIG. 5. Effects of ISO on β1-AR (A), β2-AR (B), and GH (C) mRNA levels in anterior pituitary cell aggregates cultured with/without DEX (20 nM). Data are expressed as the percentage of control values (set to 100%). Three-way GLM ANOVA was performed on log-transformed data: ***/**/*, significantly different from corresponding control condition with P = 0.001/P = 0.01/P = 0.05; and ###/##/#, significantly different from vehicle (ascorbic acid) with P = 0.001/P = 0.01/P = 0.05.

 
β1- and β2-AR is expressed at the protein level in pituitary cell aggregates and in gonadotroph cell lines
Western blotting performed on membrane extracts of pituitary cell aggregates revealed a band at approximately 64 kDa, the expected size of the β1-AR (46), that was absent when the β1-AR primary antibody was omitted (Fig. 6AGo). As analyzed semi-quantitatively by measuring optical densities of the bands using different amounts of proteins loaded, the signal was 1.5–3 times more intense for aggregates cultured in DEX-supplemented (80 nM) medium than in the steroid-free controls (range of three independent experiments).


Figure 6
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  		FIG. 6. Western blot of β1-AR in membrane extracts from anterior pituitary cell aggregates (A) and pituitary cell lines (B and C), either in steroid-free [control (CTRL)] condition or treated with 80 nM DEX during culture (DEX). Anti-PEX14P was used to check equal loading of the samples. For pituitary aggregates 50 µG protein corresponded to approximately 3 x 106 cells. A (lane 1), Seventy-five microgram protein loaded, DEX treated [primary antibody (Ab) omitted as negative control]. Lane 2, Seventy-five microgram protein loaded, control (primary antibody omitted as negative control). Lane 3, A total of 33.3 µg protein loaded, DEX treated. Lane 4, A total of 33.3 µg, control. Lane 5, Fifty microgram protein loaded, DEX treated. Lane 6, Fifty micrograms, control. B, Sixty microgram protein loaded in all lanes. Lanes 1 and 2, After preadsorption (Pre-abs) of antiserum with 33 nmol immune peptide. Lanes 3 and 4, After preadsorption of antiserum with 10 nmol immune peptide. Lanes 5 and 6, No preadsorption. Lanes 1, 3, and 5, LβT2 control. Lanes 2, 4, and 6, {alpha}T3-1 control. C, Sixty microgram protein loaded in all lanes. Lane 1, {alpha}T3-1 control. Lane 2, LβT2 control. Lane 3, GHFT DEX treated. Lane 4, GHFT control. Lane 5, GH3 DEX treated. Lane 6, GH3 control. MW, Molecular weight.

 
A band of approximately 64 kDa was also detectable in the pituitary cell lines (Fig. 6Go, B and C), except in AtT20 cells (data not shown). GH3 cells showed a considerably weaker band (Fig. 6CGo). DEX treatment did not result in an obvious increase of band intensities in the gonadotrophic or other cell lines (Fig. 6CGo; data not shown). Preadsorption of the antibody with 10 nmoles of the immune peptide, used for generating the antibody (Gly-Asp-Arg-Pro-Arg-Ala-Ser-Gly-Cys-Leu-Ala-Arg-Ala-Gly) for 8 h, resulted in a significant reduction of staining in the {alpha}T3-1 and LβT2 cell samples (Fig. 6BGo). Virtually complete disappearance of the β1-AR band was seen in the LβT2 cell samples with 33 nmoles of peptide.

Double immunostaining shows β1-AR immunoreactivity (ir) in a subpopulation of {alpha}GSU-ir and LHβ-ir cells
Localization of β1-AR-ir was examined in Vibratome sections of intact rat pituitary tissue, simultaneously stained for each of the different pituitary hormones (Fig. 7Go). β1-AR-ir cells (green color) were large (14–24 µm with an average size of 19 µm) and had a round or oval morphology. The β1-AR-ir was found in one or more compartments of the cell cytoplasm, but not at the plasma membrane area. Because β1-AR-ir staining was weak, we had to enlarge pinhole settings to enhance visibility. As a result, often a rather diffuse staining pattern was seen that makes interpretation of the cellular compartmentalization difficult. However, in some cells immunoreactive material looked particulate, plausibly the Golgi apparatus or the endoplasmatic reticulum. Preadsorption of the antirat β1-AR antibody with the peptide used to generate the antibody resulted in a clearly diminished fluorescent signal (data not shown). β1-AR-ir was colocalized with a subpopulation of LHβ-ir cells (red color) (Fig. 7Go, E and F). Colocalization was regularly encountered all over the anterior pituitary, although it was in a relatively small subpopulation, consistent with the predominant occurrence in the large gonadotrophs. Due to the relatively weak green β1-AR-ir signal, the pinhole for green had to be enlarged. The consequence of this was that most of the green signals did not colocalize with the red signal (no yellow color), and because green fluorescence was usually not seen in the periphery of the cells, the green areas were encircled by red in several of the examples shown in Fig 7Go. Consistent with the colocalization with LHβ, β1-AR was found in part of the {alpha}GSU-positive cells (Fig. 7DGo). There was no colocalization detected with GH (Fig. 7AGo), ACTH (Fig. 7BGo), and TSHβ (Fig. 7CGo). In a number of cells, some weak double staining of β1-AR-ir and PRL-ir was observed (leading to weak orange stain) (Fig. 7Go, G–J), but upon careful examination, we concluded that the orange color was due to bleed through of the red channel in the green one, which was confirmed by the finding that β1-AR-ir cells never had the morphology of these lactotrophs. Therefore, we conclude that no convincing colocalization of β1-AR with lactotrophs was present. Interestingly, β1-AR-ir cells were found adjacent to PRL-ir cells, and these lactotrophs were often cup shaped (Fig. 7Go, G–J).


Figure 7
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  		FIG. 7. Confocal microscopic photomicrographs of double-immunofluorescent staining of Vibratome sections of intact rat anterior pituitary tissue, stained for β1-AR (green) and anterior pituitary hormones (red). Nuclei stained with ToPro-3 (blue). A, GH. B, POMC. C, TSHβ. D, {alpha}GSU. E and F, LHβ. G–J, PRL (arrows indicate cup-shaped lactotrophs). Bar, 10 µm.

 
β1- and β2-AR mRNA is detectable in pituitary cell lines, with highest levels in LβT2 and {alpha}T3-1 cells
To confirm the cellular distribution of β1-AR mRNA, we investigated the presence and level of β1- and β2-AR in various pituitary cell lines representative for the different pituitary cell types. As shown in Table 2Go, β1-AR mRNA showed low levels in TtT/GF (FS cells) and GHFT cells (progenitor somatotrophs/lactotrophs), midrange levels in LβT2 cells (gonadotrophs) (factor 2 compared with GHFT cells), and high levels in {alpha}T3-1 cells (gonadotroph precursor) (factor 8 compared with LβT2 cells). No expression was found in AtT20 cells (corticotrophs). Levels of β2-AR mRNA differed according to cell line as well. Low levels were found in AtT20 cells, midrange in GHFT (factor 5 compared with AtT20), and higher levels in LβT2 and {alpha}T3-1 cells (factor 2 compared with GHFT). For both ARs expression was not markedly affected by DEX (80 nM).


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  		TABLE 2. ß1- and ß2-AR mRNA levels, normalized to 18S rRNA, in mouse pituitary cell lines cultured either in steroid-free medium or in medium with 80 nM DEX

 
DEX-dependent β1-AR mRNA is confined to a subpopulation of small cells
Because no effect of DEX on β1-AR mRNA level was found in the cell lines, we hypothesized that the up-regulation of β1-AR mRNA level by DEX in pituitary cell aggregates does not occur in gonadotrophs but in another cell type or that it is conferred by another cell type on the gonadotrophs. To test the latter hypothesis, anterior pituitary cells were fractionated into subpopulations, enriched in particular cell types, by velocity sedimentation at unit gravity through a BSA gradient, and subsequently established in aggregate cell culture and challenged with DEX. We have previously shown that cells of the top fractions of the gradient are small sized, and consist mainly of lactotrophs and nonhormonal cells, whereas cells in the bottom fraction of the gradient are large and enriched in somatotrophs and gonadotrophs (32, 33). In the present study, the proportional distribution of the cell types was verified after 5-d aggregate cell culture by detection of GH, PRL, POMC, and {alpha}GSU mRNA in individual cells after redispersion of the aggregates and fluorescence-activated cell sorting (see Materials and Methods). As shown in Table 3Go, fraction 7 cells consisted mainly of GH cells (total number of GH mRNA-positive cells: 72%), {alpha}GSU cells (12%), whereas fraction 3 cells contained mainly PRL cells (65%) and nonhormonal cells (25%).


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  		TABLE 3. Proportions of hormonal cells identified on the basis of hormone mRNA content detected by single-cell RT-PCR in anterior pituitary aggregates prepared from separated subpopulations and cultured in the absence or presence of 20 nM DEX

 
As shown in Fig. 8Go, in the absence of DEX, absolute β1- and β2-AR mRNA levels within fraction 3 and fraction 7 aggregates were comparable. Supplementation with 20 nM DEX caused a 24-fold increase in the β1-AR mRNA content in fraction 3 cell aggregates (Fig. 8AGo) but was ineffective in aggregates prepared from the bottom fraction 7. Coaggregation of fraction 3 and fraction 7 cells in a ratio 3:1 (that resembles the original ratio in the initial cell population) resulted in a DEX response of β1-AR mRNA that was weakened (8.6-fold increase in β1-AR mRNA content) compared with that found in fraction 3 cells (Fig. 8AGo, right panel). There was a small effect of DEX on the β2-AR mRNA level in fraction 3 (only 1.8-fold increase) (Fig. 8BGo). Coaggregation abolished that small effect of DEX on β2-AR (Fig. 8BGo, right panel). In contrast, DEX treatment increased GH mRNA levels in both fractions 3 and 7, but to a larger extent in fraction 3 than in fraction 7 cell aggregates (4.5- and 2.5-fold increase, respectively) (Fig. 8CGo). In the coaggregates DEX augmented GH mRNA levels with a magnitude similar to that seen in fraction 3 (3.8-fold increase).


Figure 8
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  		FIG. 8. Effect of DEX on β1-AR (A), β2-AR (B), and GH (C) mRNA levels in cultured pituitary cell aggregates obtained from subpopulations [fraction (fr) 3 and fraction 7] enriched in particular cell types. Data represent mRNA values normalized for 18S rRNA content. Three-way GLM ANOVA was performed on log-transformed data: ***, significantly different from corresponding control condition with P = 0.001; ###, significantly different from corresponding fraction 7 with P = 0.001; and °°/ °°°, significantly different from corresponding fraction 3 with P = 0.01/0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main finding of the present investigation is that the anterior pituitary expresses β1-AR mRNA and protein in a glucocorticoid-regulated manner, and that expression is found in a subpopulation of gonadotrophs. No colocalization was seen with somatotrophs, corticotrophs, thyrotrophs, and lactotrophs. In confirmation, expression of β1-AR, at the mRNA and protein level, was also found in two gonadotroph cell lines, i.e. the gonadotroph precursor {alpha}T3-1 and the more mature LβT2 cells, whereas the GHFT (somatotroph precursor), GH3 (lactosomatotrophs) cells, and TtT/GF cells (FS) showed very low mRNA expression levels. This localization is clearly distinct from that of the β2-AR, previously reported to be confined to somatotrophs, lactotrophs, and corticotrophs on the basis of pharmacological criteria (7, 11, 15, 22, 23, 24, 25). To our knowledge, the presence of β1-AR mRNA and protein has not been reported before in hormonal cells of the anterior pituitary. The failure to find reasonable expression of β1-AR mRNA in the FS cell line is at variance with pharmacological data showing bovine FS cells in culture to express both β1- and β2-ARs (27).

A striking characteristic of β1-AR mRNA expression was that its level decreased considerably after 5 d in reaggregate cell culture and that this was prevented by supplementing the cultures with glucocorticoids. DEX elevated the β1-AR mRNA level at least 10-fold. In contrast, DEX only induced a 2-fold increase in the β2-AR mRNA level. Numerous investigators have shown that glucocorticoids regulate β1-AR mRNA levels in various tissues, but in most an inhibitory effect has been observed (47, 48, 49, 50, 51, 52) through an inhibitory glucocorticoid response element located in the promoter of the rat β1-AR gene (53). To our knowledge, the only stimulatory action of glucocorticoids on β1-AR expression previously reported was by Mak et al. (54) in rat lung and by Tseng et al. (55) in ovine cardiomyocytes and receptor-transfected SK-N-MC cells. The stimulatory effect of DEX in our study could be explained by an association of the glucocorticoid receptor with corepressors or coactivators such as activator protein-1, nuclear factor-{kappa}B, and cAMP response element binding protein (55, 56, 57, 58) that may be differentially expressed in the pituitary compared with other tissues.

Glucocorticoid regulation of β1-AR mRNA expression differs from that of β2-AR in several other aspects. First, a dose as low as 1 nM already provoked a maximal effect on the β2-AR mRNA expression level (although only a 2-fold increase), whereas the β1-AR mRNA response to DEX displayed a much larger dose-response window, with 0.4 nM already effective and 20 nM provoking a 10- to 20-fold increase as a maximal effect. These differential characteristics suggest different mechanisms, direct or indirect, behind the action of the glucocorticoid receptor on these two genes. Second, up-regulation of the β1-AR mRNA level by DEX was different from that of β2-AR mRNA as a function of time. The β1-AR mRNA level already reached a 6-fold higher level within 1-h DEX exposure, and these elevated levels were maintained in the following hours, whereas β2-AR mRNA levels did not change until after 16 h, except for a small but temporary increase after 1 h. Whether the effect after 1 h is a genomic effect or is induced by nongenomic actions such as through annexin-1 release (21), remains to be studied. It has been reported that in C6 glioma cells, the effect of DEX on β2-AR expression occurs only after several days of treatment, whereas on β1-AR mRNA levels, an effect is seen within 2–4 h (47). Finally, the glucocorticoid environment influenced ISO-mediated down-regulation of both β-ARs in a different manner. DEX facilitated down-regulation of β2-AR mRNA after a 6-h exposure to a low dose of ISO. In contrast, ISO at all doses tested failed to down-regulate β1-AR mRNA levels in the absence of glucocorticoids, although this interpretation should be taken with caution because the treatment vehicle (ascorbic acid) was inhibitory on its own. These results are opposite of those of in vivo studies in rat lung, in which β2-AR but not β1-AR down-regulation could be prevented by DEX (54). This suggests tissue-specific regulation by glucocorticoids.

Immunoblotting revealed the presence of β1-AR immunoreactive protein of the expected size (64 kDa) in membrane extracts of anterior pituitary cell aggregate cultures. Estimation of the protein amount in the bands indicated only 1.5–3 times more protein in DEX-cultured compared with control aggregates, which is a magnitude considerably smaller than that induced by DEX at the mRNA level. Because expression of β1-AR-ir itself was modest on Western blot, the small changes induced by DEX should be taken with caution. The small magnitude of the DEX effect at the protein level may be trivial because the level of β1-AR mRNA in steroid-free medium could decrease faster than that of β1-AR protein. This would result in a seemingly smaller proportional effect of DEX on the protein level than on the mRNA level. Alternatively, it is possible that DEX increases the β1-AR mRNA level without (or with limited) translation into protein. It is reasonable to suspect that other regulatory factors may be required to further increase β1-AR protein expression at the translational level and that these are perhaps not present in the pituitary aggregate cell cultures.

Virtually all β1-AR-ir material was detected in the cytoplasmic compartments of relatively large cells with a round to oval morphology, and not at the cell membrane or in the peripheral areas of the cells. The lack of immunostaining at the plasma membrane may be due to diminished ir as a consequence of the folding of the protein into the membrane or of binding of regulatory proteins, particularly because the antibody was raised against a peptide from the C-terminal part of the protein. Cytoplasmic staining of the β1-AR has been described also in other tissues (59).

The finding of β1-AR expression in gonadotrophs is in striking contrast to the evidence that the up-regulation of β1-AR mRNA levels by DEX was apparently not located in the gonadotrophs. In a subpopulation of cells, separated by sedimentation at unit gravity and relatively enriched in large gonadotrophs (fraction 7), DEX had no effect on β1-AR mRNA levels, but in a subpopulation poor in gonadotrophs (fraction 3), DEX caused a vast up-regulation (24-fold increase) of the β1-AR mRNA level. Fraction 3 contains, besides lactotrophs, FS cells (60) and other nonhormonal cells (39, 61, 62) as major populations (Table 3Go), whereas corticotrophs, thyrotrophs, and somatotrophs are poorly represented. No evidence for an involvement of lactotrophs and FS cells was found because no β1-AR expression was found in lactotrophs or the lactotroph-derived GH3 cells (63) (and this study). Furthermore, only a very low to negligible β1-AR mRNA level was found in the FS cell line TtT/GF, of which expression was not inducible by glucocorticoids. There was also no effect of DEX found on the β1-AR mRNA level in the gonadotrophic cell lines {alpha}T3-1 and LβT2. Thus, it seems that the DEX-induced β1-AR mRNA up-regulation found in the small cell population (fraction 3) is located in an unknown cell type. A possibility is that these small cells are nonhormonal cells that separate in the "side population" (SP) (64). In two independent experiments, we have been able to detect β1-AR mRNA by RT-PCR in a collected population of mouse pituitary SP cells (Vankelecom, H., unpublished observations). Moreover, three independent microarray analysis experiments performed on mouse pituitary SP cells and cells from the main population showed statistically significant expression of β1-AR mRNA in both populations (Vankelecom, H., unpublished observations). The SP enriches in the upper fractions of the unit gravity sedimentation gradient (rat) (Vankelecom, H., unpublished observations). These cells express stem/progenitor cell and early embryonic markers (64). Thus, also nonhormonal cells seem to express β1-AR, at least at the mRNA level. Because in our immunofluorescent studies only gonadotrophs were immunopositive for β1-AR, it looks plausible that DEX up-regulates β1-AR mRNA in a nonhormonal cell type in fraction 3 without β1-AR mRNA sufficiently translated into β1-AR protein. Perhaps these cells are committed gonadotroph progenitors. Alternatively, the action of DEX may occur in a nonhormonal cell type of fraction 3 that in turn up-regulates β1-AR in gonadotrophs through a paracrine mechanism. In an attempt to test the latter hypothesis, fraction 3 cells were coaggregated with fraction 7 cells and the coaggregates examined for their responsiveness to DEX. It was found that fraction 3 cells did not confer the DEX responsiveness of the β1-AR to the fraction 7 cells, whereas the magnitude of the GH mRNA response to DEX seen in fraction 3 cells was indeed transduced to the fraction 7 cells. Unfortunately, we cannot exclude that fraction 7 cells may have exerted a negative feedback on the fraction 3 cells in their putative action on β1-AR expression in gonadotrophs.

A remarkable feature of the β1-AR positive cells was that they are located adjacent to lactotrophs. These lactotrophs were often found in clusters surrounding the β1-AR positive cells, and many had a cup-shaped morphology. The subpopulation of cup-shaped lactotrophs that is in close association with gonadotrophs has been described in the rat (65, 66) as well as in other species (67, 68, 69), and the selective association is reconstituted in aggregate cell culture (35). Most interestingly, also calcitonin, a paracrine factor that depresses PRL release and gene expression, has been located in gonadotrophs that are surrounded by cup-shaped lactotrophs (70). Therefore, the present findings can be related to earlier findings in our laboratory (33) and to that of others (for review, see Ref. 71) that gonadotrophs exert trophic actions on lactotrophs. Further support to the latter hypothesis is given in a companion paper (72), in which we show that in pituitary cell aggregates, the β1-AR is constitutively coupled to adenylate cyclase in a pertussis toxin-sensitive manner. The unliganded receptor constitutively activates cAMP formation, which in turn may activate communication between gonadotrophs and lactotrophs.

In conclusion, the present study shows expression of β1-AR in a subpopulation of gonadotrophs on the basis of mRNA detection, Western blot, double immunostaining, and specific cell line analysis. β1-AR mRNA expression is up-regulated by glucocorticoids, but this does not seem to occur in the main gonadotroph cell population itself. Whether this occurs within a subpopulation of small nonhormonal cells, or is transduced by the latter cells upon gonadotrophs, remains to be studied further. Because β1-AR-positive cells are often surrounded by cup-shaped lactotrophs, β1-AR may play a role in intracrine regulation in gonadotrophs or in paracrine regulation between gonadotrophs and lactotrophs. The present findings suggest a potential interference of the adrenergic system with the paracrine network in the anterior pituitary (71), which may be of relevance in the context of the negative influences of stress on reproduction.


   Acknowledgments
 
We thank Dr. A. F. Parlow (Harbor-University of California Los Angeles Medical Center, Torrance, CA) for providing guinea pig antisera against rat glycoprotein hormone {alpha}-subunit, LHβ, TSHβ, GH, and prolactin. We also thank M. Boussemaere, K. Rillaerts, and Y. Van Goethem for their skillful technical assistance.


   Footnotes
 
This work was supported by grants from the Flemish Ministry of Science Policy (Concerted Research Actions) and the Fund for Scientific Research Flanders (Belgium) (Fonds voor Wetenschappelijk Onderzoek).

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 17, 2008

Abbreviations: AR, Adrenoceptor; DEX, dexamethasone; DNase, deoxyribonuclease; FCS, fetal calf serum; FS, folliculo-stellate; GLM, generalized linear model; {alpha}GSU, glycoprotein hormone {alpha}-subunit; ir, immunoreactivity; ISO, isoproterenol; PEX14P, peroxin 14P; POMC, proopiomelanocortin; PRL, prolactin; qRT-PCR, quantitative RT-PCR; SP, side population.

Received October 11, 2007.

Accepted for publication January 10, 2008.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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K. Janssens, M. Boussemaere, S. Wagner, K. Kopka, and C. Denef
{beta}1-Adrenoceptors in Rat Anterior Pituitary May Be Constitutively Active. Inverse Agonism of CGP 20712A on Basal 3',5'-Cyclic Adenosine 5'-Monophosphate Levels
Endocrinology, May 1, 2008; 149(5): 2391 - 2402.
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