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Endocrinology Vol. 143, No. 6 2427-2436
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Adenosine-Regulated Cell Proliferation in Pituitary Folliculostellate and Endocrine Cells: Differential Roles for the A1 and A2B Adenosine Receptors

D. A. Rees, M. D. Lewis, B. M. Lewis, P. J. Smith, M. F. Scanlon and J. Ham

Departments of Medicine and Pathology (P.J.S.), University of Wales College of Medicine, Cardiff, United Kingdom CF14 4XN

Address all correspondence and requests for reprints to: Dr. D. A. Rees, Department of Medicine, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom CF14 4XN. E-mail: . reesda{at}cf.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A1 and A2 adenosine receptors have been identified in the pituitary gland, but the cell type(s) on which they are located and their effects on pituitary cell growth are not known. Therefore, we analyzed the expression of A1 and A2 receptors in primary rat anterior pituitary cells, two pituitary folliculostellate (TtT/GF and Tpit/F1) and two pituitary endocrine (GH3 and AtT20) cell lines, and compared their effects on cell proliferation. In anterior pituitary and folliculostellate cells, adenosine and adenosine receptor agonists (5'-N-ethylcarboxamidoadenosine, a universal agonist, and CGS 21680, an A2A receptor agonist) stimulated cAMP levels with a rank order of potency that indicates the presence of functional A2B receptors. This stimulation, however, was not observed in either GH3 or AtT20 cells, where adenosine and the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine inhibited VIP/forskolin-stimulated cAMP production. Expression of A2B and A1 receptors in the folliculostellate cells and that of the A1 receptor in the endocrine cells were confirmed by RT-PCR, immunocytochemistry, and ligand binding. Adenosine and 5'-N-ethylcarboxamidoadenosine dose-dependently (10 nM to 10 µM) stimulated growth in the folliculostellate, but not in the endocrine, cells, whereas in the latter, 100 µM adenosine and 2-chloro-N6-cyclopentyladenosine inhibited cell proliferation by slowing cell cycle progression. These data highlight the differential expression of A1 and A2B adenosine receptors in pituitary cells and provide evidence for opposing effects of adenosine on pituitary folliculostellate and endocrine cell growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PURINE NUCLEOSIDE adenosine modulates a number of physiological processes through interaction with G protein-coupled adenosine receptors, four of which have been described to date (A1, A2A, A2B, and A3) on the basis of convergent pharmacological, biochemical, and molecular evidence (1). The A2 receptor subtypes are positively coupled to adenylate cyclase, whereas the A1 and A3 subtypes are negatively coupled, although activation of the latter usually requires relatively high concentrations of adenosine (1). Adenosine has been identified previously in the pituitary gland (2) along with A1 and A2 (3, 4, 5, 6, 7), but not A3, receptors. However, studies on the distribution and function of these receptor subtypes are limited. Functional A1 receptors are present in GH3 and GH4 rat pituitary tumor cell lines, mediating inhibition of cAMP, calcium mobilization, and inositol phosphate generation (3, 4, 5). Also, the A1 receptor mediates the inhibition of PRL and GH production in a clonal pituitary cell line (8) and the inhibition of MSH{alpha} release in frog anterior pituitary cells (9). Adenosine receptors are important in growth stimulation (10, 11) and apoptosis (12, 13) in a variety of systems. Although adenosine has been shown to affect cell cycle kinetics in GH4 cells (14), its effects on the control of pituitary cell growth have not been explored in detail.

There are limited data describing the presence and roles of A2 receptors in the pituitary gland. Anand-Srivastava and colleagues described adenosine-sensitive adenylate cyclase in the rat anterior pituitary (6) and have suggested that in cultured anterior pituitary cells adenosine regulates the release of ACTH through activation of A2 receptors (15). In vitro stimulation of TSH and PRL release in response to A2- selective agonists has also been described (7). Despite these observations the cell types on which these receptors are located have not been precisely defined.

Folliculostellate cells, which comprise up to 10% of the cells in the anterior pituitary gland (16), play a supportive role for neighboring endocrine and endothelial cells and share similar characteristics with glial cells, including expression of the glial protein S100 (17). Several lines of evidence suggest that purines such as adenosine have trophic effects on glial cells. Rathbone and colleagues have shown that adenosine stimulates the proliferation of chick astrocytes in vitro (18) and induces a 3- to 10-fold stimulation of DNA synthesis in human astrocytoma cells (19). In addition, A2 receptor antagonists have been shown to inhibit astrogliosis and glial fibrillary acidic protein expression in vivo (20).

Based on these observations we hypothesized that A1 receptors and A2 receptors might be differentially expressed in pituitary endocrine and folliculostellate cells and could have opposing effects on proliferation. In this report we describe the presence of the A2B receptor in two folliculostellate cell lines and demonstrate its functional dominance in comparison with the other subtypes. We also show that A1 receptors are present in both folliculostellate cells and endocrine cells and confirm their existence in a low affinity state. Furthermore, we show that adenosine can have opposing effects on cell proliferation by stimulating growth, via the A2B receptor, in folliculostellate cells and inhibiting growth, via the A1 receptor, in pituitary endocrine cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Cell culture materials and reagents were obtained from Invitrogen (Paisley, UK), Autogen Bioclear (Calne, UK), Sarstedt Ltd. (Leicester, UK), and Sigma-Aldrich Corp. (Poole, UK). Adenosine, 5'-N-ethylcarboxamidoadenosine (NECA; universal adenosine receptor agonist), 2-chloro-N6-cyclopentyladenosine (CCPA; A1 receptor agonist), 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680, selective A2A receptor agonist), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; selective A1 receptor antagonist), xanthine amine congener (XAC; adenosine receptor antagonist), VIP, forskolin, Ro 20–1724 (a cAMP phosphodiesterase inhibitor that does not act as an antagonist at adenosine receptors), and calf intestinal adenosine deaminase (ADA) were obtained from Sigma-Aldrich Corp. [Methyl-3H]thymidine, [3H]CGS 21680, [3H]NECA, and [3H]DPCPX were obtained from Amersham Pharmacia Biotech (Little Chalfont, UK) or NEN Life Science Products (Dreierch, Germany). RT-PCR reagents were obtained from Promega Corp. (Southampton, UK). Affinity-purified polyclonal rabbit anti-A1 receptor (21) and rabbit anti-A2B receptor (22) antisera were obtained from Alpha Diagnostic International (San Antonio, TX). Immunocytochemistry reagents were obtained from Vector Laboratories, Inc. (Peterborough, UK). TtT/GF and Tpit/F1 cells were provided by Prof. Kinji Inoue (Department of Regulation Biology, Saitama University, Urawa, Japan).

Cell culture
Pituitary glands were removed aseptically from male Wistar rats (200–250 g). The posterior/intermediate lobes were teased away from the anterior lobes, which were chopped into small pieces and placed in an enzyme mixture containing dispase (0.1%), collagenase type II (0.5%), hyaluronidase (0.1%), and deoxyribonuclease I (0.01%) in Earle’s balanced salts solution (EBSS). The tissue was incubated for 15–30 min at 37 C with frequent trituration, and the resulting cell suspension was washed twice in EBSS. Cells (5 x 104/cm2 ) were plated into 24-well plates in DMEM supplemented with antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin) and 10% FCS. The cells were kept for 4 d at 37 C in a humidified atmosphere of 5% CO2 before experimentation. GH3 (23), AtT20 (24), TtT/GF (25), and Tpit/F1 (26) cells were cultured as described previously.

cAMP experiments
GH3 (75,000/cm2 ), AtT20 (50,000/cm2 ), and FS cells (25,000/cm2 ) were plated into 24-well plates and allowed to grow for 3–4 d. For experimentation, cells were washed in EBSS and incubated in DMEM with 20 mM HEPES (pH 7.4) and 2 U/ml ADA (to remove endogenous adenosine) for 15 min at 37 C, followed by two additional washes in EBSS. For cAMP stimulation experiments, NECA, CGS 21680, or adenosine together with 100 µM Ro 20–1724 were added for an additional 15 min. For studies investigating the inhibition of stimulated cAMP production in GH3 and AtT20 cells, CCPA or adenosine was coincubated with 100 nM VIP (GH3) or 1 µM forskolin (AtT20) for 15 min in the presence of 100 µM Ro 20–1724. At the end of the experiment, cells were extracted in 0.1 M HCl and stored at -20 C. Before assay, samples were centrifuged at 10,000 x g, evaporated to dryness, reconstituted in assay buffer (50 mM sodium acetate and 0.25% BSA, pH 5.2) and acetylated by treatment with triethylamine and acetic anhydride. cAMP levels were measured using an in-house RIA as described previously (27). The sensitivity of the assay was less than 4 fmol/tube.

RT-PCR
Total cellular RNA was prepared using TRIzol reagent (Invitrogen, Paisley, UK). After incubation with RQ1 ribonuclease-free deoxyribonuclease (Promega Corp.), 0.5 µg RNA was reverse transcribed using oligodeoxythymidilic acid [oligo(dT)15] for 1 h at 37 C, and the cDNA generated was subjected to PCR amplification using primers specific for the adenosine receptor subtypes and phosphoglucokinase-1 as a housekeeper control (Table 1Go). The primers were designed to hybridize to different exons and constructed according to the published rat and murine DNA sequences (Table 1Go). Thirty-five cycles of the reaction were performed at 94, 65, and 72 C for 30 sec, 1 min, and 2 min, respectively, followed by a final extension step of 72 C for 10 min. Amplified products were electrophoresed in 2% agarose gel and stained with ethidium bromide. Correct amplicon identity was confirmed where appropriate by automated DNA sequencing.


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  		Table 1. Primer sequences and cDNA fragment sizes

 
Immunocytochemistry
GH3, AtT20, TtT/GF, and Tpit/F1 cells were plated onto Thermanox coverslips and cultured for 2–3 d. The cells were washed in PBS (10 mM, pH 7.4) and dried after fixation in ice-cold methanol. Endogenous peroxidase activity was blocked with 0.025% periodic acid, and cells were then incubated with the appropriate primary antibody for 1 h at room temperature, followed by biotinylated goat antirabbit secondary antibody for 30 min. All antisera were used according to the manufacturer’s recommendations, and visualization was performed using an indirect immunoperoxidase procedure (Vectastain Elite ABC kit, Vector Laboratories, Inc., Burlingame, CA).

Ligand binding experiments
GH3, AtT20, TtT/GF, and Tpit/F1 cells were plated out into 24-well plates and cultured as described above. Experiments were performed at room temperature (25 C) in serum-free Ham’s F-10 (GH3) or serum-free DMEM (AtT20, TtT/GF, and Tpit/F1) buffered with 20 mM HEPES (pH 7.4) containing 2 U/ml ADA. Competition of 3 nM [3H]DPCPX (14), [3H]CGS 21680 or [3H]NECA binding was performed by incubation with different concentrations of unlabeled compound for 90 min. The cells were then washed with ice-cold PBS and extracted into 0.1 M HCl, and the cell suspension was transferred to scintillation vials containing 3 ml Safefluor scintillation fluid (Packard Bioscience, Pangbourne, UK).

Cell proliferation experiments
GH3 (30,000/cm2 ), AtT20 (15,000/cm2 ), and TtT/GF and Tpit/F1 (15,000/cm2 ) cells were seeded into 96-well plates, switched to medium with 5% FCS (GH3 and AtT20) or 2% FCS (TtT/GF and Tpit/F1) for 24 h, and incubated with the test substances for the times indicated. Cell proliferation was assessed by two different methods, a cell proliferation assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega Corp.) and [methyl-3H]thymidine incorporation. For the cell proliferation assay the number of viable cells at the end of each experiment was determined colorimetrically. Standard curves were prepared showing correlation of absorbance with cell number. For experiments looking at thymidine incorporation, [methyl-3H]thymidine (1 µCi/ml) was added to the culture medium for the final 4 h of each incubation period. Where indicated, GH3 cells were synchronized in G0/G1 by serum deprivation for 48 h. At the end of each experiment cells were disrupted and harvested in an automated cell harvester (Mach III Harvester, Tomtec, Hamden, CT) onto filters that were dried and sealed in bags with scintillation fluid before counting on an automated liquid scintillation and luminescence counter (Microbeta, Wallac, Inc., Turku, Finland).

Cell cycle kinetic studies
Flow cytometry was used to perform multidimensional analysis of cell cycle distribution and right angle light scatter characteristics of GH3 cells treated with CCPA. Approximately 5 x 105 cells in 1 ml complete culture medium were stained with 0.125 ml ethidium bromide (400 µg/ml in 1% Triton X-100) and treated with ribonuclease A (final concentration, 0.5 mg/ml). After the treatments, as indicated in Results, stained cells were analyzed using a FACS Vantage flow cytometer (Becton Dickinson and Co., San Jose, CA) incorporating an Innova Enterprise II argon ion laser (Coherent, Inc., Santa Clara, CA) emitting 488-nm and multiline UV (351–355 nm; 30 mW) wavelengths. Forward scatter and side scatter were acquired in linear mode for 10,000 cells. Ethidium bromide was excited at 488 nm, and fluorescence signals were detected using a 585/42 nm bandpass filter. CellQuest software (Becton Dickinson and Co.) was used for signal acquisition and analysis. Data were were expressed as the mean fluorescence intensity for populations of single cells. Fluorescence distributions were analyzed using a cell cycle phase-fitting program (28) that assumes normal distributions for G1 and G2/M populations and calculates a probability function for the S phase distribution based upon the means and SDs of the G1 and G2/M compartments.

Statistical analysis
Results were expressed as the mean ± SEM and were compared by ANOVA with subsequent comparisons by Tukey’s multiple comparison test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adenosine stimulates intracellular cAMP production in primary rat anterior pituitary cells via the A2B adenosine receptor
In our initial experiments we measured intracellular cAMP production in anterior pituitary cells incubated with adenosine. Adenosine caused dose-dependent stimulation of cAMP accumulation (Fig. 1Go), suggesting that A2 receptors are functionally dominant. To ascertain whether these responses were due to A2A receptor or A2B receptor activation, we stimulated the cultures with CGS 21680, a selective A2A receptor agonist, and NECA, a nonselective adenosine receptor agonist. There are no selective A2B receptor agonists currently available. At 10 µM the increases in cAMP above basal levels were 0.5- to 1.5-fold for CGS 21860, 12- to 14-fold for adenosine, and 23- to 28-fold for NECA. Thus, in terms of cAMP production (Fig. 1Go) the rank order of potency is NECA > adenosine > CGS 21680, in keeping with that reported previously for the A2B receptor (29).



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  		Figure 1. Production of cAMP in primary rat anterior pituitary cells stimulated with adenosine, NECA, and CGS 21680. The cells were stimulated for 15 min with the compounds at the indicated concentrations and were measured by an in-house RIA. All data points are represented as the mean ± SEM of quadruplicate determinations from one representative experiment. *, P < 0.05; ***, P < 0.001 (compared with the appropriate control values).

 
Functional A2B and A1 adenosine receptors are differentially expressed in pituitary folliculostellate and endocrine cells
The data from the studies of primary cells suggest functional dominance of the A2B receptor, although it is not clear which cell types possess these receptors. To address this we treated two pituitary folliculostellate (TtT/GF and Tpit/F1) and two pituitary endocrine (GH3 and AtT20) cell lines with adenosine and measured intracellular cAMP levels. Adenosine caused dose-dependent stimulation of cAMP production in both folliculostellate cell lines (Fig. 2AGo), but not in either of the pituitary endocrine cell lines, suggesting a predominance of A2 receptors in folliculostellate cells, but not in the endocrine cells. To ascertain whether these A2 receptors are either the A2A or A2B subtype, we stimulated TtT/GF cells with CGS 21680 and NECA. Both of these compounds dose-dependently stimulated cAMP production (Fig. 2BGo), with a rank order of potency of NECA > adenosine > CGS 21680, again supporting the presence of the A2B receptor subtype. Similar findings were obtained for the Tpit/F1 cells (data not shown). The lack of effect of adenosine on basal cAMP production in GH3 and AtT20 cells suggests that A2 receptors are not functionally dominant in these cells, and this was further supported by the fact that both CGS 21680 and NECA also failed to stimulate cAMP in these cell lines (data not shown). In agreement with other researchers (3, 4), 100 µM adenosine and 100 µM CCPA, a selective A1 receptor agonist, significantly inhibited VIP-stimulated cAMP accumulation in GH3 cells (by 53 ± 4% and 71 ± 6%, respectively; P < 0.001; n = 4) and forskolin-stimulated cAMP in AtT20 cells (by 38 ± 3% and 45 ± 6%, respectively; P < 0.01; n = 4). These findings are consistent with the presence of functional A1 receptors in both GH3 and AtT20 cells. In contrast, in primary cells and folliculostellate cells, 100 µM CCPA stimulated cAMP (data not shown), presumably by activating A2B receptors at high agonist concentration (30, 31); hence, additional methods to study A1 receptor expression in these cells were performed.



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  		Figure 2. cAMP production in pituitary cell lines treated with adenosine (A) and TtT/GF folliculostellate cells treated with A2 receptor agonists (B). All data points are represented as the mean ± SEM of quadruplicate determinations from one representative experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with the appropriate control values).

 
A1 and A2B adenosine receptor mRNA and protein expression in pituitary folliculostellate and endocrine cell lines
Our cAMP experiments indicate that folliculostellate cells possess predominantly A2B receptors, and pituitary endocrine GH3 and AtT20 cells possess predominantly A1 receptors. We sought to confirm these findings by investigating appropriate mRNA and protein expression in the four cell lines by RT-PCR and immunocytochemistry. Using primers specific for the murine A2B receptor, strong PCR products of the appropriate size were obtained in both folliculostellate cell lines (Fig. 3AGo, lanes 4 and 8). Only faint nonspecific amplification products of incorrect size were seen when A2A receptor primers were used (Fig. 3AGo, lanes 3 and 7) despite a strong correct size band being visible in the control sample (mouse brain; Fig. 3CGo, lane 6). Faint bands for A2A and A2B receptors were occasionally seen after high cycle number (40 cycles) in GH3 (Fig. 3BGo, lanes 3 and 4), but not in AtT20 (Fig. 3BGo, lanes 7 and 8) cells. Amplification products of the correct size for the A1 receptor were seen in rat GH3 cells (200 bp; Fig. 3BGo, lane 2) and murine AtT20 cells (385 bp; Fig. 3BGo, lane 6) and in TtT/GF and Tpit/F1 cells (Fig. 3AGo, lanes 2 and 6). Correct identification of the amplicon was confirmed in each case by DNA sequencing. Positive control amplicons for the A1, A2A, and A2B receptors in rat and mouse brains are shown in Fig. 3CGo.



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  		Figure 3. RT-PCR analysis of adenosine receptor mRNA expression in pituitary folliculostellate (A; lanes 2–5, TtT/GF; lanes 6–9, Tpit/F1) and pituitary endocrine cells (B; lanes 2–5, GH3; lanes 6–9, AtT20). Lane 1, 100-bp DNA ladder; lanes 2 and 6, A1 receptor; lanes 3 and 7, A2A receptor; lanes 4 and 8, A2B receptor; lanes 5 and 9, phosphoglucokinase-1. C, Positive controls (lanes 2–4 rat brain, lanes 5–7 mouse brain). Lane 1, 100-bp DNA ladder; lanes 2 and 5, A1 receptor; lanes 3 and 6, A2A receptor; lanes 4 and 7, A2B receptor.

 
Confirmation of protein expression was obtained by means of immunocytochemistry using polyclonal antisera specific for the A1 and A2B receptor subtypes (Figs. 4Go and 5Go). A cell surface staining pattern was obtained with the A2B receptor antibody in TtT/GF and Tpit/F1 cells (Fig. 4Go), particularly along the cytoplasmic processes, in contrast to the A1 receptor, where a more diffuse cytoplasmic pattern was apparent in all cell types (Fig. 5Go).



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  		Figure 4. Immunodetection of A2B adenosine receptors in TtT/GF and Tpit/F1 cells. A, Negative control with primary antibody omitted (Tpit/F1 cells only, TtT/GF not shown). B, A2B staining in TtT/GF cells demonstrating surface expression along the cytoplasmic processes. C, A2B staining in Tpit/F1 cells. All shown at x40 magnification.

 


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  		Figure 5. Immunodetection of A1 adenosine receptors in GH3, AtT20, TtT/GF, and Tpit/F1 cells. A, Negative control (GH3 only shown) with primary antibody omitted. B, A1 receptor staining in GH3 cells demonstrating diffuse cytoplasmic localization. C, A1 receptor staining in AtT20 cells. D, A1 receptor staining in TtT/GF cells. E, A1 receptor staining in Tpit/F1 cells. All shown at x40 magnification.

 
A1 adenosine receptors in pituitary endocrine cells are present in a low affinity conformation
To further confirm the presence of A1 receptors in pituitary folliculostellate and endocrine cells we demonstrated binding of the A1 receptor antagonist [3H]DPCPX in TtT/GF, Tpit/F1, GH3, and AtT20 cells. Competition between radiolabeled and cold DPCPX showed significant displacement in all four cell lines tested (Fig. 6Go), with an apparent Kd of approximately 0.1 µM for folliculostellate cells and 1 µM for endocrine cells. The concentrations of DPCPX required for the displacement of the radioligand suggest that A1 receptors, in particular in GH3 and AtT20 cells, are in a low affinity state. In the absence of demonstrable A2B receptors in GH3 and AtT20 cells, these data support the presence of A1 receptors in these cells. Caution has to be exercised, however, in interpreting these data in the folliculostellate cell lines, as [3H]DPCPX can also label A2B receptors (30). The differences in affinity between the folliculostellate and endocrine cells may in part reflect this. The competition experiments between radiolabeled and cold NECA and between radiolabeled and cold CGS 21680 showed no displaceable binding (data not shown). However, difficulties in labeling A2A and A2B adenosine receptors with these compounds have been reported previously (31, 32).



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  		Figure 6. Displacement curves for [3H]DPCPX binding to intact GH3, AtT20, TtT/GF, and Tpit/F1 cells. The curves show specific binding of [3H]DPCPX in the presence of cold DPCPX. All data points are represented as the mean ± SEM of quadruplicate determinations from one representative experiment.

 
Adenosine differentially regulates pituitary folliculostellate and endocrine cell proliferation
As adenosine is rapidly metabolized by ecto-adenosine deaminase on the surface of cells (33, 34), we initially chose to assess cell proliferation in response to stimulation with the nucleoside by measuring the incorporation of [methyl-3H]thymidine at 4 h. Adenosine caused dose-dependent stimulation of [methyl-3H]thymidine uptake in TtT/GF and Tpit/F1 cells at concentrations of 1–100 µM (Fig. 7Go). In contrast, 100 µM adenosine inhibited thymidine incorporation in GH3 and AtT20 cells (Fig. 7Go); the concentration of the nucleoside required for inhibition was several orders of magnitude higher than that needed to stimulate thymidine incorporation in folliculostellate cells. In addition, forskolin (10 µM), as also demonstrated previously (35), stimulated [methyl-3H]thymidine incorporation in folliculostellate and endocrine cells (data not shown), suggesting that cell growth in both cell types is enhanced by cAMP. These observations are consistent with our understanding that adenosine stimulates cell growth via a receptor linked to the stimulation of cAMP. The divergent responses of folliculostellate and endocrine cells to adenosine together with the observation that growth is stimulated by cAMP support the functional dominance of A2 receptors in folliculostellate cells but not in endocrine cells.



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  		Figure 7. Effects of adenosine on [methyl-3H]thymidine incorporation in FS and pituitary endocrine cells. The cells were treated as described in Materials and Methods. All data points are represented as the mean ± SEM of eight well replicates from one representative experiment. **, P < 0.01; ***, P < 0.001 (compared with appropriate control values).

 
To demonstrate this we treated TtT/GF, GH3, and AtT20 cells with NECA and CGS 21680 and assessed cell growth by means of a cell proliferation assay. NECA caused dose- dependent stimulation of cell proliferation in TtT/GF cells at 2 and 4 d, with a maximal response between 0.1–1 µM (data for 4 d shown, Fig. 8AGo), in contrast to CGS 21680, which had no effect on cell growth (Fig. 8AGo). At NECA concentrations greater than 10 µM there were no effects on growth of TtT/GF cells. Addition of the adenosine receptor antagonist XAC (10 µM) abolished the growth stimulation induced by NECA, confirming that these effects were mediated by activation of an adenosine receptor (Fig. 8AGo). Similar findings were obtained for Tpit/F1 cells. Neither NECA nor CGS 21680 stimulated cell proliferation in GH3 or AtT20 cells (data not shown), supporting the absence of a predominance of A2 receptors. A high concentration (100 µM) of CCPA, however, inhibited cell growth in GH3 and AtT20 cells (Fig. 8BGo).



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  		Figure 8. Effects of A2 receptor agonists on the proliferation of TtT/GF cells (A) and A1 receptor agonists (B) on the proliferation of GH3 and AtT20 cells. Cells were treated with the compounds indicated for 4 d as described in Materials and Methods. XAC was used at a concentration of 10 µM. All data points are represented as the mean ± SEM of eight well replicates of one representative experiment. *, P < 0.05; ***, P < 0.001 (compared with appropriate control values).

 
A1 adenosine receptor activation reversibly slows progression through the GH3 cell cycle
We investigated the mechanism of A1 receptor-induced growth inhibition in synchronized GH3 cells by measuring incorporation of [methyl-3H]thymidine and analyzing cell cycle kinetics in the presence of 100 µM CCPA. [Methyl-3H]thymidine uptake was inhibited by 100 µM CCPA at all time points studied (4–72 h; Fig. 9AGo), in contrast to 100 µM adenosine, which only inhibited uptake for 16 h (Fig. 9AGo). The reversibility of CCPA-induced growth inhibition was demonstrated by washing the cells in the absence of the agonist for 24 h and observing a subsequent increase in thymidine labeling (Fig. 9AGo, see 72 h point). After the washout period the level of [methyl-3H]thymidine incorporation was less than the control value due to CCPA causing a reduction in cell number (Fig. 9AGo). When the uptake of radiolabel was corrected for cell number, this was no different from the value in untreated control cells (data not shown). CCPA treatment after 24 h caused a greater number of cells to accumulate in S phase with a consequent reduction in the number of cells in Go/G1 compared with untreated cells (Fig. 9BGo). No subdiploid peak corresponding to apoptotic cells was observed in the CCPA-treated group (data not shown). These data suggest that activation of A1 adenosine receptors in GH3 cells slows progression through the cell cycle. Thus, the observed inhibition of cell growth by adenosine or CCPA in GH3 cells (Fig. 9AGo) is probably due to a slowing of cell cycle progression rather than to a stage-specific arrest.



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  		Figure 9. A, Adenosine and CCPA reversibly inhibit [methyl-3H]thymidine incorporation in synchronized GH3 cells. GH3 cells synchronized in G0/G1 by serum deprivation were incubated in complete medium in the presence or absence of 100 µM adenosine or CCPA for the time points indicated. After 48 h, where indicated treatment groups were washed and incubated in fresh medium in the absence of CCPA. All data points are represented as the mean ± SEM of eight well replicates from one representative experiment. ***, P < 0.001 compared with appropriate control values. B, Adenosine slows progression through the GH3 cell cycle. Synchronized GH3 cells were incubated in complete medium in the presence (lower panel) or absence (upper panel) of 100 µM CCPA for 24 h. DNA content was measured by flow cytometry. *, P < 0.05 compared with control population.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have shown that adenosine has opposing effects on the growth of pituitary endocrine and folliculostellate cell lines and that these actions are mediated through A1 and A2B receptors, respectively. Our data using primary anterior pituitary cells suggest that the A2B receptor predominates, and subsequent experiments using cell lines indicate that this receptor subtype is expressed principally on folliculostellate cells and not pituitary corticotropes or lactotropes. The rank order of potency in the anterior pituitary and in the folliculostellate cell lines of NECA > adenosine > CGS 21680 is in keeping with that previously described for the human (36) and murine (29) A2B receptor in transfected CHO cells and for the rat A2B receptor in primary astrocyte cultures (37). Furthermore, the Kd of the agonists (low micromolar) for the A2B receptor is of a similar order to that reported previously (36). Although adenosine stimulation of adenylate cyclase in rat anterior pituitary cells has been reported previously (6), it was not clear which of the A2 receptor subtype(s) was present and in which cells. Other investigators have suggested previously that A1 receptors might be present in both folliculostellate cells (38) and endocrine cells (3, 4, 5). Although our data confirm these findings, we show that the predominant functional adenosine receptor in folliculostellate cells is the A2B subtype. Caution, however, has to be exercised when comparing data between primary cells and cell lines. Apart from possible differences between tumor cell and normal cell function, our data suggest that pituitary cell types other than folliculostellate cells could express A2B receptors, as evidenced by differences in the magnitude of the cAMP response after NECA stimulation in primary cells (7- to 30-fold) and folliculostellate cells (2- to 8-fold). A2B receptor expression has been demonstrated previously in fibroblasts (39) and vascular endothelial cells (40), and these will undoubtedly contribute to the enhanced cAMP response in the primary cultures. Furthermore, data from in vitro experiments using hemipituitary sections have suggested that A2 receptors could be expressed in thyrotropes based on the modification of TSH release by A2-selective agonists (7). This latter study, however, did not examine the distribution of the A2 receptors present, and an indirect mediation of TSH release by other mechanisms could not be excluded. On the other hand, A1, but not A2, receptors have been described in rat gonadotropes and mediate inhibition of gonadotropin secretion (41). Although we have shown that folliculostellate cells strongly express A2B receptors, it is conceivable that some pituitary endocrine cells in addition to fibroblasts and endothelial cells also express them.

In the pituitary endocrine cell lines, neither adenosine, NECA, nor CGS 21680 elicited cAMP responses, suggesting the lack of a dominance of A2 receptors. We were, however, able to confirm previous reports (3, 4, 5) showing the presence of A1 receptors in GH3 cells by demonstrating inhibition of stimulated cAMP production. In addition, we also showed for the first time that A1 receptors are present in AtT20 corticotrope cells. In keeping with previous reports in GH4 cells (14, 42), our data indicate that the A1 receptor in GH3 and AtT20 cells is in a low affinity conformation. The Kd value for the A1 receptor may, of course, be quite different in anterior pituitary cells, as has been described previously for the dopamine D2 receptor, whose affinity is reduced by several orders of magnitude in GH3 cells compared with normal rat anterior pituitary (43). Furthermore, the expression of A1 adenosine receptors is not static and can be modified in pituitary cells by epidermal growth factor (44), suggesting that receptor number and/or affinity can be modified according to the physiological state of the cell. Nevertheless, our observation that adenosine stimulates, rather than inhibits, cAMP production in anterior pituitary cells, suggests that any high affinity A1 receptors, if they are present, are swamped by the dominance of the A2B receptor. The apparent differences in Kd values for [3H]DPCPX binding between folliculostellate and GH3/AtT20 cells may be due to the fact that it can also bind to A2B receptors (30) in folliculostellate cells. Our RT-PCR and immunocytochemistry data fully support the expression of A1 receptors in endocrine and folliculostellate cells and A2B receptors in folliculostellate cells. As A2A and A2B transcripts were sometimes detectable by RT-PCR, we cannot completely exclude the possibility that low numbers of A2A or A2B receptors do occur in GH3 cells. To detect weakly expressed transcripts, we deliberately used 35–40 cycles in our RT-PCR experiments. This may lead to nonspecific priming and the generation of faint, incorrect size amplicons, as seen particularly for the A2A receptor in our mouse cell lines.

Although A1 receptor mRNA and protein expressions were detectable in the folliculostellate cells, we were unable to demonstrate the functional relevance of the receptor, as the selective A1 receptor agonists used are also able to activate the A2B receptor at high concentrations (30, 31). Coexisting A1 and A2 receptors with opposing actions on adenylate cyclase activity have been described in a number of cells, including porcine coronary artery smooth muscle cells (45) and primary rat astrocytes (46). The expression of more than one adenosine receptor on the same cell may allow the common agonist adenosine to activate multiple signaling pathways. As the A1 and A2B receptors are, respectively, negatively and positively coupled to adenylate cyclase, this may allow reciprocal control and fine-tuning of the signaling pathway. In this regard, if the adenosine receptor subtypes in the pituitary gland have different Kd values, then the local concentrations of adenosine under physiological and pathophysiological conditions are likely to be of crucial importance in determining their activation.

The different affinities and localization of the A1 and A2B receptors in pituitary cells help to explain the different proliferative responses to adenosine between folliculostellate and endocrine cells. Adenosine stimulated DNA synthesis in folliculostellate cells at doses several orders of magnitude lower than those needed to inhibit cell growth in the pituitary endocrine cells. When NECA was incubated with folliculostellate cells, a biphasic growth response was observed, with a peak response at 0.1–1 µM and a return to control values at 10–100 µM. A possible explanation for this lack of a proliferative response at the higher concentrations of NECA is that the molecule has a broad action at all adenosine receptors, and under these conditions activation of A1 receptors could negate the stimulatory effects of the A2B receptor. The regulation of cell proliferation by purines has been studied in a number of cell types, and proliferative roles for adenosine have been demonstrated in endothelial cells, astrocytes, and microglia (46). In the pituitary gland, however, studies on adenosine-regulated cell proliferation have been limited to a description of adenosine-induced apoptosis in GH3 cells (23) and modulation of the kinetics of the cell cycle in GH4 cells (14). This latter group demonstrated that A1 receptor-selective agonists regulated progression through the phases of the cell cycle without affecting cell number. However, we were unable to confirm these findings in GH3 cells; in our study A1 receptor activation by adenosine and CCPA inhibited growth by slowing progression through the cell cycle rather than by inducing a cell cycle stage-specific arrest. In addition, the absence of a subdiploid peak corresponding to apoptotic cells suggested that apoptosis was not contributing significantly to the observed reduction in cell number. The time-related difference between the effects of adenosine and CCPA on the inhibition of [3H]thymidine incorporation (Fig. 9AGo) was probably due to the short half-life of adenosine. We were also unable to block the inhibitory effects of CCPA with the A1 antagonist DPCPX due to the lack of solubility of DPCPX in aqueous solutions at the concentrations needed to saturate the receptor. This observation has been reported previously (14).

It is tempting to speculate that the differential distribution of adenosine receptors among pituitary folliculostellate and endocrine cells and their opposing actions in mediating proliferation may have a pathological relevance to pituitary tumor growth. 5'-AMP, the immediate precursor of adenosine, has previously been shown to be the major component of the low mol wt mitogenic activity in pituitary tumor extracts (47). AMP is rapidly converted to adenosine by ecto-5'-nucleotidase, whose distribution in the nervous system shows a preferential glial cell location (48). It is conceivable, therefore, that AMP released in these circumstances might be degraded preferentially on the surface of folliculostellate cells to adenosine, leading to activation of A2B receptors and subsequent folliculostellate cell proliferation. Folliculostellate cells are present in significant amounts in GH- and PRL-secreting adenomas (49) and secrete a number of growth factors, such as basic fibroblast growth factor (16) and vascular endothelial growth factor (50), which are mitogenic for pituitary endocrine or endothelial cells. Such an indirect, paracrine action of adenosine might therefore contribute to the maintenance and growth of pituitary tumor cells.


   Acknowledgments
 


   Footnotes
 
This work was supported by a Clinical Endocrinology Trust Clinical Training Fellowship (to D.A.R.).

Abbreviations: ADA, Adenosine deaminase; CCPA, 2-chloro-N6- cyclopentyladenosine; CGS 21680, 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine; DPCPX, 1,3-dipropyl-8- cyclopentylxanthine; EBSS, Earle’s balanced salts solution; NECA, 5'-N-ethylcarboxamidoadenosine; XAC, xanthine amine congener.

Received October 18, 2001.

Accepted for publication February 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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