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Endocrinology, doi:10.1210/en.2006-1293
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Endocrinology Vol. 148, No. 6 2698-2707
Copyright © 2007 by The Endocrine Society

Tyrosine Hydroxylase and Dopamine Transporter Expression in Lactotrophs from Postlactating Rats: Involvement in Dopamine-Induced Apoptosis

Arnaud Jaubert, Guillaume Drutel, Thierry Leste-Lasserre, François Ichas and Laurence Bresson-Bepoldin

Laboratoire de Signalisation et Mécanismes Moléculaires de l’Apoptose (A.J., F.I., L.B.-B.), Institut National de la Santé et de la Recherche Médicale (INSERM), E347, Institut Bergonié, Université Bordeaux 2, 33076 Bordeaux cedex, France; and Centre de Recherche Physiopathologie de la Plasticité Neuronale (G.D., T.L.-L.), INSERM, Unité 862, Institut Européen de Chimie et de Biologie, Université Bordeaux 2, 33607 Pessac cedex, France

Address all correspondence and requests for reprints to: Laurence Bresson-Bepoldin, Laboratoire de Signalisation et Mécanismes Moléculaires de l’Apoptose, Institut National de la Santé et de la Recherche Médicale E347, Institut Bergonié, 229 cours de l’Argonne, 33076 Bordeaux cedex, France. E-mail: laurence.bresson-bepoldin{at}bordeaux.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cessation of lactation causes a massive loss of surplus lactotrophs in the rat pituitary gland. The factors and mechanisms involved in this phenomenon have not yet been elucidated. Besides its inhibitory control on prolactin secretion and lactotroph proliferation, evidence suggests that dopamine (DA) may be a proapoptotic factor for lactotrophs. We therefore tested the proapoptotic effect of DA on pituitary glands from virgin, lactating, and postlactating rats. By measuring mitochondrial membrane potential loss, caspase-3 activation, and nuclear fragmentation, we show that DA induces apoptosis specifically in lactotrophs from postlactating rats. We then determined that this effect was partly mediated by the DA transporter (DAT) rather than the D2 receptor, as corroborated by the detection of DAT expression exclusively in lactotrophs from postlactating rats. We also observed tyrosine hydroxylase (TH) expression in postlactating lactotrophs that was accompanied by an increase in DA content in the anterior pituitary gland of postlactating compared with virgin rats. Finally, we observed that cells expressing TH coexpressed DAT and cleaved caspase-3. These findings show that DA may play a role in lactotroph regression during the postlactation period by inducing apoptosis. The fact that this process requires DAT and TH expression by lactotrophs themselves suggests that it may be "autocrine" in nature.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DOPAMINE (DA) IS THE predominant catecholamine neurotransmitter in the mammalian brain, in which it controls a variety of functions, including motricity, cognition, positive reinforcement, food intake, and endocrine regulation. All of these physiological roles are mediated by the extracellular action of DA on two families of cell membrane receptors, D1 like and D2 like, coupled with Gs and Go/i proteins, respectively.

DA, its metabolic products, and 6-hydroxydopamine have also been shown to induce neurotoxicity and apoptosis in various cell types, including primary rat striatal neurons (1, 2), human neuroblastoma cells (3, 4), PC12 cells (5), and mouse thymocytes (6). Previous research has suggested that DA toxicity is mediated by the DA transporter (DAT), which may participate in the increase in intracellular concentrations of DA or DA-like molecules (3, 4, 7). The DA toxicity mechanism generally involves both auto-oxidation and monoamine oxidase-mediated oxidation of catecholamine to form reactive oxygen species (ROS) and quinones, which ultimately contribute to inducing oxidative stress in cells (8). Oxidative stress is known to trigger activation of the mitochondrial-dependent apoptosis pathway by opening the mitochondrial pore of transition, leading to caspase activation (9).

It is well documented that DA is the main lactotroph cell regulator, inhibiting prolactin (PRL) synthesis and release (10), as well as cell proliferation (11), by binding to dopamine receptor type 2 (D2R). In the hypothalamo-pituitary system, DA is synthesized by hypothalamic neurons in the arcuate and periventricular nuclei, which project their axons to the median eminence. Tyrosine, the DA synthesis precursor, is an aromatic amino acid. Two reactions transform tyrosine into DA. The first is catalyzed by the tyrosine hydroxylase enzyme (TH), which converts tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA). The second, catalyzed by L-DOPA decarboxylase, results in DA synthesis from L-DOPA. Because TH expression is finely regulated, it is considered to be the rate-limiting enzyme in this pathway (12). In the anterior pituitary (AP), the presence of TH has been described in neuronal fibers (13), as well as in lactotroph cells from adenomas or ectopic APs (14), suggesting the possibility of local DA production in the AP during specific physiological stages.

The number of pituitary cells fluctuates physiologically during postnatal life, depending on the hormonal environment (15). Thus, lactotrophs increase during gestation and parturition to provide the PRL necessary to maintain lactation (16, 17). This is followed by a massive, 50% loss of lactotrophs at the end of the lactation period, decreasing to 25% of the total number of AP cells within 1 wk (18). This process has been suggested to be apoptotic in nature, involving changes in the expression of programmed cell-death modulators, such as Bcl-2, Bax, and p53 (19). However, the signal inducing this apoptotic process in lactotrophs is still unknown.

Previous studies showed that lactotrophs were sensitized to various apoptotic inducers, such as TNF{alpha} (20) or Fas ligand (21), depending on the ovarian cycle or hormone environment. Although many studies have shown that D2R agonists, such as bromocriptine and terguride, induce apoptosis in human and rat prolactinoma (22, 23), the putative proapoptotic effect of DA on lactotrophs was still elusive. Recently, we showed that DA was able to induce apoptosis in GH3 cells, a pituitary lactotroph cell model. We established that DA oxidation generated ROS, thus causing oxidative stress. This triggered the mitochondrial apoptotic pathway, including mitochondrial membrane potential (MMP) loss, Bax relocation from the cytosol to the mitochondria, cytochrome c release, and caspase-3 activation (24). The observation of DAT expression in these cells and its implication in apoptosis was particularly important.

The aim of this work was to study the proapoptotic effect of DA in pituitary lactotroph cells from rats in various physiological stages [virgin (V), lactating (L), and postlactating animals] and determine how this mechanism takes place. In this study, we show for the first time that DA is able to induce apoptosis specifically in dispersed lactotroph pituitary cells from postlactating rats. We suggest that this process may originate in lactotrophs themselves, because we show that, in the postlactation (PL) period, they coexpress TH, which is involved in DA biosynthesis, and DAT, which partly mediates the DA-induced apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and reagents
Media and sera were purchased from Invitrogen (Cergy, France). The following drugs were obtained from the sources indicated. 3-Hydroxytyramine (DA; Sigma, L’Isle d’Abeau, France) was always prepared in the presence of an equimolar quantity of Sodium MetaBiSulfite (Sigma), to protect DA from spontaneous auto-oxidation. Norepinephrine, serotonin, haloperidol, raclopride, mazindol, GBR 12909 (1-[2-[bis(4-fluorophenyl)-methoxy]ethyl]-4-[3-phenylpropyl]piperazine), and the anti-actin polyclonal antibody were obtained from Sigma. The fluorescent probes tetramethylrhodamine methylester (TMRM) were purchased from Sigma, whereas Hoechst was from Invitrogen. Guinea pig anti-PRL, guinea pig anti-TSH, monkey anti-GH, rabbit anti-LH, and rabbit anti-FSH were kindly provided by the National Hormone Peptide Program (Torrance, CA). The anti-DAT rabbit polyclonal antibody (anti-DAT) and the anti-TH monoclonal mouse antibody (anti-TH) were from Chemicon (Temecula, CA). The anti-cleaved caspase-3 (anti-CC3) rabbit monoclonal antibody was purchased from Cell Signaling Technology (Ozyme, St. Quentin en Yvelines, France). The goat antimouse, antirabbit, or antiguinea pig fluorophore-conjugated secondary were obtained from Invitrogen. The secondary conjugated horseradish-peroxidase antibodies and the enhanced chemiluminescence kit were obtained from Amersham Biosciences (Buckinghamshire, UK).

Animals, tissue preparation, and AP cell cultures
V (6-wk-old, in diestrus), L (d 8 of lactation), and PL3 or PL24 (3 or 24 d after a 21-d lactation period) female Sprague Dawley rats (Charles River, L’Arbresle, France) were used in this study. Every effort was made to reduce the number of animals used and their suffering.

For cryostat slices, rats were anesthetized with Rompun/Imalgen (350 mg/kg) and perfused transcardially with PBS [in g/liter: 8 NaCl, 0.2 KCl, 1.15 Na2HPO4, and 0.2 KH2PO4 (pH 7.4)]. After perfusion, whole pituitaries were removed, fixed in 2% formaldehyde/0.2% picric acid for 3 h, and then cryoprotected by incubation in PBS containing 20% sucrose for several hours. Tissues were included into Cryoblock (Labonord, Templemars, Belgium), quickly frozen on dry ice, and stored at –80 C until use.

Pituitary glands were removed quickly after decapitation. Posterior lobes were discarded, whereas anterior lobes were placed in DMEM/F12 (50:50) containing 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.1% bovine serum albumin (Sigma) before protein or RNA extraction or cell dissociation.

Cells from various female rats were prepared by dispersion with trypsin as described previously (25). The cells were plated onto 12-well plates (1.5 x 105 cells per well) for flow cytometry and 25-mm round polyornithine-coated glass coverslips (3 x 105 cells per glass) for immunocytochemistry. For Western blot, cells were seeded on 60-mm Petri dishes (1.106 cells per dishes). Cells were cultured in DMEM/F12 (50:50) containing 50 IU/ml penicillin-streptomycin (Invitrogen, Cergy, France) and supplemented with 10% fetal calf serum. The cultures were maintained at 37 C in a humidified atmosphere of 95% air and 5% CO2. Treatments were performed immediately after cellular attachment to the various supports.

Protein extraction and Western blot
Before lysis, whole AP tissue was crushed in a Dounce homogenizer, and dispersed cell cultures were washed twice with cold PBS. Proteins were then extracted on ice with a lysis buffer containing 1 mM EDTA, 20 mM imidazole (pH 7.2), and 250 mM sucrose in the presence of protease inhibitors (0.1 µM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml Aprotinin, and 2.5 µg/ml pepstatin). The cell lysate was then sonicated and centrifuged (13,000 rpm, 1 min) at 4 C. The supernatant was boiled in 0.2% SDS for 90 s and kept at –20 C before immunoblotting.

The protein concentration of the lysates was quantified using the Bio-Rad (Marnes-la-Coquette, France) protein assay. Proteins were then separated on 8% (for DAT), 10% (for TH), or 12% (for CC3) SDS-PAGE and transferred to polyvinylidene difluoride membrane (Pall Corporation via VWR, Fontenay sous bois, France) by mini trans-blot electrophoretic transfer cell (Bio-Rad). After membrane blocking [1 h 30 min at room temperature with 5% milk powder in PBS containing 0.1% (vol/vol) Tween 20], proteins were probed with anti-DAT (1:100), anti-CC3 (1:500), or anti-TH or anti-actin (1:200) at 4 C overnight. The membrane was then washed, and primary antibodies were detected using appropriate secondary goat antirabbit IgG or goat antimouse IgG conjugated to horseradish peroxidase, and the bands were visualized with enhanced chemiluminescence (Amersham Biosciences).

RNA isolation and RT-PCR
After removal of the pituitary gland, the anterior lobes were quickly frozen on dry ice. RNA was immediately isolated using the Trizol reagent (Invitrogen, Karlsruhe, Germany). After extraction and ethanol precipitation, RNA was treated with Turbo DNA-free (Ambion, Austin, TX), according to the instructions of the manufacturer. The integrity of the RNA was checked by capillary electrophoresis using the RNA 6000 Nano Lab-on-a-Chip kit and the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). cDNA was synthesized from 2 µg total RNA, with or without PowerScript Reverse Transcriptase (Clontech, Palo Alto, CA), using random primers (Invitrogen, Cergy, France). Aliquots of cDNA were then subjected to PCR amplification on a PTC200 Thermal Cycler (MJResearch/Bio-Rad, Hercules, CA) with specific forward (DAT, 5'-AGACACCAGTGGAGGCTCAAGA-3'; TH, 5'-AAGCTGCTTCAGAACATCAAGGA-3') and reverse oligonucleotide primers (DAT, 5'-GCCGATGACTGATAGCAGGAA-3'; TH, 5'- AAGGACCCAGGGCTGAGAA-3'). The DAT and TH PCR products were 71 and 73 bp, respectively. The DyNAzyme II Hot Start DNA Polymerase kit (Finnzymes, Espoo, Finland) was used with the following PCR amplification cycles: initial denaturation, 95 C for 15 min, followed by 40 cycles with denaturation, 95 C for 20 sec and annealing–extension, 61 C for 35 sec. The PCR products were electrophoresed on 2% agarose gel. PCR mixes of each group (V or postlactating) were used for digestion with specific restriction enzymes to confirm the sequences of the products: DAT with BstNI and TH with HaeIII. All restriction enzymes were purchased from New England Biolabs (Hitchin, UK) and were used according to the guidelines of the manufacturer.

Apoptosis detection
MMP ({Delta}{Psi}m) was detected by flow cytometry, using a DakoCytomation (Carpinteria, CA) Galaxy flow cytometer. MMP was monitored using a fluorescent probe, TMRM, which accumulates in the mitochondria according to membrane potential (26). After treatment, the cells were trypsinized and centrifuged at 800 rpm for 5 min. They were then incubated for 30 min in HBSS [in mM: 140 NaCl, 5 KCl, 0.3 Na2HPO4, 0.4 KH2PO4, 2 MgCl2, 2 CaCl2, 10 HEPES, 5 glucose, and 4 NaHCO3 (pH 7.3)] supplemented with 200 nM TMRM at 37 C. Cells were then analyzed, by measuring the fluorescence emission in FL3 log mode. For each cytometry experiment, the minimum number of acquired cells per sample was 2 x 104.

Apoptosis was also analyzed by counting the picnotic nuclei after Hoechst staining (1 µM) and by CC3 detection using immunohistochemistry and immunocytochemistry, as described below.

DA assay
A pool of two APs from animals in each of the experimental conditions was lysed in 0.01 N HCl by sonication. After centrifugation (2 min at 13,000 x g), the supernatant was removed and kept at 4 C until DA assay.

DA assay was performed with the Dopamine Research Enzyme Immunoassay (EIA) kit distributed by BioSource (via Clinisciences, Montrouge, France), according to the information of the manufacturer (27). Briefly, DA is extracted using a cis-diol specific affinity gel, then acylated to N-acyl DA, and, after this, converted enzymatically during the detection procedure into N-acyl-3-methoxytyramine. DA is bound to the solid phase of the microtiter plate used by the competitive EIA kit. Acylated DA from the sample and solid-phase bound DA compete for a fixed number of antiserum binding sites. When the system is in equilibrium, free antigen and free antigen-serum complexes are removed by washing. The antibody bound to the solid-phase catecholamine is detected by an antirabbit IgG-peroxidase conjugate using tetramethylbenzidine as a substrate, and the reaction is monitored at 450 nm.

Indirect immunofluorescence
For indirect immunohistofluorescence, whole pituitary glands were cut on a cryostat. Slices obtained (5 µm thickness) were mounted on gelatin-coated slides and incubated in one or a combination of the following antisera: guinea pig anti-PRL (1:8000), rabbit anti-CC3 (1:100), rabbit anti-DAT (1:100), and mouse anti-TH (1:100) in PBS containing 0.2% gelatin and 1% Triton X-100 at 4 C overnight. The tissues were then rinsed in PBS and incubated in one or a combination of the appropriate secondary antibodies (1:400): rhodamine-conjugated goat anti-guinea pig or fluorescein- or rhodamine-conjugated goat antirabbit and goat antimouse in PBS for 1 h at room temperature. Unbound antibody was removed by washing several times with PBS, and slides were mounted with Fluoromount-G (Southern Biotechnology via Clinisciences) for microscope observation, after incubation with 1 µM Hoechst.

For indirect immunocytofluorescence analysis, cultured cells were rinsed with PBS and then fixed with 2% formaldehyde for 20 min. Fixed cells were washed in PBS and processed for PRL, CC3, or TH immunostaining, following the procedures described above for immunohistofluorescence.

Cells and slices were imaged using a Zeiss (Oberkochen, Germany) LSM 510 Meta confocal microscope. The green fluorescence was excited with the 488 nm line of an argon laser and detected at 527 nm. The red fluorescence was excited with the 543 nm line of a helium–neon laser and detected between 560 and 615 nm. The blue fluorescence was excited with the 405 nm line of a diode laser and detected between 420 and 480 nm. To avoid crosstalk between fluorochrome signals, the fluorescences were excited sequentially, and the emitted fluorescence was split by a 545 nm dichroic reflector and then collected by separate photomultipliers through 505–530 nm band-pass and 560 nm long-pass barrier filters.

Analysis and statistics
Flow cytometry.
Each experiment was performed in duplicate, with samples from at least three different sets of animals. Results were expressed as means ± SE and ANOVA with a Fisher’s projected least significant difference as post hoc test or unpaired t test were used for statistical analysis.

Immunocytofluorescence and counting picnotic nuclei.
In dispersed pituitary cell cultures, the percentage of cells exhibiting picnotic nuclei or immunoreactivity (ir) for the protein of interest (TH, CC3) was evaluated as follows: numbers of ir or picnotic nuclei-positive cells/total numbers of cells x 100. To determine the colocalization of PRL and TH or CC3, the number of cells containing 1) PRL ir, 2) TH or CC3 ir, and 3) colocalization of PRL and CC3 or TH ir, was analyzed for each culture. Results are presented as the percentage of PRL ir cells that contained the protein of interest. Each experiment was performed in duplicate, with samples from at least three sets of animals. A minimum of 300 cells were analyzed for each sample. Unpaired t test was used for statistical analysis.

Pituitary slices.
In pituitary slices, the percentage of cells with the protein of interest (TH, DAT, and CC3) ir was estimated as follows: number of immunoreactive cells/total number of nuclei stained by Hoechst x 100. Pituitary slices from two different sets of animals in each of the physiological stages were analyzed.

DA assay.
The experiment was performed in quadruplicate on samples from two sets of animals. The P value was obtained using the Mann–Whitney test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of caspase-3 activation in APs
To check that apoptosis occurred in pituitaries from postlactating rats, we studied caspase-3 activation, a key event in the apoptotic process. Caspase-3 activation was evaluated by the formation of CC3 in APs from V, L, and PL3 PL24 rats using immunohistochemistry. We observed CC3 ir in AP cells from PL3 and PL24 rats (0.53 ± 0.16 and 0.75 ± 0.21% of CC3-positive cells per slice, respectively), whereas no staining was detected in V and L pituitaries (Fig. 1Go). Coimmunostaining with anti-PRL antibody showed that all CC3-positive cells were lactotrophs (Fig. 1Go).


Figure 1
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  		FIG. 1. Lactotroph cell apoptosis in rat postlactating pituitary. Lactotrophs were identified using a guinea pig anti-PRL antibody. Nuclei were visualized by Hoechst staining. Representative field (x630) of AP cell clusters from slice of PL3 rats showing coimmunofluorescence for PRL and CC3. This staining was never observed in pituitaries of V female rats (n = 2).

 
This result confirms previous reports that apoptosis occurs in the rat pituitary 3–4 d after cessation of lactation (18, 19).

Proapoptotic effect of DA in AP lactotroph cells
The proapoptotic effect of DA was determined in dispersed AP cells from V, L, PL3, and PL24 rats by measuring 1) MMP loss, 2) caspase-3 activation, and 3) nuclear fragmentation.

Mitochondria are involved in many apoptosis-inducing agents, and MMP loss is recognized as an early event in the apoptotic process (28). The effect of DA on MMP was investigated by flow cytometry analysis, using TMRM as a potentiometric dye. Treatment with 200 µM DA for 24 h induced significant MMP loss in cells from PL3 (28.5 ± 2.3 vs. 13.4 ± 1.3% in untreated cells; P < 0.01) and PL24 (26.4 ± 1.4 vs. 16.0 ± 1.7% in untreated cells; P < 0.01) rats, whereas no effect was observed in V (18.1 ± 0.9 vs. 15.1 ± 1.8% in untreated cells; P > 0.05) or L (16.6 ± 0.6 vs. 15.9 ± 1.7% in untreated cells; P > 0.05) rat cells (Fig. 2AGo). This DA concentration was systematically used in all experiments, because dose-dependent assays determined that it was the lowest concentration to induce apoptosis in postlactating rat cells (Fig. 2AGo, inset), whereas no apoptosis was observed in V and L rat cells, regardless of the DA concentration tested (data not shown). To check that this effect was DA specific and not attributable to an increase in sensitivity of PL3 cells to nonspecific oxidative insult, we tested the effect of two other catecholamines on PL3 cell apoptosis. We clearly showed that neither norepinephrine nor serotonin used in the same concentration range as DA was able to trigger an apoptotic response in PL3 cells (Fig. 2AGo, inset).


Figure 2
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  		FIG. 2. DA-induced apoptosis in lactotroph dispersed cells from postlactating rats. Apoptotic cells were visualized by flow cytometry after loading with TMRM or by confocal microscopy after Hoechst staining or CC3 immunostaining. A, MMP was measured with TMRM in dispersed AP cells from V, lactating (LAC), and PL3 and PL24 rats, treated (black columns) or untreated (UNT; white columns) with 200 µM DA for 24 h. Inset, PL3 rat dispersed AP cells treated with various concentrations of DA, serotonin, and norepinephrine for 24 h. n = 3. *, P < 0.01, significant compared with untreated cells. Ba, Representative field (x630) of PL3 AP dispersed cells treated with 200 µM DA for 48 h, showing red (PRL) and/or green (CC3) fluorescence after double immunocytofluorescence. Bb, The histogram represents the number of dispersed V, lactating (Lac), and PL3 and PL24 cells showing PRL/CC3 coimmunostaining after 48 h treatment with 200 µM DA (black columns) or untreated (UNT; white columns). n = 3. *, P < 0.001 and **, P < 0.01, significant compared with untreated cells. C, The histogram represents the percentage of picnotic nuclei observed in dispersed V, lactating (Lac), and PL3 and PL24 cells incubated with 1 µM Hoechst after 48 h treatment with 200 µM DA (black columns) or untreated (UNT; white columns). n = 3. *, P < 0.001, significant compared with untreated cells.

 
Caspase-3 activation was assessed by immunocytochemistry using anti-CC3 antibody. DA treatment (200 µM, 48 h) induced an increase in the number of CC3-positive cells from PL3 (5.4 ± 0.6 vs. 0.3 ± 0.2% in untreated cells; P < 0.01) and, to a lesser extent, PL24 (1.8 ± 0.2 vs. 0.15 ± 0.1% in untreated cells; P < 0.05) rats, whereas no significant effect was observed in V and L rat cells (Fig. 2BGo).

In the same way, a measurement of DNA fragmentation, evaluated by Hoechst staining and counting the picnotic nuclei, showed a significant increase in the percentage of picnotic nuclei after DA treatment (200 µM, 48 h) in postlactating rat cells (PL3 and PL24, P < 0.01) but not in V and L rat cells (Fig. 2CGo).

To identify the cell type(s) undergoing apoptosis after DA treatment, double immunostaining using anti-CC3 and anti-hormone (PRL, GH, LH, ACTH, and TSH) antibodies was performed in dispersed PL3 rat AP cells treated with DA (200 µM, 48 h). All CC3 ir was localized in lactotrophs (Fig. 2BGo), and no coimmunostaining was observed with the other anti-hormone (GH, PRL, TSH, LH, and ACTH) antibodies (data not shown).

These results suggest that DA selectively induced apoptosis in lactotroph cells from postlactating rats.

Involvement of DAT in the proapoptotic effect of DA
In the AP gland, it is well established that DA acts through D2R. We therefore tested the effect of D2R antagonists on DA-induced apoptosis in PL3 rat pituitary cells. Surprisingly, the percentage of apoptotic cells did not change significantly when cells were incubated with haloperidol (10 µM) (11) or raclopride (40 µM) (29) for 30 min before DA treatment (200 µM, 24 h) rather than treated with DA alone (Fig. 3AGo), suggesting that D2R was not involved in DA-induced apoptosis. Moreover, Western blot analysis clearly showed a significant decrease in D2R expression in dispersed AP cells from PL3 compared with V rats (Fig. 3BGo). Although these results are surprising, they suggested that D2R was not involved in DA-induced apoptosis in lactotroph cells.


Figure 3
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  		FIG. 3. D2R is not involved in DA-induced apoptosis. A, MMP was measured by flow cytometry in dispersed PL3 AP cells treated with 200 µM DA for 24 h in the presence or absence of two D2R-specific antagonists, 10 µM haloperidol (halo) or 40 µM raclopride (raclo). n = 3. *, P < 0.001, significant compared with untreated cells. B, Western blots showing differential D2R expression in dispersed AP cells of V and PL3 rats. Whole-cell extracts (50 µg protein per lane) were subjected to immunoblot analysis with anti-D2R (top) and anti-actin (bottom) for loading control. Blots shown are typical of results obtained in two separate experiments.

 
Besides its receptor, DA is also known to act via its transporter. Because DAT expression in the AP gland was still controversial (30, 31), we investigated its expression in V and PL3 AP cells. RT-PCR analysis revealed DAT mRNA expression in the AP, regardless of physiological states of the animals studied (V or PL3) (Fig. 4AaGo), but Western blot analysis clearly showed a marked increase in DAT protein expression in dispersed AP cells from PL3 compared with V rats (Fig. 4AbGo). DAT expression in PL3 rat AP glands was then confirmed by immunohistofluorescence, showing DAT staining in 2.1 ± 0.2% of cells, localized in dispersed cell clusters that were immunoreactive for PRL, whereas only 0.38 ± 0.01% of DAT- and PRL-positive cells were found in V rat AP glands (Fig. 4BGo).


Figure 4
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  		FIG. 4. DAT expression in lactotroph cells and involvement in DA-induced apoptosis. Aa, RT-PCR product of DAT gene separated on 2% agarose electrophoresis gel. Lane 1, No PCR products were detected in the negative (–) controls, performed by omitting reverse transcriptase. Lanes 2–5 and 7–10, RT-PCR fragments from V and PL3 rats, respectively. Lane 6 and 11, Digestion of PCR mixes from each condition with BstNI. M, A 50-bp DNA marker. Ab, Western blots showing differential DAT expression in dispersed AP cells of V and PL3 rats. Whole-cell extracts (100 µg protein per lane) were subjected to immunoblot analysis with anti-DAT (top) and anti-actin (bottom) for loading control. Brain protein extract was chosen as a positive control (T+). Blots shown are typical of results obtained in three separate experiments. B, Representative fields (x630) in AP cell slices without DAT ir in V female rats (V) and cell cluster from a PL3 rat slice showing coimmunofluorescence for PRL (red) and DAT (green) (n = 2). C, Inhibition of MMP loss measured by flow cytometry in cells treated with 200 µM DA for 24 h in the presence of 5 or 50 µM mazindol (MAZ) or 5 µM GBR 12909 (GBR), two DAT inhibitors. n = 3. *, P < 0.001 and {wedge}, P < 0.005, significant compared with DA-treated cells.

 
The involvement of DAT in DA-induced apoptosis in PL3 rat AP cells was then tested using mazindol and GBR 12909, two specific DAT blockers (32). Our results showed that DA-induced apoptosis (200 µM, 24 h) was dose dependently reduced when DA was coincubated with 5 or 50 µM mazindol and completely inhibited in the presence of 5 µM GBR 12909 [13.7 ± 0.1 in cells cotreated with DA and mazindol and 9.8 ± 2.4 in cells cotreated with DA and GBR 12909 vs. 23.7 ± 0.9% in cells treated with DA alone (P < 0.01) vs. 13.7 ± 0.5 in untreated cells] (Fig. 4CGo). These data show that DA triggered apoptosis in PL3 rat pituitary cells in a DAT-dependent manner.

For the first time, our results showed that lactotrophs were able to express DAT under specific physiological conditions, such as postlactation, and suggested that DAT participated in DA-induced apoptosis in PL3 rat lactotrophs.

TH expression in postlactating rat lactotrophs
Because DAT is known to be expressed in DA-secreting cells and the DA concentrations necessary to induce apoptosis in postlactating rat lactotrophs are unlikely to be compatible with DA release in portal blood from the tuberoinfundibular nerve terminals in the median eminence, we postulated that DA was synthesized by AP cells themselves. We therefore studied expression levels of TH, the rate-limiting enzyme in DA synthesis, in AP cells from V and PL3 rats. As shown in Fig. 5AGo, TH mRNA expression was detected by RT-PCR in AP from V and PL3 rats (Fig. 5AaGo). Western blot analysis revealed one immunoreactive form of TH in the AP gland (~60 kDa), corresponding to that observed in the CNS, and we observed a strong increase in TH protein expression in AP cells from PL3 compared with V rats (Fig. 5AbGo). This last result was corroborated by immunofluorescence detection of immunoreactive cells for TH in AP glands from PL3 animals (2.2 ± 0.1% of TH-positive cells in PL3 vs. 0.4 ± 0.01% in V AP gland), as well as after cell culture (Fig. 5BGo). Double-labeled immunocytofluorescence or histofluorescence with TH and PRL antibodies showed that all TH-expressing cells were immunoreactive for PRL (Fig. 5Go, B and C). The percentage of lactotrophs expressing TH was evaluated on dispersed AP cell culture. We found that 15.5 ± 2.2% of PL3 rat lactotrophs expressed TH compared with less than 2% (1.6 ± 0.3%; P < 0.01) for V rats (Fig. 5CGo). We then determined whether TH was coexpressed with DAT in PL3 rat AP glands. Double immunolabeling revealed that all TH-positive cells were immunoreactive for DAT (Fig. 6Go). Moreover, we showed that 36.9 ± 6.3% of TH-positive cells coexpressed CC3 and that all CC3-immunoreactive cells were TH positive (Fig. 6Go).


Figure 5
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  		FIG. 5. TH expression in lactotroph cells. Aa, RT-PCR products of TH gene separated on 2% agarose electrophoresis gel. Lane 1, No PCR products were detected in the negative (–) controls, performed by omitting reverse transcriptase. Lanes 2–5 and 7–10, RT-PCR fragments from V and PL3 rats, respectively. Lanes 6 and 11, Digestion of PCR mixes from each condition with HaeIII. M, A 50-bp DNA marker. Ab, Western blot showing TH expression in dispersed AP cells from V and PL3 rats. Whole-cell extracts (50 µg protein per lane) were subjected to immunoblot analysis with anti-TH (top) and anti-actin (bottom) for loading control. Brain protein extract was chosen as a positive control (T+). Blots shown are typical of results obtained in three separate experiments. B, Representative fields (x630) of AP cell cluster from PL3 rat slices showing coimmunofluorescence for PRL (red) and TH (green) compared with a representative field from a V pituitary cell slice, in which immunostaining for TH was only observed in the post-pituitary (PP) (n = 2). Ca, Representative field (x630) of dispersed PL3 AP cells showing red (PRL) and/or green (TH) fluorescence after double immunocytofluorescence. Cb, The histogram represents the percentage of dispersed V and PL3 rat cells showing double immunostaining (black columns) and single immunostaining (white columns) for PRL. n = 3. *, P < 0.005, significant compared with V cells.

 

Figure 6
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  		FIG. 6. Coexpression of TH with DAT and CC3. Representative fields (x630) of AP cell clusters from PL3 rat slices showing coimmunofluorescence for TH (red) and DAT (top) or CC3 (bottom) in green (each experiment, n = 2).

 
Finally, to determine whether TH expression was accompanied by an increase in the DA content of PL3 rat AP glands, we compared the DA concentrations in AP glands from V and PL3 rats. An EIA measured a 4-fold higher DA concentration in AP gland from PL3 than V rats (Table 1Go). This result showed that DA levels varied in AP according to the physiological state of the animals and suggested that TH expression resulted in increased DA production in rat AP glands.


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  		TABLE 1. DA assay in anterior pituitary from V or PL3 rats

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The onset of lactation requires sustained PRL stimulation, resulting in a remarkable proliferation of lactotrophs in the AP gland during pregnancy and parturition. After the end of lactation, the expanded cell population decreases and the proportion of lactotrophs returns to prepregnancy levels (18). The mechanism responsible for this process is still controversial. Indeed, in Wistar rats, Aoki et al. (33) ascribed lactotroph regression to non-apoptotic cell death. In contrast, in Sprague Dawley rats, Ahlbom et al. (19) reported a significant increase in the expression of proapoptotic Bax protein that coincided with the peak of DNA fragmentation and a decrease in anti-apoptotic Bcl-2 protein, i.e. hallmarks of apoptosis. Our results, obtained in Sprague Dawley rats, confirm that apoptosis probably occurs in lactotrophs during the postlactation period, because the activation of caspase-3, a key event in the apoptotic process, was detected in lactotrophs from postlactating but not V or L rats. As previously described, apoptosis is detectable in the pituitary starting 3–4 d after cessation of lactation. However, at time t, we showed that only 0.5% of cells exhibited apoptotic features. This observation is quite surprising because it has been described previously that lactotroph loss occurred within the first week after weaning, suggesting a massive cellular death process (18). Several hypotheses can account for this discrepancy. 1) In physiological conditions, it is known that apoptotic cells are difficult to observe because they are quickly cleared by phagocytosis. So, the number of apoptotic cells detected in the present study could be underestimated. 2) An other process beside apoptosis could contribute to lactotroph clearance. This has been suggested by Aoki et al. (33, 34) who described the presence of "dark cells" in AP gland of end-lactating rats by electron microscopy. 3) Finally, we cannot exclude that the apoptotic process in the pituitaries of postlactating rats is not a short-lived, massive phenomenon but rather lasts several weeks because we still detected apoptotic cells in the pituitaries of PL24 rats.

Until now, the factors responsible for this apoptotic process were not known. In view of the fact that DA plays a major role in controlling lactotroph proliferation (35) and induces apoptosis in human and rat prolactinoma (22, 23), we tested the effect of DA on apoptosis in dispersed AP cells from V, L, and PL3 or PL24 rats. Several features of apoptosis were investigated for this purpose. Our results clearly showed that DA only induced MMP loss, CC3 formation, and nuclear fragmentation in AP cells from postlactating but not V or L animals. This effect seems highly DA specific, because other catecholamines, such serotonin or norepinephrine, were unable to induce apoptosis in AP cells from V or PL rats. Moreover, DA specifically triggered apoptosis in lactotrophs, as shown by coimmunostaining of CC3 and PRL. These data suggest that DA participates in lactotroph apoptosis during the postlactation period. This apoptotic effect of DA is consistent with the increase in p53 and Bax expression observed in postlactating rat AP glands (19). Indeed, DA has been shown to mediate the proapoptotic effect by inducing expression of these two proteins in the PC12 cell line (36) and neurons (37). Because cultured AP cells have been reported to express a phenotype according to their previous in vivo conditions (38, 39), the differential apoptotic response to DA according to the killed stage of the animals suggests that a specific process during the postlactation period sensitizes lactotrophs to apoptosis. In view of the fact that an estrogen surge occurs 72 h after pups are weaned and estrogens may sensitize AP glands to apoptotic stimuli, such as lipopolysaccharide or Fas (21, 40), additional investigation will be necessary to determine whether estrogens sensitize lactotrophs to DA-induced apoptosis.

All of the effects of DA described in lactotrophs were found to be mediated by D2R. We therefore tested the effect of D2R antagonists on DA-induced apoptosis. Surprisingly, neither haloperidol nor raclopride, applied during DA treatment, reduced DA-triggered apoptosis in PL3 cells. These results suggest, for the first time, that DA exerts its effect on normal lactotrophs via a D2R-independent mechanism. In view of this data, we used Western blot to study D2R expression in AP glands from V and PL3 rats, revealing a significant decrease in D2R expression in AP glands from PL3 compared with V rats. These observations indicated that D2R was not involved in the proapoptotic effect of DA, suggesting that other mechanisms were responsible for mediating DA-induced apoptosis in postlactating rats. In the central nervous system (CNS), it is clearly established that DA, its metabolic product, or 6-hydroxydopamine, trigger apoptosis via a mechanism involving DAT (4, 7, 41). Moreover, in GH3 cells, we showed that DA-induced apoptosis was mediated by a DAT rather than a D2R mechanism (24). Because DAT expression in AP glands is controversial (30, 31), we initially studied its expression in V and PL3 rat AP glands and then its involvement in the apoptotic process. DAT expression was studied by RT-PCR, Western blot, and immunohistochemistry. mRNA was detected in both V and PL3 AP glands, and a strong increase in protein expression was observed in PL3 compared with V rats, suggesting that DAT was expressed under specific physiological conditions, such as postlactation. This differential protein expression may explain why DAT was not found in AP glands in previous immunohistochemistry studies (30). Moreover, DAT is probably involved in the proapoptotic effect of DA, because apoptosis was completely inhibited in cells coincubated with DA and two specific DAT inhibitors, mazindol and GBR 12909. These findings suggest that, besides its effects on PRL synthesis and secretion and lactotroph proliferation, DA may play a new role in pituitary physiology, via a D2R-independent mechanism that, nevertheless, involves DAT. DAT-mediated apoptosis has been extensively studied. Indeed, we recently demonstrated that apoptosis was induced by DA in GH3 cells via a transduction mechanism similar to that described in CNS involving the following: DAT, oxidative stress, loss of MMP, Bax, and caspase-3 activation (24). Because the GH3 cell line is a valuable lactotroph cell model, we extrapolated from these data that DA induced apoptosis in lactotrophs in a similar way during the postlactation period.

DAT expression in the AP gland is an unusual phenomenon because distribution of this transporter seems restricted to the CNS, in which it always colocalizes with TH, the rate-limiting enzyme of the DA synthesis pathway (42). In pituitary, DA originates from tuberoinfundibular neurons, which terminate in the external zone of the median eminence. The released DA is transported via the long portal vessels to the anterior lobe of the pituitary (10). However, TH and DA may be produced in the AP under particular conditions, such as ectopic pituitaries, adenomas, or transformed lactotrophs (14). We thus investigated whether TH was expressed in AP cells during the postlactation period. We confirmed previous findings on the expression of TH mRNA in the AP gland of female rats (43), and, similar to DAT, we clearly showed that TH protein expression increased in PL3 compared with V rats. These interesting observations suggest that a pool of preexisting TH mRNA, poorly translated under basal conditions, may be actively translated in particular physiological circumstances, such as postlactation or pituitary adenoma (14). Indeed, this type of posttranscriptional TH regulation is already known in the CNS (44, 45). Although the number of TH-positive cells is relatively low in AP glands from PL rats, these cells may be responsible for the higher DA concentration measured in AP glands from PL3 than V. Indeed, in the CNS, it is known that DA is stored in secretion vesicles at extremely high concentrations (0.5–0.6 M), resulting in high postrelease concentration in the intersynaptic space. Such a release pattern could contribute to the DA content increase in AP gland and may lead to high localized concentration compatible with the DA concentration (200 µM) required to induce lactotroph apoptosis in vitro. This would also explain the decrease in D2R expression in PL3 AP, because it is well known that DA down-regulates D2R expression (46) (Jaubert, A., and L. Bresson-Bepoldin, personal observations). Nevertheless, we cannot exclude that other dopaminergic sources such as the dopaminergic shunt between the post and the AP may be involved in DA concentration variation in AP from PL rats.

Furthermore, we showed that TH was colocalized with DAT in PL3 AP glands and that all CC3-positive cells were TH-positive lactotrophs. Thus, these results show, for the first time, that, during the postlactation period, some lactotrophs express all of the protagonists necessary to trigger their own apoptosis via an "autocrine" regulation loop, including TH and DAT expression (Fig. 7Go). However, the mechanism regulating this unusual TH and DAT expression in rat AP glands requires additional investigation. In particular, the role of estrogens should be considered, because studies using various cell models have shown that they are capable of regulating TH and DAT expression and/or activity (47, 48).


Figure 7
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  		FIG. 7. Different DA effects during lactotroph cell life. In V rat lactotrophs, DA plays a physiological role via mechanisms involving D2R: PRL exocytosis is inhibited via a D2R and K+ channel interaction, leading to membrane hyperpolarization, calcium channel closure, and a subsequent decrease in intracellular calcium concentration. The effect of DA on PRL synthesis involves inhibition of the cAMP/protein kinase A (PKA) pathway. In postlactating rats, surplus lactotrophs express the TH and DAT necessary for their own apoptosis. TH expression is probably responsible for local DA synthesis and secretion by lactotrophs, leading to a locally high DA concentration. In turn, DA may undergo reuptake by DAT to exert a proapoptotic effect via ROS production and activation of the mitochondrial signaling pathway. AC, Adenylyl cyclase; PTP, permeability transition pore; Cyt c, cytochrome c; C3, caspase-3.

 
In conclusion, this study shows, for the first time, that the effect of DA on lactotroph physiology varies according to the physiological state of the animal. In V rats, DA plays a role in PRL synthesis and secretion via well-established signaling pathways involving D2R, whereas, in postlactating rats, DA triggers lactotroph apoptosis via a previously unknown mechanism involving TH and DAT expression by lactotrophs themselves (Fig. 7Go).


   Acknowledgments
 
We thank Dr. Parlow and the National Hormone and Pituitary Program for pituitary antibodies. We acknowledge Viviane Tridon for her technical help.


   Footnotes
 
First Published Online March 15, 2007

Abbreviations: AP, Anterior pituitary; CC3, cleaved caspase-3; CNS, central nervous system; D2R, DA receptor type 2; DA, dopamine; DAT, DA transporter; EIA, enzyme immunoassay; GBR 12909, 1-[2-[bis(4-fluorophenyl)-methoxy]ethyl]-4-[3- phenylpropyl]piperazine; ir, immunoreactivity; L, lactating; L-DOPA, L-3,4-dihydroxyphenylalanine; MMP, mitochondrial membrane potential; PL, postlactation; PRL, prolactin; ROS, reactive oxygen species; TH, tyrosine hydroxylase; TMRM, tetramethylrhodamine methylester; V, virgin.

This work was supported by Institut National de la Santé et de la Recherche Médicale, Université de Bordeaux 2, Etablissement Public Régional (Aquitaine Région), and La Ligue contre le Cancer. A.J. was supported by Ministère de l’Enseignement Supérieur et de la Recherche Grant 10513-2003.

The authors of this manuscript have nothing to declare.

Received September 20, 2006.

Accepted for publication February 28, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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