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Biology of Reproduction 67, 1218-1224 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regulation of Gonadotropin {alpha} Subunit Gene Expression by Dopamine D2 Receptor Agonist in Clonal Mouse Gonadotroph {alpha}T3-1 Cells1

Haruhiko Kanasaki2,a, Toshie Yoneharaa, Yoko Yamadaa, Kentaro Takahashia, Kohkichi Hataa, Rituto Fujiwakia, Hideyuki Yamamotob, Yusuke Takeuchib, Kohji Fukunagab, Eishichi Miyamotob, and Kohji Miyazakia

a Department of Obstetrics and Gynecology, Shimane Medical University, Izumo 693-8501, Japan b Department of Pharmacology, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pituitary prolactin biosynthesis is negatively regulated by hypothalamic dopamine through D2 receptors in pituitary lactotrophs, but little is known about the direct effect of dopamine on gonadotrophs. In this study, the clonal gonadotroph-derived cell line, {alpha}T3-1, was used to examine whether gene expression of the pituitary gonadotropin {alpha} subunit, stimulated with GnRH or pituitary adenylate cyclase-activating polypeptide (PACAP), was controlled by dopamine D2 receptor. Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated the presence of dopamine D2 receptors in {alpha}T3-1 cells. Both GnRH and PACAP increased {alpha} subunit gene expression. GnRH-induced {alpha} subunit gene expression was not affected by quinpirol, a specific dopamine D2 receptor agonist. In contrast, PACAP-induced gene expression was significantly lower in the presence of quinpirol. The roles of extracellular signal-regulated kinase (ERK) and cAMP in the expression of the {alpha} subunit gene were examined. GnRH activated ERK, but PACAP did not, and the activation was not inhibited by quinpirol. GnRH-induced {alpha} subunit gene expression was completely inhibited by an ERK inhibitor, PD098059. Cyclic AMP accumulation in {alpha}T3-1 cells was increased by treatment with PACAP, and quinpirol inhibited this effect. GnRH did not affect cAMP production in these cells. These results suggest that in {alpha}T3-1 cells, dopamine D2 receptors negatively regulate pituitary {alpha} subunit gene expression in association with the cAMP-dependent pathway, but not with the ERK pathway.

dopamine, gonadotropin-releasing hormone, kinases, neuroendocrinology, pituitary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gonadotropin hormones contain {alpha} and ß subunits [1]. The {alpha} subunit is common to the gonadotropin hormones, whereas the ß subunits differ from each other and confer specificity to the gonadotropin hormones. Synthesis and secretion of gonadotropins such as LH and FSH are mainly under the control of the hypothalamic peptide, GnRH. In addition to GnRH, other hypothalamic peptides such as pituitary adenylate cyclase-activating polypeptide (PACAP), endothelin, epidermal growth factor (EGF), opioids, oxytocin, and cytokines have been known to be involved in this type of regulation [2]. The initial phase of GnRH action involves G protein-mediated stimulation of phospholipase C, leading to the formation of 1,4,5-triphosphate (IP3) and diacylglycerol (DG). Subsequently, IP3 induces the release of intracellular Ca2+ from the endoplasmic reticulum, and DG activates protein kinase C (PKC), which ultimately activates extracellular signal-regulated kinase (ERK) [3–5]. The involvement of GnRH-activated PKC and ERK pathways in expression of the {alpha} subunit were strongly suggested in previous reports [6–8]. In addition, the cAMP-protein kinase A (PKA) cascade reportedly has also been involved in regulating the function of gonadotrophs [9–12]. PACAP is a hypophysiotropic factor that was first purified from ovine hypothalamus, and it has the ability to stimulate cAMP accumulation in pituitary cells [13]. Cloning of PACAP cDNA revealed that PACAP is a member of the glucagon-secretin-vasoactive intestinal polypeptide (VIP) family of peptides [13]. PACAP acts predominantly via PVR-1 receptors, which when coupled with G protein, stimulates inositol phosphate turnover with a potency approximately 1000-fold greater than VIP. PACAP also stimulates cAMP production with 100-fold more potency than VIP [14, 15]. Gonadotropin secretion and gene expression are known to be regulated by PACAP stimulation [14, 16–20].

It is well known that dopamine, which is released by hypothalamic neurons to the hypophyseal portal circulation, negatively regulates basal and stimulated prolactin secretion in pituitary lactotrophs through the D2 receptor [21]. The dopamine-D2 receptor interacts with a pertussis toxin-sensitive, inhibitory G protein to reduce adenylate cyclase activity, and to inhibit the production of several second messengers such as cAMP and inositol phosphates [22–24]. Dopamine also modulates the channel activities of K+, Ca2+, or both, and inhibits the elevation of cytosolic Ca2+ evoked by other agents [25]. In addition to the negative control of dopamine in lactotrophs, regulation of gonadotropin secretion and gene expression has been suggested in previous reports. Administration of a dopamine agonist to patients with nonfunctioning pituitary adenomas demonstrated a significant reduction of serum FSH, LH, and {alpha} subunit concentrations [26, 27]. The inhibitory effects of dopamine on gonadotropin secretion in the goldfish pituitary were also described previously [28–33]. The inhibitory effects of dopamine on gonadotropin control are believed to be due primarily to indirect mechanisms. These involve negative modulation of hypothalamic neurons, which regulate gonadotropin functions such as GnRH. But the direct mechanisms of dopamine action through its D2 receptors within the gonadotrophs are not well understood.

Studies of the regulation of pituitary gonadotropin gene expression have been aided by the development of an {alpha} subunit-producing cell line of gonadotroph lineage, {alpha}T3-1 cells [34]. These cells express the GnRH receptor and the {alpha} subunit common to LH and FSH, although they do not express the ß subunits specific to these hormones [34]. The {alpha}T3-1 cell line has proven to be a useful model for the study of gonadotropin {alpha} subunit secretion and gene expression of gonadotroph-like pituitary cells.

In the present study, using {alpha}T3-1 cells, we attempted to evaluate the gene expression of the {alpha} subunit after stimulation with GnRH and PACAP, and the possible involvement of dopamine D2 receptors in its regulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Fetal calf serum (FCS) was obtained from JRH Biosciences (Lenexa, KS); [{gamma}-32P]ATP was from DuPont-New England Nuclear (Wilmington, DE); GnRH, PACAP-38, quinpirol hydrochloride, and PD098059 were from Sigma Chemical Company (St. Louis, MO); Dulbecco modified Eagle medium (DMEM) was from Life Technologies, Inc. (Gaithersburg, MD); and dopamine D2 receptor antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cell Culture

Alpha T3-1 cells (kindly provided by Dr. P. Mellon of the University of California at San Diego) were cultured in DMEM containing 10% FCS, 50 IU/ml penicillin, and 50 µg/ml streptomycin and maintained at 37°C in an atmosphere of 95% air and 5% CO2. Two or three days before experiments, 2–3 x 105 cells were plated on a 35-mm Petri dish (Nunc, Roskilde, Denmark). Cells were then incubated at 37°C for the indicated times without (control) or with the test reagents in DMEM without serum. When quinpirol and PD098059 were used to study {alpha} subunit gene expression, these compounds were added for 30 min during preincubation and during incubation with the test reagents. After incubation, the medium was quickly aspirated off, and the cells were frozen in liquid nitrogen.

Reverse Transcription-Polymerase Chain Reaction of Dopamine D2 Receptor and {alpha} Subunit

Total RNAs were prepared from {alpha}T3-1 cells using ISOGEN Reagent (Wako Pure Chemicals, Osaka, Japan), according to the manufacturer's protocol. Messenger RNA was reverse-transcribed into single-stranded cDNA using an oligo(dT) primer (Promega, Madison, WI) and Moloney murine leukemia virus reverse transcriptase (RT; Gibco BRL, Gaithersburg, MD). The reaction mixtures were diluted 20-fold and then subjected to polymerase chain reaction (PCR) amplification of dopamine D2 receptor and gonadotropin {alpha} subunit mRNAs. The PCR primers were designed on the basis of the published sequences of dopamine D2 receptor [35] and mouse gonadotropin {alpha} subunit [36]. For amplification of cDNAs, each of the following primers were used: 5'-dopamine D2 receptor primer, 5'-AGAGCCAACCTGAAGACACCA-3'; 3'-dopamine D2 receptor primer, 5'-GATGATGAACACACCGAGAAC-3'; 5'-{alpha} subunit primer, 5'-ACTTTATTATTCAGGGGTTGC-3'; and 3'-{alpha} subunit primer, 5'-TATAAGGGATGTAACCGTAAA-3'. PCR amplification was carried out using the Program Temperature Control System PC-701 (ASTEC, Fukuoka, Japan) after samples had been denatured for 10 min. For dopamine D2 receptor amplification, cycles consisted of denaturation at 94°C for 45 sec, annealing at 52°C for 45 sec, and extension at 72°C for 45 sec. PCR was constructed for 32 cycles. For {alpha} subunit amplification, the cycles consisted of denaturation at 94°C for 45 sec, annealing at 55°C for 45 sec, and extension at 72°C for 40 sec. After amplification, the final 5-min extension step was carried out at 72°C. When a quantitative RT-PCR analysis was carried out, the PCR for the {alpha} subunit was constructed for 23 cycles. PCR products were separated by electrophoresis on a 1.0% agarose gel, visualized with ethidium bromide staining, and quantified by scanning densitometry using NIH Image software (version 1.61). The amounts of PCR products of the {alpha} subunit were normalized to those of the PCR products of ß actin in each sample.

Western Blotting

Extracts of {alpha}T3-1 cells were subjected to SDS-PAGE in a 7.5% acrylamide gel and transferred to a nitrocellulose membrane. The membrane was incubated with the anti-dopamine D2 receptor antibody (diluted 1:100) for 24 h at 4°C. After washing, the membrane was incubated for 1 h at room temperature in a horseradish peroxidase-conjugated donkey anti-goat immunoglobulin G secondary antibody (diluted 1:10 000) followed by detection on x-ray film with enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, U.K.).

Assay for ERK Activity

Two or 3 days before experiments, 2–3 x 105 cells were plated on a 35-mm Petri dish. When test reagents were added, cultured cells were preincubated with Krebs-Ringer Hepes buffer (KRH) containing 130 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM sodium phosphate, 1.2 mM MgSO4, 10 mM glucose, and 20 mM Hepes pH 7.4, and preincubated in KRH at 37°C for 60 min. Cells were then incubated at 37°C for the indicated times with no reagents (control) or the test reagents (GnRH, PACAP) in KRH. When quinpirol was used, it was added during preincubation and incubation with test reagents in KRH. After incubation for the indicated times, the medium was quickly aspirated off, and the cells were frozen in liquid nitrogen. Frozen cells were scraped off the dishes and homogenized in 150 µl of 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM orthovanadate, 0.1 mM leupeptin, 75 µM pepstatin A, and 100 nM calyculin A. After cellular debris were removed by centrifugation at 15 000 x g for 5 min, a 15-µl aliquot of the supernatant was assayed for ERK using [{gamma}-32P]ATP and synthetic EGF receptor peptide as a substrate, according to the instructions supplied by the manufacturer (p42/p44 mitogen-activated protein kinase enzyme assay system; Amersham, Buckinghamshire, U.K.). The phosphorylated peptide was separated from the unlabeled one on a peptide binding paper. After washing the paper in 75 mM phosphoric acid, phosphorylation of the peptide was determined with a liquid scintillation counter.

Cyclic AMP Accumulation

Cells were plated in 96-well plates at a density of 104 cells/well and cultured for 72 h. For long-time study, cells were preincubated with serum-free DMEM with or without 10 µM quinpirol for 30 min, and incubated for 24 h with the indicated reagents in 100 µl of DMEM. Intracellular cAMP levels were measured using the cAMP enzyme immunoassay system from Amersham Pharmacia Biotech.

Statistical Evaluation

Each experiment was conducted in at least three independent determinations for cells of separate dishes. The same experiments were repeated at least twice with reproducible results. Values were expressed as means ± SEM. Statistical analysis was performed using one-way ANOVA plus the Duncan multiple range test. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Dopamine D2 Receptor in {alpha}T3-1 Cells

In the first series of studies, total mRNAs of {alpha}T3-1 cells were analyzed by RT-PCR to determine the presence and characteristics of D2 receptors. As shown in Figure 1A, using specific dopamine D2 receptor primers, long forms of dopamine D2 receptor mRNA were detected in {alpha}T3-1 cells (Fig. 1A). Dopamine D1 receptor RNA was not amplified in these cells (data not shown). Western blotting analysis also indicated an immunoreactive band with an apparent molecular mass of about 100 kDa in {alpha}T3-1 cells (Fig. 1B), as detected by a specific dopamine D2 receptor antibody.



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  		FIG. 1. Dopamine D2 receptor expression in {alpha}T3-1 cells. A) RT-PCR analysis of dopamine D2 receptor. Total RNA (1.0 µg) prepared from {alpha}T3-1 cells were reverse transcribed, and RT-PCR was carried out using specific dopamine D2 receptor primers. The cDNAs (0.4 µg) for dopamine D2 receptor long form (D2L) and short form (D2S) inserted into pCAGGSneo vectors were used for a template as positive controls. PCR products were separated by electrophoresis on 1.0% agarose gel, and visualized with ethidium bromide staining. D2L, 459 base pairs; D2S, 372 base pairs. B) Western blotting with the antidopamine D2 receptor antibody. Western blotting was carried out with a dopamine D2 receptor antibody (diluted 1:100) as described in Materials and Methods. The immunoreactive band was visualized with enhanced chemiluminescence. D2R, dopamine D2 receptor

Effects of GnRH and PACAP on {alpha} Subunit Gene Expression in {alpha}T3-1 Cells

The effects of GnRH and PACAP on {alpha} subunit gene expression were examined by quantitative RT-PCR analysis. Analysis yielded a relationship between the relative signal and the amount of total RNA ranging from 0.5 to 4.0 µM (data not shown). Treatment of {alpha}T3-1 cells with 100 nM GnRH led to a larger increase in {alpha} subunit gene expression than it did in nonstimulated cells, and expression reached a maximal peak of 250%–300% of control levels after 12 h of stimulation (Fig. 2A). On the other hand, the effect of 100 nM PACAP on {alpha} subunit gene expression was slower than that of GnRH, and it reached a maximal peak of 200%–250% after stimulation for 24 h. In contrast to GnRH, PACAP did not increase {alpha} subunit gene expression at 6 h (data not shown) and 12 h (Fig. 2B) after stimulation.



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  		FIG. 2. Serial changes in the effects of GnRH and PACAP on the expression of {alpha} subunit gene and its time course. Alpha T3-1 cells were incubated without (control) and with 100 nM GnRH (A) and 100 nM PACAP (B) for indicated times. Total RNA (1.0 µg) from the cells were reverse transcribed, and RT-PCR was carried out using {alpha} subunit and ß actin primers. The visualized PCR products were quantified by scanning densitometry using NIH Image software. The amount of {alpha} subunit mRNA was normalized to that of the PCR product of ß actin. Values are means ± SEM (three dishes per condition in a single experiment). We repeated the same experiments at least three times with reproducible results, and representative results are shown. **P < 0.01; *P < 0.05 vs. control

Effects of Quinpirol on GnRH- and PACAP-Induced {alpha} Subunit Gene Expression

To examine the possible involvement of dopamine D2 receptors present in {alpha}T3-1 cells in {alpha} subunit gene expression, we tested the effects of the specific dopamine D2 receptor agonist, quinpirol, on the stimulatory actions of GnRH and PACAP on {alpha} subunit gene expression. Treatment of cells with GnRH for 12 h increased {alpha} subunit gene expression by up to 350% more than it did in nontreated cells. Inclusion of 10 µM quinpirol with GnRH in the medium showed no effects on GnRH-induced gene expression (Fig. 3A). However, gene expression after stimulation with PACAP for 24 h was significantly but not completely inhibited in the presence of quinpirol (Fig. 3B). PACAP-induced {alpha} subunit gene expression was lower in the presence of quinpirol in a dose-dependent manner (Fig. 4).



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  		FIG. 3. Effects of dopamine D2 receptor agonist, quinpirol, on GnRH- and PACAP-induced {alpha} subunit gene expressions in {alpha}T3-1 cells. Alpha T3-1 cells were incubated without (control) or with 100 nM GnRH (A) for 12 h and 100 nM PACAP (B) for 24 h, respectively, in the presence or absence of 10 µM quinpirol in DMEM without serum. Cells were preincubated for 60 min with 10 µM quinpirol and then incubated with GnRH or PACAP. Total RNA (1.0 µg) from the cells were reverse transcribed, and PCR was carried out using {alpha} subunit and ß actin primers. The visualized PCR products were quantified by scanning densitometry using NIH Image software. The amount of {alpha} subunit mRNA was normalized to that of the PCR product of ß actin. Values are means ± SEM (three dishes per condition in a single experiment). We repeated the same experiments at least three times with reproducible results, and representative results are shown. **P < 0.01 vs. control. Difference between PACAP and PACAP + quinpirol were statistically significant (P < 0.05)



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  		FIG. 4. Inhibition of PACAP-induced {alpha} subunit gene expression with quinpirol. Cells were preincubated for 60 min with indicated concentrations of quinpirol in DMEM without serum, and then incubated for 24 h with addition of 100 nM PACAP. Total RNA (1.0 µg) from the cells were reverse transcribed, and RT-PCR was carried out using {alpha} subunit and ß actin primers. The visualized PCR products were quantified by scanning densitometry using NIH Image software. The amount of {alpha} subunit mRNA was normalized to that of the PCR product of ß actin. Values are means ± SEM (three dishes per condition in a single experiment). We repeated the same experiments at least three times with reproducible results, and representative results are shown. **P < 0.01 vs. control

ERK Activation in {alpha}T3-1 Cells

It has been reported that activation of ERK by treatment with GnRH contributes to stimulation of {alpha} subunit gene expression. Therefore, we examined the effects of GnRH and PACAP on ERK activation and the influence of quinpirol treatment (Fig. 5). GnRH significantly raised ERK activity in assays using the synthetic EGF receptor peptide as a substrate, whereas PACAP failed to stimulate ERK activation (Fig. 5A). ERK activation (Fig. 5B) and {alpha} subunit expression (Fig. 3A) by GnRH treatment were not inhibited in the presence of quinpirol (Fig. 5B). However, GnRH-induced {alpha} subunit gene expression was completely abolished with 50 µM PD098059, a specific ERK inhibitor (Fig. 6).



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  		FIG. 5. ERK activation by stimulation with GnRH and PACAP, and the effects of quinpirol on GnRH-induced ERK activation in {alpha}T3-1 cells. A) After preincubation for 60 min in KRH, the cells were incubated for 10 min without (control) or with 100 nM GnRH or 100 nM PACAP. B) After preincubation for 60 min in KRH, the cells were incubated for 10 min without (control) or with 100 nM PACAP in the presence or absence of 10 µM quinpirol. Quinpirol was added during preincubation and incubation. Cell extracts were incubated with a synthetic peptide as a substrate in the presence of [{gamma}-32P]ATP, and phosphate incorporation into the substrate was counted with a liquid scintillation counter as described in Materials and Methods. Activities of ERK in the control were defined as 100%, and from these values, other values were calculated. Values are means ± SEM (three dishes per condition in a single experiment). We repeated the same experiments at least three times with reproducible results, and representative results are shown. **P < 0.01 vs. control



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  		FIG. 6. Effects of PD098059 on GnRH-induced {alpha} subunit gene expression. Alpha T3-1 cells were incubated in serum-free DMEM alone (control) or with 100 nM GnRH for 12 h in the presence or absence of 50 µM PD098059 (PD). After stimulation, total RNA (1.0 µg) from the cells were reverse transcribed, and RT-PCR was carried out using {alpha} subunit and ß actin primers. The visualized PCR products were quantified by scanning densitometry using NIH Image software. The amount of {alpha} subunit mRNA was normalized to that of the PCR product of ß actin. Values are means ± SEM (three dishes per condition in a single experiment). We repeated the same experiments at least three times with reproducible results, and representative results are shown. **P < 0.01 vs. control. The difference between GnRH and GnRH + PD is statistically significant (P < 0.01)

Cyclic AMP Accumulation in {alpha}T3-1 Cells

PACAP is known to stimulate adenylate-cyclase activity and to accelerate cAMP formation in cells. Indeed, treatment of cells with 100 nM PACAP for 24 h in the medium raised intracellular cAMP levels by up to 250% compared with nontreated cells, whereas GnRH had no effect on cAMP accumulation. PACAP-induced cAMP accumulation was significantly inhibited by adding quinpirol. Quinpirol alone had no effects on cAMP accumulation (Fig. 7). These results suggested that stimulation of D2 receptor in {alpha}T3-1 cells negatively regulates PACAP actions by inhibiting adenylate cyclase activation.



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  		FIG. 7. The effects of quinpirol on PACAP-induced cAMP accumulation in {alpha}T3-1 cells. Cells plated in a 96-well plates were preincubated with serum-free DMEM with or without 10 µM quinpirol for 30 min, and incubated for 24 h with indicated reagents in 100 µl of DMEM. Intracellular cAMP levels were measured as described in Materials and Methods. We repeated the same experiments at least three times with reproducible results, and representative results are shown. **P < 0.01 vs. control. The difference between PACAP and PACAP + quinpirol is statistically significant (P < 0.01)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been suggested that the secretion and gene expression of pituitary gonadotropins are controlled negatively by dopamine or dopamine agonists. Clinical trials showed that long-term treatment of patients with clinically nonfunctioning or {alpha} subunit-secreting pituitary tumors with dopamine agonists led to lower levels of serum gonadotropin concentrations [26, 27]. Furthermore, the inhibitory effects of dopamine on gonadotropin production have been reported in goldfish pituitary cells [28–33]. However, studies of the effects and mechanisms of dopamine D2 receptors in single gonadotroph cells have been hampered because the anterior pituitary gland contains at least six different cell types. Alpha T3-1 cells, which possess gonadotroph-like characteristics, were established by targeted oncogenesis of mouse gonadotrophs [34]. Although {alpha}T3-1 cells do not express the LH- and FSH-specific ß subunits, this cell line has proven a useful model in biochemical studies of the regulation of the gonadotropin {alpha} subunit, which is common to LH and FSH. RT-PCR analysis using primers for D2 receptors demonstrated the presence of the long form of D2 receptor (Fig. 1A), and Western blotting analysis showed the presence of D2 receptor at the protein level in {alpha}T3-1 cells (Fig. 1B). The presence of dopamine D2 receptors in {alpha}T3-1 cells led us to examine whether or not dopamine plays important roles in regulating gonadotropins in pituitary gonadotrophs.

In the present study, two hypothalamic peptides, GnRH and PACAP, were used. GnRH acts through Gq-coupled receptors and accelerates ERK activation in a largely PKC-dependent manner [3–5], whereas PACAP action is known to act through two types of PACAP receptors (PVR1 and PVR3) that are capable of activating both adenylate cyclase and phospholipase C in {alpha}T3-1 cells [37]. We found that both GnRH and PACAP significantly raised the expression of {alpha} subunit mRNAs in {alpha}T3-1 cells (Fig. 2). Expression reached a maximal peak after 12 h of treatment with GnRH, whereas PACAP treatment reached a maximal peak more slowly, after 24 h of treatment. This suggested that the intracellular mechanisms of GnRH and PACAP on acceleration of {alpha} subunit gene expression were distinct. In support of this idea, in experiments using {alpha}T3-1 cells, GnRH significantly increased ERK activation, whereas PACAP did not (Fig. 5A). In contrast, GnRH had no effect on cAMP accumulation, but PACAP treatment significantly raised intracellular cAMP levels (Fig. 7). These results suggested that GnRH-induced {alpha} subunit expression was conducted mainly by ERK activation. The effects of PD098059 on GnRH-induced expression of {alpha} subunit supported this conclusion (Fig. 6). In contrast, PACAP-induced gene expression was correlated with elevation of intracellular cAMP levels. In this connection, the cross-talk between intracellular signaling pathways including ERK activation and cAMP pathways has to be discussed. We previously reported that thyrotropin-releasing hormone (TRH) activated ERK, and this activation was involved in prolactin gene expression in the rat pituitary somatolactotroph cell line, GH3 cells [38]. Furthermore, we presented evidence that cAMP-induced prolactin gene expression was also dependent on ERK activation induced by cAMP [39]. In addition, the ability of cAMP to activate ERK was observed previously in the pituitary and in pituitary cell lines [40–42]. Despite the effect of cAMP pathways on ERK activation described above, ERK activation by PACAP or cAMP could not be detected in {alpha}T3-1 cells.

The dopamine D2 receptor agonist, quinpirol, significantly reduced {alpha} subunit gene expression (Fig. 3B) as well as cAMP accumulation within {alpha}T3-1 cells (Fig. 7), but only in the cells treated with PACAP. The effects of quinpirol on inhibiting PACAP-induced {alpha} subunit expression showed dose dependency (Fig. 4). In contrast to the effects of quinpirol on PACAP-induced cAMP production and {alpha} subunit expression, GnRH-induced gene expression and ERK activation were not inhibited by quinpirol. These results implied that the inhibitory effects of dopamine acting through D2 receptors were executed only during the stimulus in which cAMP accumulation is elevated. In the present study we attempted to have additional evidence using VIP, which is known as a cAMP increasing factor, however, VIP did not significantly increase {alpha} subunit expression in {alpha}T3-1 cells (data not shown). Dopaminergic control of hormone secretion and gene expression were well characterized, especially in pituitary lactotrophs [21]. The effects of dopamine, in association with D2 dopamine receptors in lactotroph cells, include inhibition of the production of several second messengers such as cAMP and inositol phosphates, and dopamine has been shown to modulate channel activities of K+, Ca2+, or both [22–25]. Similar to the action of GnRH, TRH, a prolactin-stimulating factor, increases turnover of inositol by activating Gq-coupled TRH receptors, which in turn, affect PKC pathways and Ca2+ release from Ca2+ storage sites [43], with sequential activation of ERK [38]. Recently, it was demonstrated that hormone stimulating factor-induced ERK activation plays an important role in gene expression of pituitary hormones [6–8, 38, 39]. Ohmichi et al. [44] examined the possible effects of dopamine on TRH-induced ERK activation in dispersed rat anterior pituitary cells and showed the inhibitory effect of dopamine on TRH-induced ERK activation. Those researchers suggested that cross-talk exists between the dopamine signaling pathway and the ERK activating pathway. Considering the similar signaling systems of GnRH and TRH through their receptors, the results of our experiments showing that quinpirol did not affect GnRH-induced ERK activation or gene expression led to some confusion. We could not determine whether the different results were due only to distinctions between normal pituitary cells and {alpha}T3-1 cells. Further studies of this question are ongoing in our laboratory.

The careful regulation of gonadotropin production and secretion are vital to full reproductive function. The secretion of gonadotropins in the female must occur at the right time in concert with other physiological changes that occur in the reproductive axis. Although GnRH has been known to be a key hormone responsible for regulating gonadotropins, subtle control by interaction of many factors would fulfill its functions. Our results using {alpha}T3-1 cells suggest that dopamine D2 receptors within the pituitary gonadotrophs might participate to the control of pituitary gonadotropins.

We have shown the existence of dopamine D2 receptors in gonadotroph {alpha}T3-1 cells, and that gene expression of {alpha} subunits is under the inhibitory control of dopamine D2 receptors. It is highly possible that suppressive effects of dopamine acting through D2 receptors are due to decreases in cAMP production.


   FOOTNOTES
 
1 This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan to H.K. and K.M. and by a grant from the Ministry of Health and Welfare to K.M. Back

2 Correspondence. FAX: 81-853-20-2264; kawa45{at}shimane-med.ac.jp Back

Received: 4 March 2002.

First decision: 21 March 2002.

Accepted: 25 April 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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