Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kanasaki, H.
Right arrow Articles by Miyamoto, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kanasaki, H.
Right arrow Articles by Miyamoto, E.
Agricola
Right arrow Articles by Kanasaki, H.
Right arrow Articles by Miyamoto, E.
Biology of Reproduction 62, 1486-1494 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Involvement of p38 Mitogen-Activated Protein Kinase Activation in Bromocriptine-Induced Apoptosis in Rat Pituitary GH3 Cells1

Haruhiko Kanasakia,b, Kohji Fukunagaa, Kentaro Takahashib, Kohji Miyazakib, and Eishichi Miyamoto2,a

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bromocriptine, a dopamine D2 receptor agonist, is a therapeutic agent for patients with prolactinoma and hyperprolactinemia. In this study we demonstrated that bromocriptine induced activation of p38 mitogen-activated protein (MAP) kinase, with concomitant induction of apoptosis in rat pituitary adenoma cell line GH3 cells. Treatment of GH3 cells for 48 h with bromocriptine increased the p38 MAP kinase activity up to 3- to 5-fold and simultaneously increased the number of apoptotic cells. Inclusion in the medium of SB212090 or SB203580, specific p38 MAP kinase inhibitors, completely abolished the bromocriptine-induced activation of p38 MAP kinase and significantly reduced the number of apoptotic cells. The bromocriptine-induced p38 MAP kinase activation was not prevented by S(-)-eticropride hydrochloride, a specific D2 receptor antagonist. Treatment with either epidermal growth factor (EGF) or thyrotropin-releasing hormone (TRH), which stimulates p44/42 MAP kinase, rescued cells from the bromocriptine-induced apoptosis, with concomitant inhibition of the bromocriptine-induced p38 MAP kinase activation. These results suggest that bromocriptine induces apoptosis in association with p38 MAP kinase activation, and that the p44/42 MAP kinase signaling through EGF and TRH receptors has an opposing effect on p38 MAP kinase activation as well as on apoptosis induced with bromocriptine in GH3 cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bromocriptine, a dopamine D2 receptor agonist, is widely used as a therapeutic agent for patients with prolactinoma, growth hormone (GH)-secreting pituitary adenoma, and adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma by virtue of its ability to inhibit hormone secretion and tumor development. Indeed, stimulation of the dopamine D2 receptor is known to inhibit prolactin secretion through inhibition of cAMP production and/or cytosolic Ca2+ elevation [1, 2]. Furthermore, the cytotoxic and antiproliferative effects of bromocriptine have been reported [3–5]. Ultrastructural studies following bromocriptine administration indicated nuclear pyknosis, aggregated chromatin, and reduction of volume densities of rough endoplasmic reticulum and Golgi apparatus [3, 4]. A significant reduction in the volume of the cytoplasm was observed; atrophy or swelling of mitochondria was also evident [5]. These findings suggest that bromocriptine has cytotoxic and antimitotic effects. However, the mechanisms underlying its cytotoxic and antimitotic effects have not been well understood.

Mitogen-activated protein kinases (MAP kinases), also called extracellular regulated kinases (ERKs), mainly respond to mitogens and growth factors, such as epidermal growth factor (EGF) and platelet-derived growth factor, and regulate cell proliferation and differentiation. Recently, we demonstrated that thyrotropin-releasing hormone (TRH)-stimulated MAP kinase activation is involved in inhibition of proliferation as well as stimulation of prolactin biosynthesis [6]. In contrast, the p38 MAP kinase pathway, like the c-Jun N-terminal kinase (JNK) pathway, is activated in response to cellular stress and inflammation and is involved in many fundamental biological processes. For example, it has been proposed that p38 MAP kinase functions in the regulation of cytokine production [7–9], cell cycle control [10, 11], and apoptosis [12–15]. The subfamily of p38 MAP kinase consists of p38{alpha}, p38ß, p38{gamma}, and p38{delta}. It has been reported that p38{alpha} and p38ß MAP kinase isoforms are involved in apoptosis [14, 15].

Rat pituitary cell line GH3 cells, which synthesize and secrete both prolactin and GH, are a useful model system for the study of hormonal dysfunctions. These hormones have been implicated as having a significant role in human reproduction by modulating gonadotropin secretion [16, 17]. In the present study, we found that bromocriptine induced apoptosis in GH3 cells, with concomitant activation of p38 MAP kinase. The effect of bromocriptine on apoptosis was not mediated through dopamine D2 receptors. In addition, treatment of the cells with EGF or TRH inhibited bromocriptine-induced p38 MAP kinase activation and in turn rescued cells from bromocriptine-induced apoptosis.


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

The following chemicals and reagents were obtained from the indicated sources: fetal calf serum (JRH Biosciences, Lenexa, KS); [{gamma}-32P]ATP (DuPont-New England Nuclear, Wilmington, DE); 2-bromo-{alpha}-ergocryptine and TRH (Sigma Chemical Co., St. Louis, MO); recombinant EGF (Bachem, Budendorf, Switzerland); SB202190 and SB203580 (Calbiochem, San Diego, CA); S(-)-eticropride hydrochloride (Research Biochemical International, Natick, MA); anti-p38 MAP kinase antibody and anti-phospho p38 MAP kinase antibody (New England BioLabs, Beverly, MO); and Ham's F-10 medium (ICN Biomedicals, Tokyo, Japan). Myelin basic protein was purified from bovine brain [18].

Cell Culture

GH3 cells, a rat prolactinoma cell line, were cultured in Ham's F-10 medium containing 15% horse serum, 2.5% fetal calf serum, 50 IU/ml penicillin, and 50 µg/ml streptomycin and maintained at 37°C in an atmosphere of 95% air and 5% CO2 [6]. Two or three days before experiments, 2–3 x 105 cells were plated on a 35-mm Petri dish (Nunc, Roskilde, Denmark). Then the cells were incubated at 37°C for the indicated times without (control) or with the test agents in the serum-containing medium.

Assay for p38 MAP Kinase and ERK Activity

For p38 MAP kinase assay, after GH3 cells were cultured for 3 days they were incubated for the indicated times without (control) or with 10 µM bromocriptine in the medium containing serum. Test agents such as 20 µM SB202190, 20 µM SB203580, and 20 µM S(-)-eticropride hydrochloride (eticropride) were added in the presence and absence of bromocriptine. In the case of ERK assay, GH3 cells that had been cultured for 3 days were preincubated for 60 min at 37°C in Krebs-Ringer Hepes buffer (KRH buffer). After preincubation, the cells were incubated for 10 min with 10 µM bromocriptine, 10 ng/ml EGF, and 1 µM TRH in KRH buffer. After incubation, the medium was aspirated off, and cells were washed three times with PBS and frozen in liquid N2. Frozen GH3 cells were scraped off from the dishes and solubilized in 0.2 ml of 50 mM Hepes (pH 7.4), 0.1% Triton X-100, 4 mM EGTA, 10 mM EDTA, 15 mM Na4P2O7, 100 mM ß-glycerophosphate, 25 mM NaF, 0.1 mM leupeptin, 5 µM pepstatin A, 1 mM dithiothreitol, 1 mM (p-amidinophenyl) methanesulfonyl fluoride hydrochloride (solubilization solution), 1 mM Na3VO4, and 100 nM calyculin A. The procedures for treatment of cells were carried out at 0–4°C. After sonication (Sonifier 250; Branson, Danbury, CT), the insoluble materials were removed by centrifugation at 15 000 x g for 5 min. The extracts were treated with SDS sample buffer [19] and boiled for 1.5 min. Samples containing the same amount of proteins (10–15 µg of protein) were assayed for p38 MAP kinase and ERK by SDS-PAGE containing myelin basic protein (0.5 mg/ml) as a substrate. After electrophoresis, SDS was removed by washing the gel with 20% 2-propanol. Then, after denaturation with 6 M guanidine/HCl and renaturation in a 0.04%-Triton X-100-containing buffer, the gel was preincubated at 30°C for 1 h with 30 ml of 40 mM Hepes (pH 8.0) containing 2 mM dithiothreitol, 10 mM MgCl2, and 0.1 mM sodium orthovanadate. Protein kinase reaction was then performed by incubating the gel at 30°C for 1 h with 30 ml of 40 mM Hepes (pH 8.0) containing 0.5 mM EGTA, 10 mM MgCl2 and 0.1 mM sodium orthovanadate, and 25 µM [{gamma}-32P]ATP (25 µCi) [20, 21]. After the gel was dried, the amount of 32P incorporation into myelin basic protein phosphorylated by ERK or p38 MAP kinase was quantified by a Bio-Imaging analyzer (BA100; Fujifilm, Tokyo, Japan).

Western Immunoblotting

Cells plated in a 35-mm dish were scraped off and solubilized with 100 µl of the solution used in the kinase assays. Aliquots were examined for protein concentration, and the same amount of protein was subjected to SDS-PAGE in 10% acrylamide and transferred to a polyvinylidene fluoride membrane. The membrane was incubated with the anti-p38 MAPK antibody and anti-phospho p38 MAPK antibody, each diluted 1:250. Immunoreactive proteins were visualized with 125I-labeled protein A and horseradish peroxidase-conjugated anti-rabbit antibody, respectively [22].

Immunostaining Procedures

GH3 cells were cultured without (control) or with 10 µM bromocriptine in the serum-containing culture medium. After 48 h, the cells were washed with PBS and fixed at -20°C for 10 min with cold methanol. After 10 min of dehydration at 25°C, the cells were treated with 0.05% Triton X-100 in PBS for permeabilization. Nonspecific antibody binding was blocked by preincubation with 10% goat serum in PBS (blocking solution), followed by incubation overnight with normal rabbit IgG or the rabbit anti-phospho p38 MAP kinase (Thr180/Tyr182) antibody in the blocking solution. The cells were then washed five times with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG in PBS containing 0.01% Triton X-100 for 1 h. Next the cells were stained with propidium iodide for 2 min before mounting and were examined under a confocal laser scanning light microscope (Olympus, Tokyo, Japan) [23].

Quantitation of Apoptosis (TUNEL Procedure)

Three days after plating, the GH3 cells were exposed to 10 µM bromocriptine in serum-containing F-10 medium to examine bromocriptine-induced apoptosis. SB202190 (20 µM) and SB203580 (20 µM) were added as indicated. After 48 h, cells were washed three times with PBS and fixed with 4% formaldehyde in PBS for 25 min at 4°C. Apoptotic cells were detected using the apoptosis detection system, fluorescein (Promega, Madison, WI), which measures the fragmented DNA of apoptotic cells by incorporating fluorescein-12-dUTP at the 3'-OH ends of the DNA by means of the enzyme terminal deoxynucleotidyl transferase, which forms a polymeric tail using the principle of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay [24]. The total number of cells counted was at least 500 per sample in three independent experiments.

Other Procedures

Protein concentration was determined by the method of Bradford [25] with BSA as the standard.

Statistical Evaluation

Each experiment was conducted in at least three independent determinations for cells of separate dishes. The same experiments were repeated at least two to three times. Similar results were obtained from experiment to experiment. Values were expressed as means ± SE. Statistical analysis was performed using one-way ANOVA followed by Duncan's multiple range test. Values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of p38 MAP Kinase by Stimulation with Bromocriptine

Western blot analysis with anti-p38 MAP kinase antibody indicated an immunoreactive band with a molecular mass of 38 kDa in the extract of rat pituitary adenoma cell line GH3 cells, indicating that p38 MAP kinase was present in these cells. There was no quantitative change in the amount of p38 MAP kinase between the control and bromocriptine-treated cells (Fig. 1A). When the p38 MAP kinase activity was assayed by in-gel kinase assay using myelin basic protein as substrate, increased activities of p38 MAP kinase were observed 48 and 72 h after treatment with 10 µM bromocriptine without significant changes in ERK kinase activities. Under the basal conditions, the p38 MAP kinase activity gradually increased during culture. Treatment with 10 µM bromocriptine significantly potentiated p38 MAP kinase 3- to 5-fold compared to the value in controls (Fig. 1, B and C).



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 1. Time course of p38 MAP kinase activation by treatment with bromocriptine. A) Immunoblotting with the anti-p38 MAP kinase antibody using cell extracts from cells treated for 48 h with 10 µM bromocriptine (Bromo) or without (Control). B) An autoradiograph of the time course of bromocriptine-induced p38 MAP kinase activation. Cell extracts from cells treated for indicated times without (Control) or with bromocriptine (Bromo) were prepared. The positions of ERK2 and p38 MAP kinase were determined by immunoblotting analysis with the specific antibodies and are indicated by the arrowheads. C) Time course of bromocriptine-induced activation of p38 MAP kinase. The activity of p38 MAP kinase in the control without bromocriptine was taken as 100%. Values are means ± SE (bars) (n = 3). **P < 0.01 vs. control

Immunostaining Analysis of GH3 Cells with Anti-Phospho p38 MAP Kinase Antibody

We further examined the localization of p38 MAP kinase activated with bromocriptine using anti-phospho p38 MAP kinase antibody, which recognizes phosphorylated Thr180/Tyr182. Western blotting analysis with the phospho-specific antibody revealed that the increase in the phosphorylation of Thr180/Tyr182 was correlated with the bromocriptine-induced p38 MAP kinase activation (Fig. 2A). After treatment of GH3 cells with 10 µM bromocriptine for 48 h, images from the confocal laser scanning light microscope were examined (Fig. 2B). Phase-contrast microscopy showed that apoptotic cells were round and loosely attached to the culture plates of dishes and therefore easily detached from plates (Fig. 2B, a and e). These cells were stained using the TUNEL procedure. Immunocytochemical analysis showed that apoptotic cells were strongly immunostained with the anti-phospho p38 MAP kinase antibody, especially in the cytoplasms (Fig. 2B, b and f). The nuclei of the apoptotic cells were morphologically irregular (data not shown), condensed, and fragmented (Fig. 2B, c and g). Pseudocolored confocal imaging indicated that the margins around the condensed and fragmented nuclei (Fig. 2B, g and h) were in yellow. Therefore, it is not clear that activated p38 MAP kinase was actually localized in the nuclei of apoptotic cells, but the localization of the activated kinase seemed to be observed in the nuclei by pseudocolored confocal image.



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 2. Immunoblotting and images of GH3 cells with light microscope confocal laser scanning. A) Immunoblotting with anti-phospho p38 MAP kinase antibody. GH3 cells were cultured for 48 h without (Control) or with 10 µM bromocriptine (Bromo). Cell extracts were subjected to immunoblot analysis with anti-phospho p38 MAP kinase antibody (diluted 1:250). B) Images of GH3 cells with confocal laser scanning light microscope. GH3 cells were treated for 48 h with 10 µM bromocriptine. a, e) Phase-contrast micrograph. b, f) Immunostaining with FITC-labeled anti-phospho p38 MAP kinase antibody. c, g) Staining of nuclei with propidium iodide. d, h) Pseudocolored confocal image obtained after the images recorded through the FITC and propidium iodide channels were combined. x400

Effects of Specific p38 MAP Kinase Inhibitors on Bromocriptine-Induced p38 MAP Kinase Activation

To further confirm that the increased activities measured by in-gel kinase assay were related to p38 MAP kinase activation, we tested effects of two specific p38 MAP kinase inhibitors, SB202190 and SB203580, on the bromocriptine-induced p38 MAP kinase activation. Bromocriptine induced activation of p38 MAP kinase up to 300% compared to the value in nontreated cells. Both SB202190 and SB203580 completely inhibited bromocriptine-induced p38 MAP kinase activation to control levels (Fig. 3).



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 3. Effects of specific inhibitors on bromocriptine-induced p38 MAP kinase activation. Cells were incubated for 48 h without (Control) or with 10 µM bromocriptine (Bromo) in the presence or absence of 20 µM SB202190 and 20 µM SB203580 in serum-containing medium. A) A representative autoradiograph showing the effects of SB202190 and SB203580 on bromocriptine-induced p38 MAP kinase activation; electrophotograph obtained from the same gel. B) Statistical examination of p38 MAP kinase activities. The activity is expressed as a percentage of the control. Bromocriptine treatment significantly increased p38 MAP kinase activity compared to the control value (P < 0.01). Values are means ± SE (n = 3). **P < 0.01 vs. bromocriptine treatment

Induction of Apoptosis by Stimulation with Bromocriptine and Effects of p38 MAP Kinase Inhibitors

To address whether the bromocriptine-induced p38 MAP kinase activation was related to apoptosis, we assessed apoptotic cells by measurement of DNA fragmentation using TUNEL staining. Under basal conditions, a few cells were TUNEL positive. Treatment with bromocriptine for 48 h significantly increased the number of apoptotic cells (Fig. 4, B and F) compared to the value in nontreated cells (Fig. 4, A and E). High magnification by microscopic analysis revealed that nuclear fragmentation and condensation occurred in cells in which apoptosis was induced by bromocriptine (Fig. 4, C and G). Inclusion of 20 µM SB202190 or SB203580 in the medium significantly reduced the number of apoptotic cells (Fig. 4, D and H). We summarized the ratio of apoptotic cells by TUNEL staining by counting a total of more than 500 cells in different microscopic fields. The percentage of apoptotic cells increased 4-fold following bromocriptine treatment compared to the value in nontreated cells, and the effects of bromocriptine were largely prevented by treatment with either SB202190 or SB203580 (Fig. 5).



View larger version (107K):
[in this window]
[in a new window]
  		FIG. 4. Detection by TUNEL staining of apoptosis induced in cells with bromocriptine and its inhibition with p38 MAP kinase inhibitors. GH3 cells were incubated in serum-containing medium without (Control) (A and E) or with 10 µM bromocriptine (Bromo) (B, C, F, and G). SB202190 (20 µM) (D and H) was added. Left panels show phase-contrast microscopy (A–D); right panels show fluorescence microscopy (E–H). x100 (A, B, D, E, F, and H); x400 (C and G)



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 5. Statistical examination of cells in which apoptosis was induced with bromocriptine. The ratio for the number of apoptotic cells was summarized. Treatment of GH3 cells was as described in the legend to Figure 4. Bromocriptine treatment significantly increased the ratio of apoptotic cells (P < 0.01). The total number of cells counted was at least 500 per sample in three independent experiments. Values are means ± SE in three independent experiments. **P < 0.01 vs. bromocriptine treatment

Effects of a Dopamine D2 Receptor Antagonist on Bromocriptine-Induced p38 MAP Kinase Activation

Inhibition of prolactin release by bromocriptine is known to occur through the dopamine D2 receptor. Thus, we tested the effects of a selective D2 receptor antagonist, eticropride, on p38 MAP kinase activation. Inclusion of 20 µM eticropride in the medium failed to inhibit the p38 MAP kinase activation induced by bromocriptine (Fig. 6). This suggested that the activation of p38 MAP kinase by bromocriptine is not mediated through the dopamine D2 receptor in GH3 cells. In a separate experiment, the effects of 10 µM dopamine on activation of p38 MAP kinase were examined. Incubation of the cells with dopamine for 48 h did not activate p38 MAP kinase (data not shown). These results also suggest that the effect of bromocriptine on p38 MAP kinase activation is not mediated through the dopamine D2 receptor in GH3 cells.



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 6. Effects of a D2 receptor antagonist on bromocriptine-induced p38 MAP kinase activation. Cells were incubated for 48 h without (Control) or with 10 µM bromocriptine (Bromo) in the presence or absence of 20 µM eticropride in serum-containing medium. The activity is expressed as a percentage of the control value. Values are means ± SE (n = 3). *P < 0.05

Effects of EGF and TRH on p38 MAP Kinase Activation and Apoptosis Induced by Bromocriptine

It has been reported that apoptosis is regulated by opposing actions between the ERK and the JNK/p38 MAP kinase pathways [12]. Therefore, we examined the effects of the ERK signaling pathway on bromocriptine-induced apoptosis as well as p38 MAP kinase activation. Recently, we reported that stimulation with TRH and EGF potentially activates ERK, especially ERK2, in GH3 cells and in turn stimulates prolactin biosynthesis [6]. Consistent with the previous results, treatment with either 10 ng/ml EGF or 1 µM TRH increased ERK2 activity by about 500% and 200%, respectively (Fig. 7C). Under the basal conditions, treatment with EGF or TRH alone significantly attenuated the p38 MAP kinase activity (Fig. 7B). The bromocriptine-induced p38 MAP kinase activation was abolished completely by EGF in the incubation medium and partly by TRH (Fig. 7B). The potencies of EGF and TRH with respect to inhibition of p38 MAP kinase activation induced by bromocriptine were related to their abilities with regard to ERK activation. Finally, we tested the effects of EGF and TRH on bromocriptine-induced apoptosis at 48 h. Consistent with the results of their inhibitory effects, treatment with EGF and TRH significantly attenuated the number of apoptotic cells increased by bromocriptine treatment. In contrast to findings with EGF, under basal conditions, treatment with TRH alone significantly reduced the number of apoptotic cells (Fig. 8).



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 7. Inhibition of p38 MAP kinase activity by activation of the ERK signaling pathways. Cells were incubated for 48 h without (Control) or with 10 µM bromocriptine (Bromo) in the presence or absence of 10 ng/ml EGF and 1 µM TRH in serum-containing medium. A) Representative autoradiographs showing the effects of EGF and TRH on bromocriptine-induced p38 MAP kinase activation. B) Statistical examination of p38 MAP kinase activity. The p38 MAP kinase activity in the control was taken as 100%. Bromocriptine treatment significantly increased p38 MAP kinase activity compared to the control value (P < 0.01). C) Effects of EGF and TRH on ERK activation. The activity of ERK in the control was taken as 100%. The difference between bromocriptine treatment and control was not significant. Electrophotograph obtained from the same gel. Values are means ± SE (n = 3). **P < 0.01 vs. bromocriptine treatment



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 8. Effects of EGF and TRH on bromocriptine-induced apoptosis of GH3 cells. GH3 cells were incubated in serum-containing medium without (Control) or with 10 µM bromocriptine (Bromo). Statistical examination of cells in which apoptosis was induced with bromocriptine was performed by counting the number of apoptotic cells. Bromocriptine treatment significantly increased the ratio of apoptotic cells (P < 0.01). The total number of cells counted was at least 500 per sample in three independent experiments. Values are means ± SE in three independent experiments. **P < 0.01, *P < 0.05 vs. bromocriptine treatment


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Bromocriptine is known to inhibit secretion of pituitary hormones such as prolactin and GH by inhibiting adenylate cyclase or decreasing the cytosolic Ca2+ through the dopamine D2 receptor [1, 2]. Bromocriptine has also been used as a therapeutic agent for patients with pituitary hormonal abnormalities including hyperprolactinemia, prolactinoma, acromegaly, and ACTH-secreting adenoma. However, the mechanisms underlying the anti-tumor effects have not been clarified. Johansen et al. [26] showed that prolonged treatment of GH3 cells with bromocriptine significantly reduced the production of prolactin and GH and inhibited cell growth. Treatment with bromocriptine also caused an appearance of vacuoles in the cytosol and condensation of the nucleus and the mitochondria [5]. Furthermore, long-term incubation with bromocriptine caused a cytotoxic effect in GH3 cells [27] and apoptosis in GH-secreting cell line GH1 cells [28], ACTH-secreting cell line AtT-20 cells [29], and normal rat anterior pituitary cells [30]. The present study suggests in part the mechanisms of bromocriptine effects on hormone-secreting tumors in the anterior pituitary gland.

In the present study, we demonstrated bromocriptine-induced apoptosis in GH3 cells by using TUNEL staining methods. These results were consistent with previous observations [28–30]. The bromocriptine-induced apoptosis was closely associated with activation of p38 MAP kinase. It has been proposed that the p38 MAP kinase pathway functions in the regulation of cytokine production [7, 8], proliferation and differentiation of B and T cells [31–33], and apoptosis [12–15, 34–37]. Among these functions, bromocriptine-induced p38 MAP kinase in GH3 cells was possibly implicated in apoptosis induced by bromocriptine, since the selective inhibitors for p38 MAP kinase could rescue the cells from apoptosis. Although bromocriptine-induced p38 MAP kinase activation was totally abolished by the inhibitors, the bromocriptine-induced apoptosis was only partly inhibited with the same concentration of the inhibitors. This suggests that, in addition to the p38 MAP kinase pathway, other unknown pathways are also involved in bromocriptine-induced apoptosis. The subfamily of p38 MAP kinase consists of {alpha}, ß, {gamma}, and {delta} isoforms. Among these, the {alpha} and ß isoforms were inhibited by both SB202190 and SB203580, specific p38 MAP kinase inhibitors. Therefore, it was reported that inhibitors for p38 MAP kinase inhibit apoptosis [35–37]. On the other hand, Nemoto et al. [15] reported that SB202190 induces apoptosis. According to current reports, p38{alpha} isoform induces apoptosis, while p38ß isoform induces hypertrophic responses [14] or prevents apoptosis of cells [15]. Taken together, differences in the effects of inhibitors of p38 MAP kinase on apoptosis may reflect differences in the amounts of the isoforms in the tissues.

There is a controversy about the presence of functional dopamine receptors in GH3 cells [38, 39]. In our experiments, bromocriptine-induced p38 MAP kinase activation was not elicited through the dopamine D2 receptor. This was confirmed by the fact that dopamine itself had no effect on p38 MAP kinase activation or on hormone secretion (data not shown). Dopamine has been reported to have neurotoxic effects and to induce apoptosis in cultured neurons [40–44]. The oxidative stress caused by reactive oxygen species generation has been implicated in the mechanism of neurotoxicity and apoptosis [41–43]. In future studies, we should address whether oxidative stress is underlying the bromocriptine-induced apoptosis.

Recently, Xia et al. [12] reported an opposing effect between ERK and p38 MAP kinase signaling in apoptosis. Here, we confirmed that the ERK signaling elicited by EGF and TRH rescued cells from p38 MAP kinase-induced apoptosis with concomitant inhibition of p38 MAP kinase activity in the bromocriptine-treated cells. We recently demonstrated that TRH-induced ERK activation has functions in the production of prolactin but not in the secretion process [6]. TRH elicited differentiation of GH3 cells, possibly by regulating the expression of prolactin and GH genes. In addition, TRH can inhibit proliferation of GH3 cells [6]. In contrast to EGF treatment, treatment with TRH promoted cell survival under basal conditions (Fig. 8), and inhibitory effects of TRH on the bromocriptine-induced p38 MAP kinase activation were not correlated with potency with regard to the prevention of apoptosis (Figs. 7B and 8). Phosphatidylinositol 3-kinase, which is activated by both the heterotrimeric GTP binding proteins and tyrosine kinase pathways, has been implicated in the survival signals [45, 46]. In this context, in addition to the ERK signaling, other anti-apoptotic signaling such as phosphatidylinositol 3-kinase pathway are possibly implicated in the anti-apoptotic effects elicited by TRH. These findings suggest that TRH physiologically regulates the production and secretion of hormones in pituitary cells and promotes survival in these hormone-secreting cells.

Addition of TRH or EGF increased the secretion of prolactin and decreased the secretion of GH in the medium as previously described [6, 47]. Bromocriptine decreased the secretion of both prolactin and GH in the medium (data not shown). These results were also consistent with earlier observations [26, 27]. Effects of bromocriptine on the secretion were not recovered by addition of SB202190, a specific inhibitor for p38 MAP kinase (data not shown). On the other hand, effects of bromocriptine on apoptosis were inhibited by addition of both EGF and TRH. This would suggest inhibitory and stimulatory effects of prolactin and GH, respectively, on apoptosis. However, effects of prolactin on apoptosis are still controversial [48–51], while those of GH are rather inhibitory [52, 53]. These results suggest that bromocriptine-induced apoptosis is mediated through activation of p38 MAP kinase, but not through secretion of prolactin or GH.


   FOOTNOTES
 
First decision: 15 September 1999.

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; by a Research Grant from the Human Frontier Science Program (K.F. and E.M.); and by the Grant from the Ministry of Health and Welfare (K.M.). Back

2 Correspondence: Eishichi Miyamoto, Department of Pharmacology, Kumamoto University School of Medicine, 2–2–1 Honjo, Kumamoto 860–0811, Japan. FAX: 81 96 373–5078; emiyamot{at}gpo.kumamoto-u.ac.jp Back

Accepted: December 31, 1999.

Received: August 3, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Albert PR, Neve KA, Bunzow JR, Civelli O. Coupling of a cloned rat dopamine-D2 receptor to inhibition of adenylyl cyclase and prolactin secretion. J Biol Chem 1990; 265:2098–2104.[Abstract/Free Full Text]
  2. Malgaroli A, Vallar L, Elahi FR, Pozzan T, Spada A, Meldolesi J. Dopamine inhibits cytosolic Ca2+ increases in rat lactotroph cells. Evidence of a dual mechanism of action. J Biol Chem 1987; 262:13920–13927.[Abstract/Free Full Text]
  3. Hallenga B, Saeger W, Ludecke DK. Necroses of prolactin-secreting pituitary adenomas under treatment with dopamine agonists: light microscopical and morphometric studies. Exp Clin Endocrinol 1988; 92:59–68.[Medline]
  4. Eljarmak D, Lis M, Cantin M, Carriere PD, Collu R. Effects of chronic bromocriptine treatment of an estrone-induced, prolactin-secreting rat pituitary adenoma. Horm Res 1985; 21:160–167.[Medline]
  5. Johansen PW, Clausen OPF, Haug E, Fossum S, Gautvik KM. Effects of bromocriptine on cell cycle distribution and cell morphology in cultured rat pituitary adenoma cells. Acta Endocrinol 1985; 110:319–328.
  6. Kanasaki H, Fukunaga K, Takahashi K, Miyazaki K, Miyamoto E. Mitogen-activated protein kinase activation by stimulation with thyrotropin-releasing hormone in rat pituitary GH3 cell. Biol Reprod 1999; 61:319–325.[Abstract/Free Full Text]
  7. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 1994; 372:739–746.[CrossRef][Medline]
  8. Beyaert R, Cuenda A, Berghe WV, Plaisance S, Lee JC, Haegeman G, Cohen P, Fiers W. The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis in response to tumor necrosis factor. EMBO J 1996; 15:1914–1923.[Medline]
  9. Rincon M, Enslen H, Raingeaud J, Recht M, Zapton T, Su MS, Penix LA, Davis RJ, Flavell RA. Interferon-gamma expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway. EMBO J 1998; 17:2817–2829.[CrossRef][Medline]
  10. Molnar A, Theodoras AM, Zon LI, Kyriakis JM. Cdc42Hs, but not Rac1, inhibits serum-stimulated cell cycle progression at G1/S through a mechanism requiring p38/RK. J Biol Chem 1997; 272:13229–13235.[Abstract/Free Full Text]
  11. Takenaka K, Moriguchi T, Nishida E. Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest. Science 1998; 280:599–602.[Abstract/Free Full Text]
  12. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995; 270:1326–1331.[Abstract/Free Full Text]
  13. Juo P, Kuo CJ, Reynolds SE, Konz RF, Raingeaud J, Davis RJ, Biemann HP, Blenis J. Fas activation of the p38 mitogen-activated protein kinase signaling pathway requires ICE/CED-3 family proteases. Mol Cell Biol 1997; 17:24–35.[Abstract]
  14. Wang Y, Huang S, Sah VP, Ross J Jr, Brown JH, Han J, Chien KR. Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38/mitogen-activated protein kinase family. J Biol Chem 1998; 273:2161–2168.[Abstract/Free Full Text]
  15. Nemoto S, Xiang J, Huang S, Lin A. Induction of apoptosis by SB202190 through inhibition of p38ß mitogen-activated protein kinase. J Biol Chem 1998; 273:16415–16420.[Abstract/Free Full Text]
  16. Steger RW, Chandrashekar V, Zhao W, Bartke A, Horseman ND. Neuroendocrine and reproductive functions in male mice with targeted disruption of the prolactin gene. Endocrinology 1998; 139:3691–3695.[Abstract/Free Full Text]
  17. Chandrashekar V, Bartke A, Coschigano KT, Kopchick JJ. Pituitary and testicular function in growth hormone receptor gene knockout mice. Endocrinology 1999; 140:1082–1088.[Abstract/Free Full Text]
  18. Deibler GE, Martenson RE, Kies MW. Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Prep Biochem 1972; 2:139–165.[Medline]
  19. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685.[CrossRef][Medline]
  20. Geahlen RL, Anostario M Jr, Low PS, Harrison ML. Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 1986; 153:151–158.[CrossRef][Medline]
  21. Gotoh Y, Nishida E, Yamashita T, Hoshi M, Kawakami M, Sakaki H. Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells: identity with the mitogen-activated MAP kinase of fibroblastic cells. Eur J Biochem 1990; 193:661–669.[Medline]
  22. Matsumoto K, Fukunaga K, Miyazaki J, Shichiri M, Miyamoto E. Ca2+/calmodulin dependent protein kinase II and synapsin I-like protein in mouse insulinoma MIN6 cells. Endocrinology 1995; 136:3784–3793.[Abstract]
  23. Fukunaga K, Soderling TR, Miyamoto E. Activation of Ca2+/calmodulin dependent protein kinase II and protein kinase C by glutamate in cultured rat hippocampal neurons. J Biol Chem 1992; 267:22527–22533.[Abstract/Free Full Text]
  24. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119:493–501.[Abstract/Free Full Text]
  25. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248–254.[CrossRef][Medline]
  26. Johansen PW, Haug E, Gautvik KM. Effects of bromocriptine on hormone production and cell growth in cultured rat pituitary cells. Acta Endocrinol 1985; 110:200–206.
  27. Lahteenmaki P, Marrs RP, Schechter J, Zacharias S, Kletzky OA. Hormonal and morphologic effects of bromocriptine on normal rat pituitary and GH3 tumor cells. Am J Obstet Gynecol 1985; 153:349–357.[Medline]
  28. Yin D, Kondo S, Takeuchi J, Morimura T. Induction of apoptosis in rat somatotrophin-secreting pituitary adenoma cells by bromocriptine. Oncol Res 1993; 5:383–387.[Medline]
  29. Yin D, Kondo S, Takeuchi J, Morimura T. Induction of apoptosis in murine ACTH-secreting pituitary adenoma cells by bromocriptine. FEBS Lett 1993; 339:73–75.
  30. Drewett N, Jacobi JM, Willgoss DA, Lloyd HM. Apoptosis in the anterior pituitary gland of the rat: studies with estrogen and bromocriptine. Neuroendocrinology 1993; 57:89–95.[Medline]
  31. Crawley JB, Rawlinson L, Lali FV, Page TH, Saklatvala J, Foxwell BM. T cell proliferation in response to interleukins 2 and 7 requires p38 MAP kinase activation. J Biol Chem 1997; 272:15023–15027.[Abstract/Free Full Text]
  32. Craxton A, Shu G, Graves JD, Saklatvala J, Krebs EG, Clark EA. p38 MAPK is required for CD40-induced gene expression and proliferation in B lymphocytes. J Immunol 1998; 161:3225–3236.[Abstract/Free Full Text]
  33. Sugawara T, Moriguchi T, Nishida E, Takahama Y. Differential roles of ERK and p38 MAP kinase pathways in positive and negative selection of T lymphocytes. Immunity 1998; 9:565–574.[CrossRef][Medline]
  34. Huang S, Jiang Y, Li Z, Nishida E, Mathias P, Lin S, Ulevitch RJ, Nemerow GR, Han J. Apoptosis signaling pathway in T cells is composed of ICE/Ced-3 family proteases and MAP kinase 6b. Immunity 1997; 6:739–749.[CrossRef][Medline]
  35. Yue TL, Ni J, Romanic AM, Gu JL, Keller P, Wang C, Kumar S, Yu GL, Hart TK, Wang X, Xia Z, DeWolf WE Jr, Feuerstein GZ. TL1, a novel tumor necrosis factor-like cytokine, induces apoptosis in endotherial cells. Involvement of activation of stress protein kinases (stress-activated protein kinase and p38 mitogen-activated protein kinase) and caspase-3-like protease. J Biol Chem 1999; 274:1479–1486.[Abstract/Free Full Text]
  36. Horstmann S, Kahle PJ, Borasio GD. Inhibitors of p38 mitogen-activated protein kinase promote neuronal survival in vitro. J Neurosci Res 1998; 52:483–490.[CrossRef][Medline]
  37. Schwenger P, Bellosta P, Vietor I, Basilico C, Skolnik EY, Vilcek J. Sodium salicylate induces apoptosis via p38 mitogen-activated protein kinase but inhibits tumor necrosis factor-induced c-Jun N-terminal kinase/stress-activated protein kinase activation. Proc Natl Acad Sci USA 1997; 94:2869–2873.[Abstract/Free Full Text]
  38. Johnston JM, Wood DF, Read S, Johnston DG. Dopamine regulates D2 receptor gene expression in normal but not in tumorous rat pituitary cells. Mol Cell Biol 1993; 92:63–68.
  39. Missale C, Castelletti L, Boroni F, Memo M, Spano P. Epidermal growth factor induces the functional expression of dopamine receptors in the GH3 cell line. Endocrinology 1991; 128:13–20.[Abstract/Free Full Text]
  40. Coronas V, Feron F, Hen R, Sicard G, Jourdan F, Moyse E. In vitro induction of apoptosis or differentiation by dopamine in an immortalized olfactory neuronal cell line. J Neurochem 1997; 69:1870–1881.[Medline]
  41. Gabby M, Tauber M, Porat S, Simantov R. Selective role of glutathione in protecting human neuronal cells from dopamine-induced apoptosis. Neuropharmacology 1996; 35:571–578.[CrossRef][Medline]
  42. Hoyt KR, Reynolds IJ, Hasting TG. Mechanisms of dopamine-induced cell death in cultured rat forebrain neurones: interactions with and differences from glutamate-induced cell death. Exp Neurol 1997; 143:269–281.[CrossRef][Medline]
  43. Luo Y, Umegaki H, Wang X, Abe R, Roth GS. Dopamine induces apoptosis through an oxidation-involved SAPK/JNK activation pathway. J Biol Chem 1998; 273:3756–3764.[Abstract/Free Full Text]
  44. McLaughlin BA, Nelson D, Erecinska M, Chesselet MF. Toxicity of dopamine to striatal neurons in vitro and potentiation of cell death by a mitochondrial inhibitor. J Neurochem 1998; 70:2406–2415.[Medline]
  45. Wymann MP, Pirola L. Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta 1998; 1436:127–150.[Medline]
  46. Crowder RJ, Freeman RS. Phosphatidylinositol 3-kinase and akt protein kinase are necessary and sufficient for the survival of nerve growth factor dependent sympathetic neurons. J Neurosci 1998; 18:2933–2943.[Abstract/Free Full Text]
  47. Schonbrunn A, Krasnoff M, Westendorf JM, Tashjian AH Jr. Epidermal growth factor and thyrotropin-releasing hormone act similarly on a clonal pituitary cell strain. J Cell Biol 1980; 85:786–797.[Abstract/Free Full Text]
  48. Krumenacker JS, Buckley DJ, Leff MA, McCormack JT, de Jong G, Gout PW, Reed JC, Miyashita T, Magnuson NS, Buckley AR. Prolactin-regulated apoptosis of Nb2 lymphoma cells: pim-1, bcl-2, and bax expression. Endocrine 1998; 9:163–170.[CrossRef][Medline]
  49. Gaytan F, Bellido C, Morales C, Sanchez-Criado JE. Both prolactin and progesterone in proestrus are necessary for the induction of apoptosis in the regressing corpus luteum of the rat. Biol Reprod 1998; 59:1200–1206.[Abstract/Free Full Text]
  50. Leff MA, Buckley DJ, Krumenacker JS, Reed JC, Miyashita T, Buckley AR. Rapid modulation of the apoptosis regulatory genes, bcl-2 and bax by prolactin in rat Nb2 lymphoma cells. Endocrinology 1996; 137:5456–5462.[Abstract]
  51. Matsuyama S, Chang KT, Kanuka H, Ohnishi M, Ikeda A, Nishihara M, Takahashi M. Occurrence of deoxyribonucleic acid fragmentation during prolactin-induced structural luteolysis in cycling rats. Biol Reprod 1996; 54:1245–1251.[Abstract]
  52. Haeffner A, Deas O, Mollereau B, Estaquier J, Mignon A, Haeffner-Cavaillon N, Charpentier B, Senik A, Hirsch F. Growth hormone prevents human monocytic cells from Fas-mediated apoptosis by up-regulating Bcl-2 expression. Eur J Immunol 1999; 29:334–344.[CrossRef][Medline]
  53. Eisenhauer KM, Chun SY, Billig H, Hsueh AJ. Growth hormone suppression of apoptosis in preovulatory rat follicles and partial neutralization by insulin-like growth factor binding protein. Biol Reprod 1995; 53:13–20.[Abstract]



This article has been cited by other articles:


Home page
 Eur J EndocrinolHome page
R. Pivonello, D. Ferone, G. Lombardi, A. Colao, S. W J Lamberts, and L. J Hofland
Novel insights in dopamine receptor physiology
Eur. J. Endocrinol., April 1, 2007; 156(suppl_1): S13 - S21.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
J. Fombonne, S. Reix, R. Rasolonjanahary, E. Danty, S. Thirion, G. Laforge-Anglade, O. Bosler, P. Mehlen, A. Enjalbert, and S. Krantic
Epidermal Growth Factor Triggers an Original, Caspase-independent Pituitary Cell Death with Heterogeneous Phenotype
Mol. Biol. Cell, November 1, 2004; 15(11): 4938 - 4948.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
A. Bahar, D. J. Simpson, S. J. Cutty, J. E. Bicknell, P. R. Hoban, S. Holley, M. Mourtada-Maarabouni, G. T. Williams, R. N. Clayton, and W. E. Farrell
Isolation and Characterization of a Novel Pituitary Tumor Apoptosis Gene
Mol. Endocrinol., July 1, 2004; 18(7): 1827 - 1839.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. O. Velardez, A. H. Poliandri, J. P. Cabilla, C. C. A. Bodo, L. I. Machiavelli, and B. H. Duvilanski
Long-Term Treatment of Anterior Pituitary Cells with Nitric Oxide Induces Programmed Cell Death
Endocrinology, April 1, 2004; 145(4): 2064 - 2070.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
Y. Yamada, H. Yamamoto, T. Yonehara, H. Kanasaki, H. Nakanishi, E. Miyamoto, and K. Miyazaki
Differential Activation of the Luteinizing Hormone {beta}-Subunit Promoter by Activin and Gonadotropin-Releasing Hormone: A Role for the Mitogen-Activated Protein Kinase Signaling Pathway in L{beta}T2 Gonadotrophs
Biol Reprod, January 1, 2004; 70(1): 236 - 243.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
B. Banihashemi and P. R. Albert
Dopamine-D2S Receptor Inhibition of Calcium Influx, Adenylyl Cyclase, and Mitogen-Activated Protein Kinase in Pituitary Cells: Distinct G{alpha} and G{beta}{gamma} Requirements
Mol. Endocrinol., October 1, 2002; 16(10): 2393 - 2404.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
P. R. Albert
G Protein Preferences for Dopamine D2 Inhibition of Prolactin Secretion and DNA Synthesis in GH4 Pituitary Cells
Mol. Endocrinol., August 1, 2002; 16(8): 1903 - 1911.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
N. Ben-Jonathan and R. Hnasko
Dopamine as a Prolactin (PRL) Inhibitor
Endocr. Rev., December 1, 2001; 22(6): 724 - 763.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
E. J. Lee, W. R. Duan, M. Jakacka, B. D. Gehm, and J. L. Jameson
Dominant Negative ER Induces Apoptosis in GH4 Pituitary Lactotrope Cells and Inhibits Tumor Growth in Nude Mice
Endocrinology, September 1, 2001; 142(9): 3756 - 3763.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
T. Yonehara, H. Kanasaki, H. Yamamoto, K. Fukunaga, K. Miyazaki, and E. Miyamoto
Involvement of Mitogen-Activated Protein Kinase in Cyclic Adenosine 3',5'-Monophosphate-Induced Hormone Gene Expression in Rat Pituitary GH3 Cells
Endocrinology, July 1, 2001; 142(7): 2811 - 2819.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
I. Munir, K. Fukunaga, H. Kanasaki, K. Miyazaki, T. Ohba, H. Okamura, and E. Miyamoto
Expression of Cyclooxygenase 2 by Prostaglandin E2 in Human Endometrial Adenocarcinoma Cell Line HEC-1B
Biol Reprod, September 1, 2000; 63(3): 933 - 941.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kanasaki, H.
Right arrow Articles by Miyamoto, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kanasaki, H.
Right arrow Articles by Miyamoto, E.
Agricola
Right arrow Articles by Kanasaki, H.
Right arrow Articles by Miyamoto, E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS