Estradiol Enhances the Neurotoxicity of Glutamate in GT1–7 Cells Through an Estrogen Receptor-Dependent Mechanism

NeuroToxicology 24 (2003) 65–73

Estradiol Enhances the Neurotoxicity of Glutamate in GT1–7 Cells Through an Estrogen Receptor-Dependent Mechanism Rei-Cheng Yang1, Huei-Chuan Shih2, Hseng-Kuang Hsu1, Hwei-Chiu Chang1, Chin Hsu1,* 1

Department of Physiology, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan, ROC 2 School of Nursing, Mei-Ho Institute of Technology, Pingtung, Taiwan, ROC Received 16 November 2001; accepted 17 July 2002

Abstract Glutamate plays an important role in neuroendocrine regulation of reproduction through acting on the N-methyl-Daspartate receptor (NMDAR) in the preoptic area (POA). However, a larger dose of glutamate is neurotoxic. Estradiol (E2) increases the responsiveness of neurons to glutamate through activation and/or expression of NMDAR. In order to investigate whether estradiol modulates the neurotoxic effect of glutamate on the neurons through estrogen receptor (ER), immortalized GT1–7 cells, which simultaneously express ER and NMDAR were used. Tamoxifen and ICI 182,780, ER antagonist, were used to investigate whether the ER is involved in the effect of estradiol on glutamate-induced neurotoxicity. MK-801, a NMDAR antagonist, was used to confirm the enhancement of NMDAR-mediated neurotoxicity by estradiol. Neurotoxicity was evaluated by cell viability and LDH efflux. Cell death was observed by flow cytometry and DNA fragmentation. The results showed that: (1) estradiol (10 nM, incubated for 3 days) significantly enhanced the glutamate-induced neuronal death; (2) the percentages of necrosis and apoptosis were elevated after glutamate treatment, and estradiol significantly enhanced the glutamate-induced cell death; (3) glutamate-induced DNA fragmentation was enhanced by E2-pretreatment; (4) the induction of cell death and increase of LDH efflux after glutamate treatment were also enhanced by E2-pretreatment; (5) both the tamoxifen and ICI 182,780 abolished the estradiol-enhanced NMDAR expression and neurotoxicity of glutamate; (6) higher dose of MK-801 (2 mM) was needed in E2-pretreated cells than in non-E2-pretreated group to block the glutamate-induced neurotoxicity. These results suggested that pretreatment of estradiol might enhance the expression of NMDAR and subsequent glutamate-induced neurotoxicity on the GT1–7 cells through an ER-dependent manner. # 2002 Elsevier Science Inc. All rights reserved.

Keywords: Estradiol; NMDA receptor; Cell death

INTRODUCTION Glutamate regulates the reproductive function (Brann and Mahesh, 1992, 1997) through stimulation of GnRH at the hypothalamic level (Saitoh et al., 1991; Hsu et al., 1993). Activation of NMDA receptor, a subtype of glutamate receptor, affects sexual behavior * Corresponding author. Tel.: þ886-7-3121101x2309; fax: þ886-7-3234687. E-mail address: [email protected] (C. Hsu).

and LH surge through stimulation of GnRH release at the preoptic area (POA) (Hsu et al., 1993). However, neonatal rat treated with a pharmacological dose of glutamate exhibit a more severe neuronal damage in POA of male rat than that of females (Hsieh et al., 1997). N-methyl-D-aspartate receptor (NMDAR), which predominantly mediates the neurotoxicity of glutamate (Mody and MacDonald, 1995), is a subtype of ionotropic glutamate receptors and composed of one NR1 and several NR2 subunits. In the developing rat CNS, the NR1 gene is expressed in virtually all neurons,

0161-813X/02/$ – see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 1 - 8 1 3 X ( 0 2 ) 0 0 1 0 8 - 0

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whereas the four NR2 transcripts display distinct expression patterns (Monyer et al., 1994). In comparison with other NR2 subunits, NR2D is a particularly interesting subunit because both its mRNA (Monyer et al., 1994) and protein (Dunah et al., 1996, 1998) are highly expressed in the prenatal and early postnatal brains, suggesting an important role for NMDA receptor containing NR2D subunit in brain development. NMDAR is implicated as a mediator of effects of estradiol on morphological plasticity and related physiological and cognitive processes in the brain (Moriyoshi et al., 1991; Monyer et al., 1992; Sucher et al., 1993; Follesa and Ticku, 1996). Previous reports indicated that estradiol treatment increases the neuronal responsiveness (Weiland, 1992), dendritic spine density (Gould et al., 1990) and synapses via a mechanism dependent on NMDAR activation (Woolly and McEwen, 1992, 1994). Results of intracellular recording also revealed that estradiol treatment increased the duration of NMDAR mediated EPSPs and long-term potentiation (Wong and Moss, 1992; Foy et al., 1999). Recent report indicated that estradiol treatment increased the number of NMDAR binding sites (Woolly et al., 1997) and expression of NR1 subunit protein in the rat hippocampus (Gazzaley et al., 1996). Our recent results showed that the expression of NMDA receptor in POA of neonatal male rats was higher than that of females (Hsu et al., 1999). It is reasonable to suspect that the sex-specific neuronal damage induced by glutamate may be due to estradiol, which is converted from testosterone evoked only in male rats (Weisz and Ward, 1980). In the present study, the immortalized GT1–7 cells which derived from hypothalamic neurons and carrying GnRH promoter (Mellon et al., 1990) and simultaneously express estrogen receptor (ER) and NMDAR (Lawson et al., 1995) were used as a model of hypothalamic neurosecretory neurons in POA in the present study to investigate: (1) Whether estradiol enhances the neuronal death (apoptosis or necrosis) induced by glutamate through affecting the expression of NR1 subunit protein?; (2) Whether estradiol modulates the neurotoxic effect of glutamate on the neurons in an ER-dependent manner?

MATERIALS AND METHODS

GT1–7 Cell Culture GT1–7 cells, generously provided by Dr. Ke-Wen Dong (The Jone Institute for Reproductive Medicine,

East Virginia Medical School), were cultured at 37 8C in a humidified air containing 5% CO2. The culture medium comprised phenol red free Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4.5 mg/ml glucose, 0.6 mg/ml L-glutamate, 100 U/ml penicillin, 100 mg/ml streptomycin, 3.7 mg/ml sodium bicarbonate and 15% fetal bovine serum, which is prestripped of steroid by charcoal (Urbanski et al., 1994). Assessment of Neuronal Cell Viability Cells were plated at 2:0  104 cells/ml. Same number of cells was applied in each well and neuronal feed was replaced with Mg2þ-free Kerbs–Ringer HEPES buffer (KRH) before treatment. KRH buffer contains 128 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 2.7 mM CaCl2, 10 mM glucose, 20 mM HEPES, pH 7.4. In the first part of the experiment, KRH buffer was replaced with KRH buffer without or with 10 nM E2-treatment for 3 days (Takagi and Kawashima, 1993). The reason we choose the 3 days pretreatment of estradiol is because of the simulation purpose of prenatal testosterone peak, which is evoked during prenatal days 17–19 and is converted into estradiol by aromatase (Davis et al., 1996). In the second part of the experiment, co-incubation of E2 and 10 mM tamoxifen (estrogen receptor antagonist) or 50 nM ICI 182,780 for 3 days was performed (Chowen et al., 1992; Nuttall et al., 2000). In the third part of the experiment, co-incubation of E2 and 1 or 2 mM MK-801 for 3 days was performed (Ankarcrona et al., 1995). Then, cells were treated with or without 1.0 mM glutamate for 16 h (Ankarcrona et al., 1995). The adhered cells were counted by a cytometer (CAD-500, Sysmex) after trypsinization and were measured as viable cells. Annexin V and Propidium Iodide (PI) Staining Cells (including the floating cells, which may include the cell populations of necrosis or apoptosis) were harvested and washed with cold incubation buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) and centrifuged at 200  g for 5 min. One million cells were re-suspended in 100 ml of staining solution (2 ml annexin V fluorescein labeling reagent and 2 ml propidium iodide in 100 ml HEPES buffer). The cells were gently mixed and then incubated for 10– 15 min at 20–25 8C in darkness. HEPES buffer (0.8 ml) was added following the incubation and analyzed by flow cytometer using 488 nm excitation and a 516 nm band pass filter for fluorescence detection and a filter >600 nm for PI detection. Twenty thousand of cells were evaluated in each sample. Electronic

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compensation of the instrument was required to exclude overlapping of the two emission spectra (Homburg et al., 1995). Isolation of DNA and Gel Electrophoresis Isolation of DNA was performed according to the manufacturers procedure for apoptotic DNA Ladder Kit (Cat. No. 1835246, Boehringer Mannheim). DNA yield was quantified by measuring the OD 260 of an aliquot of each sample dissolved in distilled water. Gel electrophoresis of the DNA (8 mg per well) was performed at 6 V/cm for 2 h on a 2% agarose gel using a buffer containing 1 mM ethidium bromide, 0.04 M Tris acetate and 1 mM EDTA (pH 7.4). Loading buffer contained 0.25% xylene cyanole FF, 0.25% bromophenol blue, 40% sucrose, and 1 mM ethidium bromide. The gel was visualized by an ultraviolet light fluorescence and photographed using a photo-documentation camera (Patel et al., 1994). Estimation of the Lactate Dehydrogenase (LDH) Efflux

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(Bradford, 1976). Samples were heated for 5 min in boiling water. Equal amounts of protein (15 mg) were separated by 7.5% SDS-polyacrylamide gel. The gels were transferred onto polyvinylidene difluoride (PVDF) membrane by electroblotting for 1 h (100 V), and the membrane was blocked overnight at 4 8C with the Tween–Tris buffer saline solution (t-TBS; 20 mM Tris base, 0.44 mM NaCl, 0.1% Tween 20, pH 7.6) containing 5% non-fat dry milk and 0.1% Tween 20. The blot was incubated with NR1 antibody (monoclonal; PharMingin, San Diego, CA 92121, USA) at a 1:1000 dilution in t-TBS containing 5% non-fat milk and 0.1% Tween 20 for 1 h, then, incubated for 1 h with goat anti-mouse IgG (HRP conjugated, Santa Cruz) diluted to 1:2000 in t-TBS containing 5% nonfat dry milk and 0.1% Tween 20. The blot was finally washed for 1 h with the t-TBS. In addition, b-tubulin (Birkett et al., 1985) was blotted as an internal control. Immunoreactive protein was visualized by enhanced chemiluminescence (ECL, Amersham) according to the manufacturer’s specifications (Siegel et al., 1994). Statistics

LDH bioassay was done by cytotoxicity detection kit (Boehringer Mannheim). GT1–7 cells were plated in 24 wells (2  104 cells per well) containing 0.5 ml DMEM supplemented with 15% FCS and cultured with 10 nM estradiol for 3 days. The medium was changed into DMEM supplemented with 5% FCS and 1.0 mM glutamate for 16 h. The delayed neuronal death was estimated by measuring the LDH efflux by damaged cells into the bathing medium 24 h following the glutamate exposure (Zeevalk and Nicklas, 1994). Assay medium (DMEM supplemented with 5% FCS) was used as low control, and total cell lysate as high control. All the assays were duplicated. cytotoxicity ð%Þ ¼

experiment value  low control  100 high control  low control

Western Blot Analysis of NMDAR The cell membranes were prepared as described previously (Hsu et al., 1999). To each sample, nine volumes of dissecting buffer (50 mM Tris acetate, pH 7.4, 10% sucrose, 5 mM EDTA) was added. After homogenization, the suspension was subsequently centrifuged at 16,000  g for 30 min, then, the resulting pellets were re-suspended, re-homogenized and stored at 70 8C. The protein concentration was estimated using the Bio-Rad protein microassay procedure

The statistical analysis of the data was performed by using 2  2 factorial analysis of variance followed by Scheffe’s test (Steel and Torrie, 1981). A 95% confidence limit was accepted as statistically significant.

RESULTS Effect of Estradiol on Glutamate-Induced Neurotoxicity The result was transferred to 2  2 factorial analysis and showed that the difference between simple effects of glutamate or estradiol treatment was significant. As showed in Fig. 1, glutamate (1 mM) induced the cell death in GT1–7 cells by 29%, while cell number was significantly increased after estradiol treatment. Estradiol pretreatment for 72 h significantly enhanced the glutamate-induced cell death by 58% (P < 0:01). The significant interaction indicated glutamate and estradiol treatments are not independent. After re-examining the simple effects, sum of squares within other factor are all significantly different for the simple effects. It means any simple effect is dependent on the level of the other factor in the experiment.

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Fig. 1. Effect of estradiol (E2) on glutamate-induced cell death. Cells were pretreated with 10 nM E2 for 3 days, followed by 1.0 mM glutamate treatment for 16 h. Cytometry was used to measure cell viability. The data shown indicates mean  S:D: of four samples in each group. Single asterisk indicates P < 0:05; double asterisks indicate P < 0:01.

The result of flow cytometry analysis showed that, only small population of cells showed necrosis and apoptosis (Fig. 2a). After glutamate treatment, both the necrotic and apoptotic cell populations were significantly increased to 24:1  1:2 and 7:9  0:8%, respectively (Fig. 2b). Although the distribution of cell population was unaltered by E2-pretreatment (Fig. 2c), the glutamate-induced necrosis and apoptosis were significantly increased to 32:9  2:3 and 14:1  0:9%, respectively, by E2-pretreatment (Fig. 2d). Estradiol did not cause DNA fragmentation, while DNA integrity caused by glutamate in the E2-pretreatment group was more prominent than that of non-E2pretreated group (Fig. 3). LDH efflux was increased by glutamate treatment and E2-pretreatment significantly (P < 0:01) enhanced the LDH efflux induced by glutamate (Fig. 4).

Fig. 2. Effect of estradiol (E2) on the apoptotic and necrotic cell populations after glutamate treatment: (a) non-treated control; (b) glutamate-treated; (c) E2-pretreated; (d) E2-pretreated combined with glutamate treatment. Cells were pretreated with 10 nM E2 for 3 days and then treated with 1.0 mM glutamate for 16 h. Cells were stained with annexin V fluorescein and propidium iodide, then 20,000 cells were evaluated in each sample. Flow cytometry was used to estimate the cell population. Cell population located in quadrants II and IV indicates necrotic and apoptotic cells, respectively. The data shown was a representative of four independent experiments.

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Fig. 3. Effect of estradiol on DNA fragmentation caused by glutamate. Cells were pretreated with 10 nM E2 for 3 days, then, treated with 1.0 mM glutamate for 16 h. DNA was prepared and separated by 2% agarose gel. The data shown was a representative of four independent experiments.

Effects of Tamoxifen and ICI 182,780 on Estradiol-Enhanced Neurotoxicity of Glutamate The glutamate-induced cell death was significantly enhanced by E2-pretreatment. No effect of tamoxifen (10 mM) or ICI 182,780 was observed on the glutamate-induced cell death. However, the enhancement of glutamate-induced neurotoxicity by estradiol was completely reversed by tamoxifen or ICI 182,780 co-incubation (Fig. 5).

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Fig. 4. Effect of estradiol (E2) on the glutamate-induced LDH efflux. GT1–7 cells were pretreated with E2 (10 nM) for 3 days, then treated with 1.0 mM glutamate for 16 h. Data showed were expressed as mean  S:D: of six samples. Statistic significance was evaluated by 2  2 factorial analysis followed by Scheffe’s test. Single asterisk indicates P < 0:05; double asterisks indicate P < 0:01.

Effects of Tamoxifen and ICI 182,780 on the Promotion of NR1 Expression by E2 The result of Western blotting analysis showed that E2-pretreatment significantly (P < 0:05) promoted the expression of the NR1 subunit protein and the enhancing effect of estradiol on NR1 subunit protein expression was abolished by tamoxifen or ICI 182,780 coincubation (Fig. 6).

Fig. 5. Effect of tamoxifen and ICI 182,780 on the enhancement of glutamate-induced cell death by estradiol. Cells were co-incubated with 10 mM tamoxifen (a) or 50 nM ICI 182,780 (b) and 10 nM estradiol for 3 days, followed by 1.0 mM glutamate treatment for 16 h. Cell number was counted by a cytometer. The data were expressed as mean  S:D: of six samples. Double asterisks indicate P < 0:01; N.S. indicates non-significant difference between two groups.

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Fig. 6. Effect of tamoxifen and ICI 182,780 on the estradiol modulated expression of NR1 subunit protein. Cells were co-incubated with 10 mM tamoxifen (a) or 50 nM ICI 182,780 (b) and 10 nM estradiol for 3 days. Then, cells were harvested and 15 mg protein sample was separated by 7.5% SDS-polyacrylamide gel. The expression of NR1 subunit protein (MW ¼ 116 kDa) was semi-quantified by Western blot analysis. The b-tubulin (MW ¼ 56 kDa) was used as an internal control. The data were normalized by b-tubulin and were expressed as mean  S:D: of four samples.

Fig. 7. Dosage of MK-801, a NMDA receptor antagonist, efficiently blocked the glutamate-induced cell death. Cells were co-incubated with or without E2 (10 nM) and MK-801 (1 or 2 mM) for 3 days. Then treated with 1.0 mM glutamate for 16 h. Cell number was counted by a cytometer. The data shown indicate mean  S:D: of four samples in each group. Single asterisk indicates P < 0:05; double asterisks indicate P < 0:01; N.S. indicates no significant difference between two groups.

Dosage of MK-801 Efficiently Blocked the Glutamate-Induced Cell Death in GT1–7 Cells Pretreated With or Without Estradiol The 2  2 factorial analysis of variance was individually performed within non-E2-treated or E2-treated groups. The result showed that glutamate induced a 31% cell death, which was efficiently reversed by 1 mM MK-801 (a non-competitive NMDAR antagonist) coincubation (Fig. 7a). While, when GT1–7 cells were pretreated with 10 nM estradiol for 3 days, a higher dose (2 mM) of MK-801 was needed to reverse the cell death caused by glutamate (Fig. 7b).

DISCUSSION According to the present results, two forms of neuronal cell death (necrosis and apoptosis) occur after exposure to glutamate (1.0 mM), and estradiol enhanced both types of cell death induced by glutamate as showed in Fig. 2. Ankarcrona et al. (1995) indicated that a rapid necrotic cell killing occurs immediately after glutamate exposure and is associated with loss of mitochondrial function and a delayed apoptotic type of neuronal death occurs subsequently in the surviving neurons that recover their mitochondrial function and cellular energy levels (Ankarcrona et al., 1995). A

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growing body of evidence indicated that overstimulation of the NMDA receptor, caused the overload of intracellular Ca2þ (Orrenius et al., 1989, 1992; Mody and MacDonald, 1995), deficiency of cellular Kþ (Perregaux and Gabel, 1994), and subsequently leading to cell death including programmed cell death (Ankarcrona et al., 1995; Finiels et al., 1995; Yu et al., 1997, 1999). Our results showed that estradiol pretreatment significantly promoted the protein expression of NR1 subunit protein (Fig. 6), which is mandatory for channel activity (Moriyoshi et al., 1991; Nakanishi, 1992). Moreover, a higher dose of MK-801 was needed to block the glutamate-induced cell death when GT1–7 cells were pretreated with estradiol (Fig. 7). It suggested that estradiol might enhance the glutamate-induced neurotoxicity through increasing the expression of NR1 subunit protein. It also provides evidence to support our previous suggestion that the sex-specific neuronal damage in preoptic area (POA) of hypothalamus is caused by estradiol through modulating NMDA receptor (Hsu et al., 1999). The appreciation that estrogens are important neurotrophic and neuroprotective factors has grown rapidly (Wise et al., 2001). However, the present result showed that estradiol enhances the glutamate-induced neurotoxicity. Estradiol can act via mechanisms that require classical intracellular receptor that affect transcription, via mechanisms that may involve membrane receptors or channels and/or via mechanisms that include crosstalk between ER and second messenger pathways (Weiland, 1992). In the present results, both the effects of estradiol on NR1 expression and glutamate-induced cell death were blocked by ER antagonist, i.e. tamoxifen and ICI 182,780. It suggested that activation of ER is necessary for enhancement of estradiol on glutamateinduced neurotoxicity. Although some reports showed neuroprotective effects of estrogen on glutamate toxicity (Zaulyanov et al., 1999; Singer et al., 1999), the neuronal protection afforded by estradiol seems to be estrogen receptor independent. Moreover, this neuronal protection was afforded only at a high concentration of estradiol (10 mM, for 20 h), but not in lower concentrations (0.1 mM, for 20 h) (Behl et al., 1995, 1997). In the present study, low concentration of estradiol (10 nM) was used and treated for 3 days. It is possible that, the difference of dosage, exposure duration or signaling pathway of estradiol may contribute to the contradictory effects of estrogen. Accordingly, since there is areaspecific and sex-specific distribution of estrogen receptor (Brown et al., 1988; Yuri and Kawata, 1991), it can be expected that the regulation of NMDA receptor expression by estrogen might be also area-specific

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and sex-specific. Recent report indicated that estradiol functions as a neuroprotective agent or an inducer of apoptosis depending on the estrogen receptorsubtype present in the cell. ERa has a neuroprotective effect, while ERb mediates the induction of neuronal apoptosis (Nilsen et al., 2000). Moreover, effects of estrogen include membrane effect, cytoplasmic interaction and classic nuclear effect (Nadal et al., 2001). Therefore, further detail evaluations of estradiol considering the estrogen trinity will be needed to delineate the molecular and cellular mechanisms responsible for the apoptosis-potentiating effect of estradiol on glutamate-induced neurotoxicity. Except for the modulation of glutamate-induced neurotoxicity by estradiol, the neurotrophic effect of estradiol was observed in the present result as showed in Fig. 1. It showed that cell number was significantly increased after estradiol treatment. Previous report indicated that estradiol affects the development of the hypothalamus, at least in part, by acting as a trophic factor to modulate the number of neurons in the hypothalamus (Chowen et al., 1992). It has been postulated that part of the neurotrophic effects of estradiol on the brain may be mediated by trophic factors, such as insulin-like growth factor I (IGF-I) (Duenas et al., 1996) or brain-derived neurotrophic factor (BDNF) (Liu et al., 2001). Moreover, both estradiol and IGF-I use the ER to mediate their trophic effects on hypothalamic cells (Garcia-Segura et al., 1996). Furthermore, estrogen treatment increased the BDNF both at the levels of gene and protein expression (Liu et al., 2001). Since BDNF markedly potentiated the neuronal death induced by exposure to NMDA (Koh et al., 1995), estradiol may interact with the signaling pathways of IGF-I and/or BDNF leading to increase of neuronal number. However, the trophic effect of estradiol did not overcome the glutamateinduced neuronal cell death in the present result. It suggested that the enhancement of glutamate-induced neurotoxicity by estradiol might be more prominent than the neurotrophic effect of estradiol per se. Recent evidence suggested that the functional NMDAR is probably a pentamer (Premkumar and Auerbach, 1997; Hawkins et al., 1999), containing at least one NR1 subunit and one or more NR2 subunits (Monyer et al., 1992; Buller et al., 1994). Few studies have addressed the mechanisms underlying regulation of NMDAR by E2. Gazzaley et al. indicated that estradiol enhances the function of NMDAR via posttranscriptional regulation of NR1 expression (Gazzaley et al., 1996). However, the post-transcriptional modification by estradiol seems unable to be explained by

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the classical cellular mechanism of steroid hormone regulation in gene transcription. On the other hand, one genomic DNA fragment, corresponding to the putative 30 -untranslated region of NR2D gene, contained at least four half palindromic estrogen responsive elements (Watanabe et al., 1999). Because, the NR2 subunits require the NR1 subunit to form a functional complex (Monyer et al., 1992), estradiol may modulate the expression of NR1 subunit protein indirectly via regulation of NR2D and the assembly of NMDAR. But the exact mechanism needs further investigation. In conclusion, the present result showed that pretreatment of estradiol at 10 nM for 3 days enhanced the glutamate-induced neurotoxicity on the GT1–7 cells through increasing the NR1 subunit protein expression in an estrogen receptor-dependent manner.

ACKNOWLEDGEMENTS This work was supported by The National Science Council, ROC under grant NSC-89-2320-B037-047.

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