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Endocrinology, doi:10.1210/en.2004-0149
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Endocrinology Vol. 145, No. 8 3788-3795
Copyright © 2004 by The Endocrine Society

A Membrane Estrogen Receptor Mediates Intracellular Calcium Release in Astrocytes

Victor V. Chaban, Alexander J. Lakhter and Paul Micevych

Laboratory of Neuroendocrinology of the Brain Research Institute, Department of Neurobiology, Mental Retardation Research Center, and Center for Neurovisceral Sciences and Woman’s Health, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California 90095-1763

Address all correspondence and requests for reprints to: Victor V. Chaban, Department of Neurobiology, David Geffen School of Medicine at University of California, Los Angeles, 10833 Le Conte Avenue, Los Angeles, California 90095-1763. E-mail: chaban{at}ucla.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Appreciating the physiology of astrocytes and their role in brain functions requires an understanding of molecules that activate these cells. Estradiol may influence astrocyte functions. We now report that estrogen altered intracellular calcium concentration ([Ca2+]i) in neonatal astrocytes that expressed estrogen receptor (ER) mRNA in vitro. Western blotting revealed both ER{alpha} and ERß proteins in both the nuclear fractions and plasma-membrane fractions. Application of 17ß-estradiol (20 nM) to fura 2-loaded astrocytes in vitro stimulated [Ca2+]i in 75% of astrocytes with an EC50 of 12.7 ± 3.1 nM. This rapid action of estradiol was blocked by the ER antagonist, ICI 182,780. The membrane-impermeable estradiol-BSA induced a [Ca2+]i flux that was statistically similar to estradiol. Removal of extracellular Ca2+ did not alter the effect of estradiol, but phospholipase C inhibitor U73122 (10 µM) and 2-aminoethoxydiphenyl borate (5 µM), an inhibitor of the inositol-1,4,5,-trisphosphate-gated intracellular Ca2+ channel, significantly decreased the estradiol-induced [Ca2+]i flux. Estradiol was unable to induce [Ca2+]i flux in thapsigargin-depleted cells. These results indicate that estradiol mediates [Ca2+]i flux in astrocytes through a membrane-associated ER that activates the phospholipase C pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CLASSICALLY, ESTRADIOL BINDS to nuclear receptors that act as ligand-activated transcription factors. Additionally, estradiol activates extranuclear membrane receptors that stimulate intracellular signaling pathways through interaction with G proteins (1, 2). Research into the effects of estradiol on intracellular signaling has largely concentrated on neurons (3, 4), where rapid actions of estradiol activate transcriptionally important signaling cascades and modulate ion channels (5, 6). For example, estrogen attenuates activated L-type voltage-gated Ca2+ channels (7, 8), decreases glutamate-induced rise in intracellular calcium concentration ([Ca2+]i) (9), uncouples opioid receptors from intracellular signaling cascades (10), and modulates inositol-1,4,5,-trisphosphate (IP3) signaling and the release of [Ca2+]i stores (11, 12, 13).

Astrocytes appear to express estrogen receptors (ER) in vivo and in vitro (14, 15) and may mediate a number of estradiol-induced effects in the brain. For example, ERs acting through astrocytes have been implicated in estrogen action on synaptic plasticity and neural repair (16, 17). Moreover, astrocytes may have a central role in sexual differentiation of the brain (18) and in reproduction through modulation of the estrogen-positive feedback in the LH surge (19).

In addition to the transcriptional effects of estradiol via nuclear ERs, estradiol may rapidly activate cells by increasing cytoplasmic [Ca2+]i levels. Astrocyte excitability has a Ca2+ component that is similar to estradiol-induced [Ca2+]i flux in pancreatic and epithelial cells (20, 21), suggesting that rapid effects of estrogen may be mediated by membrane ERs that activate the phospholipase C (PLC) pathway to increase [Ca2+]i flux. Membrane ERs have not been described in astrocytes, but a major form of astrocyte excitability is based on [Ca2+]i transients (22, 23, 24, 25, 26), suggesting that estradiol may also mediate Ca2+-induced intracellular signaling in astrocytes.

The purpose of the present study was to establish the subcellular distribution of ER and to determine whether ER is coupled to the PLC/IP3-signaling cascade in astrocytes. RT-PCR was used to detect ER{alpha} and ERß mRNA in cultured astrocytes. Western blots of membrane fractions from cultures had ER{alpha} and ERß immunoreactivity, indicating that both proteins were present in the membrane fraction. Estradiol stimulated [Ca2+]i transients in astrocyte cultures incubated with the Ca2+-sensitive dye, fura 2. Seventy-five percent of the astrocytes in vitro responded to 17ß-estradiol with a rapid [Ca2+]i transient that was dependent on activation of the IP3-sensitive smooth endoplasmic reticulum Ca2+ channel. In 25% of the estradiol-responsive astrocytes, the initial [Ca2+]i transient was followed by a series of [Ca2+]i spikes. The estradiol effects were stereospecific and inhibited by ER antagonists. These results have been previously reported in a preliminary form (27).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary cell culture
Enriched astrocyte cultures were derived from the Long-Evans rat neonatal cortex (1–2 d old) using a technique modified from McCarthy and de Vellis (28) that favors the survival and proliferation of astrocytes over neurons. Briefly, glial cells dissociated with papain were grown in DMEM/F12 supplemented with 10% fetal calf serum (Globepharm, Deerfield, IL). The initial cortical cells were cultured for 9 d. Periods shorter than this were found to be insufficient for stratification of astrocytes and oligodendrocytes, and longer periods can result in the clustering of astrocytes above the bed layer. Oligodendrocyte precursors (top cells) and microglia were separated from the glia by shaking after 24 h in vitro. This process was repeated until the purity of the cell culture was more than 95% astrocytes, assessed immunocytochemically with the astrocyte markers galactocerebroside and glial fibrillary acidic protein (GFAP) as previously described (29). The remaining 5% of non-GFAP-positive cells could include astrocytes, oligodendrocytes, and microglia but not neurons because neuronal-specific enolase immunocytochemistry did not label any cells in the cultures. For Ca2+ imaging studies, the cultures were grown to confluence and subcultured using an EDTA-trypsin wash. DMEM medium was removed from flasks, and cells were washed twice with 5 ml EDTA-trypsin, 2.5% trypsin solution, and then resuspended in 8 ml DMEM/F12–10% fetal calf serum. Astrocytes were centrifuged for 5 min at 80 x g and plated onto Matrigel (Beckton Dickinson, Franklin Lakes, NJ)-coated, 15-mm coverslips (Life Technologies, Inc., Grand Island, NY). Before experiments, all cells were gently washed with serum- and phenol red-free DMEM/F12 (free DMEM) and then incubated overnight in the same media.

RT-PCR analysis
Total RNA was isolated from several cultures of Long-Evans rat neonatal astrocytes using Trizol Reagent (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s protocol. RNA was further purified using the Absolutely RNA RT-PCR Miniprep Kit (Stratagene, La Jolla, CA) with an addition of a deoxyribonuclease I digestion step to remove genomic DNA. RNA integrity was confirmed by 2% agarose gel electrophoresis. cDNA was synthesized from the total RNA using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) and subjected to PCR using specific primers for ER{alpha} and ERß genes. Primers were as follows: 1) ER{alpha}, sense primer, 5'-TACGAAGTGGGCATGATGAA-3' and antisense primer, 5'-AAGGTTGGCAGCTCTCATGT-3'; and 2) ERß, sense primer, 5'-TATCTCCTCCCAGCAGCAGT-3' and antisense primer, 5'-CTCCAGCAGCAGGTCATACA-3'. Primers were designed using Primer3 software (The Whitehead Institute, Boston, MA), and their specificity was confirmed by a BLAST software-assisted search of a nonredundant nucleotide sequence database (National Library of Medicine, Bethesda, MD). PCR experiments were conducted on iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) using Brilliant SYBR Green QPCR Master Mix (Stratagene). Each cycle consisted of the following three steps: denaturation for 45 sec at 95 C, annealing for 1 min at 56 C, and 1 min of elongation at 72 C. Data were collected in real time during the elongation step after each cycle of the 40-cycle reaction. The dissociation/melting curve analysis performed after the reaction was completed indicated presence of a single product with a melting temperature of 83 C in the samples processed with reverse transcriptase. Negative controls without reverse transcriptase and water controls were included in each reaction. In addition to real-time and melting curve analysis of the reactions, amplified products were separated electrophoretically in 3% agarose gels with ethidium bromide and visualized and photographed under UV light to confirm the proper size and the absence of nonspecific products. As an additional control, samples were purified using Rapid PCR Purification System (Marligen Biosciences, Ijamsville, MD) and sequenced at the University of California, Los Angeles, DNA Sequencing Core Facility.

Western blotting
Western blots were probed with rabbit monoclonal primary antibody to ER{alpha} (clone SP1, Lot no. 9101-50-26; Lab Vision Corp., Fremont, CA) and rabbit polyclonal antibody to ERß (Lot no. 24916; Upstate Biotechnology, Inc, Charlottesville, VA). Membrane proteins were separated from the intracellular proteins using Mem-PER(r) Eukaryotic Membrane Protein Extraction Reagent Kit (Pierce Biotechnology, Rockford, IL). Thirty microliters of each fraction were used for immunoblot analysis. The detection was done using a donkey antirabbit IgG (H+L) secondary antibody (Jackson ImmunoResearch, West Grove, PA) and developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). Both ER{alpha} and ERß staining was blocked by preincubating each antibody with its immunizing peptide as described in the enclosed literature. Plasma membrane fractions were also isolated using differential centrifugation. Briefly, astrocyte cultures were washed three times with PBS, and then cells were homogenized at 2000 rpm in 50 mM Tris-HCl (pH 7.5) containing general-purpose protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO.) Nuclear pellets were collected through low-speed centrifugation. The remaining supernatant was centrifuged at 49,000 x g for 15 min at 4 C. The supernatant, containing nonmembrane proteins, was collected. The pellet was washed with 300 µl of 50 mM Tris containing protease inhibitors and centrifuged at 49,000 x g for 15 min at 4 C to pellet the membranes. Eighty micrograms of total protein from each fraction were blotted onto Invitrolon PVDF Membrane (Invitrogen) and probed with antibodies against ER{alpha} and ERß.

Digital fluorescence videomicroscopy
[Ca2+]i was measured by the ratiometric method (30). Coverslips were mounted in an Attofluor recording chamber, and changes in [Ca2+]i were measured by the Attofluor Ratio Vision Digital Fluorescence Microscopy System (Atto Instruments, Rockville, MD), as previously described (31, 32). After incubation with the fluorescent marker (fura 2, 5 µM; Molecular Probes, Eugene, OR) for 45 min at 37 C, the cells were washed and kept in Hanks’ balanced salt solution (HBSS; Life Technologies) for the length of the experiment. Astrocytes were bathed and perfused (1–5 ml/min) with HBSS buffered with HEPES (20 mM) using a peristaltic pump (Rainin Instrument, Woburn, MA). Water-soluble 17ß-estradiol (cyclodextrin-encapsulated 17ß-estradiol; Sigma), thapsigargin (Sigma), and 1,3,5(10)-estratrien-3,17{alpha}-diol-6-one:BSA (E-6-BSA; Steraloids Inc., Newport, RI) were applied by brief superfusion (~10 sec) of the experimental chamber with HBSS until an increase in [Ca2+]i was detected. A [Ca2+]i increase that exceeded 50 nM was considered a [Ca2+]i transient. ICI 182,780 (Tocris Cookson, Ellisville, MO) and 2-aminoethoxydiphenyl borate (2-APB; Sigma) were added 5 min before the addition of estradiol. In the experiments testing the contribution of extracellular Ca2+, cells were incubated in a Ca2+-free HBSS medium with 10 mM of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (Sigma) replaced the 1.8 mM CaCl2. To demonstrate that a membrane-associated ER induces [Ca2+]i flux, E-6-BSA, filtered through a 3-kDa cut-off filter (Amicon, Beverly, MA) to remove free 17ß-estradiol, was immediately applied to cultured astrocytes by rapid perfusion (8, 33). All experiments were performed at room temperature (20–23 C).

Statistics
Data are presented as means ± SE. The amplitude of [Ca2+]i change was calculated as the difference between baseline concentration and peak response to stimulation. Statistical comparisons were made using the Student’s t test or using ANOVA to compare groups followed by Tukey’s posttest comparisons (GraphPad Prizm, San Diego, CA). Differences of P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of ER{alpha} and ERß in neonatal astrocytes
RT-PCR analysis of astrocytic mRNA demonstrated the expression of ER{alpha} and ERß. Agarose gel electrophoresis of the PCR products indicated the presence of the appropriately sized bands in samples with reverse transcriptase and the absence of amplicons in the samples processed without reverse transcriptase and in water controls (Fig. 1Go). Samples were normalized to 18S rRNA. Real-time RT-PCR analysis determined the threshold cycle to be between cycles 30 and 32 for both ER{alpha} and ERß. RNA samples of ovarian tissue, used for positive control, had a threshold cycle between cycles 25 and 26. Compared with the threshold cycle for ovary, this result suggests that astrocytes have a low level of ER{alpha} and ERß mRNA. Combined GFAP immunocytochemistry and in situ hybridization for ER{alpha} and ERß demonstrates that both ERs are expressed in GFAP-positive astrocytes (Romeo, H., and P. Micevych, unpublished data).



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  		FIG. 1. RT-PCR analysis of ER{alpha} and ERß gene expression in rat neonatal astrocytes. Samples with reverse transcriptase show a band of 138 bp corresponding to the amplicon of ER{alpha} (A) and a band of 145 bp corresponding to ERß (B), which are absent from the same samples processed without the reverse transcriptase and in the water controls. Lane M, 100 bp DNA marker; lanes 1 and 2, sample 1; lanes 3 and 4, sample 2; lanes 5 and 6, sample 3; and lane 7, water control.

 
To further characterize the expression and location of ERs in neonatal astrocytes, plasma membrane proteins were extracted and compared with proteins from cytosol and nuclear fractions. Recombinant ER{alpha} and ERß proteins (Invitrogen) were used as positive controls. Cytosol/nuclear protein fraction and membrane protein fraction were subjected to SDS-PAGE, transferred onto Invitrolon PVDF membrane (Invitrogen), and then stained with 0.5% Ponceau S stain (Acros Organics, Morris Plains, NJ) to confirm efficient protein transfer. Both ER{alpha} and ERß were consistently detected in the membrane and cytosol/nuclear fractions from independently grown astrocyte cultures (n > 4; Fig. 2Go). Similar results were obtained with cytosol/nuclear fractions and plasma membrane fractions collected using the commercially available membrane protein extraction kit, MEM-PER Eucariotic Membrane Protein Extraction Kit (Pierce).



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  		FIG. 2. Western blot analysis of ERs in rat neonatal astrocytes. A, Protein expression of ER{alpha} and ERß is consistent with plasma membrane localization. B, Nonmembrane protein fractions of ER{alpha} and ERß. Each corresponds to an independently grown sample or positive control. Lane 1, positive control [recombinant ER protein (0.1 pmol) ER{alpha}, 67 kDa; ERß, 49 kDa]; lane 2, sample 1; lane 3, sample 2; and lane 4, sample 3. (The nonspecific doublet at 75–77 kDa appears as indicated in the antibody manufacturer’s literature.)

 
Effects of estradiol on [Ca2+]i in neonatal astrocytes in vitro
Approximately 75% of the astrocytes responded to 20 nM 17ß-estradiol stimulation within 10–30 sec of treatment. The initial [Ca2+]i transient peaked at 252.9 ± 18.3 nM (mean ± SEM; n = 53). Although there was variability in the shape of the falling phase, typical [Ca2+]i transients rapidly reached a maximum and then slowly decayed to baseline levels (t1/2 = 21.5 ± 3.6 sec; Fig 3Go). The estradiol-induced [Ca2+]i levels in astrocytes were dose responsive (1 nM, 20 nM, 50 nM, and 100 nM), with an EC50 of 12.7 ± 3.1 nM (n > 10) and a maximum response at 50 nM estradiol. Based on preliminary studies, a 5- to 10-min period between stimuli was sufficient to allow for refilling of the [Ca2+]i stores and to induce a similar [Ca2+]i increase with the estradiol. After a 5- to 10-min wash-out period, the second estradiol-induced [Ca2+]i transient was statistically the same as the first (P < 0.05; Fig. 3AGo). A comparison between the first and second stimuli was used to measure and compare the amplitude of the drug effects as previously described (8).



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  		FIG. 3. Effect of 17ß-estradiol (E2) on [Ca2+]i in astrocytes. A, Representative [Ca2+]i levels in cortical astrocytes stimulated with E2 (20 nM); the arrows in each trace indicate time of addition. The effect of E2 (20 nM) was fully reversible after 10 min of washout. B, In a subpopulation of astrocytes (25%), E2 stimulated Ca2+ spikes. C, Representative trace of [Ca2+]i in astrocytes stimulated with E2 in Ca2+-free medium (in the presence of 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) indicates Ca2+ release from intracellular stores. D, Endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (1 µM), induced [Ca2+]i increase and abolished subsequent E2 (20 nM) stimulation.

 
Estradiol-induced [Ca2+]i transients (Fig. 3Go) were heterogeneous in terms of amplitude, induction, and frequency of oscillations, an observation that is consistent for [Ca2+]i flux induced by other stimuli such as ATP (23) and glutamate (34). In 25% of the estradiol-responsive astrocytes, a single pulse of estradiol induced a series of [Ca2+]i transients composed of two or more [Ca2+]i spikes with an average duration of 3–5 min (Fig. 3BGo). The [Ca2+]i spikes in these oscillations had a periodicity of 57.05 ± 3.9 sec. Of the astrocytes in which estradiol induced oscillations, 70% had successively smaller transients (Fig. 3BGo), and 30% had oscillatory transients that had spikes all of similar amplitude.

Estradiol releases Ca2+ from intracellular stores
To determine whether estradiol-induced [Ca2+]i transients resulted from Ca2+ influx of extracellular sources or from mobilizing intracellular [Ca2+]i stores, the response to estradiol was tested in astrocytes perfused with HBSS containing 10 mM of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid but no Ca2+. The estradiol-induced [Ca2+]i spikes were not significantly different in Ca2+-free media from those in Ca2+-containing media (224.5 ± 22.7 nM, n = 12, Figs. 3CGo and 5AGo vs. 252.9 ± 18.3 nM, n = 53, P > 0.05, Figs. 3AGo and 5AGo, respectively), suggesting that estradiol predominantly mobilized Ca2+ from intracellular stores. However, in Ca2+-free media, there were no [Ca2+]i oscillations, suggesting that the initial [Ca2+]i spikes were dependent on intracellular [Ca2+]i stores and subsequent [Ca2+]i oscillations required extracellular Ca2+ to sustain them (35, 36). Further support for this interpretation was obtained from astrocytes in which thapsigargin (1 µM) depleted intracellular [Ca2+]i stores. After intracellular [Ca2+]i depletion, estradiol did not elicit a [Ca2+]i spike (Fig. 3DGo).



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  		FIG. 5. Pharmacological profile of the effect of estrogen on [Ca2+]i in neonatal cortical astrocytes. A, Stimulation of astrocyte with estradiol (E2) in the presence or absence of extracellular Ca2+ or with E-6-BSA produced a similar [Ca2+]i increase. B, Inhibition of the ER with ICI 182,780 (E2 + ICI) and the IP3-induced Ca2+ channel with 2-APB prevented the E2-induced [Ca2+]i increase. The number of experiments is indicated in each bar. *, Significant difference, P < 0.05.

 
To test whether the PLC/IP3 pathway is involved in the estradiol-induced [Ca2+]i flux, the PLC inhibitor U73122 (37) and IP3 receptor inhibitor 2-APB (38) were used. For this study, we used a double stimulation protocol. When estradiol was applied twice to the astrocytes, there was no difference between the first and second stimulation under control conditions (P < 0.05; Fig. 3AGo). To test the effects of blockers, astrocytes were exposed to estradiol once, as a control, and then a second time to the drug. The PLC inhibitor U73122 (5 min, 10 µM) inhibited the 17ß-estradiol-induced release of [Ca2+]i (71.1 ± 11.9 nM vs. 244.6 ± 30.1 nM with control, n = 7, P < 0.05; Figs. 4AGo and 5BGo). Similarly, when IP3-sensitive Ca2+ channels on smooth endoplasmic reticulum were blocked with 2-APB (5 µM) for 5 min before stimulation with 17ß-estradiol, 2-APB inhibited the estradiol-induced [Ca2+]i transients (85.7 ± 13.6 nM vs. 260.1 ± 25.9 nM with control, n = 10, P < 0.05; Figs. 4BGo and 5BGo). None of the inhibitors alone altered basal Ca2+ level. Taken together these results support the hypothesis that the main source of the initial estradiol-induced [Ca2+]i transient was intracellular and mediated through the PLC/IP3 pathway.



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  		FIG. 4. Estradiol (E2) induces [Ca2+]i flux through the membrane-associated ER by PLC/IP3 activation. A, PLC inhibitor U73122 (10 µM) added for 5 min inhibited subsequent E2 (20 nM) stimulation. B, The inhibitor of IP3-induced Ca2+ release, 2-APB (5 µM, 5 min), inhibited E2-induced [Ca2+]i increase. C, The pure ER antagonist ICI 182,780 (1 µM) inhibited E2-stimulated [Ca2+]i increase. D, E2-induced [Ca2+]i fluxes are mediated by membrane ER. Membrane-impermeable E-6-BSA (100 nM) filtered immediately before use induced a Ca2+ increase that was similar to E2.

 
Estradiol acts via specific membrane-associated ERs
To investigate whether a specific receptor mediated estradiol action, astrocytes were superfused with the inactive stereoisomer 17{alpha}-estradiol (50 nM). No changes in [Ca2+]i flux were observed after 17{alpha}-estradiol treatment. In another set of experiments, 5-min exposure of the pure ER antagonist ICI 182,780 (1 µM) before 17ß-estradiol blocked the increase of [Ca2+]i levels (108.3 ± 15.4 nM, n = 26; Figs. 4CGo and 5Go). These results were consistent with Western blot results, which indicated that ER{alpha} and ERß were associated with the plasma membrane. Further support for a membrane ER was obtained when filtered E-6-BSA (100 nM) rapidly induced [Ca2+]i transients. E-6-BSA-induced Ca2+ spikes were not significantly different in amplitude compared with those induced by 17ß-estradiol alone; however, there was a trend that E-6-BSA might be less effective based on a probability that was very close to significance (198.6 ± 18.2 nM, n = 27 vs. 252.9 ± 18.3 nM, n = 53, P = 0.06; Figs. 4DGo and 5Go). To control for potential BSA effects, 100 nM BSA was applied to the astrocytes. BSA alone did not induce [Ca2+]i flux (data not shown). These data suggest that estradiol, acting on a membrane-associated ER, rapidly stimulated [Ca2+]i flux in astrocytes via a PLC/IP3-sensitive mechanism.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main findings from our experiments are that estradiol directly and rapidly induces [Ca2+]i transients and oscillations in cultured neonatal astrocytes. Our results that astrocytes in vitro express both ER{alpha} and ERß mRNA agreed with previous studies (14, 15, 39, 40), but the current situation extends these earlier studies by showing that both ER{alpha} and ERß proteins are associated with the plasma membrane and can be activated to modulate [Ca2+]i. The membrane ERs were characterized as ER{alpha} and ERß based on Western blotting and by establishing that the effect of estradiol was stereospecific and antagonized by the type I ER blocker ICI 182,780. Additional evidence that the ER-mediating [Ca2+]i flux in astrocytes is associated with the plasma membrane was that E-6-BSA mimicked the effect of 17ß-estradiol.

Western blots of the membrane fraction indicate that the membrane ER in astrocytes is either ER{alpha} or ERß, which is in contrast to a recent report that indicated that the predominant ER isotype located on the plasma membrane of cultured astrocytes was ER{alpha} (15). These membrane receptors appear to be biochemically and pharmacologically similar to classical ERs that have been demonstrated to be associated with the plasma membrane in other cells (reviewed in Refs. 1, 2 , and 41). Moreover, the time course of estradiol activation of [Ca2+]i transients in the present experiments was similar to that of embryonic and fetal neurons, brain synaptosomes, pituitary cells, and GnRH neurons (12, 42, 43, 44). The time course of the present effect, however, was more rapid than that observed for membrane ER-mediated inhibition of the L-type voltage-gated Ca2+ channel in striatal and dorsal root ganglion neurons (7, 8).

Several lines of evidence suggest that the membrane- associated ERs appear to be G protein-coupled (13, 41). First, the estradiol-triggered rise in [Ca2+]i did not require extracellular Ca2+ and was abolished in cells that were depleted of intracellular [Ca2+]i stores by thapsigargin, which suggests that the Ca2+ mobilized by estradiol was released from intracellular stores. Second, the effect of estradiol on [Ca2+]i is dependent on the activation of the PLC pathway and opening of IP3-sensitive Ca2+ channels on the smooth endoplasmic reticulum based on the inhibition of the estradiol-induced [Ca2+]i flux with U73122 and 2-APB. These results are consistent with the idea that the membrane ER{alpha}/ß in astrocytes activates a PLC signaling cascade that generates IP3. The IP3 binds to a receptor mediating the release of Ca2+ from smooth endoplasmic reticulum stores (45). In our experiments, we observed a delay of several seconds, which is consistent with the time needed to generate sufficient IP3 to activate its receptors (35). Thus, estradiol produces a rapid stereospecific rise in [Ca2+]i from intracellular stores upon stimulation of the PLC/IP3 cascade, which probably represents the first physiological cellular response defining further down-stream signaling events such as calcium-dependent protein kinase activation (46, 47).

On the other hand, the present results were not consistent with the antioxidant- (48) or ER-X-mediated actions of estrogen (49). The levels of estradiol (EC50 = 12.7 ± 3.1 nM) in the present study were significantly lower than the dose needed for antioxidant actions (48) but higher than those needed to activate ER-X (49). Neither antioxidant- nor ER-X-mediated events are stereospecific, but the ER-mediating astrocyte [Ca2+]i transients were stereospecific as evidenced by a lack of effect with 17{alpha}-estradiol. Moreover, ER-X and antioxidant effects were reported to be nonreversible by ICI 182,780, whereas the effect of estradiol on [Ca2+]i flux, transients, and oscillations was blocked by this pure ER antagonist.

New insights have been accumulated supporting functions of astrocytes in the regulation of neuronal activity. Astrocytes may regulate neurons by integrating synaptic signals (35, 47, 52) and providing feedback responses based on variations in [Ca2+]i (53). Astrocytes have been reported to control synaptogenesis, and an increase in [Ca2+]i could serve as a mechanism to stabilize new circuitry in the adult brain (50). Neurons and glia in neocortex display coherent activity in membrane depolarization (51). Astrocyte-derived TGF-ß and IGF-I can protect neurons and may be involved in neuroendocrine regulation by sex steroids (16, 17). In addition, estrogen-induced synaptic plasticity depends on astrocytic-neuronal interaction.

Recently, estrogen has been shown to have numerous effects on astrocytes including expression and release of trophic factors (54), modification of cellular morphology, and response to injury (55, 56). The mechanisms of these estradiol actions have not been elucidated, and it is reasonable that some of them are mediated through estradiol-induced increases in [Ca2+]i.

As in luteal cells, estradiol dramatically increases [Ca2+]i and facilitates the synthesis of progesterone (57, 58); one interesting estrogen-astrocyte interaction is the synthesis of the neurosteroid in hypothalamus (19). The source of this estradiol-induced progesterone synthesis is astrocytic (59). It is intriguing to speculate that the increase in neuroprogesterone may be dependent on estradiol-induced [Ca2+]i spikes in astrocytes. This neuroprogesterone has been implicated in the regulation of LH release, the development of the brain, the response to injury, and in learning and memory (19, 60, 61).

Although estradiol can elicit a release of Ca2+ through the formation of IP3 in neurons (12, 62), granulose cells (63), and astrocytes (present study), the predominant effect of estradiol in neurons has been to inhibit L-type voltage-gated Ca2+ channels. This has been demonstrated in neostriatal, hippocampal, and dorsal root ganglia neurons (7, 8, 9, 31, 64). In neurons, estradiol did not activate [Ca2+]i directly, but rather, it attenuated the induced [Ca2+]i flux. Several different stimuli were used including ATP (8) and glutamate (9). The pharmacology of the astrocytic ER and the neuronal ER that mediate rapid [Ca2+]i transients were similar; the receptors were stereospecific and blockable by ICI 182,780. In neurons, however, the [Ca2+]i flux is dependent on extracellular Ca2+, but in astrocytes, estradiol stimulated the release of [Ca2+]i. Several possible explanations can account for these differences. Currently, there is evidence for both membrane-associated ER{alpha} and ERß (65); however, it is possible that in astrocytes ERß may be the predominant ER expressed in the membrane. Thus, rapid action of estradiol may be mediated by different ERs. For example, a hypothesis based on this idea is that ER{alpha} mediates inhibitory effects, and ERß mediates stimulatory responses such as [Ca2+]i flux in astrocytes and the excitability of GnRH cells (5). Another explanation is that ERs may be coupled to different signaling cascades in astrocytes vs. neurons. Currently, neither hypothesis can be formally excluded. Regardless of which ER mediated these actions, the present results demonstrate direct, rapid, and reversible estradiol-mediated [Ca2+]i signaling in astrocytes, supporting the conclusion that there is a mechanism through which estradiol can modulate astrocyte excitability.


   Acknowledgments
 
We thank Dr. J. DeVellis, Ms. R. Cole, and the Mental Retardation Research Center Cell Culture Core for providing astrocytes for this project. We are grateful to Dr. E. Mayer for the use of his imaging system and Drs. K. Sinchak and S. Tiwari-Woodruff for their discussions during the writing of the manuscript.


   Footnotes
 
This work was supported by National Institutes of Health Grant HD 042635.

Abbreviations: 2-APB, 2-Aminoethoxydiphenyl borate; [Ca2+]i, intracellular calcium concentration; E-6-BSA, 1,3,5,(10 )-estratrien-3,17{alpha}-diol-6-one:BSA; ER, estrogen receptor; GFAP, glial fibrillary acidic protein; HBSS, Hanks’ balanced salt solution; IP3, inositol-1,4,5,-trisphosphate; PLC, phospholipase C.

Received February 6, 2004.

Accepted for publication April 28, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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