Intracellular signaling involved in estrogen regulation of serotonin reuptake

Molecular and Cellular Endocrinology 226 (2004) 33–42

Intracellular signaling involved in estrogen regulation of serotonin reuptake Nina Koldzic-Zivanovic, Patricia K. Seitz, Cheryl S. Watson, Kathryn A. Cunningham, Mary L. Thomas∗ Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031, USA Received 5 May 2004; received in revised form 6 July 2004; accepted 8 July 2004

Abstract 17␤-Estradiol (E2 ) regulates neuronal activity via genomic and rapid, non-genomic mechanisms. The rat serotonergic neuronal cell line (RN46A) was used to investigate the rapid effects of E2 on serotonin (5-HT) reuptake and on potential intracellular signaling pathways. RN46A cells express the serotonin transporter (SERT) and estrogen receptor (ER)␤, but not ER␣. Fifteen minute E2 treatment (10−9 M) decreased 5-HT uptake. Intracellular cAMP levels were not increased by 15 min E2 treatment; however, E2 caused an increase in intracellular Ca2+ levels, with a maximum response within the first minute. The response was E2 specific, since other steroids (17␣-estradiol, testosterone, and progesterone) had no effect. The ER antagonist ICI 182,780 blocked the rapid E2 effects on intracellular Ca2+ levels as did the selective ER modulator tamoxifen. In summary, changes in intracellular Ca2+ levels caused by E2 and mediated through ER␤ may be responsible for observed rapid effects of E2 on SERT activity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Estrogen; Calcium; Serotonin transporter; Non-genomic effects; RN46A cells

1. Introduction The majority of drugs currently used to treat depression (selective serotonin reuptake inhibitors and tricyclic antidepressants) block serotonin (5-HT) reuptake through their actions to inhibit 5-HT uptake via the serotonin transporter (SERT). Dysfunction of SERT-mediated uptake of 5-HT has been implicated in depression [reviewed by Siever et al. (1991)], which exhibits a notable gender difference in its prevalence (Kessler et al., 1993). In females, depression is more common during periods of fluctuating estrogen levels (e.g., peri-menopausal, post-partum, premenstrual) (Hunter et al., 1986; Gotlib et al., 1989; Wittchen et al., 2002).

Abbreviations: 5-HT, serotonin; SERT, serotonin transporter; E2 , 17␤estradiol; ER␣, estrogen receptor ␣; ER␤, estrogen receptor ␤; PR, progesterone receptor; P, progesterone; T, testosterone; 17␣-E2 , 17␣-estradiol; TMX, tamoxifen; ICI, ICI 182,780 ∗ Corresponding author. Tel.: +1 409 772 9641; fax: +1 409 772 9642. E-mail address: [email protected] (M.L. Thomas). 0303-7207/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2004.07.017

Estrogens, including the most active endogenous estrogen, 17␤-estradiol (E2 ), have been shown to play an important role in numerous brain functions [reviewed by McEwen (2002)]. The regulation of these functions has been considered to be primarily mediated through the genomic pathway which involves the interaction of estrogen with a nuclear receptor (either ER␣ or ER␤) [reviewed by Hall et al. (2001)]. However, rapid E2 effects observed in the brain (occurring within seconds to minutes) cannot be attributed to the classical genomic pathway [reviewed by McEwen and Alves (1999)]. These rapid, non-genomic effects of E2 , such as changes in Ca2+ currents (Mermelstein et al., 1996; Nikezic et al., 1996; Kurata et al., 2001), activation of MAP kinase (Singer et al., 1999; Singh et al., 1999; Bulayeva et al., 2004), changes in the cAMP pathway (Aronica et al., 1994; Gu and Moss, 1996; Kelly et al., 1999), and activation of PKC (Qiu et al., 2003; Kelly et al., 1999; Cordey et al., 2003) can profoundly affect neuronal survival and transmission [reviewed by Toran-Allerand et al. (1999) and Joels (1997)].

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Genomic effects of estradiol (E2 ) on the serotonergic system in the brain and specifically on the SERT have been primarily studied using in vivo models [reviewed by Bethea et al. (2002a)]. It has been shown that chronic E2 treatment of ovariectomized animals downregulates SERT mRNA levels (Pecins-Thompson et al., 1998; Bethea et al., 2002b; Zhou et al., 2002), but increases [3 H]5-HT uptake (Lu et al., 2003; Rehavi et al., 1987) in both primates and rodents. However, in vivo models are not suitable for addressing the molecular events involved in rapid regulation of 5-HT uptake by E2 . RN46A cells, derived from rat embryonal raphe neurons (White et al., 1994), endogenously express SERT and ER␤, but not ER␣ (Bethea et al., 2003). The goals of our study were to: 1) determine whether E2 rapidly regulates SERT function and 2) investigate what intracellular signaling pathways are involved in E2 regulation of SERT. Using this cell model system, we demonstrated that 15-minute treatment with E2 decreases SERT activity, and that E2 rapidly increases intracellular Ca2+ , but does not increase cAMP levels.

mouse monoclonal antibody (ER␤-14C8, GeneTex, San Antonio, TX) or anti-rat SERT rabbit antibody (ImmunoStar Inc., Hudson, WI) were applied to the cells in a 1:500 dilution with 0.1% HIGS in DPBS and incubated overnight at 4 ◦ C. The slides were then washed three times with DPBS over 1 h. Primary antibody binding was revealed by incubation of the sections with a 1:1000 dilution of secondary antibodies: Alexa Fluor 488 goat anti-mouse IgG or Alexa Fluor 555 goat anti-rabbit IgG (Molecular Probes, Eugene, OR) made up in antibody diluent (0.1% HIGS in DPBS) for 1 h at room temperature. The cells were washed again for 1 h with five changes of DPBS and mounted in the aqueous mounting medium, Fluoromount G (Electron Microscopy Sciences, Hatfield, PA), coverslipped and sealed with nail polish. Cells were viewed with an Olympus BX51 fluorescent microscope (Melville, NY) equipped with a digital camera, and imaged using Simple PCI imaging software (Compix, Inc., Cranberry Township, PA). For double labeling, the two individual color images were digitally overlaid. 2.3. [3 H] 5-HT uptake

2. Materials and methods 2.1. Cells RN46A cells, derived from embryonic day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV 40 large T antigen (White et al., 1994), were kindly provided by Dr. Scott Whittemore, School of Medicine, University of Louisville. For all experiments, the cells were grown to 50% density in phenolred free 1:1 solution of DMEM/F12 (GIBCO, Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 100 U/ml penicillin, 100 ␮g/ml streptomycin (GIBCO, Invitrogen, Carlsbad, CA) for 24 h at 33 ◦ C in 5% CO2 . For the next 24 h, the cells were maintained in serum-free conditions: B27 supplement (GIBCO, Invitrogen, Carlsbad, CA) in DMEM/F12 with 1 ␮M 5-HT, 100 U/ml penicillin and 100 ␮g/ml streptomycin at 33 ◦ C in 5% CO2 . All experiments were carried out in serum-free conditions (B27/DMEM/F12 + 5-HT) with cells between passage numbers 20 and 30.

Cells were plated in 24-well plates and maintained in serum-free conditions for 24 h, as described earlier. The uptake medium consisted of B27/DMEM/F-12 containing 1 ␮M 5-HT with 100 ␮M pargyline and 100 ␮M Na-ascorbate. The cells were preincubated for 30 min in uptake medium with or without 100 ␮M imipramine before initiating uptake by the addition of 1 ␮M [3 H]5-HT (27.1 Ci/mmol, Perkin Elmer Life Sciences Inc., Boston, MA). During the uptake experiment, the cells were treated with vehicle (ethyl alcohol, final concentration 0.01%), specified concentrations of 17␤estradiol (range 10−11 to 3 × 10−9 M) or the ER antagonist ICI 182,780 (10−8 M). Uptake was allowed to proceed for 15 min at 37 ◦ C and was stopped by three washes with 1 ml/well of cold DPBS. The cells were then hydrolyzed with 1 M NaOH, and the hydrolysate from each well was analyzed for 3 H and for protein (Bio-Rad Protein Assay, Bio-Rad Laboratories, Hercules, CA) according to Bradford (1976). Specific uptake was calculated as the difference between total uptake and uptake in the presence of imipramine; data are presented as cpm/␮g protein.

2.2. Immunofluorescent staining 2.4. cAMP measurements For immunofluorescent staining, RN46A cells were grown in four-well chambered slides (Nalge Nunc International, Naperville, IL). Immunostaining was carried out as previously described by Clarke et al. (2000). Briefly, cells were washed twice in Dulbecco’s phosphate-buffered saline (DPBS), fixed in acetone at −20 ◦ C for 5 min, then washed three times with DPBS. Reactive aldehyde groups were minimized by incubating the cells with 50 mM ammonium chloride for 15 min at room temperature. Non-specific binding was blocked by incubating the samples in DPBS containing 4% heat-inactivated goat serum (HIGS, GIBCO, Invitrogen, Carlsbad, CA) for 1 h at room temperature. Anti-human ER␤

Production of cAMP by whole cells was measured as previously described (Seitz et al., 1990). Briefly, the cells were plated in 24-well plates and maintained in serum-free conditions, as described for 5-HT uptake. Levels of cAMP were measured in binding buffer (DMEM/F12 with 1 ␮M 5-HT) containing 1 mM isobutylmethylxanthine to inhibit phosphodiesterase activity. Cells were rinsed twice with 1 ml PBS, and then 250 ␮l of buffer with vehicle or buffer plus specified concentrations of E2 (10−11 to 10−8 M) were added. Forskolin (10−5 M, Sigma, St. Louis, MO) was used as a positive control. After 15 min at room temperature, cells were quickly

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rinsed three times with 1 ml ice-cold PBS, and cellular proteins were precipitated with 1 ml cold 10% tricloroacetic acid (TCA) for later determination of protein concentration (by BioRad). TCA supernatants were frozen at −80 ◦ C until assayed. After neutralization of TCA by addition of excess CaCO3 powder (Tihon et al., 1977), cAMP content was measured by RIA, as previously described (Brooker et al., 1979). Reference standards and unknown samples were acetylated to enhance assay sensitivity; 125 I-cAMP was purchased from Perkin Elmer Life Sciences Inc., Boston, MA. Goat antiserum to cAMP (kindly provided by G.A. Nickols) was used at a final dilution of 1:200,000. Data are presented as fmol cAMP/␮g protein. 2.5. Spectrofluorometry for intracellular calcium detection RN46A cells, grown on round glass coverslips (Fisher Scientific, Pittsburgh, PA), were maintained in serum-free conditions as described for uptake experiments. Intracellular Ca2+ measurements were carried out using the ratiometric method of intracellular Ca2+ imaging. Briefly, the cells were loaded with the Ca2+ indicator Fura-2 acetoxymethyl ester (Fura2 AM, Molecular Probes, Eugene, OR) by incubating them in 5 ␮M Fura-2 AM in B27/DMEM/F12/5-HT for 30 min at room temperature. Coverslips were mounted on an open style recording chamber inserted on a peltier-controlled stage microincubator system (HCMIS and PTC-20, ALA Scientific Instruments Inc., Westbury, NY). Cells were maintained at 33 ◦ C in B27/DMEM/F12/5-HT throughout the experiment. E2 and other chemicals in B27/DMEM/F12/5-HT were added directly to the chamber; no perfusion was used. Indicatorloaded cells were imaged on an inverted microscope (Nikon TE200, Nikon Inc., Melville, NY) using a 40×, 1.3 numerical aperture superfluor oil immersion objective (Nikon) and a cooled digital camera (Coolsnap HQ monochrome 12 bit digital camera, Roper Scientific, Tucson, AZ). Fluorescence excitation was provided by an illumination system (DG4 Illumination System, Sutter Instruments, Novato, CA), which includes a 150 W xenon arc lamp with rapid filter switching and shutter mechanism. Recordings were done using a Fura2 filter set (71000a set, Chroma Technology Corp., Rockingham, VT). At each time point image-pairs were acquired alternating excitation at 340 and 380 nm while recording at a single emission of 510 nm. Image acquisition and processing were done using the software package MetaFluor, Version 5.7 (Universal Imaging Corporation, West Chester, PA) running on a personal computer. After proper background correction, the ratio of the measurements obtained at 340 nm over the corresponding measurements at 380 nm (Ex340/Ex380) was used as an index of intracellular Ca2+ changes (R); no calibration was performed. At each time point (images were taken every 5 s), the data were internally normalized by calculating the ratio
R/R0 , where R0 is a basal ratio, obtained during vehicle treatment, while
R is a ratio at each time point – R0 . Data are presented as mean
R/R0 ± SEM.

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2.6. Statistical analysis Statistical significance in [3 H] 5-HT uptake experiments, cAMP and intracellular Ca2+ measurements was determined using ANOVA followed by Dunnett post-test (InStat, GraphPad, San Diego, CA). Differences at the P < 0.05 level were considered statistically significant. 2.7. Chemicals All drugs were purchased from Sigma (St. Louis, MO) unless stated otherwise. ICI 182,780 was obtained from AstraZeneca Pharmaceuticals (Wilmington, DE). 17␤Estradiol, testosterone, progesterone, 17␣-estradiol, ICI 182,780, and tamoxifen were dissolved in ethanol. The final concentration of vehicle in the medium was 0.1%.

3. Results 3.1. Expression of SERT and ER␤ in RN46A cells Using reverse transcription-polymerase chain reaction (RT-PCR), we determined that RN46A cells express mRNAs for SERT and ER␤ but not for ER␣, progesterone receptor (PR) or dopamine transporter (DAT) (data not shown). This finding was confirmed by immunofluorescent staining (Fig. 1), which demonstrated the presence of SERT and ER␤ proteins (panels A and C, respectively). No immunofluorescence was detected in the absence of the primary antibodies (panels B and D). While the distribution of SERT protein in RN46A cells is relatively uniform throughout the cell (panel A), ER␤ protein is primarily distributed in cell bodies and processes (with the area of more intense staining in the perinuclear region), but not in the nucleus (panel C). Panel E shows SERT and ER␤ double labeling with overlapping signals shown in yellow. This profile of expression (SERT+, ER␤+, ER␣−) was also shown for rat serotonergic raphe neurons (Lu et al., 2001; Alves et al., 1998). 3.2. E2 rapidly inhibits 5-HT uptake Cells were kept in serum-free medium containing 1 ␮M 5-HT for 24 h prior to the experiment. The cells were then treated with either vehicle or the indicated E2 concentrations (10−11 to 10−9 M) for 15 min after which [3 H]5-HT uptake was measured. Specific, SERT-mediated uptake was calculated by subtracting the uptake in the presence of the SERT blocker imipramine from total uptake (uptake in the absence of imipramine). Only 10−9 M E2 significantly inhibited SERT activity (Fig. 2A). Additional experiments using a narrower dose range of E2 showed that neither 3 × 10−10 nor 3 × 10−9 M of E2 had statistically significant effects on 5-HT uptake (Fig. 2B).

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Fig. 1. RN46A cell immunostaining for SERT and ER␤. The cells were kept in serum-free medium (DMEM/F12+B27) with 1 ␮M 5-HT for 24 h before the immunofluorescent staining. (A) The expression of SERT protein. (B) Secondary only Ab, primary Ab for SERT omitted. (C) The expression of ER␤ protein. (D) Secondary only Ab, primary Ab for ER␤ omitted. (E) Double labeling for SERT and ER␤ protein. Yellow indicates co-localization of red SERT and green ER␤ fluorescence.

3.3. ER antagonist ICI 182,780 blocks rapid E2 effects on 5-HT uptake To determine the involvement of ER in rapid E2 effects on 5-HT uptake, we used the ER antagonist ICI 182,780. The cells were kept in serum-free medium containing 1 ␮M

5-HT for 24 h and then treated with vehicle, E2 (10−9 M, 15 min), ICI 182,780 (10−8 M, 45 min), or both E2 and ICI 182,780. [3 H]5-HT uptake was measured as described earlier. As shown in Fig. 3, ICI 182,780 blocked E2 -induced inhibition of 5-HT uptake. ICI 182,780 alone had no effect on 5-HT uptake.

Fig. 2. Effects of 15 min E2 treatment on [3 H]5-HT uptake in RN46A cells. The cells were kept in serum-free medium (DMEM/F12 + B27) with 1 ␮M 5-HT for 24 h before the E2 treatment. [3 H]5-HT uptake was initiated by the addition of 1 ␮M [3 H]5-HT and lasted 15 min. (A) E2 concentrations: 10−11 , 10−10 , 10−9 M. Vehicle (VEH) = 0.1% alcohol. [3 H]5-HT uptake in control (vehicle treated) group = 72.5 ± 7.8 cpm/␮g protein. * P < 0.05, N = 10–11. (B) Narrow range of E2 concentrations: 3 × 10−10 , 10−9 , and 3 × 10−9 M. Vehicle (VEH) = 0.1% alcohol. [3 H]5-HT uptake in control (vehicle treated) group = 119.6 ± 5.61 cpm/␮g protein. ** P < 0.01, N = 9–11.

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Fig. 3. Effects of E2 and ER antagonist ICI 182,780 on [3 H]5-HT uptake in RN46A cells. The cells were kept in serum-free medium (DMEM/F12 + B27) with 1 ␮M 5-HT for 24 h before the E2 treatment. [3 H]5-HT uptake was initiated by the addition of 1 ␮M [3 H]5-HT and lasted 15 min. E2 treatment: 15 min, 10−9 M; ICI 182,780 (ICI) treatment: 45 min, 10−8 M. Vehicle (VEH) = 0.1% alcohol. [3 H]5-HT uptake in control (vehicle treated) group = 45.2 ± 3.5 cpm/␮g protein. * P < 0.05, N = 8–10.

3.4. E2 does not increase intracellular cAMP levels Phosphorylation of SERT has been shown to modulate (decrease) its ability to remove 5-HT from the synaptic space. Both PKA (Ramamoorthy et al., 1998) and PKC have been shown to phosphorylate SERT (Qian et al., 1997; Ramamoorthy et al., 1998). To determine whether E2 treatment causes changes in the cAMP pathway that could be associated with SERT phosphorylation level, intracellular cAMP levels were measured following E2 treatment. The cells were kept in serum-free conditions and with 1 ␮M 5-HT for 24 h before the experiment, identical to the protocol for measuring SERT activity. The cells were treated with vehicle, forskolin (10−5 M, positive control) or E2 (10−11 to 10−8 M) for 15 min. Cellular cAMP content was measured by RIA. Although forskolin significantly increased (approximately 20-fold) intracellular cAMP levels in RN46A cells compared with the vehicle group, none of the E2 treatment groups differed from those treated with vehicle alone (Fig. 4). 3.5. E2 rapidly increases intracellular Ca2+ levels Next, we measured intracellular Ca2+ in RN46A cells following E2 treatment in order to determine whether intracellular Ca2+ might provide a transduction pathway for PKCmediated SERT phosphorylation which in turn could lead to decreased 5-HT uptake (Ramamoorthy et al., 1998; Qian et al., 1997). The cells were kept in serum-free conditions with 1 ␮M 5-HT for 24 h (as for the 5-HT uptake experiments) before loading with the Ca2+ -sensitive indicator Fura-2. Intracellular Ca2+ measurements were carried out using the

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Fig. 4. Effects of 15 min E2 treatment on intracellular cAMP levels in RN46A cells. The cells were kept in serum-free medium (DMEM/F12 + B27) with 1 ␮M 5-HT for 24 h before the E2 treatment. Cellular cAMP content was measured by RIA. E2 concentrations: 10−11 , 10−10 , 10−9 and 10−8 M. Vehicle (VEH) = 0.1% alcohol. Forskolin (FSK, 10−5 M) was used as a positive control. N = 4.

ratiometric method of intracellular Ca2+ imaging. Only cells with stable intracellular Ca2+ levels were used. RN46A cells rapidly responded to E2 by increasing intracellular Ca2+ levels in a cell population-dependent manner. In the predominant cell population (2/3 of the cells), E2 treatment (10−11 to 10−8 M) rapidly increased intracellular Ca2+ levels in a dose dependent manner, with a maximum response within the first minute of E2 treatment (Fig. 5A). The dose-response to E2 during the first minute of treatment is presented as a bar graph in Fig. 5B. A second cell population (1/3 of the cells) also responded to E2 ; however, in this case the response was not dose-dependent (Fig. 6A). The E2 response during the first minute of E2 treatment is also presented in Fig. 6B. 3.6. An increase in intracellular Ca2+ levels in response to E2 is specific to 17␤-estradiol Next we investigated whether the E2 -induced increase in the intracellular Ca2+ levels was 17␤-estradiol-specific. RN46A cells were kept in serum-free conditions with 1 ␮M 5-HT for 24 h before the experiment. Intracellular Ca2+ measurements were carried out as described above. The cells were treated with 10−8 M 17␤-estradiol, progesterone, testosterone, and 17␣-estradiol. Of these hormones, only 17␤-estradiol induced changes in intracellular Ca2+ levels (Fig. 7). 3.7. ER antagonist ICI 182,780 and tamoxifen block intracellular Ca2+ response to E2 To further investigate the pharmacology of the E2 -induced response on Ca2+ in RN46A cells, we tested the effects of a pure ER antagonist, ICI 182,780, and a selective ER modulator (SERM), tamoxifen. The cells were kept in

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Fig. 5. Effects of E2 treatment on intracellular Ca2+ levels in the predominant population of RN46A cells. The cells were kept in serum-free medium (DMEM/F12 + B27) with 1 ␮M 5-HT for 24 h before the E2 treatment. Intracellular Ca2+ measurements were carried out using the ratiometric method of intracellular Ca2+ measurements. E2 concentrations: 10−11 , 10−10 , 10−9 and 10−8 M. Vehicle (VEH) = 0.1% alcohol. 14/21 = no. of responsive cells/total no. of cells. (A) E2 dose-response and time course. (B) The mean
R/R0 during the first minute of treatment. *** P < 0.0001.

Fig. 6. Effects of E2 treatment on intracellular Ca2+ levels in secondary population of RN46A cells. The cells were kept in serum-free medium (DMEM/F12 + B27) with 1 ␮M 5-HT for 24 h before the E2 treatment. Intracellular Ca2+ measurements were carried out using the ratiometric method of intracellular Ca2+ measurements. E2 concentrations: 10−11 , 10−10 , 10−9 and 10−8 M. Vehicle (VEH) = 0.1% alcohol. 7/21 = no. of responsive cells/total no. of cells. (A) E2 response and time course. (B) The mean
R/R0 during the first minute of treatment.

serum-free conditions with 1 ␮M 5-HT for 24 h before the experiment. Intracellular Ca2+ measurements were carried out as described above. Both ICI 182,780 and tamoxifen blocked the intracellular Ca2+ response to E2 , while causing no changes in intracellular Ca2+ levels by themselves (Fig. 8). 3.8. The intracellular Ca2+ response to E2 does not occur under Ca2+ -free conditions

Fig. 7. Effects of E2 and other steroids (progesterone, testosterone, and 17␣estradiol) on intracellular Ca2+ levels in RN46A cells. The cells were kept in serum-free medium (DMEM/F12 + B27) with 1 ␮M 5-HT for 24 h before the experiment. Intracellular Ca2+ measurements were carried out using the ratiometric method of intracellular Ca2+ measurements. 17␤-Estradiol (E2 ), progesterone (P), testosterone (T) and 17␣-estradiol (17␣-E2 ) concentrations: 10−8 M. Vehicle (V) = 0.1% alcohol. 6/18 = no. of responsive cells/total no. of cells.

In order to determine if the E2 -induced intracellular Ca2+ response resulted from influx of extracellular Ca2+ , the experiments were performed in Ca2+ -free medium. As for previous experiments, the cells were kept in serum-free conditions with 1 ␮M 5-HT for 24 h before the experiment. Intracellular Ca2+ measurements were carried out as described above. Ca2+ -free conditions were achieved by adding the Ca2+ chelator EGTA (2 mM) to the medium. EGTA treatment decreased the intracellular Ca2+ levels in RN46A cells and blocked the Ca2+ response to E2 . The intracellular Ca2+ response to

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Fig. 8. Effects of E2 , ER agonist/antagonist (tamoxifen, TMX) and ER antagonist (ICI 182,780) on intracellular Ca2+ levels in RN46A cells. The cells were kept in serum-free medium (DMEM/F12 + B27) with 1 ␮M 5-HT for 24 h before the experiment. Intracellular Ca2+ measurements were carried out using the ratiometric method of intracellular Ca2+ measurements. E2 , tamoxifen (TMX) and ICI 182,780 (ICI) concentrations: 10−8 M. Vehicle (V) = 0.1% alcohol. 6/18 = no. of responsive cells/total no. of cells.

Fig. 9. Effects of Ca2+ -free medium on E2 -induced changes in intracellular Ca2+ levels in RN46A cells. The cells were kept in serum-free medium (DMEM/F12 + B27) with 1 ␮M 5-HT for 24 h before the experiment. Medium additions depicted by “V” and “E2 ” are in normal (Ca2+ -containing) medium. Intracellular Ca2+ measurements were carried out using the ratiometric method of intracellular Ca2+ measurements. E2 concentration: 10−8 M. EGTA concentration: 2 mM. Vehicle (V) = 0.1% alcohol. 11/30 = no. of responsive cells/total no. of cells.

E2 was restored by re-addition of Ca2+ -containing medium (Fig. 9).

4. Discussion The results of our experiments demonstrate that RN46A cells of serotonergic neuronal origin rapidly respond to E2 with distinct patterns of increased intracellular Ca2+ concentrations. These cells also demonstrate decreased 5-HT uptake in response to acute exposure to E2 . These findings are consistent with the hypothesis that changes in intracellular Ca2+ may play a role in the rapid E2 -mediated modulation of 5-HT reuptake in serotonergic neurons.

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We used RN46A cells (which endogenously express SERT and ER␤, but not ER␣ or PR) as an in vitro cell model system of serotonergic neurons to address questions about the rapid regulation of 5-HT uptake by E2 . RN46A cells respond rapidly to E2 treatment by increasing intracellular Ca2+ levels. The maximum response to E2 occurs within the first minute of treatment. The intracellular Ca2+ response to E2 does not occur in a Ca2+ -free environment, indicating that the influx of Ca2+ from the extracellular space is necessary for the E2 -induced increase in intracellular Ca2+ in our cell model system. Additionally, our preliminary data suggest that the L-type calcium channel blocker, nifedipine (10−5 M), also blocks E2 effects on intracellular Ca2+ (data not shown) pointing toward the possibility that E2 -induced activation (either direct or indirect) of calcium channels might be the mechanism of rapid E2 effects on intracellular Ca2+ in RN46A cells. However, additional experiments are needed in order to address this question, as well as the possibility that mobilization of Ca2+ from intracellular stores could also contribute to the observed rapid E2 effects on intracellular Ca2+ . In some other neuronal systems E2 has been shown to rapidly increase intracellular Ca2+ levels releasing it from intracellular stores (Beyer and Raab H, 1998), while rapid E2 effects on calcium channels have been shown to be predominantly inhibitory. For example, E2 rapidly inhibits L-type Ca2+ currents in rat neostriatal neurons (Mermelstein et al., 1996), hippocampal neurons (Kurata et al., 2001), and dorsal root ganglia cells (Chaban et al., 2003) as well as N-type Ca2+ currents in dorsal root ganglia cells (Lee et al., 2002). However, in some non-neuronal cell model systems, such as intestinal epithelial cells (Harvey et al., 2002; Picotto et al., 1996), endometrial cells (Perret et al., 2001), and pancreatic ␤ cells (Nadal et al., 1998) E2 has been shown to rapidly activate calcium channels. The fact that unlike other neuronal cell model systems, RN46A cells express only ER␤ could explain this discrepancy. Interestingly, the Ca2+ response in our cell model system is population-dependent. Our study has shown that one population of RN46A cells responded to E2 in a dose-dependent fashion. However, there was also a population of cells that responded to E2 in a non-dose related manner. This kind of a population-dependent rapid response to E2 also has been observed in other cell model systems, such as GH3 /B6/F10 rat pituitary tumor cells (Watson et al., 1999a) and cultured astrocytes (Chaban et al., 2004). One possible explanation for this phenomenon could be the differences in cell cycle stages and/or degrees of differentiation among the RN46A cells, with different cell stages expressing sufficient variations in components of the signaling cascade to account for the different patterns of intracellular Ca2+ responses to E2 . The Ca2+ response was also 17␤-E2 specific; progesterone, testosterone, and 17␣-estradiol did not alter intracellular Ca2+ levels. Additionally, the ER antagonist ICI 182,780 blocked the rapid E2 effects on both intracellular Ca2+ levels and 5-HT uptake. The effectiveness of the ER antagonist in blocking the E2 effect on the intracellular Ca2+ levels

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indicates an ER-mediated mechanism (in this case ER␤, as RN46A cells do not express ER␣). The finding that tamoxifen blocked the E2 -induced increase in intracellular Ca2+ levels suggests that this SERM compound acts as an ER antagonist in RN46A cells. We have shown that E2 rapidly decreased 5-HT uptake. The observed effect of E2 on SERT activity occurred within 15 min of E2 treatment, presumably through non-genomic effects, since 15 min is not enough time for genomic effects to occur (Shang et al., 2000). Other in vitro studies have also shown that E2 rapidly inhibits SERT activity; for example, E2 caused competitive inhibition of 5-HT uptake in synaptosomal preparations (Michel et al., 1987) and non-competitive inhibition in transfected mouse fibroblasts (Chang and Chang, 1999). However, the E2 concentrations used in both of these studies were in the micromolar range, compared to the 10−9 M used in our study, which is closer to physiologic levels of E2 (Honda et al., 1998). Interestingly, in our study, 10−9 M was the only concentration of E2 that induced a significant inhibition of 5-HT uptake. Such an unusual dose-response curve for non-genomic effects of E2 , with only a very narrow dose range of E2 causing an inhibition has also been demonstrated by E2 -induced prolactin release by GH3 /B6 cells (Watson et al., 1999b). Phosphorylation of SERT is one of the mechanisms shown to modulate its ability to remove 5-HT from the synaptic space, and both PKA (Ramamoorthy et al., 1998) and PKC have been shown to phosphorylate SERT (Qian et al., 1997; Ramamoorthy et al., 1998). However, it is only PKCmediated SERT phosphorylation that causes the removal of the transporter from the plasma membrane and subsequently decreases 5-HT uptake (Ramamoorthy et al., 1998; Qian et al., 1997). Also, it has been shown that Ca2+ /calmodulindependent protein kinase does not phosphorylate SERT (Ramamoorthy et al., 1998). In our cell model system, an E2 -induced rise in intracellular Ca2+ levels could lead to activation of PKC (Battaini, 2001), which would phosphorylate SERT, causing its removal from the plasma membrane, and consequently decreasing 5-HT uptake. Although we have shown that E2 increases intracellular Ca2+ over much broader dose range compared with E2 -induced inhibition of 5-HT uptake, it is possible that increased intracellular Ca2+ provides a “trigger” signal, while downstream signaling events “filter” it, giving as a net result a very narrow dose range of E2 effects on 5-HT uptake. Additionally, there is also the possibility of direct interaction between ER␤ and SERT through protein–protein interaction, as SERT and ER␤ double labeling has shown that the signals for SERT and ER␤ overlap in the cell bodies and the processes, though not in the nuclear region. Moreover, E2 has been shown to rapidly activate PKC in other cell model systems (Kelly et al., 1999; Cordey et al., 2003; Mize and Alper, 2002), providing another explanation for rapid E2 regulation of 5-HT uptake. However, further experiments are needed to address this issue. E2 has been shown to rapidly increase intracellular cAMP levels in a number of neuronal systems (Aronica et al., 1994;

Gu and Moss, 1996; Kelly et al., 1999). However, in our cell model system E2 failed to induce an increase in intracellular cAMP levels. Thus, E2 -induced downregulation of 5-HT uptake does not appear to be mediated via the cAMP pathway in this model. We have shown that E2 can regulate 5-HT uptake nongenomically through ER␤. Our in vitro studies were carried out in a biologically relevant system: RN46A cells endogenously express SERT and ER␤ (as do serotonergic neurons in vivo), and only physiologic levels of E2 were used. We have found that E2 rapidly increases intracellular Ca2+ levels through influx of Ca2+ from extracellular space via a time course and at a dose consistent with a role for E2 to regulate 5-HT uptake. We found no increase in cAMP associated with E2 -mediated regulation of 5-HT uptake. As SERT is the primary means by which serotonergic neurons control extracellular 5-HT levels (O’Reilly and Reith, 1988), it contributes to the strength of the serotonergic transmission by indirectly controlling the number of receptors activated and the number of neurons receiving the signal (Hoffman et al., 1998; Blakely et al., 1994). Thus, the rapid, non-genomic effects of E2 on SERT may be important in the modulation of serotonergic transmission, particularly in women in whom fluctuating estrogen levels may contribute to the higher prevalence of depression.

Acknowledgements We would like to thank Dr. Scott Whittemore for providing RN46A cells and Dr. Leoncio Vergara for his help with Ca2+ measurement experiments. This research was supported by NIDA grants DA11428 and DA00260.

References Alves, S.E., Weiland, N.G., Hayashi, S., McEwen, B.S., 1998. Immunocytochemical localization of nuclear estrogen receptors and progestin receptors within the rat dorsal raphe nucleus. J. Comp. Neurol. 391, 322–334. Aronica, S.M., Kraus, W.L., Katzenellenbogen, B.S., 1994. Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription. Proc. Natl. Acad. Sci. U.S.A. 91, 8517–8521. Battaini, F., 2001. Protein kinase C isoforms as therapeutic targets in nervous system disease states. Pharmacol. Res. 44, 353–361. Bethea, C.L., Lu, N.Z., Gundlah, C., Streicher, J.M., 2002a. Diverse actions of ovarian steroids in the serotonin neural system. Front Neuroendocrinol. 23, 41–100. Bethea, C.L., Lu, N.Z., Reddy, A., Shlaes, T., Streicher, J.M., Whittemore, S.R., 2003. Characterization of reproductive steroid receptors and response to estrogen in a rat serotonergic cell line. J. Neurosci. Meth. 127, 31–41. Bethea, C.L., Mirkes, S.J., Su, A., Michelson, D., 2002b. Effects of oral estrogen, raloxifene and arzoxifene on gene expression in serotonin neurons of macaques. Psychoneuroendocrinology 27, 431–445. Beyer, C., Raab, H., 1998. Nongenomic effects of oestrogen: embryonic mouse midbrain neurones respond with a rapid release of calcium from intracellular stores. Eur. J. Neurosci. 10, 255–262.

N. Koldzic-Zivanovic et al. / Molecular and Cellular Endocrinology 226 (2004) 33–42 Blakely, R.D., De Felice, L.J., Hartzell, H.C., 1994. Molecular physiology of norepinephrine and serotonin transporters. J. Exp. Biol. 196, 263–281. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72, 248–254. Brooker, G., Harper, J.F., Terasaki, W.L., Moylan, R.D., 1979. Radioimmunoassay of cyclic AMP and cyclic GMP. Adv. Cyclic Nucleotide Res. 10, 1–33. Bulayeva, N.N., Gametchu, B., Watson, C.S., 2004. Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways. Steroids 69, 181– 192. Chaban, V.V., Lakhter, A.J., Micevych, P., 2004. A membrane estrogen receptor mediates intracellular calcium release in astrocytes. Endocrinology 10.1210/en, 2004–2149. Chaban, V.V., Mayer, E.A., Ennes, H.S., Micevych, P.E., 2003. Estradiol inhibits ATP-induced intracellular calcium concentration increase in dorsal root ganglia neurons. Neuroscience 118, 941–948. Chang, A.S., Chang, S.M., 1999. Nongenomic steroidal modulation of high-affinity serotonin transport. Biochim. Biophys. Acta 1417, 157–166. Clarke, C.H., Norfleet, A.M., Clarke, M.S., Watson, C.S., Cunningham, K.A., Thomas, M.L., 2000. Perimembrane localization of the estrogen receptor alpha protein in neuronal processes of cultured hippocampal neurons. Neuroendocrinology 71, 34–42. Cordey, M., Gundimeda, U., Gopalakrishna, R., Pike, C.J., 2003. Estrogen activates protein kinase C in neurons: role in neuroprotection. J. Neurochem. 84, 1340–1348. Gotlib, I.H., Whiffen, V.E., Mount, J.H., Milne, K., Cordy, N.I., 1989. Prevalence rates and demographic characteristics associated with depression in pregnancy and the postpartum. J. Consult. Clin. Psychol. 57, 269–274. Gu, Q., Moss, R.L., 1996. 17Beta-estradiol potentiates kainate-induced currents via activation of the cAMP cascade. J. Neurosci. 16, 3620–3629. Hall, J.M., Couse, J.F., Korach, K.S., 2001. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J. Biol. Chem. 276, 36869–36872. Harvey, B.J., Doolan, C.M., Condliffe, S.B., Renard, C., Alzamora, R., Urbach, V., 2002. Non-genomic convergent and divergent signalling of rapid responses to aldosterone and estradiol in mammalian colon. Steroids 67, 483–491. Hoffman, B.J., Hansson, S.R., Mezey, E., Palkovits, M., 1998. Localization and dynamic regulation of biogenic amine transporters in the mammalian central nervous system. Front Neuroendocrinol. 19, 187–231. Honda, H., Isihara, H., Kogo, H., 1998. A variation in acetylcholineinduced relaxation of rat aorta in pregnancy. Physiol. Behav. 65, 409–412. Hunter, M., Battersby, R., Whitehead, M., 1986. Relationships between psychological symptoms, somatic complaints and menopausal status. Maturitas 8, 217–228. Joels, M., 1997. Steroid hormones and excitability in the mammalian brain. Front Neuroendocrinol. 18, 2–48. Kelly, M.J., Lagrange, A.H., Wagner, E.J., Ronnekleiv, O.K., 1999. Rapid effects of estrogen to modulate G protein-coupled receptors via activation of protein kinase A and protein kinase C pathways. Steroids 64, 64–75. Kessler, R.C., McGonagle, K.A., Swartz, M., Blazer, D.G., Nelson, C.B., 1993. Sex and depression in the National Comorbidity Survey. I. Lifetime prevalence, chronicity and recurrence. J. Affect Disord. 29, 85–96. Kurata, K., Takebayashi, M., Kagaya, A., Morinobu, S., Yamawaki, S., 2001. Effect of beta-estradiol on voltage-gated Ca(2+) channels in rat hippocampal neurons: a comparison with dehydroepiandrosterone. Eur. J. Pharmacol. 416, 203–212.

41

Lee, D.Y., Chai, Y.G., Lee, E.B., Kim, K.W., Nah, S.Y., Oh, T.H., Rhim, H., 2002. 17Beta-estradiol inhibits high-voltage-activated calcium channel currents in rat sensory neurons via a non-genomic mechanism. Life Sci. 70, 2047–2059. Lu, H., Ozawa, H., Nishi, M., Ito, T., Kawata, M., 2001. Serotonergic neurones in the dorsal raphe nucleus that project into the medial preoptic area contain oestrogen receptor beta. J. Neuroendocrinol. 13, 839–845. Lu, N.Z., Eshleman, A.J., Janowsky, A., Bethea, C.L., 2003. Ovarian steroid regulation of serotonin reuptake transporter (SERT) binding, distribution, and function in female macaques. Mol. Psychiatry 8, 353–360. McEwen, B.S., 2002. Estrogen actions throughout the brain. Recent Prog. Horm. Res. 57, 357–384. McEwen, B.S., Alves, S.E., 1999. Estrogen actions in the central nervous system. Endocr. Rev. 20, 279–307. Mermelstein, P.G., Becker, J.B., Surmeier, D.J., 1996. Estradiol reduces calcium currents in rat neostriatal neurons via a membrane receptor. J. Neurosci. 16, 595–604. Michel, M.C., Rother, A., Hiemke, C., Ghraf, R., 1987. Inhibition of synaptosomal high-affinity uptake of dopamine and serotonin by estrogen agonists and antagonists. Biochem. Pharmacol. 36, 3175–3180. Mize, A.L., Alper, R.H., 2002. Rapid uncoupling of serotonin-1A receptors in rat hippocampus by 17beta-estradiol in vitro requires protein kinases A and C. Neuroendocrinology 76, 339–347. Nadal, A., Rovira, J.M., Laribi, O., Leon-quinto, T., Andreu, E., Ripoll, C., Soria, B., 1998. Rapid insulinotropic effect of 17beta-estradiol via a plasma membrane receptor. FASEB J. 12, 1341–1348. Nikezic, G., Horvat, A., Nedeljkovic, N., Martinovic, J.V., 1996. 17Betaestradiol in vitro affects Na-dependent and depolarization-induced Ca2+ transport in rat brain synaptosomes. Experientia 52, 217–220. O’Reilly, C.A., Reith, M.E., 1988. Uptake of [3H]serotonin into plasma membrane vesicles from mouse cerebral cortex. J. Biol. Chem. 263, 6115–6121. Pecins-Thompson, M., Brown, N.A., Bethea, C.L., 1998. Regulation of serotonin re-uptake transporter mRNA expression by ovarian steroids in rhesus macaques. Brain Res. Mol. Brain Res. 53, 120–129. Perret, S., Dockery, P., Harvey, B.J., 2001. 17Beta-oestradiol stimulates capacitative Ca2+ entry in human endometrial cells. Mol. Cell Endocrinol. 176, 77–84. Picotto, G., Massheimer, V., Boland, R., 1996. Acute stimulation of intestinal cell calcium influx induced by 17 beta-estradiol via the cAMP messenger system. Mol. Cell Endocrinol. 119, 129–134. Qian, Y., Galli, A., Ramamoorthy, S., Risso, S., DeFelice, L.J., Blakely, R.D., 1997. Protein kinase C activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression. J. Neurosci. 17, 45–57. Qiu, J., Bosch, M.A., Tobias, S.C., Grandy, D.K., Scanlan, T.S., Ronnekleiv, O.K., Kelly, M.J., 2003. Rapid signaling of estrogen in hypothalamic neurons involves a novel G-protein-coupled estrogen receptor that activates protein kinase C. J. Neurosci. 23, 9529–9540. Ramamoorthy, S., Giovanetti, E., Qian, Y., Blakely, R.D., 1998. Phosphorylation and regulation of antidepressant-sensitive serotonin transporters. J. Biol. Chem. 273, 2458–2466. Rehavi, M., Sepcuti, H., Weizman, A., 1987. Upregulation of imipramine binding and serotonin uptake by estradiol in female rat brain. Brain Res. 410, 135–139. Seitz, P.K., Nickols, G.A., Nickols, M.A., McPherson, M.B., Cooper, C.W., 1990. Radioiodinated rat parathyroid hormone-(1–34) binds to its receptor on rat osteosarcoma cells in a manner consistent with two classes of binding sites. J. Bone Miner. Res. 5, 353–359. Shang, Y., Hu, X., DiRenzo, J., Lazar, M.A., Brown, M., 2000. Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 103, 843–852. Siever, L.J., Kahn, R.S., Lawlor, B.A., Trestman, R.L., Lawrence, T.L., Coccaro, E.F., 1991. Critical issues in defining the role of serotonin in psychiatric disorders. Pharmacol. Rev. 43, 509–525.

42

N. Koldzic-Zivanovic et al. / Molecular and Cellular Endocrinology 226 (2004) 33–42

Singer, C.A., Figueroa-Masot, X.A., Batchelor, R.H., Dorsa, D.M., 1999. The mitogen-activated protein kinase pathway mediates estrogen neuroprotection after glutamate toxicity in primary cortical neurons. J. Neurosci. 19, 2455–2463. Singh, M., Setalo Jr., G., Guan, X., Warren, M., Toran-Allerand, C.D., 1999. Estrogen-induced activation of mitogen-activated protein kinase in cerebral cortical explants: convergence of estrogen and neurotrophin signaling pathways. J. Neurosci. 19, 1179–1188. Tihon, C., Goren, M.B., Spitz, E., Rickenberg, H.V., 1977. Convenient elimination of trichloroacetic acid prior to radioimmunoassay of cyclic nucleotides. Anal. Biochem. 80, 652–653. Toran-Allerand, C.D., Singh, M., Setalo Jr., G., 1999. Novel mechanisms of estrogen action in the brain: new players in an old story. Front Neuroendocrinol. 20, 97–121. Watson, C.S., Campbell, C.H., Gametchu, B., 1999a. Membrane oestrogen receptors on rat pituitary tumour cells: immuno-identification and

responses to oestradiol and xenoestrogens. Exp. Physiol. 84, 1013– 1022. Watson, C.S., Norfleet, A.M., Pappas, T.C., Gametchu, B., 1999b. Rapid actions of estrogens in GH3/B6 pituitary tumor cells via a plasma membrane version of estrogen receptor-alpha. Steroids 64, 5–13. White, L.A., Eaton, M.J., Castro, M.C., Klose, K.J., Globus, M.Y., Shaw, G., Whittemore, S.R., 1994. Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons. J. Neurosci. 14, 6744–6753. Wittchen, H.U., Becker, E., Lieb, R., Krause, P., 2002. Prevalence, incidence and stability of premenstrual dysphoric disorder in the community. Psychol. Med. 32, 119–132. Zhou, W., Koldzic-Zivanovic, N., Clarke, C.H., de Beun, R., Wassermann, K., Bury, P.S., Cunningham, K.A., Thomas, M.L., 2002. Selective estrogen receptor modulator effects in the rat brain. Neuroendocrinology 75, 24–33.