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. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: J Neurochem. 2008 May 19;106(4):1525–1533. doi: 10.1111/j.1471-4159.2008.05491.x

The roles of membrane estrogen receptor subtypes in modulating dopamine transporters in PC-12 cells

RA Alyea 1, SE Laurence 1, SH Kim 2, BS Katzenellenbogen 3, JA Katzenellenbogen 2, CS Watson 1,*
PMCID: PMC2574842  NIHMSID: NIHMS56845  PMID: 18489713

Abstract

The effects of 17β-estradiol (E2) on dopamine (DA) transport could explain gender and life-stage differences in the incidence of some neurological disorders. We tested the effects of E2 at physiological concentrations on DA efflux in NGF-differentiated rat pheochromocytoma (PC12) cells that express estrogen receptors ERα, ERβ and GPR30, and DA transporter (DAT). DAT efflux was determined as the transporter-specific loss of 3H-DA from preloaded cells; a 9–15 min 10−9M E2 treatment caused maximal DA efflux. Such rapid estrogenic action suggests a nongenomic response, and an E2-dendrimer conjugate (limited to non-nuclear actions) caused DA efflux within 5 mins. Efflux dose-responses for E2 were non-monotonic, also characteristic of nongenomic estrogenic actions. ERα siRNA knockdown abolished E2-mediated DA efflux, while ERβ knockdown did not, and GPR30 knockdown increased E2-mediated DA efflux (suggesting GPR30 is inhibitory). Use of ER-selective agonists/antagonists demonstrated that ERα is the predominant mediator of E2-mediated DA efflux, with inhibitory contributions from GPR30 and ERβ. E2 also caused trafficking of ERα to the plasma membrane, trafficking of ERβ away from the plasma membrane, and unchanged membrane GPR30 levels. Therefore, ERα is largely responsible for nongenomic estrogenic effects on DAT activity.

Introduction

In the central nervous system 17β-estradiol (E2) regulates feedback in hypothalamic and pituitary hormone secretion, and also affects cognition, mood, memory, and mental state (Chakraborty and Gore 2004;Dhandapani and Brann 2002;Dluzen 2000;Sawada et al. 1998a). Many gender-biased neurodegenerative and neuropsychiatric disorders such as Parkinson’s (PD), Alzheimer’s, schizophrenia, (Cho et al. 2003) and drug addictions involve alterations in dopamine (DA)-driven function via modulation of production, packaging, release, and reuptake of dopamine, or selective viability of dopamine transporter (DAT)-bearing cells (Bellgrove et al. 2005;Niznik et al. 1991;Porritt et al. 2005;Yue et al. 2005). The DAT belongs to a family of Na+-Cl− co-transporters whose primary function is to transport DA from the extracellular space into DA neurons resulting in signal termination. Although the incidence of these diseases is more prevalent in males, females become more susceptible during periods of hormone fluctuations (for example in adolescence, premenopausal female cycling, and in peri-menopause), followed by postmenopausal disease levels that resemble male incidence patterns. This further points to the role of E2 in neuroprotection and neuromodulation of dopamine-related activity (McEwen and Alves 1999). Neuroprotective actions of E2 recently have gained a new focus on acute rapid responses to E2 via nongenomic membrane estrogen receptors (mERs) (Cztonkowska et al. 1986;Sawada et al. 1998b;Beyer 1999;Czlonkowska et al. 2003;Zhao and Brinton 2007;Bosse et al. 1997b;Toran-Allerand et al. 2002b), though there is some confusion over whether supraphysiological doses are required for this protection, and little is known about the mechanism and ER subtype involvement in these processes. In addition, chronic E2 treatment increases DAT transporter densities without affecting mRNA levels, also suggesting a nongenomic effect on the DAT (Bosse et al. 1997a).

The classical genomic actions of E2 are initiated by its binding to the nuclear steroid receptors estrogen receptor-α (ERα) or ERβ, followed by dimerization, and subsequent binding to estrogen response elements. Because of the subsequent multiple macromolecular syntheses (RNA and protein) involved, these actions require hours to days. However, E2 can also rapidly affect many cellular signaling responses by binding to mERs [reviewed in (Watson and Gametchu 2003b;Watson et al. 2005)]. ERα and ERβ are differentially expressed in the adult rat brain [reviewed in (Zhang et al. 2002)], and another ER called GPR30, recently identified, is widely expressed in the brain (Brailoiu et al. 2007); however, little is known about the acute effects of E2 on dopamine activity through mERs.

We have previously reported that E2 rapidly inhibits DA uptake through the DAT in NGF-differentiated PC12 cells, which endogenously express three mERs: ERα, ERβ, and GPR30 (Watson et al. 2006a). NGF-differentiated PC12 cells are a well-known cell model to examine DAT function and neuronal cellular regulation. In this current study we demonstrate that E2, through a membrane-initiated event, causes apparent rapid reversal of DAT, resulting in increased extracellular DA. We used an E2-conjugated dendrimer which cannot easily enter cells (and therefore does not bind nuclear ERs) to examine the membrane localization of ERs participating in E2-mediated DA efflux. We also used siRNA technology and subtype-selective agonists and antagonists to examine different ER subtypes’ (ERα, ERβ, and GPR30) contributions. Along with receptor trafficking to sites of action after E2 treatment, our results demonstrate that ERα residing in the plasma membrane primarily mediates E2-induced DA efflux, but that GPR30 and ERβ can exert inhibitory control when multiple ER subtypes are involved.

Materials and Methods

PC12 cell culture

PC12 cells were grown in medium containing modified high-glucose, phenol red-free RPMI 1640 (Sigma) with 5% equine serum (HS) and 5% fetal bovine serum (FBS). Before experimentation, cells were transferred to medium supplemented with 0.5% of 4X charcoal-stripped HS and FBS totaling 1% 4x charcoal-stripped serum for 48 hrs to minimize the effects of endogenous hormones. During this time 20ng/ml NGF-β was added to promote differentiation for the 2 days preceding the experiments.

Dopamine efflux assay

We adapted a published method in Current Protocols in Neuroscience (7.9) for our efflux assay. PC12 cells were plated on poly-D-lysine (10 μg/ml)-coated 48-well plates at a density of 15,000 cells per well. After 48 hrs the 1% serum (4X charcoal-stripped) medium with 20ng/ml NGF-β was replaced with uptake buffer containing 25mM HEPES, 120mM NaCl, 5mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1μM pargyline, 2mg/ml glucose, 0.2mg/ml ascorbic acid, and desipramine (50nM), pH 7.4. GBR 12909, 100nM, was used to define selective efflux by DAT. Cells were incubated in uptake buffer in the presence of stabilizers (pargyline and ascorbic acid) and inhibitors (desipramine and +/−GBR12909) for 60 min at 37°C. Uptake was initiated by addition of 3H-DA (20nM) and allowed to progress for 10 min to load the cells. Cells were then washed twice in release buffer containing 25mM HEPES, 120mM NaCl, 5mM KCl, 1.2 mM MgSO4, 1μM pargyline, 2mg/ml glucose, 0.2mg/ml ascorbic acid, and desipramine (50nM). Release buffer containing treatments and +/− GBR12909 was added and extracellular fluid was collected at the indicated time points to assess efflux. Triplicate aliquots were counted in 2ml Hydroflour scintillant using a Beckman LS600SE scintillation counter. Specific efflux was defined by averaging the DPMs due to efflux with desipramine and GBR 12909, and subtracting these values from the efflux observed with only desipramine. We subtracted background (vehicle controls) from treatment groups and determined statistical significance as p<0.05 using SigmaStat software program.

siRNA transfection

PC12 cells were collected and washed in serum-free medium. Cells were counted using a 1:1 dilution of trypan blue and 3x 106 cells were placed in 400μl serum-free medium, and then 100nM of each Dharmacon pooled siRNA transcript was added. (Dharmacon ON-TARGETplus duplex ESR1 L-091219-00 for ERα, ESR2 L-097837-00 for ERβ, GPR30 L-093123-00 for GPR30, and non-targeting control D-001810-01).The cells were allowed to sit at room temperature for 10 mins and then electroporated at 300V. After electroporation cells were plated on poly-D-lysine (10μg/ml)-coated 48-well plates in normal growth medium for 24 hrs before a 48 hr 20ng/ml NGF-β treatment in 1% 4X charcoal-stripped serum.

Protein extraction and western blot analysis

PC12 cells transfected with siRNA for 72 hrs were washed once with ice-cold PBS containing 2mM KCl, 1.4mM KH2PO4, 136mM NaCl, and 8mM Na2HPO4 pH to 7.3. Cells were collected in 5ml ice-cold PBS and centrifuged at 200 x g at 4°C for 5 mins. PBS was removed and 500μl 1x cell lysis buffer (Cell Signaling) plus 1mM Phenylmethanesulfonyl fluoride (PMSF, Sigma) and 1mM Dithiothreitol (DTT, Sigma) were added. Cells were homogenized by passing through a 22 gauge needle 15 times and centrifuged at 1500 x g at 4°C for 10 mins. The supernatant was removed and 50μg/ml cell protein was added to SDS sample buffer and boiled for 10 mins. Samples were loaded onto a 7.5% acrylamide SDS-PAGE gel and transferred to a nitrocellulose membrane. Blots were blocked using 2.5% BSA and 2.5% milk in 10mM Tris-buffered saline pH 7.4 for 1 hr before overnight incubation with primary antibodies (Abs), to ERα (1:1000 Mc-20, Santa Cruz: sc-542), ERβ (1:2000 Clone 9.88, Sigma: E1276), GPR30 (1:1000 Novus: NLS4271) at 4°C. Blots were washed three times for 10 mins with 0.05% TBST and incubated for 1 hr with peroxidase-conjugated anti-mouse IgG (Amersham Biosciences) for ERα and ERβ or peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences) for GPR30. Immunoreactivity was detected by enhanced chemiluminescence (Amersham Biosciences) on x-ray film.

Use of ER subtype-selective agonists and antagonists and extranuclear selective E2 conjugate

PC12 cells were prepared for the DA efflux assay and treated in efflux buffer with either vehicle, 10−9M E2, the ERα-selective agonist 4,4',4"-(4-propyl-(1H)-pyrazole-1,3,5 triyl)trisphenol (PPT), ERα-selective antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylet hoxy)phenol]-1H-pyrazole dihydrochloride (MPP dihydrochloride), the ERβ-selective agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN, all from Tocris), or the GPR30 agonist G-1 compound from Chemidiv Inc. for 9 min. 10−9M ER antagonist ICI 182 780 (Tocris) use added for 30 mins in uptake buffer before a 9 min treatment with 10−9M E2 in efflux buffer. Ethanol (0.005%) was the vehicle for PPT, E2, and DPN, and 0.005% DMSO plus 0.005% ethanol was the vehicle for G1. A nondegradable poly(amido)amine E2-dendrimer was synthesized and characterized by the Katzenellenbogen laboratories (Harrington et al. 2003a).

Quantitative immunoreactive plate assay

Briefly, PC12 cells were plated on poly-D-lysine (10vg/ml)-coated Costar/Corning 96-well plates at 5000 cells per well, as previously described (Watson et al. 2006a). Cells were left in normal growth media for 24 hrs after plating before a 48 hr 20ng/ml NGF-β treatment in 4x 1% charcoal stripped serum. Cells were washed with PBS once and treatments were added in the previously described uptake buffer with 50nM dopamine for 9 mins. Cells were fixed for 30 at room temperature with 50vl 2% paraformaldehyde, and 0.2% gluteraldehyde +/− NP-40 for non-permeabilized and permeabilized cells. Cells were washed twice with PBS and blocked with 0.1% fish gelatin/PBS for 45mins at room temperature. The diluted 1° Abs were added for 10 hrs at 4°C; 2μg anti-clathrin Ab was added to one row (8 wells) to control for cell permeabilization. Cells were washed three times in PBS and incubated in appropriate biotinylated 2° Ab for 1 hr, then cells were washed three times before a 1 hr incubation with ABC-alkaline phosphatase (AP) solution. Cells were washed five times with PBS, and substrate para-Nitro-phenol phosphate (pNpp) plus 0.5mM levamisole was added in 100mM sodium bicarbonate solution for 50 mins at 37°C. Plates were read at A405nm and then rinsed and stained with 50vl/well 0.1% crystal violet for 30 mins at room temperature. Cells were washed with ddH20 and dried overnight. Dye extracted from each well (using 50vl 10% acetic acid) was read at A590 nm and used to estimate cell number per well).

Results

Time course and concentration curve of E2-mediated DA efflux

We have previously demonstrated that a 5 min 10−8M E2 treatment significantly inhibits specific DA uptake during a 30 min uptake assay (Watson et al. 2006a). This led us to explore the possibility that E2 could also reverse the transporter and cause DA efflux, as has been reported for drugs of abuse such as amphetamine [reviewed in (Fleckenstein et al. 2007)]. Fig. 1A shows that a 10−9M E2 treatment at the earliest time points (1, 3, 5, and 7 mins) did not cause DA efflux. However, starting at 9 and continuing at 15 min, a significant efflux of preloaded 3H-DA was observed, followed by a return to the basal level (no efflux) at 20 and 30 mins. Our results suggest that E2-mediated dopamine efflux is time-dependent, and may rapidly follow the inhibition of dopamine uptake at 5 mins (Watson et al. 2006a).

Figure 1.

Figure 1

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E2 dopamine efflux time course and dose-response curve. (A) 10−9M E2 time course (B) physiologically relevant E2 dose response curve at 9 minutes; *=significant compared to 0.005% ethanol control (p<0.05); n=24 in four experiments.

An extensive dose-response curve within physiological concentration ranges is important for assessing the non-genomic actions of steroids because these responses typically display non-monotonic response patterns (Watson et al. 1999;Bulayeva et al. 2004c). Therefore, to further characterize these rapid actions of E2, we chose a 9 min time point optimum for the DA efflux assay and examined a detailed and physiologically relevant E2 dose-response curve (Fig. 1B). At 10−14M, E2 caused a significant increase in DA efflux. While intermediary concentrations (10−13 to 10−11) did not cause DA efflux, we observed another DA efflux response at 10−10 to 10−8M E2 concentrations – a non-monotonic, non-conventional dose-response. Each response was similar to the others in the level of DA efflux.

Effect of cell-impermeable E2-dendrimer on DA efflux

The rapidity with which E2 causes DA efflux suggests that our response is nongenomic, and thus initiated at the plasma membrane, consistent with many other assessments of these activities [reviewed in (Watson and Gametchu 1999;Watson and Gametchu 2003a)]. Previous studies have used cell-impermeable E2-BSA and E2-peroxidase conjugates to demonstrate non-genomic actions of E2 via binding to mER, with the limitation that preparations must be freshly cleared of free E2, and used shortly thereafter (Gaetjens and Pertschuk 1980;Stevis et al. 1999a). We used a new impeded ligand construct that has been reported to be more stable, and which avoids any artifacts due to effects of the proteins to which the E2 is conjugated (Harrington et al. 2006a). This nondegradable poly(amido)amine E2-dendrimer does not bind to nuclear estrogen receptors in intact cells or activate transcription of estrogen target genes (Harrington et al. 2003b). Fig. 2 shows that E2-dendrimer (with a conjugated E2 concentration equivalent to 10−9M free E2) caused significant DA efflux as early as 5 mins (faster than with E2). Like free E2, a 9–15 min E2-dendrimer application, caused efflux. The resulting 9 min E2-dendrimer DA efflux was 3 fold higher than the DA efflux caused by free E2 at 9 mins. The ability of E2-dendrimer to mediate DA efflux further supports a mechanism by which this action on the DAT occurs through a rapid nongenomic response mediated by a mER.

Figure 2.

Figure 2

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E2-dendrimer-elicited dopamine efflux time course. (●) 10−9 M E2 dendrimer equivalents in mins compared to (○) 10−9 M E2 dopamine efflux time course. *=p<0.05 significance of control (0.005% ethanol) compared to non-conjugated dendrimer (the E2 dendrimer control), #=p<0.05 from E2 treatment. n=18 in three experiments

Effects of siRNA knockdowns of ERα, ERβ, and GPR30 on E2-mediated dopamine efflux

We have previously shown that 2 day, 20ng/ml NGF-differentiated PC12 cells express membrane ERα and β, and elevated whole-cell ERα levels. These cells also express the transcript for the alternative GPCR-related mER, GPR30 (Watson et al. 2006b). In the present study we tested for the contribution of each of these ERs to E2-mediated DA efflux. We transfected groups of PC12 cells with Dharmacon On-target Plus rat siRNA transcripts for ERα, ERβ, and GPR30. Electroporation at 300V resulted in optimal transfection with little cell death (data not shown). We also confirmed substantial decreases in protein expression for ERα, ERβ, and GPR30 by immunoblot analysis (Fig. 3A). As shown in Fig. 3B, PC12 cells transfected with ERα siRNA showed significant inhibition of DA efflux during a 9 min 10−9M E2 treatment, compared to cells transfected with non-targeting random sequence siRNA. However, ERβ knockdown did not abrogate the E2-mediated DA efflux, while GPR30 knockdown caused a significant increase in E2-mediated DA efflux. These data suggest that ERα is primarily driving E2-mediated DA efflux, but that GPR30 could play an inhibitory role in the regulation of this DAT activity.

Figure 3.

Figure 3

Figure 3

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Effects of siRNA on protein expression and DA efflux. (A) Immunoblot analysis of ERα, ERβ, and GPR30 proteins due to siRNA knockdown. PC12 cells were electroporated without (−) and with (+) Dharmacon SMART pool rat siRNA transcripts for ERα, ERβ, and GPR30. Protein expression was examined in whole cell lysates after 72 hrs. (B) Effect of 10−9 M E2 treatment for 9 mins on DA efflux from siRNA-transfected cells compared to ethanol control, non-transfected, and random sequence-transfected cells. *=p<0.05 significance compared to 0.005% ethanol control, #=p<0.05 from non-transfected, ^=p<0.05 from random sequence pool. n=18 in three experiments

Effects of selective ERα, ERβ, and GPR30 agonists and antagonists on dopamine efflux

Next we used receptor-selective agonists and antagonists to further test the involvement of the three different types of ERs (or their combinations) that exist in PC12 cells on DA efflux. Although these selective ligands have primarily been used to test genomic estrogenic responses in the past (Frasor et al. 2003;Meyers et al. 2001a;Stauffer et al. 2000b), we tested their ability to elicit nongenomic responses to membrane-resident ERs over rapid time courses. PPT, a selective agonist for ERα, is 410-fold more potent in binding to ERα than to ERβ, and selectively activates ERα within the dose ranges of 10−7 to 5x10−7M (Stauffer et al. 2000a). In our system PPT caused an increase in DA efflux 500–2700% higher than that caused by E2 (shown by horizontal line with dashed line error ranges on graph Fig. 4A). DPN binds to nuclear ERβ with a 72-fold higher affinity than to ERα (Meyers et al. 2001b); we observed that it caused dopamine efflux comparable to 10−9 M E2 at 5x10−10 and 10−9 M concentrations, and significantly increased efflux compared to E2 at 5x10−9 M (outside its range of best selectivity). The GPR30-selective agonist, G-1 (Bologa et al. 2006), caused significantly less DA efflux than E2, at 10−9 M. Using two or all three selective agonists in combination (G1+PPT+DPN or PPT+ DPN) caused efflux comparable to E2 alone as expected, since E2 binds to all three receptors. Combinations also eliminated PPT’s maximal efflux response, suggesting inhibition via ERβ and GPR30. Altogether, these data suggest that both ERβ and GPR30 can mount a response when acting in the absence of other receptors, but they act as potent inhibitory mediators to stimulation caused by ERα.

Figure 4.

Figure 4

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Effects of selective agonists and antagonists for ERα, ERβ, and GPR30 on 9 min dopamine efflux compared with % of 10−9 M E2 treatment. Horizontal dashed lines represent standard error around 100%; solid line represents the mean of 10−9 M E2 treatment set at 100%. (A) PPT is an ERα-, DPN an ERβ-, and G-1 a GPR30-selective ligand. (B) Effect of selective ligands in combination with E2 compared to E2 alone. MPP is an ERα- and ICI 182 780 an ERα- and ERβ-antagonist. The G-1 vehicle control is DMSO+EtOH, 0.005% ethanol is the control for PPT, and 0.005% ethanol is the control for both E2 and DPN. *=p<0.05 significance compared to control, #=p<0.05 from E2 treatment. n=18 in three experiments

We then tested the effects of selectively activating receptor subtypes in combination with E2 (which activates all of these receptors). Fig. 4B shows that 10−7 M PPT, in combination with 10−9 M E2, caused efflux comparable to E2 alone, but lower relative to that caused by the α-selective PPT alone. DPN (10−9 M) in combination with 10−9 M E2 caused significantly decreased dopamine efflux compared to either E2 or DPN alone (panel A). The efflux induced by G-1 in combination with E2 was significantly lower that the E2 response, and comparable to the attenuated efflux seen with G-1 alone. PPT and DPN when in combination with E2, all at their most selective concentrations, caused significantly lowered DA efflux compared to E2. Therefore, again is appears that ERβ and GPR30 are inhibitory when ERα is activated by another hormone.

To address these hypotheses in another way, we then used selective antagonists in combination with E2 (Fig. 4B). The selective ERα inhibitor MPP, or an inhibitor of both ERα and ERβ (ICI 182 780), both caused significant inhibition of efflux in combination with E2, again suggesting that ERα was the predominant mediator of efflux when all receptors are activated by E2. Although the ability of each selective agonist(s) and antagonist to act to some extent on the other receptors complicates our understanding of each ER’s contribution, these results seem to be most consistent with ERα being the predominant mediator of E2-mediated DA efflux, with inhibitory contributions from GPR30 or ERβ being dependent upon the simultaneous activation of ERα.

Membrane ER trafficking after E2 treatment

To explore one possible mechanism of E2 action on DA transport, we used our quantitative immunoreactive plate assay to monitor the levels of membrane vs. total ERs after E2 treatment (Campbell and Watson 2001). Fig. 5 shows that a 9 min E2 treatment caused a significant increase in membrane ERα levels, while causing a decrease in membrane ERβ levels, and unchanged membrane GPR30 levels. E2 also resulted in decreased total ER levels for ERα and ERβ. Others have reported that after ligand association, ERs rapidly become degraded resulting in decreased total levels (Alarid et al. 1999;Bosse et al. 1997c;Horwitz and McGuire 1978). It is interesting that GPR30 measured on the membrane represents only 50% of the total GPR30 population. This protein has been found in either the plasma membrane or the endoplasmic reticulum by different laboratories in other tissues and cell types (Sakamoto et al. 2007;Filardo and Thomas 2005).

Figure 5.

Figure 5

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Quantitative plate assay measuring membrane and total ERα, ERβ, and GPR30 immunoreactive protein levels after a 9 minute 10−9 M E2 treatment. *=p<0.05 significance compared to 0.005% ethanol control, #=p<0.05 from membrane levels. n=50 in 3 experiments

Discussion

The cellular mechanisms that underlie estrogenic neuroprotective effects, particularly in physiological concentrations ranges, are largely unknown. During periods of hormonal fluctuation females become more susceptible to neurological disorders and diseases, some of which are associated with DA dysregulation. Postmenopausal women receiving estrogen replacement therapy (ERT) show increased levels of DAT available at the plasma membrane and therefore available for regulation of DA signaling (Gardiner et al. 2004). Decreased synaptic DA is the acute pathological manifestation of many DA-associated gender-biased neurological disorders and diseases. Ours is the first study to examine the nongenomic effects of E2 on DA efflux via contributions of three well-characterized estrogen receptors.

In our current studies, E2 at physiological concentrations caused rapid DA efflux (at 9 and 15 mins), indicative of a nongenomic action. Reports of non-monotonic dose-response curves are increasingly common for the nongenomic actions of E2, and we have again observed this phenomenon here in our analysis of estrogenic effects on DAT. Various functional assays including ERK1/2 activation and prolactin release in a pituitary cell line (Bulayeva et al. 2004b;Bulayeva et al. 2005;Wozniak et al. 2005) are examples of this type of dose-response behavior. When such complex dose relationships are evident, it becomes extremely important to carefully examine a wide dose range for actions of the many estrogens and compounds that mimic them.

The E2-dendrimer is a large molecule consisting of a scaffold onto which many E2 molecules are conjugated (Harrington et al. 2006b). It is the latest in a long line of impeded ligands (E2 bound to fibers, E2-BSA, and E2-peroxidase) designed to prevent the rapid cellular uptake of estrogens through the plasma membrane, thus separating actions at the membrane from genomic actions in the nuclear compartment. We previously noted altered time courses when impeded E2 ligands were used (Bulayeva et al. 2004a;Watson et al. 1999), which is consistent with our results here. There has been some debate as to whether these compounds behave the same biologically or retain the same receptor-binding characteristics as E2 (Stevis et al. 1999b), though many of them are quite active in nongenomic responses, as again shown here. Multivalent binding of E2-dendrimers with the receptor could possibly alter the time course of hormone-induced dopamine efflux by immobilizing or sterically hindering receptor movement (including toward interacting/enabling factors, or into signaling compartments like caveolae) in the plasma membrane, or trafficking into the cell; this could cause disrupted signaling. In addition, we do not know the binding properties of the E2-dendrimer to GPR30 receptors, which are also present in our PC12 cells (Watson et al. 2006b), or other less well-defined receptors for estrogen (Toran-Allerand et al. 2002a).

Several of our lines of evidence presented here suggest that in PC12 cells, ERα is the predominant subtype regulating E2-mediated DA efflux through the DAT. The decreased response after siRNA knockdown of ERα suggests that a minimum protein level of ERα is required for this function. Knockdown of the other receptors, ERβ and GPR30 either did not affect, or elevated, the responses (respectively). We also showed that E2 caused increased trafficking of ERα to the membrane during the same time frame as these actions on DAT, which suggests that membrane-resident ERα is involved; certainly it places ERα at the right place and time to influence the DAT by either interaction between ERα and DAT, or between ERα and a variety of signaling mediators responsible for second messengers and response cascades affecting DAT.

Our selective ligand results for ERα also support a major role for this receptor subtype in DAT-meditated DA efflux, with inhibitory roles for ERβ and GPR30 in the presence of ERα activity. In other systems where ERα mediates signaling and functional responses, co-expression of ERβ usually results in compensatory or inhibitory regulation (Hall and McDonnell 1999;Paech et al. 1997). These examples come from the actions of these receptors in transcriptional regulation. Our results suggest that ERα and GPR30 interact in a similar way for estrogen-induced nongenomic actions on DAT. Also, if we compare the response to the selective ERβ ligand DPN to the much larger ERα-selective ligand-mediated (PPT) response, DPN falls short (though it is as effective as E2, which binds to all three receptors). However, ERβ was still able to elicit a response in the absence of any stimulation of ERα, an observation that has also been made before in knockout animals (Hewitt et al. 2005). Our results show that the ligand selective for GPR30 has a similar but less robust effect. In conjunction with these functional responses, we also show that E2 causes trafficking of ERβ away from the plasma membrane, indicating that movement of ERβ away from the site of nongenomic actions might allow stimulation of efflux.

There were other interesting results from these receptor-selective manipulations. Knockdown of GPR30 actually increases E2-mediated DA efflux, while GPR30 membrane levels remain unchanged during the time frame of E2 effects on transport. It is thus possible for GPR30 to play its inhibitory role, even though its compartmental localization does not change. However, other studies have shown that other G-protein coupled receptors (GPCRs) involved in other ligand activations can directly associate with the DAT (e.g. GPR37), and knockouts of this GPCR cause altered DAT trafficking to and from the plasma membrane (Marazziti et al. 2007), though the mechanism(s) involved are unknown. In our experiments the unchanged levels of membrane-resident GPR30 and concomitant GPR30 inhibitory action in E2-mediated DA efflux (based on siRNA and selective agonist data) suggest that GPR30 could instead exert inhibitory control by causing trafficking of the DAT, alteration of protein associations with DAT within the membrane, or post-translational modifications of the DAT protein, which we will explore in future studies.

Our studies suggest that rapid nongenomic effects of E2 on DA transport via the DAT could be similar to those observed for amphetamines. This regulation could occur by direct interactions between the receptor and the DAT, or via indirect actions mediated by kinases activated by the hormones. Amphetamine, a drug of abuse, has long been known to cause reversal of the DAT resulting in DA efflux. This action involves phosphorylation of serines, threonines, and tyrosines located within the intracellular N and C termini by various kinases, including prominently protein kinase C; this modification is also important for DAT trafficking to and from the plasma membrane. Amphetamines also redistribute and/or inhibit VMATs (vesicular monoamine transporters), whose function is to sequester and protect DA from degradation inside the cell. Our previous study showed a decrease in membrane DAT levels after a 5 min E2 treatment (Watson et al. 2006b). In vivo studies have shown that E2 causes both increases and decreases in DAT densities in the presynaptic membrane of different brain regions, depending on the circulating E2 levels (Bosse et al. 1997d;Datla et al. 2003;Morissette et al. 2008). Therefore, these E2 effects on PC12 cells represent a cell model that can be used to provide us with some insights into the similarities or differences with the regulatory mechanisms of other agents that cause DA efflux.

Neurodegenerative diseases often first occur in women during life stages when physiological estrogen levels are beginning to fluctuate and then finally decline. This could present a situation where estrogens previously maintaining DA concentrations in the synapse by promoting efflux cease to support this activity at menopause or other times of estrogen loss (eg. changing to a new phase of the menstrual cycle, the end of pregnancy, ovariectomy). The DA in the synapse thus falls, promoting diseases featuring low DA stimulation. Selective action of ligands on specific ER subtype populations may present a therapeutic opportunity to correct for these estrogen losses in a way that most effectively and selectively treats this deficit. With the advent of this new understanding and development of new pharmacological ER subtype-selective agonists and antagonists, we could begin to manipulate these mechanisms to address gender-biased neurological diseases of many kinds.

Acknowledgments

We thank Dr. David Konkel for skillful editing of this manuscript. Financial support was provided by NIEHS (T32 ES07254) and NIDA (T32 DA07287) training grants, NIH ES015292 (to C. S. W.) and DK15556 (to J. A. K.), and the Center for Addiction Research at the University of Texas Medical Branch.

Abbreviations used

Ab

antibody

E2

17β-estradiol

DAT

dopamine transporter

DA

dopamine

ERα

estrogen receptor α

ERβ

estrogen receptor β

GPR30

G-protein coupled receptor 30

mER

membrane estrogen receptor

NGF

nerve growth factor

PC12 cells

pheochromocytoma cell line

PPT

4,4',4"-(4-propyl-(1H)-pyrazole-1,3,5 triyl)trisphenol

MPP dihydrochloride

1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylet hoxy)phenol]-1H-pyrazole dihydrochloride

DPN

2,3-bis(4-hydroxy-phenyl)-propionitrile

THC

RR-diethyl-tetrahydrochrysene

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