PC12 cell culture
PC12 cells were grown in high-glucose, phenol red-free RPMI 1640 medium containing 5% fetal bovine serum (FBS) and 5% equine serum (HS). To promote PC12 differentiation and minimize the effects of endogenous hormones respectively, 20 ng/ml NGF-β was added in medium supplemented with 0.5% of 4× charcoal-stripped FBS and HS for 48 hrs prior to experiments.
Dopamine efflux assay
We measured 3H-dopamine efflux using selective catecholamine transporter inhibitors to define specific dopamine transport via the DAT as previously described in . PC12 cells were plated on poly-D-lysine (10 μg/ml)-coated 48-well plates and uptake buffer (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1 μM pargyline, 2 mg/ml glucose) containing 0.2 mg/ml ascorbic acid, and desipramine (50 nM), pH 7.4 ± GBR 12909 was added for 60 min at 37°C. In experiments containing 50 nM reserpine, a VMAT inhibitor, a 120 min preincubation in uptake buffer preceded the 60 min GBR 12909 preincubation. GBR 12909 (100 nM) was added to define selective efflux by DAT. In experiments containing kinase inhibitors 10 μM U0126 (a MAPK inhibitor) or 10 μM Ly294002 (a PI3K inhibitor) were also added during the 60 min uptake buffer addition. 10 μM H89 (a PKA inhibitor) and 100 nM Ro32-0432 (a PKC inhibitor) were added to the uptake buffer for 30 min of preincubation. For experiments testing Ca2+involvement, 1 μM thapsigargin was added for a 15 min preincubation to empty intracellular Ca2+ stores, or cells were incubated for 10 min in 0 Ca2+ medium (5 mM EGTA, 10 mM HEPES, 130 mM NaCl, 5.5 mM KCl, 2 mM MgCl2, 7 mM glucose, 30 mM sucrose, pH 7.4) and washed twice in 0 Ca2+ medium. For all assays cells were loaded with 3H-DA (20 nM) for 10 min prior to two washes in release buffer (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1 μM pargyline, 2 mg/ml glucose, 0.2 mg/ml ascorbic acid, and 50 nM desipramine). Release buffer containing treatments, +/- GBR12909, was then added, and extracellular fluid was collected at 9 min to assess3H-DA efflux. Triplicate aliquots were counted in 2 ml Scintiverse II scintillant using a Beckman LS600SE scintillation counter. Specific efflux was defined by averaging the disintegrations per minute due to efflux in the presence of desipramine and GBR 12909, and then subtracting these values from the efflux observed with desipramine alone. We subtracted background (vehicle controls) from treatment groups and represented the data as 3H-DA efflux compared to % of 9 min 10-9 M E2-induced efflux (set as 100%).
PC12 cells were collected from five, 150 cm2 Corning tissue culture flasks by scraping, and then centrifuged at 1500 × g, 4°C for 5 min, and resuspended in 2 ml homogenizing buffer (10 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, pH 7.4). Cells were then sonicated 15 times using a pulse probe sonicator, and further processed using a Dounce homogenizer, on ice, until the majority of cells appeared broken by microscopic examination. The resulting broken cell preparation was then centrifuged at 1500 × g at 4°C to remove the nuclear pellet. The supernatant was then centrifuged at 120,000 × g at 4°C to obtain the plasma membrane pellet, which was then resuspended in membrane buffer (50 mM Tris-HCl, 1 M NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 250 mM sucrose, 1 mM EDTA, 1 mM DTT, 1:1000 protease inhibitor cocktail, pH 7.4) by stirring 8 hours at 4°C and then re-pelleted by centrifugation for 45 min at 45,000 × g, 4°C. The Bradford Bio-rad assay was used to determine protein concentration in the supernatant per manufacturer's instructions. Protein samples were incubated with 40 μl protein G agarose (Sigma, P-7700) beads for 10 min at 4°C, then centrifuged using a microfuge for 1 min. The supernatant was incubated overnight at 4°C with 2.5 μg DAT antibody (C-20, Santa Cruz: sc-1433). 50 μl of protein G agarose beads were washed 3 times in phosphate buffered saline (PBS) and samples containing antibody were incubated with these beads for 4 hours at 4°C on a rotator. Beads were then washed 4 times with PBS for 10 min, each wash. Samples were eluted using 50 mM glycine buffer pH 2.5, added to SDS sample buffer and heated at 67°C for 10 min, and then electrophoresed on a 7.5% acrylamide SDS-PAGE gel followed by transfer to a nitrocellulose membrane. Blots were blocked using 2.5% BSA and 2.5% milk in 10 mM 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), and DAT (1:1000, Santa Cruz: sc-33056) at 4°C. Blots were washed three times for 15 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, or peroxidase-conjugated anti-goat (Amersham Biosciences) for DAT. Immunoreactivity was detected by enhanced chemiluminescence (Amersham Biosciences) on Hyperfilm (Amersham) film.
Quantitative plate immuno-assay
Briefly, PC12 cells were plated on poly-D-lysine (10 μg/ml)-coated 96-well plates at 5000 cells per well, as previously described . NGF-differentiated, serum-deprived cells were washed with PBS for 5 min, and treatments were added in the above uptake buffer with 50 nM dopamine for 9 min. Cells were fixed for 30 min at room temperature with 50 μl 2% paraformaldehyde, and 0.2% gluteraldehyde +/- NP-40 to permeabilize or not permeabilize cells, respectively. Cells were then washed twice (5 min each) with PBS and blocked with 0.1% fish gelatin/PBS for 45 mins at 22°C. Diluted 1° Abs, to ERα (1:1000 Mc-20, Santa Cruz: sc-542), ERβ (1:1500 Clone 9.88, Sigma: E1276), GPR30 (1:1000 Novus: NLS4271), and DAT (1:2000 W-17, Sigma: sc-33056) were added overnight at 4°C; 2 μg anti-clathrin Ab provided a control for cell permeabilization . Cells were washed three times in PBS and incubated in appropriate biotinylated 2° Ab for 1 hr, then washed three times prior to 60 min incubation with ABC-alkaline phosphatase (AP) solution. Cells were washed five times with PBS, and the substrate para-nitro-phenol phosphate (pNpp) plus 0.5 mM levamisole was added in 100 mM sodium bicarbonate solution for 30 mins at 37°C. Plates were read at A405 nm and then rinsed and stained with 0.1% crystal violet for 30 mins at room temperature, then washed with ddH20 and dried overnight. Dye was then extracted from each well with 50 μl 10% acetic acid, read at A590, and used to estimate cell number per well. Data are plotted as % of vehicle-treated control levels (A405 nm/A590 nm).
Statistical analyses for all assays were performed using SigmaStat software (Chicago, IL, USA), and statistical significance was accepted at p < 0.05. Figure legends contain the n for each experimental set and the specific statistical analysis applied. All experiments were repeated 3 times.