Estradiol effects on the dopamine transporter - protein levels, subcellular location, and function - PubMed (original) (raw)
Estradiol effects on the dopamine transporter - protein levels, subcellular location, and function
Cheryl S Watson et al. J Mol Signal. 2006.
Abstract
Background: The effects of estrogens on dopamine (DA) transport may have important implications for the increased incidence of neurological disorders in women during life stages when hormonal fluctuations are prevalent, e.g. during menarche, reproductive cycling, pregnancy, and peri-menopause.
Results: The activity of the DA transporter (DAT) was measured by the specific uptake of 3H-DA. We found that low concentrations (10(-14) to 10(-8) M) of 17beta-estradiol (E2) inhibit uptake via the DAT in PC12 cells over 30 minutes, with significant inhibition taking place due to E2 exposure during only the last five minutes of the uptake period. Such rapid action suggests a non-genomic, membrane-initiated estrogenic response mechanism. DAT and estrogen receptor-alpha (ERalpha) were elevated in cell extracts by a 20 ng/ml 2 day NGFbeta treatment, while ERbeta was not. DAT, ERalpha and ERbeta were also detectable on the plasma membrane of unpermeabilized cells by immunocytochemical staining and by a fixed cell, quantitative antibody (Ab)-based plate assay. In addition, PC12 cells contained RNA coding for the alternative membrane ER GPR30; therefore, all 3 ER subtypes are candidates for mediating the rapid nongenomic actions of E2. At cell densities above 15,000 cells per well, the E2-induced inhibition of transport was reversed. Uptake activity oscillated with time after a 10 nM E2 treatment; in a slower room temperature assay, inhibition peaked at 9 min, while uptake activity increased at 3 and 20-30 min. Using an Ab recognizing the second extracellular loop of DAT (accessible only on the outside of unpermeabilized cells), our immunoassay measured membrane vs. intracellular/nonvesicular DAT; both were found to decline over a 5-60 min E2 treatment, though immunoblot analyses demonstrated no total cellular loss of protein.
Conclusion: Our results suggest that physiological levels of E2 may act to sequester DAT in intracellular compartments where the transporter's second extramembrane loop is inaccessible (inside vesicles) and that rapid estrogenic actions on this differentiated neuronal cell type may be regulated via membrane ERs of several types.
Figures
Figure 1
DAT proteins are present in the membranes of PC12 cells and are elevated by 2–7 day NGFβ treatment. A. Cell lysate immunoblots with Ab to the second extracellular loop of DAT, showing an increase in DAT protein levels due to a 20 ng/ml NGFβ treatment over a time course of 2 (NGF 2d), 4 (NGF 4d) and 7 (NGF 7d) days, compared to controls without NGFβ treatment (2d, 4d, and 7d). Representative of 2 experiments. B. Staining of nonpermeablized fixed cells with the same DAT e2 Ab. Fluorescent images viewed with an FITC filter were photographed. Vector Red appears as orange-red signal on a background of yellow-green autofluorescence (typically seen with this cell fixation protocol). Note the staining of cell bodies, especially at the growth cones (overexposed), and the smaller amount of staining on processes of these NGFβ-differentiated cells. The bar represents 2 μm. C. DAT levels are stimulated ~8-fold on day 2 of NGFβ treatment, as demonstrated by the plate immunoassay of fixed cells using the same e2 DAT-specific Ab; 2° Ab conjugated to alkaline phosphatase was used to generate paranitrophenol (pNp) colorimetric signals, which were normalized to the cell number determined in each well by the crystal violet (CV) assay. Symbols are +NGF (■); -NGF (○). Controls: NGF-treated cells probed with a nonspecific IgG Ab is labeled as "+NGF IgG" (▼) "-NGF IgG" IgG (△); clathrin (◇); no primary Ab (no 1° Ab) and no primary or secondary Ab (no 1°, no 2° Abs) are at the origin. A very low clathrin Ab signal under these nonpermeabilizing conditions demonstrated the lack of inadvertent permeabilization of the cells in these assays. This graph represents the average values from 3 experiments ± S.E.M.
Figure 2
DA uptake is inhibited by 10 nM E2 in PC12 cells. Estradiol (E2), vs. ethanol vehicle (Control) was added to the cells together with 3H-DA for a 30 minute incubation, * = significantly different from vehicle control at p < 0.05. A. Cells were serum-starved for 48 hours with no NGFβ treatment. Total cellular uptake of DA over a 30 min period ± E2 treatment was monitored. B. Cells were serum-starved while being treated for 2 days ± 20 ng/ml NGFβ. DAT-specific DA uptake was measured over a 30 min period ± E2 treatment. NET- and DAT-blocking drugs were included to evaluate DAT-specific uptake of DA. # = significantly different vs. NGFβ-treated control (p < 0.05). C. The rapid effects of E2 treatment on 3H-DA uptake were shown when E2 treatment was added only during the last 5 minutes of the 30 min uptake assay. # = significantly different vs. NGFβ-treated vehicle control (p < 0.05).
Figure 3
PC12 cells have both membrane and intracellular ERα that is increased by NGFβ treatment. ERα was detected with C542 Ab. A. Protein levels of ERα in whole-cell extracts were determined by immunoblot analysis at 2, 4 and 7 days ± NGFβ treatment in medium lacking serum. Representative of 3 experiments. B. Micrograph of immunocytochemical staining of ERα using nonpermeabilized cells that had been serum-starved for 48 hours while being treated with 20 ng/ml NGFβ. The bar represents 2 μm. Fluorescent images viewed with an FITC filter were photographed. Left panel: Transmission micrograph of middle panel. Middle panel: Immunocytochemistry of ERα shown in red (Vector Red product), while the autofluorescent background is green. Right panel: Punctate ERα staining is irregularly distributed on the cell surface of a more highly magnified cell. C. The fixed cell microplate immunoassay shows that ERα is present in PC12 cells grown in serum-containing medium. The values for nonpermeabilized cells are shown by symbols: ● different ERα Ab concentrations, △ combined control conditions (IgG isotype control, no 1°, and no 1°/no 2°), superimposed at the origin as their values are all very low and do not differ from each other significantly, ◇ clathrin. The values for permeabilized cells are shown by bars at the appropriate Ab concentrations. The crosshatched bar is ERα, the gray bar at the origin represents combined controls (see above), and the white bar is clathrin (the permeabilization indicator). This graph represents average values from 3 experiments ± S.E.M.
Figure 4
PC12 cells have both intracellular and membrane ERβ. Monoclonal Ab clone 9.88 was used for detection. A. Protein levels of ERβ in whole-cell extracts were determined by immunoblot analysis. ERβ is present in cells grown in serum-free medium, but is not induced by NGFβ over a 7-day period. Representative of 3 experiments. B. Immunocytochemistry of non-permeabilized cells treated with 20 ng/ml NGFβ for 2 days before staining. The bars represent 2 μm. Fluorescent image micrographs were viewed with an FITC filter were photographed. Left panel: Transmission image of middle panel. Middle panel: membrane ERβ immunofluorescence present heterogeneously on cells. Right panel: Punctate ERβ staining is irregularly distributed on the cell surface of a more highly magnified cell. C. Both nonpermeabilized cells (symbols) and permeabilized cells (bars) were assessed for ERβ by the plate assay over an Ab saturation curve. Controls (C) are as previously described in Figures 1 and 3. The cross-hatched bar is clathrin from permeabilized cells; ▼ = clathrin signal from unpermeabilized cells. The solid gray bar represents combined controls (IgGκ, no1°, no2° Abs) from permeabillzed cells and ▲ = the same combined controls in unpermeabilized cells. The white bars represent values for ERβ Ab 9.88 detection in permeabilized cells at the concentrations shown on the X axis. This graph represents average values from 2 experiments (each with multiple samples) ± S.E.M.
Figure 5
GPR30 RNA is present in PC12 cells. The presence of GPR30 ER RNA was detected by RT-PCR using two different primer sets compared to the human breast cancer cell line MCF-7, previously shown to express this receptor. Markers (1 kb DNA Ladder, Invitrogen) were used to determine that the amplimers were of the correct size of 680 and 585 bp, for primer set 1 and 2, respectively.
Figure 6
Inhibition of DA uptake by E2 is regulated by cell density, and dose and time of E2 treatment. Cells were serum-starved while being treated for 2 days with 20 ng/ml NGFβ. NET- and DAT-blocking drugs were included to define DAT-specific uptake. A. DA uptake inhibition by 10 nM E2 is robust at 10,000 and 15,000 cells/well, but reversed at densities as high as 20,000 cells/well. DAT-specific DA uptake was measured over a 30 min period ± E2 treatment during the last 5 minutes of the assay. * = significant difference between control and E2-treated samples at the level of p < 0.05. # = significant difference in DA uptake due to cell density conditions. B. All doses of E2 from 10-14 to 10-8 M caused inhibition of DA uptake, though with different levels of effectiveness, and in a nonconventional dose-response pattern. * = significant difference between control and E2-treated samples at the level of p < 0.05. C. The oscillating effects of 10 nM E2 on DA uptake at room temperature. Ethanol control background was subtracted from these values.
Figure 7
Effects of acute E2treatment on DAT protein levels in the membrane vs. intracellular compartment using the fixed cell immunoplate assay. After the cells had been serum-starved and NGFβ-treated for 2 days, 10 nM E2 or ethanol vehicle (ETOH) was added for the times indicated, before cell fixation and assay. Nonpermeabilizing conditions of fixation are shown in the left panel and permeabilizing conditions are shown in the right panel. ◇ represents clathrin Ab signal for monitoring the cell permeabilization status, as in previous figures. All E2-treated samples had DAT values significantly lower than ethanol-treated controls (p < 0.05).
Figure 8
DAT levels in whole-cell extracts are not affected by E2 treatment for 5–60 min. Cells were serum-starved and NGFβ-treated for 2 days, then treated with 1 nM E2 vs. ethanol vehicle (C). Cell lysates were processed for immunoblot analysis with DAT Ab e2. DAT protein levels did not change due to E2 treatment over a 5–60 min time course. Representative of 2 experiments.
References
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