Fig. 2.
The effect of atorvastatin treatment and cholesterol replenishment on DA uptake in N2A cells stably expressing hDAT. (A) Cells were treated with increasing concentrations of atorvastatin (Ator) for 24 hr in 10% lipoprotein-deficient serum followed by [3H]DA (15 nM) uptake in the presence of increasing concentrations of unlabeled DA (100 nM − 30 μM) for 10 min at room temperature. Treatment with atorvastatin at 0.3 μM (N=6), 1 μM (N=6) or 10 μM (N=6) reduced DA uptake in a concentration-dependent manner when compared to vehicle treatment (N=11). (B & C) The values of the maximal DA uptake rate (Vmax) and Km were significantly lower in cells treated with 1 and 10 μM atorvastatin when compared to vehicle treatment. (D) Replenishment of water-soluble cholesterol (250 and 500 μM, 2 hr at 37°C) to cells following treatment with atorvastatin (1 μM, 24 hr, N=8/group) restored DA uptake. For non-atorvastatin treated cells, addition of 500 μM and 1000 μM cholesterol significantly reduced DA uptake (N=5/group). (E) Following atorvastatin treatment for 24 hr, surface DAT was biotinylated using membrane non-permeable sulfo-NHS-SS-biotin (1.5 mg/ml) for 1.5 hr at 4°C. The biotinylated proteins were pulled down using streptavidin beads. The biotinylated proteins were eluded from the beads with SDS-PAGE loading buffer containing DTT. Representative western blot images for biotinylated DAT, total DAT and GAPDH are presented. (F & G) Quantitation of surface DAT and total DAT expression levels. For surface DAT levels, data were calculated as the surface DAT levels vs. the total DAT levels, and presented as relative to the vehicle treatment. Total DAT levels were normalized to GAPDH levels and presented as relative to vehicle treatment. Data are presented with mean ± SEM *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs. vehicle