Figure 2.
Effect of APD treatments on cellular levels of the D2R. A, time-dependent haloperidol-induced enhancement of cell-surface D2R expression. HEK293T cells transiently expressing the extracellular N-terminal FLAG-tagged D2R construct, FLAG-D2R, were treated with haloperidol (10 μm) for the indicated times. The levels of cell-surface D2R were then quantified by probing the intact cells with anti-FLAG antibody. The haloperidol-induced enhancement of D2R surface expression is depicted as a percentage of the signal from vehicle-treated cells. Significant enhancements of cell-surface D2R expression levels were observed at each measurement time point following haloperidol treatment, both with respect to vehicle and with respect to the previous time point (bar representing the mean; n = 6; Tukey; p < 0.001 for the 6-h treatment time point versus vehicle, and p < 0.0001 for all other comparisons). B, effect of APD treatment on the cell-surface levels of N-terminal FLAG-tagged D2SR. HEK293T cells transiently expressing the extracellular N-terminal FLAG-tagged D2SR construct, FLAG-D2SR, were treated with either haloperidol or clozapine (10 μm, 24 h). The levels of cell-surface D2SR were then quantified as in A and are reported as a percentage of the signal from vehicle-treated cells. The levels of cell-surface D2SR measured after haloperidol treatment were significantly greater than after clozapine or vehicle treatment (n = 12, Tukey, p < 0.0001). C, concentration–response curves of APD-induced enhancement of cell-surface D2R expression. HEK293T cells transiently expressing the extracellular N-terminal FLAG-tagged D2R construct, FLAG-D2R, were treated for 24 h with the indicated concentrations of haloperidol (halo), olanzapine (olanz), or clozapine (cloz). The levels of cell-surface D2R were then quantified by probing the intact cells with anti-FLAG antibody. The APD-induced enhancement of D2R surface expression was calculated as the percentage increase in cell-surface receptor levels over vehicle-treated cells and is plotted as a percentage of the response to 10 μm haloperidol (mean ± S.E. (error bars), n = 38 for 10 μm olanzapine, 6 for all other concentrations; n = 44 for 10 μm halperidol, 7 for all other concentrations; n = 50 for 10 μm clozapine, 12 for 3 and 30 μm clozapine, 6 for all others). Cell-surface levels of D2R became significantly different (Dunnett's multiple-comparison test) from vehicle after treatment with 100 nm haloperidol (p < 0.001), 1 μm olanzapine (p < 0.0001), and 3 μm clozapine (p < 0.01). Cell-surface D2R levels after treatment with 10 μm concentrations of each drug were significantly different from each other (Tukey, p < 0.0001). There was no significant difference in cell-surface D2R levels between the 3, 10, and 30 μm clozapine treatments. D, comparison of the effect of multiple APD treatments on cell-surface expression of D2R. HEK293T cells transiently expressing FLAG-D2R were treated with the indicated APDs (24 h, 30 μm for remoxipride and 10 μm for all other APDs). The levels of cell-surface D2R were then quantified as in A and are reported as a percentage of the signal of vehicle-treated cells (bar representing the median, whiskers representing the full range of data; n = 7 for amisulpride, 8 for remoxipride, 16 for tiapride, 31 for droperidol and ziprasidone, and 32 for all other drugs). Relative D2R surface expression after treatment with all APDs was significantly greater than vehicle except for the APDs clozapine and aripiprazole (Dunnett, p < 0.01). E, effect of APD treatments on total cellular expression of D2R. HEK293T cells transiently expressing FLAG-D2R were treated with the indicated APDs (10 μm, 24 h) and were then fixed and permeabilized with methanol. Total cellular D2R was then assessed by probing with anti-FLAG antibody, and levels are reported as a percentage of the signal from vehicle-treated cells (n = 6 for aripiprazole, 22 for haloperidol, 16 for all other drugs). Treatment with aripiprazole produced significantly less enhancement of total cellular receptor levels compared with the other APDs (Tukey, p < 0.005), and all APDs, except for aripiprazole, significantly enhanced total receptor levels compared with vehicle (Tukey, p < 0.0001). F, APD-mediated increase in cell-surface D2R normalized to increase in total cellular D2R. Cell-surface D2R normalized to total cellular D2R levels after clozapine treatment was significantly lower than after treatment with all other APDs or vehicle (n = 32, Dunnett, p < 0.005).