Skip to main content
. 2022 Feb 22;7(4):e151892. doi: 10.1172/jci.insight.151892

Figure 3. NET and DAT on human macrophages can work in uptake mode.

Figure 3

(A) Schematic of the experimental design employing various conditions to measure DAT- versus NET-specific IDT307 uptake. (B) Representative 40× images of human macrophages following perfusion with IDT307 under conditions in A. (C and E). Quantification of IDT307 uptake as fold change in fluorescence in the presence of nomifensine (C), desipramine (E), or both antagonists (gray trace). (D and F) The nonspecific values (gray trace) were subtracted to show fold increase in NET- or DAT-mediated IDT307 uptake. (G and H) Blockade of either NET or DAT significantly decreased IDT307 uptake (average slope: control versus DAT-specific [P < 0.0001], control versus NET-specific [P < 0.0001]; AUC: control versus DAT-specific [P < 0.0001], control versus NET-specific [P < 0.0001]), and the multiantagonist cocktail further decreased IDT uptake (average slope: all block versus DAT-specific [P < 0.0001], all block versus NET-isolated; AUC: all block versus DAT-isolated, all block versus NET-specific [P = 0.0016]). Images and data in B–H are from n = 88–294 cells/group from 3 independent experiments. Statistical analysis in G was by Kruskal-Wallis test with Dunn’s test for multiple comparisons. Statistical analysis in H was by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparison’s test. (I) Nomifensine-sensitive uptake of tritiated dopamine (3H-DA) by cultured human macrophages shown as nM/min with a KM of approximately 3.2 μM. Data from 4 experiments from 2 donors. (J) Representative traces of inward currents on human macrophages measured via whole-cell voltage-clamp with vehicle, after amphetamine (AMPH), and after nomifensine, a DAT antagonist. The bath solution contained NET and SERT antagonists. (K) The DAT-mediated inward currents were calculated by subtracting the nomifensine current from the current in the no drug recording (baseline) or amphetamine recording (AMPH). (L) Bar graph compares basal and amphetamine-induced DAT-mediated inward current at –120mV (right, P = 0.01, Mann-Whitney U test). Data are from 9 experiments/group.