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. Author manuscript; available in PMC: 2018 Feb 1.
Published in final edited form as: Neuropharmacology. 2016 Oct 12;113(Pt A):281–292. doi: 10.1016/j.neuropharm.2016.10.011

Figure 1. Regulation of 5-HT uptake (SERT activity) by KOR ligands in EM4 cells.

Figure 1

EM4 cells were transfected with hSERT plus either pcDNA3 (empty vector) or KOR. After 24 hrs, cells were treated with KOR ligands or the vehicle as described under “Materials and Methods”. Non-specific 5-HT uptake in the presence of 0.1 μM fluoxetine was subtracted from the total 5-HT uptake to obtain SERT mediated 5-HT transport. The results were expressed as percentage of 5-HT uptake by vehicle-E or vehicle-B (100%) treated cells and provided as mean ± S.E.M of three independent experiments performed in duplicates or triplicates. A, B. Dose dependent effect of KOR-agonists U69,593 and U50,488: Cells were pre-incubated with indicated concentrations of U69,593 or U50,488 or corresponding vehicle (Vehicle-E or Vehicle-B) for 15 min at 37°C followed by 5-HT uptake assays. Both U69,593 and U50,488 decreased SERT activity (One-way ANOVA: F(7,34) = 12.12, P <0.0001 for U69,595 and F(7,48) = 120.1, P <0.0001 for U50,488). Bonferroni’s post hoc test: *p <0.05; **p < 0,01; ***p <0.001 U69,593 or U50,488 versus Vehicle-E (U69,593) or Vehicle-B (U50,488). C, D. Time dependent effect of KOR-agonists U69,593 and U50,488: Cells were pre-incubated with vehicle or 10 μM U69,593 or U50,488 for indicated times prior to 5-HT uptake assay. (One-way ANOVA: F(7,40) = 4.94, P <0.0004 for U69,595 and F(4,30) = 30.38, P <0.0001 for U50,488). Bonferroni’s post hoc test: *p <0.05; **p < 0,01; ***p <0.001 U69,593 or U50,488 versus control vehicle (Vehicle-E (U69,593) or Vehicle-B (U50,488). E, Effect of KOR antagonist BNI on U69,593 or U50,488-mediated down regulation of SERT function: Pre-incubation of BNI (1 μM, 60 min) prior to U69,593 or U50,488 (10 μM, 15 min) exposure prevents U69,593 or U50,488-mediated SERT down regulation (One-way ANOVA: F(5,47) = 8.16, P <0.0001). Bonferroni’s post hoc test: **p <0.01; ***p < 0,001 U69,593 or U50,488 versus control vehicle (contained both ethanol and buffer vehicle); ^^^p <0.001 verses U69,593; #p < 0.05 versus U50,488). F. U69,593 and U50,488 decreases SERT activity through KOR activation: EM4 cells expressing SERT with out the expression of KOR were treated with U69,593 or U50,488 (10 μM) or corresponding vehicle (contained both ethanol and buffer vehicle) for 15 min at 37°C followed by 6 min 5-HT uptake assays. In the absence of KOR coexpression, U69,593 and U50,488 failed to decrease SERT-mediated 5-HT uptake in cells expressing SERT. (One-way ANOVA: F(5=2,15) = 0.12, P <0.089).