They share a number of overlapping substrates including metformin, cisplatin, lamivudine, and entecavir, as well as select 5-HT3 antagonist drugs [15,16,17,18,19,20,24]. > dolasetron (IC50: 85.4 M) and the inhibition of ASP+ uptake by MATE1 in order of potency was ondansetron (IC50: 0.1 M) > palonosetron = tropisetron > granisetron > dolasetron (IC50: 27.4 M). Ondansetron (0.5C20 M) inhibited the basolateral-to-apical transcellular transport of ASP+ up to 64%. Higher concentrations (10 and 20 M) of palonosetron, tropisetron, and dolasetron similarly reduced the transcellular transport of ASP+. In double-transfected OCT2-MATE1 MDCK cells, ondansetron at concentrations of 0.5 and 2.5 M caused significant intracellular accumulation of ASP+. Taken together, these data suggest that 5-HT3 antagonist drugs may inhibit the renal secretion of cationic drugs by interfering with OCT2 and/or MATE1 function. = 3). To ensure these conditions reflected active transport by each transporter, the IC50 values of cimetidine, a well-established OCT2 and MATE1 inhibitor, were determined (Figure 3 and Table 1). The IC50 for cimetidine was 24.5 4.0 M in OCT2-expressing cells and 0.23 0.2 M in MATE1-expressing cells, in agreement with published data showing inhibition of MATE1 at lower concentrations [18,20]. Cimetidine had no influence on ASP+ uptake in EV cells. Open in a separate window Figure 3 5-HT3 Antagonist Inhibition of ASP+ Transport in HEK293 Cells Overexpressing OCT2 and MATE1 following 5-HT3 Antagonist Treatment. Cells were incubated with ASP+ (10 M) in the presence and absence of various concentrations of 5-HT3 antagonist or the positive control inhibitor, cimetidine. Fluorescence was quantified and normalized to protein concentration. Fluorescence quantified in empty vector, OCT2, and MATE1 treated with vehicle control (no inhibitor) was set to 100%. Data are expressed as mean SE (= 3). * < 0.05 compared to the vehicle. Table 1 5-HT3 antagonist inhibition of in vitro ASP+ transport by OCT2 and MATE1 in HEK293 cells 1. = 3). 2.2. Inhibition of OCT2- and MATE1-Mediated Transport by Antiemetic Drug in HEK293 Cells Five different 5-HT3 antagonists (ondansetron, palonosetron, granisetron, Vibunazole tropisetron, and dolasetron) were evaluated for their inhibition of OCT2 and MATE1 transport in HEK293 cells using ASP+ as a substrate (Figure 3). A concentration-dependent decrease in ASP+ uptake was observed in OCT2- and MATE1-expressing cells in the presence of all five 5-HT3 antagonists tested across a range of concentrations. IC50 values for the inhibition of ASP+ accumulation by 5-HT3 antagonists using the concentration ranges tested are shown in Table 1. With the exception of granisetron, the other 5-HT3 antagonists inhibited MATE1 more potently than they did OCT2. OCT2-mediated transport was inhibited up to Vibunazole ~90% while MATE1-mediated transport was inhibited up to ~70% at the concentrations tested. Vibunazole In general, the uptake of ASP+ by EV cells was not altered to a large degree by the 5-HT3 antagonists. However, it was noted that palonosetron and tropisetron stimulated additional ASP+ uptake in EV cells and the highest concentration of granisetron caused a small decrease in ASP+ accumulation. 2.3. Characterization of the Transcellular Transport and Intracellular Accumulation of ASP+ in OCT2/MATE1-Expressing MDCK Cells To investigate the LATS1 combined contribution of OCT2 and MATE1 in transepithelial secretion, subsequent experiments were performed in MDCK cells that polarize with basolateral (OCT2) and apical (MATE1) surfaces. The expression of the OCT2 and MATE1 protein was confirmed in double-transfected MDCK cells using Western blotting (Figure 4A). The transcellular transport of the cationic probe substrate ASP+ (25 M) was tested in these cells using Transwell inserts. The basolateral-to-apical (B-to-A) transport of ASP+ was much greater (up to 2.8-fold at 120 min) than the apical-to-basolateral (A-to-B) transport in the OCT2/MATE1 double transfected cells (Figure 4B). The B-to-A/A-to-B efflux ratio at 120 min was estimated to be 2.7 for OCT2/MATE1 cells supporting the active secretion of ASP+. In contrast, control cells exhibited much lower ASP+ transport in both directions compared to OCT2/MATE1 cells. The B-to-A transport of ASP+ was only significantly higher compared to the A-to-B transport in control cells at 90 (1.3-fold) and 120 min (1.7-fold). All further inhibition assays were performed in the B-to-A direction. Open in a separate window Figure 4 Transcellular Flux of ASP+ in Control and OCT2/MATE1-Transfected MDCK cells. (A). Protein expression of OCT2 (~63 kDa,) and MATE1 (~54 kDa) in vector control MDCK cells (lanes 1C2) and OCT2/MATE1 double transfected (lanes 3C4). (B). Cells were incubated with ASP+ (25 M) in either apical or basolateral media for 120 min and fluorescence in apical or basolateral media was quantified (A-to-B: apical-to-basolateral; B-to-A: basolateral-to-apical). ? < 0.05 compared to A-to-B. ?.