Probenecid is currently used as a nephroprotectant during clinical therapy with intravenous cidofovir in the treatment of cytomegalovirus retinitis in AIDS patients. and flavonolignans), around the uptake of is the concentration of flavonoid, is the percentage of the specific uptake of [14C]PAH, is the Hill coefficient. For each flavonoid, the IC50 value (expressed as mean S.D.) was decided from three individual experiments and each experiment experienced triplicate measurements. Flavonoid Cellular Uptake Studies The intracellular uptake of fisetin, luteolin, morin, and quercetin was examined with or without OAT1 inhibitor (probenecid, 200 values of molecular ion and product ion of luteolin were 285.2 and 132.8, respectively. The values of molecular ion and product ion of morin were 301.3 and 150.8, respectively. The values of parent ion and product ion of quercetin were 301.1 and 151.1, respectively. The lower limit of quantification of these four flavonoids was 1 ng/ml. The Eprotirome calibration curve was linear over the concentration range of 1C500 ng/ml for all those compounds. Statistical Analysis A commercially available bundle (SPSS 11.0; SPSS Inc., Chicago, IL) was used for all statistics. The differences between the mean values were analyzed for significance using a Students values were <0.05. Results Time-Dependent Uptake of [14C]PAH in LLC-PK1 Cells. As shown in Fig. 1, the uptake of [14C]PAH in OAT1-expressing cells was linear over a 5-minute time period. Therefore, we selected 5 minutes as an appropriate time for the following [14C]PAH uptake studies. It should be noted that after 5 minutes, the uptake of PAH decreased with the increase in the incubation time. Ueo et al. (2005) evaluated the time course of PAH in a different cell collection (HEK-hOAT1), and they also reported the time-dependent decrease of PAH. The reason for the time-dependent decrease of PAH uptake is usually unclear. Open in a separate windows Fig. 1. Mouse monoclonal to COX4I1 Time-dependent uptake of [14C]PAH in hOAT1-transfected LLC-PK1 cells and corresponding hOAT1-unfavorable control cells. Effects of Flavonoids on OAT1-Mediated [14C]PAH Uptake. To determine whether flavonoids have modulatory effects on hOAT1-mediated transport, uptake studies were conducted with PAH, a well known OAT1 substrate, in OAT1-expressing and OAT1-unfavorable LLC-PK1 cells in the presence or absence of flavonoids (50 < 0.001). Probenecid (200 < 0.001; percentage switch in mean value, ?95.4%), while no significant effect of probenecid was observed on [14C]PAH uptake in OAT1-negative cells (> 0.05; percentage switch in mean value, +9.4%). It should be noted that in the presence of probenecid, the intracellular Eprotirome concentration of PAH in OAT1-expressing cells decreased to a level that is very close to that observed in OAT1-unfavorable cells, indicating that OAT1 activity was completely inhibited with 200 < 0.001; percentage changes in mean value ranging from ?53.7 to ?94.8%). In contrast, [14C]PAH uptake in OAT1-unfavorable cells was slightly increased in the presence of these flavonoids (Table 1). The Eprotirome results indicated that these flavonoids are OAT1 inhibitors. Among these eight flavonoids, fisetin, luteolin, and morin produced the greatest inhibitory effect on OAT1, resulting in substantial decreases in [14C]PAH uptake (< 0.001; percentage decreases in mean Eprotirome value of 93.6, 94.8, and 92.3%, respectively), which is comparable to that caused by probenecid in OAT1-expressing cells. In addition, in the presence of fisetin, luteolin, and morin, the intracellular concentration of PAH in Eprotirome OAT1-expressing cells decreased to a level that is comparable to that observed in OAT1-unfavorable cells (Table 1), indicating that OAT1-mediated PAH transport was almost completely blocked with 50 = 4. The values in the Switch in Mean.