Common drugs inhibit human organic cation transporter 1 (OCT1)-mediated neurotransmitter uptake

Abstract

The human organic cation transporter 1 (OCT1) is a polyspecific transporter involved in the uptake of positively charged and neutral small molecules in the liver. To date, few endogenous compounds have been identified as OCT1 substrates; more importantly, the effect of drugs on endogenous substrate transport has not been examined. In this study, we established monoamine neurotransmitters as substrates for OCT1, specifically characterizing serotonin transport in human embryonic kidney 293 cells. Kinetic analysis yielded a Km of 197 micomolar and a Vmax of 561 pmol/mg protein/minute for serotonin. Furthermore, we demonstrated that serotonin uptake was inhibited by diphenhydramine, fluoxetine, imatinib, and verapamil, with IC50 values in the low micromolar range. These results were recapitulated in primary human hepatocytes, suggesting that OCT1 plays a significant role in hepatic elimination of serotonin and that xenobiotics may alter the elimination of endogenous compounds as a result of interactions at the transporter level.

Fig. 1.

Transport of serotonin, dopamine, and norepinephrine by human OCT1. (A) HEK293 cells transiently transfected with empty vector and OCT1 plasmid were incubated with 100 µM serotonin, dopamine, or norepinephrine (0.8 µCi/ml) at 37°C for 5 minutes. (B) OCT-mediated uptake (net uptake) was determined by subtracting uptake into empty vector cells from that of OCT1-expressing cells. Mean + S.D. of three independent experiments is shown.

Fig. 2.

Kinetics of OCT1-mediated serotonin transport. Empty vector- and OCT1-transfected HEK293 cells were incubated with increasing concentrations of serotonin, ranging from 50 µM (0.8 µCi/ml) to 2 mM (2.0 µCi/ml), for 5 minutes at 37°C. Net uptake was fit to the Michaelis-Menten equation to obtain the affinity constant, K_m = 197 ± 42 µM, and maximum transport velocity, V_max = 561 ± 36 pmol/mg of protein/min. Values ± S.E.M. are the result of three independent experiments.

Fig. 3.

Inhibition of serotonin transport by common drugs. Transport of 200 µM serotonin (1.2 µCi/ml) was measured in HEK293 cells transfected with empty vector or OCT1 plasmid cDNA in the presence of 10 µM or 100 µM drug for 5 minutes at 37°C. Dimethylsulfoxide controls were included at concentrations equivalent to those of imatinib preparations. Mean + S.D. of net uptake is shown. *P < 0.05.

Fig. 4.

Concentration-dependent inhibition of serotonin uptake by OCT1. In HEK293 cells transfected with empty vector or OCT1, uptake of 100 µM serotonin (0.4 µCi/ml) was measured in the presence of increasing concentrations of (A) diphenhydramine, (B) fluoxetine, (C) imatinib, or (D) verapamil. Uptake values are expressed as percent of control and the mean + S.D. of three independent experiments were subjected to nonlinear regression analysis for IC₅₀ determination.

Fig. 5.

Serotonin transport in primary human hepatocytes. (A) Between 20 and 24 hours after plating, freshly isolated primary human hepatocytes were incubated for 5 minutes with 1.2 µCi/ml (radiolabeled only, 42 nM) serotonin. Carrier-mediated transport was inhibited by the presence of 1 mM unlabeled serotonin (right). (B) OCT1 expression was confirmed in hepatocytes used to measure serotonin uptake. 50 µg of protein isolated from hepatocytes was resolved by SDS-PAGE and blotted with anti-OCT1 antibody; 0.5 µg of protein isolated from HEK293 cells transfected with empty vector (HEK-EV) and OCT1 (HEK-OCT1) was included as control. (C) Drug-mediated inhibition of serotonin transport was conducted as in (A) in the presence of 10 µM and 100 µM diphenhydramine, fluoxetine, imatinib, or verapamil. Dimethylsulfoxide controls were included at sufficient concentrations to match those in imatinib preparations. Net uptake is represented as the mean + S.D. of three independent experiments.*P < 0.05.