Article Figures & Data
Figures
- Fig. 1.
Inhibition by methamphetamine, amphetamine, and p-OHMA of hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-K. Uptake of [14C]metformin (11 μM) in the absence and presence of inhibitor was measured in both transporter-expressing and control human embryonic kidney cells. Transporter-specific uptake was obtained by subtracting the uptake in vector-transfected cells from the uptake in transporter-expressing cells. Incubations were performed at 2, 0.5, 2, 5, and 0.5 minutes for of hOCT1 (A), hOCT2 (B), hOCT3 (C), hMATE1 (D), and hMATE2-K (E), respectively, which are within the linear initial rate of uptake. Activity in the absence of inhibitor (100%) corresponds to 28.2, 373, 60.2, 52.9, and 100 pmol/min/mg protein for hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-K, respectively. Each data point represents the mean ± S.D. from one representative experiment in triplicate. Curves from two additional independent repeats are displayed in Supplemental Fig. 2. The IC50 values shown in Table 1 are mean ± S.D. of the IC50 values from the three independent experiments.
- Fig. 2.
Uptake of 1 μM methamphetamine, amphetamine, and p-OHMA by hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-K. Uptake was measured after 5-minute incubation at 37°C. Data are illustrated as the mean ± S.D. from three independent experiments performed in triplicate. Uptake in transporter-expressing cells was compared with that in control cells (**P < 0.01; ***P < 0.001).
- Fig. 3.
Interactions of methamphetamine, amphetamine, and p-OHMA with renal hOAT1 and hOAT3. Effect of methamphetamine, amphetamine, and p-OHMA on para-aminohippurate (PAH) (1 µM) uptake by hOAT1 (A) and estrone sulfate (0.06 μM) uptake by hOAT3 (B) was measured at 1 minute after incubation at 37°C. Transporter-specific uptake was obtained by subtracting the uptake in vector-transfected cells from the uptake in transporter-expressing cells. The classic organic anion transporter inhibitor probenecid was used as the control. Activity in the absence of an inhibitor (100%) corresponded to 36.6 and 2.1 pmol/min/mg protein for PAH and estrone sulfate uptake, respectively. Uptake of methamphetamine (C), amphetamine (D), and p-OHMA (E) by hOAT1 and hOAT3 was measured after 5-minute incubation at 37°C. Data are illustrated as the mean ± S.D. from three independent experiments performed in triplicate. Uptake in transporter-expressing cells was compared with that in control cells (**P < 0.01; ***P < 0.001).
- Fig. 4.
Methamphetamine, amphetamine, and p-OHMA uptake kinetics by hOCTs. Concentration-dependent uptake of substrate was measured in both transporter-expressing and control cells at 37°C after 1-minute incubations. Transporter-specific uptake was obtained by subtracting the uptake in vector-transfected cells from the uptake in transporter-expressing cells. Panels display saturation curves (v vs. s) and Eadie-Hofstee transformations (v vs. v/s) for the kinetic data. Based on the Eadie-Hofstee plots, the kinetics for hOCT2-mediated methamphetamine transport (A) and hOCT- and hOCT3-mediated p-OHMA transport (C and E) were fitted with the standard Michaelis-Menten equation. hOCT2-mediated amphetamine transport (B) was fitted to a biphasic Michaelis-Menten equation (eq. 5). hOCT2-mediated p-OHMA transport (D) was fitted to the Michaelis-Menten equation with a Hill slope (eq. 4). Each data point represents the mean ± S.D. from one representative experiment in triplicate. Curves from two additional independent repeats are displayed in Supplemental Fig. 4. The kinetic parameters in Table 3 are mean ± S.D. of the values from three independent experiments.
- Fig. 5.
Methamphetamine and metabolite uptake kinetics by hMATE1 and hMATE2-K. Methamphetamine, amphetamine, and p-OHMA uptake in hMATE1-expresing cells was performed during the initial linear uptake time at 5, 2, and 2 minutes, respectively (A–C). Methamphetamine and amphetamine uptake in hMATE2-expressing cells was performed during initial linear uptake time at 5 minutes (D and E). Incubations were performed at 37°C and data were fitted with a Michaelis-Menten equation with a nonsaturable passive diffusion component (eq. 3). Each data point represents the mean ± S.D. from one representative experiment in triplicate. Curves from two additional independent repeats are displayed in Supplemental Fig. 5. The kinetic parameters in Table 4 are mean ± S.D. of the values from three independent experiments.
Tables
- TABLE 1
IC50 values of methamphetamine, amphetamine, and p-OHMA for hOCT1–3 and hMATE1/2-K determined by one-binding site fitting
Results represent mean ± S.D. of three independent experiments each run in triplicate.
Transporter One-Binding Site Methamphetamine Amphetamine p-OHMA IC50 Hill Slope IC50 Hill Slope IC50 Hill Slope μM μM μM hOCT1 21.1 ± 8.8 0.55 ± 0.02 96.7 ± 37 0.61 ± 0.28 12.0 ± 3.4 1.17 ± 0.14 hOCT2 15.0 ± 6.8 0.56 ± 0.07 20.3 ± 16.9 0.44 ± 0.1 83.8 ± 22.3 1.55 ± 0.32 hOCT3 300 ± 139 1.42 ± 0.68 363 ± 56.4 1.1 ± 0.2 44.4 ± 25.5 1.1 ± 0.26 hMATE1 107 ± 38 0.79 ± 0.09 94.0 ± 25.3 0.89 ± 0.2 59.1 ± 14.3 0.84 ± 0.12 hMATE2-K 84.3 ± 12.9 1.63 ± 0.14 158 ± 48 1.9 ± 0.6 234 ± 86.8 1.21 ± 0.11 - TABLE 2
EC50 values of methamphetamine and amphetamine for hOCT1 and hOCT2 determined by two-binding site fitting
The EC50 values were obtained by fitting inhibition data in Fig. 1 and Supplemental Fig. 2 using eq. 2 described in Materials and Methods with the fit shown in Supplemental Fig. 3. The last column lists the P values obtained by comparing eqs. 1 and 2 with an extra sum-of-squares F test. Results represent mean ± S.D. of three independent experiments each run in triplicate.
Inhibitor Two-Binding Site Two-Binding versus One-Binding Site Transporter EC50 Value High Affinity Low Affinity μM μM Methamphetamine hOCT1 5.29 ± 0.66 400 ± 229 P < 0.0001 hOCT2 1.21 ± 0.19 58.2 ± 23.4 P < 0.0001 Amphetamine hOCT1 NA NA P = 0.55 hOCT2 0.72 ± 0.29 145 ± 104 P < 0.0001 NA, not applicable.
- TABLE 3
Kinetic parameters of methamphetamine and metabolites determined from modeling the data in Fig. 4 and Supplemental Fig. 4
Models were chosen based on examination of Eadie-Hofstee plots. Methamphetamine was fit to a standard Michaelis-Menten equation. Amphetamine uptake kinetics was fit to a biphasic Michaelis-Menten equation (eq. 5). p-OHMA hOCT1- and hOCT3-mediated transport were fit to a standard Michaelis-Menten equation. Sigmoidal kinetics of hOCT2-mediated p-OHMA transport was obtained by fitting transporter-mediated uptake to the Michaelis-Menten equation with a Hill slope (eq. 5) for the substrate concentration and half-maximal transport concentration (K1/2 in place of Km). Results represent mean ± S.D. of three independent experiments each run in triplicate.
Compound Transporter Km1 Vmax1 Km2 Vmax2 μM pmol/mg/min μM pmol/mg/min Methamphetamine hOCT2 2.09 ± 0.88 49.7 ± 12.2 ND ND Amphetamine hOCT2 0.830 ± 0.55 34.6 ± 23.7 534 ± 350 853 ± 474 p-OHMA hOCT1 14.5 ± 8.7 312 ± 163 NA NA hOCT2 K1/2: 31.8 ± 9.3; H: 1.64 ± 0.15 1780 ± 718 NA NA hOCT3 53.3 ± 6.2 1290 ± 830 NA NA NA, not applicable; ND, not determined (due to high diffusion).
- TABLE 4
Apparent kinetic transport parameters for methamphetamine, amphetamine, and p-OHMA for hMATE1 and hMATE2-K from simultaneously modeling active and passive accumulation
Results represent Mean ± S.D. of three independent experiments each run in triplicate (Fig. 5; Supplemental Fig. 5).
Transporter Methamphetamine Amphetamine p-OHMA Km Vmax Km Vmax Km Vmax μM pmol/mg/min μM pmol/mg/min μM pmol/mg/min hMATE1 20.6 ± 4.5 86.6 ± 54 14.1 ± 4.9 238 ± 141 49.8 ± 26 257 ± 190 hMATE2-K 18.1 ± 11 97.7 ± 25.3 16.4 ± 12.2 92.9 ± 8.5 NA NA NA, not applicable.
Data Supplement
- Supplemental Data -
Supplemental Figure 1 - Metformin uptake by hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-K in Flp-in HEK293 cells in the presence or absence of the prototypical inhibitor cimetidine
Supplemental Figure 2 - Replica studies of methamphetamine, amphetamine, and p-OHMA inhibition of hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-K
Supplemental Figure 3 - Inhibition of hOCT1 and hOCT2 by methamphetamine and amphetamine fitted to a two binding site model
Supplemental Figure 4 - Replica studies on methamphetamine, amphetamine, and p-OHMA uptake kinetics by hOCTs
Supplemental Figure 5 - Replica studies of methamphetamine (A-D), amphetamine (E-F), and p-OHMA (I & J) uptake kinetics by hMATE1 and hMATE2-K
- Supplemental Data -
