Interaction of organic cation transporter 3 (SLC22A3) and amphetamine

Abstract

The organic cation transporter (OCT) 3 is widely expressed in various organs in humans, and involved in the disposition of many exogenous and endogenous compounds. Several lines of evidence have suggested that OCT3 expressed in the brain plays an important role in the regulation of neurotransmission. Relative to wild-type (WT) animals, Oct3 knockout (KO) mice have displayed altered behavioral and neurochemical responses to psychostimulants such as amphetamine (AMPH) and methamphetamine. In the present study, both in vitro and in vivo approaches were utilized to explore potential mechanisms underlying the disparate neuropharmacological effects observed following AMPH exposure in Oct3 KO mice. In vitro uptake studies conducted in OCT3 transfected cells indicated that dextroamphetamine (d-AMPH) is not a substrate of OCT3. However, OCT3 was determined to be a high-capacity and low-affinity transporter for the neurotransmitters dopamine (DA), norepinephrine (NE), and serotonin (5-HT). Inhibition studies demonstrated that d-AMPH exerts relatively weak inhibitory effects on the OCT3-mediated uptake of DA, NE, 5-HT, and the model OCT3 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide. The IC(50) values were determined to be 41.5 +/- 7.5 and 24.1 +/- 7.0 microM for inhibiting DA and 5-HT uptake, respectively, while 50% inhibition of NE and 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide uptake was not achieved by even the highest concentration of d-AMPH applied (100 microM). Furthermore, the disposition of d-AMPH in various tissues including the brain, liver, heart, kidney, muscle, intestine, spleen, testis, uterus, and plasma were determined in both male and female Oct3 KO and WT mice. No significant difference was observed between either genotypes or sex in all tested organs and tissues. Our findings suggest that OCT3 is not a prominent factor influencing the disposition of d-AMPH. Additionally, based upon the inhibitory potency observed in vitro, d-AMPH is unlikely to inhibit the uptake of monoamines mediated by OCT3 in the brain. Differentiated neuropharmacological effects of AMPHs noted between Oct3 KO and WT mice appear to be due to the absence of Oct3 mediated uptake of neurotransmitters in the KO mice.

Fig. 1

Uptake of dextroamphetamine (d-AMPH), 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP), and 1-methyl-4-phenylpyridinium (MPP⁺) by organic cation transporter 3 (OCT3). Intracellular accumulation of d-AMPH, 4-Di-1-ASP, and MPP⁺ in OCT3 and vector transfected cells were measured after incubation 30 min at 37 C. Data are represented as the mean with the error bar indicating the SEM of four independent measurements.

Fig. 2

OCT3 specific uptake of dopamine (DA), norepinephrine (NE), and serotonin (5-HT). Intracellular accumulation of the neurotransmitters DA, NE, and 5-HT in organic cation transporter 3 (OCT3) and vector transfected cells were determined utilizing an established high-performance liquid chromatography assay. OCT3 specific uptake was calculated by subtracting the uptake in vector cells from that in OCT3 transfected cells. Data are presented as mean ± SEM from three independent experiments.

Fig. 3

Dextroamphetamine (d-AMPH) inhibition of organic cation transporter 3-mediated transport of dopamine (DA), norepinephrine (NE), serotonin (5-HT), and 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP). Organic cation transporter3-mediated uptake of DA, NE, 5-HT, and 4-Di-1-ASP were measured in the absence and presence of various concentrations of d-AMPH. The uptake of the substrates without the treatment of d-AMPH was defined as 100%. Data are presented as mean ± SEM (n = 3).

Fig. 4

Pharmacokinetic study of dextroamphetamine (d-AMPH) in Oct3 knockout and wild-type mice. The concentrations of d-AMPH in plasma and the major organs including the brain, liver, heart, kidney, muscle, intestine, spleen, testis, and uterus were analyzed in both male and female Oct3 knockout and wild-type mice at 20 and 40 min after i.p. injection of 3.0 mg/kg d-AMPH. No statistic differences were found between either genotypes or sex. Data are presented as mean ± SEM (n = 3). OCT3, organic cation transporter 3.

Fig. 5

Disposition of dextroamphetamine (d-AMPH) in plasma and the brain of Oct3 knockout (KO) and wild-type mice. Plasma and brain concentrations of d-AMPH were measured 30 min post-administration of d-AMPH in Oct3 KO and wild-type males at a dose of 1.5 mg/kg. Each group contains six animals. Both plasma and brain concentrations did not differ between Oct3 KO and wild-type mice. Data are the mean with the error bars presenting the SEM. OCT3, organic cation transporter 3.