A receptor mechanism for methamphetamine action in dopamine transporter regulation in brain

Abstract

This study reveals a novel receptor mechanism for methamphetamine action in dopamine transporter (DAT) regulation. Trace amine-associated receptor 1 (TAAR1) is expressed in brain dopaminergic nuclei and is activated by methamphetamine in vitro. Here, we show that methamphetamine interaction with TAAR1 inhibits [(3)H]dopamine uptake, enhances or induces [(3)H]dopamine efflux, and triggers DAT internalization. In time course assays in which methamphetamine and [(3)H]dopamine were concurrently loaded into cells or synaptosomes or in pretreatment assays in which methamphetamine was washed away before [(3)H]dopamine loading, methamphetamine caused a distinct inhibition in [(3)H]dopamine uptake in TAAR1 + DAT-cotransfected cells and in wild-type mouse and rhesus monkey striatal synaptosomes. This distinct uptake inhibition was not observed in DAT-only transfected cells or in TAAR1 knockout mouse striatal synaptosomes. In [(3)H]dopamine efflux assays using the same cell and synaptosome preparations, methamphetamine enhanced [(3)H]dopamine efflux at a high loading concentration of [(3)H]dopamine (1 muM) or induced [(3)H]dopamine efflux at a low loading concentration of [(3)H]dopamine (10 nM) in a TAAR1-dependent manner. In DAT biotinylation assays using the same cell and synaptosome preparations, we observed that 1 muM methamphetamine induced DAT internalization in a TAAR1-dependent manner. All these TAAR1-mediated effects of methamphetamine were blocked by the protein kinase inhibitors H89 [N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline] and/or 2-{8-[(dimethylamino) methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-(1-methylindol-3-yl)maleimide (Ro32-0432), suggesting that methamphetamine interaction with TAAR1 triggers cellular phosphorylation cascades and leads to the observed effects of methamphetamine on DAT. These findings demonstrate a mediatory role of TAAR1 in methamphetamine action in DAT regulation and implicate this receptor as a potential target of therapeutics drugs for methamphetamine addiction.

Fig. 1.

TAAR1 response to methamphetamine and effects of concurrent loading of methamphetamine on [³H]dopamine uptake in transfected cells and brain synaptosomes. A, TAAR1 expression and its activation by methamphetamine in transfected cells. HEK293 and HEK293-derived stable TAAR1 cells were transiently transfected with human DAT to generate cell lines labeled as DAT and TAAR1-DAT, respectively. CRE-Luc and pGL4.73 were simultaneously introduced into the cells to monitor cAMP variation, which was measured as percentage change in relative light units by luciferase assay after exposure to vehicle or 1 μM methamphetamine. Data are values of mean ± S.E.M. for three independent experiments performed in triplicate. **, p < 0.01 by Student's t test. Robust TAAR1 expression in TAAR1-DAT cells was detected by SDS-PAGE and Western bolting. B and C, role of TAAR1 in methamphetamine-induced inhibition of [³H]dopamine uptake. Transfected cells and striatal synaptosomes were loaded with 10 nM [³H]dopamine alone ([³H]DA) or 10 nM [³H]dopamine plus 100 nM methamphetamine ([³H]DA + METH) for different time periods. Shown are the uptake curves over 10 min, generated in TAAR1-DAT and DAT cells (B), and in brain striatal synaptosomes generated from wild-type (WT) mouse, TAAR1 knockout (TAAR1-/-) mouse, and rhesus monkey (Rhesus) (C). Data are values of mean ± S.E.M. for three independent experiments performed in triplicate. D, TAAR1 expression levels detected by SDS-PAGE and Western blotting in the synaptosomes relative to untransfected HEK293 cells.

Fig. 2.

Effects of methamphetamine pretreatment on [³H]dopamine uptake in transfected cells and brain striatal synaptosomes. A and B, dose-response evaluation of the effect of methamphetamine on [³H]dopamine uptake. TAAR1-DAT and DAT cells (A) or wild-type (WT) and TAAR1 knockout (TAAR1-/-) mouse striatal synaptosomes (B) were pretreated with methamphetamine (1 nM–10 μM) for 10 min, washed twice, and then loaded with 10 nM [³H]dopamine for 5 min. *, p < 0.05; **, p < 0.01 by one-way ANOVA/Turkey post hoc test, compared with the baseline [DMEM (A) or buffer pretreatment (B)]. Note that methamphetamine was washed away after pretreatment and, consequently, is not in competition with [³H]dopamine during [³H]dopamine uploading. C and D, influence of phosphorylation inhibitors on the effects of methamphetamine. Cells and mouse striatal synaptosomes were pretreated with vehicle, 1 μM methamphetamine alone, or 1 μM methamphetamine plus 10 μM H89 or Ro32-0432 for 10 min, washed twice, and then loaded with 10 nM [³H]dopamine for 5 min. **, p < 0.01 by one-way ANOVA/Turkey post hoc test. All data shown are values of mean ± S.E.M. for three independent experiments performed in triplicate.

Fig. 3.

Effects of methamphetamine on [³H]dopamine efflux in transfected cells. A, DAT cells were preloaded with various concentrations of [³H]dopamine for 20 min, washed twice, and then exposed to vehicle or 1 μM methamphetamine for 30 min. **, p < 0.01 by one-way ANNOVA/Turkey post hoc test, compared with baseline. B, DAT and TAAR-DAT cells were preloaded with 1 μM[³H]dopamine for 20 min and then washed twice and treated with 1 μM methamphetamine for a time course of 1, 5, 10, 20, and 30 min. *, p < 0.05 by two-way ANOVA/Bonferroni post hoc test, compared with the level at the same time point in DAT cells. C, DAT and TAAR1-DAT cells were preloaded with 10 nM [³H]dopamine for 20 min, washed twice, and then treated with 1 μM methamphetamine for a time course of 1, 5, 10, 20, and 30 min. *, p < 0.05; **, p < 0.01 by two-way ANOVA/Bonferroni post hoc test, compared with the level at the same time point in DAT cells. All data shown are values of mean ± S.E.M. for three independent experiments performed in triplicate.

Fig. 4.

Characterization of methamphetamine-induced [³H]dopamine efflux at the preloading concentration of 1 μM[³H]dopamine. TAAR-DAT and DAT cells (A) and wild-type (WT) and TAAR1 knockout (TAAR1-/-) mouse striatal synaptosomes (B) were preloaded with 1 μM[³H]dopamine for 20 min, washed twice, and then exposed to vehicle, 1 μM methamphetamine alone, or 1 μM methamphetamine plus 10 μM MPH, H89, or Ro32-0432 for 30 min. **, p < 0.01 by two-way ANOVA/Bonferroni post hoc test. Data shown are values of mean ± S.E.M. for three independent experiments performed in triplicate.

Fig. 5.

Characterization of methamphetamine-induced [³H]dopamine efflux at the preloading concentration of 10 nM [³H]dopamine. A and B, dose-response evaluation of the effect of methamphetamine on [³H]dopamine efflux in TAAR-DAT and DAT cells (A) and wild-type (WT) and TAAR1 knockout (TAAR1-/-) mouse striatal synaptosomes (B). Cells and synaptosomes were preloaded with 10 nM [³H]dopamine for 20 min, washed twice, and then exposed to methamphetamine (1 nM∼100 μM) for 30 min. *, p < 0.05; **, p < 0.01 by one-way ANOVA/Turkey post hoc test, compared with baseline [DMEM (A) or buffer (B) treatment]. C to E, TAAR-DAT and DAT cells (C), wild-type (WT) and TAAR1 knockout (TAAR1-/-) mouse striatal synaptosomes (D), and rhesus monkey striatal synaptosomes (E) were preloaded with 10 nM [³H]dopamine for 20 min, washed twice, and then exposed to vehicle, 1 μM methamphetamine alone, or 1 μM methamphetamine plus 10 μM MPH, H89, or Ro32-0432 for 30 min. **, p < 0.01 by two-way ANOVA/Bonferroni post hoc test (C and D), or by one-way ANOVA/Turkey post hoc test (E). All data shown are values of mean ± S.E.M. for three independent experiments performed in triplicate.

Fig. 6.

Role of TAAR1 in methamphetamine-induced dopamine transporter internalization. A and B, transfected cells and synaptosomes were treated with 1 μM methamphetamine for different time periods before the biotinylation reaction. Shown are the changes in the level of biotinylated dopamine transporter in DAT versus TAAR1-DAT cells (A) and wild-type (WT) versus TAAR1 knockout (TAAR1-/-) mouse striatal synaptosomes (B) after methamphetamine exposure. *, p < 0.05; **, p < 0.01 by one-way ANOVA/Turkey post hoc test, compared with untreated cells or synaptosomes. Data shown are values of mean ± S.E.M. for three independent experiments performed in triplicate. C, shown are the changes in the level of biotinylated dopamine transporter rhesus monkey striatal synaptosomes after 1 μM methamphetamine treatment versus vehicle treatment. Data shown are values derived from a single experiment. D, representative data for the unbiotinylated dopamine transporter levels detected in the flow-through after 1 μM methamphetamine treatment in A and B. E, TAAR1-DAT cells and WT mouse striatal synaptosomes were exposed to vehicle, 1 μM methamphetamine, or 1 μM methamphetamine plus 10 μM H89 or 10 μM Ro32-0432 for 10 min before the biotinylation reaction. Data from a representative experiment (left) and analysis of mean ± S.E.M. for three independent experiments (right) is shown. **, p < 0.01 by one-way ANOVA/Turkey post hoc test.