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 [3H]dopamine uptake, enhances or induces [3H]dopamine efflux, and triggers DAT internalization. In time course assays in which methamphetamine and [3H]dopamine were concurrently loaded into cells or synaptosomes or in pretreatment assays in which methamphetamine was washed away before [3H]dopamine loading, methamphetamine caused a distinct inhibition in [3H]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 [3H]dopamine efflux assays using the same cell and synaptosome preparations, methamphetamine enhanced [3H]dopamine efflux at a high loading concentration of [3H]dopamine (1 μM) or induced [3H]dopamine efflux at a low loading concentration of [3H]dopamine (10 nM) in a TAAR1-dependent manner. In DAT biotinylation assays using the same cell and synaptosome preparations, we observed that 1 μM 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.
Footnotes
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This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA022323, DA016606]; and by the National Institutes of Health National Center for Research Resources [Grant RR00168].
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.109.153775.
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ABBREVIATIONS: TAAR1, trace amine-associated receptor 1; DAT, dopamine transporter; DMEM, Dulbecco's modified Eagle's medium; CRE, cAMP-response element; Luc, luciferase; PLB, passive lysis buffer; PK, protein kinase; PAGE, polyacrylamide gel electrophoresis; ANOVA, analysis of variance; HEK, human embryonic kidney; MPH, methylphenidate; Ro32-0432, 2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-(1-methylindol-3-yl)maleimide; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received March 18, 2009.
- Accepted April 10, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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