title: Intracellular Ca2+ regulates amphetamine-induced dopamine efflux and currents mediated by the human dopamine transporter.
abstract: Although it is clear that amphetamine-induced dopamine (DA) release mediated by the dopamine transporter (DAT) is integral to the behavioral actions of this psychostimulant, the mechanism of this release is not clear. In this study, we explored the requirement for intracellular Ca(2+) in amphetamine-induced DA efflux and currents mediated by the human DAT. The patch-clamp technique in the whole-cell configuration was used on Na(+) and DA-preloaded human embryonic kidney 293 cells stably transfected with the human DAT (hDAT cells). Chelation of intracellular Ca(2+) by inclusion of 50 microM BAPTA in the whole-cell pipette reduced the voltage-dependent amphetamine-induced hDAT current, with the greatest effect seen at positive voltages. Likewise, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) reduced amphetamine-induced DA efflux as measured by amperometry. Furthermore, preincubation of the cells with 50 microM BAPTA acetoxy methyl ester (AM) or thapsigargin also blocked amphetamine-induced release of preloaded N-methyl-4-[(3)H]phenylpyridinium from superfused hDAT cells. BAPTA-AM also reduced DA release from striatal synaptosomes. Amphetamine also led to an increase in intracellular Ca(2+) that was blocked by prior treatment with 5 microM thapsigargin or 10 microM cocaine. These studies demonstrate that amphetamine-induced DAT-mediated currents and substrate efflux require internal Ca(2+) and that amphetamine can stimulate dopamine efflux by regulating cytoplasmic Ca(2+) levels through its interaction with DAT.
authors.affiliation: Department of Pharmacology, 2220E MSRB III, University of Michigan Medical School, Ann Arbor, MI 48109-0632, USA. pgnegy@umich.edu
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Gnegy
authors.forename: Habibeh
authors.initials: H
authors.lastname: Khoshbouei
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Berg
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
authors.forename: William P
authors.initials: WP
authors.lastname: Clarke
authors.forename: Minjia
authors.initials: M
authors.lastname: Zhang
authors.forename: Aurelio
authors.initials: A
authors.lastname: Galli
mesh_terms: D015655:1-Methyl-4-phenylpyridinium / Q000494:pharmacology; D000662:Amphetamines / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D004594:Electrophysiology; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051381:Rats; D014162:Transfection; D014316:Tritium
chemical_list: D000662:Amphetamines; D002614:Chelating Agents; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; C493216:Slc6a3 protein, rat; D014316:Tritium; D015655:1-Methyl-4-phenylpyridinium; D002118:Calcium; D004298:Dopamine