Abstract
Dopamine (DA) retention and drug-induced release kinetics were characterized in human embryonic kidney (HEK)-293 cells stably coexpressing the human DA transporter (hDAT) and human vesicular monoamine transporter (hVMAT2). Cofunction of hDAT and hVMAT2 caused greater retention of [3H]DA at 20 min (37°C), or 45 min (22°C) compared with cells that were treated with dihydrotetrabenazene (DHTB) to block the hVMAT2. In hDAT- and hVMAT2-coexpressing cells treated with DHTB during [3H]DA loading, methamphetamine (METH)-induced efflux was only 20% of preloaded [3H]DA, compared with 50 to 60% efflux in the absence of DHTB. Interestingly, the presence of DHTB (during release only) increased the potency and efficacy of METH at inducing [3H]DA release (without DHTB: EC50 = 33.8 μM, maximal release 51%; release with DHTB: EC50 = 3.2 μM, maximal release 61%), suggesting that the effects of METH and DHTB on vesicular storage are additive. High concentrations of lobeline induced a statistically significant release of [3H]DA from HEK-hDAT-hVMAT2 cells, but only in the absence of DHTB, suggesting an hVMAT2-mediated effect. Likewise, lobeline did not induce a significant release of [3H]DA from HEK-hDAT cells. The substrates DA and p-tyramine induced robust release of preloaded [3H]DA from cotransfected cells. Cocaine was somewhat effective at blocking substrate-induced [3H]DA efflux. These results suggest that coexpression of the hDAT and hVMAT2 can be used as a model system to distinguish functional pools of DA and to quantify differences in drug effects on DA disposition. In addition, cotransfected cells can be used to determine mechanisms of simultaneous drug interactions at multiple sites.
Footnotes
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This work was supported by the Department of Veterans Affairs Merit Review and Research Career Scientist Programs (to A.J.), a National Institutes of Health/Veterans Administration interagency agreement (to A.J. and A.E.), and by the Portland Alcohol Research Center (P50AA10760) (to A.E.).
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doi:10.1124/jpet.104.067314.
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ABBREVIATIONS: METH, methamphetamine; DA, dopamine; DAT, dopamine transporter; VMAT, vesicular monoamine transporter; hVMAT, human vesicular monoamine transporter; rVMAT, rat vesicular monoamine transporter; HEK, human embryonic kidney; DHTB, dihydrotetrabenazine; MSR, macrophage scavenger receptor; RTI-55, 3β-(4-iodophenyl)tropane-2β-carboxylic acid methyl ester; MAO, monoamine oxidase; COMT, catechol-o-methyl transferase; ANOVA, analysis of variance.
- Received February 19, 2004.
- Accepted April 21, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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