The endogenous cannabinoid, anandamide, inhibits dopamine transporter function by a receptor-independent mechanism

P>The endocannabinoid, anandamide (AEA), modulates the activity of the dopamine transporter (DAT) in heterologous cells and synaptosomal preparations. The cellular mechanisms mediating this effect are unknown. The present studies employed live cell imaging techniques and the fluorescent, high affinity DAT substrate, 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP+), to address this issue. AEA addition to EM4 cells expressing yellow fluorescent protein-tagged human DAT (hDAT) produced a concentration-dependent inhibition of ASP+ accumulation (IC50: 3.2 +/- 0.8 mu M). This effect occurred within 1 min after AEA addition and persisted for 10 min thereafter. Pertussis toxin did not attenuate the effects of AEA suggesting a mechanism independent of G(i)/G(o) coupled receptors. The amidohydrolase inhibitor, phenylmethylsulfonyl fluoride (0.2 mM), failed to alter the AEA-evoked inhibition of ASP+ accumulation. Methanandamide (10 mu M), a metabolically stable analogue of AEA inhibited accumulation but arachidonic acid (10 mu M) was without effect suggesting that the effects of AEA are not mediated by its metabolic products. The extent of AEA inhibition of ASP+ accumulation was not altered in cells pre-treated with 1 mu M URB597, a specific and potent fatty acid amide hydrolase inhibitor, and the cyclooxygenase inhibitor, indomethacin (5 mu M) Live cell imaging revealed a significant redistribution of hDAT from the membrane to the cytosol in response to AEA treatment (10 mu M; 10 min). Similarly biotinylation experiments revealed that the decrease in DAT function was associated with a reduction in hDAT cell surface expression. These results demonstrate that AEA modulates DAT function via a cannabinoid receptor-independent mechanism and suggest that AEA may produces this effect, in part, by modulating DAT trafficking.