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

Abstract

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 (IC(50): 3.2 +/- 0.8 microM). 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 microM), a metabolically stable analogue of AEA inhibited accumulation but arachidonic acid (10 microM) 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 microM URB597, a specific and potent fatty acid amide hydrolase inhibitor, and the cyclooxygenase inhibitor, indomethacin (5 microM) Live cell imaging revealed a significant redistribution of hDAT from the membrane to the cytosol in response to AEA treatment (10 microM; 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.

Fig. 1

The effect of AEA on ASP⁺ uptake in EM4 cells transiently transfected with YFP-hDAT. Confocal images presented in panel (a) shows an image of cells expressing YFP-hDAT. YFP-hDAT (green) and ASP⁺ (red) localization prior to (time 0: left panel) or 5 (middle panel) and 40 s (right panel) after ASP⁺ addition are shown. YFP-DAT is indicated by green fluorescence and ASP⁺ by red fluorescence. Co-localization of the two fluorphores is indicated by the merged images (yellow). These images show that ASP⁺ rapidly binds to hDAT located on the cell surface followed by a slower phase of intracellular ASP⁺ accumulation. (b) Time course of mean ASP⁺ uptake in control and AEA-treated cells (n = 9–11 cells/treatment) is shown. Note the initial rapid binding phase after ASP⁺ addition followed by the linear uptake phase. Addition of AEA (10 μM) decreases the rate of ASP⁺ uptake relative to vehicle (0.001% ethanol) values. The changes in DAT function is presented as the change in the slope of uptake measured for 1 min immediately before and 10 min after AEA addition. Each data point represents the mean ± SEM fluorescent intensity which was calculated as arbitrary fluorescent units (AFU). (c) The bar graph compares changes in the ASP⁺ accumulation rates calculated after 10 min application of vehicle (0.001% ethanol) or AEA. The data is from three different experiments. The numbers of cells are presented above each bar. * indicates statistically significant difference at the level of p < 0.01.

Fig. 2

AEA inhibits ASP⁺ accumulation in a time and concentration-dependent manner. (a) Time course of the effect of AEA on ASP⁺ accumulation. Each data point represents the means ± SEM of 9–11 cells. * indicates data points significantly different (at the level of p < 0.01) from the mean of the four control measurement taken before the addition of AEA. (b) Concentration-dependent effect of AEA on ASP⁺ accumulation. Each concentration of AEA was applied for 10 min and changes in ASP⁺ uptake rates are plotted. Data points represent the mean ± SEM of three different experiments. The number of cells used to calculate these values are presented above each data point.

Fig. 3

Lack of involvement of PTX-sensitive G-proteins or intracellular Ca²⁺ in mediating AEA-evoked functional hDAT regulation. (a) The effects of 10 min application of 10 μM AEA on slope values in control [(−))-PTX] and PTX pre-treated cells [(+) PTX)]. Data points represent the mean ± SEM of three different experiments. The numbers of cells are presented above each data point. * indicates statistically significant difference relative to vehicle at the level of p < 0.001. Grey bars for each condition indicate the mean slope values before the addition of vehicle or AEA (pre-treatment slope value). Black bars for each condition represent the mean slope values calculated 10 min after the addition of vehicle or AEA. Slope values normalized to the mean slope value calculated before the vehicle treatment in (−))-PTX group. (b) The effect of 10 min application of 10 μM AEA on intracellular Ca²⁺ levels in Fluo-3-AM (10 μM) loaded cells. Data points indicate the mean ± SEM fluorescent intensity (AFU) from 11 cells. Inset shows percent changes in the presence of AEA (10 min, 10 μM) or A23187 (5 min, 10 μM) compared to baseline values at 4 min. * indicates statistically significant difference at the level of p < 0.01.

Fig. 4

AEA inhibition of ASP⁺ accumulation rates in EM4 cells expressing hDAT is not mediated by AEA metabolites. (a) Methanandamide (mAEA) but not arachidonic acid (AA) inhibits ASP⁺ accumulation at equimolar AEA concentrations. (b) PMSF (0.2 mM) does not alter AEA inhibition of ASP⁺ accumulation rates. (c) URB597 (1 μM) does not alter AEA inhibition of ASP⁺ accumulation rates. (d) Indomethacin (5 μM) does not alter AEA inhibition of ASP⁺ accumulation rates. In the figure, the number of cells analyzed are presented above each bar. Results are expressed as the mean ± SEM Data are from three separate experiments and in each experiment at least three different dishes were analyzed. * indicates statistically significant difference at the level of p < 0.01.

Fig. 5

Confocal images of EM4 cells expressing YFP-hDAT. Images show the effect of 10-min anandamide treatment on the cellular distribution of YFP-hDAT expression in EM4 cells. The areas with enhanced internalization of YFP-hDAT fluorescence were indicated by arrows in the image shown on the left.

Fig. 6

Anandamide down-regulates hDAT by increasing the internalization and decreasing surface expression of hDAT in EM4 cells. (a) The ratio of intracellular to surface fluorescent intensity of YFP-hDAT expressed in EM4 cells before and after 10 min pre-incubation with 10 μM AEA. The numbers of cells analyzed are indicated above each bar. Results are expressed as the mean ± SEM. Data are from three separate experiments and in each experiment at least two different dishes were analyzed. * indicates statistically significant difference at the level of p < 0.01. (b) AEA pre-incubation decreases membrane ASP⁺ fluorescence in the cell surface without altering its affinity for hDAT expressed in EM4 cells. Cells were pre-treated with either vehicle (0.001% ethanol) or 10 μM AEA for 10 min at room temperature (22–24°C) in Krebs solution. Subsequently, 10 μM ASP⁺ (in the continuing presence of vehicle or AEA) was included and the corrected (see Methods) ASP⁺ fluorescence (excitation, 488 nm; emission, 607–652 nm) intensity (CAFU) was presented as a function of time. B_T: Total binding (maximal CAFU value). τ_1/2 = time to reach 50% of maximal CAFU. Each data point is the mean ± SEM from 8 to 11 cells/treatment.

Fig. 7

AEA application decreases DAT cell surface expression. (a) EM4 cells were transfected with FLAG-hDAT. After 48 h, cells were washed once with KRH buffer and incubated with biotinylating reagent with or without AEA (10 μM) for 10 min. Isolation of biotinylated DAT and detection of DAT were performed as described under Methods. Western blots of DAT and calnexin from total lysates, streptavidin beads eluates, and streptavidin beads unbound fractions are shown at the top. Each blot is a representative of four separate experiments. (b) Quantitative analysis of DAT band densities from four experiments (means ± SEM) is shown in bar presentation. *Significant changes in DAT band intensities were compared between vehicle and AEA treatment groups (p ≤ 0.05, one-way ANOVA with Bonferroni post hoc analysis).