Prolonged Amphetamine Treatments Cause Long-Term Decrease of Dopamine Uptake in Cultured Cells

Abstract

Amphetamine (AMPH) is a systemic stimulant used to treat a variety of diseases including Attention Deficit Hyperactive Disorder, narcolepsy and obesity. Previous data showed that by binding to catecholamine transporters, AMPH prevents the reuptake of the neurotransmitters dopamine (DA) and norepinephrine (NE). Because AMPH, either used therapeutically at final concentrations of 1-10 µM or abused as recreational drug (50-200 µM), is taken over long periods of time, we investigated the prolonged effects of this drug on the uptake of DA. We found that, in LLC-PK1 cells stably expressing the human DA transporter (hDAT), pretreatments with 1 or 50 µM AMPH caused significant reduction in DA uptake right after the 15-h pretreatment. Remarkably, after 50 but not 1 µM AMPH pretreatment, we observed a significant reduction in DA uptake also after one, two or three cell divisions. To test whether these long-term effects induced by AMPH where conserved in a model comparable to primordial neuronal cells and native neurons, we used the human neuroblastoma cell line SH-SY5Y cells, which were reported to endogenously express both hDAT and the NE transporter. Pretreatments with 50 µM AMPH caused a significant reduction of DA uptake both right after 15 h and 3 cell divisions followed by neuro-differentiation with retinoic acid (RA) for 5 days. Under these same conditions, AMPH did not change the intracellular concentrations of ATP, ROS and cell viability suggesting, therefore, that the reduction in DA uptake was not cause by AMPH-induced toxicity. Interestingly, while 1 µM AMPH did not cause long-term effects in the LLC-PK1 cells, in the SH-SY5Y cells, it decreased the DA uptake after one, two, but not three, cell divisions and 5-day RA differentiation. These data show that besides the well-known acute effects, AMPH can also produce long-term effects in vitro that are maintained during cell division and transmitted to the daughter cells.

Prolonged Amphetamine Exposures Increase the Endogenous Human Dopamine Receptors 2 at the Cellular Membrane in Cells Lacking the Dopamine Transporter

“…It should be noted that our imaging data showed that the 15-h treatment with Amph did not alter the Na + /K + -ATPase expression (yellow stain) with respect to control-treated cells (2798 ± 423 and 2353 ± 296 max fluorescence, respectively) suggesting, therefore, that Amph does not alter the expression of every protein at the cellular membrane. Moreover, as previously shown (Ferdous et al, 2020), we did not observe any obvious cytotoxic effect induced by the 15 h treatment with both Amph concentrations. In fact, cell morphology (Figures 2A-C) and the number of cells in controltreated samples (14 ± 3 cells/microslide) were comparable to those treated with 1 or 50 µM Amph (13 ± 2 or 13 ± 3 cells/microslide, respectively).…”