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.
The dopamine transporter (DAT) is a plasma membrane transporter that clears released dopamine from the synaptic cleft; therefore it is an important regulator of extracellular levels of dopamine. DAT is also the main molecular target of many drugs of abuse, most notably the psychostimulants cocaine and amphetamine. Amphetamine (AMPH) is a substrate of DAT and competes for reuptake with dopamine. Several groups have shown that acute AMPH treatments alter the function and the number of DAT on the cell membrane. However, no data is available regarding the long‐term effects of AMPH on DAT activity. Data collected in our laboratory suggest that parental AMPH exposure affects dopamine uptake in C. elegans dopaminergic neurons isolated from progeny. These results led us to hypothesize that reduced dopamine reuptake in subsequent generations, as a result of parental AMPH exposure, is caused by down‐regulation of DAT. In order to overcome the complexity of multicellular organisms and the challenges of primary cell isolation and culture, where very few cells differentiate into dopaminergic neurons, we investigated the long‐term effects caused by AMPH in simplified systems. Initial experiments were carried out in LLC‐PK1 cells, stably transfected with human DAT. Cells were pre‐exposed to AMPH for 15 hours and uptake assays, carried out after cells had doubled, showed a significant decrease in dopamine uptake compared to control (untreated cells). These data suggest that changes caused by AMPH were maintained after one cell cycle. We repeated the same experiment using the human neuroblastoma SH‐SY5Y cells which can be differentiated into authentic dopaminergic neurons. Prior to the uptake assay one group of undifferentiated SH‐SY5Y cells was exposed to AMPH for 15 hours and the other group was treated with control solution. The cells were then allowed to grow and cross generations before they were treated with Retinoic Acid (RA) to induce differentiation. Uptake assays carried out 5 days after RA initiated differentiation revealed a significant decrease in dopamine uptake in AMPH treated groups with respect to control. These results suggest that parental AMPH pretreatment down regulates the expression or activity of DAT. We are currently investigating whether chronic exposure to AMPH in progenitor cells alters DAT expression in daughter cells.Support or Funding InformationCOBRE P20 GM104360
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