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.
Highlights
The neurotransmitters dopamine (DA) and norepinephrine (NE) belong to the catecholamine and phenylethylamine families of organic compounds and play an important role in fine-tuning a variety of animal behaviors such as movement, reward, cognition and attention
Using Elisa kits, we found that the concentration of AMPH in cells pretreated with AMPH was 0.013 ± 0.001 pM and this value was comparable to the amount of AMPH, 0.01 ± 0.003 pM, measured in cells pretreated with control solution which never encountered AMPH
Low concentrations of AMPH (5–30 mg/pill) are successfully used to treat patients affected with narcolepsy, chronic fatigue syndrome and those affected with attention deficit disorders
Summary
The neurotransmitters dopamine (DA) and norepinephrine (NE) belong to the catecholamine and phenylethylamine families of organic compounds and play an important role in fine-tuning a variety of animal behaviors such as movement, reward, cognition and attention. Most of the released catecholamines diffuse away from the synapse [1], a good portion binds to the DA and/ or NE transporters (DAT and NET) [2, 3]. This step prevents further stimulation of the receptors. When DAT moves DA inside the neurons, it causes cell-membrane depolarization affecting, neuronal excitability [4, 5]
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