Abstract
The dopamine 2 receptors (D2R) are G-protein coupled receptors expressed both in pre- and post-synaptic terminals that play an important role in mediating the physiological and behavioral effects of amphetamine (Amph). Previous studies have indicated that the effects of Amph at the D2R mainly rely on the ability of Amph to robustly increase extracellular dopamine through the dopamine transporter (DAT). This implies that the effects of Amph on D2R require the neurotransmitter dopamine. However, because of its lipophilic nature, Amph can cross the cellular membrane and thus potentially affect D2R expression independently of dopamine and DAT, e.g., in post-synaptic terminals. Here we used an in vitro system to study whether Amph affects total expression, cellular distribution, and function of the human D2R (hD2R), endogenously expressed in HEK293 cells. By performing Western blot experiments, we found that prolonged treatments with 1 or 50 μM Amph cause a significant decrease of the endogenous hD2R in cells transfected with human DAT (hDAT). On the other hand, in cells lacking expression of DAT, quantification of the hD2R-mediated changes in cAMP, biotinylation assays, Western blots and imaging experiments demonstrated an increase of hD2R at the cellular membrane after 15-h treatments with Amph. Moreover, imaging data suggested that barbadin, a specific inhibitor of the βarrestin-βadaptin interaction, blocked the Amph-induced increase of hD2R. Taken together our data suggest that prolonged exposures to Amph decrease or increase the endogenous hD2R at the cellular membrane in HEK293 cells expressing or lacking hDAT, respectively. Considering that this drug is often consumed for prolonged periods, during which tolerance develops, our data suggest that even in absence of DAT or dopamine, Amph can still alter D2R distribution and function.
Highlights
Amphetamine (Amph) is a psychostimulant broadly prescribed as long-term therapy for attention deficit hyperactivity disorder (ADHD)
Using a cell line which endogenously expresses human dopamine 2 receptors (D2R), we found that when cells were transfected with human DAT (hDAT), 15 h of continuous exposure to Amph significantly decreased the expression of endogenous D2R
Similar to previous data obtained in Human embryonic kidney 293T (HEK293) cells transfected with D2R (Free et al, 2007), cell lysates from parental HEK293 cells probed with the human D2R (hD2R) antibody showed multiple bands (Figure 1A, panel 1)
Summary
Amphetamine (Amph) is a psychostimulant broadly prescribed as long-term therapy for attention deficit hyperactivity disorder (ADHD). In vivo and in vitro experiments have shown that proteins expressed in dopaminergic neurons, such as the dopamine transporter (DAT) and the type 2 dopamine receptors (D2R) are altered during and after Amph treatments (Sulzer, 2011; Ashok et al, 2017). Because dopamine is highly involved in the action of addictive drugs – all drugs of abuse increase synaptic dopamine – it is not surprising that this drug alters the function of DAT and D2R. These two proteins are key players of dopamine transmission. The intracellular accumulation of Amph causes several detrimental consequences e.g., reduction of the transporters at the cellular membrane (Saunders et al, 2000), depletion of vesicular dopamine stores and reverse transport of dopamine through DAT (Carvelli et al, 2010; Sulzer, 2011), causing an increase of DAT- and vesicle-mediated (Daberkow et al, 2013) dopamine release
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