Nawaratne Vindhya, McLaughlin Sean P, Mayer Felix P, Gichi Zayna, Mastriano Alyssa, Carvelli Lucia
Department of Biology, Harriet L. Wilkes Honors College, Florida Atlantic University, Jupiter, FL, United States.
Brain Institute, Florida Atlantic University, Jupiter, FL, United States.
Front Cell Neurosci. 2021 Aug 26;15:681539. doi: 10.3389/fncel.2021.681539. eCollection 2021.
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, , in post-synaptic terminals. Here we used an 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.
多巴胺2受体（D2R）是一种G蛋白偶联受体，在突触前和突触后终末均有表达，在介导苯丙胺（Amph）的生理和行为效应中起重要作用。先前的研究表明，Amph对D2R的作用主要依赖于其通过多巴胺转运体（DAT）强力增加细胞外多巴胺的能力。这意味着Amph对D2R的作用需要神经递质多巴胺。然而，由于其亲脂性，Amph可以穿过细胞膜，从而可能独立于多巴胺和DAT影响突触后终末中D2R的表达。在这里，我们使用一种系统来研究Amph是否会影响内源性表达于HEK293细胞中的人D2R（hD2R）的总表达、细胞分布和功能。通过进行蛋白质免疫印迹实验，我们发现用1或50μM的Amph进行长时间处理会导致转染了人DAT（hDAT）的细胞中内源性hD2R显著减少。另一方面，在缺乏DAT表达的细胞中，对hD2R介导的环磷酸腺苷（cAMP）变化的定量分析、生物素化分析、蛋白质免疫印迹和成像实验表明，用Amph处理15小时后，细胞膜上的hD2R增加。此外，成像数据表明，β抑制蛋白-β衔接蛋白相互作用的特异性抑制剂 barbardin 可阻断Amph诱导的hD2R增加。综合我们的数据表明，长时间暴露于Amph分别会使表达或缺乏hDAT的HEK293细胞中细胞膜上的内源性hD2R减少或增加。考虑到这种药物经常被长期服用，在此期间会产生耐受性，我们的数据表明，即使在没有DAT或多巴胺的情况下，Amph仍然可以改变D2R的分布和功能。
