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.

中文翻译：

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长期接触安非他明会增加缺乏多巴胺转运蛋白的细胞细胞膜上的内源性人类多巴胺受体 2。

多巴胺 2 受体 (D2R) 是在突触前和突触后末梢表达的 G 蛋白偶联受体，在介导安非他明 (Amph) 的生理和行为效应中发挥重要作用。先前的研究表明，Amph对D2R的作用主要依赖于Amph通过多巴胺转运蛋白（DAT）强劲增加细胞外多巴胺的能力。这意味着 Amph 对 D2R 的影响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，因此可能独立于多巴胺和 DAT 影响 D2R 表达，例如在突触后末端。在这里，我们使用体外系统来研究 Amph 是否影响 HEK293 细胞中内源表达的人 D2R (hD2R) 的总表达、细胞分布和功能。通过进行蛋白质印迹实验，我们发现用 1 或 50 μM Amph 长时间处理会导致转染人 DAT (hDAT) 的细胞中内源性 hD2R 显着减少。另一方面，在缺乏 DAT 表达的细胞中，hD2R 介导的 cAMP 变化的定量、生物素化测定、蛋白质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，巴巴丁（βarrestin-βadaptin 相互作用的特异性抑制剂）可阻断 Amph 诱导的 hD2R 增加。总而言之，我们的数据表明，长期暴露于 Amph 会分别减少或增加表达或缺乏 hDAT 的 HEK293 细胞细胞膜上的内源性 hD2R。考虑到这种药物通常需要长期服用，在此期间会产生耐受性，我们的数据表明，即使没有 DAT 或多巴胺，Amph 仍然可以改变 D2R 的分布和功能。