Volz Trent J, Farnsworth Sarah J, King Jill L, Riddle Evan L, Hanson Glen R, Fleckenstein Annette E
Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah 84112, USA.
J Pharmacol Exp Ther. 2007 Nov;323(2):738-45. doi: 10.1124/jpet.107.126888. Epub 2007 Aug 10.
In vivo methylphenidate (MPD) administration increases vesicular monoamine transporter-2 (VMAT-2) immunoreactivity, VMAT-2-mediated dopamine (DA) transport, and DA content in a nonmembrane-associated (referred to herein as cytoplasmic) vesicular subcellular fraction purified from rat striatum: a phenomenon attributed to a redistribution of VMAT-2-associated vesicles within nerve terminals. In contrast, the present study elucidated the nature of, and the impact of MPD on, VMAT-2-associated vesicles that cofractionate with synaptosomal membranes after osmotic lysis (referred to herein as membrane-associated vesicles). Results revealed that, in striking contrast to the cytoplasmic vesicles, DA transport velocity versus substrate concentration curves in the membrane-associated vesicles were sigmoidal, suggesting positive cooperativity with respect to DA transport. Additionally, DA transport into membrane-associated vesicles was greater in total capacity in the presence of high DA concentrations than transport into cytoplasmic vesicles. Of potential therapeutic relevance, MPD increased DA transport into the membrane-associated vesicles despite rapidly decreasing (presumably by redistributing) VMAT-2 immunoreactivity in this fraction. Functional relevance was suggested by findings that MPD treatment increased both the DA content of the membrane-associated vesicle fraction and K(+)-stimulated DA release from striatal suspensions. In summary, the present data demonstrate the existence of a previously uncharacterized pool of membrane-associated VMAT-2-containing vesicles that displays novel transport kinetics, has a large sequestration capacity, and responds to in vivo pharmacological manipulation. These findings provide insight into both the regulation of vesicular DA sequestration and the mechanism of action of MPD, and they may have implications regarding treatment of disorders involving abnormal DA disposition, including Parkinson's disease and substance abuse.
体内给予哌醋甲酯（MPD）可增加从大鼠纹状体纯化的非膜相关（本文称为细胞质）囊泡亚细胞组分中的囊泡单胺转运体2（VMAT-2）免疫反应性、VMAT-2介导的多巴胺（DA）转运以及DA含量：这一现象归因于VMAT-2相关囊泡在神经末梢内的重新分布。相比之下，本研究阐明了MPD对渗透裂解后与突触体膜共分离的VMAT-2相关囊泡（本文称为膜相关囊泡）的性质及影响。结果显示，与细胞质囊泡形成鲜明对比的是，膜相关囊泡中DA转运速度与底物浓度曲线呈S形，表明DA转运存在正协同性。此外，在高DA浓度存在时，DA向膜相关囊泡的转运总能力大于向细胞质囊泡的转运。具有潜在治疗相关性的是，尽管MPD迅速降低了该组分中VMAT-2的免疫反应性（推测是通过重新分布），但它仍增加了DA向膜相关囊泡的转运。MPD治疗增加了膜相关囊泡组分中的DA含量以及纹状体悬浮液中K⁺刺激的DA释放，这些发现提示了其功能相关性。总之，本数据证明存在一个以前未被表征的含VMAT-2的膜相关囊泡池，其表现出新颖的转运动力学，具有很大的隔离能力，并对体内药理学操作有反应。这些发现为囊泡DA隔离的调节以及MPD的作用机制提供了见解，并且可能对涉及异常DA处置的疾病（包括帕金森病和药物滥用）的治疗有影响。
