Volz Trent J, Farnsworth Sarah J, Hanson Glen R, Fleckenstein Annette E
Department of Pharmacology and Toxicology, University of Utah, 30 South 2000 East, Room 201, Salt Lake City, UT 84112, USA.
J Neurosci Methods. 2009 Feb 15;177(1):177-82. doi: 10.1016/j.jneumeth.2008.10.004. Epub 2008 Oct 17.
In vivo methylphenidate (MPD) administration decreases vesicular monoamine transporter-2 (VMAT-2) immunoreactivity in membrane-associated vesicles isolated from the striata of treated rats while concurrently kinetically upregulating VMAT-2-mediated vesicular dopamine (DA) sequestration. The functional consequences of these MPD-induced effects include an increase in both vesicular DA content and exocytotic DA release. This report describes experiments designed to develop and validate an in vitro MPD model to further elucidate the molecular mechanism(s) underlying the effects of MPD on the VMAT-2 in membrane-associated vesicles. Method development experiments revealed that in vitro MPD incubation of striatal homogenates, but not striatal synaptosomes, increased DA transport velocities and decreased VMAT-2 immunoreactivity in membrane-associated vesicles. An incubation time of 30min with a MPD concentration of 10mM was optimal. Method validation experiments indicated that in vitro MPD incubation kinetically upregulated VMAT-2 in membrane-associated vesicles, increased vesicular DA content, and increased exocytotic DA release. These results reveal that the in vitro MPD incubation model successfully reproduced the salient features of in vivo MPD administration. This in vitro MPD incubation model may provide novel insights into the receptor-mediated mechanism(s) of action of in vivo MPD in the striatum as well as the physiological regulation of vesicular DA sequestration and synaptic transmission. Accordingly, this in vitro model may help to advance the treatment of disorders involving abnormal DA disposition including Parkinson's disease, attention-deficit hyperactivity disorder, and substance abuse.
体内给予哌醋甲酯（MPD）可降低从经治疗大鼠纹状体分离的膜相关囊泡中囊泡单胺转运体2（VMAT - 2）的免疫反应性，同时在动力学上上调VMAT - 2介导的囊泡多巴胺（DA）摄取。这些MPD诱导效应的功能后果包括囊泡DA含量增加和DA胞吐释放增加。本报告描述了旨在开发和验证体外MPD模型的实验，以进一步阐明MPD对膜相关囊泡中VMAT - 2作用的分子机制。方法开发实验表明，纹状体匀浆而非纹状体突触体的体外MPD孵育增加了膜相关囊泡中的DA转运速度并降低了VMAT - 2免疫反应性。MPD浓度为10mM孵育30分钟是最佳时间。方法验证实验表明，体外MPD孵育在动力学上上调了膜相关囊泡中的VMAT - 2，增加了囊泡DA含量，并增加了DA胞吐释放。这些结果表明，体外MPD孵育模型成功再现了体内给予MPD的显著特征。这种体外MPD孵育模型可能为体内MPD在纹状体中的受体介导作用机制以及囊泡DA摄取和突触传递的生理调节提供新的见解。因此，这种体外模型可能有助于推进对涉及DA异常处置的疾病的治疗，包括帕金森病、注意力缺陷多动障碍和药物滥用。
