Pharmacology of Dopamine Release by Amphetamine

安非他明释放多巴胺的药理学

• 批准号: 7127155
• 负责人: MARGARET E GNEGY
• 金额: $ 37.54万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7127155
• 关键词: amphetamines; behavior test; cell membrane; dopamine; dopamine agonists; dopamine transporter; enzyme activity; fluorescence microscopy; gene targeting; genetically modified animals; isozymes; laboratory mouse; laboratory rat; neuropharmacology; phosphorylation; protein kinase C; synaptosomes; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): The dopamine transporter (DAT) is the fundamental regulator of dopamine (DA) content and duration in the synapse, and is a predominant site of action of the psychostimulant drug, amphetamine (AMPH). Signal transduction mechanisms, especially ones altering protein kinase C (PKC), profoundly alter DAT activity through direct phosphorylation and by altering trafficking. Short exposures to PKC activators elicit DAT- mediated efflux. The overall goal of this proposal is to examine the regulation of AMPH-induced DA efflux through DAT by phosphorylation. The hypothesis will be tested that actions of PKC on DAT can be distinguished by a consideration of specific PKC isozymes and by time and that influx and efflux can be regulated independently. Specific Aim 1: Test the hypothesis that PKCbeta, particularly PKCbetaII, promotes AMPH-induced DA efflux. PKC isozyme knockout mice and shRNA against specific PKC isozymes will differentiate the effects of PKCbetaII, PKCbetaI and PKCalpha on AMPH-stimulated DA efflux. Active shRNAs against the PKC isozymes will be incorporated into a lentivirus and injected into rat ventral tegmentum. AMPH-stimulated DA efflux and AMPH-stimulated locomotor behavior will be measured in the rats. Specific Aim 2. Using biochemical and cell biological approaches with confocal microscopy and total internal reflectance fluorescent microscopy (TIRFM), we will test the hypothesis that AMPH has a rapid effect to increase the DAT-containing vesicles at the plasmalemmal membrane. This hypothesis will be tested in rat primary neuronal cultures and neuronal cultures from mice endogenously containing EGFP-hDAT. The hypothesize that PKCa plays a role in the rapid effects of AMPH will be tested. Specific Aim 3. The hypothesis that T62 in DAT is important for DA efflux and that its phosphorylation status alters the affinity for DA at the inward-facing transporter will be tested. The possibility that T62 is phosphorylated in response to AMPH will be examined. The role of T62 in DAT trafficking will be investigated using the T62D-DAT and T62A-DAT mutants. The initial action of AMPH at the dopamine transporter initiates a series of events that can lead to drug addiction. Knowledge of the mechanism of action and regulation of these transporters are integral to understanding the regulation of synaptic dopamine and controlling the action of AMPH. In addition, acute AMPH serves as a useful model for mania so knowledge of AMPH's mechanism of action could aid in understanding of that disease.

描述（由申请人提供）：多巴胺转运蛋白（DAT）是突触中多巴胺（DA）含量和持续时间的基本调节剂，是精神兴奋剂药物安非他明（AMPH）的主要作用部位。信号转导机制，特别是改变蛋白激酶C（PKC）的机制，通过直接磷酸化和改变运输而深刻地改变DAT活性。短时间暴露于PKC激活剂引起DAT介导的外排。这个建议的总体目标是检查AMPH诱导的DA流出的调节通过DAT的磷酸化。将检验PKC对DAT的作用可以通过考虑特定的PKC同工酶和时间来区分，并且流入和流出可以独立调节的假设。具体目标1：检验PKC β（特别是PKC β II）促进AMPH诱导的DA外排的假设。PKC同工酶敲除小鼠和针对特定PKC同工酶的shRNA将区分PKC β II、PKC β I和PKCalpha对AMPH刺激的DA流出的影响。将针对PKC同工酶的活性shRNA掺入慢病毒中并注射到大鼠腹侧被盖中。将在大鼠中测量AMPH刺激的DA流出和AMPH刺激的运动行为。具体目标2。使用生物化学和细胞生物学方法与共聚焦显微镜和全内反射荧光显微镜（TIRFM），我们将测试的假设，AMPH具有快速的效果，以增加含DAT囊泡质膜。将在大鼠原代神经元培养物和来自内源性含有EGFP-hDAT的小鼠的神经元培养物中测试该假设。PKCa在AMPH的快速效应中起作用的假设将被检验。具体目标3。假设DAT中的T62对DA外排很重要，并且其磷酸化状态改变了内向转运蛋白对DA的亲和力。将检查T62响应于AMPH而磷酸化的可能性。将使用T62 D-DAT和T62 A-DAT突变体研究T62在DAT运输中的作用。 AMPH在多巴胺转运体上的最初作用引发了一系列可能导致药物成瘾的事件。了解这些转运体的作用机制和调节机制对于理解突触多巴胺的调节和控制AMPH的作用是不可或缺的。此外，急性AMPH作为躁狂症的有用模型，因此了解AMPH的作用机制有助于了解该疾病。