Pharmacology of Dopamine Release by Amphetamine

安非他明释放多巴胺的药理学

• 批准号: 7847038
• 负责人: MARGARET E GNEGY
• 金额: $ 2.07万
• 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 2010-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7847038
• 关键词: Acute; Affect; Affinity; Alanine; Amphetamines; Aspartate; Behavior; Biochemical; Biological; Catecholamines; Cells; Cocaine; Confocal Microscopy; Corpus striatum structure; Disease; Dopamine; Drug Addiction; Event; Exposure to; Face; Goals; Isoenzymes; Knockout Mice; Knowledge; Laboratories; Lead; Manic; Measures; Mediating; Membrane; Microscopy; Modeling; Mus; Mutation; Neurons; PC12 Cells; PKC-betaII; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Play; Protein Kinase C; Protein Kinase C Inhibitor; Rattus; Regulation; Role; Series; Signal Transduction; Site; Small Interfering RNA; Subfamily lentivirinae; Synapses; Synaptosomes; Testing; Threonine; Time; Ventral Tegmental Area; Vesicle; citrate carrier; dopamine transporter; mutant; noradrenaline transporter; psychostimulant; response; small hairpin RNA; trafficking

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，并且是精神兴奋药物安非他明（AMPH）的主要作用位点。信号转导机制，尤其是改变蛋白激酶 C (PKC) 的机制，通过直接磷酸化和改变运输来深刻改变 DAT 活性。短时间暴露于 PKC 激活剂会引起 DAT 介导的外流。该提案的总体目标是检查磷酸化通过 DAT 对 AMPH 诱导的 DA 流出的调节。将测试以下假设：PKC 对 DAT 的作用可以通过考虑特定 PKC 同工酶和时间来区分，并且可以独立调节流入和流出。具体目标 1：检验 PKCbeta（尤其是 PKCbetaII）促进 AMPH 诱导的 DA 流出的假设。 PKC 同工酶敲除小鼠和针对特定 PKC 同工酶的 shRNA 将区分 PKCbetaII、PKCbetaI 和 PKCalpha 对 AMPH 刺激的 DA 流出的影响。针对 PKC 同工酶的活性 shRNA 将被整合到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以及共焦显微镜和全内反射荧光显微镜 (TIRFM)，我们将测试 AMPH 对增加质膜上含有 DAT 的囊泡具有快速作用的假设。该假设将在大鼠原代神经元培养物和内源性含有 EGFP-hDAT 的小鼠神经元培养物中进行测试。 PKCa 在 AMPH 快速作用中发挥作用的假设将得到检验。具体目标 3. 将测试以下假设：DAT 中的 T62 对于 DA 外流很重要，并且其磷酸化状态会改变向内转运蛋白处对 DA 的亲和力。将检查 T62 响应 AMPH 磷酸化的可能性。将使用 T62D-DAT 和 T62A-DAT 突变体研究 T62 在 DAT 贩运中的作用。 AMPH 对多巴胺转运蛋白的最初作用会引发一系列可能导致药物成瘾的事件。了解这些转运蛋白的作用机制和调节对于理解突触多巴胺的调节和控制 AMPH 的作用至关重要。此外，急性 AMPH 是躁狂症的有用模型，因此了解 AMPH 的作用机制有助于了解该疾病。