Effects of arachidonic acid on the dopamine transporter

花生四烯酸对多巴胺转运蛋白的影响

• 批准号: 6648226
• 负责人: Dove Keith
• 金额: $ 3.3万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-11 至 2005-06-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6648226
• 关键词: G protein; SDS polyacrylamide gel electrophoresis; adenylate cyclase; arachidonate; biological signal transduction; cell migration; central nervous system stimulants; confocal scanning microscopy; dopamine receptor; dopamine transporter; drug abuse; immunocytochemistry; laboratory rat; neurochemistry; neuropsychology; phospholipase A2; predoctoral investigator; radiotracer; tissue /cell culture

DESCRIPTION (provided by applicant): The dopamine (DA) transporter (DAT) is the primary mechanism of removing released dopamine from the synaptic cleft. Improper DAT function or expression has been correlated with many clinical pathologies, including schizophrenia and Parkinson's disease. Of particular interest is regulation due to substance abuse since the DAT is the primary site of action of psychostimulants such as cocaine and amphetamines. Activation of the DA D2 type receptor initiates many signaling cascades, including G-protein mediated stimulation of phospholipase A2 (PLA2) and adenylyl cyclase. PLA2 liberates arachidonic acid (AA) from membrane phospholipids. The AA signaling cascade has been implicated in mediating psychostimulant induced sensitization, thus it is important to examine the role of AA in DAT regulation. In this proposal we focus on the hypothesis that PLA2 generated AA regulates DAT function and cellular localization in rat primary neuronal DA cell cultures. The specific aims of this proposal are: 1) to characterize the effect of exogenously added A.A on DAT function and localization, 2) examine the effect of PLA2 activation and production of endogenous AA on DAT regulation, and 3) determine the effect of DA receptor activation on PLA2 mediated DAT regulation. The proposed experiments will use radioligand uptake assays to measure AA mediated changes in DAT function. Cellular expression of the DAT will be determined using fluorescent confocal microscopy and live cell imaging will be used to visualize membrane trafficking. These studies will be important for determining the role of the AA signaling cascade in regulating dopaminergic transmission via the DAT. Additional understanding concerning mechanisms of substance abuse will be gained through these experiments.

描述（由申请人提供）： 多巴胺（DA）转运蛋白（DAT）是从突触裂隙中释放多巴胺的主要机制。 DAT功能或表达不当与许多临床病理相关，包括精神分裂症和帕金森氏病。特别令人感兴趣的是由于药物滥用而引起的调节，因为DAT是可卡因和苯丙胺等心理刺激剂的主要作用部位。 DA D2受体的激活启动了许多信号级联反应，包括G蛋白介导的磷脂酶A2（PLA2）和腺苷酸环化酶的刺激。 PLA2从膜磷脂中释放了花生四烯酸（AA）。 AA信号传导级联反应与介导心理刺激诱导的敏化有关，因此检查AA在DAT调节中的作用很重要。在此提案中，我们关注以下假设：PLA2产生的AA调节大鼠原发性神经元DA细胞培养物中的DAT功能和细胞定位。该提案的具体目的是：1）表征外源添加的A.A对DAT功能和本地化的影响，2）检查PLA2激活和内源性AA对DAT调节的影响； 3）确定DA受体激活对PLA2介导的DAT调节的影响。提出的实验将使用放射性摄取测定法来测量AA介导的DAT功能变化。 DAT的细胞表达将使用荧光共聚焦显微镜确定，并且活细胞成像将用于可视化膜运输。这些研究对于确定AA信号传导级联对通过DAT调节多巴胺能传播的作用至关重要。通过这些实验将获得有关滥用药物滥用机制的进一步理解。