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) 是从突触间隙去除释放的多巴胺的主要机制。 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病。特别令人感兴趣的是对药物滥用的监管，因为 DAT 是可卡因和安非他明等精神兴奋剂的主要作用部位。 DA D2 型受体的激活启动许多信号级联反应，包括 G 蛋白介导的磷脂酶 A2 (PLA2) 和腺苷酸环化酶的刺激。 PLA2 从膜磷脂中释放花生四烯酸 (AA)。 AA 信号级联与介导精神兴奋剂诱导的致敏有关，因此检查 AA 在 DAT 调节中的作用非常重要。在本提案中，我们重点关注以下假设：PLA2 产生的 AA 调节大鼠原代神经元 DA 细胞培养物中的 DAT 功能和细胞定位。该提案的具体目标是：1) 表征外源添加的 A.A 对 DAT 功能和定位的影响，2) 检查 PLA2 激活和内源 AA 的产生对 DAT 调节的影响，3) 确定 DA 受体的影响PLA2 介导的 DAT 调节的激活。拟议的实验将使用放射性配体摄取测定来测量 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，活细胞成像将用于可视化膜运输。这些研究对于确定 AA 信号级联在通过 DAT 调节多巴胺能传递中的作用非常重要。通过这些实验将获得有关药物滥用机制的更多了解。