MOLECULAR ANALYSIS OF ENDOGENOUS CANNABINOID TRANSPORT

描述：（申请人摘要） 这项R21应用的广泛、长期目标是推进 对内源性大麻素调节分子机制的全面认识 通过推定的内源性大麻素摄取系统进行信号传导。内生 大麻素如大麻酰胺是脂肪酸衍生的分子， 代谢和信号终止似乎依赖于转运回到 释放细胞用于随后被细胞内酰胺水解酶分解 酵素因此，吸收过程将在动态中发挥关键作用， 内源性大麻模拟物活性的调节。具体目标是：（1） 分离并鉴定负责内源性蛋白质摄取的蛋白质， 大麻素，和（2）为了表征与大麻素相关的转运活性， 花生四烯酸摄取该项目与健康直接相关， 大麻素系统涉及调节各种生理和 病理过程，包括精神病、运动、炎症、血液 压力和疼痛。基于表明以下各项之间的功能相似性的数据， 花生四烯酸转运和长链脂肪酸摄取的研究 设计是为了探索各种脂肪酸的潜在贡献 使用异源表达的内源性大麻素转运中的转运蛋白 系统.此外，内源性大麻素摄取的药理学概况， 将测定天然和转染的细胞，并测定细胞的组织分布。 已鉴定的大麻素转运体研究，以更好地了解 转运蛋白在内源性大麻素信号传导中的作用。应采用的方法 包括分子克隆技术、转运蛋白表征 使用放射性标记的底物摄取测定， 内源性大麻素底物的积累，并在原位 杂交以揭示转运蛋白分布。这些研究将提供一个 未来研究的基础，研究与 内源性大麻素转运的结构/功能和调节。

DESCRIPTION: (Applicant's Abstract) The broad, long-term objective of this R21 application is to advance the overall understanding of molecular mechanisms regulating endogenous cannabinoid signaling by a putative endocannabinoid uptake system. The endogenous cannabinoids such as anandamide are fatty acid-derived molecules whose metabolism and signal termination appear to rely upon transport back into releasing cells for subsequent breakdown by an intracellular amidohydrolase enzyme. Thus, the uptake process would play a critical role in the dynamic regulation of endogenous cannabimimetic activity. The Specific Aims are (1) To isolate and identify the protein(s) responsible for the uptake of endogenous cannabinoids, and (2) To characterize the transport activity associated with anandamide uptake. This project has direct health-relatedness as the endogenous cannabinoid system has been implicated in modulating various physiological and pathological processes including psychosis, motor movement, inflammation, blood pressure, and pain. Based upon data indicating functional similarities between anandamide transport and the uptake of long-chain fatty acids, the Research Design is to explore the potential contributions of various fatty acid transporters in endocannabinoid transport using heterologous expression systems. In addition, the pharmacologic profile of endocannabinoid uptake in native and transfected cells will be determined, and the tissue distribution of the identified anandamide transporter investigated to better understand the role of transport proteins in endocannabinoid signaling. The Methods to be used include molecular cloning techniques, characterization of transport proteins using radiolabeled substrate uptake assays, determinations of intracellular accumulation of endocannabinoid substrates using mass spectrometry, and in situ hybridization to reveal transporter distribution. These studies will provide a basis for future studies examining the molecular mechanisms associated with both structure/function and regulation of endogenous cannabinoid transport.