VR1 receptor-induced synthesis of anandamide in caveolae

VR1 受体诱导小窝内 anandamide 的合成

• 批准号: 6806616
• 负责人: ERIC L BARKER
• 金额: $ 15.2万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6806616
• 关键词: anandamide; biosensor device; biosynthesis; calcium flux; calcium indicator; capsaicin; caveolas; cell membrane; drug receptors; eicosanoid metabolism; immunofluorescence technique; membrane channels; posttranslational modifications; protein localization; protein structure function; receptor expression; western blottings

DESCRIPTION (provided by applicant): The broad long-term objective of this proposal is to characterize possible mechanisms involved in the initiation of biosynthesis/release of the endocannabinoid anandamide. Anandamide has been shown to display the same physiological effects as plant derived cannabinoids by acting as an agonist at the brain and peripheral cannabinoid receptors (CB1 and CB2 respectively). A better understanding of the mechanisms that initiate the synthesis/release of anandamide may reveal new drug targets that provide therapeutic benefits in multiple conditions. Interestingly, anandamide has been recently shown to exhibit endovanilloid activity by activating the Ca2+-permeable vanilloid receptor (VR1), a member of the TRP family of receptors. The proposed studies examine the cellular localization of VR1 receptors and their potential link to anandamide synthesis/release. The hypothesis to be explored is that VR1 receptors are found in the lipid raft/caveolae domains of the plasma membrane and that their activation by noxious stimuli, and possibly anandamide itself, acts to stimulate the synthesis/release of anandamide from caveolae. The Specific Aims are (1) To determine if the VR1 receptor is localized to a specific domain of the plasma membrane and (2) To determine if VR1 receptor activation will stimulate the synthesis/release of anandamide from caveolae. Several TRP channels have been shown to localize to the caveolin-rich domains of cells, and evidence suggests that the precursors for anandamide are enriched in caveolin-rich membranes. The Research Design will seek to show that VR1 receptors are localized in the caveolae domains of the plasma membrane using subcellular fractionation techniques, Western blot analysis, and immunofluorescence. A novel use of the fluorescent calcium biosensor, yellow cameleon proteins will be exploited as a molecular marker of VR1-induced Ca 2. level increases in caveolae/lipid raft domains in living cells. The ability of VR1 to stimulate the synthesis/release of anandamide in a Ca2*-dependent fashion will be measured by quantification of anandamide accumulated in either the subcellular fractions or the assay buffer following VR1 stimulation. The use of the emerging technology of fluorescent biosensors to explore yet unidentified mechanisms associated with endocannabinoid biosynthesis will provide new opportunities to understand the cellular biology of the system modified by cannabinoids.

描述（由申请人提供）： 该提案的长期长期目标是表征与内源性大麻丁字母生物合成/释放启动有关的可能机制。已证明，通过充当大脑和周围大麻素受体（分别为CB1和CB2）的激动剂来表现出与植物衍生的大麻素相同的生理作用。更好地理解启动合成/释放anandamide的机制，可能会揭示在多种条件下提供治疗益处的新药物靶标。有趣的是，最近已证明Anandamide通过激活TRP受体家族的成员Ca2+可渗透的香草素受体（VR1）来表现出内烷基活性。 拟议的研究检查了VR1受体的细胞定位及其与Anandamide合成/释放的潜在联系。要探讨的假设是，在质膜的脂质筏/小窝域中发现了VR1受体，并且它们通过有害刺激及其可能是anandamide本身的激活，可以刺激阿诺丹胺从caveolae中刺激/释放。具体目的是（1）确定VR1受体是否定位于质膜的特定结构域，（2）确定VR1受体激活是否会刺激从小窝中刺激/释放Anandamide的合成/释放。已经证明了几个TRP通道可以定位于富含小窝蛋白的细胞结构域，证据表明，anandamide的前体富含富含小窝素的膜。该研究设计将试图证明使用亚细胞分级技术，蛋白质印迹分析和免疫荧光局力，VR1受体位于质膜的小窝结构域中。荧光钙生物传感器的新型使用，黄色的Cameleon蛋白将被用作VR1诱导的Ca 2的分子标记。在活细胞中，小窝/脂质筏结构域中的水平升高。 VR1在Ca2*依赖性时尚中刺激Anandamide的合成/释放的能力将通过定量在VR1刺激后的亚细胞分数或测定缓冲液中累积的Anandamide来衡量。 荧光生物传感器的新兴技术使用与内源性大麻素生物合成相关的未识别机制将为了解大麻素修饰的系统的细胞生物学提供新的机会。