VR1 receptor-induced synthesis of anandamide in caveolae

VR1 受体诱导小窝内 anandamide 的合成

• 批准号: 6917934
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6917934
• 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）： 该提案的广泛长期目标是描述内源性大麻素 anandamide 生物合成/释放启动所涉及的可能机制。 Anandamide 已被证明通过充当大脑和外周大麻素受体（分别为 CB1 和 CB2）的激动剂，表现出与植物源大麻素相同的生理作用。更好地理解启动 anandamide 合成/释放的机制可能会揭示在多种情况下提供治疗益处的新药物靶点。有趣的是，anandamide 最近被证明通过激活 Ca2+ 通透性香草酸受体 (VR1)（TRP 受体家族的成员）来表现出内香草素活性。 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。待探索的假设是，VR1 受体存在于质膜的脂筏/小凹结构域中，并且它们被有害刺激以及可能的 anandamide 本身激活，从而刺激小凹中 anandamide 的合成/释放。具体目标是 (1) 确定 VR1 受体是否定位于质膜的特定区域，以及 (2) 确定 VR1 受体激活是否会刺激小窝中 anandamide 的合成/释放。一些 TRP 通道已被证明定位于细胞中富含小窝蛋白的区域，并且有证据表明 anandamide 的前体在富含小窝蛋白的细胞膜中富集。研究设计将试图利用亚细胞分级分离技术、蛋白质印迹分析和免疫荧光来证明 VR1 受体定位于质膜的小凹结构域。黄驼蛋白是荧光钙生物传感器的一种新用途，将被用作 VR1 诱导的活细胞中小窝/脂筏域 Ca 2. 水平增加的分子标记。 VR1 以 Ca2* 依赖性方式刺激 anandamide 合成/释放的能力将通过对 VR1 刺激后亚细胞级分或测定缓冲液中积累的 anandamide 进行定量来测量。 使用新兴的荧光生物传感器技术来探索与内源性大麻素生物合成相关的尚未确定的机制，将为了解大麻素修饰系统的细胞生物学提供新的机会。

项目成果

Mechanisms for recycling and biosynthesis of endogenous cannabinoids anandamide and 2-arachidonylglycerol.

内源性大麻素 anandamide 和 2-arachidonylglycerol 的回收和生物合成机制。

• DOI: 10.1111/j.1471-4159.2008.05659.x
• 发表时间: 2008
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: Placzek,EkaterinaA;Okamoto,Yasuo;Ueda,Natsuo;Barker,EricL
• 通讯作者: Barker,EricL

Activation of TRPC6 channels promotes endocannabinoid biosynthesis in neuronal CAD cells.

• DOI: 10.1016/j.neuint.2010.05.002
• 发表时间: 2010-08
• 期刊: NEUROCHEMISTRY INTERNATIONAL
• 影响因子: 4.2
• 作者: Bardell, Tamera K.;Barker, Eric L.
• 通讯作者: Barker, Eric L.

Membrane microdomains and metabolic pathways that define anandamide and 2-arachidonyl glycerol biosynthesis and breakdown.

定义 anandamide 和 2-arachidonyl 甘油生物合成和分解的膜微区和代谢途径。

• DOI: 10.1016/j.neuropharm.2008.07.047
• 发表时间: 2008
• 期刊: Neuropharmacology
• 影响因子: 4.7
• 作者: Placzek,EkaterinaA;Okamoto,Yasuo;Ueda,Natsuo;Barker,EricL
• 通讯作者: Barker,EricL

Lipidomic metabolism analysis of the endogenous cannabinoid anandamide (N-arachidonylethanolamide).

内源性大麻素 anandamide（N-花生四烯乙醇酰胺）的脂质代谢分析。

• DOI: 10.1016/j.jpba.2010.03.035
• 发表时间: 2010
• 期刊: Journal of pharmaceutical and biomedical analysis
• 影响因子: 3.4
• 作者: Placzek,EkaterinaA;Cooper,BruceR;Placzek,AndrewT;Chester,JuliaA;Davisson,VJo;Barker,EricL
• 通讯作者: Barker,EricL