VR1 receptor-induced synthesis of anandamide in caveolae

描述（由申请人提供）： 该提案的广泛长期目标是表征参与内源性大麻素anandamide生物合成/释放启动的可能机制。已显示大麻素通过作为脑和外周大麻素受体（分别为CB 1和CB 2）的激动剂而表现出与植物来源的大麻素相同的生理作用。更好地理解引发合成/释放anandamide的机制可能会揭示新的药物靶点，在多种条件下提供治疗益处。有趣的是，花生四烯酸最近已被证明通过激活钙渗透性香草素受体（VR1），TRP受体家族的成员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花生四烯酸合成/释放的潜在联系。要探讨的假设是，VR1受体中发现的脂筏/小窝结构域的质膜和它们的激活的有害刺激，并可能anandamide本身，刺激合成/释放的anandamide从小窝。具体目的是（1）确定VR1受体是否定位于质膜的特定结构域和（2）确定VR1受体活化是否会刺激小囊合成/释放花生四烯酸。几个TRP通道已被证明定位于细胞的小窝蛋白丰富的领域，有证据表明，花生四烯酸的前体富集在小窝蛋白丰富的膜。研究设计将试图表明，VR1受体定位在质膜的小窝域使用亚细胞分级分离技术，蛋白质印迹分析，和免疫荧光。一个新的使用的荧光钙生物传感器，黄cameleon蛋白将被开发作为一个分子标记的VR1诱导的钙2。活细胞中小窝/脂筏结构域的水平增加。VR1以Ca2 * 依赖性方式刺激花生四烯酸合成/释放的能力将通过在VR1刺激后对亚细胞级分或测定缓冲液中积累的花生四烯酸进行定量来测量。 使用荧光生物传感器的新兴技术，探索尚未确定的机制与内源性大麻素的生物合成将提供新的机会，了解大麻素修饰的系统的细胞生物学。

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.