VR1 receptor-induced synthesis of anandamide in caveolae

描述(由申请人提供)： 这项提议的广泛的长期目标是描述参与启动内源性大麻素的生物合成/释放的可能机制。阿南达胺已被证明通过在大脑和外周大麻素受体(分别为CB1和CB2)上起激动剂的作用，显示出与植物来源的大麻素相同的生理作用。更好地了解启动花生胺合成/释放的机制可能会揭示在多种情况下提供治疗益处的新的药物靶点。有趣的是，阿南达胺最近被证明通过激活钙离子可渗透的香草素受体(VR1)显示出血管内皮细胞的活性，VR1是色氨酸受体家族的成员。这项拟议的研究考察了VR1受体的细胞定位及其与ANANDIAME合成/释放的潜在联系。有待探索的假设是，VR1受体存在于质膜的脂筏/小凹结构域中，它们被伤害性刺激激活，可能还有ANANDAME本身，起到刺激小窝合成/释放ANANDAME的作用。其具体目的是(1)确定VR1受体是否定位于质膜的特定区域；(2)确定VR1受体激活是否会刺激腺泡合成/释放花生胺。几个Trp通道已被证明定位于细胞富含小窝蛋白的区域，证据表明，花青胺的前体在富含小窝蛋白的膜上富含。这项研究设计将利用亚细胞分离技术、Western印迹分析和免疫荧光技术，试图证明VR1受体定位于质膜的小凹区域。作为钙荧光生物传感器的一种新用途，黄色骆驼蛋白将被开发为VR1诱导活细胞内小凹/脂筏结构域钙水平升高的分子标记。VR1以依赖于钙离子的方式刺激ANANDAME合成/释放的能力将通过量化VR1刺激后亚细胞组分或分析缓冲液中积累的ANANDAME来衡量。利用新兴的荧光生物传感器技术探索与内源性大麻素生物合成相关的尚未确定的机制，将为理解被大麻素修饰的系统的细胞生物学提供新的机会。

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.