LIPID PHOSPHATASES AND CANNABINOID BIOSYNTHESIS

脂质磷酸酶和大麻素生物合成

• 批准号: 6164357
• 负责人: Shelley B Hooks
• 金额: $ 1.79万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6164357
• 关键词: active sites; brain; brain metabolism; cannabinoid receptor; cell line; enzyme activity; enzyme mechanism; enzyme substrate; high performance liquid chromatography; in situ hybridization; isozymes; laboratory rat; lipid biosynthesis; liquid chromatography; mass spectrometry; messenger RNA; phosphatidate phosphatase; phospholipase C; protein localization; radiotracer; thin layer chromatography

Cannabinoid receptors mediate the psychotropic effects of marijuana, as well as those of therapeutic potential, including antinociceptive and immunomodulatory responses. Acylethanolamide (AEA) and 2-monoacyl glycerol (2-MAG) are endogenous cannabinomimetics, and are simply the nonphosphorylated counterparts of the LPA receptor agonists N-acyl ethyl phosphoric acid (NAEPA) and sn-2 LPA, respectively. Further, the structural similarity between the two classes of agonists suggests that sn-2 LPA and NAEPA may be cannabinoid precursors. The characterization of membrane bound phosphatidic acid phosphatases (PAP-2) suggests that these enzymes may provide the enzymatic link between the two classes of lipids. This proposal is designed to test the hypothesis that the biosynthesis of the cannabinomimetics 2-MAG and AEA occurs via PAP-2 mediated hydrolysis of LPA and NAEPA, and the latter is in turn the product of phospholipase C cleavage of N-acyl phosphatidylethanolamine (NAPE). First, the pharmacological and biochemical characterization of PAP-2 isoforms will be extended by determining if sn-2 LPA and NAEPA with long chain, polyunsaturated fatty acyl moities are PAP-2 substrates. Additionally, the predicted extracellular orientation of the PAP-2 active site will be tested experimentally, and structural analogs of PAP-2 substrates will be screened as blockers of PAP-2 activity. Secondly, mass spectroscopy will be employed to determine if NAEPA is detectable in rat brain and if it is present at sufficiently high quantities to support the proposed role as LPA receptor agonist and AEA precursor. Next, neuronal cell lines homogenates will be assayed for the presence of a PLC activity using radiolabelled NAPE as substrate. Finally, to examine whether the generation of cannabinoid ligands by PAP-2s may be physiologically relevant, in situ hybridization will be used to determine if the phosphatases are expressed in the same brain regions as cannabinoid receptors. Herein, a novel biosynthetic route for endogenous cannabinoid ligands is proposed, which may be functionally coupled to a second family of bioactive lipids, the LPA receptor agonists.

大麻素受体介导大麻的精神作用，作为 以及具有治疗潜力的那些，包括抗伤害感受和 免疫调节反应。 乙酰乙醇酰胺（AEA）和2-单一酰胺 甘油（2-MAG）是内源性大麻仪，仅仅是 LPA受体激动剂N-酰基乙基的非磷酸化对应物 磷酸（NAEPA）和SN-2 LPA分别。此外， 两类激动剂之间的结构相似性表明 SN-2 LPA和NAEPA可能是大麻素前体。 表征 膜结合的磷脂酸磷酸酶（PAP-2）表明 这些酶可以提供两类的酶促连接 脂质。 该建议旨在检验以下假设 大麻仪2-MAG和AEA的生物合成通过PAP-2发生 LPA和NAEPA的介导的水解，后者又是 N-酰基磷脂酰乙醇胺的磷脂酶C裂解的产物 （颈背）。 首先，药理学和生化表征 PAP-2同工型将通过确定SN-2 LPA和NAEPA是否扩展 与长链相关，多不饱和脂肪酰基是Pap-2 基材。另外，预测的细胞外向 PAP-2活动位点将进行实验测试，结构类似物 PAP-2底物的构造将被筛选为PAP-2活动的阻滞剂。 其次，将采用质谱来确定NAEPA是否为 在大鼠大脑中可检测到，如果存在足够高 支持拟议角色作为LPA受体激动剂和AEA的数量 前体。 接下来，将分析神经元细胞系匀浆 使用放射性标记的NAPE作为底物的PLC活性的存在。 最后，检查是否通过 PAP-2在生理上可能是相关的，原位杂交将是 用于确定磷酸酶是否在同一大脑中表达 区域为大麻素受体。这里是一种新颖的生物合成途径 提出了内源性大麻素配体，可能在功能上是 与第二个生物活性脂质家族结合，LPA受体 激动剂。