LIPID PHOSPHATASES AND CANNABINOID BIOSYNTHESIS

大麻素受体介导大麻的精神作用，如 以及具有治疗潜力的药物，包括抗伤害性药物和 免疫调节反应。乙酰乙醇胺(AEA)和2-单酰基 甘油(2-MAG)是内源性大麻仿制药，简单地说就是 LPA受体激动剂N-酰乙基的非磷酸化对应物 磷酸(NAEPA)和sn-2 LPA。此外， 这两类激动剂的结构相似性表明 SN-2、LPA和NAEPA可能是大麻素前体。人物刻画 膜结合磷脂酸磷酸酶(PAP-2)的表达提示 这些酶可能提供两类酶之间的酶联系 脂类。这一提议旨在检验这样一种假设，即 大麻仿制物2-MAG和AEA的生物合成通过PAP-2进行 介导LPA和NAEPA的水解，而NAEPA又是 N-酰基磷脂酰乙醇胺的磷脂酶C裂解产物 (后颈)。首先，药理和生化特性 PAP-2亚型将通过确定sn-2 LPA和NAEPA 对于长链，多不饱和脂肪酰基是PAP-2 底物。此外，预测的细胞外取向 将对PAP-2活性中心进行实验测试，并进行结构模拟 的PAP-2底物将被筛选为PAP-2活性的阻断剂。 其次，将使用质谱学来确定NAEPA是否 在大鼠大脑中可检测到，如果存在足够高的 支持LPA受体激动剂和AEA作用的数量 前身。接下来，将对神经细胞系匀浆进行检测 以放射性标记的NAPE为底物的PLC活性的存在。 最后，为了检查大麻素配体的产生是否通过 PAP-2可能与生理相关，原位杂交将是 用于确定磷酸酶是否在同一个大脑中表达 大麻素受体的区域。在此，一种新的生物合成路线 内源性大麻素配体被提出，这可能是功能上的 与第二个生物活性脂类家族偶联，LPA受体 激动剂。

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.