PDF(Pubmed)
Abstract：
The endocannabinoid system plays a critical role in modulating both peripheral and central nervous system function. Despite being present throughout the animal kingdom, there has been relatively little investigation of the endocannabinoid system beyond the traditional animal model systems. In this study, we report on the identification and characterization of a fatty acid aminohydrolase (FAAH) in the medicinal leech, Hirudo verbana. FAAH is the primary enzyme responsible for metabolizing the endocannabinoid signaling molecule arachidonoyl ethanolamide (anandamide or AEA) and therefore plays a critical role in regulating AEA levels in the nervous system. This Hirudo FAAH (HirFAAH) is expressed in the leech central nervous system (CNS) and is an orthologue of FAAH-2 observed in vertebrates. Functionally, HirFAAH has serine hydrolase activity based on activity-based protein profiling (ABPP) studies using the fluorophosphonate probe TAMRA-FP. HirFAAH also hydrolyzes arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a substrate specific to FAAH. Hydrolase activity during both the ABPP and AAMCA assays was eliminated by mutation at a conserved activity-binding site. Activity was also blocked by the known FAAH inhibitor, URB597. Treatment of Hirudo ganglia with URB597 potentiated synapses made by the pressure-sensitive mechanosensory neuron (P cell), mimicking the effects of exogenously applied AEA. The Hirudo CNS has been a useful system in which to study properties of endocannabinoid modulation of nociception relevant to vertebrates. Therefore, this characterization of HirFAAH is an important contribution to comparative studies of the endocannabinoid system.
摘要：
内源性大麻素系统在调节外周和中枢神经系统功能中起关键作用。尽管存在于整个动物王国，除了传统的动物模型系统外，对内源性大麻素系统的研究相对较少。在这项研究中,我们报告了药用水蛭中脂肪酸氨基水解酶（FAAH）的鉴定和表征，HirudoVerbana.FAAH是负责代谢内源性大麻素信号分子花生四酰基乙醇酰胺（anandamide或AEA）的主要酶，因此在调节神经系统中的AEA水平中起关键作用。这种水蛭FAAH（HirFAAH）在水蛭中枢神经系统（CNS）中表达，并且是在脊椎动物中观察到的FAAH-2的直向同源物。功能上,基于使用氟膦酸酯探针TAMRA-FP的基于活性的蛋白质谱分析(ABPP)研究,HirFAAH具有丝氨酸水解酶活性。HirFAAH还水解花生四酰基7-氨基，4-甲基香豆素酰胺(AAMCA),FAAH特有的底物。ABPP和AAMCA测定期间的水解酶活性通过保守活性结合位点处的突变而消除。活性也被已知的FAAH抑制剂阻断，URB597.用URB597治疗Hirudo神经节增强了由压敏机械感觉神经元（P细胞）产生的突触，模仿外源应用AEA的效果。HirudoCNS是研究与脊椎动物相关的伤害性感受的内源性大麻素调节特性的有用系统。因此,HirFAAH的这种表征是对内源性大麻素系统比较研究的重要贡献。
