Division of Basic Biomedical Sciences, Center for Brain and Behavior Research (CBBRe), Sanford School of Medicine, University of South Dakota, Vermillion, SD, 57069, USA.
Neurochem Res. 2024 Nov;49(11):3015-3029. doi: 10.1007/s11064-024-04216-7. Epub 2024 Aug 2.
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 traditional animal models. In this study, we report on the identification and characterization of a putative fatty acid amide hydrolase (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. mRNA encoding Hirudo FAAH (HirFAAH) is expressed in the leech central nervous system (CNS) and sequence analysis suggests that this 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 a mutation at a conserved catalytic serine. 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.
内源性大麻素系统在调节外周和中枢神经系统功能方面起着关键作用。尽管内源性大麻素系统存在于整个动物界，但除了传统的动物模型外，对其的研究相对较少。在这项研究中，我们报告了在医用水蛭 Hirudo verbana 中鉴定和表征一种假定的脂肪酸酰胺水解酶（FAAH）。FAAH 是负责代谢内源性大麻素信号分子花生四烯酰乙醇胺（大麻素或 AEA）的主要酶，因此在调节神经系统中 AEA 水平方面起着关键作用。编码 Hirudo FAAH（HirFAAH）的 mRNA 在水蛭中枢神经系统（CNS）中表达，序列分析表明这是脊椎动物中观察到的 FAAH-2 的同源物。基于使用氟膦酸盐探针 TAMRA-FP 的基于活性的蛋白质谱（ABPP）研究，HirFAAH 具有丝氨酸水解酶活性。HirFAAH 还水解花生四烯酰 7-氨基，4-甲基香豆素酰胺（AAMCA），这是 FAAH 的特异性底物。在 ABPP 和 AAMCA 测定中，通过突变保守的催化丝氨酸消除了水解酶活性。已知的 FAAH 抑制剂 URB597 也阻断了活性。URB597 处理 Hirudo 神经节增强了由压力敏感机械感觉神经元（P 细胞）形成的突触，模拟了外源性应用 AEA 的作用。Hirudo CNS 一直是一个有用的系统，可用于研究与脊椎动物有关的内源性大麻素对伤害感受的调制特性。因此，HirFAAH 的这种特征是对内源性大麻素系统比较研究的重要贡献。
