Biosynthesis and metabolism of anandamide as an endogenous ligand for cannabinoid receptors

大麻素的特异性受体早先在大脑（CB1）和外周组织（CB2）中被发现。作为这些受体的内源性配体，花生四烯酰基乙醇胺（anandamide）和2-花生四烯酰基甘油（2-AG）最近被发现，目前正在积极研究中。本课题旨在研究这些内源性大麻素的生物合成和代谢，以及它们的检测方法和受体激动剂和拮抗剂的进展。研究小组的个别成员报告了以下实验结果。1)酶的研究：在大脑和肝脏中发现了能水解阿南达明并失去生物活性的阿南达明酰胺酰胺水解酶。S.Yamamoto等人对猪脑酶进行了部分纯化，证明该酶反应具有广泛的底物特异性和可逆性。用大鼠重组酶证实花生四烯酸和乙醇胺合成了花生四烯酰胺。值得注意的是，同样的酶也能水解2-AG。I.Yamamoto等人报道该酶被多种大麻素抑制。由于乙醇胺的高Km， anandamide酰胺水解酶逆反应的生理作用值得怀疑。Waku et al.和Kudo et al.提出磷脂酶D参与水解n -花生四烯酮酰磷脂乙醇胺，释放出anandamide。2)阿南达明胺检测方法的建立：建立了阿南达明胺荧光衍生物的高效液相色谱检测方法。同时对阿南达胺进行GC-MS分析。3)生物活性研究：Waku等人发现2-AG是CB1的另一种内源性配体，并研究了其作为CB1配体的构效关系。Kudo等人发现anandamide对胶原诱导的血小板聚集有抑制作用。Miyamoto等人开发了不影响CB1的CB2新激动剂。

Specific receptors for cannabinoids were earlier found in brain (CB1) and peripheral tissues (CB2). As endogenous ligands for these receptors, arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG) were recently identified, and they are now under active investigations. The purpose of this research project is to study on the biosynthesis and metabolism of these endocannabinoids together with the developments of their assays and receptor agonists and antagonists. The following experimental results have been reported by individual members of the research group.1) Studies on the enzymes : Anandamide amidohydrolase which hydrolyzed anandamide and lost its biological activities was found in brain and liver. The porcine brain enzyme was partially purified by S.Yamamoto et al., who demonstrated a broad substrate specificity and the reversibility of the enzyme reaction. The anandamide synthesis from arachidonic acid and ethanolamine was confirmed with a rat recombinant enzyme. It should be noted that the same enzyme hydrolyzes 2-AG.It was reported by I.Yamamoto et al. that the enzyme was inhibited by various cannabinoids. Because of a high Km for ethanolamine, the physiological role of the reverse reaction of anandamide amidohydrolase is questionable. The particifation of phospholipase D was suggested by Waku et al. and Kudo et al. to hydrolyze N-arachidonoylphosphatidy lethanolamine releasing anandamide.2) Development of anandamide assay : A sensitive HPLC method was developed for fluorescent derivative of anandamide. GC-MS analysis was also performed with anandamide.3) Studies on biological activities : Waku et al. identified 2-AG as another endogenous ligand for CB1, and studied its structure-activity relationship as CB1 ligand. Kudo et al. found the inhibitory activity of anandamide on the collagen-induced aggregation of platelets. New agonists for CB2 without effect on CB1 were developed by Miyamoto et al..