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

早期在大脑(CB1)和外周组织(CB2)中发现了大麻素的特异性受体。作为这些受体的内源性配体，花生四烯酰乙醇胺和2-花生四烯基甘油(2-AG)是最近被发现的两种受体的内源性配体，目前正在积极研究中。本研究的目的是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发展。研究小组成员报道了以下实验结果：1)酶的研究：在脑和肝脏中发现了水解胺的氨基水解酶，该酶使氨基水解酶失去生物活性。S.Yamamoto等人对猪脑酶进行了部分纯化，证明了该酶具有广泛的底物专一性和酶反应的可逆性。用大鼠重组酶证实了花生四烯酸和乙醇胺的合成。值得注意的是，同样的酶可以水解2-AG。这是由I.Yamamoto等人报道的。这种酶被各种大麻素抑制。由于乙醇胺的Km很高，因此，氨基水解酶的反向反应的生理作用是值得怀疑的。Waku等人提出了磷脂酶D的分段化。和工藤等人。水解N-花生四烯基磷脂酰乙醇胺释放的脱氧核糖胺2)脱氧核糖胺分析方法的建立：建立了测定脱氢表雄胺荧光衍生物的高效液相色谱方法。3)生物活性研究：Waku等人。鉴定了2-AG为CB1的另一个内源性配体，并研究了其构效关系。工藤等人。发现花生胺对胶原诱导的血小板聚集有抑制作用。新的CB2激动剂不影响CB1，是由Miyamoto等人开发的。

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..