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

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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..