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Studies on an enzyme hydrolyzing anandamide, an endogenous ligand for cannabinoid receptors

水解大麻素受体内源性配体大麻素的酶的研究

基本信息

批准号：
09670157
负责人：
UEDA Natsuo
金额：
$ 1.98万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670157/
关键词：
Cannabinoid Marijunana Anandamide 2-Arachidonoylglycerol Hydrolase Arachidonic acid Ethanolamine

项目摘要

Cannabinoids are psychoactive components contained in Cannabis sativa L.and their specific receptors are expressed in brain and other organs. Anandamide (arachidonoylethanolamide) was found as an endogenous ligand for the cannabinoid receptors, and reported to lose its cannabimimetic biological activities by the hydrolysis to arachidonic acid and ethanolamine. In order to elucidate the catalytic properties of anandamide amidohydrolase catalyzing this reaction, COS-7 cells were transfected with cDNA for the rat liver enzyme, and the recombinant enzyme was expressed. The enzyme showed not only the anandamide hydrolase activity but also its reverse reaction, namely, the anandamide synthase activity by the condensation of arachidonic acid with ethanolamine. Both the reactions did not occur in the control COS-7 cells. These results demonstrated the reversibility of the anandamide hydrolysis. However, due to a high Km for ethanolamine, the enzyme seemed to act as an anandamide hydrolase rather than an anandamide synthase under the physiological conditions. In addition to the amidase activity, the enzyme showed an esterase activity for methyl arachidonate.Therefore, I examined whether or not the enzyme hydrolyzes the ester bond of 2-arachidonoylglycerol (2-AG), another ligand for the cannabinoid receptors. The results showed that the hydrolysis of 2-AG proceeded about 4-fold faster than the anandamide hydrolysis with a Km value as low as 6muM and an optimal pH of IO.This hydrolytic reaction was hardly detected with the control cells. Two compounds inhibited the hydrolysis of both anandamide and 2-AG in parallel, and anandamide inhibited the hydrolysis of 2-AG dose-dependently. These results indicate that anandamide and 2-AG can be degraded by the same enzyme.

大麻素是大麻中含有的精神活性成分，其特定受体在大脑和其他器官中表达。 Anandamide（花生四烯酰乙醇酰胺）被发现是大麻素受体的内源性配体，据报道通过水解成花生四烯酸和乙醇胺而失去其大麻模拟生物活性。为了阐明anandamide酰胺水解酶催化该反应的催化特性，用大鼠肝酶的cDNA转染COS-7细胞，并表达重组酶。该酶不仅表现出花生四烯酸乙醇胺水解酶活性，而且还表现出其逆反应，即花生四烯酸与乙醇胺缩合的花生四烯酸乙醇胺合酶活性。这两种反应均未在对照 COS-7 细胞中发生。这些结果证明了花生四烯酸乙醇胺水解的可逆性。然而，由于乙醇胺的 Km 高，该酶在生理条件下似乎充当花生四烯乙醇胺水解酶而不是花生四烯乙醇胺合酶。除了酰胺酶活性外，该酶还显示出花生四烯酸甲酯的酯酶活性。因此，我检查了该酶是否水解大麻素受体的另一种配体 2-花生四烯酰甘油 (2-AG) 的酯键。结果表明，2-AG的水解速度比anandamide水解快约4倍，Km值低至6μM，最佳pH为IO。在对照细胞中几乎检测不到这种水解反应。两种化合物同时抑制 anandamide 和 2-AG 的水解，并且 anandamide 剂量依赖性地抑制 2-AG 的水解。这些结果表明 anandamide 和 2-AG 可以被相同的酶降解。