Department of Chemistry, University of Athens, Panepistimioupolis, 15771 Athens, Greece.
Platelets. 2009 Sep;20(6):376-85. doi: 10.1080/09537100903121813.
The endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA) are produced by neurons and other cells, including platelets, in a stimulus-dependent manner and act as signaling molecules; they are then inactivated through transport into cells followed by enzymatic degradation. A number of studies showed that monoacylglycerol lipase (MAGL) plays an important role in the degradation of 2-AG. In this study we investigated the enzymatic degradation of 2-acylglycerols in rabbit platelets and we characterized the responsible enzyme(s). [(3)H]2-AG and [(3)H]2-oleoylglycerol (2-OG) were both metabolized to [(3)H]glycerol and the respective fatty acid in a time and protein concentration-dependent manner, apparently by the action of MAGL activity. In the presence of the specific fatty acid amide hydrolase (FAAH) inhibitors URB597 and AM374, though, 2-OG hydrolysis was inhibited up to 55% in a concentration-dependent manner (IC(50) = 129.8 nM and 20.9 nM respectively). These results indicate the involvement of both MAGL and FAAH on 2-acylglycerol hydrolysis. MAGL was further characterized in the presence of URB597 and it was found that 2-monoacylglycerols were hydrolyzed in a time, pH and protein concentration-dependent manner and hydrolysis followed Michaelis-Menten kinetics, with an apparent K(M) of 0.11 microM and V(max) of 1.32 nmol/min*mg protein. Subcellular fractionation of platelet homogenate showed that MAGL activity was present in both the cytosolic and membrane fractions. In conclusion, the endocannabinoid 2-AG, as well as other 2-acylglycerols, are substrates of both FAAH and MAGL; the latter was characterized for the first time in platelets. In human platelets, under the same experimental conditions, the hydrolysis of 2-acylglycerols was higher and MAGL activity showed a different sensitivity against the inhibitors mentioned above. Finally, immunoblot analysis revealed the presence of MAGL, both in rabbit and human platelets, with a molecular mass of approximately 33 kDa.
内源性大麻素 2-花生四烯酰甘油（2-AG）和花生四烯酰乙醇胺（N-花生四烯酰乙醇胺，AEA）由神经元和其他细胞（包括血小板）以刺激依赖性方式产生，并作为信号分子发挥作用；然后，它们通过转运进入细胞并随后通过酶促降解而失活。多项研究表明，单酰基甘油脂肪酶（MAGL）在 2-AG 的降解中起着重要作用。在这项研究中，我们研究了兔血小板中 2-酰基甘油的酶促降解，并对负责的酶（s）进行了表征。[（3）H]2-AG 和 [（3）H]2-油酰甘油（2-OG）均以时间和蛋白浓度依赖的方式代谢为[（3）H]甘油和各自的脂肪酸，显然是通过 MAGL 活性的作用。然而，在存在特异性脂肪酸酰胺水解酶（FAAH）抑制剂 URB597 和 AM374 的情况下，2-OG 水解以浓度依赖性方式被抑制高达 55%（IC50 分别为 129.8 nM 和 20.9 nM）。这些结果表明 MAGL 和 FAAH 均参与 2-酰基甘油水解。在 URB597 的存在下进一步表征了 MAGL，发现 2-单酰甘油以时间、pH 和蛋白浓度依赖的方式水解，并遵循米氏动力学，表观 K（M）为 0.11 μM，V（max）为 1.32 nmol/min*mg 蛋白。血小板匀浆的亚细胞分级显示，MAGL 活性存在于胞质和膜部分中。总之，内源性大麻素 2-AG 以及其他 2-酰基甘油都是 FAAH 和 MAGL 的底物；后者在血小板中首次被表征。在相同的实验条件下，人血小板中 2-酰基甘油的水解更高，MAGL 活性对上述抑制剂的敏感性不同。最后，免疫印迹分析显示 MAGL 存在于兔和人血小板中，分子量约为 33 kDa。
