Abstract
Cannabinoids are psychoactive components in marijuana, and their specific membrane-bound receptors are present in the brain and other animal tissues. Arachidonoylethanolamide was found as an endogenous ligand for the cannabinoid receptors, and referred to as anandamide. Anandamide is enzymatically hydrolyzed to arachidonic acid and ethanolamine resulting in the loss of its biological activity. We partially purified "anandamide amidohydrolase" from microsomes of porcine brain. The purified enzyme also catalyzed the reverse reaction, namely, the condensation of arachidonic acid and ethanolamine to form anandamide. This reversibility was confirmed with the recombinant anandamide amidohydrolase of rat liver which was overexpressed in COS-7 cells. The recombinant enzyme showed a wide substrate specificity hydrolyzing primary amides and esters of unsaturated fatty acids as well as anandamide. 2-Arachidonoylglycerol, which was recently identified as another endogenous ligand for cannabinoid receptors, was also hydrolyzed efficiently by the same enzyme. The enzyme was distributed in various organs such as liver, brain, alimentary tract, and eye tissues. The enzyme activity of small intestine was underestimated by the presence of endogenous lipid inhibitors, and was increased by the acetone precipitation of the enzyme to remove lipids. In addition to the hydrolysis, anandamide was oxygenated at C-12 or C-15 of the arachidonate moiety by mammalian 12- and 15-1ipoxygenases. These results suggest the in vivo functions of these enzymes to regulate the biological activity of anandamide.
