The ATP- and CoA-independent synthesis of arachidonoylethanolamide. A novel mechanism underlying the synthesis of the endogenous ligand of the cannabinoid receptor.

Recently, arachidonoylethanolamide was identified as the endogenous ligand of the delta 9 tetrahydrocannabinol receptor. Herein, we demonstrate that the synthesis of arachidonoylethanolamide is catalyzed by a novel CoA- and ATP-independent pathway, which is highly selective for arachidonic acid as the aliphatic constituent, is specific for ethanolamine as the polar moiety and occurs through a mechanism that utilizes a critical sulfhydryl residue. The production of arachidonoylethanolamide was calcium-independent and heat-labile and was selectively catalyzed by brain microsomal and cytosolic proteins among the tissues examined. Four independent lines of evidence demonstrate that the synthesis of arachidonoylethanolamide occurs through an ATP- and CoA-independent process. 1) The depletion of endogenous rabbit brain cytosolic or microsomal CoA and ATP by dialysis, or depletion of ATP by apyrase treatment, did not diminish the production of arachidonoylethanolamide; 2) the addition of exogenous ATP or CoA to cofactor depleted cytosol or microsomes did not stimulate arachidonoylethanolamide production; 3) the synthesis of arachidonoylethanolamide occurred in the absence of detectable arachidonoyl-CoA and did not correlate with either the amount of arachidonoyl-CoA in the incubation medium or the amount of arachidonoyl-CoA incorporated into polar and nonpolar lipids; 4) the addition of a 20-fold molar excess of unlabeled arachidonoyl-CoA to incubations containing [3H]arachidonic acid and ethanolamine did not attenuate the production of [3H]arachidonoylethanolamide by rabbit brain cytosolic or microsomal proteins. Collectively, these results demonstrate a novel chemical paradigm for the ATP- and CoA-independent conjugation of the carboxylate moiety of arachidonic acid to yield arachidonoylethanolamide, the first member of a new class of biologically active eicosanoid second messengers.