Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist

Abstract

Enzymatic activities have been identified which catalyze both the hydrolysis and synthesis of arachidonylethanolamide (anandamide). Anandamide was taken up by neuroblastoma and glioma cells in culture, but it did not accumulate since it was rapidly degraded by an amidase activity that resided mainly in the membrane fractions. This amidase activity was expressed in brain and the majority of cells and tissues tested. Phenylmethylsulfonyl fluoride (PMSF) was found to be a potent inhibitor of this amidase. A catalytic activity for the biosynthesis of anandamide from ethanolamine and arachidonic acid was readily apparent in incubations of rat brain homogenates. The stability of anandamide in serum and its rapid breakdown in cells and tissues are consistent with the observation that it is active when administered systemically, and its duration of action will be regulated by its rate of degradation in cells. The psychoactive marijuana plant-derived cannabinoid, Δ 9-tetrahydrocannabinol, and numerous synthetic derivatives have been shown to bind to a specific brain receptor [1,2,3,4]. Arachidonylethanolamide has been identified recently as a naturally occurring brain constituent that binds to this cannabinoid receptor [5]. Furthermore, using a series of behavioral tests to evaluate cannabinoid analogs [6], anandamide has been shown to be a cannabinoid receptor agonist exhibiting pharmacological activity in mice parallel to that of other psychotropic cannabinoids [7]. In this study, the uptake and breakdown of anandamide in neuroblastoma and glioma cell cultures were characterized. Subcellular fractions of these cultured cells and crude homogenates of other cultured cells and of rat tissues were assayed for enzymatic breakdown of arachidonylethanolamide to arachidonic acid. It was observed that phenylmethylsulfonyl flouride (PMSF) was a potent inhibitor of the breakdown activity (anandamide amidase). Employing ethanolamine and arachidonic acid as substrates, a synthetic activity (anandamide synthase) was readily demonstrated in crude homogenates of rat brain.