Abstract
Inhibition of the metabolism of the endocannabinoids, anandamide (AEA) and 2-arachidonyl glycerol (2-AG), by their primary metabolic enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively, has the potential to increase understanding of the physiological functions of the endocannabinoid system. To date, selective inhibitors of FAAH, but not MAGL, have been developed. The purpose of this study was to determine the selectivity and efficacy of N-arachidonyl maleimide (NAM), a putative MAGL inhibitor, for modulation of the effects of 2-AG. Our results showed that NAM unmasked 2-AG activity in a tetrad of in vivo tests sensitive to the effects of cannabinoids in mice. The efficacy of 2-AG (and AEA) to produce hypothermia was reduced compared with Delta(9)-tetrahydrocannabinol; however, 2-AG differed from AEA by its lower efficacy for catalepsy. All tetrad effects were partially CB(1) receptor-mediated because they were attenuated (but not eliminated) by SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide HCl] and in CB(1)(-/-) mice. In vitro, NAM increased endogenous levels of 2-AG in the brain. Furthermore, NAM raised the potency of 2-AG, but not AEA, in agonist-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assay, a measure of G-protein activation. These results suggest that NAM is an MAGL inhibitor with in vivo and in vitro efficacy. NAM and other MAGL inhibitors are valuable tools to elucidate the biological functions of 2-AG and to examine the consequences of dysregulation of this endocannabinoid. In addition, NAM's unmasking of 2-AG effects that are only partially reversed by SR141716A offers support for the existence of non-CB(1), non-CB(2) cannabinoid receptors.