The novel endogenous cannabinoid 2-arachidonoylglycerol (2-AG) was rapidly inactivated by intact rat basophilic leukaemia (RBL-2H3) and mouse neuroblastoma (N18TG2) cells through diffusion/hydrolysis/reacylation processes. The hydrolysis of 2-AG was inhibited by typical esterase inhibitors and by more specific blockers of ‘fatty acid amide hydrolase ’ (FAAH), the enzyme catalysing the hydrolysis of the other ‘endocannabinoid ’, anandamide (AEA). No evidence for a facilitated-diffusion process was found. A 2-AG-hydrolysing activity was detected in homogenates from both cell lines, with the highest levels in membrane fractions. It exhibited an optimal pH at 10, and recognized both 2- and 1(3)- isomers of monoarachidonoylglycerol with similar efficiencies. The apparent Km and Vmax values for [3H]2-AG hydrolysis were 91 µM and 29 µM and 2.4 and 1.8 nmol·min-1·mg of protein-1 respectively in N18TG2 and RBL-2H3 cells. [3H]2-AG hydrolysis was inhibited by Cu2+, Zn2+ and p-hydroxymercuribenzoate, and by 2- or 1(3)-monolinoleoyl- and -linolenoyl-glycerols, but not by the oleoyl, palmitoyl and myristoyl congeners. Purified fractions from solubilized membrane proteins catalysed, at pH 9.5, the hydrolysis of 2-AG as well as AEA. Accordingly, AEA as well as FAAH inhibitors, including arachidonoyltrifluoromethyl ketone (ATFMK), blocked [3H]2-AG hydrolysis by N18TG2 and RBL-2H3 membranes, whereas 2-AG inhibited [14C]AEA hydrolysis. FAAH blockade by ATFMK preserved from inactivation the 2-AG synthesized de novo by intact N18TG2 cells stimulated with ionomycin. These data suggest that FAAH may be one of the enzymes deputed to the physiological inactivation of 2-AG, and create intriguing possibilities for the cross-regulation of 2-AG and AEA levels.
Affiliated with the National Institute for the Chemistry of Biological Systems, CNR.