The cellular uptake of anandamide is coupled to its breakdown by fatty-acid amide hydrolase

Anandamide is an endogenous compound that acts as an agonist at cannabinoid receptors, It is inactivated via intracellular degradation after its uptak e into cells by a carrier-mediated process that depends upon a concentratio n gradient. The fate of anandamide in those cells containing an amidase cal led fatty-acid amide hydrolase (FAAH) is hydrolysis to arachidonic acid and ethanolamine. The active site nucleophilic serine of FAAH is inactivated b y a variety of inhibitors including methyl-arachidonylfluorophosphonate (MA FP) and palmityl-sulfonyl fluoride. In the current report, the net uptake o f anandamide in cultured neuroblastoma (N18) and glioma (C6) cells, which c ontain FAAH, was decreased by nearly 50% after 6 min of incubation in the p resence of MAFP, Uptake in laryngeal carcinoma (Hep2) cells, which lack FAA H, is not inhibited by MAFP. Free anandamide was found in all MAFP-treated cells and in control Hep2 cells, whereas phospholipid was the main product in N18 and C6 control cells when analyzed by TLC, The intracellular concent ration of anandamide in N18, C6, and Hep2 cells was up to 18-fold greater t han the extracellular concentration of 100 nM, which strongly suggests that it is sequestered within the cell by binding to membranes or proteins. The accumulation of anandamide and/or its breakdown products was found to vary among the different cell types, and this correlated approximately with the amount of FAAH activity, suggesting that the breakdown of anandamide is in part a driving force for uptake. This was shown most clearly in Hepa cells transfected with FAAH. The uptake in these cells was 2-fold greater than i n vector-transfected or untransfected Hepa cells. Therefore, it appears tha t FAAH inhibitors reduce anandamide uptake by cells by shifting the anandam ide concentration gradient in a direction that favors equilibrium. Because inhibition of FAAH increases the levels of extracellular anandamide, it may be a useful target for the design of therapeutic agents.