Anandamide is an endogenous compound that acts as an agonist at cannabinoid receptors. It is inactivated via intracellular degradation after its uptake into cells by a carrier-mediated process that depends upon a concentration gradient. The fate of anandamide in those cells containing an amidase called fatty-acid amide hydrolase (FAAH) is hydrolysis to arachidonic acid and ethanolamine. The active site nucleophilic serine of FAAH is inactivated by a variety of inhibitors including methylarachidonylfluorophosphonate (MAFP) and palmitylsulfonyl fluoride. In the current report, the net uptake of anandamide in cultured neuroblastoma (N18) and glioma (C6) cells, which contain FAAH, was decreased by nearly 50% after 6 min of incubation in the presence of MAFP. Uptake in laryngeal carcinoma (Hep2) cells, which lack FAAH, 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 concentration of anandamide in N18, C6, and Hep2 cells was up to 18-fold greater than 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 Hep2 cells transfected with FAAH. The uptake in these cells was 2-fold greater than in vector-transfected or untransfected Hep2 cells. Therefore, it appears that FAAH inhibitors reduce anandamide uptake by cells by shifting the anandamide 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.Endocannabinoids, such as anandamide (arachidonyl ethanolamide) and 2-arachidonyl glycerol, are endogenous ligands that bind to the cannabinoid receptors (1-3). Emerging evidence suggests that they are involved in many physiological phenomena such as pain, locomotion, memory, learning, blood pressure, immunity, sleep, reproduction, mood, perception, response to stress, and so forth (for review see Ref. 4). ⌬ 9 -Tetrahydrocannabinol, the active component of marijuana, appears to mimic many of the physiological and pharmacological effects of the endogenous cannabinoids, in some cases to an extreme degree (e.g. a marijuana "high"). Anandamide is transported into the neuroblastoma, glioma, brain neuron, brain astrocyte, cerebellar granule cells, leukocyte, macrophage, leukemia, and lymphoma cells in culture (5-10). The driving force for uptake is substrate concentration (facilitated diffusion) rather than an active cotransport system (11,12). The transport appears to be carrier-mediated, and specific transport inhibitors have been described (9,11,(13...

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