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 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.
Deutsch
D G
DG
Department of Biochemistry and Cell Biology and Physiology and Biophysics, State University of New York at Stony Brook, 11974-5215, USA. ddeutsch@notes.cc.sunysb.edu
Glaser
S T
ST
Howell
J M
JM
Kunz
J S
JS
Puffenbarger
R A
RA
Hillard
C J
CJ
Abumrad
N
N
D000581:Amidohydrolases / Q000737:chemistry* / Q000378:metabolism*; D001095:Arachidonic Acids / Q000493:pharmacokinetics* / Q000494:pharmacology; D001665:Binding Sites; D002855:Chromatography, Thin Layer; D063388:Endocannabinoids; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D063065:Organophosphonates / Q000494:pharmacology; D053284:Polyunsaturated Alkamides; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D063065:Organophosphonates; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C104810:methyl arachidonylfluorophosphonate; C078814:anandamide