Abstract
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.
Highlights
Endocannabinoids, such as anandamide and 2-arachidonyl glycerol, are endogenous ligands that bind to the cannabinoid receptors [1,2,3]
We show that the net movement of anandamide into the cells is coupled to the activity of intracellular fatty-acid amide hydrolase (FAAH)
To study the effects of palmitylsulfonyl fluoride (PSF), the other hydrolase inhibitor used in these studies, on anandamide uptake, identical experiments were conducted in N18 and C6 cell lines
Summary
Cell Culture—N18TG2 neuroblastoma, C6 glioma (kindly provided by Allyn Howlett and Joel Levine, respectively), and human laryngeal carcinoma cells (Hep2), provided by our in-house cell culture facility, were grown in 35 ϫ 10-mm dishes in 2 ml of Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Gemini Bioproducts, Calabasas, CA), 1% penicillin/streptomycin, and L-glutamine (Life Technologies, Inc.). All cells were grown at 37 °C with 5% CO2.
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