Date Published: July 31, 2014
Publisher: Public Library of Science
Author(s): Emmelie Björklund, Anders Blomqvist, Joel Hedlin, Emma Persson, Christopher J. Fowler, Patrizia Campolongo.
The endocannabinoid ligand anandamide (AEA) is removed from the extracellular space by a process of cellular uptake followed by metabolism. In many cells, such as the RBL-2H3 cell line, inhibition of FAAH activity reduces the observed uptake, indicating that the enzyme regulates uptake by controlling the intra- : extracellular AEA concentration gradient. However, in other FAAH-expressing cells, no such effect is seen. It is not clear, however, whether these differences are methodological in nature or due to properties of the cells themselves. In consequence, we have reinvestigated the role of FAAH in gating the uptake of AEA.
The effects of FAAH inhibition upon AEA uptake were investigated in four cell lines: AT1 rat prostate cancer, RBL-2H3 rat basophilic leukaemia, rat C6 glioma and mouse P19 embryonic carcinoma cells. Semi-quantitative PCR for the cells and for a rat brain lysate confirmed the expression of FAAH. No obvious expression of a transcript with the expected molecular weight of FLAT was seen. FAAH expression differed between cells, but all four could accumulate AEA in a manner inhibitable by the selective FAAH inhibitor URB597. However, there was a difference in the sensitivities seen in the reduction of uptake for a given degree of FAAH inhibition produced by a reversible FAAH inhibitor, with C6 cells being more sensitive than RBL-2H3 cells, despite rather similar expression levels and activities of FAAH. The four cell lines all expressed FABP5, and AEA uptake was reduced in the presence of the FABP5 inhibitor SB-FI-26, suggesting that the different sensitivities to FAAH inhibition for C6 and RBL2H3 cells is not due to differences at the level of FABP-5.
When assayed using the same methodology, different FAAH-expressing cells display different sensitivities of uptake to FAAH inhibition.
The endogenous cannabinoid ligand anandamide (arachidonoylethanolamide, AEA) is produced “on demand”  and removed from the synaptic cleft by a process of cellular uptake followed by metabolism, primarily by the intra-cellular enzyme fatty acid amide hydrolase (FAAH) . The process of the cellular clearance has been widely discussed in the literature (for review, see ) and several intracellular AEA transporters such as fatty acid binding protein 5 and 7, heat shock protein 70 and albumin have been proposed , . An FAAH-like AEA transporter (FLAT) has also been proposed as an intracellular carrier protein , although this has been disputed .
Although the metabolic pathways for AEA are well characterized, the mechanisms responsible for the cellular uptake of AEA still remain to be completely explained (review, see ). One point of contention is the degree to which FAAH regulates the uptake of AEA. In the present study, we have investigated this in some detail using cells with different expression levels of the enzyme. Two approaches were taken: in the first, time courses of the reduction of the rate of uptake following irreversible FAAH inhibition were investigated. For all three cell lines, 1 µM URB597 (which completely inhibits hydrolysis in the C6 cells) produced a 70% reduction in the rates of uptake between 1 and 7 min incubations. It is notable that for the C6 cells, a significant effect of URB597 is seen even at the 1 minute incubation time. This would suggest that sufficient AEA has reached FAAH for it to be metabolised by 1 min of incubation; thereby at this time point, the enzyme contributes to the intra: -extracellular concentration gradient. This is reinforced by our finding that the FABP-5 inhibitor SB-FI-26 also produces a significant reduction in uptake at the 1 min time point, assuming, of course, that FABP5 plays a part in the shuttling of AEA to FAAH in this cell line. More conclusively, it has been shown that in RBL-2H3 and U937 human monocytic leukaemia cells, AEA hydrolysis can be demonstrated at very short incubation times (25–30 sec) , , and so this explanation seems feasible.