Pharmacological characterization of endocannabinoid transport and fatty acid amide hydrolase inhibitors

Abstract

: 1. The mechanism of anandamide uptake and disposal has been an issue of considerable debate in the cannabinoid field. Several compounds have been reported to inhibit anandamide uptake or fatty acid amide hydrolase (FAAH; the primary catabolic enzyme of anandamide) activity with varying degrees of potency and selectivity. We recently reported the first evidence of a binding site involved in the uptake of endocannabinoids that is independent from FAAH. There are no direct comparisons of purported selective inhibitory compounds in common assay conditions measuring anandamide uptake, FAAH activity and binding activity. 2. A subset of compounds reported in the literature were tested in our laboratory under common assay conditions to measure their ability to (a) inhibit [(14)C]-anandamide uptake in cells containing (RBL-2H3) or cells lacking (HeLa) FAAH, (b) inhibit purified FAAH hydrolytic activity, and (c) inhibit binding to a putative binding site involved in endocannabinoid transport in both RBL and HeLa cell membranes. 3. Under these conditions, nearly all compounds tested inhibited (a) uptake of [(14)C]-anandamide, (b) enzyme activity in purified FAAH preparations, and (c) radioligand binding of [(3)H]-LY2183240 in RBL and HeLa plasma membrane preparations. General rank order potency was preserved within the three assays. However, concentration response curves were right-shifted for functional [(14)C]-anandamide uptake in HeLa (FAAH(-/-)) cells. 4. A more direct comparison of multiple inhibitors could be made in these three assay systems performed in the same laboratory, revealing more information about the selectivity of these compounds and the relationship between the putative endocannabinoid transport protein and FAAH. At least two separate proteins appear to be involved in uptake and degradation of anandamide. The most potent inhibitory compounds were right-shifted when transport was measured in HeLa (FAAH(-/-)) cells suggesting a requirement for a direct interaction with the FAAH protein to maintain high affinity binding of anandamide or inhibitors to the putative anandamide transport protein.

Fig. 1.

Hydrolytic activity of purified ΔTM-FAAH is inhibited by reported FAAH inhibitors, but also with compounds reported as selective anandamide uptake inhibitors. Data are expressed as mean ± S.E.M. of at least three separate experiments.

Fig. 2.

Uptake of [¹⁴C]-anandamide in RBL-2H3 and HeLa cells. (A) [¹⁴C]-anandamide uptake is inhibited in RBL-2H3 cells by all compounds tested. (B) Uptake of [¹⁴C]-anandamide in HeLa cells is also inhibited by all compounds tested with the exception of OMDM-1 and OMDM-2. Relative inhibition of potent compounds in RBL-2H3 cells is right-shifted in HeLa cells. Data are expressed as mean ± S.E.M. of duplicates repeated in at least three separate experiments.

Fig. 3.

Significant levels of [¹⁴C]-anandamide are accumulated in both RBL-2H3 and HeLa cells (p < 0.0001), with overall levels significantly less in HeLa compared to RBL-2H3. All data are expressed as mean ± S.E.M. (nM) of at least 3 independent experiments.

Fig. 4.

Displacement of specific binding of [³H]-LY2183240 to a binding site involved in transport of endocannabinoids. (A) Reported anandamide uptake and FAAH activity inhibitors displace binding of [³H]-LY2183240 in RBL-2H3 cell membranes with potency similar to that observed for functional [¹⁴C]-anandamide uptake in whole cells. (B) Binding of [³H]-LY2183240 is also displaced in HeLa cell membranes with similar potency to that observed in RBL-2H3 cell membranes. Data are expressed as mean ± S.E.M. of duplicates repeated in at least three separate experiments.