Studies of Anandamide Accumulation Inhibitors in Cerebellar Granule Neurons: Comparison to Inhibition of Fatty Acid Amide Hydrolase

The endocannabinoid, N-arachidonylethanolamine (AEA) is accumulated by neurons via a process that has been characterized biochemically but not molecularly. Inhibitors of AEA accumulation have been characterized individually but have not been compared in a single study. Our purpose was to compare the potency of five previously described compounds (AM404, AM1172, VDM11, OMDM-2, and UCM707) both as inhibitors of AEA and N-palmitoylethanolamine (PEA) accumulation by cerebellar granule neurons and as inhibitors of AEA hydrolysis. The compounds all inhibited AEA accumulation; AM404, VDM11 and OMDM-2 with IC₅₀ values of approximately 5 μM, whereas AM1172 and UCM707 exhibited IC₅₀ values of 24 and 30 μM, respectively. The compounds also inhibited PEA accumulation; AM404 being the most potent with an IC₅₀ of 6 μM, whereas the other compounds had IC₅₀ values in the range of 30–70 μM. All of the compounds potently inhibited AEA hydrolysis by brain membranes; the K _I values for AM404, VDM11, and UCM707 were less than 1 μM; AM1172 and OMDM-2 exhibited K _I values of 3 and 10 μM, respectively. The IC₅₀ values for inhibition of AEA accumulation were compared to the IC₅₀ values for PEA accumulation and AEA hydrolysis using linear regression. None of the regressions were significant. These data indicate that inhibition of AEA accumulation by neurons is not a result of the inhibition of endocannabinoid hydrolysis and is a process different from the accumulation of PEA. These studies support the hypothesis that the cellular AEA accumulation beyond simple equilibrium between intracellular and extracellular concentrations occurs because AEA binds to an intracellular protein that is not FAAH but that also recognizes the AEA uptake inhibitors.