Studies of anandamide accumulation inhibitors in cerebellar granule neurons: comparison to inhibition of fatty acid amide hydrolase

Abstract

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 IC(50) values of approximately 5 microM, whereas AM1172 and UCM707 exhibited IC(50) values of 24 and 30 microM, respectively. The compounds also inhibited PEA accumulation; AM404 being the most potent with an IC(50) of 6 microM, whereas the other compounds had IC(50) values in the range of 30-70 microM. All of the compounds potently inhibited AEA hydrolysis by brain membranes; the K(I) values for AM404, VDM11, and UCM707 were less than 1 microM; AM1172 and OMDM-2 exhibited K(I) values of 3 and 10 microM, respectively. The IC(50) values for inhibition of AEA accumulation were compared to the IC(50) 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.

Figure 1

Structures of the compounds used in this study.

Figure 2

Concentration response curves for the inhibition of AEA accumulation by CGN. Cells were washed, equilibrated in buffer for 30 min, then preincubated for 5 min with the inhibitor. [³H]AEA was added (final concentration, 0.65 nM) and the incubation was continued for exactly 2 min. Buffer was removed and cells were scraped in 0.5 ml of water. Buffer and cells were counted and the fraction accumulated by the cells was calculated as: (dpm in cells)/(dpm in cells + dpm in media). Nonspecific association was defined as the fraction accumulated in the presence of 100 μM AEA. Each experiment was carried out in triplicates\ and the entire concentration responses curves were repeated in at least 3 different preparations of CGN. Points represent the mean, vertical lines represent the S.E.M. Lines drawn are the best fit of the data points to a single site, competition equation using non-linear regression.

Figure 3

Concentration response curves for the inhibition of AEA hydrolysis by mouse brain membrane-derived FAAH. Membranes were preincubated for 5 min with the inhibitor. [³H]AEA was added (final concentration, 2 nM) and the incubation was continued for exactly 10 min. The reaction was stopped by the addition of chloroform:methanol (1:2) followed by extraction and separation of aqueous and organic phases. Radioactivity was determined in both phases and the percent hydrolysis was calculated. Each experiment was carried out in triplicate and the entire concentration responses curves were repeated in at least 3 different membrane preparations. Points represent the mean, vertical lines represent the S.E.M. Lines drawn are the best fit of the data points to a single site, competition equation using non-linear regression.