Figure 1.

Mode of inhibition of FAAH by Flu-AM4 showing a reversible and competitive mode of FAAH inhibition. Inhibition of rat (unless otherwise shown) brain [³H]AEA hydrolysis by Flu-AM4. Panel A shows the inhibition curves for both rat and mouse brain homogenates from which the IC₅₀ values were derived. In Panel B, rat brain homogenates were preincubated for the times shown prior to addition of substrate and incubation for a further 10 min. Panel C shows the kinetics of the inhibition of rat brain [³H]AEA hydrolysis by Flu-AM4. The data were better fitted by a model assuming competitive inhibition (K_i value 0.013 µM) than by a mixed model inhibition. All data are means ± s.e.m. (unless hidden by the symbols) n = 3–4. In Panel D, rat brain homogenates (at 20-fold normal strength) were preincubated with either vehicle, 0.1, 0.2 or 0.3 µM Flu-AM4 for 60 min. Aliquots were then diluted 20-fold and assayed for FAAH activity. These are shown as 0.1 → 0.005, 0.2 → 0.01 and 0.3 → 0.015. Concomitantly, Flu-AM4 was added to vehicle-preincubated aliquots to give concentrations of 0.005, 0.01 and 0.015 µM (representing free concentrations after a 20-fold dilution), 0.1, 0.2 and 0.3 µM final concentrations. The panel shows the data as % of corresponding control. For a fully reversible inhibitor, the preincubated + diluted samples should match the corresponding “diluted” rather than “concentrated” concentrations added after the preincubation phase (i.e. the 0.1 → 0.005 samples should match the 0.005 and not the 0.1 µM samples). Data are means ± s.e.m. (unless hidden within the symbols), n = 3–4.