Fig. 5.

Degradation of anandamide, 20-HETE-EA. and 14,15-EET-EA by rat brain homogenates. In A, each substrate (5 μM) was incubated in the presence of rat brain homogenate (0.5 mg protein/reaction) in phosphate buffer, pH 7.4, at 37°C for 0 to 64 min. At the designated time points, aliquots (500 μl) were removed, and, after the addition of internal standard, the reaction mixtures were extracted with 3 volumes of ethyl acetate and analyzed by ESI-LC-MS as described under Materials and Methods. The amounts of substrate remaining at each time point were plotted as a percentage of the starting amount (at time 0). The data were fitted to a one-phase exponential decay model using Prism 5 software, from which the respective half-lives were obtained. In B, each substrate (5 μM) was incubated in the presence of rat brain homogenate (0.5 mg protein/reaction) in phosphate buffer, pH 7.4, at 37°C for 0 to 90 min in the presence of the FAAH inhibitor, PMSF, at a concentration of 50 μM. At the designated time points aliquots (500 μl) were removed and after the addition of internal standard, the reaction mixtures were extracted with 3 volumes of ethyl acetate and analyzed by ESI-LC-MS as described under Materials and Methods. The amounts of substrate remaining at each time point were plotted as a percentage of the starting amount (at time 0). The data were fitted to a one-phase exponential decay model using Prism 5 software and are representative of three individual experiments. C, determination of the ratios between the epoxide hydrolase-generated product 14,15-DHET-EA and 14,15-EET-EA in the presence and absence of PMSF (50 μM) from the data in B. The data show single measurements at each time point and are representative of the at least three individual experiments.