For the assays, the tissue homogenate (175 μg) was mixed with radiolabeled [3H]anandamide (specific activity 60 Ci/mmol) or 2-oleoyl-[3H]glycerol (specific activity 60 Ci/mmol) (containing 10 mg/ml fatty acid free bovine serum albumin) and unlabeled anandamide and 2-oleoyl-[3H]glycerol to produce samples of 200 μl total volume. Labeled and unlabeled chemicals were obtained from American Radiolabeled Chemicals (St Louis) and Cayman Chemical Company, respectively. Samples were incubated at 37 °C with continuous shaking in a water bath. ‘Blank’ samples that contained assay buffer, instead of the homogenate, were incubated in the same manner. Incubation was stopped after 15 min by placing the tubes on ice, and 400 μl of 1:1 chloroform/methanol was added to the samples. Samples were then vortexed three times, followed by centrifugation to produce phase separation. A measure of 200 μl of the upper aqueous phase was removed and analyzed for radioactivity by liquid scintillation counting. The blank sample values were subtracted from each count. Data were expressed as the percent of the 0 time point value (for 1 mg/kg AM3506) and the effect of time point analyzed using repeated measures analysis of variance (ANOVA).
