Reactions were in triplicate for each sample and data were normalized by GAPDH levels

reased linearly to 100% over 7 min and kept at this level for 4 min, followed by an equilibration period at 30% for 2 min. The electrospray interface was operating in the positive ion mode at 550uC and the ion spray voltage was set to 5000 volts. The mass transitions used were the following: 396.2/110.1 for AM404, 374.2/110.0 for HPODA, 400.2/114.0 for AM404-D4, 440.3/137.1 for arvanil and 418.3/137.1 for olvanil. The declustering potentials were between 96 and 130 volts and the collision energies between 25 and 41 volts. Quantification of 4-aminophenol and HMBA. Sample aliquots of 5 ml were Amezinium metilsulfate injected into a Hypercarb column. All mobile phases were water-acetonitrile gradients, containing 0.1% formic acid. The acetonitrile content was initially 2%, then increased linearly to 75% over 5 min and then kept at 100% for 1 min, followed by an equilibration period at 2% for 1.5 min. The electrospray ion source was set at 550uC and used in the positive ion mode with an ion spray voltage of 5000. The mass transitions was 110.1/93.0 for 4-aminophenol. HMBA loses the amino group already in the source of the MS and therefore the mass Experiments on brain homogenates in vitro. transition used was 137.0/94.0. The declustering potentials were 100 and 90 volts and the collision energies 21 and 29 volts. No internal standard was used for these analyses. Quantification of anandamide and 2-arachidonoylglycerol. Anandamide, anandamide-D8 and 2arachidonoylglycerol were determined as previously described. Since 2-arachidonoylglycerol is nonenzymatically converted to 1-arachidonoylglycerol, the content of 2-arachidonoylglycerol was estimated as the sum of these lipids. The contents of the compounds were expressed either in moles or as normalized peak areas and related to the protein content in the samples. 22988107 The nPA was obtained by dividing the peak area of the analyte with the peak area for the internal standard in the same sample. The detection limits were calculated as the concentrations corresponding to three times the standard deviation of the blanks. When levels were below detection limit, numerical values of half the detection limit were used in the calculations. Behavioural Tests The behavioural tests were performed in a quiet room and evaluated by a single investigator in a blinded manner. Treatments and/or genotypes were randomized in blocks and the number of animals in each block corresponded to the number of treatments and/or genotypes compared. For example, when the effect of capsazepine on the antinociceptive responses to 4-aminophenol was tested, four animals were included in each block, corresponding to the following treatments: Vehicle+vehicle, vehicle +4aminophenol, capsazepine+vehicle and capsazepine +4-aminophenol. Each animal was exposed to only one treatment. All behavioral tests except the rat paw pressure test were performed 10 18194435 min after the intraperitoneal injection of 4-aminophenol, HMBA, dipyrone or vehicle. PMSF and capsazepine were administrated 20 min and 10 min before 4-aminophenol, respectively. Capsazepine, tropisetron and WAY100635 were injected 5 min before 4-aminophenol or HMBA. Assessment of locomotor activity mouse. Mice were placed in actimetry boxes and spontaneous motor activity was assessed by determining the number of crossings of light beams during 15 min. Formalin test mouse and rat. The animals received an intraplantar injection of a 2.5% formalin solution into a hind paw. The time spent biting and licking of the inj