animals were then given water but nothing else all animal procedures were conducted in strict adherence to the european directive for protection of vertebrate animals used for experimental and other scientific purposes 86 609cee and portuguese legislation decreto lei 129 92 portarias 1005 92 e 1131 97 the number of animals used was the minimum possible in compliance with current regulations and scientific integrity anandamide ethanolamine 1 3 h 40 60 ci mmol was obtained from american radiochemicals all other reagents were obtained from sigma aldrich optiphase supermix was obtained from perkin elmer and activated charcoal were obtained from sigma aldrich tissues were thawed on ice and were homogenized in 10 volumes of membrane buffer 3 mm mgcl 2 1 mm edta 50 mm tris hcl ph 7 4 with either potter elvejhem brains u2014 8 strokes at 500 rpm or heidolph diax livers u2014 2 strokes at position 5 for 20 sec with 30 sec pauses total protein in tissues was determined with the biorad protein assay biorad using a standard curve of bsa 50 250 u03bcg ml reaction mix total volume of 200 u03bcl contained 2 u03bcm aea 2 u03bcm aea 5 nm 3 h aea 0 1 fatty acid free bsa 15 u03bcg brain 5 u03bcg liver or 50 u03bcg lung protein in 1 mm edta 10 mm tris ph 7 6 after a 15 min pre incubation period at 37 u00b0 c reaction was started by the addition of the substrate solution cold aea radiolabelled aea bsa reaction was carried out for 10 min brain and lung or 7 min liver before termination by the addition of 400.