ANANDAMIDE HYDROLYSIS BY HUMAN-CELLS IN CULTURE AND BRAIN

Anandamide (arachidonylethanolamide; AnNH) has important neuromodulato ry and immunomodulatory activities. This lipid is rapidly taken up and hydrolyzed to arachidonate and ethanolamine in many organisms. As yet , AnNH inactivation has not been studied in humans. Here, a human brai n fatty-acid amide hydrolase (FAAH) has been characterized as a single protein of 67 kDa with a pi of 7.6, showing apparent K-m and V-max va lues for AnNH of 2.0 +/- 0.2 mu M and 800 +/- 75 pmol min-l mg of prot ein-l, respectively. The optimum pH and temperature for AnNH hydrolysi s were 9.0 and 37 degrees C, respectively, and the activation energy o f the reaction was 43.5 +/- 4.5 kJ.mol(-1). Hydro(pero)xides derived f rom AnNH or its linoleoyl analogues by Lipoxygenase action were compet itive inhibitors of human brain FAAH, with apparent K-i values in the low micromolar range. One of these compounds, linoleoylethanolamide is the first natural inhibitor (K-i = 9.0 +/- 0.9 mu M) of FAAH as yet d iscovered. An FAAH activity sharing several biochemical properties wit h the human brain enzyme was demonstrated in human neuroblastoma CHP10 0 and lymphoma U937 cells. Both cell lines have a high affinity transp orter for AnNH, which had apparent K-m and V-max values for AnNH of 0. 20 +/- 0.02 mu M and 30 +/- 3 pmol.min(-1).mg of protein(-1) (CHP100 c ells) and 0.13 +/- 0.01 mu M and 140 +/- 15 pmol.min(-1).mg of protein (-1) (U937 cells), respectively. The AnNH carrier of both cell lines w as activated up to 170% of the control by nitric oxide.