Anandamide (arachidonylethanolamide; AnNH) has important neuromodulatory 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 brain fatty-acid amide hydrolase (FAAH) has been characterized as a single protein of 67 kDa with a pI of 7.6, showing apparent Km and Vmax values for AnNH of 2.0 ± 0.2 μm and 800 ± 75 pmol·min−1·mg of protein−1, respectively. The optimum pH and temperature for AnNH hydrolysis were 9.0 and 37 °C, respectively, and the activation energy of the reaction was 43.5 ± 4.5 kJ·mol−1. Hydro(pero)xides derived from AnNH or its linoleoyl analogues by lipoxygenase action were competitive inhibitors of human brain FAAH, with apparent Kivalues in the low micromolar range. One of these compounds, linoleoylethanolamide is the first natural inhibitor (Ki = 9.0 ± 0.9 μm) of FAAH as yet discovered. An FAAH activity sharing several biochemical properties with the human brain enzyme was demonstrated in human neuroblastoma CHP100 and lymphoma U937 cells. Both cell lines have a high affinity transporter for AnNH, which had apparent Km and Vmax values for AnNH of 0.20 ± 0.02 μm and 30 ± 3 pmol·min−1·mg of protein−1 (CHP100 cells) and 0.13 ± 0.01 μm and 140 ± 15 pmol·min−1·mg of protein−1 (U937 cells), respectively. The AnNH carrier of both cell lines was activated up to 170% of the control by nitric oxide.
This work was supported in part by the Istituto Superiore di Sanità (X AIDS Program) and the Ministero dell’Universitàe della Ricerca Scientifica e Tecnologica, Rome (to A. F. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.