Biosynthesis and Inactivation of N-Arachidonoylethanolamine (Anandamide) and N-Docosahexaenoylethanolamine in Bovine Retina T. Bisogno,* ,1 I. Delton-Vandenbroucke,† A. Milone,* ,1 M. Lagarde,† and V. Di Marzo* ,1,2 *Istituto per la Chimica di Molecole di Interesse Biologico, CNR, Via Toiano 6, 80072 Arco Felice, Napoli, Italy; and †Institut National de la Sante ´ et de la Recherche Me ´dicale, U352 Biochimie and Pharmacologie, INSA de Lyon, 20, avenue A. Einstein, 69621 Villeurbanne (Cedex), France Received June 10, 1999, and in revised form July 26, 1999 N-Arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), the two proposed endogenous agonists of cannabinoid receptors, and the putative AEA biosynthetic precursor, N-arachido- noylphosphatidylethanolamine (NArPE), were identi- fied in bovine retina by means of gas chromatography– electron impact mass spectrometry (GC-EIMS). This technique also allowed us to identify N-docosahex- anoylethanolamine (DHEA) and 2-docosahexanoylg- lycerol (2-DHG), two derivatives of docosahexaenoic acid (DHA), one of the most abundant fatty acids es- terified in retina phospholipids and necessary for op- timal retinal function. N-Docosahexaenoylphosphati- dylethanolamine (NDHPE), the potential biosynthetic precursor for DHEA, was also found. The fatty acid composition of the sn-1 and sn-2 positions of bovine retina’s most abundant phospholipid classes, also de- termined here, were in agreement with a phospholip- id-dependent mechanism for 2-AG, 2-DHG, AEA, and DHEA biosynthesis, as very high levels of polyunsatu- rated fatty acids, including DHA, were found on the sn-2 position of phosphatidylcholine (PC) and -etha- nolamine (PE), and measurable amounts of di-docosa- hexanoyl-PC and -PE, two potential biosynthetic pre- cursors of NDHPE, were detected. Accordingly, we found that isolated particulate fractions from bovine retina could release AEA and DHEA in a time-depen- dent fashion. Finally, a fatty acid amide hydrolase (FAAH)-like activity with subcellular distribution and pH dependency similar to those reported for the brain enzyme was also detected in bovine retina. This activ- ity was inhibited by FAAH inhibitors, phenylmethyl- sulfonyl fluoride and arachidonoyltrifluoromethyl- ketone, and appeared to recognize DHEA with a lower efficiency than AEA. These data indicate that AEA and its congeners may play a physiological role in the mammalian eye. © 1999 Academic Press Key Words: cannabinoids; anandamide; 2-arachido- noylglycerol; bovine retina; docosahexaenoic acid; eye. Two receptor subtypes for  9 -tetrahydrocannabinol (THC) 3 , the active constituent of Cannabis sativa, have been identified and named CB 1 and CB 2 . While the former protein is expressed in both nervous and non- nervous tissues, the CB 2 receptor subtype is mostly confined to cells of the immune system (1, 2). Anand- amide ( N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) were isolated from cen- tral and peripheral tissues (3–5) and suggested to act as endogenous cannabinoid receptor ligands (hereafter referred to as “endocannabinoids”). Pathways for AEA and 2-AG biosynthesis and inactivation by intact cells have been identified (as reviewed in (6)). It was sug- 1 Affiliated with the National Institute for the Chemistry of Bio- logical Systems, CNR. 2 To whom correspondence should be addressed. E-mail: VDM@TRINC.ICMIB.NA.CNR.IT. Fax: +39-081-8041770. 3 Abbreviations used: THC,  9 -tetrahydrocannabinol; AEA, N-arachidonoylethanolamine; 2-AG, 2-arachidonoylglycerol; NAE, N-acylethenolamine; NArPE, N-arachidonoylphosphatidyletha- nolaminde; PE, phosphatidylethanolamine; AA, arachidonic acid; PC, phosphatidylcholine; FAAH, fatty acid amide hydrolase; IOP, intraocular blood pressure; DHA, docosahexaenoic acid; NDHPE, N-docosahexanoyl-PE; DHEA, N-docosahexanoylethanolamine; 2-DHG, 2-docosahexanoylglycerol; NP–HPLC, normal phase– high pressure liquid chromatography; GC, gas chromatography; EIMS, electron impact mass spectrometric; PBS, phosphate-buffered saline; DGBZ, diglycerobenzoate; PMSF, phenylmethylsulfonyl fluoride; ATFMK, arachidonoyltrifluoromethylketone. 300 0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 370, No. 2, October 15, pp. 300 –307, 1999 Article ID abbi.1999.1410, available online at http://www.idealibrary.com on