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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

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-arachidonoylphosphatidylethanolamine (NArPE), were identified in bovine retina by means of gas chromatography– electron impact mass spectrometry (GC-EIMS). This technique also allowed us to identify N-docosahexanoylethanolamine (DHEA) and 2-docosahexanoylglycerol (2-DHG), two derivatives of docosahexaenoic acid (DHA), one of the most abundant fatty acids esterified in retina phospholipids and necessary for optimal retinal function. N-Docosahexaenoylphosphatidylethanolamine (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 determined here, were in agreement with a phospholipid-dependent mechanism for 2-AG, 2-DHG, AEA, and DHEA biosynthesis, as very high levels of polyunsaturated fatty acids, including DHA, were found on the sn-2 position of phosphatidylcholine (PC) and -ethanolamine (PE), and measurable amounts of di-docosahexanoyl-PC and -PE, two potential biosynthetic precursors of NDHPE, were detected. Accordingly, we found that isolated particulate fractions from bovine retina could release AEA and DHEA in a time-dependent 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 activity was inhibited by FAAH inhibitors, phenylmethylsulfonyl fluoride and arachidonoyltrifluoromethylketone, 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-arachidonoylglycerol; 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 nonnervous tissues, the CB
2

receptor subtype is mostly confined to cells of the immune system (1, 2). Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) were isolated from central 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 sug1 Affiliated with the National Institute for the Chemistry of Biological 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-arachidonoylphosphatidylethanolaminde; 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.

gested that AEA, analogous to other N-acylethanolamines (NAEs), is released from a preformed membrane phospholipid, N-arachidonoylphosphatidylethanolamine (NArPE) through the action of a microsomal phospholipase D (7). NArPE, in turn, was shown to derive, by analogy with other N-acyl-PEs, from the N-arachidonoylation of phosphatidylethanolamine (PE), catalyzed by a Ca2 -dependent microsomal transacylase using arachidonic acid (AA) derived from the sn-1 position of phosphoglycerides (8–11). An alternative AEA biosynthetic pathway, through the energy-free condensation between AA and ethanolamine, was also suggested to occur in some reproductive tissues (11). 2-AG biosynthesis depends on sn-2–arachidonate-containing phospholipid precursors such as phosphatidylcholine (PC), phosphatidylinositol, and phosphatidic acid (9, 12, 13). An enzyme catalyzing the hydrolysis of AEA to AA and ethanolamine was recently characterized and named “fatty acid amide hydrolase” (FAAH) (14). This enzyme catalyzes the hydrolysis also of other fatty acid amides and esters, including 2-AG (see (15) for a review). AEA exhibits numerous THC-like properties (recently reviewed in (16, 17), including an inhibitory effect on intraocular blood pressure (IOP) in normotensive rabbits (18–21). A series of chiral -substituted AEA derivatives were synthesized in order to minimize the degradation and increase the effect of the endocannabinoid on IOP. These compounds, unlike AEA, produced hypotensive effects that were not preceded by an initial elevation of IOP (22), thus suggesting that the arachidonate produced from AEA hydrolysis could be responsible for this undesired side-effect. Evidence both in favor and against the involvement of cannabinoid receptors in the IOP-lowering effects of cannabinoids have been reported. High levels of CB
1

mRNA were found in the ciliary body of the eye (23), but one study failed to demonstrate any potent effect for the synthetic cannabinoid receptor agonist, WIN-55,212-2, or for the metabolically stable AEA congener, (R)-( -methanandamide (24). In another study (25), however, unilateral administration of AEA, its metabolically stable R- isopropyl analogue, or the synthetic cannabinoid CP55,940 did reduce the IOP. Only the effect of the latter two compounds was prevented by pretreatment of the animals with the CB
1

receptor antagonist SR141716A, suggesting that AEA hypotensive action could be mediated also by other mechanisms, possibly due to AEA hydrolysis to AA. In fact, AEA hydrolase activities have been identified in porcine ocular tissues (26), thus prompting a physiological role of AEA, and possibly 2-AG, in the eye. Among the ocular tissues analyzed, the retina showed the highest specific enzymatic activity. In this tissue, an endogenous cannabinoid tone leading to inhibition of dopamine release was recently suggested based on the finding that a CB
1

antagonist 301 ENDOCANNABINOIDS IN BOVINE RETINA facilitates, whereas a synthetic cannabinoid agonist inhibits, the release of the neurotransmitter from this tissue (27). However, despite the increasing evidence for a possible physiological role of AEA in the retina, no study has addressed so far the question of whether this or the other endocannabinoid, 2-AG, is synthesized by this tissue. The retina contains a high level of docosahexaenoic acid (22:6 n 3; DHA), a polyunsaturated fatty acid that is essential for optimal retinal function as shown by the observation that low levels of DHA were found in red blood cells from patients with retinis pigmentosa (28, 29) or in the plasma of dogs affected by progressive rod–cone degeneration (30). The fatty acids composition of retina phosphoglycerides has been reported for rat and bovine species (31, 32), and DHA was shown to be among the most abundant fatty acids in all phospholipids, with high levels being found in PC and PE. Furthermore, unusually high levels of di-docosahexanoyl-PC and -PE species, i.e., two of the potential fatty acid donors for the formation of a putative Ndocosahexanoyl-PE (NDHPE) and, subsequently, of Ndocosahexanoylethanolamine (DHEA), have been reported for rat retina (31). Starting from this background, the objective of the present study was to provide an answers to the following two open questions: (i) Are 2-AG, AEA, and AEA putative biosynthetic precursor, NArPE, produced and metabolized by the retina? (ii) In view of the high levels of phospholipid-bound DHA found in this tissue, is it possible that retinas also produce and metabolize 2-docosahexanoylglycerol (2-DHG), DHEA, and DHEA putative precur-

sor, NDHPE?
MATERIALS AND METHODS DHEA was synthesised by mixing equimolar amounts of docosahexaenoyl-chloride (obtained by reacting docosahexaenoic acid (Sigma) with an excess of oxalyl chloride) and ethanolamine for 20 min at 4°C in anhydrous dichloromethane. The mixture was then brought to dryness under a flow of N 2 and purified by normal-phase high-pressure liquid chromatography (NP-HPLC) carried out with a semipreparative column (Spherisorb S5W) eluted with a 40-min linear gradient from 90 to 80% of n-hexane in 2-propanol (flow rate 2 ml/min). Under these conditions DHEA was eluted after 26 min. NP-HPLC fractions containing DHEA were pooled, and the solvent was evaporated under a flow of N 2 . Gas chromatography–electron impact mass spectrometric (GC-EIMS) analysis of the tris-methylsilyl-ether, carried out as previously described (33) on a HP-5989B quadrupole mass analyzer equipped with an electron impact source operating at 70 eV and 250°C, confirmed the chemical structure of the compound, which was eluted from the GC column after 20.5 min. The GC column was a capillary fused silica column (Optima-1-MS, 0.25 m, Macherey–Nagel; length, 30 m; i.d., 0.25 mm) and was eluted with a programmed temperature gradient from 200 to 300°C (5°C/min; flow rate, 1 ml/min; injector and detector temperature, 260°C). Freshly enucleated bovine eyes were obtained from Lyon– Corbas slaughterhouse. The retinas were removed and extracted with chloroform/methanol (2/1, by vol). d 8 -2-AG and d 8 -AEA (5 nmol), synthesized from d 8 -AA and ethanolamine or glycerol (3, 34), were

FIG. 1. Characterization of N-docosahexaenoylethanolamine and 2-docosahexaenoylglycerol in bovine retina. (A) Total ion monitoring EIMS spectrum of synthetic DHEA (1 nmol), (B) selected ion monitoring EIMS spectrum of native DHEA, and (C) total ionmonitoring EIMS spectrum of native 2-DHG from bovine retina. 302 Abundance BOO 7000 6000 5000 4000 300 2000 1000 7000 3 6000134 A 150 200 250 300 350 400 B 175 428 Abundance 10000 J 4000-1 sooof 2000 1000": 6 103 147 280 50 100 150 200 250 300 350 400 450 S00

added to extracting mixture. The lipids were purified by silica gel

column chromatography and NP-HPLC as described (34). NP-HPLC fractions with the same retention time of monoacylglycerols (17–22 min) and NAEs (24–28 min) were liophylized and trimethylsilylated as previously described (33). The samples were analyzed by GCEIMS under conditions previously shown to allow the separation of the components of each family of lipids (34). In order to detect AEA and DHEA, the EIMS was run in the selected ion-monitoring mode to improve sensitivity. For other NAEs, 2-AG and 2-DHG, which were more abundant, the EIMS was run in the total ion current mode. The amounts of AEA and 2-AG were calculated from the area ratio between the peaks at m/z 404 and 412 (loss of methyl group from undeuterated and d 8 -AEA) and m/z 507 and 515 (loss of methyl group from undeuterated and d 8 -2-AG), respectively. The amounts of endogenous DHEA and 2-DHG were calculated by using calibration curves obtained with external synthetic standards of DHEA and 2-AG, respectively. NArPE and NDHPE were identified as the corresponding NAEs released from the digestion of NArPElike chromatographic fractions with S. chromofuscus PLD (Sigma, UK) (33). For analysis of phosphoglyceride fatty acid composition, the retinas were removed from freshly enucleated bovine eyes and placed in cold PBS, minced into small pieces, and homogenized in a Dounce homogenizer in the presence of butylated hydroxytoluene (50 M) to prevent oxidation. After extraction with chloroform/methanol (2/1, by vol), phospholipids were separated by two-step TLC by using the developing system chloroform/methanol (80/8, by vol) followed by the developing system chloroform/methanol/methylamine (60/20/5, by vol). Phospholipids were visualized by spraying with 0.02% 2 ,7 dichlorofluorescein in 95% aqueous ethanol and extracted from the silica gel with chloroform/methanol/water (5/5/1, by vol). To analyze their fatty acid composition, phospholipids were transmethylated for 90 min at 100°C with 5% concentrated sulfuric acid in methanol in vials sealed under N 2 to prevent oxidation. The resulting fatty acid methyl esters were analyzed by GC using a Supelco SP2380 capillary column (0.2 m, 30 m 0.25 mm) with helium as a carrier gas and identified by comparison with commercial standards. PE and PC were converted to their respective diradylglycerols by digestion with phospholipase C (35). Diradylglycerols were derivatized with 3,5dinitrobenzoylchloride in anhydrous pyridine and separated into diacylglycerobenzoates (DGBZ) and ether-linked glycerobenzoates by TLC on silica gel using a solvent system of toluene/hexane/diethyl ether (50/45/5, by vol) (36). The DGBZ were extracted from the silica gel with hexane and fractionated into molecular species by HPLC on a Superspher RP-18 column (5 mm 25 cm 4.6 mm) using acetonitrile/isopropanol (90/10, v/v) at a flow rate of 1 ml/min. Molecular species were detected at 230 nm and identified by comparison with standards or by GC analysis of the fatty acid methyl esters. The former method allowed us to identify and quantify the fatty acids on the sn-1 (or sn-3) and sn-2 position. Experiments on NAE biosyn-

thesis were performed by using a procedure similar to that previously described for rat testes (37). Aliquots (3.2 mg of total proteins) of pooled particulate fractions from a 280,000g centrifugation of retina homogenates were resuspended in 1 ml Tris–HCl 50 mM, pH 7.4, and incubated at 37°C for different intervals of time (0, 15, or 30 min). Control incubations were carried out with boiled membranes or with aliquots of 280,000g supernatants. After incubation the mixtures were extracted with chloroform/methanol (2/1, by vol) containing 5 nmol of d 8 -AEA and d 4 -palmitoyl-, d 4 -stearoyl-, d 4 -oleoyl-, and d 4 -linoleoylethanolamides. The organic extracts were purified and analyzed as described above. The amounts of NAEs were calculated by the isotope-dilution GC-EIMS methods described previously (33, 37). Finally, fatty acid amide hydrolase assays were performed as described previously (38). Bovine retinas were homogenized in 50 mM Tris–HCl, pH 7.4, and centrifuged sequentially at 1500g (10 min), 80,000g (30 min), and 280,000g (30 min). Pellets from the three centrifugation steps and the supernatant from the last centrifugation were incubated in 50 mM Tris, pH 9, at 37°C for 30 min in

303 ENDOCANNABINOIDS IN BOVINE RETINA
TABLE I Fatty Acid Composition of Bovine Retina Phosphatidylcholine (PC) and Phosphatidylethanolamine (PE) Fatty acid PC PE 14:0 0.73 0.18 0.10 0.01 16:0 DMA 0.05 0.02 0.62 0.10 16:0 42.36 3.86 9.26 0.84 16:1 n-9 0.98 0.07 0.23 0.03 16:1 n-7 0.41 0.05 0.12 0.03 18:0 DMA 0.13 0.07 3.46 0.58 18:0 18.07 1.30 33.28 2.19 18:1 n-9 16.08 1.55 6.64 0.59 18:1 n-7 3.20 0.43 1.50 0.29 18:2 n-6 1.10 0.38 0.85 0.30 20:0 0.16 0.03 0.59 0.10 18:3 n-3 0.08 0.02 0.05 0.01 20:2 n-6 0.31 0.03 0.13 0.04 20:3 n-6 0.68 0.16 0.65 0.06 20:4 n-6 4.37 1.13 9.48 1.17 20:5 n-3 0.20 0.09 0.17 0.05 22:4 n-6 0.43 0.16 2.08 0.46

22:5 n-6 0.36 0.32 1.33 0.01 22:5 n-3 0.59 0.12 1.74 0.46 22:6 n-3 10.50 1.59 27.85 2.32 Note. Phospholipids were transmethylated and analyzed by GC. Data are in mol% SD, n 4. presence of 50,000 cpm (12.5 M) of [14C]AEA synthesized from arachidonoylchloride and [14C]ethanolamine as described (3). Incubations were also carried out in buffers at different pH values (38) or in the presence of arachidonoyltrifluoromethylketone (ATFMK, Biomol), phenylmethylsulfonyl fluoride (PMSF, Sigma) or AEA and DHEA, synthesised as described above. RESULTS AND DISCUSSION

In the present study we investigated the occurrence, in bovine retinas, of the two endocannabinoids AEA and 2-AG together with their (i) DHA homologues, (ii) possible phospholipid precursors, and (iii) biosynthetic and hydrolytic enzymes. The EIMS spectrum of a synthetic standard of DHEA and of a bovine retina lipid component with the same chromatographic behavior in HPLC (r
t

26 min) and GC-EIMS (r
t

20.42 min) analyses are shown in Figs. 1A and 1B. Synthetic DHEA displayed a typical EI fragmentation pattern, with a molecular ion at m/z 443, and main fragment ions at m/z 428 (loss of a methyl group), m/z 284 and 159 ( -cleavage followed by rearrangement), m/z 268 and 175 ( cleavage), and m/z 116 (cleavage between C1 and C2 of the ethanolamine moiety). The native component exhibited all the selected ion signals at m/z 443, 428, 175, and 116 (Fig. 1B). These data indicate that DHEA is a component of lipid extracts from bovine retina. The amount of DHEA obtained from the area of the GC peaks compared to those of the external stan-

TABLE II

Molecular Species Analysis of Bovine Retina Phosphatidylcholine (PC) and Phosphatidylethanolamine (PE) sn-1–sn-2 PC PE 22:6 n-3–22:6 n-3 1.31 0.23 4.16 0.39 Dipolyunsaturated 1.51 0.18 6.70 1.05 18:1–22:6 n-3 1.22 0.05 4.35 0.10 16:0–22:6 n-3 8.05 0.43 14.03 0.17 18:1–20:4 n-6 0.47 0.20 0.86 0.13 16:0–20:4 n-6 2.93 0.60 1.29 0.01 16:0–22:5 n-6 0.83 0.29 0.83 0.28 18:0–22:6 n-3 15.19 1.79 47.83 1.30 18:0–20:4 n-6 4.18 0.31 9.35 1.55 18:0–22:5 n-6 1.11 0.56 2.05 0.45 16:0–18:1 25.66 0.36 2.16 0.76 16:0–16:0 20.52 0.22 2.61 0.33 18:0–18:1 7.24 0.53 1.76 0.56 16:0–18:0 7.64 0.26 0.48 0.15 Others 2.22 0.05 1.54 0.05 Note. Phospholipids were converted into diradylglycerols by treatment with phospholipase C, derivatized with 3,5-dinitrobenzoylchloride, and analyzed by HPLC. Data are in mol% SD, n 3. Since AA is the most abundant polyunsaturated fatty acid after DHA in retina phospholipids (see Table I and (31, 32)), the presence of sn-1 arachidonoyl-PC and -PE in bovine retina can be assumed as being very likely (the HPLC system used for the separation of diradylglycerol does not allow us to identify these molecular species).

dard was 39.9 19.0 pmol/g (n 2). We also found measurable amounts of AEA, as detected from the presence of a GC peak (r
t

18.00 min) with ion signals at m/z 419, 404, 175, and 116, typical of the trismethyl-silyl-ether of AEA. The amount of AEA was 64.0 9.6 pmol/g (n 2). Finally, the tris-methylsilyl-ethers of other typical NAEs were also found, including the C16:0 (r
t

13.78 min), C18:0 (r
t

16.78 min), C18:1 (r
t

16.30 min), and C18:2 (r
t

16.20 min) homologues, as well as the more unusual C22:4 congener (r
t

20.75 min). These compounds were
TABLE III Biosynthesis of N-Acylethanolamines (NAEs) in Bovine Retina Membranes

Time 0 min 15 min 30 min N-Palmitoylethanolamide (C 16:0 ) 52.4 2.6 60.1 8.2 70.8 11.1 N-Stearoylethanolamide (C 18:0 ) ND 172.9 31.2 330.9 30.0 N-Oleoylethanolamide (C 18:1,n 9 ) 159.6 31.2 165.9 7.8 188.9 16.7 N-Linoleoylethanolamide (C 18:2,n 6 ) 12.2 5.6 31.3 4.1 35.2 1.8 N-arachidonoylethanolamide (C 20:4 ) ND 288.8 20.3 774.3 58.4 N-Docosahexaenoylethanolamide (C 22:6 ) 92.0 15.2 342.0 142.0 873.4 247.8 Note. NAE release from 280,000g isolated pellets incubated at 37°C in 50 mM Tris–HCl for increasing periods of time. After the incubation, the organic extracts of each incubate were purified by HPLC, trimethylsilylated, and analyzed by isotope-dilution GC-EIMS. Data are expressed as fmol mg protein 1 SE, n 3. ND, not detectable.

304 BISOGNO ET AL. identified on the basis of their typical fragmentation patterns in total ion-current EIMS analyses (data not shown), but could not be quantified in this experiment due to the absence of the appropriate deuterated standards in the lipid extraction solvent. An estimate of the relative amounts in the retina of these NAE species, however, can be obtained from the experiments carried out with membrane preparations (see below and Table III). When the silica column fraction eluted with chloroform/methanol (1/1, by vol) was digested with S. chromofuscus phospholipase D, it released NAEs that, once derivatized and analyzed by GC-EIMS, showed the presence of AEA and DHEA (51.2 31.1 and 26.6 18.1 pmol/g, respectively, n 2) as well as of the C16:0, C18:0, C18:1, C18:2, and C22:4 NAEs. These data indicate for the first time the presence in bovine retina of the NAE family of lipids, including AEA and DHEA, and of their putative biosynthetic precursors according to the “phospholipid” pathway (6), i.e., the corresponding N-acylPEs. Of the several tissues where the presence of NAEs has been reported so far (reviewed in (6, 11)), only rat brain was shown previously

to contain DHEA—in amounts slightly higher than AEA—as well as the corresponding N-acyl-PE, NDHPE (8). GC-EIMS also revealed that bovine retina lipid extracts contained 2-AG (data not shown) and its docosahexaenoyl homologue, 2-DHG (Fig. 1C). Typical signals of the EIMS spectrum of the bis-tris-methylsilyl-ether of the latter metabolite were the molecular ion at m/z 546, the peak at m/z 531 (loss of a methyl group), and the peak at m/z 456 (loss of one moiety of tris-methyl-silyl-alcohol). The EIMS spectrum of the bis-tris-methyl-silyl-ether of 2-AG has been previously reported (12) and exhibited the same fragmentation pattern described above for 2-DHG, with typical ions at m/z 522, 507, and 432. The amounts of 2-AG and 2-DHG were 1.63 0.31 and 0.34 0.11 nmol/g, respectively (n 2). It should be pointed out that the GC-EIMS method used here allows to separate

FIG. 2. Fatty acid amide hydrolase (FAAH)-like enzymatic activity

in bovine retina. FAAH activity was measured by quantifying [14C]ethanolamine released from [14C]AEA hydrolysis. (A) Subcellular localization of [14C]AEA-hydrolyzing activity in bovine retina homogenate. (B) pH dependency of the [14C]AEA-hydrolyzing activity from bovine retina 280,000g pellets, expressed as a percentage of the

305 ENDOCANNABINOIDS IN BOVINE RETINA 2-monoacylglycerols from their 1(3)-isomers, which are usually eluted from the column half a minute later. Occasionally, small amounts of 1(3)-AG and 1(3)-DHG were found to accompany the corresponding 2-isomers. The finding of the ethanolamides, N-acyl-PEs and 2-glycerol esters of AA and DHA in the retina, although described here for the first time, is not surprising if one looks at the fatty acid composition of the two most abundant phosphoglyceride classes in this tissue, i.e., PC and PE (Tables I and II). In both phospholipids we found, in agreement with previous studies (for example (31, 32)), very high levels of DHA and AA, whereas other polyunsaturated fatty acids were less abundant. These two fatty acids are esterified at both the sn-2 and, to a lesser extent, sn-1 positions of PE and PC, thus indicating that the potential phospholipid precursors for either the 2-glycerol ester or the N-acylPE, respectively, of AA and DHA are available in bovine retina. The hypothesis that AEA and DHEA could be biosynthesized in the retina like in nervous tissue (7–10), i.e., directly from their N-acyl-PE precursors, is in agreement with the observation that total membrane preparations from this tissue—which supposedly contain both these precursors and the phospholipase D enzyme necessary for their conversion into NAEs (6, 11)—released increasing amounts of AEA and DHEA when incubated at 37°C in a physiological buffer (Table III). Also the other NAEs found here in lipid retina extracts were produced under these conditions, although in lower amounts, whereas no NAE was released from heat-inactivated membrane preparations or from aliquots of the 280,000g supernatant incubated under the same conditions (data not shown). However, under the conditions used here the activation of membrane-bound phospholipase A
2

and D enzymes that could catalyze the release of free AA and ethanolamine from phospholipids and PE, respectively, cannot be ruled out. Therefore, although we did not add exogenous arachidonate or DHA and ethanolamine to the

incubation mixtures, it is still possible that AEA and DHEA are also produced from the condensation of the corresponding fatty acids with ethanolamine, as it has been suggested for AA in some reproductive tissues (11, 37). Further studies will be required in order to investigate further the biosynthetic mechanism for AEA and DHEA in the retina. Interestingly, however,
activity at pH 10 (maximal activity). (C) Effect of various substances on [14C]AEA hydrolysis by bovine retina 280,000g pellets. The effect is expressed as a percentage of the activity in the absence of inhibitors. Data are means SE from three separate experiments carried out in duplicate. The asterisk indicates values statistically different from controls (P 0.05, as determined by the unpaired Student’s t test). AEA, N-arachidonoylethanolamine; DHEA, N-docosahexaenoylethanolamine; PMSF, phenylmethylsulfonyl fluoride; ATFMK, arachidonoyltrifluoromethylketone. % Maximal activity % Mßximal activity'

the N-acyl composition of NAEs produced from membrane incubations seems to reflect the fatty acid composition of PE rather than PC (Table I), thus suggesting that PE may act as the source of the N-fatty acids of NAEs. Within the PE class, the fatty acid composition on the sn-2 position rather than the sn-1 position reflected more closely the composition of NAEs released after 30-min incubations, thus suggesting that, under these conditions, the remodeling of phospholipids may occur or that NAEs are produced in part through the “condensation” pathway (11, 37). Indeed, synthesis of AEA through this pathway, very probably through reversal of the action of AEA hydrolase, was shown to occur in porcine retina when using a very high concentration (250 mM) of ethanolamine (26). Porcine ocular tissues, including the retina, were recently shown to express a membrane-bound AEA hydrolase whose pH dependency, sensitivity to inhibitors, and specificity for polyunsaturated NAEs other than AEA was not determined (26). As shown in Fig. 2A, also bovine retina homogenates contain a [14C]AEA-hydrolyzing activity mostly associated with particulate fractions (e.g., pellets from 80,000g and 280,000g centrifugations). The enzyme activity in these fractions was lower than that reported for porcine retina, possibly also because we did not use a saturating concentration of the substrate. However, bovine retina AEA hydrolase displayed optimal activity at pH 10 (Fig. 2B), very similar to that previously observed with FAAH from mammalian brain and rat liver (15). Furthermore, the enzymatic hydrolysis of [14C]AEA by microsomal membranes was not only counteracted by 100 M AEA and 200 M PMSF, but also by 100 M ATFMK, a more specific inhibitor of FAAH (Fig. 2C). DHEA (100 M) also significantly inhibited [14C]AEA hydrolysis, although to a lesser extent than that observed with 100 M AEA. This latter finding suggests that the enzyme recognizes as substrate also the ethanolamide of docosahexaenoic acid, albeit with a lower efficiency. This may indicate that the half-life of DHEA in bovine retina is relatively longer than that of AEA, thus possibly compensating for the lower activity of DHEA at CB
1

receptors (39). However, the apparent K
m

and V
max

values for the hydrolysis of DHEA should be calculated by using the appropriate labeled compound before drawing any conclusion on the efficiency with which this NAE species is recognized by the bovine retina enzyme. In conclusion, the data reported herein have shown for the first time that the retina contains AEA and DHEA and their putative direct biosynthetic precursors, NArPE and NDHPE, as well as precursors for these latter phospholipids, i.e., diarachidonoyl- and didocosahexaenoyl-PC and -PE. We showed that isolated membranes from bovine retina release enzymatically AEA and DHEA and contain a FAAH-like activity. 306 BISOGNO ET AL. Finally, this tissue also contains 2-AG and 2-DHG, as well as some of the potential phospholipid precursors for these compounds. The finding, in bovine retina, of the endocannabinoids: (i) provides biochemical grounds to the previously reported pharmacological evidence of an endocannabinoid tone controlling dopamine release in this tissue (27), and (ii) suggests that AEA and 2-AG may play a role as local hypotensive agents in the eye. Future studies will be required in order to assess not only if the levels of AEA and 2-AG change with the onset of pathological conditions, but also to understand the role in the eye of DHEA and 2-DHG. In fact, both these metabolites are weak agonists at the CB
1

cannabinoid receptors (39, 40), and it is unlikely that they exert a hypotensive action, unless it is conclusively proven that AEA activity on IOP is also due to noncannabinoid receptor-mediated effects. DHEA and 2-DHG might also act as local inhibitors of AEA and 2-AG hydrolysis, thereby enhancing the local effects of the two endocannabinoids, as previously suggested for other NAEs and monoacylglycerols (17, 41). In any event, the findings reported in the present study should spark further research efforts aimed at developing new AEA-derived hypotensive drugs for the cure of glaucoma.
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