bisogno 99, bio synthesis and inactivation an and amide, arch b

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Archives of Biochemistry and Biophysics

Vol. 370, No. 2, October 15, pp. 300307, 1999


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Article ID abbi.1999.1410, available online at http://www.idealibrary.com on

Biosynthesis and Inactivation ofN-Arachidonoylethanolamine

(Anandamide) and N-Docosahexaenoylethanolamine

in Bovine RetinaT. 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;

andInstitut National de la Sante et de la Recherche Medicale, 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). Thistechnique also allowed us to identifyN-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 thesn-1 andsn-2 positions of bovine

retinas 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.


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Two receptor subtypes for9-tetrahydrocannabinol

(THC)3, the active constituent ofCannabis sativa, have

been identified and named CB

1

and CB

2

. While theformer 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 (35) 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:

[email protected]. 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; NPHPLC, normal phasehighpressure 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.


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gested that AEA, analogous to otherN-acylethanol-

amines (NAEs), is released from a preformed mem-

brane phospholipid,N-arachidonoylphosphatidyletha-

nolamine (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

thesn-1 position of phosphoglycerides (811). An al-

ternative AEA biosynthetic pathway, through the en-

ergy-free condensation between AA and ethanolamine,

was also suggested to occur in some reproductive tis-

sues (11). 2-AG biosynthesis depends onsn-2arachi-

donate-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 re-cently characterized and named fatty acid amide hy-

drolase (FAAH) (14). This enzyme catalyzes the hy-

drolysis 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 (1821).

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 pro-

duced 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 canna-

binoid receptor agonist, WIN-55,212-2, or for the met-

abolically stable AEA congener, (R)-( -methanand-

amide (24). In another study (25), however, unilateral

administration of AEA, its metabolically stableR- -

isopropyl analogue, or the synthetic cannabinoid CP-

55,940 did reduce the IOP. Only the effect of the latter

two compounds was prevented by pretreatment of the

animals with the CB

1


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receptor antagonist SR141716A,

suggesting that AEA hypotensive action could be me-

diated 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 activ-

ity. 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 progressiverodcone degeneration (30). The fatty acids composi-

tion 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 phos-

pholipids, with high levels being found in PC and PE.

Furthermore, unusually high levels of di-docosahex-

anoyl-PC and -PE species, i.e., two of the potential

fatty acid donors for the formation of a putativeN-

docosahexanoyl-PE (NDHPE) and, subsequently, ofN-

docosahexanoylethanolamine (DHEA), have been re-

ported for rat retina (31). Starting from this back-

ground, the objective of the present study was to

provide an answers to the following two open ques-

tions: (i) Are 2-AG, AEA, and AEA putative biosyn-

thetic precursor, NArPE, produced and metabolized by

the retina? (ii) In view of the high levels of phospho-

lipid-bound DHA found in this tissue, is it possible that

retinas also produce and metabolize 2-docosahexanoyl-

glycerol (2-DHG), DHEA, and DHEA putative precur-


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sor, NDHPE?

MATERIALS AND METHODS

DHEA was synthesised by mixing equimolar amounts of docosa-

hexaenoyl-chloride (obtained by reacting docosahexaenoic acid

(Sigma) with an excess of oxalyl chloride) and ethanolamine for 20

min at 4C in anhydrous dichloromethane. The mixture was then

brought to dryness under a flow of N2

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% ofn-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 chromatographyelectron

impact mass spectrometric (GC-EIMS) analysis of the tris-methyl-

silyl-ether, carried out as previously described (33) on a HP-5989B

quadrupole mass analyzer equipped with an electron impact sourceoperating at 70 eV and 250C, 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, MachereyNagel; length, 30 m; i.d., 0.25 mm) and was

eluted with a programmed temperature gradient from 200 to 300C

(5C/min; flow rate, 1 ml/min; injector and detector temperature,

260C). 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


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FIG. 1. Characterization ofN-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 ion-

monitoring EIMS

spectrum of native 2-DHG from bovine retina.

302

Abundance

BOO 7000 6000 5000

4000 300 2000 1000

7000 3

6000-

134 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


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


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column chromatography and NP-HPLC as described (34). NP-HPLC

fractions with the same retention time of monoacylglycerols (1722

min) and NAEs (2428 min) were liophylized and trimethylsilylated

as previously described (33). The samples were analyzed by GC-

EIMS 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 modeto 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/z404 and 412 (loss of methyl group

from undeuterated and d

8

-AEA) and m/z507 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 identifiedas the corresponding NAEs released from the digestion of NArPE-

like 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 analyzetheir fatty acid composition, phospholipids were transmethylated for

90 min at 100C 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 3,5-

dinitrobenzoylchloride in anhydrous pyridine and separated into

diacylglycerobenzoates (DGBZ) and ether-linked glycerobenzoates

by TLC on silica gel using a solvent system of toluene/hexane/diethylether (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. Mo-

lecular 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

thesn-1 (orsn-3) andsn-2 position. Experiments on NAE biosyn-


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thesis were performed by using a procedure similar to that previ-

ously described for rat testes (37). Aliquots (3.2 mg of total proteins)

of pooled particulate fractions from a 280,000gcentrifugation of

retina homogenates were resuspended in 1 ml TrisHCl 50 mM, pH

7.4, and incubated at 37C for different intervals of time (0, 15, or 30

min). Control incubations were carried out with boiled membranes or

with aliquots of 280,000gsupernatants. After incubation the mix-tures were extracted with chloroform/methanol (2/1, by vol) contain-

ing 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 andanalyzed 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 TrisHCl, 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 centrifuga-

tion were incubated in 50 mM Tris, pH 9, at 37C for 30 min in


TABLE IFatty 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 0.2918:2 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


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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 fromarachidonoylchloride and [14C]ethanolamine as described (3). Incu-

bations were also carried out in buffers at different pH values (38) or

in the presence of arachidonoyltrifluoromethylketone (ATFMK, Bi-

omol), 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 ofDHEA 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/z443, and main fragment ions at m/z428 (loss

of a methyl group), m/z284 and 159 ( -cleavage

followed by rearrangement), m/z268 and 175 ( -cleavage), and m/z116 (cleavage between C1 and

C2 of the ethanolamine moiety). The native component

exhibited all the selected ion signals at m/z443,

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-


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TABLE II


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Molecular Species Analysis of Bovine

Retina Phosphatidylcholine (PC)

and Phosphatidylethanolamine (PE)

sn-1sn-2 PC PE

22:6 n-322:6 n-3 1.31 0.23 4.16 0.39

Dipolyunsaturated 1.51 0.18 6.70 1.05

18:122:6 n-3 1.22 0.05 4.35 0.1016:022:6 n-3 8.05 0.43 14.03 0.17

18:120:4 n-6 0.47 0.20 0.86 0.13

16:020:4 n-6 2.93 0.60 1.29 0.01

16:022:5 n-6 0.83 0.29 0.83 0.28

18:022:6 n-3 15.19 1.79 47.83 1.30

18:020:4 n-6 4.18 0.31 9.35 1.55

18:022:5 n-6 1.11 0.56 2.05 0.45

16:018:1 25.66 0.36 2.16 0.76

16:016:0 20.52 0.22 2.61 0.33

18:018:1 7.24 0.53 1.76 0.56

16:018:0 7.64 0.26 0.48 0.15

Others 2.22 0.05 1.54 0.05

Note. Phospholipids were converted into diradylglycerols by treat-ment with phospholipase C, derivatized with 3,5-dinitrobenzoyl-

chloride, 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 dira-

dylglycerol 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/z419, 404, 175, and 116, typical of the tris-

methyl-silyl-ether of AEA. The amount of AEA was

64.0 9.6 pmol/g (n 2). Finally, the tris-methyl-

silyl-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 ofN-Acylethanolamines (NAEs) in Bovine Retina Membranes


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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 15.2 342.0 142.0 873.4 247.8Note.NAE release from 280,000gisolated pellets incubated at 37C in 50 mM TrisHCl 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 stan-

dards 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 chlo-

roform/methanol (1/1, by vol) was digested with S.

chromofuscusphospholipase 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. Thesedata 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

correspondingN-acylPEs. Of the several tissues where

the presence of NAEs has been reported so far (re-

viewed in (6, 11)), only rat brain was shown previously


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to contain DHEAin amounts slightly higher than

AEAas well as the correspondingN-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-methyl-

silyl-ether of the latter metabolite were the molecular

ion at m/z546, the peak at m/z531 (loss of a

methyl group), and the peak at m/z456 (loss of one

moiety of tris-methyl-silyl-alcohol). The EIMS spec-

trum of the bis-tris-methyl-silyl-ether of 2-AG has been

previously reported (12) and exhibited the same frag-

mentation pattern described above for 2-DHG, with

typical ions at m/z522, 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


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


20/28

in bovine retina. FAAH activity was measured by quantifying

[14C]ethanolamine released from [14C]AEA hydrolysis. (A) Subcellu-

lar localization of [14C]AEA-hydrolyzing activity in bovine retina

homogenate. (B) pH dependency of the [14C]AEA-hydrolyzing activity

from bovine retina 280,000gpellets, expressed as a percentage of the


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, al-

though described here for the first time, is not surpris-

ing 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 phospholipidswe found, in agreement with previous studies (for ex-

ample (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

thesn-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-acyl-

PE, respectively, of AA and DHA are available in bo-

vine retina. The hypothesis that AEA and DHEA could

be biosynthesized in the retina like in nervous tissue

(710), i.e., directly from theirN-acyl-PE precursors, is

in agreement with the observation that total mem-

brane preparations from this tissuewhich suppos-

edly contain both these precursors and the phospho-

lipase D enzyme necessary for their conversion into

NAEs (6, 11)released increasing amounts of AEA

and DHEA when incubated at 37C in a physiological

buffer (Table III). Also the other NAEs found here in

lipid retina extracts were produced under these condi-

tions, although in lower amounts, whereas no NAE

was released from heat-inactivated membrane prepa-

rations or from aliquots of the 280,000gsupernatant

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 ethanol-

amine from phospholipids and PE, respectively, cannot

be ruled out. Therefore, although we did not add exog-

enous arachidonate or DHA and ethanolamine to the


21/28

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 280,000gpellets. The effect

is expressed as a percentage of the activity in the absence of inhib-

itors. Data are means SE from three separate experiments carried

out in duplicate. The asterisk indicates values statistically different

from controls (P0.05, as determined by the unpaired Students t

test). AEA,N-arachidonoylethanolamine; DHEA,N-docosa-

hexaenoylethanolamine; PMSF, phenylmethylsulfonyl fluoride; AT-

FMK, arachidonoyltrifluoromethylketone.

% Maximal activity % Mximal activity'


22/28


23/28

theN-acyl composition of NAEs produced from mem-

brane incubations seems to reflect the fatty acid com-

position of PE rather than PC (Table I), thus suggest-

ing that PE may act as the source of theN-fatty acids

of NAEs. Within the PE class, the fatty acid composi-

tion on thesn-2 position rather than thesn-1 position

reflected more closely the composition of NAEs re-

leased after 30-min incubations, thus suggesting that,

under these conditions, the remodeling of phospholip-

ids 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 inhibi-tors, 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 80,000gand

280,000gcentrifugations). The enzyme activity in

these fractions was lower than that reported for por-

cine retina, possibly also because we did not use a

saturating concentration of the substrate. However,

bovine retina AEA hydrolase displayed optimal activ-

ity 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 ex-

tent than that observed with 100 M AEA. This latter

finding suggests that the enzyme recognizes as sub-

strate also the ethanolamide of docosahexaenoic acid,

albeit with a lower efficiency. This may indicate that

the half-life of DHEA in bovine retina is relativelylonger than that of AEA, thus possibly compensating

for the lower activity of DHEA at CB

1

receptors (39).

However, the apparentK

m

and V

max


24/28

values for the

hydrolysis of DHEA should be calculated by using the

appropriate labeled compound before drawing any con-

clusion 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 precur-

sors, NArPE and NDHPE, as well as precursors for

these latter phospholipids, i.e., diarachidonoyl- and di-

docosahexaenoyl-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 dopa-

mine 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 ago-nists 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 sug-

gested for other NAEs and monoacylglycerols (17, 41).

In any event, the findings reported in the present study

should spark further research efforts aimed at devel-

oping new AEA-derived hypotensive drugs for the cure

of glaucoma.

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