Anandamide degradation and N-acylethanolamines level in wild-type and CB1 cannabinoid receptor knockout mice of different ages: Anandamide degradation in CB1 knockout mice

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 339±348 339

Journal of Neurochemistry, 2001, 78, 339±348

Anandamide degradation and N-acylethanolamines level in

wild-type and CB1 cannabinoid receptor knockout mice of

different ages

Mauro Maccarrone,* Marina AttinaÁ,²,1 Monica Bari,* Antonella Cartoni,² Catherine Ledent³ andAlessandro Finazzi-AgroÁ*

Departments of *Experimental Medicine and Biochemical Sciences and ²Chemical Sciences and Technologies, University of Rome

`Tor Vergata', Italy

³IRIBHN, Universite Libre de Bruxelles, Brussels, Belgium

Abstract

CD1 mice lacking the CB1 receptors (knockout, KO) were

compared with wild-type littermates for their ability to degrade

N-arachidonoylethanolamine (anandamide, AEA) through a

membrane transporter (AMT) and a fatty acid amide hydro-

lase (FAAH). The regional distribution and age-dependence of

AMT and FAAH activity were investigated. Anandamide

membrane transporter and FAAH increased with age in

knockout mice, whereas they showed minor changes in

wild-type animals. Remarkably, they were higher in all brain

areas of 6-month-old knockout versus wild-type mice, and

even higher in 12-month-old animals. The molecular mass

(<67 kDa) and isoelectric point (<7.6) of mouse brain FAAH

were determined and the FAAH protein content was shown to

parallel the enzyme activity. The kinetic constants of AMT and

FAAH in the cortex of wild-type and knockout mice at different

ages suggested that different amounts of the same proteins

were expressed. The cortex and hippocampus of wild-type

and knockout mice contained the following N-acylethanola-

mines: AEA (8% of total), 2-arachidonoylglycerol (5%),

N-oleoylethanolamine (20%), N-palmitoylethanolamine (53%)

and N-stearoylethanolamine (14%). These compounds were

twice as abundant in the hippocampus as in the cortex. Minor

differences were observed in AEA or 2-arachidonoylglycerol

content in knockout versus wild-type mice, whereas the other

compounds were lower in the hippocampus of knockout

versus wild-type animals.

Keywords: anandamide hydrolase, anandamide transporter,

CB1 receptor knockout, CD1 mouse, endocannabinoids.

J. Neurochem. (2001) 78, 339±348.

Endocannabinoids are an emerging class of lipid mediators,

isolated from brain and peripheral tissues (Devane et al.

1992; Pop 1999). They are amides and esters of long-chain

polyunsaturated fatty acids. N-Arachidonoylethanolamine

(anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are

the main endogenous agonists of cannabinoid receptors

described to date (for reviews, see Pop 1999; Salzet et al.

2000). They bind to both brain (CB1) and peripheral (CB2)

cannabinoid receptors, thus mimicking some of the

psychotropic and analgesic effects of D9-tetrahydrocanna-

binol, the psychoactive principle of hashish and marijuana.

Moreover, endocannabinoids inhibit gap junction commu-

nication in glial cells (Venance et al. 1995), regulate sleep

induction (Boger et al. 1998), interact with GABAergic

(Tsou et al. 1998), serotonergic (Cheer et al. 1999) and

dopaminergic (Beltramo et al. 2000) neurotransmission, and

are also formed during glutamate-induced neurotoxicity

(Hansen et al. 1999). The activity of AEA at CB receptors

depends on its life-span in the extracellular space, which is

regulated by AEA degradation. This normally occurs in a

Received March 12, 2001; revised manuscript received April 19, 2001;

accepted April 20, 2001.

Address correspondence and reprint requests to Alessandro Finazzi-

AgroÁ or Mauro Maccarrone, Department of Experimental Medicine and

Biochemical Sciences, University of Rome `Tor Vergata', Via di Tor

Vergata 135, I-00133 Rome, Italy. E-mail: [email protected] or

[email protected] October 26, 2000.

Abbreviations used: AEA, N-arachidonoylethanolamine (ananda-

mide); 2-AG, 2-arachidonoylglycerol; AMT, AEA membrane transpor-

ter; AM404, N-(4-hydroxyphenyl)-arachidonoylamide; FAAH, fatty

acid amide hydrolase; GC/MS, gas chromatography-mass spectrometry;

NAE, N-acylethanolamines; OEA, N-oleoylethanolamine; PBS, phosphate-

buffered saline; PEA, N-palmitoylethanolamine; pI, isoelectric point;

SDS2PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis;

SEA, N-stearoylethanolamine; SIN-1, 3-morpholinosydnonimine;

SNAP, S-nitroso-N-acetylpenicillamine; SNP, sodium nitroprusside.

two-step process, including: (i) cellular uptake by the AEA

membrane transporter (AMT; Maccarrone et al. 2000a), and

(ii) intracellular degradation by the enzyme anandamide

hydrolase (fatty acid amide hydrolase, FAAH; Maccarrone

et al. 2000d). Recently, a functional link has been

demonstrated in vitro between CB1 receptors and AMT,

suggesting that activation of the receptors triggers AEA

degradation by enhancing AMT activity (Maccarrone et al.

2000a). Mutant mice lacking the CB1 receptor gene

represent a powerful tool to investigate the role of a putative

CB12AMT coupling in vivo, and to ®nd out whether the

CB1-dependent regulation of AEA degradation might be

compensated when the CB1 gene is deleted. Here, we

studied the regional distribution of AMT and FAAH in

different brain areas of CB1 knockout (KO) and wild-type

mice (Ledent et al. 1999). We also measured the levels of

AEA and 2-AG, as well as of the other N-acylethanolamines

[N-oleoylethanolamine (OEA), N-palmitoylethanolamine

(PEA) and N-stearoylethanolamine (SEA)], and looked for

a possible age dependence of endocannabinoid degradation

by wild-type and CB1 knockout animals.

Materials and methods

Materials and animals

All chemicals were of the purest analytical grade. Anandamide,

oleic, palmitic and stearic acids, ethanolamine and sodium

nitroprusside (SNP) were from Sigma Chemical Co. (St. Louis,

MO, USA). The N-acylethanolamines (NAE) of oleic (OEA),

palmitic (PEA) and stearic (SEA) acids were synthesized by

reacting the corresponding fatty acyl chloride with ethanolamine,

as described previously (Van der Stelt et al. 1997). [3H]AEA

(223 Ci/mmol) was purchased from NEN Dupont de Nemours

(KoÈln, Germany). Deuterated anandamide (AEAd4) was synthe-

sized from d4-ethanolamine (isotopic purity � 98%; Isotec Inc.,

Miamisburg, OH, USA) according to Schmid et al. (1995). The

unlabelled/labelled ratio of AEAd4 was � 0.02. 2-Arachidonoyl-

glycerol (2-AG), S-nitroso-N-acetylpenicillamine (SNAP) and

N-(4-hydroxyphenyl)-arachidonoylamide (AM404) were from

Research Biochemicals International (Natick, MA, USA).

3-Morpholinosydnonimine (SIN-1) was from Alexis Corporation

(LaÈufel®ngen, Switzerland). Linvanil (a-linolenoyl-vanillyl-amide)

was a kind gift of Dr V. Di Marzo (Consiglio Nazionale delle

Ricerche, Arco Felice, Italy). The identity and chemical purity of

NAEs and 2-AG standards were determined by 1H NMR and mass

spectrometric analysis under electron impact mode (Maccarrone

et al. 2001). Anti-FAAH polyclonal antibodies were elicited in

rabbits against the conserved FAAH sequence VGYYETDNY

TMPSPAMR (Giang and Cravatt 1997), conjugated to ovalbumin,

and were prepared by Primm S.r.l. (Milan, Italy). Goat anti-rabbit

alkaline phosphatase conjugates were purchased from Bio-Rad

(Richmond, CA, USA). Non-immune rabbit serum was from

Nordic Immunology (Tilburg, The Netherlands).

The generation of CD1 mice lacking the cannabinoid CB1

receptors (knockout mutants) was described earlier (Ledent et al.

1999). In order to homogenize the genetic background of the mice,

®rst-generation heterozygotes (CB1 1/2) were bred for ®ve

generations on a CD1 background, with selection for the mutant

CB1 gene at each generation. Fifth-generation heterozygotes were

bred together, to generate the homozygous mice, either wild-type

(CB11/1, WT) or knockout (CB12/±, KO), used in this study.

Mice were maintained for the indicated periods under standard

animal housing conditions in a 12 h dark/light cycle with free

access to food and water. Equal numbers of male and female

animals were tested in all biochemical analysis and the data were

pooled, because there were no statistically signi®cant differences in

the results obtained with males and females. Animal care was in

accordance with ethical guidelines (Directive 86/609/EEC; Council

of Europe November 24, 1986) and all experimental procedures

were approved by the local animal care committees.

Determination of anandamide uptake

Synaptosomes were prepared from the different areas of mouse

brain as described (Barbaccia et al. 1983). Tissues were

resuspended in ice-cold 0.32 m sucrose, 5 mm Tris2HCl buffer

(pH 7.4) and were gently disrupted by 10 up-and-down strokes in a

Te¯on-glass homogenizer (1 : 20 w/v). The homogenates were

centrifuged at 1000 g for 5 min, at 48C, the supernatants were then

centrifuged again at 17 000 g for 15 min, at 48C. The ®nal pellets

were resuspended in 136 mm NaCl, 5 mm KCl, 0.16 mm CaCl2,

0.1 mm EGTA, 1.3 mm MgCl2, 10 mm glucose, 10 mm Tris2HCl

buffer (pH 7.4), at a protein concentration of 3 mg/mL. The

activity of the AEA membrane transporter (AMT) was measured as

described (Maccarrone et al. 2000a). Synaptosomes (100 mL/test)

were incubated for different time intervals, at 378C or 48C, with

300 nm [3H]AEA, they were washed three times in 2 mL

phosphate-buffered saline (PBS) containing 1% bovine serum

albumin and were ®nally resuspended in 200 mL PBS. Membrane

lipids were then extracted (Maccarrone et al. 2000a), resuspended

in 0.5 mL methanol, mixed with 3.5 mL Sigma-Fluor liquid

scintillation cocktail for non-aqueous samples (Sigma), and radio-

activity was measured in a LKB1214 Rackbeta scintillation counter

(Amersham Pharmacia Biotech, Uppsala, Sweden). To discriminate

non-carrier-mediated from carrier-mediated transport of AEA

through cell membranes, [3H]AEA uptake at 48C was subtracted

from that at 378C (Hillard et al. 1997). Incubations (15 min) were

also carried out with different concentrations of [3H]AEA, in the

range 0±1000 nm, in order to determine apparent Km and Vmax

values of the uptake (also in this case, the uptake at 48C was

subtracted from that at 378C). The experimental points were

analysed by non-linear regression with a prism 3 program

(GraphPAD Software for Science, San Diego, CA, USA).

Anandamide membrane transporter activity was expressed as

pmol of AEA taken up per min per mg protein. The Q10 value

was calculated as the ratio of AEA uptake at 30 and 208C

(Hillard et al. 1997). The effect of various compounds on AMT

activity was determined by adding directly each substance to the

assay buffer, at the indicated concentrations, and incubating for

15 min at 378C.

Anandamide hydrolase assay

Anandamide hydrolase (arachidonoylethanolamide amidohydro-

lase, EC 3.5.1.4) activity was assayed in mouse brain homogenates

by RP-HPLC, incubating the samples for 15 min with 5 mm

[3H]AEA (Maccarrone et al. 1998). Anandamide hydrolase activity

340 M. Maccarrone et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 339±348

was expressed as pmol of arachidonate released per min per mg

protein. Kinetic studies were performed using different concentra-

tions of [3H]AEA (in the range 0±15 mm), and the kinetic constants

(Km, Vmax) were calculated by ®tting the experimental points to a

non-linear regression plot, as described above for [3H]AEA uptake.

For immunochemical analysis, sodium dodecyl sulfate±poly-

acrylamide gel electrophoresis (SDS2PAGE; 12%) was performed

under reducing conditions in a Mini Protean II apparatus (Bio-Rad),

with 0.75 mm spacer arms (Maccarrone et al. 1998). Rainbow

molecular mass markers (Amersham Pharmacia Biotech) were

phosphorylase b (97.4 kDa), bovine serum albumin (66.0 kDa) and

ovalbumin (46.0 kDa). Native IEF was performed in the Mini

Protean II apparatus, using a 5% polyacrylamide gel containing

ampholytes in the pH range 5.0±9.0 (Sigma), as described

(Robertson et al. 1987). IEF was calibrated by running the

following isoelectric point (pI) markers (Sigma): lentil (Lens

culinaris) lectin (8.8, 8.6 and 8.2), myoglobin from horse heart (7.2

and 6.8), carbonic anhydrase I from human erythrocytes (6.6) and

carbonic anhydrase II from bovine erythrocytes (5.9). Mouse brain

homogenates (20 mg/lane) were subjected to either SDS2PAGE or

IEF, slab gels were then electroblotted onto 0.45 mm nitrocellulose

®lters (Bio-Rad), using a Mini Trans Blot apparatus (Bio-Rad) as

reported (Maccarrone et al. 1998). Immunodetection of FAAH on

nitrocellulose ®lters was performed with anti-FAAH polyclonal

antibodies (diluted 1 : 200), which have previously been shown to

speci®cally recognize FAAH in human brain (Maccarrone et al.

Fig. 1 Anandamide hydrolysis and uptake

in wild-type and CB1 receptor knockout

(KO) mice of different ages. Anandamide

hydrolase (FAAH) activity (a) and protein

content (b), and anandamide membrane

transporter (AMT) activity (c) were mea-

sured in different mouse brain areas. Anan-

damide hydrolase activity and AMT activity

were measured using 5 mM and 300 nM

[3H]AEA, respectively, with 15 min incuba-

tions. In (b), 100% � 0.250 ^ 0.030 absor-

bance units at 405 nm. Vertical bars

represent SD values. *p , 0.05 versus

corresponding WT; **p , 0.01 versus cor-

responding WT (n � 4 in all cases).

Anandamide degradation in CB1 knockout mice 341

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 339±348

1998), human lymphocytes (Maccarrone et al. 2000d) and mouse

uterus (Maccarrone et al. 2000b). Goat anti-rabbit alkaline

phosphatase conjugates (diluted 1 : 2000) were used as the second

antibody. Immunoreactive bands were stained with the alkaline

phosphatase staining solution according to the manufacturer's

instructions (Bio-Rad). ELISA was performed by coating the plate

with mouse brain homogenates (20 mg/well), reacted with anti-

FAAH polyclonal antibodies (diluted 1 : 300) and then with goat

anti-rabbit alkaline phosphatase conjugate (diluted 1 : 2000).

Colour development of the alkaline phosphatase reaction was

measured at 405 nm, using p-nitrophenylphosphate as substrate.

Controls were carried out by using non-immune rabbit serum and

included wells coated with different amounts of bovine serum

albumin.

GC/MS analysis

Immediately after decapitation, mouse brains were washed in PBS,

pre-cooled at 48C, dissected, frozen in liquid nitrogen, and kept at

2708C until processed. A maximum of 8 min elapsed between

mouse decapitation and freezing of dissected tissues, a time not

suf®cient to cause arti®cial increases in endocannabinoid levels (Di

Marzo et al. 2000a; Maccarrone et al. 2001). Lipids were extracted

from frozen tissues and analysed by gas chromatography-electron

impact mass spectrometry (GC/MS), as described (Maccarrone

et al. 2001). Lipid extracts were injected into a Carlo Erba model

HRGC5160 gas chromatograph (Rome, Italy), equipped with a BP5

silica capillary column (30 m � 0.25 mm i.d.) from SGE (Milan,

Italy), and interfaced with a VG Micromass model QUATTRO

spectrometer (Manchester, UK). Analyses were performed in

`splitless' mode at temperatures increasing from 70 to 2508C, at a

rate of 308C/min. The identity of NAEs was assessed by

comparison of the retention times and the mass spectra recorded

at 70 eV with those of authentic standards. A full scan spectrum of

NAEs and 2-AG in brain samples, and electron impact mass spectra

of these compounds and the corresponding standards, have been

recently reported (Maccarrone et al. 2001). Quanti®cation of AEA

was achieved by isotope dilution with AEAd4, whereas the other

NAEs and 2-AG were quanti®ed by the internal standard method

with AEAd4. Calibration solutions were prepared by adding a ®xed

amount of AEAd4 (5 nmol) to samples containing various amounts

of NAEs (up to 80 nmol) or 2-AG (up to 40 nmol), all analysed in

the selected ion monitoring (SIM) mode. Calibration curves were

obtained by plotting the area ratios of each characteristic selected

ion (m/z � 85, 98 for AEA; 85, 98, 154 for NAEs; 79, 91, 105 for

2-AG) to the fragment of the internal standard AEAd4 (m/z � 89 or

102), as a function of the nmol amount of AEA, of the other NAEs

or of 2-AG (Maccarrone et al. 2001). The quantitative data for each

compound were calculated after correction for the recovery of all

NAEs and 2-AG from the extraction procedure, which was < 60%

in all cases. Moreover, for each sample all characteristic selected

ions of each compound were monitored simultaneously, and each

injection of samples into GC was followed by injection of pure

solvent, in order to avoid signal disturbance by the background

noise. The detection limit, calculated with standard solutions of

NAEs or of 2-AG, was 20 ^ 10 pmoles (Maccarrone et al. 2001).

Statistical analysis

The data reported here are the mean (^ SD) of at least

four independent determinations, each performed in duplicate.

Statistical analysis was performed by the non-parametric Mann±

Whitney test, elaborating experimental data by means of the Instat

program (GraphPad Software Inc., San Diego, CA, USA).

Results

Anandamide degradation in CB11/1 and CB1±/± mice

of different ages

Anandamide hydrolase activity was measured in different

areas of mouse brain, and was found in the order

hippocampus, cerebellum, cortex, striatum (Fig. 1a). The

same regional distribution of FAAH activity was observed

in CB1 wild-type and in CB1 knockout animals at all ages

tested (2, 6 or 12 months). Such a distribution is in keeping

with previous reports on rat brain FAAH (Hillard et al.

1995) and correlates with the distribution of cannabinoid

receptor binding sites (Hillard et al. 1995; Tsou et al. 1998).

In wild-type mice, FAAH activity was not age dependent,

whereas it increased signi®cantly with ageing in knockout

animals (Fig. 1a). In this latter group, hippocampus showed

the most remarkable increase in FAAH activity compared

with wild-type mice of the same age, followed by cortex,

cerebellum and striatum (Fig. 1a). The increase of FAAH

activity in knockout versus wild-type mice reached

statistical signi®cance in 6-month-old animals and was

even higher at 12 months (Fig. 1a).

Western blotting showed that anti-FAAH polyclonal

antibodies speci®cally recognized a single immunoreactive

band in homogenates of whole mouse brain, corresponding

to a molecular mass of < 67 kDa and an pI of < 7.6 (Fig. 2).

These molecular properties of mouse brain FAAH, reported

here for the ®rst time, resemble those of FAAH in human

brain (Maccarrone et al. 1998). The anti-FAAH antibodies

were used to quantify FAAH protein content in the different

Fig. 2 Electrophoretic properties of mouse brain FAAH. Mouse

brain extracts (20 mg/lane) were subjected to either SDS2PAGE

(left) or IEF (right). Slab gels were then electroblotted on nitrocellu-

lose ®lters and FAAH was detected with speci®c anti-FAAH polyclo-

nal antibodies. Molecular mass markers and pI markers are shown.

342 M. Maccarrone et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 339±348

areas of mouse brain by ELISA (Fig. 1b). Anandamide

hydrolase protein was shown to follow the same regional

distribution as the enzyme activity (compare Fig. 1a and b),

suggesting that the same FAAH was differently expressed in

the different brain areas.

In pilot experiments, synaptosomes prepared from the

cortex of wild-type, 2-month-old mice were found to take up

[3H]AEA in a dose-, temperature- (Q10 < 1.5) and time- (t1/2

< 5 min) dependent manner (Fig. 3a, b and data not shown),

suggesting that an AMT was responsible for [3H]AEA

uptake (Bisogno et al. 1997; Hillard et al. 1997; Maccarrone

et al. 1998, 2000a; Jarrahian et al. 2000). The uptake of

300 nm [3H]AEA by AMT was inhibited by 10 mm AM404

or 10 mm linvanil (Fig. 3c), previously shown to selectively

inhibit AEA uptake by cultured cells (Piomelli et al. 1999;

Melck et al. 1999). Conversely, nitric oxide donors SNP and

SNAP, and even more peroxynitrite donor SIN-1, activated

AMT in cortex synaptosomes (Fig. 3c). Sodium nitroprus-

side, SNAP and SIN-1 were used at 5, 2.5 and 1 mm,

respectively, concentrations shown previously to activate

AMT in human cells in culture (Maccarrone et al. 2000a). In

contrast, 1 mm 2-AG inhibited almost completely the uptake

of 300 nm [3H]AEA by AMT in cortex synaptosomes

(Fig. 3c), again extending previous observations on cultured

cells (Maccarrone et al. 2000a). To our knowledge, this is

the ®rst report showing AMT activity ex vivo, i.e. in syn-

aptosomes prepared from brain areas. The same methodol-

ogy was used to investigate the regional distribution of AMT

Fig. 3 Anandamide membrane transporter

(AMT) in the brain cortex of wild-type,

2-month-old mice. Dependence of AMT

activity on anandamide (AEA) concentration

(a) and temperature (b). (c) Effect of differ-

ent compounds on the activity of AMT at

378C. *p , 0.01 versus controls; **p , 0.05

versus controls (n � 4). In (b) and (c),

300 nM [3H]AEA was used as substrate. In

all experiments synaptosomes were incu-

bated with [3H]AEA for 15 min.

Anandamide degradation in CB1 knockout mice 343

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 339±348

activity, which was found to follow the order striatum .

hippocampus . cortex � cerebellum (Fig. 1c). In wild-type

animals, AMT activity decreased (striatum and hippocam-

pus) or remained unchanged (cortex and cerebellum) with

ageing, whereas in knockout mice it increased remarkably at

older ages (Fig. 1c). The molecular properties of AMT are

unknown and there are no anti-AMT antibodies available.

Therefore, it was not possible to quantify AMT protein in

mouse brain areas. However, from saturation curves such as

that shown in Fig. 3(a), it was possible to calculate the

kinetic parameters, i.e. apparent Michaelis2Menten constant

(Km) and maximum velocity (Vmax), of AMT in the cortex of

wild-type and knockout mice of different ages (Table 1).

Anandamide membrane transporter in wild-type mice was

found to have the same Vmax values at all ages

(<150 ^ 15 pmol/min/mg protein), whereas in knockout

animals Vmax values increased from 150 ^ 15 pmol/min/mg

protein (at 2 months) to 420 ^ 40 pmol/min/mg protein (at

12 months). Km values of AMT activity were always

< 280 ^ 30 nm in all animals (Table 1). A similar trend

was observed with FAAH activity in the cortex, which

showed almost the same Vmax values in wild-type mice at all

ages (<1900 ^ 200 pmol/min/mg protein), but increasing

Vmax values in ageing knockout animals (from 1900 ^

200 pmol/min/mg protein at 2 months to 2600 ^ 250 pmol/

min/mg protein at 12 months). Km values for FAAH activity

were < 4.0 ^ 0.5 mm in all animals (Table 1).

N-Acylethanolamines level in CB11/1 and CB12/2

mice of different ages

GC/MS analysis of rapidly frozen samples showed that

mouse cortex and hippocampus contain ®ve NAEs: AEA,

2-AG, OEA, PEA and SEA (Tables 2 and 3). No traces of

1(3)-AG were detected either in standard 2-AG solutions or

in brain samples, suggesting that no appreciable isomeriza-

tion occurred under our experimental conditions, as recently

observed in human brain (Maccarrone et al. 2001). In wild-

type, 2-month-old animals, AEA represented < 8% of all

NAEs, 2-AG 5%, OEA 20%, PEA 53% and SEA 14%, both

in cortex (Table 2) and hippocampus (Table 3). However,

the amount of total NAEs in the hippocampus was

< twofold higher than the amount in the cortex (Tables 2

and 3). In this latter area NAEs, except OEA and 2-AG, did

Table 1 Kinetic constants of AEA membrane transporter (AMT) and

AEA hydrolase (FAAH) in the brain cortex of wild-type (WT) or

knockout (KO) mice

Mouse

cortex

AMT

Kma Vmax

b

FAAH

Kmc Vmax

b

WT (2 months) 280 �̂ 30 180 �̂ 20 4.2 �^ 0.5 1700 �̂ 180

KO (2 months) 280 �^ 30 150 �̂ 15 4.0 �^ 0.5 1900 �̂ 200

WT (6 months) 280 �̂ 30 150 �̂ 15 4.0 �^ 0.5 1900 �̂ 200

KO (6 months) 300 �^ 30 400 �̂ 40* 3.8 �^ 0.5 2300 �̂ 250**

WT (12 months) 290 �̂ 30 150 �̂ 15 4.1 �^ 0.5 2000 �̂ 200

KO (12 months) 300 �̂ 30 420 �̂ 40* 4.0 �^ 0.5 2600 �̂ 250**

aValues expressed as nM. bValues expressed as pmol/min/mg protein.cValues expressed as mM. *p , 0.01 versus WT (2 months);

**p , 0.05 versus WT (2 months) (n � 4 in all cases).

Table 2 Quantitative analysis of N-acylethanolamines and 2-arachidonoylglycerol in the cortex of wild-type (WT) or knockout (KO) mice

Compound

(nmol/mg protein)

WT cortex

(2 months)

KO cortex

(2 months)

WT cortex

(6 months)

KO cortex

(6 months)

AEA 0.2 �̂ 0.1 0.2 �^ 0.1 0.2 �^ 0.0 0.2 �^ 0.0

(100%) (100%) (100%) (100%)

2-AG 0.1 �̂ 0.0 0.1 �^ 0.0 0.1 �^ 0.0 0.1 �^ 0.0

(100%) (100%) (100%) (100%)

OEA 0.6 �̂ 0.2 0.8 �^ 0.2* 0.9 �^ 0.2* 0.8 �^ 0.1*

(100%) (133%) (150%) (133%)

PEA 1.5 �̂ 0.4 1.6 �^ 0.7 1.6 �^ 0.2 1.9 �^ 0.4

(100%) (107%) (107%) (127%)

SEA 0.4 �̂ 0.1 0.4 �^ 0.1 0.5 �^ 0.1 0.5 �^ 0.0

(100%) (100%) (125%) (125%)

Total amount 2.8 3.1 3.3 3.5

(100%) (111%) (118%) (125%)

AEA, N-arachidonoylethanolamine (anandamide); 2-AG, 2-arachidonoylglycerol; OEA, N-oleoylethanolamine; PEA, N-palmitoylethanolamine;

SEA, N-stearoylethanolamine. Values in brackets represent percentage of the wild-type (2 months) animals. *p , 0.05 versus WT (2 months)

(n � 4 in all cases).

344 M. Maccarrone et al.

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not signi®cantly change in wild-type (6 months) versus

wild-type (2 months), or in knockout (6 months) versus

knockout (2 months) mice, or in knockout versus wild-type

mice at either age (Table 2). However, OEA increased

signi®cantly in wild-type (6 months) versus wild-type

(2 months) and in knockout (2 months) versus wild-type

(2 months) mice (150 and 133% of the control, respec-

tively), but not in knockout (6 months) versus knockout (2

months) or in knockout (6 months) versus wild-type

(6 months) animals (Table 2). Interestingly, mouse hippo-

campus showed a marked decrease in OEA, PEA and

SEA content in knockout (2 months) versus wild-type

(2 months) mice, and an even larger decrease in

knockout (6 months) versus wild-type (6 months) animals

(Table 3). The decrease in OEA, PEA and SEA was also

signi®cant in wild-type (6 months) versus wild-type (2

months) and in knockout (6 months) versus knockout

(2 months) mice (Table 3). However, AEA decreased

signi®cantly only in knockout (6 months) versus wild-type

(6 months) animals, whereas the content of 2-AG in the

hippocampus was the same in all groups of mice tested

(Table 3).

Discussion

This study describes presence and kinetic properties of the

AMT in synaptosomes from different brain areas, con®rm-

ing ex vivo the model of AEA transport already described in

cultured cells. The activity of AMT from cortex synapto-

somes was enhanced by nitric oxide donors SNP and SNAP,

and even more by peroxynitrite donor SIN-1 (Fig. 3c). This

®nding, which con®rms previous in vitro data (Maccarrone

et al. 2000a), needs to be emphasized, because nitric oxide

and peroxynitrite have already been shown to couple CB1

receptor and AMT in human endothelial cells, where AEA

binding to CB1 induces release of nitric oxide, which in turn

activates AEA uptake by AMT and intracellular degradation

by FAAH (Maccarrone et al. 2000a). A functional link

between the AEA receptor and AEA degradation might form

a regulatory loop, because activation of CB1 stimulates AEA

removal from the extracellular space, thus terminating AEA

signalling at the receptor itself. Mutant mice lacking the

gene encoding for CB1 receptors (knockout mice) represent

a useful model in which this putative CB12AMT coupling

is disrupted. In contrast, AEA has been shown to have

neurotoxic potential, because of its ability to induce

programmed cell death of neuronal cells in vitro (Maccar-

rone et al. 2000c) and in vivo (Galve-Roperh et al. 2000).

Endocannabinoid formation has been observed consistently

during glutamate-induced neurotoxicity (Hansen et al.

1999). Therefore, it can be speculated that suppressing the

CB1 receptor gene, and thus CB12AMT coupling, might

reduce neuron survival, unless some kind of compensatory

mechanism takes place. Such a compensation should keep

AEA levels under control, thus explaining how CB1

knockout mice remain healthy and fertile (Ledent et al.

1999; Zimmer et al. 1999). The results reported here suggest

that indeed a more ef®cient degradation of AEA occurs

when the CB1 gene is disrupted (Fig. 1). Both AMT and

FAAH activity increased in knockout but not in wild-type

mice, and kinetic analysis (Table 1) suggests that this was

caused by increased amounts of just the same AMT and

Table 3 Quantitative analysis of N-acylethanolamines and 2-arachidonoylglycerol in the hippocampus of wild-type (WT) or knockout (KO) mice

Compound

(nmol/mg protein)

WT hippocampus

(2 months)

KO hippocampus

(2 months)

WT hippocampus

(6 months)

KO hippocampus

(6 months)

AEA 0.5 �^ 0.1 0�.4 ^ 0.0 0�.4 ^ 0.0 0�.3 ^ 0.0**²

(100%) (80�%) (80�%) (60�%)

2-AG 0.3 �^ 0.1 0�.3 ^ 0.1 0�.3 ^ 0.1 0�.3 ^ 0.1

(100%) (100�%) (100�%) (100�%)

OEA 1.3 �^ 0.3 1�.0 ^ 0.3 0�.9 ^ 0.1** 0�.3 ^ 0.1*³

(100%) (77�%) (69�%) (23�%)

PEA 3.0 �^ 1.0 1�.1 ^ 0.3* 1�.4 ^ 0.3* 0�.3 ^ 0.0*³

(100%) (37�%) (47�%) (10�%)

SEA 0.8 �^ 0.2 0�.4 ^ 0.0** 0�.5 ^ 0.1** 0�.1 ^ 0.0*³

(100%) (50�%) (62�%) (13�%)

Total amount 5.9 3�.2** 3�.5** 1�.3*³

(100%) (54�%) (59�%) (22�%)

AEA, N-arachidonoylethanolamine (anandamide); 2-AG, 2-arachidonoylglycerol; OEA, N-oleoylethanolamine; PEA, N-palmitoylethanolamine;

SEA, N-stearoylethanolamine. Values in brackets represent percentage of the WT (2 months) animals. *p , 0.01 versus WT (2 months);

**p , 0.05 versus WT (2 months); ²p , 0.05 versus WT (6 months); ³p , 0.01 versus WT (6 months) (n � 4 in all cases). The statistics of KO

(6 months) versus KO (2 months) showed p , 0.01 for OEA, PEA and SEA, and was omitted for the sake of clarity.

Anandamide degradation in CB1 knockout mice 345

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 339±348

FAAH, rather than to different isoforms. The observation

that FAAH expression at the protein level was enhanced in

KO mice (Fig. 1b) strengthens this concept. Remarkably,

AMT and FAAH activity increased further in ageing

animals, suggesting that AEA degradation might become

more critical. This observation can be explained by recalling

that AEA can be converted into noxious hydroperoxy-

derivatives by lipoxygenase and cyclooxygenase (Burstein

et al. 2000), which are more active in the brain of ageing

animals (Manev et al. 2000).

Quantitative analysis of NAEs and of 2-AG content in the

cortex and hippocampus of wild-type and knockout mice of

different ages yielded the results shown in Tables 2 and 3.

We were not able to extend our GC/MS analysis to other

brain areas or ages, because of the limited number of

animals available. We chose to normalize the amounts of

NAEs and 2-AG to the protein content of the samples, in

order to better compare different tissues, and also in

consideration of the fact that determination of protein is

much more signi®cant than that of fresh weight. Cortex and

hippocampus showed a similar composition in AEA and

congeners in wild-type, 2-month-old mice: PEA was the

most abundant NAE (53% of total), AEA (8%) and 2-AG

(5%) were in the lowest amounts (Tables 2 and 3). It is

noteworthy that AEA and 2-AG were detected in similar

nmol/mg protein concentrations, extending to mouse pre-

vious observations on human and rat brains (Maccarrone

et al. 2001). This is at variance with pmole per g values for

AEA and nmole per g values for 2-AG reported in rat (Stella

et al. 1997) and mouse (Di Marzo et al. 2000a) brain,

although it is in keeping with similar nmole per g values for

AEA and 2-AG reported in rat substantia nigra and globus

pallidus (Di Marzo et al. 2000b). We do not have an

explanation for this discrepancy on NAE quanti®cation, but

we have already ruled out that arti®cial increases in

endocannabinoid level might occur during our sample

preparation and analysis (Maccarrone et al. 2001), e.g.

because of post-mortem lipid breakdown and AEA accu-

mulation (Schmid et al. 1995; Stella et al. 1997). In this

context, it seems noteworthy that the amounts found here are

compatible with a role for AEA and 2-AG in the CNS not

only as modulatory substances (Cadas et al. 1997), but also

as true neurotransmitters (Self 1999). At any rate, an

important ®nding of this investigation is the observation that

AEA and 2-AG showed only minor changes (if any) in

ageing wild-type or knockout mice and, most notably, in

knockout versus wild-type animals (Tables 2 and 3).

Anandamide decreased in a statistically signi®cant way

only in the hippocampus of knockout versus wild-type mice

aged 6 months (Table 3). This seems interesting, if one

recalls that AEA and 2-AG are the most biologically active

endocannabinoids known to date (Pop 1999; Salzet et al.

2000) and can also exert non-CB receptor-mediated actions

(Pertwee 1997). In contrast, OEA, PEA and SEA decreased

markedly in the hippocampus of ageing mice (either wild-

type or knockout) and, most notably, in the hippocampus of

knockout versus wild-type animals. These ®ndings are

consistent with the increased FAAH activity in the

hippocampus of knockout animals (Fig. 1a), because OEA

and PEA are hydrolysed by FAAH (Boger et al. 2000).

However, because AEA and 2-AG are also hydrolysed by

FAAH, the steady level of these endocannabinoids might

imply an increased synthesis. The enhancement of synthesis

in CB1 knockout mice remains to be con®rmed, as does

its mechanism. Indeed, it is not yet clear whether

N-acylethanolamine phospholipid-speci®c phospholipases

D or N-acyltransferases might represent the rate-limiting

step of AEA synthesis (Hansen et al. 2000; references

therein), and even FAAH may play a role in this process

(Kurahashi et al. 1997). Therefore, further work is required

to address the contribution of these synthetic activities to

AEA homeostasis in CB1 knockout animals. The picture is

complicated further by the fact that the endocannabinoid

system plays a role in GABAergic (Tsou et al. 1998),

serotonergic (Cheer et al. 1999) and dopaminergic

(Beltramo et al. 2000) neurotransmission, and in pain

initiation (Calignano et al. 1998), making it likely that

endocannabinoid homeostasis is under the control of a

complex network of signalling molecules. At any rate, the

above data seem to represent a useful biochemical back-

ground to understanding the molecular mechanism(s)

responsible for the control of the activity of endocannabi-

noids in brain, as well as for the different behaviour of

CB1 knockout versus wild-type mice. During the

preparation of this manuscript, an article appeared showing

that FAAH activity, as well as AEA and 2-AG levels, were

unchanged in 2-month-old CB1 knockout versus wild-type

mice (Di Marzo et al. 2000a). Our investigation con®rms

and extends these data to the analysis to all NAEs known to

date, and to the activity of FAAH and AMT at different

ages.

Acknowledgements

Dr Francesca Klinger and Mr Giuseppe D'Arcangelo are

gratefully acknowledged for their excellent technical

assistance. Dr Vincenzo Di Marzo (Istituto per la Chimica di

Molecole d'Interesse Biologico, CNR, Arco Felice, Italy) is

gratefully acknowledged for the kind gift of linvanil. This

investigation was supported by Istituto Superiore di SanitaÁ (III

AIDS Program) and by Ministero dell'UniversitaÁ e della

Ricerca Scienti®ca e Tecnologica, Rome. CL was supported

by the Interuniversity Poles of Attraction (Belgian State,

Prime Minister's Of®ce, Federal Service for Science,

Technology and Culture), by the Fondation MeÂdicale Reine

Elisabeth, by the Fonds de la Recherche Scienti®que MeÂdicale

and by the EU BIOMED II program. Marina AttinaÁ is sadly

missed.

346 M. Maccarrone et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 339±348

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