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Experimental Gerontology 55 (2014) 134–142

Contents lists available at ScienceDirect

Experimental Gerontology
journal homepage: www.elsevier.com/locate/expgero

Cannabinoid receptor-dependent metabolism of 2-arachidonoylglycerol

during aging
Ana C. Pascual, Virginia L. Gaveglio, Norma M. Giusto, Susana J. Pasquaré ⁎
Instituto de Investigaciones Bioquímicas de Bahía Blanca, Universidad Nacional del Sur - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Edificio E1. Camino La Carrindanga
Km 7, 8000 Bahía Blanca, Argentina

a r t i c l e i n f o a b s t r a c t

Article history: 2-Arachidonoylglycerol (2-AG) is one of the principal endocannabinoids involved in the protection against neu-
Received 6 January 2014 rodegenerative processes. Cannabinoids primarily interact with the seven-segment transmembrane cannabinoid
Received in revised form 27 March 2014 receptor 1 (CB1) and cannabinoid receptor 2 (CB2), both of which are expressed in the central nervous system
Accepted 16 April 2014
(CNS). The level of 2-AG is controlled through key enzymes responsible for its synthesis or degradation. We
Available online 24 April 2014
have previously observed a deregulation of 2-AG metabolism in physiological aging. The aim of this study was
Section Editor: Christian Humpel to analyze how 2-AG metabolism is modulated by CB1/CB2 receptors during aging. To this end, both CB1 and
CB2 receptor expression and the enzymatic activities (diacylglycerol lipase (DAGL), lysophosphatidate
Keywords: phosphohydrolase (LPAase) and monoacylglycerol lipase (MAGL)) involved in 2-AG metabolism were analyzed
Cannabinoid receptors in the presence of cannabinoid receptor (CBR) agonists (WIN and JWH) and/or antagonists (SR1 and SR2) in syn-
2-Arachidonoylglycerol aptosomes from adult and aged rat cerebral cortex (CC). Our results demonstrate that: (a) aging decreases the
Aging expression of both CBRs; (b) LPAase inhibition, due to the individual action of SR1 or SR2, is reverted in the pres-
Lysophosphatidate phosphohydrolase ence of both antagonists together; (c) LPAase activity is regulated mainly by the CB1 receptor in adult and in aged
Diacylglycerol lipase
synaptosomes while the CB2 receptor acquires importance when CB1 is blocked; (d) modulation via CBRs of
Monoacylglycerol lipase
DAGL and MAGL by both antagonists occurs only in aged synaptosomes, stimulating DAGL and inhibiting
MAGL activities; (e) only DAGL stimulation is reverted by WIN. Taken together, the results of the present
study show that CB1 and/or CB2 receptor antagonists trigger a significant modulation of 2-AG metabolism,
underlining their relevance as therapeutic strategy for controlling endocannabinoid levels in physiological aging.
Published by Elsevier Inc.

1. Introduction 2-AG biosynthesis in neurons occurs through various possible

Several natural lipids called endocannabinoids have recently been
shown to bind to and activate cannabinoid receptors (CBRs). Ananda-
mide (AEA) and 2-arachidonoylglycerol (2-AG) are the most active en-
dogenous cannabinoids described to date (Devane et al., 1992; Stella
et al., 1997). The biological actions of AEA and 2-AG are controlled
through key enzymes responsible for either their synthesis or their deg-
radation (Basavarajappa, 2007).
Abbreviations: ABHD6, serine hydrolase alpha/beta-hydrolase domain-containing 6;
2-AG, 2-Arachidonoylglycerol; AEA, anandamide; BSA, bovine serum albumin; CBRs, CB1
and CB2 receptors; CC, cerebral cortex; CNS, central nervous system; DAG, diacylglycerol;
DAGL, diacylglycerol lipase; DTT, dithiotreitol; EDTA, ethylenediaminetetraacetic acid;
EGTA, ethylene glycol bis (β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid; FAAH, fatty
acid amide hydrolase; HEPES, N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid];
LPA, lysophosphatidic acid; LPAase, lysophosphatidate phosphohydrolase; MAG,
monoacylglycerol; MAGL, monoacylglycerol lipase; TLC, thin-layer chromatography.
⁎ Corresponding author at: Instituto de Investigaciones Bioquímicas de Bahía Blanca
(INIBIBB), Centro Científico y Tecnológico CONICET-Bahía Blanca and Universidad
Nacional del Sur, Edificio E1, Camino La Carrindanga Km 7, 8000 Bahía Blanca, Argentina.
Tel.: +54 291 4861201; fax: +54 291 4861200.
E-mail address: pasquare@criba.edu.ar (S.J. Pasquaré).
routes: i) phospholipase C (PLC)/diacylglycerol lipase (DAGL)
(Prescott and Majerus, 1983; Sugiura et al., 1995); ii) Mg2+ and Ca2+
dependent phosphatidic acid (PA) phosphohydrolase/DAGL (Bisogno
et al., 1999; Carrier et al., 2004) and iii) phospholipase A1 (PLA1)/
lysophospholipid phosphohydrolase (LPLase). PLA1 acting on PA
generates lysophosphatidic acid (LPA), which is a substrate of LPA
phosphohydrolase (LPAase) (Nakane et al., 2002; Pascual et al., 2013;
Sugiura et al., 2002). This endocannabinoid is inactivated by a two-
step mechanism whereby it is firstly carried into cells and subsequently
hydrolyzed by monoacylglycerol lipase (MAGL), alpha-beta-hydrolase
domain 6 (ABHD6) and/or fatty acid amide hydrolase (FAAH) (Ahn
et al., 2008).
The CBRs CB1 and CB2 are localized in the central nervous system
and in peripheral tissues (Howlett, 2002; Van Sickle et al., 2005). CB1
is most prevalent in the brain (Herkenham et al., 1991; Svizenska
et al., 2008). Its distribution is ubiquitous and it is therefore involved
in numerous processes that impact on neuronal functions such as
motor activity, modulation of memory and learning processes, emotion,
sensory perception and various autonomous and endocrine functions
(Mackie, 2008; Zanettini et al., 2011). The CB2 receptor is mainly
present in the immune system, thus being involved in the modulation

http://dx.doi.org/10.1016/j.exger.2014.04.008
0531-5565/Published by Elsevier Inc.
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A.C. Pascual et al. / Experimental Gerontology 55 (2014) 134–142 135

of immune response (Klein, 2005). Recent reports have suggested that Rats were killed by decapitation and CC was immediately dissected
CB2 can also be found in neurons (at a lower expression level than (2–4 min after decapitation).
CB1 receptors) and in microglia, and that it can impact on a variety of
neuronal functions such as proliferation and survival (Maldonado 2.3. Preparation of synaptosomes
et al., 2006; Onaivi, 2011).
A number of reports have highlighted the importance of CC homogenates were prepared in the following way: 20% (w/v) in
endocannabinoid signaling through CBRs in neuronal synapses within 0.32 M sucrose, 1 mM EDTA, 5 mM buffer HEPES-Na (pH 7.4). The CC
the central nervous system (CNS) and thus its involvement in the regu- homogenate was centrifuged at 1300 g for 7 min and the supernatant
lation of various physiological and pathophysiological processes, such as was carefully transferred into another tube. The nuclear pellet was re-
neuronal plasticity and protection against neurological insult and age- suspended with the isolation medium and subsequently spun at 1300 g
related neurodegenerative disorders, most of which are mediated by for 7 min. The combined supernatant was then centrifuged at 17,000 g
CBRs (Bahr et al., 2006; Freund et al., 2003; Mulder et al., 2011). Our for 10 min to obtain the crude mitochondrial pellet (CM). The latter
analysis of the enzymes involved in 2-AG metabolism showed a low was re-suspended with the isolation medium and layered onto a two-
availability of 2-AG in cerebral cortex (CC) membrane fractions and step gradient of 7.5 to 13% Ficoll solution prepared in the isolation
synaptic terminals during aging (Pascual et al., 2013). Our findings con- medium. The sample layered onto Ficoll discontinuous gradient was
firm important changes in endocannabinoid metabolism and lead us to centrifuged at 99,000 g for 30 min using a model LK-90 Beckman ultra-
hypothesize that regulation of the endocannabinoid system is central to centrifuge with an SW41 swinging bucket rotor. The myelin fraction
preventing damage during aging such as occurs in neurodegenerative band is at the interphase between the isolation medium and 7.5% Ficoll
diseases (Sanchez and Garcia-Merino, 2012). The purpose of the present medium, the synaptosomal fraction band is at the interphase between
study was therefore to examine whether the enzymes involved in 2-AG 7.5% and 13% Ficoll medium, and the free mitochondrial fraction is the
metabolism could be regulated through CBRs in synaptic terminals dur- pellet below 13% Ficoll medium (Cotman, 1974).
ing aging.
2.4. Preparation of rat CC membrane fraction
2. Material and methods
Cerebral cortices were homogenized (10%) in Tris–HCl (20 mM,
pH 8) containing 0.32 M sucrose and protease inhibitors (0.1 mM
2.1. Materials
PMSF, 1 mg/ml aprotinin, and 2 mg/ml leupeptin) and centrifuged to
eliminate nuclei and cellular debris (1000 g, 10 min, 4 °C). The pellet
[2-3H]glycerol (200 mCi/mmol or 2 Ci/mmol) and omnifluor
was discarded. Supernatants were ultracentrifuged (100,000 g, 60 min,
were obtained from New England Nuclear-Dupont (Boston, MA); 2-
4 °C) to obtain membrane fraction (Blankman et al., 2007).
arachidonoylglycerol [glycerol-1,2,3-3H] (40 Ci/mmol), unlabeled 2-
arachidonoylglycerol, and lysophosphatidic acid, 1-oleoyl [oleoyl-9,10-
3 2.5. Preparation of radioactive 1,2-diacyl-sn-glycerol
H(N)]-(54 Ci/mmol) were obtained from American Radiolabeled
Chemicals, Inc. (Saint Louis, MO). Oleoyl-L-α-lysophosphatidicacid,
DAG was synthesized from bovine retinas incubated with [2-3H]
and BSA were obtained from Sigma-Aldrich (St. Louis, MO).
glycerol (2 Ci/mmol) as previously described (Pasquare de Garcia and
Antibodies against CB1 (generously supplied by Dr. María L. de
Giusto, 1986), extracted from the tissue and isolated as described else-
Ceballos) and CB2 (purchased from Alomone labs, cat # ACR-002)
where (Folch et al., 1957; Pascual et al., 2013). It was subsequently ex-
receptors were used. The secondary antibody used for CB1 and CB2
tracted from silica gel with n-hexane:2-propanol (3:2 v/v) to avoid
detection was horse radish peroxidase (HRP)-conjugated anti-rabbit
isomerization and stored at − 20 °C. [3H]DAG specific activity was
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
2.35 mCi/mmol.
Spectra Multicolor Broad Range Protein Ladder (purchased from Ther-
mo Scientific, cat # 26634) was used as a molecular weight marker for
2.6. LPAase activity assay
Western blot analyses.
WIN55212-2 (CB1 and CB2 agonist) and JWH-133 (CB2 agonist) were
Incubations contained unlabeled 1-oleoyl LysoPtdOH and
from Tocris Bioscience (Bristol, UK). N-piperidino-5-(4-chlorophenyl)-l-
LysoPtdOH,1-oleoyl [oleoyl-9,10-3H(N)]- (20 μM, 6 × 10− 4 DPM),
(2, 4-dichlorophenyl)-4-methylpyrazole-3-carboxamide [SR141716
100 mM Tris–HCl (pH 7.4), 1.2 mM DTT, 2 mM EDTA, and synapto-
(SR1)] (CB1 antagonist), and N-[(1S)-endo-1,3,3-trimethyl bicyclo
somes (50 μg protein preincubated in the presence of 4.4 mM NEM
[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4methylbenzyl)-
and 1 μM of MAGL inhibitor, KML29) in a final volume of 100 μl
pyrazole-3-carboxamide [SR144528 (SR2)] (CB2 antagonist) were
(Fleming and Yeaman, 1995). The substrate was added in phosphate
synthesized and kindly donated by Sanofi-Synthelabo (Montpellier,
buffer saline solution (pH 7.4) containing fatty acid free bovine serum
France) to Dr. Maria L de Ceballos (Dept. Cellular, Molecular and Devel-
albumin (BSA) 0.1%.
opmental Neuroscience and CIBERNED, Cajal Inst., CISC, Madrid, Spain)
who generously supplied it to us. KML29, a more specific MAGL inhibi-
2.7. DAGL activity assay
tor, was generously supplied by Dr. Cravatt (Dept. of Chemical Physiol-
ogy, La Jolla, USA). All the other chemicals used were of the highest
Assays using exogenously added [3H]DAG as substrate were per-
purity available.
formed in 50 mM MOPS buffer (pH 7.4) containing 0.25% BSA in a final
volume of 100 μl. The DAGL assay was initiated with the addition of
2.2. Animals [3H]DAG suspensions (300 μM, 10,000 DPM) to synaptosomes (50 μg
protein per assay). [3H]DAG suspensions were prepared by separately
Wistar-INIBIBB stock adult (4 months old) and aged (24–28 months sonicating with equimolecular concentrations of lysoPtdCho in 50 mM
old) rats were kept under constant environmental conditions and fed on MOPS buffer (pH 7.4).
a standard pellet diet. All procedures with animals were carried out
following the guidelines issued by the Animal Research Guide for the 2.8. 2-AG hydrolysis assay
Care and Use of Laboratory Animals (NIH, 2010) prepared according
to the procedures followed by the Institute for Laboratory Animal Re- 2-AG hydrolysis was assessed by incubating synaptosomes (50 μg
search (ILAR), National Academy of Sciences (Bethesda, MD, 1996). protein) in a buffer solution of Tris–HCl (50 mM, pH 7.5) containing
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136 A.C. Pascual et al. / Experimental Gerontology 55 (2014) 134–142
1 mM EDTA in a final volume of 200 μl. 2-AG was prepared using [3H]2-
AG ([1,2,3-3H]glycerol) plus unlabeled 2-AG (10 μM, 5000 DPM) in ace-
tonitrile (Blankman et al., 2007).
Agonists were used at a concentration of 5 μM and were solubilized
in enzyme assay buffer. Antagonists were used at a concentration of
1 μM and were solubilized in DMSO at a concentration that did not af-
fect the enzyme activities (Breivogel et al., 2004; Ramirez et al., 2005).
When the effect of agonist or antagonist was individually evaluated,
the synaptosomes were preincubated with the agonist or antagonist for
10 min. They were subsequently incubated with the respective substrate
of each enzyme. In order to assess the antagonist plus agonist condition,
synaptosomal membranes were pre-incubated with the antagonist for
10 min and subsequently incubated in the presence of the agonist plus
the respective substrate of the enzyme activity to be determined.
All enzymatic reactions were conducted at 37 °C for 20 min. The
enzymatic reactions were stopped by adding chloroform:methanol
(2:1, v/v) or chloroform:methanol (1:1, v/v) when 2-AG hydrolysis
was assayed. Blanks were prepared identically to each enzymatic
assay except that the synaptosomes were either boiled for 10 min or
inactivated by the addition of chloroform:methanol (2:1, v/v) before
use. Blanks without protein were also prepared and no differences
were observed among the different blanks. Blank values were
subtracted from each enzyme activity. Lipid products derived from
DAGL and LPAase activities were extracted with chloroform:methanol
(2:1, v/v) and washed with 0.2 volume of CaCl2 (0.05%) (Folch et al.,
1957).
2.9. Separation of enzymatic reaction products
The DAGL product, MAG, was separated by thin layer chromatogra-
phy (TLC) on a silica gel G plate and developed with hexane:diethyl
ether:acetic acid (45:55:1.5, v/v) (Giusto and Bazan, 1979). In this
solvent system, DAG migrates with an Rf of 0.65 and MAG remains
near the origin. LPAase products, MAG and free fatty acids were
chromatographed by TLC on a silica gel H plate and developed with
chloroform:acetone:methanol:acetic acid:water (30:40:10:10:4, v/v)
up to the middle of the plate. The chromatogram was subsequently
rechromatographed up to the top of the plate using hexane:diethyl
ether:acetic acid (45:55:1.5, v/v) as developing solvent. Chromato-
grams were visualized by exposure to iodine vapors and scraped off
for counting by liquid scintillation.
Glycerol, the 2-AG hydrolysis product, was obtained in the upper
phase after interrupting the enzymatic reaction. The aqueous phase
containing radiolabel glycerol was concentrated to dryness and counted
by liquid scintillation. Radiolabel samples were counted after the addi-
tion of 0.4 ml water and 10 ml 5% Omnifluor in toluene/Triton X-100
(4/1, v/v).
2.10. SDS-PAGE and immunoblot
Proteins (40 μg) were boiled in Laemmli buffer, resolved in SDS-
PAGE using 10% gels according to Laemmli (Laemmli, 1970). Resolved
proteins were transferred to immobilon PDVF membranes using a
Mini Trans-Blot cell electro blotter (BIO-RAD Life Science Group, CA)
for 75 min. Membranes were blocked for 2 h with Tris-buffered saline
(20 mM Tris–HCl, 150 mM NaCl) pH 7.5, containing 0.1% Tween 20
(TTBS) and 5% BSA. Incubations with primary antibodies (anti-CB1
(1:3000) and anti-CB2 (1:1000)) and anti-Actin (1:1000) were carried
out at 4 °C overnight. Membranes were washed with TTBS and subse-
quently exposed to the appropriate HRP-conjugated secondary
antibody (anti-rabbit or anti-goat) for 2 h. The membranes were
rewashed with TTBS and immunoreactive bands were detected by en-
hanced chemiluminescence (ECL, Amersham Biosciences, NJ, USA)
using standard X-ray film (Kodak X-Omat AR). Spectra Multicolor
Broad Range Protein Ladder was used as a molecular weight marker.
2.11. Other methods
Protein content was determined following Lowry et al. (1951).
2.12. Statistical analysis
Three pools (two adult or aged animals per pool) were prepared and
each one was used to assay three replicates per condition. Each pool was
considered as an individual sample (n = 3).
Statistical analyses were carried out using GraphPad software (San
Diego, CA, USA, www.graphpad.com) and corroborated using InfoStat
software, 2009p version (FCA — Universidad Nacional de Córdoba —
Argentina, www.infostat.com.ar).
For immunoblot assays, results were analyzed by Student's t-test.
Statistical significance was set at p b 0.05.
Data from the different enzymatic activities were analyzed by two-
way ANOVA, which showed interaction between age and treatments
for each of the three enzymatic activities. To determine differences
among our experimental conditions a post-test (Bonferroni test) was
used. Statistical significance was set at p b 0.05, thus, considering 0.05
global error (α).
All figures are mean values ± standard error (SE) from three indi-
vidual samples.
3. Results
3.1. Immunoblot analysis in CC synaptosomal and membrane fraction from
adult and aged rats
CB1 and CB2 receptor expression was assayed by Immunoblot.
The specificity of the CB2 receptor antibody used was demonstrated as
shown in Supplementary Fig. 1 (Supplementary data). Results are
shown in Fig. 1. Synaptosomal CB1 and CB2 receptor expression
(Fig. 1A) decreased by 36% (p b 0.001) and 24% (p b 0.05), respectively,
with aging whereas their expression underwent no changes in mem-
brane fractions from aged animals (Fig. 1B). Thus, aging diminished
CBR expression only in synaptosomal endings.
3.2. LPAase activity in the presence of CB1 and CB2 agonist and/or
antagonist in adult and aged synaptosomes
In order to determine whether 2-AG synthesis is regulated through
CBRs, we studied how CB1 and CB2 receptor agonists and/or antagonists
modify LPAase and DAGL activities. To this end, WIN (CB1 and CB2 ago-
nist), JWH (CB2 agonist), SR1 (CB1 antagonist) and SR2 (CB2 antago-
nist) were used. With the aim of inhibiting MAGL activity during the
LPAase assay, KML 29 inhibitor was used (Chang et al., 2012). CBR ago-
nists and/or antagonists were observed to exert a differential effect on
adult and aged synaptosomal LPAase activity (p = 0.0029).
WIN was found to inhibit LPAase activity by a similar percentage
(24%) in adult (p b 0.001) and aged (p b 0.001) synaptosomes whereas
JWH did not significantly modify the enzymatic activity. Furthermore,
no significant differences were observed in WIN and JWH effects be-
tween adult and aged groups (Fig. 2A and B). The CB1 antagonist
inhibited LPAase activity by 10% (Fig. 2A) and 23% (Fig. 2B) in adult
(p b 0.001) and aged (p b 0.001) synaptosomes, respectively. The SR1
effect was significantly more potent in aged animals (p b 0.01). LPAase
activity, however, was inhibited by 22% in the presence of CB2 antago-
nist in both adult (p b 0.001) and aged (p b 0.001) synaptosomes
(Fig. 2A and B). SR2 showed the same effect in adult and aged animals.
Interestingly, the effect of CB2 antagonist was more pronounced than
the effect of CB1 antagonist on adult synaptosomes (p b 0.01). When
a preincubation of adult and aged synaptosomes in the presence of
both antagonists was assayed, a reversion of the effect was observed
with respect to SR1 (p b 0.01 in adult and p b 0,001 in aged animals)
and to SR2 (p b 0.001 in adult and in aged animals), considered
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A.C. Pascual et al. / Experimental Gerontology 55 (2014) 134–142 137

Fig. 1. Immunoblot analysis in synaptosomes (A) and membrane (B) CC fraction from adult and aged rats. Numbers on the right indicate molecular weights and the data shown represent
the results of three independent experiments. The bar graph shows relative density corresponding to protein expression indicated as a ratio of loading control (actin). ***p b 0.001,
and *p b 0.05, with respect to adult condition.

separately (Fig. 2A and B). When adult synaptosomes were incubat-
ed with WIN after a 10 min-preincubation with CB1 antagonist,
LPAase activity increased 11% with respect to the antagonist condi-
tion (p b0.01), reaching control values. Under preincubation condi-
tions with CB2 antagonist, incubation with WIN stimulated LPAase
activity by 9% with respect to CB2 antagonist (p b 0.05) although it
was significantly different from control (p b 0.001) (Fig. 2C). Howev-
er, this enzymatic activity was stimulated by 17% (p b 0.001) and
underwent no changes when aged synaptosomes were preincubated
with either CB1 or CB2 antagonist and subsequently incubated with
WIN (Fig. 2D). Summing up, results on LPAase activity show i) a de-
crease caused by the action of WIN and SR2 in both adult and aged
synaptosomes; ii) a greater decrease caused by SR1 in aged synapto-
somes; and iii) a reversion of the effect of SR1 but not of SR2 by WIN
in aged synaptosomes.
CBR agonists or antagonists did not modify DAGL activity in adult syn-
aptosomes (Fig. 3A). Whereas this enzymatic activity showed no chang-
es in the presence of CBR agonists, DAGL activity in aged synaptosomes
was stimulated by SR1 and SR2 antagonists added either individually
(p b 0.001) or simultaneously (p b 0.05). This stimulation was similar
under all the above-mentioned antagonist conditions (Fig. 3B). Incuba-
tion with WIN after a 10 min-preincubation with CB1 or CB2 antagonist
did not modify DAGL activity in adult synaptosomes (Fig. 3C). In
contrast, under preincubation conditions with CB1 or CB2 antagonist,
incubation with WIN significantly reverted the antagonist stimulatory
effects (p b 0.01) (Fig. 3D) in aged synaptosomes. Our results on
DAGL activity show changes only in aged synaptosomes, resulting in
i) stimulation by SR1 and/or SR2 action and ii) a reversion of the antag-
onist effects by WIN.

3.3. DAGL activity in the presence of CB1 and CB2 agonist and/or antagonist
in adult and aged synaptosomes
CBR agonists and/or antagonists exert a differential effect on adult
and aged synaptosomal DAGL activity (p = 0.035). The presence of
3.4. 2-AG hydrolysis in the presence of CB1 and CB2 agonist and/or
antagonist in adult and aged synaptosomes
As stated in Sections 3.2 and 3.3, in order to elucidate the possible
regulation of 2-AG hydrolysis through CBRs, we investigated whether
CB1 and CB2 agonists and/or antagonists were able to modify the
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138 A.C. Pascual et al. / Experimental Gerontology 55 (2014) 134–142

Fig. 2. LPAase activity in the presence of CB1 and CB2 agonist and/or antagonist in adult and aged synaptosomes. Synaptosomes (50 μg protein) were preincubated with WIN55212-2 (CB1
and CB2 agonist), JWH-133 (CB2 agonist), SR141716/SR1 (CB1 antagonist) or SR144528/SR2 (CB2 antagonist) for 10 min and subsequently incubated with the substrate (A and B); or
preincubated with SR1 or SR2 for 10 min and then incubated with WIN plus the substrate (C and D). Results in adult (A and C) and aged (B and D) synaptosomes are expressed as a per-
centage of the corresponding control values (control without agonist or antagonist represents 100%) and represent the mean ± SE of three individual samples. ***p b 0.001 with respect to
the corresponding control condition; +++p b 0.001 and ++p b 0.01 with respect to SR1 condition; ###p b 0.001 and #p b 0.05 with respect to SR2 condition.

enzymatic activities involved in 2-AG hydrolysis. CBR agonists and/or
antagonists were observed to exert a differential effect on 2-AG hydro-
lytic activity (p = 0.0086) in adult and aged synaptosomes. WIN and
JWH exerted no effect on adult (Fig. 4A) or aged (Fig. 4B) synaptosome
2-AG hydrolysis. Furthermore, CB1 and CB2 antagonists were not
found to produce changes in 2-AG hydrolysis in adult synaptosomes
(Fig. 4A), though they did reduce hydrolytic activity in the aged synap-
tosomal fraction (p b 0.001 for SR1 and p b 0.01 for SR2) (Fig. 4B). This
effect (15%) was similar for both antagonists. The simultaneous pres-
ence of both antagonists reinforces the observed inhibitory effect of
each antagonist resulting in an inhibition of 50% (p b 0.001) in aged
synaptosomes with respect to the control condition (Fig. 4B). Further-
more, no changes were observed in 2-AG hydrolysis by WIN after
preincubation with CB1 or CB2 antagonist in adult synaptosomes
(Fig. 4C), nor were the inhibitory effects exerted by CB1 and CB2 antag-
onists reverted after incubation with WIN in aged synaptosomes
(Fig. 4D). Thus, SR1 + WIN and SR2 + WIN conditions resulted in a
20% 2-AG hydrolysis inhibition with respect to the control condition
(p b 0.01). Our observations on 2-AG hydrolysis showed changes only
in aged synaptosomes, in particular i) a decrease with SR1 and SR2,
which was potentiated in the presence of both antagonists together
and ii) the inability of WIN to reverse the inhibitory effects of CBR
antagonists.
4. Discussion
In the present study we describe how 2-AG metabolism in synaptic
endings is modulated by its own receptors. This modulation is exerted
mainly by CB1/CB2 antagonists in aged animals. Our findings point to
the possible use of cannabinoid receptor antagonists in therapeutic
strategies for increasing the level of the neuroprotective 2-AG in physi-
ologically aged brain.
Chronological aging predisposes the brain to great sensitivity to neu-
rodegenerative disease (Ledesma et al., 2012). Many of the deleterious
events that occur in the aging process could be at least partially attenu-
ated by the action of endocannabinoids (Bilkei-Gorzo, 2012). Though
previous studies had revealed that the levels of endocannabinoids,
their metabolic enzymes and CBRs are modified under several physio-
logical and pathological conditions (Di Marzo and Petrosino, 2007;
Ludanyi et al., 2008; Pertwee, 2005; Wang and Ueda, 2008), it was
only recently that the first study on endocannabinoid metabolism and
aging was conducted (Pascual et al., 2013).
Interestingly, our findings in the present study demonstrated that
the expression of both CBRs is markedly diminished during aging in
CC synaptic endings, an effect which, according to observations of the
CC membrane, appears to be exclusive to this neuronal region. This is
in line with a paper by Berrendero et al. (1998), who demonstrated re-
duced cannabinoid receptor binding in CC of aged rats without changes
in CBR mRNA levels. A co-expression of CB1 and CB2 receptors has been
observed in some brain regions (Callen et al., 2012; Golech et al., 2004;
Gong et al., 2006). Together with our findings, these results suggest that
both endocannabinoid receptors can work independently and/or coop-
eratively in different neuronal populations. We have also previously
demonstrated that 2-AG availability decreases during aging (Pascual
et al., 2013) which, together with the diminished CBR expression ob-
served in aged synaptosomes, could be a consequence of neuronal de-
generation and/or could partly contribute to the synaptic impairment
observed in aging (Bilkei-Gorzo, 2012).
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A.C. Pascual et al. / Experimental Gerontology 55 (2014) 134–142 139

Fig. 3. DAGL activity in the presence of CB1 and CB2 agonist and/or antagonist in adult and aged synaptosomes. The assay conditions with agonists and/or antagonists are indicated in Fig. 2.
Results in adult (A and C) and aged (B and D) synaptosomes are expressed as a percentage of the corresponding control values (control without agonist or antagonist represents 100%) and
represent the mean ± SE of three individual samples. ***p b 0.001 and *p b 0.05 with respect to the corresponding control condition; ++p b 0.01 with respect to SR1 condition; ##p b 0.01
with respect to SR2 condition.

The profile of 2-AG metabolism in the presence of CBR agonists and/
or antagonists is different in aged with respect to adult synaptosomes.
In adults, LPAase alone is responsible for balancing the levels of 2-AG,
keeping them low, through the modulation of CB1 and CB2 receptors.
Though in aged synaptosomes LPAase was also observed to diminish
the availability of 2-AG via CB1 or CB2 receptor antagonists, this effect
was strongly reversed through increased DAGL activity and a marked
decrease in 2-AG hydrolysis, resulting in higher availability of the can-
nabinoid. CBR blockage in aged synaptosomes could therefore be per-
ceived as a low endocannabinoid level, which is then compensated by
increasing 2-AG availability as observed in the presence of CB1 and/or
CB2 antagonists. Although CB1 is more abundant in the CNS than CB2
(Govaerts et al., 2004; Zanettini et al., 2011), the effect of the two antag-
onists on DAGL and MAGL activities appears to be practically the same.
This could be due to the fact that CB1 and CB2 receptors colocalize,
working independently or cooperatively, in several cerebral structures
(Gong et al., 2006). It was also observed that CB1 and CB2 receptors
can form heteromers in neurons, exhibiting a bidirectional cross-talk
phenomenon (Callen et al., 2012). Furthermore, low mRNA levels are
not necessarily associated with low protein expression and low protein
biological activity (Berrendero et al., 1998).
In order to discern whether 2-AG metabolism was modulated by
CB1 and/or CB2 receptors, enzymatic assays were performed blocking
CBRs with a CB1-selective agent, SR141716, or a CB2-selective agent,
SR144528 (Rinaldi-Carmona et al., 1995, 1998) prior to incubation
with the agonist of both receptors (WIN). The low 2-AG availability ob-
served in adult synaptosomes in the presence of antagonists was ei-
ther completely or partially reversed by the action of WIN through
CB2 or CB1 receptors. This conclusion derives from the fact that
LPAase activity increased when WIN was redirected either to the CB1
or the CB2 receptor. A different regulation pattern was observed in
aged synaptosomes. The reduced 2-AG availability as a consequence
of diminished LPAase activity through the action of CBR antagonists
was only reversed by the CB2 receptor. It is important to note that
even though the CB2-specific agonist did not modify LPAase activity,
the action of WIN by CB2 was able to reverse the inhibition exerted by
the CB1 antagonist. The CB2 receptor therefore acquires significance
when CB1 is blocked.
Interestingly, though WIN was found to reverse the blockade of CB1
or CB2 receptors, so that 2-AG reached basal levels via DAGL inhibition,
this cannabinoid was observed to remain high at the expense of its hy-
drolysis inhibition, an effect mediated by both receptors. These results
strongly support the need to maintain high levels of this neuroprotec-
tive molecule during aging.
Our results reveal that 2-AG metabolism is modulated mainly by
CB1/CB2 antagonist. However, it has not yet been possible to determine
whether these effects occur only through the direct action of CBR antag-
onists or whether they also occur through a non-receptor mediated
mechanism. Any direct action of CBR modulators on 2-AG metabolism
enzymes seems unlikely, as indicated by earlier research showing
that the effects of other CBR ligands, such as pure cannabinoids and
cannabinoid-enriched cannabis extracts, inhibit MAGL with IC50 values
between 30 and 50 μM (De Petrocellis et al., 2011). These IC50 values
are much higher than the concentrations of agonists (5 μM) and antag-
onists (1 μM) used in our present study.
5. Conclusion
The role of the endocannabinoid system in neuroprotection is re-
ceiving increasing attention in the literature. In this context, the ability
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140 A.C. Pascual et al. / Experimental Gerontology 55 (2014) 134–142

Fig. 4. 2-AG hydrolysis in the presence of CB1 and CB2 agonist and/or antagonist in adult and aged synaptosomes. The assay conditions with agonists and/or antagonists are indicated in
Fig. 2. Results in adult (A and C) and aged (B and D) synaptosomes are expressed as a percentage of the corresponding control values (control without agonist or antagonist represents
100%) and represent the mean ± SE of three individual samples. ***p b 0.001 and **p b 0.01 with respect to the corresponding control condition; +++p b 0.001 with respect to SR1
condition; ###p b 0.001 with respect to SR2 condition.

Fig. 5. Modulation of 2-AG metabolism by CBRs in aged synaptosomes. CB1 and CB2 antagonists increase 2-AG availability in aged synaptosomes by stimulating its synthesis (DAGL) and
inhibiting its hydrolysis (MAGL). LPA: 1-oleoyl lysophosphatidic acid; DAG: diacylglycerol; 2-AG: 2-arachydonoyl glycerol; LPAase: lysophosphatidic acid phosphohydrolase; DAGL: diac-
ylglycerol lipase; MAGL, monoacylglycerol lipase.
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A.C. Pascual et al. / Experimental Gerontology 55 (2014) 134–142
to increase 2-AG availability by modulating CBR mainly via the action of
antagonists (summarized in Fig. 5) suggests that these cannabinoid re-
ceptors are good therapeutic targets for attenuating synaptic dysfunc-
tion and/or protecting the nervous system from some of the damage
inflicted by the aging process.
Conflict of interest
The authors have no conflict of interest to declare.
Acknowledgements
This work was supported by the Fundación Florencio Fiorini, the
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)
(Grant no. 11220110100437), the Agencia Nacional de Promoción
Científica y Tecnológica (FONCyT) (Grant no. 01-14527), and the
Secretaría General de Ciencia y Tecnología of the Universidad Nacional
del Sur (UNS), Argentina (Grant no. 24/B207). Polyclonal antibodies
against CB1 receptor, CB1 and CB2 agonists and antagonists were gener-
ously supplied by Dr. María L. de Ceballos from the Department of Cellu-
lar, Molecular and Developmental Neuroscience and CIBERNED, Cajal
Institute, CISC, Madrid, Spain. KML29 inhibitor was generously supplied
by Dr. Cravatt (Dept. of Chemical Physiology, La Jolla, USA).
S.J. Pasquaré is a research member of UNS. N.M. Giusto is a research
member of CONICET. A.C. Pascual and V.L. Gaveglio are research fellows
of CONICET.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.exger.2014.04.008.
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