Neurobiology of Disease 5, 386–404 (1998)

Article No. NB980214

Biochemistry of the Endogenous Ligands

of Cannabinoid Receptors

Vincenzo Di Marzo* and Dale G. Deutsch†

*Istituto per la Chimica di Molecole di Interesse Biologico, CNR, Via Toiano 6, 80072 Arco
Felice, Naples, Italy; and †Department of Biochemistry and Cell Biology, State University of
New York at Stony Brook, Stony Brook, New York 11794-5215

In 1992 the discovery of the first endogenous ligand of cannabinoid receptors, anandamide, provided
conclusive support to the hypothesis that an ‘‘endogenous cannabinoid regulatory system’’ exists in
mammalian nervous tissue. Anandamide (N-arachidonoyl-ethanolamine) was the first of a series of
long-chain fatty acid derivatives, including two other polyunsaturated N-acylethanolamines and
2-arachidonoyl-glycerol, found to exert cannabimimetic properties in either central or peripheral
tissues. Here we review the current knowledge on the biochemical bases of the formation and
inactivation of endogenous cannabinoid ligands as well as of their interaction with cannabinoid
receptor subtypes. r 1998 Academic Press

INTRODUCTION discovered to date are all lipid derivatives and not

The discovery, cloning, and molecular characteriza-
tion, between 1988 and 1990, of the brain cannabinoid
receptor, later named CB1 (1, 2), and the more recent
discovery of a ‘‘peripheral,’’ CB2 subtype of cannabi-
noid receptors in the spleen and immune cells (3),
explained several of the pharmacological actions ex-
erted by D9-tetrahydrocannabinol (THC) or synthetic
cannabimimetics in both the CNS and peripheral
tissues. These important breakthroughs, however,
would have merely added two new members to the
family of orphan receptors had not endogenous sub-
stances, capable of functionally activating these two
receptors, been isolated shortly afterward. The puta-
tive endogenous ligands of cannabinoid receptors
1Abbreviations used: 2-AG, 2-arachidonoyl-glycerol; CB , cannabi-
1
noid receptor 1; CB2, cannabinoid receptor 2; SAR, structure–activity
relationship; N-ArPE, N-arachidonoylphosphatidylethanolamine;
DAG, sn-2-arachidonate containing diacylglycerols; FAAH, fatty
acid amide hydrolase; PMSF, phenylmethylsulfonyl fluoride; NEM,
N-ethylmaleimide; ATFMK, arachidonoyltrifluoromethyl ketone;
MAFP, methylarachidonoyl fluorophosphonate; cannabimimetic, act-
ing cannabinoid-like; endocannabinoids, compounds which occur
endogenously (i.e., anandamide) and act cannabimimetically by
binding to cannabinoid receptors.
386
peptides, as was the case for the receptors of another
plant-derived drug, morphine. However, if one consid-
ers the high lipophilicity of the psychoactive cannabi-
noids, it is not surprising that anandamide, the first
endogenous cannabinoid receptor ligand to have been
identified and characterized (4), is a non-water-soluble
substance. Although there have been some reports in
the brain of functionally active peptide-like ligands for
CB1 receptors (5–7), their structure has not yet been
elucidated. By contrast, following the discovery of
anandamide, other lipophilic molecules, i.e., amides or
esters of long-chain fatty acids (Fig. 1), have been
shown to exhibit some cannabimimetic properties both
in vitro and in vivo and, in some cases, through more
than one mechanism of action. Here we review the
discovery of these compounds, the chemical bases of
their pharmacological actions, the pathways proposed
for their biosynthesis and inactivation, and the possible
ways of regulating these pathways in order to modu-
late the levels of endogenous cannabimimetic ligands
for pharmacological and therapeutical purposes. In
analogy with the nomenclature for the ‘‘endorphins’’
(morphine within) we shall refer to the endogenous
ligands of cannabinoid receptors as ‘‘endocannabi-
noids’’ (cannabinoids within).
0969-9961/98 $25.00
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Endocannabinoid Biochemistry 387

FIG. 1. Chemical structures of cannabimimetic fatty acid derivatives discovered to date. The putative targets for the cannabimimetic actions of
these metabolites, which may act either at one or both of the two cannabinoid receptor subtypes identified so far, or at yet to be discovered
non-CB1-non-CB2 cannabinoid receptors (CBn), or by competing with anandamide for the enzyme fatty acid amide hydrolase (FAAH), thereby
enhancing endogenous levels of anandamide, are shown in parentheses.

DISCOVERY OF THE amide was named anandamide, from the Sanskrit

ENDOCANNABINOIDS AND THEIR
OCCURRENCE IN ANIMAL CNS
In December 1992 Devane et al. (5) reported the
isolation and structure elucidation of a porcine brain
lipid component that selectively displaced the binding
of a high-affinity cannabinoid receptor ligand to brain
membrane preparations. This metabolite exhibited a
typical cannabimimetic response, i.e., the inhibition of
electrically evoked mouse vas deferens contractions.
The chemical structure of the compound was deter-
mined to be, by a combination of nuclear magnetic
resonance spectroscopy, mass spectrometry, and chemi-
cal synthesis, the amide formed from arachidonic acid
and ethanolamine. As a consequence of its cannabimi-
metic and chemical properties arachidonoylethanol-
word for ‘‘bliss,’’ ananda. Immediately after its discov-
ery, anandamide was shown to be active in the ‘‘tet-
rad’’ of mouse behavioral tests highly indicative of
cannabinoid-like substances, i.e., inhibition of locomo-
tor and rearing activity, hypothermy, catalepsy, and
analgesia (8, 9). Anandamide also mimicks THC in
vitro, by inhibiting forskolin-induced adenylate cy-
clase and N-type Ca21 channels in cells transfected
with CB1 receptors and in mouse neuroblastoma N18TG2
cells, respectively (10, 11). Following these early stud-
ies several other cannabinoid-like responses have been
reported for anandamide in both central and periph-
eral tissues [reviewed in 12–14]. Subsequently, several
possible physiological roles have been suggested for
this endocannabinoid in the CNS and the reproduc-
tive, cardiovascular, renal, and immune systems [re-

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388
viewed in 15–17]. In these systems and in numerous
tissues and cell types anandamide was quantified, and
the pathways for its inactivation and stimulus-induced
biosynthesis were characterized (see below). Thus,
anandamide was found, in levels ranging between 4.3
and 105 pmol/g wet tissue weight, in rat brain, testes,
and kidneys [18–20, 25]; in human heart and spleen
and in several areas of human and rat brain, with the
highest concentrations in the hippocampus, parahippo-
campal cortex, thalamus, striatum, and cerebellum
(18); in bovine and ovine brain (21); in stimulated
murine N18TG2, basophilic-like cells, and macrophages
(22–24, 122); and in invertebrate tissues, such as sea
urchin ovaries and mussels (26, 27). Very high amounts
of anandamide (up to 20 nmol/g wet tissue weight)
were only recently reported for mouse uterus in the
preimplantation period (28).
Although the physiological significance of the occur-
rence of anandamide in so many different tissues and
cells has not yet been fully clarified, the outcome of
these pharmacological and analytical studies can be
summarized as follows: (i) compared to classical neuro-
transmitters, anandamide basal levels in the brain are
similar to those of dopamine and serotonin but at least
10-fold lower than those of GABA and glutamate (18);
(ii) the central cannabimimetic actions of anandamide
in vivo are weaker than those of THC and, although
they can be detected with a more rapid onset, are
usually of shorter duration (8, 9, 29); (iii) some CB1
receptor-mediated actions of anandamide in vitro, such
as inhibition of adenylate cyclase, are exerted at the
same potency as classical cannabinoids; however, anan-
damide, at doses lower than 1 mg/kg, exhibits a
‘‘stimulatory’’ rather than ‘‘inhibitory’’ effect in some
in vivo tests (30); (iv) anandamide can behave also as a
partial agonist, for example, in the inhibition of N-type
Ca21 currents, in cells with a low density of CB1
receptors (31) and can antagonize some THC-induced
effects both in vitro and in vivo (30); (v) although
anandamide is capable of binding to CB2 receptors, it is
not very efficient in eliciting CB2-mediated functional
responses, and it behaves as an antagonist at CB2 (32,
33). From these observations one can postulate that
anandamide may not be stored in tissues as such but
synthesized on demand; that its synthesis, as well as its
release, may be triggered by cell stimulation; that
anandamide is rapidly degraded by cells; that other
endogenous metabolites may potentiate its actions
either by acting in concert at CB1 receptors or by
protecting it against degradation; and that other endo-
cannabinoids, some of which selective for CB2 recep-
tors, may be present in tissues. These suggestions
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Di Marzo and Deutsch
provided the rationale for continuing the search for
other putative endocannabinoids. Two anandamide
congeners, the ethanolamides of di-homo-g-linolenic
and docosatetraenoic acids, both with CB1 binding
activity, were isolated from porcine brain. They were
found to inhibit forskolin-induced adenylate cyclase
(with IC50s slightly higher than those previously ob-
served with anandamide), as well as the mouse vas
deferens twitch response, and to be active in the tetrad
of mouse behavioral tests (11, 34–36).
Another member of the family of long-chain acyletha-
nolamides, palmitoylethanolamide, which does not
bind significantly to CB1 receptors (4), was shown to
mimick synthetic cannabinoids by: (i) down-regulat-
ing mast cell and rat basophilic leukemia (RBL-2H3)
cell degranulation and (ii) protecting cerebellar gran-
ule cells against glutamate-induced excitotoxicity (32,
37). Both actions were antagonized by anandamide
and it has been suggested that they are mediated by
CB2 receptors, based upon the finding that palmitoy-
lethanolamide was shown to displace the binding of
[ 3H]WIN 55,212-2, a high-affinity cannabinoid receptor
ligand, from RBL-2H3 cell and cerebellar granule cell
membranes and that CB2 transcripts were found in
both cell types. However, subsequent studies, con-
ducted with cells transfected with CB2 receptor comple-
mentary DNA (cDNA), did not confirm the high-
affinity binding of palmitoylethanolamide to the
‘‘peripheral’’ receptor subtype (38, 39), thus raising the
possibility that this saturated fatty acid ethanolamide
could be binding to a non-CB1, non-CB2 cannabinoid
receptor. Whatever its mode of action, palmitoyletha-
nolamide is likely to play a physiological role in both
the CNS and the immune system. Interestingly, its
synthesis was found to be induced, together with that
of other acylethanolamides, by the same stimuli lead-
ing to anandamide formation in both neurons (40) and
some immune blood cells (24), and its co-occurrence
with anandamide has been shown in all tissues and
cells studied (19–27).
While the possible role of palmitoylethanolamide as
a ligand for CB2 receptors, as well as the presence of
the latter in the brain, were being investigated, another
arachidonic acid derivative, 2-arachidonoyl-glycerol
(2-AG), was isolated from canine gut during a study
aimed at finding peripheral ligands for cannabinoid
receptors (41). 2-AG was previously known to occur in
several cells as a possible product of diacylglycerol
degradation and/or an alternative precursor for arachi-
donic acid liberation (75). 2-AG, like anandamide, is
active in the tetrad of mouse behavioral assays. It
inhibits forskolin-induced adenylate cyclase in spleno-
Endocannabinoid Biochemistry
cytes and neurons and displaces high-affinity cannabi-
noid ligands from membrane preparations from brain
synaptosomes or cells transfected with either CB1 or
CB2 receptors, albeit with relatively high IC50 and Ki
values (41–43). Monoarachidonoylglycerol species were
described in rat brain (42), although the first full
chemical characterization of 2-AG in a neuronal cell
was obtained in ionomycin-stimulated mouse neuro-
blastoma N18TG2 cells (44). Subsequently, the 2-isomer
was also found in rat brain in amounts 170-fold higher
than anandamide, and its synthesis was observed in
electrically stimulated rat hippocampal slices (43).
CB1- or CB2-mediated effects, i.e., inhibition of hippo-
campal long-term potentiation (43), modulation of
intracellular Ca21 concentration in both undifferenti-
ated and differentiated neuroblastoma 3 glioma hy-
brid cells (45–47), and inhibition of lymphocyte prolif-
eration (48), respectively, have also been reported for
this substance. However, the characterization of the
behavioral and pharmacological effects of 2-AG have
not been conducted as thoroughly as for anandamide,
probably owing to the instability of this compound
which can easily be converted to the 1(3)-isomer.
STRUCTURE–ACTIVITY RELATIONSHIP
(SAR) STUDIES ON
ENDOCANNABINOIDS
With the aim of designing novel drugs that are
potentially useful in therapeutical applications and
resistant to enzymatic degradation, SAR studies have
been performed on anandamide. This compound,
apart from its overall lipophilicity, shares with psycho-
tropic cannabinoids two moieties previously sug-
gested to constitute the chemical bases for the cannabi-
noid pharmacophore, i.e., the n-pentyl chain and the
hydroxy group. The affinity of acylethanolamide con-
geners of anandamide for the CB1 receptor was shown
to vary depending on the length and degree of unsat-
uration of the fatty acid chain. Aliphatic chains with
less than three double bonds and with more or less
than 20 carbon atoms yielded compounds which were
less potent or inactive (11). Also the presence of an v3
double bond in the fatty acid chain, probably acting by
disrupting the n-pentyl chain of anandamide and
other v6 acylethanolamides, appeared to weaken the
interaction with the receptor. Enzymatic oxidation of
the fatty acid chain, depending on its regioselectivity,
resulted in either unmodified or impaired binding
activity (see below). Chemical modification of the
N-(2-hydroxy-ethyl)-moiety of anandamide appeared
389
to cause more interesting effects on the binding to CB1
receptors. Its substitution with GABA and L-serine (49)
or with amides, ethers, or esters of various length (50)
decreased or abolished the binding activity. N-2-(4-
Hydroxy-phenyl)-ethyl- and N-2-(4-hydroxy-phenyl)-
anandamide were less active than anandamide but
more resistent to enzymatic hydrolysis of the amide
bond (51), while the substitution of the hydroxyl group
with a fluorine atom increased the activity (52). The
introduction of a methyl group, as in N-(2-hydroxy-
isopropyl)-arachidonoyl-ethanolamide (methanan-
damide), produced a compound less sensitive to enzy-
matic hydrolysis (53) which was also more active than
anandamide provided that the absolute configuration
of the new chiral center was R. Surprisingly, the
hydroxyl group of the alkanolamine moiety was not
found to be essential for CB1 binding activity, since
arachidonoyl-n-propylamide and -isopropylamide
were more active than anandamide (38, 50). Con-
versely, the secondary amide group in the molecule
was shown to be a necessary prerequisite for activity
(38). Finally introduction of one or two methyl groups
on the carbon atom a to the carbonyl group in
anandamide did not cause any change in the binding
activity, whereas analogous derivatizations of arachi-
donoyl-n-propylamide and -isopropyl-amides pro-
duced the two most active anandamide derivatives
described to date (38). There have been two attempts to
fit these data in conformational low-energy models for
the relationships between anandamide and the canna-
binoid pharmacophore by means of computer-assisted
molecular dynamics studies (54, 55). It was suggested
that a looped, low-energy conformation of anan-
damide, similar to the cannabinoid tricyclic structure,
can overlap to the three-dimensional conformation of
classical cannabinoids through the superimposition of
the hydroxyl group of the ethanolamide and the
phenolic group of THC, the oxygen of the carboxyam-
ide and the pyran oxygen in THC, and the v6 aliphatic
region of anandamide and the C3 pentyl chain of THC
(54). This model explains why palmitoylethanolamide,
which has a short alkyl chain and no double bonds to
help the molecule to assume a looped conformation,
has very low affinity for the CB1 receptor, but does not
explain why the phenolic group in THC is necessary
for activity whereas the hydroxy group of anandamide
is not. Another model, however, based, inter alia, on the
observation that ethanolamides of prostaglandins are
completely inactive (50), favors a linear conformation
of anandamide as the lowest-energy three-dimen-
sional structure best fitting the cannabinoid receptor
binding site (56).
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390
Another SAR study (57) showed that some of the
most active compounds at the CB1 receptor, i.e., anan-
damide, 28-fluoro-anandamide and (R)-methanan-
damide, bind very weakly to CB2 receptors, in agree-
ment with the original finding by Munro et al. (3), and
with the suggestion that anandamide, like THC, be-
haves as a weak ligand at CB2 receptors (32). In
separate studies, however, anandamide was equipo-
tent in CB1 and CB2 binding assays carried out with
transfected cells (58, 59). It is likely that criteria
different from those outlined above regulate the inter-
actions between anandamide-like compounds and CB2
receptors. Even if capable of binding to this receptor,
anandamide does not seem to elicit a typical CB2-
mediated response, i.e., adenylate cyclase inhibition
(32, 48), thus differing substantially from 2-AG, which
has a lower affinity than anandamide for CB2 receptors
but can efficiently inhibit forskolin-induced cAMP
formation in cells selectively expressing these recep-
tors (41, 48).
BIOSYNTHESIS OF THE
‘‘ENDOCANNABINOIDS’’
One of the prerequisites for an endogenous sub-
stance to be classified as a neuromodulator, is the
presence in the nervous system of molecular apparati
for its synthesis and inactivation which are regulated
by neuronal activity/stimulation. Several studies were
undertaken to elucidate such mechanisms for the two
cannabimimetics, anandamide and 2-AG. As both
compounds belonged to two lipid classes thoroughly
studied in previous decades, the N-acyl-ethanolamines
and the monoacylglycerols, the major difficulty encoun-
tered during these studies was to understand which of
the previously discovered pathways, and under which
physiopathological conditions, would contribute to
the in vivo levels of anandamide and 2-AG. This issue
has now been settled, at least in part, for anandamide,
for which two possible biosynthetic pathways had
been proposed in analogy with the pioneering work
carried out in the 1970s and 1980s on N-acyl-ethanol-
amines with saturated and monounsaturated fatty
acid chains [for a review see 60]. According to these
studies, anandamide would be produced either from
the ATP- and conzyme A-independent condensation
between arachidonic acid and ethanolamine or through
the phospholipase D-catalyzed hydrolysis of a pre-
formed membrane precursor, N-arachidonoyl-phospha-
tidyl-ethanolamine (N-ArPE) (Fig. 2). Early studies
exploring anandamide synthesis (61–63), by describ-
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Di Marzo and Deutsch
ing the presence, in cell-free preparations from brain or
neuronal cells, of a membrane-bound ‘‘anandamide
synthase’’ enzyme selective for arachidonic acid,
seemed to favor the condensation reaction. The major
drawback of these studies, however, was the very high
concentrations of arachidonic acid and, particularly,
ethanolamine (up to 160 mM) required in order to
observe the anandamide synthase-mediated formation
of anandamide. This led to postulate that the prior
activation of both phospholipases A2 and D (the two
enzymes mostly responsible for the liberation of the
two substrates of anandamide synthase) was necessary
for such a pathway to be operative in vivo. However,
when the synthesis and release of anandamide (as well
as other N-acyl-ethanolamines) from intact rat central
neurons (but not astrocytes) was induced by treatment
with membrane depolarizing stimuli, such as ionomy-
cin, high K1, kainate, and 4-aminopyridine (40), the
formation of this compound was not dependent on
phospholipase A2 activation, but rather on the phospho-
diesterase-mediated hydrolysis of a membrane phos-
pholipid, i.e., N-ArPE. The latter was isolated from rat
neurons and brain, and its biosynthesis was found to
be induced by the same stimuli leading to anandamide
formation (40, 64). Moreover, a phosphodiesterase of
the D-type capable of catalyzing N-ArPE conversion
into anandamide was also identified in several neuro-
nal as well as peripheral systems (19, 20, 23, 24, 40,
64–66). This enzyme was partially characterized (19)
and found to be similar to the dog brain phospholipase
D previously reported to catalyze the hydrolysis of
N-oleoyl-phosphatidylethanolamine to the correspond-
ing N-acyl-ethanolamine (67). Subsequently, it was
demonstrated that N-ArPE was synthesized from the
Ca21-dependent N-transacylation of phosphatidyletha-
nolamine (PE) with arachidonic acid stereoselectively
donated from the sn-1 position of di-arachidonoyl-
phosphatidylcholine (19, 20, 65, 66), a minor compo-
nent of membrane phospholipids (Fig. 2). On the basis
of these findings, the following sequence of events
leading to anandamide biosynthesis in stimulated
neurons is proposed. The Ca21 influx that follows
membrane depolarization stimulates the formation of
N-ArPE, whose levels can be enhanced further by
adenylate cyclase-activating mediators such as vasoac-
tive intestinal peptide (64). Up-regulation of N-ArPE
levels then stimulates a N-acyl-phosphatidylethanol-
amine-selective phospholipase D (67) to convert this
phospholipid into anandamide (19, 40, 65, 66). The
latter may then negatively feed back on its own release
by inhibiting adenylate cyclase (10, 11). A similar
pathway is likely to be operative also for the stimulus-
 Endocannabinoid Biochemistry

FIG. 2. Possible biochemical pathways for the biosynthesis of anandamide and 2-arachidonoyl-glycerol. As for other lipid mediators, biosynthetic routes
occurring either through the remodeling of membrane phosphoglycerides (top) or the de novo synthesis (bottom) have been suggested for the two
endocannabinoids. In particular, some of these pathways may connect the biosynthesis of anandamide to that of 2-arachidonoyl-glycerol.

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391

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392
induced synthesis of the other N-acyl-ethanolamines,
including palmitoylethanolamide, found to be core-
leased with anandamide in rat central neurons and
murine leukocytes (40, 23, 24), as well as for the
formation of other cannabimimetic members of this
family of lipids (11, 34). However, further studies are
needed to clarify the biosynthesis of the sn-1,2-di-
arachidonoyl phospholipid precursors for N-ArPE and
to fully characterize the enzymes involved in this
pathway as well as their regulation. It is worthwhile
mentioning that a recent study showed that, in iono-
phore-stimulated mouse peritoneal macrophages, sn-
1,2-arachidonoyl-phosphatidylcholine can be formed
from arachidonic acid released by phospholipids
through a Ca21-dependent phospholipase A2 (68).
Whether this pathway also occurs in nervous tissue
remains to be assessed.
If synthesis of anandamide occurs as described,
what role does anandamide synthase play? Based
upon: (i) the observation that the enzyme pH depen-
dency, tissue distribution, sensitivity to inhibitors, and
chromatographic behavior were identical to those
observed for the enzyme catalyzing anandamide hydro-
lysis to arachidonic acid and ethanolamine (‘‘anan-
damide amidohydrolase,’’ see next section) and (ii) the
previous suggestion that N-acyl-ethanolamine synthe-
sis could be catalyzed from high concentrations of
fatty acids and ethanolamine by the amidase cata-
lyzing N-acylethanolamine hydrolysis (69), it was hy-
pothesized that anandamide synthase was anan-
damide amidohydrolase catalyzing the reverse reaction
from nonphysiological concentrations of ethanol-
amine. This hypothesis was recently confirmed when
anandamide amidohydrolase was cloned and tran-
siently expressed in COS-7 cells (see next section). The
recombinant enzyme was in fact shown to catalyze
anandamide synthesis in the presence of arachidonic
acid and high millimolar concentrations of ethanol-
amine (70, 71). Does this mean that anandamide
synthase does not contribute to the physiological
production of anandamide? Examples of lipid media-
tors with more than one physiological biosynthetic
pathway have been reported (for example, the platelet
activating factor [72]). Data obtained in neuronal cells
and leukocytes, where inhibitors of anandamide syn-
thase/amidohydrolase do not block the stimulus-
induced formation of anandamide (23, 24), and basal
anandamide levels are lower than those of preformed,
granule-stored mediators, support a phospholipid pre-
cursor-mediated pathway for the formation of anan-
damide. However, the recent finding in mouse uterus
of distinct anandamide synthase and amidohydrolase
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Di Marzo and Deutsch
enzymes, as well as of very high levels of anandamide
in the ‘‘preimplantation’’ phase (28), and preliminary
experiments carried out in rat testes suggest that a
Ca21-independent condensation pathway may also be
operative in certain peripheral tissues (73, 74).
The physiological biosynthetic pathway for 2-AG
remains to be fully elucidated. The most obvious route
for the formation of this compound is by the stereoselec-
tive hydrolysis of the sn-1 ester bond of sn-2-arachidon-
ate-containing diacylglycerols (DAGs). An enzyme for
this reaction, sn-1-specific DAG lipase, has been de-
scribed in the nervous system (75). DAGs, in turn, are
produced upon the activation of two separate path-
ways both postulated to participate in widespread
receptor-induced intracellular signal transduction
events, i.e., (a) the activation of phosphatidylinositol
(PI)- or phosphatidilcholine (PC)-specific phospholi-
pase C enzymes or (b) the activation of phospholipase
D with formation of phosphatidic acid followed by the
hydrolysis of the latter catalyzed by a specific phospho-
hydrolase. Once formed, DAGs activate protein kinase
C enzymes, and, often in synergy with phosphoinosi-
tols, mediate the intracellular actions of several neuro-
transmitters, which, for example, may modulate synap-
tic plasticity (76). This raises the following questions: Is
it plausible that 2-AG, a putative primary messenger
with cannabinoid receptors as extracellular targets, be
produced within a framework of intracellular messen-
ger-generating pathways? Should not the routes respon-
sible for the trans-membrane signalling of neurotrans-
mitters be kept segregated from those leading to the
formation of other neuromodulators? In the case of
anandamide, for example, the phospholipase D cata-
lyzing N-ArPE hydrolysis is very probably different
from the PC- and PE-specific phospholipases D which
participate in receptor signal transduction (67). If the
cannabimimetic 2-AG is produced from the second
messenger DAGs, would not this result in an interfer-
ence with the protein kinase C-mediated signalling
coupled to several other neuronal signals? To provide
a possible solution to the issue of 2-AG biosynthesis,
one can again turn to the literature. One possibility is
the phospholipase A1-catalyzed formation of sn-1-lyso-
phospholipids followed by their hydrolysis by phos-
pholipases C distinct from those involved in signal
transduction (77). Another is the de novo synthesis of
DAGs through phosphatidic acid resulting from the
reaction between 1-monoacylglycerol-phosphate and
arachidonoyl-coenzyme A (Fig. 2). Taking these alterna-
tives into consideration, studies of the stimulus-
induced biosynthesis of 2-AG were undertaken in
Endocannabinoid Biochemistry
neuronal cells. In mouse N18TG2 cells ionomycin stimu-
lation led to approximately a sevenfold increase of
2-AG levels in a Ca21-dependent fashion (44). About
20% of the metabolite synthesized de novo was found to
be released in the cell culture medium, in support of its
putative role as a cannabinoid receptor ligand. Ionomy-
cin-induced 2-AG synthesis occurred concomitantly
with DAG formation, and an abundant sn-1 DAG
lipase activity was detected in N18TG2 cell homoge-
nates, suggesting a role for DAGs as precursors for the
monoglyceride. 2-AG formation, however, was not
inhibited by neomycin sulfate, a phospholipase C
inhibitor, nor by pretreatment of cells with phospholi-
pase A2, which enhanced the effect of ionomycin, in
support of a non-phospholipase C-mediated pathway
for the synthesis of the cannabimimetic glycerol ester
(44). Such a pathway, if operative in vivo, would
probably segregate DAG-mediated intracellular signal-
ling from the DAG-dependent formation of 2-AG. On
the other hand, three distinct phospholipase C-like
enzymes capable of converting either PC or sn-1-lyso-
2-arachidonoyl-PC and -PI into 2-AG have been identi-
fied in N18TG2 cells in culture and in rat brain homoge-
nates (66, 77). This suggests that upon stimulation with
agonists coupled to phospholipase C activation, neuro-
nal cells may also release the monoglyceride from
endogenous (lyso)phospholipids. The phospholipid-
mediated biosynthesis of 2-AG, apart from raising the
possibility of cross-talk with signal-induced and DAG/
protein kinase C-mediated intracellular signalling, also
raised the issue of interactions with anandamide bio-
synthesis. Di-arachidonoyl-PC and sn-1-lyso-2-arachi-
donoyl-PC, a precursor and a by-product of N-ArPE
biosynthesis, respectively, were in fact shown to be
converted into 2-AG in vitro (66). Hence, the latter
endocannabinoid may be produced in stimulated neu-
rons either in competition or concomitantly with anan-
damide (Fig. 2). A more recent study (43), carried out
with ionomycin-treated cultured central neurons and
electrically stimulated hippocampal slices, confirmed
that 2-AG can be released in a Ca21-dependent manner
through the action on DAGs of a DAG lipase. In this
case, however, 2-AG biosynthesis was counteracted by
an inhibitor of PI-selective phospholipase C. This
finding indicates that the phospholipid-mediated path-
way is indeed a candidate route for the in vivo
formation of DAGs which are used as precursors for
the cannabimimetic monoglyceride. However, it raises
three important inter-related questions for future re-
search: (i) is the phospholipase C enzyme involved in
this pathway identical or distinct from that participat-
ing in phosphoinositide-mediated intracellular signal-
393
ling? (ii) How much of the estimated 2–4 nmol/g wet
weight of 2-AG in rat brain (42, 43) is due to degrada-
tion of DAGs (and therefore to termination of phospho-
inositide/DAG/protein kinase C-mediated responses)
and how much is used for initiation of a cannabimi-
metic signal? (iii) Are there PI/DAG pools uniquely
devoted to 2-AG biosynthesis? These questions need
now to be addressed also in view of other possible
biosynthetic pathways suggested for 2-AG by the
studies carried out in N18TG2 cells (44, 66).
INACTIVATION OF THE
ENDOCANNABINOIDS
Another important requirement for an endogenous
substance to be considered as a neuromodulator is that
there exists a mechanism(s) for the termination of the
modulatory signal within the CNS or peripheral tis-
sues. Indeed, since the discovery of anandamide,
considerable progress has been achieved in the identi-
fication of the proteins responsible for its inactivation.
In early studies it was shown that anandamide could
be sequestered by cells and/or enzymatically hydro-
lyzed to arachidonic acid and ethanolamine in rat
brain as well as various tumoral cell lines (61). Subse-
quently, the rapid inactivation by rat central neurons
and astrocytes was found to be effected by both
reuptake and enzymatic hydrolysis (40). Ethanolamine
and, particularly, arachidonic acid produced from
anandamide hydrolysis were readily reincorporated
into membrane phosphoglycerides (Fig. 3). Anan-
damide uptake by central neurons was shown to be
rapid (t1/2 5 2.5 min), saturable, selective, and tempera-
ture dependent, thus implying the presence of a
specific membrane transporter for the facilitated diffu-
sion of anandamide (40). Following these studies, the
enzyme catalyzing anandamide hydrolysis, named
anandamide amidohydrolase or anandamide amidase,
was partially characterized from rat, pig, and mouse
whole brains (78–81) as well as from N18TG2 cells (82).
In all these tissues and cells, the enzyme exhibited: (a)
a subcellular distribution mostly confined to micro-
somal (100,000g pellets) and mitochondrial (10,000g
pellets) membrane preparations; (b) a typical pH depen-
dency curve, with maximal activity at 8.5 , pH , 10;
(c) a selectivity toward anandamide with respect to
other acylethanolamides; and (d) a sensitivity to typi-
cal Ser- and Cys-protease inhibitors and/or sulfhydryl-
selective reagents such as PMSF, p-hydroxy-mercuri-
benzoate, N-ethylmaleimide (NEM), p-bromo-phenacyl-
Copyright r 1998 by Academic Press
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394 Di Marzo and Deutsch

FIG. 3. Mechanisms for the inactivation of anandamide and 2-arachidonoylglycerol in the CNS. FAAH contributes to the physiological
degradation of both compounds. The degradation products of the two compounds are basic constituents of membrane phospholipids.
2-Arachidonoylglycerol can also be reacylated into membrane phospholipids prior to hydrolysis.

Copyright r 1998 by Academic Press

All rights of reproduction in any form reserved.
Endocannabinoid Biochemistry
bromide, di-isopropyl-fluoro-phosphate, and thi-
merosal. Moreover, the enzyme appeared to be distinct
from that previously shown to catalyze the hydrolysis
of another bioactive fatty acid amide, ceramide (79).
These observations, together with the finding of
high levels of anandamide amidohydrolase in rat liver
and kidneys (78), led to the suggestion that this
enzyme could be the same amidohydrolase shown
previously to catalyze the hydrolysis of saturated and
monounsaturated fatty acid ethanolamides (69). More-
over: (a) the partially purified ‘‘neuronal’’ amidohy-
drolase from N18TG2 cells was shown to also recognize
the sleep-inducing factor cis-9,10-octadecenoamide
(oleamide [83]) as a substrate, albeit with a lower
catalytic efficiency (82) and (b) anandamide amidohy-
drolase partially purified from porcine brain was
found to catalyze also the synthesis of anandamide in
the presence of high concentrations of arachidonic acid
and ethanolamine (79). Within the CNS, the distribu-
tion of the enzyme reflected the distribution of the CB1
receptor, with the highest activity found in the globus
pallidus, hippocampus, substantia nigra, cerebral cor-
tex, and cerebellum and the lowest activity in the brain
stem and medulla (78). Subsequent studies confirmed
that the enzyme was present also in peripheral cell
types, such as murine basophil-like and monocyte/
macrophage cell lines (24) and porcine ocular tissues
(84), while a recent investigation elegantly managed to
unmask a potent anandamide amidohydrolase activity
also in rat small intestine (85). Hence, a regulatory role
appeared to emerge for anandamide amidohydrolase
in many different tissues, which prompted the design
of selective inhibitors (see next section), the develop-
ment of new assay procedures for its identification (86,
87), and further efforts toward its full purification and
molecular characterization. Based upon the finding
that acyl-trifluoro-methyl-ketones could act as potent
reversible inhibitors of anandamide amidohydrolase
(88), and the fact that the latter protein could also
catalyze the hydrolysis of oleamide (82), the purifica-
tion and molecular characterization of the enzyme was
achieved by Cravatt and co-workers (89). They had
developed a series of transition-state inhibitors for
‘‘oleamide hydrolase’’ (90). One of these inhibitors,
oleoyl-trifluoro-methyl-ketone, was used to prepare an
affinity chromatography resin which turned out to
provide the decisive step for the isolation of the
inactive oleamide hydrolase. The amino acid se-
quences, obtained from tryptic digests of this inactive
preparation eluted from the column, were used to
construct degenerate oligonucleotides. These oligo-
395
nucleotides were then used to synthesize, by polymer-
ase chain reaction, a 350-bp complementary DNA
fragment from rat liver mRNA. This partial clone was
used as a probe to screen a rat liver cDNA library for
full-length clones. Two of these clones were fused
through an internal restriction site to generate the
putative full-length cDNA of 2473 bp. This cDNA was
ligated into pcDNA3 and was transfected into COS-7
cells which expressed the enzyme catalyzing the hydro-
lysis not only of oleamide but also of anandamide and
other fatty acid amides. The expressed enzyme exhib-
ited a molecular weight of 60–65 kDa, in agreement
with the amino acid sequence deduced from the
cDNA. The primary sequence indicated: (i) a highly
hydrophobic transmembrane domain, (ii) an extensive
signature sequence similar to several other amidases,
and (iii) a consensus class II SH3-domain-binding
sequence (89). Moreover, the preferential substrate for
the recombinant enzyme was anandamide, and this,
together with the tissue distribution of its transcript,
which was found to be highly expressed in the brain,
indicated that oleamide hydrolase and anandamide
amidohydrolase are the same enzyme. Given the
variety of fatty acid amides that this enzyme recog-
nized as substrates, the more general name of ‘‘(long-
chain) fatty acid amide hydrolase’’ (FAAH) was pro-
posed as an alternative to anandamide amidohydrolase
(82, 89). Subsequent studies carried out in the rat
confirmed the identity of the latter enzyme and FAAH
(85) and showed that FAAH mRNA distribution in the
brain well correlated with the presence of CB1 recep-
tors by being predominantly present in the neocortex,
hippocampus, amygdala, and thalamus and sparsely
expressed also in the hypothalamus, brain stem, and
cerebellum (91). FAAH was also recently cloned from
human and mouse liver (92). The mouse enzyme
displayed a 91% amino acid identity with rat FAAH,
while human FAAH shared with the mouse and rat
enzymes 84 and 82% sequence homology, respectively.
These small differences may explain why human
FAAH appeared slightly less selective than the rat
enzyme toward saturated fatty acid amides (92) or
why the catalytic efficiency of rat ‘‘basophilic’’ anan-
damide amidohydrolase with palmitoylethanolamide
was higher than that observed with the mouse neuro-
nal enzyme from N18TG2 cells (24). Finally, expression
of rat FAAH cDNA in COS-7 cells proved that this
enzyme could also catalyze the reverse reaction that is
the condensation of fatty acids with ethanolamine or
ammonia, thus behaving as an anandamide/oleamide
synthase (70, 71).
To date, there is no evidence for anandamide oxida-
Copyright r 1998 by Academic Press
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396
tion in the CNS, although such an alternative pathway
for anandamide catabolism may occur in peripheral
cells and tissues (Fig. 3). Enzymes of the arachidonic
acid ‘‘cascade,’’ such as some lipoxygenases, cyto-
chrome P450 oxidases, and cycloxygenase-2, but not
cycloxygenase-1, in a purified form, in cell-free homog-
enates, or transfected into baculovirus, were shown to
recognize anandamide as a substrate (93–96). This
resulted in the formation of hydroxy-anandamide
derivatives, which in some cases exhibited CB1-
receptor binding activity comparable to anandamide,
and of the noncannabimimetic ‘‘prostaglandin E2-
ethanolamide.’’ The only example of lipoxygenase-
catalyzed oxidation of anandamide by intact cells
reported to date was recently described in human
platelets and polymorphonuclear leukocytes, which
converted the endocannabinoid into its 12(S)- and
15(S)-hydroxy-derivatives. These metabolites showed
little affinity for anandamide amidohydrolase and, in
the case of 12(S)-anandamide, relatively high CB2-
receptor binding activity (97).
Evidence for the rapid inactivation of anandamide
in vivo has recently been shown in mice (98). [ 3H]Anan-
damide was injected intravenously, and the distribu-
tion of radioactivity in various tissues was analyzed.
The highest levels of radioactivity were found in
plasma and the adrenal gland 1 and 5 min after
administration, whereas, in brain, anandamide, albeit
still clearly detectable, was mostly transformed into
arachidonic acid and more polar metabolites. No
anandamide was observed in brain 15 and 30 min after
administration.
As FAAH seems to be mostly present in intracellular
membranes (92), the degradation of anandamide is not
likely to occur in vivo without a previous uptake step
by cells (Fig. 3), as hypothesized in early studies on
anandamide inactivation (40, 61). Some progress has
been recently made in the characterization of the
facilitated transport system(s) responsible for anan-
damide uptake. Such a transporter was reported in
RBL-2H3 cells, where a selective, temperature-depen-
dent, and rapid (t1/2 5 7 min) uptake mechanism,
displaying apparent Km and Vmax values for anan-
damide of 33 µM and 0.6 nmol min21 per 106 cells, and
sensitive to the protein-alkylating agents PMSF and
NEM, was described (24). More recently, two reports
have appeared in the literature on the characterization
of Na1-independent anandamide transporters in the
CNS (99, 100). In cerebellar granule neurons, apparent
Km and Vmax values for anandamide were very similar
to those reported for RBL-2H3 cells, i.e., 41 µM and
0.61 nmol min21 per 106 cells, respectively, while the
Copyright r 1998 by Academic Press
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Di Marzo and Deutsch
t1/2 was 2.6 min. The accumulation of anandamide was
inhibited by phloretin, N-oleoyl-ethanolamine, N-
arachidonoyl-benzylamine, and N-arachidonyol-pro-
pylamine, but not by arachidonic acid (99). In rat
cortical neurons and astrocytes the transporter exhib-
ited higher affinity for anandamide (apparent Km
values were 1.2 and 0.32 µM, respectively) was slightly
less rapid (t1/2 5 4 min) and was selectively inhibited
by N-(4-hydroxyphenyl)arachidonylamide (AM404)
(100).
Data on the existence of inactivating mechanisms for
extracellular, cannabimimetic 2-AG are now also avail-
able (Fig. 3). Evidence for the presence of membrane-
bound lipases selective for monoacylglycerols with
polyunsaturated fatty acid chain in either the sn-1(3) or
2 position was recently obtained in N18TG2 and RBL-
2H3 cells (101). A major contribution to this lipase
activity comes from FAAH, which catalyzes 2-AG
hydrolysis in transfected cells, as well as in N18TG2 and
RBL-2H3 cells (102, 103). 2-AG may be also inactivated
via reacylation into membrane phospholipids prior to
hydrolysis (103), whereas no evidence exists as yet for
its facilitated diffusion into cells.
NATURAL AND SYNTHETIC INHIBITORS
OF ENDOCANNABINOID INACTIVATION
In view of the possible many physiological roles
suggested for the endogenous cannabinoid system in
both the CNS and peripheral systems (12–17), it is
likely that synthetic drugs capable of modulating
either the levels or the action of endocannabinoids in
vivo may provide the basis for the development of new
therapies for several nervous and peripheral dysfunc-
tions. These illnesses range from depression, nausea,
lack of appetite, sleeplessness, and locomotor diseases
to hyperalgesia, inflammation, and hypertension. Due
to the advances made in characterizing anandamide
amidohydrolase, attention has been focussed on the
regulation of this enzyme. Inhibitors of anandamide
breakdown are likely to be of interest in those cases
where there is an inbalance in endocannabinoid homeo-
stasis (decreased synthesis or excessive degradation).
The design of the first inhibitors of anandamide hydro-
lysis was based on the assumption that anandamide
amidohydrolase behaved as a Cys- or Ser-protease-like
enzyme (see above). Hence, possible transition-state
inhibitors, forming a reversible tetrahedral (thio)hemik-
etal intermediate with active site -SH or -OH groups,
Endocannabinoid Biochemistry
e.g., acyl-trifluoro-methyl-ketones, acyl-a-keto-esters,
and acyl-a-keto-amides with fatty acid chains of vary-
ing length and degree of insaturation, were synthe-
sized (88). The most effective classes of compounds
were the trifluoromethyl-ketones and the a-keto-
esters, whose inhibitory effect on anandamide hydroly-
sis by both particulate fractions and intact N18TG2 cells
were maximal when arachidonic acid was the fatty
acid substituent. More recently, palmitoyl-trifluoro-
methyl-ketone was shown to be as effective as arachi-
donoyl-trifluoromethyl-ketone (ATFMK) toward the
rat basophilic amidohydrolase (24). Later, the need for
possible affinity-labeling reagents for the hydrolytic
enzyme prompted the development of possible irre-
versible inhibitors. Again, these were designed on the
basis of previous knowledge of Ser- and Cys-protease
inhibitors. Basically, five classes of irreversible inhibi-
tors are known for this large class of enzymes: the
sulfonyl-fluorides (e.g., PMSF), the fluoro-phospho-
nates (e.g., di-isopropyl-fluoro-phosphate), the mercuri-
benzoates and bromo-phenacyl-bromides, the diazo-
methyl- (and chloromethyl-) ketones, and the N-acyl-
hydroxamates [for example, see 104, 105]. Hence, fatty
acid derivatives containing some of the above highly
reactive chemical moieties (Fig. 4) were synthesized
and tested for their capability of inhibiting anan-
damide/oleamide amidohydrolase (and, therefore,
FAAH) from various sources (86, 90, 106–108). Interest-
ingly, only arachidonoyl-methyl-fluoro-phosphonate
(MAFP) and the acyl-sulfonyl-fluorides were found to
potently and irreversibly inhibit the solubilized en-
zyme (with IC50 values in the medium to low nanomo-
lar range [106–108]). In theory, these electrophilic
inhibitors behaved in this fashion because they easily
lose their fluoride upon forming a covalent adduct
with the nucleophilic, enzymatically reactive -SH/OH
groups (Fig. 4). On the other hand, the acyl-diazo-
methyl- and -chloro-methyl-ketones and the N-hy-
droxamates behaved as transition-state, reversible in-
hibitors of FAAH (90, 106). This suggests that the
stereochemistry of the active site of FAAH does not
allow these compounds to undergo the same transposi-
tion suggested to occur for Ser- and Cys-proteases in
the presence of the peptidyldiazomethylketones, -chlo-
romethylketones, and -N-hydroxamates and necessary
for the covalent modification of the enzyme ([104, 105]
and Fig. 4). Of the acyl-diazomethyl- and -chloro-
methyl-ketones, the arachidonic acid derivatives ap-
peared to be much more potent than the oleic acid
analogues, whereas the N-acyl-hydroxamates were
only weak inhibitors, whether they were O-acetylated
or not (90, 106).
397
Ideally, in order to be useful as therapeutic agents,
inhibitors should be selective for FAAH vs other
esterases-amidases and should fail to appreciably bind
to cannabinoid receptors. Unfortunately this is not the
case for most of the above compounds. MAFP, the
most potent FAAH inhibitor developed so far, and
ATFMK also inhibit cytosolic phospholipase A2, even
though the former compound does so only at concen-
trations 500–1000 times higher than those required for
FAAH inhibition (107, 108). These two compounds
also bind to CB1 receptors (MAFP irreversibly), albeit
with lower potencies than those exhibited for the
inhibition of anandamide hydrolysis (88, 108). More
disappointing were the findings that the bromoenol
lactone, a well-known irreversible blocker of cytosolic
phospholipase A2, and ibuprofen, an inhibitor of cy-
cloxygenases, inhibit anandamide amidohydrolase at
concentrations similar to those required to inactivate
the enzymes they were originally targeted to (109, 110).
Arachidonoyl-diazo-methyl-ketone, which is as potent
as ATFMK as a reversible inhibitor of the amidohydro-
lase (IC50 5 3–6 µM), also inhibited leukocyte 5-lipoxy-
genase (106, 111). As of yet this inhibitor was not tested
on phospholipase A2. More promising seem to be the
sulfonyl-fluorides of saturated fatty acids with 14, 16,
and 18 carbon atoms, which exhibited high selectivity
for anandamide amidohydrolase than toward CB1
receptors, even though they were not tested on other
esterases (108). Recently, arachidonoyl serotonin was
shown to inhibit FAAH with a relatively good potency
(IC50 between 5 and 12 µM) without affecting cytosolic
phospholipase A2 or binding to CB1 receptors (123). In
conclusions, further efforts will be required in order to
obtain highly selective anandamide amidohydrolase
(FAAH) inhibitors. The understanding of the molecu-
lar mechanism of action of this enzyme, together with
the identification of the amino acid residues involved
in its catalytic activity, will probably be of help toward
the achievement of this task. On the other hand, some
success has been obtained recently in the search for
selective inhibitors of anandamide uptake by cells.
N-(4-Hydroxyphenyl)arachidonylamide (AM404), a
compound with no FAAH inhibitory activity, was in
fact shown to block the facilitated transport of anan-
damide into rat cortical neurons and astrocytes and,
more importantly, to potentiate some in vitro (i.e.,
adenylate cyclase inhibition) and in vivo (i.e., analgesia
on a hot plate) effects of anandamide (100). SAR
studies for the interaction of endocannabinoids with
the corresponding hydrolytic enzymes and transport-
ers still need to be performed in order to dictate the
design either of selective inhibitors of inactivation or of
Copyright r 1998 by Academic Press
All rights of reproduction in any form reserved.
FIG. 4. Chemical structures and likely mechanisms of actions of FAAH inhibitors. For compounds 2, 3, and 4, an irreversible inhibition, based
on a mechanism similar to that shown in the figure, was predicted, but a reversible inhibition was found instead. X, sulfur or oxygen atom; E,
strong electrophilic groups; Y, phosphorus or sulfur atom; L, good leaving group.

398
Endocannabinoid Biochemistry
noninactivable analogs to be employed as therapeuti-
cally useful drugs. The recent finding that FAAH may
contribute to the physiological degradation of 2-AG
(102, 103) will allow the use of new FAAH inhibitors
also in studies on the cannabimimetic glyceride.
While the development of synthetic modulators of
endocannabinoid levels is still in progress, recent
studies seem to point at natural congeners of anan-
damide and 2-AG as possible ‘‘physiological’’ inhibi-
tors of the inactivation of these two endocannabinoids.
These studies originated from the observation that
several fatty acid amides and monoacylglycerols, with
very little affinity for cannabinoid receptor subtypes,
are present in cells and, in some cases, cosynthesized
with anandamide and 2-AG, respectively (40, 42, 101).
Moreover, it was thought that neuronal cells may have
a means to enhance and prolong the activity of the two
arachidonate derivatives, which in vivo is shorter-
lasting and weaker than that of synthetic cannabi-
noids. It seemed plausible that noncannabimimetic
fatty acid derivatives, by being present often in amounts
higher than cannabimimetic congeners, were competi-
tive substrate inhibitors for the inactivation of 2-AG
and anandamide, thus enhancing their levels and
prolonging their action. In an analogous manner, v3
fatty acids are competitive substrates for the enzymes
responsible for v6 fatty acid metabolism and, there-
fore, can modulate the levels of both v6 fatty acids and
their derivatives [for a recent review see 112]. Accord-
ingly, it was found that fatty acid ethanolamides, such
as palmitoyl-, oleoyl-, and linoleoyl-ethanolamides,
whose formation is induced in most cell types by the
same stimulus leading to anandamide formation, can
inhibit the hydrolysis and/or the uptake of anan-
damide by both cell-free and whole cell systems (24,
40, 99, 113). Oleamide, which is also more abundant
than anandamide in mouse neuronal cells (114), is not
active at cannabinoid receptors (115) and is a substrate
for FAAH (70, 89, 92), exerts a similar inhibitory action
on anandamide hydrolysis (115). Likewise, monoacyl-
glycerols, which accompany 2-AG in both the brain
and peripheral tissues (42, 116), inhibit 2-AG uptake/
hydrolysis by both cell membranes and intact cells
(116) and enhance 2-AG functional activation of CB1
and CB2 receptors (116). Whether these ‘‘natural’’
inhibitors of anandamide and 2-AG inactivation also
operate under physiological conditions remains to be
assessed, but it is worthwhile mentioning that, in the
tetrad of mouse in vivo tests: (i) oleamide, oleoyletha-
nolamide, and linoleoyl-ethanolamide are signifi-
cantly, albeit weakly, active (115, 117); (ii) a submaxi-
399
mal dose of oleamide strongly potentiates anandamide
action (115); and (iii) 2-palmitoyl-glycerol and 2-lino-
leoyl-glycerol, which are themselves inactive, signifi-
cantly potentiate the activity of a subliminal dose of
2-AG (116). It will be interesting to determine, in future
studies, which of the pharmacological actions reported
for noncannabimimetic fatty acid derivatives are due
to an increase of the levels of the two endocannabi-
noids.
CONCLUSIVE REMARKS AND FUTURE
DEVELOPMENTS
From the reports on the biochemistry of endogenous
cannabinoid receptor ligands that have appeared to
date and are discussed herein, a few conclusions can be
drawn. First, two types of cannabimimetic metabolites
have been discovered: those directly activating the
cannabinoid receptors, represented by the polyunsatu-
rated acylethanolamides and the 2-monoglycerol ester
of arachidonic acid, and those possibly acting by
prolonging and potentiating the effect of these two
types of metabolites, represented by their nonpolyun-
saturated analogues. Only the former compounds,
which are obviously the most active ones, can be
considered as true endocannabinoids, whereas those
of the second type may rather behave as endogenous
‘‘enhancers’’ of anandamide and 2-AG action. Second,
studies will be needed in order to understand the
mechanisms regulating the enzymes responsible for
endocannabinoid biosynthesis and inactivation and to
correlate them with both physiological and pathologi-
cal conditions. The recent finding of anandamide
formation in rat macrophages following haemorrhagic
shock (122) provides a prototypical example of such
studies. The development of techniques for the rapid
quantitation of endocannabinoids, such as a selective
radioimmunoassay, as opposed to the time-consuming
and relatively insensitive HPLC and MS techniques
currently used for the analysis of anandamide and
2-AG (20, 21, 118–121), is likely to improve our knowl-
edge of the physiopathological regulation of the levels
of these metabolites. Once that this knowledge has
been extended, SAR studies for the interaction of
endocannabinoids with either their receptors or their
inactivating enzymes, on the one hand, and the finding
of inhibitors of endocannabinoid biosynthesis, on the
other hand, will hopefully provide new drugs for the
treatment of those nervous illnesses that may be due to
mulfunctions of the endogenous cannabinoid sys-
tem.
Copyright r 1998 by Academic Press
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400
ACKNOWLEDGMENTS
This work was partly supported by a grant from the Human
Frontier Science Program Organization (RG 26/95) to VDM and a
grant from the National Institute of Drug Abuse (DA07318) to
DGD.
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