An Introduction to the Endocannabinoid System: From the Early to the Latest Concepts

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An introduction to the endocannabinoid system: from theearly to the latest concepts

Luciano De Petrocellis, Doctor, Vincenzo Di Marzo, PhD, Doctor *

Endocannabinoid Research Group, Institute of Biomolecular Chemistry and Institute of Cybernetics,

Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, Comprensorio Olivetti, 80078 Pozzuoli, Naples, Italy

Keywords:

anandamide

2-arachidonoylglycerol

cannabinoid

N-acyl-ethanolmine

CB1CB2

A rather complex and pleiotropic endogenous signalling system

was discovered in the late 1990s, starting from studies on the

mechanism of action of D9-tetrahydrocannabinol, the major

psychoactive principle of the hemp plant Cannabis sativa. This

system includes: (1) at least two G-protein-coupled receptors,

known as the cannabinoid CB1 and CB2 receptors; (2) the endog-

enous agonists at these receptors, known as endocannabinoids, of

which anandamide and 2-arachidonoylglycerol are the bestknown; and (3) proteins and enzymes for the regulation of

endocannabinoid levels and action at receptors. The number of the

members of this endocannabinoid signalling system seems to be

ever increasing as new non-CB1 non-CB2 receptors for endo-

cannabinoids, endocannabinoid-related molecules with little

activity at CB1 and CB2 receptors, and new enzymes for endo-

cannabinoid biosynthesis and degradation are being identified

every year. The complexity of the endocannabinoid system and of

its physiological and pathological function is outlined in this

introductory chapter, for a better understanding of the subsequent

chapters in this special issue.

2008 Elsevier Ltd. All rights reserved.

The endocannabinoid system: the early view

For centuries the biological and molecular bases of the recreational and medicinal use of prepa-

rations from the hemp plant Cannabis sativa have remained unexplained. It took, in fact, much longer to

identify the natural components responsible for the pharmacological effects of marijuana and hashish

* Corresponding author. Tel.: 39 081 8675093; Fax: 39 081 8041770.E-mail address: [email protected](V. Di Marzo).

Contents lists available atScienceDirect

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Endocrinology & Metabolismj o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / lo c a t e / b e e m

1521-690X/$ see front matter 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.beem.2008.10.013

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than it had taken to assign a chemical identity to the active principle of opium. The identification of

cannabidiol (CBD) first, and of D9-tetrahydrocannabinol (THC) shortly after, in the 1960s1,2 were

therefore major breakthroughs, since these two compounds, being the two most abundant Cannabis

secondary metabolites, were likely to explain most of its pharmacological actions. Early studies focused

more on THC because of its clear psychotropic activity and the social implications of it. The strong

hydrophobic nature of this compound suggested that its effects might be due to a general non-specificperturbation of cell membranes rather than to a specific interaction with selective binding sites. It was

only thanks to the synthesis of enantiomers of THC and its synthetic analogues3,4, and to the subse-

quent discovery that its principal pharmacological actions were enantioselective, that the idea of

a cannabinoid receptor was put forward. Thus, the first specific binding sites for a radiolabelled and

enantiomerically pure synthetic analogue of THC were identified in the brain only in 19885, and this

major achievement opened the way for the identification of the first THC-specific receptor, named CB1,

and screened out of several previously cloned orphan G-protein-coupled receptors (GPCRs).6 The

second cannabinoid receptor, named CB2, was then identified by homology cloning, and, quite inter-

estingly, it turned out to be rather different from CB1in its amino acid sequence.7 While CB1was found

to be extremely abundant in the brain, and was immediately suggested to be responsible for THC

psychoactivity, CB2was more abundant in immune cells.The molecular characterization of THC binding sites, whilst still leaving open the question of the

mechanism of action of non-psychotropic plant cannabinoids (particularly CBD, which exhibits several

therapeutically interesting pharmacological activities)8, opened the way for the identification of the

true raison detre of these receptors, i.e., of their endogenous ligands. The first such compound to be

discovered was anandamide (arachidonoyl ethanolamide, from the Sanskrit wordanandafor bliss)9,

and this finding was soon to be followed by the identification of the cannabimimetic properties of an

already known endogenous metabolite, 2-arachidonoylglycerol (2-AG).10,11 Although other chemically

similar endocannabinoids (Fig. 1) were identified during the last 10 years, including, in chronological

order, 2-arachidonyl-glycerol ether (noladin ether)12, N-arachidonoyl-dopamine (NADA)13,14, and

virodhamine15, anandamide and 2-AG have remained the only ones of which the pharmacological

activity and metabolism have been most thoroughly investigated. Therefore, these two compounds arestill referred to as the major endocannabinoids.

Studies on the biosynthetic and catabolic pathways and enzymes for anandamide and 2-AG started

immediately after their discovery (Fig. 2). N-arachidonoyl-phosphatidylethanolamine (NArPE) was

identified as the biosynthetic precursor of anandamide16, and diacylglycerols (DAGs) with arachidonic

NH

O

OH NH

O

OH

OH

O

OH

OH

O

O

NH2

O

O

OH

OH

Anandamide N-arachidonoyldopamine

Noladin-ether

Virodhamine 2-Arachidonoylglycerol

Fig. 1. Chemical structures of the proposed endocannabinoids.

L. De Petrocellis, V. Di Marzo / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 1152


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acid on the 2-position as the most likely compounds from which 2-AG is generated.1719

NArPE wasshown to be produced from the transfer of arachidonic acid from the sn-1 position of phospholipids to

the nitrogen atom of phosphatidylethanolamine.20 whereas DAG precursors for 2-AG were shown to be

produced either from the phospholipase-C-catalysed hydrolysis of phosphatidylinositol19 or from the

hydrolysis of phosphatidic acid.21 Both anandamide and 2-AG were found to be inactivated mostly by

enzymatic hydrolysis of the amide and ester bonds, respectively, and the major enzymes responsible

for these reactions were cloned and named fatty acid amide hydrolase (FAAH)22 and monoacylglycerol

lipase (MAGL)23,24, respectively. As to the proposed enzymes for anandamide and 2-AG biosynthesis

from their direct precursors, they were cloned only in the new century. Two sn-1-selective DAG lipases,

named DAGL-a and DAGL-b, were identified as clearly responsible for 2-AG biosynthesis in cells and

tissues25, whereas the enzyme catalysing the direct conversion of N-acylethanolamines, including

NArPE, intoN-acylethanolamines, including anandamide, was cloned a year later.

26

Finally, a specificprocess through which endocannabinoids are either taken up by cells following cannabinoid receptor

activation, or released from cells following endocannabinoid biosynthesis, and therefore functioning in

the direction of the gradient of endocannabinoid concentration across the plasma membrane, was

identified.16,2729 This mechanism was suggested to be pharmacologically distinct from FAAH or

Fig. 2. Biosynthesis, action, and inactivation of the two best-studied endocannabinoids, anandamide and 2-arachidonoylglycerol (2-

AG). Several pathways might exist for both the formation and catabolism of anandamide and 2-AG. The former originates from

a phospholipid precursor, N-arachidonoyl-phosphatidyl-ethanolamine (NArPE), formed from the N-arachidoylation of phosphati-

dylethanolamine via N-acyltransferases (NATs). NArPE is transformed into anandamide via four possible alternative pathways, the

most direct of which is catalysed by an N-acyl-phosphatidylethanolamine-selective phosphodiesterase (NAPE-PLD). 2-AG is

produced almost exclusively via the hydrolysis of diacylglycerols (DAGs) viasn-1-selective DAG lipases (DAGLs)aandb. After cellular

re-uptake via a specific and yet-to-be characterized mechanism (EMT), which appears to also mediate the release of de-novo

biosynthesized endocannabinoids, anandamide is metabolized via fatty acid amide hydrolase-1 (FAAH) and 2-AG via several

monoacylglycerol lipases (MAGLs). 2-AG can also be degraded by FAAH. Both endocannabinoids activate CB1and CB2receptors with

different affinities (anandamide being the one with highest affinity in both cases) and efficacies (2-AG being the one with highest

efficacy in both cases). Anandamide can also activate transient receptor potential vanilloid type-1 (TRPV1) channels at an intra-

cellular site, and interact with several other molecular targets, whereas both compounds were recently reported by some authors,

but not by others, to interact with GPR55, an orphan G-protein-coupled receptor. Abh4,6,12, a-b-hydrolases 4, 6, 12; PLD, phos-

pholipase D; PLA1/2, phospholipase A1/A2; PTPN22, protein tyrosine phosphatase N22. Biosynthetic pathways are shown in black,

degradative ones in blue. Thick arrows denote movement or action.

L. De Petrocellis, V. Di Marzo / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 115 3


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MAGL30,31 or CB1 receptors32, but has not yet been identified from a molecular point of view, to the

point that some authors still feel sceptical about its existence.33,34

Non-CB1 non-CB2 receptors for endocannabinoids

Especially when compared to the several GPCRs for, e.g., histamine and glutamate, only tworeceptors for endocannabinoids looked like too little of a good thing. However, homology cloning

could not identify other THC receptors with some sequence similarity to CB1 and CB2, and the screening

of the several tens of orphan GPCRs, the sequences of which are already available, initially yielded

negative results (but see below). On the other hand, several pharmacological studies, reviewed by Di

Marzo and De Petrocellis35 and Begg et al36, suggested the existence of non-CB1non-CB2receptors for

endocannabinoids. For example, in both endothelial cells and the brain of transgenic mice lacking the

two cloned receptors, anandamide was found to induce pharmacological effects that were indicative of

its capability to activate other GPCRs.37,38 Therefore, it was hypothesized that, if other molecular targets

existed for endocannabinoids, these had to be either already discovered GPCRs for other mediators or

exhibit very little homology to CB1 and CB2, and even belong to different classes of receptors. Both

hypotheses have gained experimental support. In fact, endocannabinoids, and anandamide in partic-ular, were found to interact positively or negatively with serotonin and muscarinic receptors, on the

one hand, and with glycine and nicotinic acetylcholine gated channels on the other hand.3944

Furthermore, anandamide was also found to inhibit several types of Ca2 and K channels.35,45

However, to date, none of these interactions (mostly investigated in vitro) has been conclusively shown

to contribute to the in-vivo pharmacology of endocannabinoids, and therefore their physiopathological

relevance has yet to be confirmed.

Perhaps the best established non-CB1 non-CB2 receptor for anandamide is the transient receptor

potential vanilloid type-1 (TRPV1) receptor, a non-selective cation channel belonging to the large

family of the six-transmembrane-domain transient receptor potential (TRP) channels, and activated by

noxious heat (>42 C), low pH (


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has yet to be demonstrated, and therefore their biological relevance still needs to be substantiated by

further experimentation. The possibility that CB1 also makes heterodimers with one of its two

previously reported splicing variants61 needs also to be considered, as this was shown to occur for

prostaglandin FP receptors and to create the conditions for the specific recognition of putative

cyclooxygenase-2 derivatives of anandamide that are otherwise very weak FP agonists (see below).

Finally, some evidence is accumulating to suggest that anandamide and 2-AG also activate theperoxisome proliferator activated receptors (PPAR)-aand -g, a class of nuclear receptors that control

the expression of several genes involved particularly in metabolism and immune/inflammatory

responses.62 Unlike the interactions described above, however, usually high concentrations (10mM)

of either anandamide or 2-AG seem to be required to exert PPAR-mediated effects in vitro. 6366

Furthermore, unlike, for example, the case of TRPV1 channels, several chemically unrelated synthetic

and plant cannabinoid receptor ligands also share this property with endocannabinoids 67,68, thus

hinting at the possibility that this effect might be rather unspecific.

Endogenous bioactive endocannabinoid-related molecules

Since their discovery as endocannabinoids, it was immediately clear that both anandamide and 2-AG are often accompanied in cells, tissues and biological fluids by congeners that are less active, or even

inactive, at cannabinoid receptors. Thus, it is now well established that other long-chain N-acyletha-

nolamines, very probably biosynthesized from molecules similar to NArPE, the N-acyl-phosphatidyl-

ethanolamines, and, like anandamide, degraded to the corresponding fatty acid and ethanolamine by

FAAH, are often more abundant in tissues than anandamide, and play a biological function by activating

non-CB1 non-CB2 receptors.69 Of these compounds, oleoylethanolamide (OEA) is known to inhibit food

intake, reduce body weight and affect lipid and glucose metabolism via TRPV1- and/or, particularly,

PPAR-a-mediated mechanisms.7072 A role for the orphan GPCR, GPR119, in some of these effects has

also been suggested73,74 but not yet confirmed in GPR119 knockout mice. Palmitoylethanolamide,

exerts anti-inflammatory actions via a variety of molecular mechanisms, including direct activation of

PPAR-a75

and, possibly, GPR5552

receptors, and potentiation of anandamide actions at CB176,77

,TRPV17780 or PPRg77 receptors. In the case of 2-AG congeners, no specific molecular target has yet

been identified for compounds such as 2-palmitoyl-, 2-oleoyl- and 2-linoleoyl-glycerol, and the only

endocannabinoid-related biological activity described for these molecules is their ability, in mixture, to

enhance some of the CB1-mediated 2-AG actions in vitro and in vivo.81,82

Several other types of bioactive fatty acid amides were discovered in the wake of anandamide

isolation in 1992, exploiting the development of the lipidomic approach and of lipid profiling tech-

niques. First it was the turn of the N-acyl-glycines and N-acyl-serines, of which N-arachidonoyl-glycine

was the first83 andN-arachidonoyl-serine the latest84 to be identified. Although several members of

these two classes of potentially bioactive lipids which appear to exert analgesic or vasodilatory

effects, respectively83,84 have been identified in tissues85; the potential molecular targets have been

investigated for only a few of them, but not yet characterized from a molecular point of view.84,86

Another class of long-chain fatty acid amides that is currently being studied is theN-acyl-dopamines.14

While the aforementioned NADA, like anandamide, binds to and activates both CB1 and TRPV1

receptors13 and antagonizes the TRPM8 receptor50, the unsaturated members of this family of

compounds selectively activate TRPV187 in a way enhanced by saturatedN-acyl-dopamines, which are

inactive per se at this target.88 Interestingly, bothN-acyl-glycines andN-acyl-dopamines seem to be

biosynthesized by direct condensation between the corresponding fatty acids and amino acids,

whereas the involvement of FAAH in their inactivation has been investigated so far only for the latter,

and ruled out. Bioactive fatty acid amides that are certainly substrates for FAAH-catalysed degradation

are instead the N-acyl-taurines89 which, like the N-acyl-dopamines, seem to interact with TRP

channels.90

In conclusion, the identification of the endocannabinoids in the late 1990s opened the way to thediscovery of a whole class of related lipid mediators, the biological significance of which still remains to

be investigated. It can be predicted that, with the ever more advanced methods for lipid profiling that

are being described in the literature, more and more fatty acid amides and esters will be identified that

share with the endocannabinoids either metabolic pathways or molecular targets or both.



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epoxyeicosatetraenoyl-anandamides.112,113 Since the metabolites obtained are, in most cases, still

active at cannabinoid receptors, the biological relevance of these reactions remains to be established,

also in view of the fact that no evidence has been reported to date for the presence of these compounds

in live animals.

Tools for the study of endocannabinoid biology and new potential leads for drug development

Comprehensive reviews of the most widely used pharmacological tools for the study of the

endocannabinoid system have been published very recently.114,115 These tools include: (1) inhibitors of

endocannabinoid cellular uptake, such as AM404, LY-2183240, VDM11, UCM707, OMDM-1, OMDM-2

and AM1172, in increasing order of selectivity; (2) inhibitors of FAAH, such as URB-597, OL-135, BMS-1,

SA-47, PF-750 andN-arachidonoyl-serotonin (which also antagonizes TRPV1 receptors); (3) inhibitors

of MAGL, such as URB602 and N-arachidonoyl-maleimide; (4) dual CB1/CB2 agonists, such as WIN-

55,512-2, CP-55940 and HU-210; (5) anandamide analogues that are more metabolically stable than

the parent compound and more suitable for in vivo studies, such as methanandamide and met-

fluoroanandamide; (6) selective CB1 agonists, such as arachidonoylchloroethanolamide and arach-

idonoylcyclopropylamide; (7) selective CB2 agonists, such as HU-308, JWH-015, JWH-133 and AM1241;(8) selective antagonists/inverse agonists for CB1 receptors, such as SR141716A (rimonabant), SR147778

(surinabant), AM251, AM281, MK-0363 (Taranabant), LY320135 and AVE1625; (9) neutral CB1antag-

onists, such as AM4113; (10) selective CB2 antagonist/inverse agonists, i.e. SR144528, AM630

and JTE907; and (11) allosteric modulators of CB1 receptors, including Org27596, Org29647 and

PSNCBAM-1.116 The chemical structures of the most widely used of these compounds is shown in Fig. 3.

The issue of the selectivity of some of these tools has been thoroughly reviewed elsewhere115 and will

not be discussed here. It is important to note, however, that several of these compounds have been

used in experimental models of disorders in which the endocannabinoids were shown to either have

a protective function or contribute to disease symptoms and progress.114 In particular, and more

relevant to other articles in this special issue: (1) CB1antagonists are already being used in the clinic or

in clinical trials as anti-obesity agents (rimonabant, now marketed as Acomplia in more than 55countries as an anti-obesity agent, is prescribed in the EU as an aid to caloric restriction and exercise to

reduce body weight in patients with body mass index (BMI) > 30, or with BMI>27 and metabolic

complications such as dyslipidaemia and type-2 diabetes)117; (2) inhibitors of endocannabinoid uptake

and/or hydrolysis have been used recently to induce appetite after central administration118,119, and to

reduce tumour cell growth in vitro and in vivo120122; (3) endocannabinoid uptake and/or hydrolysis

inhibitors have been suggested also to reduce chronic pain (although they have not been tested against

cancer pain), colitis, and anxiety and depression in experimental models of these disorders.123125

Further tools might be developed in the future based on the recent discovery of proteins that

specifically interact with cannabinoid receptors and modulate their activity. This is the case of the two

structurally related CB1cannabinoid receptor interacting proteins (CRIP1a and CRIP1b) that bind to the

distal C-terminal tail of CB1.126

These proteins are generated by alternative splicing of a gene located onchromosome 2 in humans. CRIP1a co-immunoprecipitates with CB1 in rat-brain homogenates, indi-

cating that CRIP1a and CB1might interact in vivo. Furthermore, in superior cervical ganglion neurons

co-injected with CB1 and CRIP1a or CRIP1b cDNA, the former suppressed the CB1-mediated tonic

inhibition of voltage-gated Ca2 channels. The authors suggested that the discovery of the CRIP

proteins may lead to the development of novel drugs to treat disorders where modulation of CB1activity has therapeutic potential (e.g. chronic pain, obesity, and epilepsy). Also previously discovered

proteins might be used to regulate cannabinoid receptor activity. For example, recent data indicate that

the heat-shock protein Hsp90 may serve as a scaffold to keep the CB2 receptor and its signalling

components, including Ga(i2), in proximity, thus facilitating CB2-mediated cell migration.127

Another recent discovery, that might at the same time expand further the members of the endo-

cannabinoid system and serve as the basis for the development of new drugs, is the finding of the firstendogenous antagonist/inverse agonist of CB1 receptors. This is a nonapeptide known as haemo-

pressin, isolated from various tissues including the brain128, and previously found to induce hypo-

tensive effects that would not be entirely in agreement with the similar activity described for CB1agonists. However, the authors showed that another pharmacological activity of haemopressin, the



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ability to reduce pain, which again would be paradoxical for a CB 1 inverse agonists but had been

previously reported also for synthetic such compounds, was mediated by its interaction with the CB1receptor. Further studies on the pharmacology and regulation of the levels of this peptide during

physiopathological conditions are required in order to substantiate its role as endogenous CB1blocker.

Anatomy of the endocannabinoid system, its general strategy of action and

its pathological disruption

Studies carried out immediately after the molecular characterization of CB1 and CB2 receptors

established the distribution of their mRNAs in several mammalian tissues, with a very high abundance

of CB1in the brain and of CB2in immunocompetent cells and tissues.129 We now know, however, that

OHN

NH2O

O

O

N

O

N

URB-597

OL-135

OMDM-2 and OMDM-2

OH

NOH

O

H

OH

N

O

H

AM1172

OH

NH

OAM404N N

N

N N

O

OH

N

O

VDM11

NH

O O

LY-2183240

UCM707

OH

NH

OHO

N

O

H

Arachidonoyl serotonin

URB602

O

NO

N

O

WIN-55,512-2

OH

OH

OH

CP-55940

O

CH2OH

OH

HU-210

NH

OH

O

(R)-Met-anandamide

Met-fluoro-anandamide

NH

F

O

NH

Cl

O

O

H

H

N

O

I

NO2

O

NN

H3C

AM1241

arachidonoylchloroethanolamide

JWH133

JWH-015

N

HN

N

N

O

Cl

ClClSR141716A

(rimonabant)

I

N

N

Cl

Cl

N

N

O H

AM251HN O

N

N

Cl

H3CO

O

N

IN

O

AM630

SR144528

Cl

NH

NH

O

N N

NH

Cl

NH

O N

PSNCBAM-1

Org29647

O

Fig. 3. Chemical structures of some of the pharmacological tools used to investigate the endocannabinoid system.



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both receptors, and CB1in particular, are much more widely distributed than originally believed. For

example, the liver, initially used as a negative control to validate probes and antibodies designed to

detect the CB1 receptor, is now known to express a low but nevertheless functionally important

amount of this protein.130 On the other hand, the CB2receptors, the existence of which in the brain had

been initially ruled out, have been shown to be expressed in low amounts also in this organ during

neuroinflammatory conditions131, and even in central neurons under physiological conditions.132,133 Asa consequence, the original idea that CB1receptors played a role almost uniquely in the brain, and CB2in the immune system, has now evolved into the concept that either cannabinoid receptor type might

control several general central and peripheral functions, including neuronal development, trans-

mission and inflammation, cardiovascular, respiratory and reproductive functions, hormone release

and action, bone formation and energy metabolism, as well as cellular functions such as cell archi-

tecture, proliferation, motility, adhesion and apoptosis.134137

It has also been shown that both the levels of the major endocannabinoids and those of CB1and

CB2 undergo strong changes following several physiological and pathological stimuli.114,138,139 This

plasticity of the endocannabinoid system is particularly evident in the central nervous system, where

it controls adaptive and pro-homeostatic responses to chronic stress, neuronal excitotoxicity and

damage, and neuroinflammation140, but also more physiological phenomena such as synaptic strengthin cognitive, motivational and affective processes, as well as its pathological perturbations.141 The on-

demand character of endocannabinoid biosynthesis, action and degradation, and the pro-homeostatic

effects of cannabinoid receptor activation, allow this signalling system to exert a general protective

function and are in turn made possible, and restricted in time and space, by the lipophilic nature of

endocannabinoids, their phospholipid-dependent biosynthetic pathways, and the Ca2-sensitive

activity of some of their biosynthetic enzymes. Furthermore, in the brain, the biosynthetic and

degradative enzymes at least for 2-AG are anatomically distributed with respect to CB1 receptors in

a way that the activity of post-synaptic neurons, which express the DAGL-a in proximity to the

dendrites and synapses, can control, by producing and releasing this endocannabinoid, the activity of

the corresponding pre-synaptic neurons, where in most cases the CB1 receptor is expressed selec-

tively.142

This retrograde modulatory action is terminated by MAGL expressed selectively on the pre-synaptic axon terminal. Therefore, the anatomical distribution of some of the components of the

endocannabinoid system, together with the property of CB1 activation to reduce the activity of voltage-

activated Ca2 channels and enhance the activity of inwardly-rectifying K channels, thus reducing the

release of neurotransmitters143,144, offers a unique opportunity to re-establish an excessive activity of

the post-synaptic neurons, such as after certain acute or chronic perturbations of neuronal homeo-

stasis.142 As to anandamide, its general strategy of action appears to be more complicated due to the

following early and recent findings: (1) unlike MAGL, FAAH is mostly located post-synaptically and in

intracellular membranes, and this localization might not allow a rapid inactivation of anandamide

action at pre-synaptic neurons; (2) unlike DAGL-a, NAPE-PLD is mostly located pre-synaptically and in

intracellular membranes145,146, although there are several exceptions to this rule147, and this protein is

probably not the only biosynthetic enzyme for anandamide; (3) anandamide also activates TRPV1, thepresence and functional activity of which in the brain, in both pre- and post-synaptic neurons, is now

widely accepted148151; and (4) activation of post-synaptic TRPV1 inhibits DAGL-a, thereby depressing

2-AG levels and retrograde signalling activity at CB1.152 These experimental data indicate a potential

role for anandamide as an intracellular mediator acting at TRPV1 on a cytosolic binding site153, and

controlling Ca2 homeostasis and/or 2-AG biosynthesis, in addition to its potential anterograde

activity at the post-synaptic targets of this compound, or of other NAPE-PLD-generated molecules and

FAAH-substrates (see above).

Finally, the tight time- and space-selectivity of endocannabinoid action might be lost during chronic

conditions, in which endocannabinoids might start acting for a longer time or at targets located in cells

that they were not initially supposed to activate, thus contributing to the late symptoms and progress

of diseases. This might explain why, often for the same type of pathological conditions, not onlyenhancers of endocannabinoid action (such as FAAH and MAGL inhibitors) but also cannabinoid

receptor antagonists might exert beneficial actions.114 A typical example of dysregulation of endo-

cannabinoid action is concerned with the control of energy metabolism, and this will be the subject of

most of the forthcoming articles of this special issue.



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Summary

The endocannabinoid system is a complex and pleiotropic endogenous signalling system discov-

ered in the late 1990s from studies on the mechanism of action ofD9-tetrahydrocannabinol. It includes:

(1) at least two G-protein-coupled receptors, known as the cannabinoid CB1and CB2receptors; (2) the

endogenous ligands of these receptors, known as endocannabinoids, of which anandamide and 2-arachidonoylglycerol are the best studied; and (3) proteins and enzymes for the regulation of endo-

cannabinoid levels and action at receptors. However, the number of members of the endocannabinoid

system is still increasing and might soon include non-CB1 non-CB2 receptors for endocannabinoids,

endocannabinoid-related molecules with little activity at CB1and CB2receptors, and new enzymes for

the biosynthesis and degradation of these molecules. The endocannabinoid system can be described as

a pleiotropic and locally acting pro-homeostatic signalling system activated on demand following

perturbation of cell homeostasis.

Research agenda

the biosynthesis, inactivation and pharmacology of the minor endocannabinoids and of

endocannabinoid-related molecules need to be investigated

the occurrence and biological relevance of non-CB1, non-CB2, non-TRPV1 receptors for

endocannabinoids needs to be fully assessed

the mechanisms regulating cannabinoid receptor and endocannabinoid metabolic enzyme

expression during development and aging, or following pathological conditions, still need to

be investigated

the role of the endocannabinoid system in cell biology needs to be studied in more depth more selective pharmacological and biochemical tools for studies on the endocannabinoid

system need to be developed, and their action investigated in experimental models of

diseases, together with parallel studies using transgenic animals

Practice points

an endocannabinoid system, composed of G-protein-coupled receptors, their endogenous

ligands and proteins controlling ligand and receptor levels and activity, was discovered

following studies on the mechanism of action of D9-tetrahydrocannabinol, the major

psychoactive component of the hemp plantCannabis sativa

recent studies have expanded the endocannabinoid system by including targets other than

CB1 and CB2, signalling molecules other than anandamide and 2-arachidonoylglycerol, and

novel metabolic enzymes for the latter compounds; however, the biological role of these new

members is still to be ascertained

tools have been developed that target the proteins of the endocannabinoid system, and some

of these synthetic compounds have been useful to investigate the physiological and patho-

logical functions of the endocannabinoid system, and as templates for the development ofnew therapeutic drugs

the endocannabinoid system can be described as a pleiotropic but locally acting signalling

system, activated on demand following perturbation of the local homeostasis to help re-

establish the latter; the anatomical (cellular and subcellular) distribution of the proteins of

the endocannabinoid system in various organs and tissues supports this general pro-

homeostatic role

under physiological conditions the action of the endocannabinoid system is tightly regulated

in time and space; however, under some pathological conditions, this system can become

dysregulated and start contributing to disease progress and/or symptoms



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An Introduction to the Endocannabinoid System: From the Early to the Latest Concepts