Endocannabinoidome

Abstract

The classical definition of the endocannabinoid system (ECS), at the turn of the century, was that of a complex pleiotropic system composed by: (1) the two cannabinoid receptors (CB1 and CB2); (2) their endogenous ligands, the ‘endocannabinoids’ (EC) and (3) the five enzymes believed at that time to be uniquely responsible for EC biosynthesis and degradation. However, studies carried out during the last 10 years have revealed the potential existence of a high degree of redundancy for both the molecular targets and metabolic routes, and corresponding enzymes, of the ECs. Therefore, these new discoveries suggested that the time had come to expand our view of the ECS. In fact, other bioactive long chain fatty acid amides were identified, and their biosynthesis, inactivation and function investigated. Since this plethora of novel mediators has something more than a few chemical features in common with ECs, the name of ‘endocannabinoidome’ (eCBome) was proposed.

Key Concepts

• The endocannabinoid system is an ever‐expanding pleiotropic signalling system with key role in several physiopathological conditions.
• Studies carried out during the last 10 years have revealed the potential existence of a high degree of redundancy for both the molecular targets and metabolic routes, and corresponding enzymes, of the endocannabinoids.
• Several N‐acyl‐ethanolamines (NAEs), monoacylglycerols, N‐acyl amino acids, N‐acyldopamines/taurines/serotonines were suggested to be part of this system.
• These endocannabinoid‐like molecules may activate other molecular targets independently from cannabinoid receptors.
• All these recent findings together with the discovery of new endocannabinoid‐like molecules have led us to expand the classical view of the eCB system and to look at it as the ‘endocannabinoidome’.

Full text

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Endocannabinoidome
Iannotti Fabio Arturo, Istituto di Chimica Biomolecolare Consiglio Nazionale
delle Ricerche, Pozzuoli, Italy
Piscitelli Fabiana, Istituto di Chimica Biomolecolare Consiglio Nazionale delle
Ricerche, Pozzuoli, Italy
Advanced article
Article Contents
•Introduction
•Redundancy and Promiscuity of eCB Metabolic
Pathways and Enzymes
•N-acyl Amino Acids
•N-acyl-dopamines and N-acyl-serotonins
•Products of the Oxidative Metabolism of eCBs
and Related Lipids
•Fatty Acid Esters of Hydroxyl Fatty Acids
•Conclusions: How to Investigate and Manage
the eCBome
Online posting date: 16th November 2018
The classical definition of the endocannabinoid sys-
tem (ECS), at the turn of the century, was that of
a complex pleiotropic system composed by: (1) the
two cannabinoid receptors (CB1and CB2); (2) their
endogenous ligands, the ‘endocannabinoids’ (EC)
and (3) the five enzymes believed at that time to
be uniquely responsible for EC biosynthesis and
degradation. However, studies carried out during
the last 10 years have revealed the potential exis-
tence of a high degree of redundancy for both the
molecular targets and metabolic routes, and cor-
responding enzymes, of the ECs. Therefore, these
new discoveries suggested that the time had come
to expand our view of the ECS. In fact, other bioac-
tive long chain fatty acid amides were identified,
and their biosynthesis, inactivation and function
investigated. Since this plethora of novel medi-
ators has something more than a few chemical
features in common with ECs, the name of ‘endo-
cannabinoidome’ (eCBome) was proposed.
Introduction
The discovery of the main psychotropic component of Cannabis
Sativa,theΔ9-tetrahydrocannabinol (THC) and the chemical
synthesis of its analogues provided the foundation for cannabis
research and the following discovery of two G-protein-coupled
receptors (GPCRs), named cannabinoid receptors. Therefore,
growing body of evidence has emerged on the role of the
eLS subject area: Biochemistry
How to cite:
Fabio Arturo, Iannotti and Fabiana, Piscitelli (November 2018)
Endocannabinoidome. In: eLS. John Wiley & Sons, Ltd:
Chichester.
DOI: 10.1002/9780470015902.a0028301
endocannabinoid system (ECS) in numerous diseases and
disorders from this research. Initially, the ECS was dened as
the endogenous lipid signalling system comprised of (1) the
two most potent endogenous agonists of cannabinoid recep-
tors, anandamide (N-arachidonoylethanolamine, AEA) and
2-arachidonoyl-glycerol (2-AG), also named endocannabi-
noids; (2) several endocannabinoid-related molecules, of which
N-oleoylethanolamine (OEA) and N-palmitoylethanolamine
(PEA) are the most studied; (3) the enzymes regulating the
endocannabinoid biosynthesis (NAPE-PLD, ABDH4, GDE1,
PTPN22 for AEA, and DAGLαand DAGLβfor 2-AG) and
degradation (FAAH for AEA, and MAGL, ABDH6, ABDH12
and FAAH for 2-AG); and (4) the two endocannabinoids
responsive GPCRs known as cannabinoid receptor of type
1 (CB1) and cannabinoid receptor of type 2 (CB2) and the
cation permeant transient receptor potential vanilloid type-1
(TRPV1) (Figure 1a,see also:Endocannabinoid System;
Endocannabinoid System: An Update;Cannabinoids and
Their Receptors). Notably, AEA and 2-AG were also shown to
have afnity for noncannabinoid receptors including GABA-A,
PPARγ, adenosine A3 and GPR55 (Baggelaar et al., 2018). The
ECS is one of the most pleiotropic signalling systems in verte-
brates by playing a key role in several aspects of the mammalian
physiology and pathology that led researchers to investigate on
it as it represents a challenging and fascinating target to develop
new therapeutic drugs (see also:Endocannabinoid System;
Endocannabinoid System: An Update;Cannabinoids and
Their Receptors). However, the ECS is more complicated than
the one described initially, at the turn of the century. In fact,
other endogenous AEA and 2-AG analogues, including other
N-acyl-ethanolamines (NAEs), monoacylglycerols, N-acyl amino
acids, N-acyl-dopamines/taurines/serotonines, were suggested
to be part of this system (Di Marzo and Wang, 2015). Mainly,
because eCBs share biosynthetic and degradative pathways with
other endogenous signals and are biosynthesised and degraded
through redundant routes (Rahman et al., 2014); moreover, they
may activate other receptors, independently from cannabinoid
receptors (De Petrocellis and Di Marzo, 2010). These nd-
ings together with the discovery of new endocannabinoid-like
molecules have led us to expand our view of the eCB system and
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Endocannabinoidome
«Old» view of the endocannabinoid system
The ever expanding «Endocannabinoidome»
Phosphatidylethanolamine (PE)
Phosphatidylethanolamine (PE)
Phosphatidic acid (PA) Phospholipid
Phosphatidic acid (PA) Phospholipid
PLCβ
PLCβ
PA phosphohydrolase
PA phosphohydrolase
sn-1-arachidonate containing
diacylglycerol
sn-1-arachidonate containing
diacylglycerol
sn-2-arachidonoyl- lysophosphatidic acid (LPA)
Kinase
Phosphatase
DAGL α/β
DAGL α/β
sn-1-arachidonate containing
phospolipid
sn-1-arachidonate containing
phospolipid
NATs
NATs
N-arachidinoyl-
phosphatidylethanolamine (PE)
N-arachidinoyl-phosphatidylethanolamine (PE)
ABDH4
ABDH4
PTPN22 NAPE-PLD GDE-1
PTPN22 NAPE-PLD GDE-1
FAAH
COX2 PMs
15-LOX
15 HAEA
FAAH
ABDH6/12
Glycerol
MAGL
Arachidonic acidArachidonic acid
N-arachidonoyl-dopamine
Ethanolamide
2-arachidonoyl-glycerol (2-AG)
Dopamine PG-GEs COX2
N
H
OH
O
N
H
OH
O
OOH
OOH
OOH
OOH
N
HCOOH
O
N
H
O
N-arachidonoyl-glycine
Glycine
N-arachidonoylethanolamine (Anandamide, AEA)
FAAH
N-arachidonoylethanolamine (Anandamide, AEA) 2-arachidonoyl-glycerol (2-AG)
Arachidonate
+
Ethanolamide
Arachidonate
+
Glycerol
MAGL
FAAH
ABDH6/12
CB1 > CB2
CB1 > CB2
Inactivation
Inactivation
Oxidation
TRPV1
TRPV1
EMT EMT
CB1 = CB2
CB1 = CB2
TRPV1
TRPV1
GPR55
GPR55
GABAA
ADENOSINE A3
PPARγ
OH
OH
OH
HO
HO
H2NOH
O
OO
OOH
POH
O
O−
(a)
(b)
Figure 1 The evolution of the endocannabinoid system from the classical description (a) to the new definition as the ‘endocannabi-
noidome’(b). ABH4/6/12, αβ-hydrolase 4/6/12; CB1/2, cannabinoid receptor 1/2; COX2, cyclooxygenase 2; DAG, diacylglycerol; EMT, ‘endocannabinoid
membrane transporter’; FAAH, fatty acid amide hydrolase; GDE1, glycerophosphodiester phosphodiesterase 1; GPR55, G-protein-coupled receptor 55;
MAGL, monoacylglycerol lipase; NAPE-PLD, N-acylphosphatidylethanolamine-selective phosphodiesterase; NATs, N-acyltransferases; PA, phosphatidic acid;
PLCβ, phospholipase Cβ; PLD, phospholipase D; PTPN22, protein tyrosine phosphatase, nonreceptor type 22; TRPV1, transient receptor potential, vanilloid
subtype 1 receptor; GABAA,type-Aγ-aminobutyric acid receptors; adenosine A3, adenosine A3 receptor; PPARγ, peroxisome proliferator-activated receptor
gamma; 15-LOX, lipoxygenase-15; PMs, prostaglandin-ethanolamides/prostamides; PG-GEs, prostaglandin-glyceryl esters.
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Endocannabinoidome
to look at it as a new ‘ome’ in its own right, the ‘endocannabi-
noidome’ (eCBome, Figure 1b) (Piscitelli et al., 2011; Iannotti
et al., 2013; Guida et al., 2018).
Redundancy and Promiscuity of
eCB Metabolic Pathways and
Enzymes
The two rst discovered and the most studied eCBs are the
AEA or anandamide and the 2-arachidonoylglycerol (2-AG)
belonging to the N-acylethanolamine (NAE) and monoacyl-
glycerol (MAGs) families, respectively. AEA and other NAEs
are biosynthesised via a phospholipid-dependent pathway
consisting of the enzymatic hydrolysis of the corresponding
N-acyl-phosphatidylethanolamines (NAPEs) (Bisogno et al.,
2005) (Figure 1). These precursors are generated by transferring
the acyl group esteried on the sn-1 position of other phos-
pholipids to the nitrogen atom of phosphatidylethanolamine,
via an as-yet unidentied Ca2+-dependent N-acyl-transferase,
although the existence of Ca2+-independent N-acyl-transferase
suggests the possibility that other enzymes are also capa-
ble of catalysing this reaction. The enzyme catalysing the
hydrolytic reaction (release of N-acylethanolamine from
N-acyl-phosphatidylethanolamines) was identied as a phos-
pholipase D selective for NAPEs (NAPE-PLD), which exhibits
catalytic properties different from other PLD enzymes. AEA is
one of the least abundant NAEs in comparison to N-stearoyl-,
N-palmitoyl- and N-oleoylethanolamine found in tissues and
cells so far (Iannotti et al., 2016) because arachidonic acid
(AA) is one of the least abundant esteried fatty acids on the
sn-1 position of phospholipids. Therefore, AEA is biosyn-
thesised together with other NAEs, usually more abundant.
On the other hand, the major pathway for the biosynthesis of
2-AG comprises the hydrolysis of diacylglycerols containing
arachidonate moiety in the 2 position (DAGs) catalysed by
DAG lipases selective for the sn-1 position (Figure 1). In this
case, the arachidonate moiety is esteried as one of the most
abundant fatty acids. However, 2-AG may be released together
with its congener 2-acylglycerols, although these are usually
less abundant. Regarding inactivation pathways, the fatty acid
amide hydrolase (FAAH) recognises as substrates all NAEs and
it also recognises as substrates other long chain fatty acid amides,
including other NAEs, fatty acid primary amides, N-acyl amino
acids and N-acyltaurines (Saghatelian et al., 2004; Mulder and
Cravatt, 2006). In some cases, FAAH is also responsible for
2-AG hydrolysis to AA and glycerol (Bisogno et al., 2002), and
likewise, monoacylglycerol lipase (MAGL) is nonselective for
2-AG but can hydrolyse also other MAGs. Although, MAGL is
the major enzyme responsible for 2-AG degradation in the brain,
there are other two enzymes, the α/β-hydrolase-6 (ABDH6) and
the α/β-hydrolase-12 (ABDH12), that can inactivate this eCB,
unselectively (Blankman et al., 2007) (Figure 1). The presence
of an arachidonate moiety in both AEA and 2-AG raised the
possibility that these molecules might function also as substrate
for the same enzymes implicated in AA metabolism. These
include the cyclooxygenase-2 (COX-2) (Kozak and Marnett,
2002), the lipoxygenases-12 and 15 (LOX-12, LOX-15) (Kozak
and Marnett, 2002) and cytochrome P450 oxygenases (Kozak
and Marnett, 2002), known to be involved in eicosanoid pro-
duction from AA (Figure 1b). However, these enzymes can
nevertheless metabolise other members of the two families,
for example the ω-3 and ω-6 congeners, which are enriched
following certain diets. Moreover, AEA and 2-AG can be pro-
duced from, and/or catabolised to, other lipid mediators, i.e.
sn-2-arachidonoyl-lysophosphatidic acid (LPA) (Fukushima and
Chun, 2001), which acts preferentially on specic GPCRs for
LPAs, can act as both biosynthetic precursor and metabolic prod-
uct for 2-AG (Nakane et al., 2002) (Figure 1b). These evidences
suggested that great care must be taken when manipulating
pharmacologically the levels of eCB anabolic and catabolic
enzymes. Future studies have to include not only, the observa-
tion of the consequent phenotypes, but also by the quantitative
analysis of the tissue levels of both the eCBs and their related
mediators.
N-acyl Amino Acids
N-acyl amino acids encompass a large family of recently
discovered lipids mediators whose importance has still not
been fully elucidated. Among them, N-arachidonoyl-l-serine;
N-arachidonoyl glycine; N-arachidonoyl taurine, N-oleoyl serine
and N-oleoyl glycine are the most studied ones.
N-arachidonoyl-l-serine (ARA-S) was isolated for the rst time
from bovine brain in 2006. In spite of its structural afnity with
AEA (Figure 2), ARA-S shows very low binding afnity to
CB1 (<1% of that of AEA) and does not bind CB2 or TRPV1
receptors (Piscitelli and Bradshaw, 2017). Surprisingly, at least
some of the biological effects exerted by ARA-S resemble those
of AEA and counteracted by cannabinoid receptors antagonists
(Kino et al., 2016). To this regard, there are two sequential studies
demonstrating that ARA-S exerts neuroprotective effects follow-
ing the traumatic brain injury, and this action is dependent on
TRPV1, but also on CB2 and CB1 receptors and BK K+chan-
nels (Cohen-Yeshurun et al., 2011; Cohen-Yeshurun et al., 2013).
In addition, Milman and colleagues demonstrated that ARA-S
shows similar effects to abnormal cannabidiol (Abn-CBD), a syn-
thetic analogous of cannabidiol, which acts as agonist of the third
putative cannabinoid receptors (GPR18) producing vasodilator
effects, lowers blood pressure, and induces cell migration, prolif-
eration and mitogen-activated protein kinase (MAPK) activation
in microglia (Piscitelli and Bradshaw, 2017). The biological role
of N-oleoyl serine (Figure 2) is still mostly unknown except for
its role in the bone remodelling by promoting the osteoblast cells
proliferation (Smoum et al., 2010).
N-arachidonoyl glycine (NAGly) is a carboxylic derivative of
anandamide (Figure 2). In 2002, (Burstein et al., 2002) demon-
strated that NAGly may serve as an endogenous enhancer of
tissue anandamide concentrations in vivo.However,thiseffect
was not reproduced in vitro (Piscitelli and Bradshaw, 2017). The
orphan receptors GPR18, GPR72, GPR92 represent the most well
targets of NAGly (Piscitelli and Bradshaw, 2017). Its endoge-
nous production may occur through two proposed distinct biosyn-
thetic pathways: (1) The rst is via an enzymatically regulated
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N-oleoyl-taurine
N-arachidonoyl-L-serine (ARA-S)
N-acyl-aminoacids
N-oleoyl-glycine (OlGly)
N
H
O
OH
O
N-oleoyl-serine
N
H
OCOOH
OH
R=Alkyl chain
R1=Amino acids
O
N
H
R1
OH
R
O
N
H
OCOOH
OH
N-arachidonoyl-glycine (NAGly)
N
H
O
OH
O
N-arachidonoyl-taurine
N
H
O
S OH
O
O
N
H
O
SOH
O
O
Figure 2 Chemical structures of different N-acyl amino acids.
conjugation between the AA and glycine, in a FAAH-dependent
reaction. (2) The second one is mediated by an alcohol dehydro-
genase that produces NAGly as an oxidative metabolite of AEA
(Bradshaw et al., 2009). In mammalian, the highest concentra-
tion of NAGly has been found in the spinal cord and brain. The
biological activity of NAGly is still poorly understood.
Nevertheless, it has been shown by different research groups
that NAGly plays an important antinociceptive and antiinam-
matory role in vivo. However, the exact mechanism of action
through which NAGly exerts these effects remains unknown. The
afnity for the cannabinoid receptors and TRPV1 is very low
(Sheskin et al., 1997; Huang et al., 2002; Succar et al., 2007;
Vuong et al., 2008). Another N-acyl glycine that has been most
characterised is N-oleoyl glycine (OLGly) (Figure 2). As for
other N-acyl glycines, OLGLy was detected in partially puried
rat brain lipid extracts using liquid chromatography combined
with tandem mass spectrometry (LC/MS/MS) Besides the brain,
OLGLy was detected in peripheral organs and tissues including
skin, lung, spinal cord, ovaries, kidney, liver and spleen (Piscitelli
and Bradshaw, 2017). OLGly was shown to regulate body tem-
perature and locomotion in rats (Chaturvedi et al., 2006). Most
recent evidences demonstrated that OLGLy promote adipogene-
sis murine 3 T3-L1 cells through the activation of CB1 receptor
and the enhancement of insulin-mediated Akt signalling path-
way (Wang et al., 2015). In addition, OlGly was demonstrated
to reduce the nicotine reward and withdraw by directly activat-
ing peroxisome proliferator-activated receptor alpha (PPAR-α)
(Donvito et al., 2018).
N-acyl taurines (Figure 2) were discovered by Saghatelian
and Cravatt (2005) that identied them in the liver and kid-
neys of FAAH(−/−) mice using a large scale mass spectrometry
lipidomics analysis (Saghatelian and Cravatt, 2005). In a subse-
quent study, the same authors demonstrated that NATs are reg-
ulated by FAAH and activate multiple members of the transient
receptor potential (TRP) family of calcium channels, including
TRPV1 and TRPV4 (Saghatelian et al., 2006).
More recently, N-arachidonoyl taurine and N-oleoyl taurine
(Figure 2) were demonstrated to possess signicant antipro-
liferative effects in human prostate cancer cells (Chatzakos
et al., 2012). Whilst, in pancreatic β-cell lines (HIT-T15) and
rat islet cell lines (INS-1), N-arachidonoyl-taurines and N-oleoyl
taurine resulted in a signicant increase in insulin secretion
(Waluk et al., 2013), which was then demonstrated by others
to imbalance the insulin homeostasis favouring type 2 diabetes
(Aichler et al., 2017). Finally, Sasso et al. (2016) demonstrated
that NATs are novel targets to accelerate the skin repair and
enhance healing-associated responses in human keratinocytes
and broblasts, through a molecular mechanism that involves the
epidermal growth factor receptor (EGFR) and TRPV-1. Finally,
in a recent studies, it has been demonstrated that other N-acyl
amino acids including N-docosahexaenoyl-, N-arachidonoyl-
and N-linoleoyl-GABA, N-docosahexaenoyl-serine,
N-docosahexaenoyl-glycine and N-docosahexaenoyl-aspartic
acid (DAsA), as agonists; and N-docosahexaenoyl proline, bind
TRPV1 channels as an antagonist (Piscitelli and Bradshaw,
2017).
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Endocannabinoidome
N
H
O
N-palmitoyl-dopamine
(PALDA)
OH
OH N
H
O
N-stearoyl-dopamine
(STEARDA)
OH
OH
N
H
O
N-oleoyl-dopamine
(OLDA)
OH
OH
N
H
O
OH
OH
N-arachidonoyl-dopamine
(NADA)
OH
OH
N
H
R
O
N-acyl-dopamines
R=Alkyl chain
Figure 3 Chemical structures of different N-acyldopamines.
N-acyl-dopamines and
N-acyl-serotonins
N-arachidonoyl-dopamine (NADA),
N-Oleoyl dopamine (OLDA), N-palmitoyl
dopamine (PALDA) and stearoyl
dopamine (STEARDA)
Among long-chain N-acyl dopamines, the N-arachidonoyl-
dopamine (NADA, Figure 3) is the most studied due to of its
well-characterised activity at TRPV1 and CB1 receptors and its
ability to inhibit T-type Ca2+channels (Piscitelli and Bradshaw,
2017). The metabolic pathways of NADA as well as of other
N-acyl dopamines have not been fully claried yet. So far, three
alternative biosynthetic pathways have been hypothesised or
demonstrated. First mechanism consists of the conversion by
aromatic l-amino acid hydroxylase of N-arachidonoyl tyrosine
(NA-Tyr) to N-arachidonoyl-L-DOPA, which is subsequently
converted to NADA by aromatic l-amino acid decarboxylase,
although it remains to be established how NA-Tyr is biosyn-
thesised. The second mechanism, supported by the fact that
dopamine seems to be necessary to observe the presence of
NADA, consists instead in the direct condensation of AA
with dopamine (Hu et al., 2009). Alternatively, an enzyme
recently identied in Drosophila melanogaster, arylalkylamine
N-acyltransferase-like 2, catalyses the transfer of fatty acid
chains to the amine group of dopamine (Dempsey et al., 2014).
However, no mammalian orthologue of this enzyme has been
identied yet. Also, the mechanisms of NADA catabolism are
largely unknown. NADA was identied for the rst time in
the bovine brain where it appeared particularly abundant in the
striatum, hippocampus and cerebellum. In contrast, lower levels
of NADA were detected in rat dorsal root ganglia (Huang et al.,
2002). More recent ndings showed that NADA is specically
expressed in the cell bodies and terminals of dopaminergic
neurons. Furthermore, NADA modulates GABAergic neuro-
transmission via a mechanism dependent on both CB1 and
TRPV1 activation (Piscitelli and Bradshaw, 2017). Interest-
ingly, as with the endocannabinoids AEA and 2-AG, NADA is
produced ‘on demand’, responding to specic stimuli.
In C6 glioma cells, NADA is rapidly taken up by the putative
endocannabinoid membrane transporter and it is slowly hydrol-
ysed by FAAH to AA and dopamine. However, the afnity of
FAAH for NADA appears much lower than that for AEA, and
there is still no evidence of whether the manipulation of FAAH
activity affects NADA levels. On the other hand, NADA is a
substrate for O-catecholamine-methyl-transferase (COMT), the
enzyme involved in catecholamine degradation. COMT converts
NADA to O-methyl-NADA, that in turn possesses a potency at
least 100 times lower than that of NADA at activating TRPV1
(Huang et al., 2002; Almasi et al., 2008). NADA competes with
[14C]AEA for the putative endocannabinoid membrane trans-
porter (Ki =17.3 ±7.3 μM; compared with Ki =11.7 ±1.0 μM
for anandamide inhibition of [14C]anandamide uptake) (Chu
et al., 2003). This has led to suggest that also NADA can
be taken up by cells through the same mechanisms used for
endocannabinoids.
Therefore, due to its peculiarity to activate both CB1 and
TRPV1, NADA has been proposed to play a role as an endo-
cannabinoid and as the rst endovanilloid with a structure and
potency similar to capsaicin. However, the mechanism through
which NADA activates CB1 is still not entirely clear. In summary,
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N
H
ONH
OH
N-palmitoyl-serotonin
(PA-5-HT)
N
H
ONH
OHN-arachidonoyl-serotonin
(AA-5-HT)
RN
H
ONH
OH
N-acyl-serotonins
N
H
ONH
OH
N-oleoyl-serotonin
(OA-5-HT)
N
H
ONH
OH
N-stearoyl-serotonin
R=Alkyl chain
Figure 4 Chemical structures of different N-acylserotonins.
taken together, these studies indicate that NADA behaves as either
a TRPV1 or CB1 agonist depending on its tissue concentration,
and, therefore like AEA, it is believed to be both an endocannabi-
noid and an endovanilloid.
N-Oleoyl dopamine (OLDA, Figure 3) is structurally similar to
NADA, and, although never investigated, its biosynthetic path-
way is likely similar to that of the latter compound. Detectable
levels of this compound, and higher than those of NADA, are
found in the brain (Huang et al., 2002; Chu et al., 2003). In
a recent study, Zajac and colleagues provided evidence for a
dopamine-like catabolic pathway of OLDA, revealing that OLDA
is methylated by COMT leading to O-methylated products (Zajac
et al., 2014). OLDA shows afnity for the putative endocannabi-
noid membrane transporter in RBL-2H3 cells while being a poor
substrate for FAAH (Chu et al., 2003).
Unlike NADA, OLDA, whilst being an agonist at TRPV1,
shows very low afnity for CB1. In contrast, two other endoge-
nous N-acyldopamines, PALDA and STEARDA (Figure 3), were
found to be inactive per se on TRPV1. However, both these com-
pounds enhanced the effects of NADA on TRPV1 through an
‘entourage’ mechanism comparable to that previously observed
for PEA on AEA and 2-AG (Iannotti et al., 2016).
N-acyl-serotonins
N-arachidonoyl-serotonin (AA-5-HT, Figure 4) was origi-
nally synthetised as a FAAH inhibitor (Bisogno et al., 1998)
and has been shown to increase AEA levels both at periph-
eral and central sites in vivo (Capasso et al., 2005; de Lago
et al., 2005). Subsequently, AA-5-HT was found to act as a
competitive antagonist of TRPV1 receptors, and to be quite
effective, also through this mechanism, in models of both inam-
matory and neuropathic pain (Maione et al., 2007) and/or in
the treatment of anxiety and stress-related disorders (Micale
et al., 2009; Navarria et al., 2014). Only in 2011, however,
Verhoeckx et al. (2011) demonstrated that AA-5H-T and other
N-acyl-serotonins (N-oleoyl-serotonin, N-palmitoyl-serotonin
and N-stearoyl-serotonin; Figure 4) are present in the ileum and
jejunum of the gastrointestinal tract of pigs and mice, where they
act to regulate the glucagon-like peptide-1 (GLP-1) secretion.
Thus, AA-5-HT was the rst endogenous antagonist of TRPV1
to be identied, a function only recently extended to oleic acid
at ten-fold higher concentrations (Morales-Lazaro et al., 2016).
Indeed, N-oleoyl-serotonin (OA-5-HT), which is also found in
the pig and mouse gastrointestinal tract (Verhoeckx et al., 2011),
antagonises TRPV1 at micromolar concentrations, similar to
those needed to oleic acid to exert the same effect (Ortar et al.,
2007; Morales-Lazaro et al., 2016). Very recently, we showed
that the levels of AA-5HT and OA-5-HT were signicantly
reduced in the small intestine in a mouse model of dysbiosis
and these alterations might be partly responsible for the effects
of antibiotics and the ensuing dysbiosis on CNS function and
subsequent depression-like signs in mice (Guida et al., 2018).
Subsequent administration of a probiotic counteracted the
antibiotic-induced behavioural and CNS functional alterations
and concomitantly restored near-physiological levels of intestinal
N-acyl-serotonins (Guida et al., 2018).
Products of the Oxidative
Metabolism of eCBs and Related
Lipids
AA is a substrate of several oxidising agents, as cytochromes
P-450, lipoxygenases (LOX) and cyclooxygenases (COX)
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Endocannabinoidome
HO
O
O
OH
HO
O
OH
PGE2-glyceryl ester (PGE2-GE)
O
OH
O
OH
HO
O
OH
PGD2-glyceryl ester (PGD2-GE)
HO
OH
NOH
O
OH
PGE2 ethanolamide (PME2)
O
O
R2
O
OH
COX-2 derivatives
OH
OH
-O
R=Alkyl chain
R2=-H-CH2-NCH2-OH or
O
HO H
NOH
O
OH
PGD2 ethanolamide (PMD2)
HO
OH H
NOH
O
OH
PGF2α ethanolamide (PMF2α)
HO
OH
O
OH
HO
O
OH
PGF2α-glyceryl ester (PGF2α-GE)
OO
OH
O
OO
OH
O
Palmitic acid-hydroxy palmitic acid (PAHPA)
OO
OH
O
Oleic acid-hydroxy stearic acid (OAHSA)
Palmitic acid-hydroxy stearic acid (PAHSA)
OO
OH
O
PalmitOleic acid-hydroxy stearic acid (POHSA)
(a)
(b)
Figure 5 Chemical structures of different COX-2 derivatives (a) and fatty acid esters of hydroxy fatty acids (b).
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Endocannabinoidome
that, respectively, introduce one atom, one molecule and two
molecules of O2in the carbon framework. The presence of
the arachidonoyl group in both 2-AG and AEA makes them
likely to be oxidised by the same enzymes implicated in the AA
metabolism and producing metabolites analogous to derivatives
produced by AA except for the presence of the glycerol ester
or ethanolamide moiety (Kozak and Marnett, 2002). The most
interesting oxidative products are those produced by COX-2 for
their biological and pharmacological effects.
COX-2 oxidises 2-AG and AEA, whereas they are oxidised by
COX-1 to a much less extent (see also:Cyclooxygenase-2: Biol-
ogy of Prostanoid Biosynthesis and Metabolism). They are con-
verted into prostaglandin endoperoxides containing ethanolamide
or glycerol functionalities and the most studied are the glyc-
erol esters and ethanolamide of PGE2, PGD2, PGF2a and PGI2
(Kozak and Marnett, 2002) (Figure 5a).COX-2isalsoable
to oxidise NAGly, NAla, NAGABA and NADA (Kozak and
Marnett, 2002).
It is noteworthy, however, that the biological signicance of
the aforementioned lipoxydative metabolism is still unclear and
needs to be further investigated. The more simplistic explanation
would be to classify these pathways just as alternative endo-
cannabinoid inactivation modalities. Another, more intriguing
possibility, would be to consider EC oxidation as an unusual
activation pathway due to the fact that these reactions lead the pro-
duction of cannabimimetic-derivatives with an enhanced stability
compared to their parental endocannabinoids. Moreover, given
that the two main endocannabinoids undergo to different oxida-
tive transformations, it might be possible consider that these oxy-
genated products represent unique signal mediators with potent
activities distinct from their cannabimimetic precursors or, addi-
tionally, that the oxidised endocannabinoids may act as prodrugs
since these products retain amide or ester functionalities.
Fatty Acid Esters of Hydroxyl Fatty
Acids
The existence of branched fatty acid esters of hydroxy fatty acids
(FAHFAs) was revealed for the rst time by Yore et al. (2014) in
serum and many tissues in humans and rodents and was found to
be associated with type 2 diabetes. In particular, they were able
to identify 16 FAHFA family members by using an untargeted
lipidomics approach, including palmitic acid-hydroxy palmitic
acid (PAHPA), oleic acid-hydroxy stearic acid (OAHSA), palmi-
toleic acid-hydroxy stearic acid (POHSA) (Figure 5b) and oleic
acid-hydroxy palmitic acid (OAHPA). In 2015, an in silico tan-
dem MS/MS library was developed to allow users to anno-
tate FAHFAs from accurate tandem mass spectra easily and the
method was validate by identied new FAHFAs species in egg
yolk (Ma et al., 2015). Lee et al. (2016) demonstrated that a
specic family of FAHFA, the branched palmitic acid esters of
hydroxystearic acids (PAHSAs, Figure 5b), has benecial effects
in murine colitis by exerting antiinammatory effects partially
regulated by GPR120 (Lee et al., 2016). Very recently, a new
study clearly demonstrated that chronic treatment of chow-fed
and HFD-mice with PAHSAs enhances insulin sensitivity and
glucose tolerance and reduces adipose inammation (Syed et al.,
2018). Moreover, these effects were GPR40-mediated (Syed
et al., 2018).
Conclusions: How to Investigate
and Manage the eCBome
Given the redundancy and promiscuity of eCB metabolic path-
ways and enzymes described above, the use of canonical pharma-
cological tools might not be sufcient to completely distinguish
endocannabinoid functions from other endocannabionoid-related
molecules. Also, the development of new therapeutic drugs to
treat disorders in which endocannabinoids play a key role might
be challenging if this complexity is not taken into account. In
summary, the eCBome opens new questions:
1. With so many organ, cellular and molecular targets, how
do we know if and in what direction the eCB system is
dysregulated during a specic disease, and how can we
predict the consequences of such dysregulation?
2. With so many biosynthetic enzymes, how do we know which
one we need to inhibit in order to reduce eCB tone, and
how do we cope with possible compensatory effects of such
redundancy?
3. With so many eCB-like mediators being either produced
together, or degraded by the same enzyme as, the eCBs, and
so many potential targets being concomitantly dysregulated,
how do we know that a dysfunctional production or inactiva-
tion of eCBs is really concurring to a specic disease state,
and how do we decide that it is really the case to intervene
with?
4. How do we make sure that the intervention is really acting
via the correction of the eCB system?
The answer to all these questions is just one: through the tar-
geted proling of the transcriptional, proteic and metabolic output
of the eCBome in various tissues and organs involved in the
pathology. This methodology, called ‘endocannabinoidomics’,
and extensively reviewed elsewhere (Piscitelli and Bradshaw;
Piscitelli; Bisogno Piscitelli and Di Marzo), could allow us to
shed new light into the study of this complex system and could lay
the foundations for the development of novel cannabinoid-based
drugs.
Glossary
Endocannabinoid system A complex signalling system
composed by the two cannabinoid receptors (CB1 and CB2),
their endogenous ligands, named endocannabinoids, and the
large plethora of enzymes regulating the metabolism of these
latter.
Endocannabinoidome The plethora of novel identied
molecules that have chemical and functional similarity with
classical endocannabinoids and that can affect with their
activity.
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N-acylaminoacids Class of endogenous fatty acids
characterized by a fatty acyl group linked to a primary amine
metabolite by an amide bond.
N-acyl-dopamines Class of endogenous fatty acids
characterized by a fatty acyl group linked to dopamine.
N-acyl-serotonins Class of endogenous fatty acids
characterized by a fatty acyl group linked to serotonin.
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