2.1 Endogenous cannabinoid ligands, their synthesis and
inactivation
Endocannabinoids are N-acylethanolamines synthesized from membrane
lipids in response to specific signals such as inflammatory signals
(Barrie and Manolios, 2017; Katona and Freund, 2012). Of these
endogenous signaling molecules, anandamide and 2-arachidonoylglycerol
(2-AG) represent the two most investigated ligands (Devane et al., 1992;
Mechoulam et al., 1995). Anandamide (arachidonoyl ethanolamine, AEA) was
initially isolated from porcine brain, and so named due to its ability
to increase motivation and pleasure (derived from Ᾱnanda, a Sanskrit
word for bliss) (Monteleone et al., 2015). It binds to both
CB1 and CB2 receptors and is responsible
for maintaining basal endocannabinoid signaling. This alludes to the
capacity of anandamide to increase motivation and pleasure. On the other
hand, 2-AG, initially isolated from canine intestines, binds to
CB1 and CB2 with greater affinity than
anandamide since it functions as a full agonist for these receptors
(Sugiura et al., 2000; Argenziano et al., 2019). In addition to these
ligands, a number of other
biochemically similar endocannabinoids, including virodhamine, 2-AG
ether and N-arachidonoyl dopamine have been identified. Knowledge on
their exact biological functions is, however, yet to be fully elucidated
(Argenziano et al., 2019). The chemical structures of most common
endocannabinoids are presented in Figure 1. For illustration purposes,
figure 1 also presents the chemical structures of synthetic analogs and
phytocannabinoids.
Although anandamide and 2-AG are both lipid molecules synthesized from
the breakdown of arachidonic acid (AA) liberated from the cell membrane,
their biosynthetic pathways are largely dissimilar (Argenziano et al.,
2019). N-acyltransferase catalyses the transfer of AA from the sn-1
position of a donor phospholipid to the primary amine
phosphatidylethanolamine, resulting in the formation ofN -arachidonoylphosphatidylethanolamine (NAPE) (Sharke and Wiley,
2016). NAPE is then hydrolysed byN -acylphosphatidylethanolamine-hydrolyzing phospholipase D to
yield anandamide (Di Marzo et al., 1994). Other biosynthetic pathways
for the production of anandamide such as sequential O-deacylation ofN -arachidonoylphosphatidylethanolamine by the lyso (N-acyl
phosphatidylethanolamine)-lipase α-β hydrolase 4 and cleavage of
the phosphodiester bond by the glycerophosphodiesterase GDE1 (Simon and
Cravatt, 2006; 2008), direct liberation by N-acyl
phosphatidylethanolamine-selective phospholipase D enzyme (Okamoto et
al., 2004), among others, have been suggested and also reviewed
extensively by Blankman and Cravatt, 2013. During the synthesis of 2-AG,
phosphoinositol PLC β is first activated leading to hydrolyses of
arachidonoyl phosphatidylinositol 4,5-bisphosphate (PIP2) species at the
sn-2 position with subsequent production of diacylglycerol
(Hashimotodani et al., 2005). Diacylglycerol is then hydrolysed by
sn-1-selective diacylglycerol lipases-α and -β
(DAGLα and -β) resulting in the
production of 2-AG (Ledent et al., 1999). The biosynthetic pathways for
endocannabinoids are summarized in figure 1.
Unlike most hormones and other neurotransmitters, endocannabinoids are
believed not to be transported into vesicles for storage following
synthesis due to their hydrophobicity (Katona and Freund, 2012). Rather,
they are thought to be mobilized in a process referred to as “on
demand” biogenesis, where endocannabinoids are liberated from membrane
phospholipid precursors and/or storage sites in an activity-dependent
manner (Min et al., 2010; Alger and Kim, 2011). Upon release, these
endocannabinoids diffuse, and regulate the release of multiple
presynaptic messengers by acting locally as retrograde messengers
(Barrie and Manolios, 2017). Soon after cellular uptake from the
synaptic space, these endogenous molecules are inactivated by specific
enzymes within the intracellular environment. For instance, in the
nervous system, 2-AG and anandamide are inactivated primarily by the
serine hydrolase enzymes monoacylglycerol lipase (MAGL) and fatty acid
amide hydrolase (FAAH), respectively (Burstein and Zurier, 2009;
Blankman and Cravatt, 2013). Although 2-AG is mostly inactivated by
MAGL, α/β hydrolase domain-containing protein-6 and -12 are known to
account for about 15% of the enzyme degradation (Blankman et al.,
2007). In addition to the enzymatic transformation, cyclooxygenase-2
(COX2) also oxygenates 2-AG to form biologically active prostaglandin
glyceryl esters, involved in the regulation of inflammation (Hermanson
et al., 2014; Alhouayek and Muccioli, 2014). Enzymatic inactivation of
anandamide yields arachidonic acid and ethanolamine while the
degradation of 2-AG by respective enzyme yields glycerol as shown in
figure 2 (Wang and Ueda, 2009).