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).