ReviewThe endocannabinoid signalling system: Biochemical aspects
Introduction
The finding, in the early 1990s, of specific G-protein-coupled receptors for the psychoactive component of Cannabis sativa (−)-Δ9-tetrahydrocannabinol (THC) (Gaoni and Mechoulam, 1971), led to the discovery of a whole endogenous signaling system now known as the endocannabinoid system. This consists of the cannabinoid receptors, endocannabinoids and the proteins for their synthesis and inactivation.
Cannabinoid receptors are seven-transmembrane-domain proteins coupled to G-proteins of the Gi/o type. Mammalian tissues contain at least two types of cannabinoid receptors, CB1 and CB2. CB1 receptors, cloned in 1992, are mostly expressed in the central nervous system but also in most peripheral tissues including immune cells, the reproductive system, the gastrointestinal tract and the lung, while CB2 receptors, cloned in 1993, are most abundant in the immune system, i.e. in tonsils, spleen, macrophages and lymphocytes (B-cells and natural killer cells) (Devane et al., 1988, Matsuda et al., 1990, Munro et al., 1993). Inside the brain, CB1 distribution accounts for the pharmacological properties reported for THC and psychotropic cannabinoids. CB1 and CB2 receptors share 44% overall identity and 68% identity within the transmembrane domains. Both receptors are coupled to pertussis toxin-sensitive inhibition of cAMP formation, implicating Gi/o proteins as transducers, and to stimulation of p42/p44 mitogen-activated protein kinase activity (Vogel et al., 1993, Bouaboula et al., 1995). CB1, but not CB2, receptors signal also via ion channels by inhibiting N- and P/Q-type calcium channels and by activating A-type and inwardly rectifying potassium channels (Mackie and Hille, 1992, Mackie et al., 1995, McAllister et al., 1999). Furthermore, CB1 activation stimulates phosphatidylinositol 3-kinase and protein kinase B (Gomez del Pulgar et al., 2000, Molina-Holgado et al., 2002).
By definition, endocannabinoids are endogenous compounds capable of binding to and functionally activating these two receptors (Di Marzo and Fontana, 1995). Anandamide (AEA), the first endogenous ligand to be reported at the end of 1992, is the amide between arachidonic acid and ethanolamine, and it acts as a partial CB1 agonist (Devane et al., 1992) and only as a weak CB2 agonist (Fig. 1). This compound belongs to the family of the N-acyl-ethanolamines (NAEs) already known for their pharmacological properties; other members of this family, such as homo-γ-linolenylethanolamide (HEA) and docosatetraenylethanolamide (DEA), are produced by neurons and bind to CB1 receptors. In the past 10 years, other endocannabinoids, all derived from arachidonic acid, were identified. First came the finding of 2-arachidonoylglycerol (2-AG), the arachidonate ester of glycerol, which activates both CB1 and CB2 receptors (Mechoulam et al., 1995, Sugiura et al., 1995), and, more recently, 2-arachidonyl-glyceryl ether (noladin, 2-AGE), a selective CB1 agonist, O-arachidonoyl-ethanolamine (virhodamine, OAE), a partial CB2 agonist and a CB1 antagonist, and N-arachidonoyl-dopamine (NADA), a selective CB1 agonist and a potent agonist of vanilloid receptors, were discovered (Hanus et al., 2001, Porter et al., 2002, Bisogno et al., 2000, Huang et al., 2002) (Fig. 1). While the physiological role of virhodamine, NADA and 2-AGE has not been clarified yet, the endocannabinoids AEA and 2-AG, since their finding, have been implicated in a wide range of physiological and pathological processes. The full characterization of most of the proteins involved in AEA and 2-AG metabolism, i.e. of the enzymes responsible of their biosynthesis and degradation, will open a new area of research aimed at developing potential therapeutic strategies for the pharmacological treatment of diseases in which the endocannabinoid system seems to be involved. The purpose of this article is to overview the endocannabinoid signalling system in order to provide information as complete and as updated as possible regarding its biochemical aspects.
Section snippets
Biosynthesis of endocannabinoids
AEA and 2-AG are not stored in resting cells but, unlike other mediators, they are synthesized and released only “on demand”, i.e. when and where necessary, following physiological or pathological stimuli, in a way depending upon Ca2+-dependent phospholipid remodeling (Di Marzo and Deutsch, 1998). Furthermore, the synthesis of AEA and 2-AG is associated with the formation of non-cannabimimetic, or weakly cannabinoid receptor active, related compounds, i.e. of cannabinoid receptor-inactive N
Biosynthesis of AEA
The family of the N-acyl-ethanolamines (NAEs), to which AEA belongs, has been long investigated before the identification of AEA, and these studies led to the conclusion that those compounds are biosynthesized via a phospholipid-dependent pathway consisting of the enzymatic hydrolysis of the corresponding N-acyl-phosphatidylethanolamines (NAPEs) (Schmid et al., 1983, Schmid et al., 1990, Schmid et al., 1996, Schmid and Berdyshev, 2002, Hansen et al., 1998). The enzyme catalyzing this reaction
Biosynthesis of 2-arachidonoylglycerol
In unstimulated tissues and cells the levels of 2-AG are higher than those of AEA, although they are probably overestimated due, for example, to the rapid increase of 2-AG formation that follows rat decapitation (Sugiura et al., 2001). This simple observation suggests that only a part of 2-AG found in tissues is used to activate cannabinoid receptors. In fact this endocannabinoid is an important precursor and/or degradation product of phospho-, di- and tri-glyceride pathways. Several stimuli
Inactivation of endocannabinoids
The endocannabinoids, as any other endogenous mediator of physiological and pathological responses, need mechanisms for their rapid removal from their molecular targets and subsequent degradation. Because they are lipophilic compounds, the endocannabinoids can diffuse through the cell membrane. In order to be rapid, selective and subject to regulation, the diffusion of the endocannabinoids through the plasma membrane needs to be facilitated by a carrier or to be driven by a mechanism capable of
Hydrolysis
Once inside the cell, the endocannabinoids are degraded through mechanisms depending on their chemical nature (Fig. 4). One enzyme, FAAH, has been identified as mostly responsible of AEA and, in some cases, 2-AG hydrolysis to arachidonic acid and ethanolamine or glycerol, respectively (Cravatt et al., 1996, Cravatt and Lichtman, 2002, Bisogno et al., 2002). FAAH was originally purified and cloned from rat liver, and catalyzes the hydrolysis also of long chain primary fatty acid amides and
Other mechanisms of inactivation
Ethanolamine, arachidonic acid and glycerol, the hydrolysis products of AEA and 2-AG, are recycled into membrane phospholipids in order to be used again, at least in part, in the biosynthetic pathways of the two endocannabinoids. Furthermore 2-AG, unlike AEA, can be re-esterified into phospholipids also before being enzymatically hydrolyzed, and this re-esterification occurs through mechanisms involving phosphorylation or acylation of its hydroxyl groups (Sugiura et al., 2002). This metabolic
Inhibitors
The knowledge of the mechanisms underlying the biosynthesis and inactivation of the endocannabinoids contributed to a better understanding of the effects mediated by cannabinoid receptors when they are activated by their endogenous ligands, and opened the way to the hypothesis that compounds able to regulate endocannabinoid metabolism might become potential therapeutic agents for the treatment of diseases where the endocannabinoid system is involved. Since AEA and 2-AG biosynthetic enzymes have
Other molecular targets for the endocannabinoids
Although great progress has been made towards the understanding of the biochemical and molecular mechanisms that underlie to the actions of the endocannabinoids, several findings suggest that those compounds may act also on non-cannabinoid receptor targets. First of all, pharmacological and biochemical data suggest the existence of non-CB1 non-CB2 receptors activated in vitro by physiologically relevant concentrations of AEA (Di Marzo et al., 2002c, Pertwee, 2004). The first example of such
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