More surprises lying ahead. The endocannabinoids keep us guessing

Abstract

The objective of this review is to point out some important facts that we don't know about endogenous cannabinoids - lipid-derived signaling molecules that activate CB1 cannabinoid receptors and play key roles in motivation, emotion and energy balance. The first endocannabinoid substance to be discovered, anandamide, was isolated from brain tissue in 1992. Research has shown that this molecule is a bona fide brain neurotransmitter involved in the regulation of stress responses and pain, but the molecular mechanisms that govern its formation and the neural pathways in which it is employed are still unknown. There is a general consensus that enzyme-mediated cleavage, catalyzed by fatty acid amide hydrolase (FAAH), terminates the biological actions of anandamide, but there are many reasons to believe that other as-yet-unidentified proteins are also involved in this process. We have made significant headway in understanding the second arrived in the endocannabinoid family, 2-arachidonoyl-sn-glycerol (2-AG), which was discovered three years after anandamide. Researchers have established some of the key molecular players involved in 2-AG formation and deactivation, localized them to specific synaptic components, and showed that their assembly into a multi-molecular protein complex (termed the '2-AG signalosome') allows 2-AG to act as a retrograde messenger at excitatory synapses of the brain. Basic questions that remain to be answered pertain to the exact molecular composition of the 2-AG signalosome, its regulation by neural activity and its potential role in the actions of drugs of abuse such as Δ(9)-THC and cocaine. This article is part of a Special Issue entitled 'NIDA 40th Anniversary Issue'.

Keywords: Anandamide; Cannabinoid receptor; Cocaine; Drugs of abuse; Endocannabinoid.

Figure 1. Formation and deactivation of 2-AG in brain neurons

Receptor-operated phospholipase C-β (PLC-β) converts phosphatidylinositol-4,5-bisphosphate (PIP₂) into 1,2-diacylglycerol (DAG). DAG is hydrolyzed by diacylglycerol lipase-α (DGL-α) forming 2-AG. 2-AG is subjected to hydrolytic cleavage catalyzed by either monoacylglycerol lipase (MGL) or α/β hydrolase domain-containing protein 6 (ABHD-6). Additionally, 2-AG can be dioxygenated by cyclooxygenase-2 (Cox-2) to yield a family of non-endocannabinoid prostaglandin (PG) glyceryl esters. Free arachidonic acid (AA) is converted into the eicosanoid family of compounds, for example prostacyclin (PGI₂), by cyclooxygenase or lipoxygenase enzymes.

Figure 2. Formation and deactivation of anandamide in brain neurons

The ‘canonical route’ of anandamide biosynthesis is shown in the center. According to this model, anandamide is released by hydrolysis of the phospholipid precursor, N-arachidonoyl-phosphatidylethanolamine (N-arachidonoyl-PE), catalyzed by a selective phospholipase D (PLD). N-arachidonoyl-PE is produced through a two-step reaction in which arachidonic acid (AA) is transferred from the sn-2 position of a phospholipid to the sn-1 position of lyso-phosphatidylcholine (PC), producing diarachidonoyl-PC. The sn-1 arachidonoyl chain of diarachidonoyl-PC is then transferred to the free amino group of PE, generating N-arachidonoyl-PE. Two additional routes of anandamide biosynthesis have been described. Left: an as-yet-uncharacterized phospholipase C (PLC) converts N-arachidonoyl-PE into phospho-anandamide, which is then dephosphorylated by a phosphatase (P-ase) forming anandamide. Right: N-arachidonoyl-PE is hydrolyzed by α/β hydrolase domain-containing protein 4 (ABHD-4), forming glycerophospho-anandamide, which generates anandamide after losing the glycerophosphate group. Anandamide is degraded intracellularly by the serine amidase, fatty acid amide hydrolase (FAAH).

Figure 3. Schematic model of the 2-AG signalosome

This supra-molecular complex, selectively localized to the perisynaptic zone of the dendritic spine, connects in a single functional unit three key proteins involved in 2-AG production at excitatory synapses of the brain – mGluR5 metabotropic glutamate receptors, phospholipase C-β (PLC-β) and diacylglycerol-α (DGL-α). Evidence indicates that these proteins may be held together by the scaffolding proteins Homer-cc and Homer 1a. The proximity of mGluR5 to PLC-β and DGL-α allows for the rapid accumulation of 2-AG, which travels across the synaptic cleft to activate CB₁ receptors on axon terminals. The 2-AG that reaches presynaptic terminals may be quickly hydrolyzed by monoacylglycerol lipase (MGL), while the 2-AG that fails to reach the terminals may be degraded by α/β hydrolase domain-containing protein 6 (ABHD-6). Other abbreviations: AMPAR, AMPA receptors; NMDAR, NMDA receptors.