Review
Biology of endocannabinoid synthesis system

https://doi.org/10.1016/j.prostaglandins.2008.12.002Get rights and content

Abstract

Endocannabinoids (endogenous ligands of cannabinoid receptors) exert diverse physiological and pathophysiological functions in animal tissues. N-Arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG) are two representative endocannabinoids. Both the compounds are arachidonic acid-containing lipid molecules generated from membrane glycerophospholipids, but their biosynthetic pathways are totally different. Anandamide is principally formed together with other N-acylethanolamines (NAEs) in a two-step pathway, which is composed of Ca2+-dependent N-acyltransferase and N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD). cDNA cloning of NAPE-PLD and subsequent analysis of its gene-disrupted mice led to the discovery of alternative pathways comprising multiple enzymes. As for the 2-AG biosynthesis, recent results, including cDNA cloning of diacylglycerol lipase and analyses of phospholipase Cβ-deficient mice, demonstrated that these two enzymes are responsible for the in vivo formation of 2-AG functioning as a retrograde messenger in synapses. In this review article, we will focus on recent progress in the studies on the enzymes responsible for the endocannabinoid biosyntheses.

Introduction

Molecular cloning and characterization of two G protein-coupled cannabinoid receptors, CB1 and CB2, in the early 1990s [1], [2] led to the discovery in animal tissues of an important endogenous signaling system known as the endocannabinoid system. This system consists of the cannabinoid receptors, their endogenous ligands (the endocannabinoids), and the enzyme proteins catalyzing the endocannabinoid formation and degradation [3]. CB1 is predominantly expressed in the central nervous system but also present in a variety of peripheral tissues at much lower levels [4]. CB2 is primarily expressed in immune and blood cells, although it was recently found in various brain areas [5] and other tissues [6], [7]. Moreover, pharmacological studies strongly suggested the existence of novel cannabinoid receptor subtypes [8], [9].

N-Arachidonoylethanolamine (anandamide) [10] and 2-arachidonoylglycerol (2-AG) [11], [12] have been well-studied as endocannabinoids although several other arachidonic acid derivatives were also suggested to be endocannabinoids [13]. Binding of endocannabinoids as well as cannabinoids to cannabinoid receptors results in the decrease in intracellular cyclic AMP level and the activation of mitogen-activated protein kinase through the coupled Gi/o proteins. In addition, the activation of cannabinoid receptors modulates ion channels through Gi/o proteins, leading to the activation of A-type and inwardly rectifying potassium channels and the inhibition of N-type and P/Q-type calcium channels. CB1 can also stimulate the formation of cyclic AMP through Gs under certain conditions [14]. Anandamide acts as a partial agonist for CB1 and a weak agonist for CB2 [15], [16], while 2-AG acts as a full agonist for both the receptors [17], [18]. Pharmacological interactions in vivo between anandamide and 2-AG remain unclear.

In animal tissues, anandamide co-exists with ethanolamides of other fatty acids, collectively referred to as N-acylethanolamines (NAEs). Anandamide is usually a relatively minor component, while saturated and mono-unsaturated long-chain NAEs are major components [19]. Although these major NAEs do not bind to or activate cannabinoid receptors [20], they have been reported to exhibit a variety of biological activities such as antinociceptive effect (N-palmitoylethanolamine) [21], anti-inflammatory effect (N-palmitoylethanolamine and N-stearoylethanolamine) [22], [23], anorexic effect (N-oleoylethanolamine and N-stearoylethanolamine) [24], [25], and pro-apoptotic effect (N-stearoylethanolamine) [26]. Recent studies suggested that peroxisome proliferator-activated receptor (PPAR)-α [27], [28], transient receptor potential vanilloid type 1 (TRPV1) [29], and the G protein-coupled receptors GPR55 [30] and GPR119 [31] are involved in these effects. Regarding the endogenous ligand of GPR55, contradictory reports have been published, and very recently 2-arachidonoyl-lysophosphatidylinositol was shown to be one of the candidates for the endogenous ligands [32].

The endocannabinoid system is well-known to be involved in a broad range of physiological functions, such as emotion, cardiovascular regulation, energy metabolism, and reproduction, and in a growing number of pathophysiological conditions [6]. Modulating the activity of this system turned out to hold therapeutic promise under a variety of physiological and pathophysiological conditions including motor coordination, pain modulation, memory processing, control of appetite, neuroprotection, and reproduction [16], [18]. Since 2006 the CB1 antagonist rimonabant has been available in Europe as a clinical drug for antiobesity and smoking cessation [4]. Several other cannabinoid receptor-related compounds are being developed as drugs at preclinical stage [6], [33], [34]. For example, the CB1 antagonists taranabant, otenabant, and surinabant are expected as anti-obesity drugs or for the treatment of nicotine-dependence, and the CB2 antagonists SR144528, JTE-907, and Sch.336 as anti-inflammatory drugs. Moreover, enzymes responsible for the endocannabinoid biosynthesis and degradation also drew much attention as therapeutic targets [6], [33], [35], [36]. In particular, specific inhibitors of fatty acid amide hydrolase (FAAH), which hydrolyzes anandamide and other NAEs, are expected to be used in the treatment of anxiety, depression and pain [34], [37].

Unlike classical neurotransmitters and neuropeptides, endocannabinoids are not stored in vesicles in the cell, rather they are produced on demand from membrane phospholipids by a series of intracellular enzymes and released from cells, followed by immediate action as signaling molecules [38], [39]. After reuptake by cells, they are rapidly inactivated by enzymatic hydrolysis. Fig. 1 shows outline of the major pathways through which anandamide (A) and 2-AG (B) are produced and degraded. Recent cDNA cloning of the key enzymes such as N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) [40] and diacylglycerol lipase (DAGL) [41] accelerated molecular biological studies on the endocannabinoid biosyntheses. The intention of this short review is to discuss the latest advances in research on the pathways and enzymes responsible for the biosyntheses of anandamide and 2-AG.

Section snippets

Outline

It is generally accepted that in animal tissues anandamide and other NAEs are principally biosynthesized from membrane phospholipids through a common two-step pathway, termed ‘the transacylation-phosphodiesterase pathway’ (Fig. 1A) [42], [43], [44], [45]. The first reaction in this pathway is N-acylation of phosphatidylethanolamine (PE) in which an acyl group is transferred from the sn-1 position of a glycerophospholipid molecule to the amino group of PE. This reaction is catalyzed by Ca2+

PLC and DAGL

The major pathway for the biosynthesis of 2-AG comprises sequential hydrolysis of arachidonic acid-containing inositol phospholipids by PLC and DAGL (Fig. 1B) [12], [94]. Members of the phosphoinositide-specific PLC family are confirmed to be key enzymes in cell signaling [95], [96], [97]. In response to many extracellular stimuli, such as hormones, neurotransmitters, antigens, and growth factors, PLCs catalyze the hydrolysis of phosphatidylinositol-4,5-bisphosphate, thereby generating two

Perspectives

The two major endocannabinoids anandamide and 2-AG are arachidonic acid-containing lipid molecules derived from glycerophospholipids, but their biosynthetic pathways are totally different. Recent cDNA cloning of NAPE-PLD and generation of its deficient mice led to the discovery of alternative pathways for the biosyntheses of anandamide and other NAEs. On the other hand, cDNA cloning of DAGL and application of PLCβ-disrupted mice to this research area demonstrated that the PLCβ-DAGL pathway

Acknowledgements

This work was partially supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Japan Society for the Promotion of Science, and grants from Medical Institution Union Foundation, the Japan Foundation for Applied Enzymology, and Kagawa University Specially Promoted Research Fund 2008.

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