After the discovery, in the early 1990s, of specific G‐protein‐coupled receptors for marijuana's psychoactive principle Δ 9 ‐tetrahydrocannabinol, the cannabinoid receptors, and of their endogenous agonists, the endocannabinoids, a decade of investigations has greatly enlarged our understanding of this altogether new signalling system. Yet, while the finding of the endocannabinoids resulted in a new effort to reveal the mechanisms regulating their levels in the brain and peripheral organs under physiological and pathological conditions, more endogenous substances with a similar action, and more molecular targets for the previously discovered endogenous ligands, anandamide and 2‐arachidonoylglycerol, or for some of their metabolites, were being proposed. As the scenario becomes subsequently more complicated, and the experimental tasks to be accomplished correspondingly more numerous, we briefly review in this article the latest ‘additions’ to the endocannabinoid system together with earlier breakthroughs that have contributed to our present knowledge of the biochemistry and pharmacology of the endocannabinoids.

Abbreviations:

AEA N‐arachidonoyl‐ethanolamine (anandamide) 2‐AG 2‐arachidonoyl‐glycerol 2‐AGE 2‐arachidonyl‐glyceryl ether (noladin) AMT anandamide membrane transporter DAG diacylglycerol FAAH fatty acid amide hydrolase GPCRs G‐protein‐coupled receptors MAGLs monoacylglycerol lipase NADA N‐arachidonoyl‐dopamine NAPE N‐acyl‐phosphatidylethanolamines NAPE‐PLD NAPE‐selective PLD NArPE N‐arachidonoyl‐phosphatidylethanolamine PA phosphatidic acid PI phosphoinositides PI‐PLC PI‐selective phospholipase C PLD phospholipase D sn‐1 DAGL sn‐1‐selective DAG lipase THC (‐)‐Δ9‐tetrahydrocannabinol TRPV1 transient receptor potential vanilloid type 1 receptor virhodamine O‐arachidonoyl‐ethanolamine

Introduction The discovery in the early 1990s of specific membrane receptors of marijuana's psychoactive component (‐)‐Δ9‐tetrahydrocannabinol (THC) opened the way to the revelation of a whole endogenous signaling system now known as the endocannabinoid system. Apart from the cannabinoid CB 1 and CB 2 receptors (Pertwee, 1997), this system comprises also their endogenous ligands (the endocannabinoids) and the proteins for their synthesis and inactivation, as well as other molecular targets for the endocannabinoids. However, as new findings on the regulation of the levels and action of the endocannabinoids, and new data on their possible physiological and pathological role, are reported every day in the literature, it is easy to understand that the story of the endocannabinoid system is far from set. For example, while until the end of the 20th century only two endocannabinoids, anandamide (N‐arachidonoyl‐ethanolamine, AEA) and 2‐arachidonoyl‐glycerol (2‐AG) had been discovered (Devane et al., 1992; Mechoulam et al., 1995; Sugiura et al., 1995), in just a couple of years, three more candidates to the role of cannabinoid receptor agonists have been proposed: 2‐arachidonyl‐glyceryl ether (noladin, 2‐AGE), O‐arachidonoyl‐ethanolamine (virhodamine) and N‐arachidonoyl‐dopamine (NADA) (Bisogno et al., 2000; Huang et al., 2002; Porter et al., 2002). These findings not only suggest that the endocannabinoid family is larger than initially thought but also enlarge our view on the possible molecular mechanisms for the biosynthesis, action and inactivation of these lipid mediators. This brief article aims at giving a picture as much updated as possible on the ‘old’ and ‘new’ components of the endocannabinoid system, while highlighting the latest and most important findings in this field.

The endocannabinoid system: a lot done, and yet much more to do In consideration of the fact that the cloning of the first cannabinoid receptor was only reported in 1990, and the first endocannabinoid discovered only 2 years later, considerable progress has been made, in little more than a decade, towards the understanding of the regulation of the ‘cannabinergic’ signal. The endocannabinoid system appears to be conserved at least in all vertebrate phyla, and to be present, possibly with some major differences in the structure of receptors and in their function, also in invertebrates (Salzet & Stefano, 2002; McPartland & Glass, 2003), thus implying its participation in vital functions. Also from the pharmacological and pharmaceutical points of view, great breakthroughs in this field have been achieved, last but not least the fact that drugs or preparations targeting the cannabinoid receptors might soon be on the market. Sativex®, an extract of Cannabis strains with a known concentration of THC and cannabidiol, in the form of a sublingual spray, useful in the treatment of symptoms of multiple sclerosis, and rimonabant® (SR141716A), a selective CB 1 antagonist that might be marketed in the future as an antiobesity drug, are the two examples of a possible entirely new generation of products to be developed from the endocannabinoid system. New therapeutic drugs might be obtained in the future also from selective inhibitors of endocannabinoid biosynthesis and inactivation, but first several other milestones will have to be reached. In particular, it will be necessary: to clone the key enzymes catalyzing AEA biosynthesis and to understand their regulation at the molecular level;

to find biosynthetic pathways for virodhamine, NADA and 2‐AGE, and to clarify their regulation;

to establish transgenic mice lacking functional genes for endocannabinoid biosynthesis, and to study their phenotype;

to develop selective and potent inhibitors of endocannabinoid biosynthesis and of 2‐AG degradation that can be used in vivo ;

to clone the novel receptors proposed for AEA and to establish their actual role in endocannabinoid biology;

to understand conclusively the relationships between the cannabinoid receptor‐ and noncannabinoid receptor (e.g. TRPV1)‐mediated actions of AEA (and NADA);

to carry on identifying, first in animal models and then possibly in the clinic, those pathological conditions that can be caused, at least in part, by a malfunctioning endocannabinoid system, or whose onset, progress and/or symptoms are controlled tonically by the endocannabinoids. The joint multidisciplinary effort of many scientists will be necessary to fulfill these task in the shortest time possible, in order to understand fully the physiological and pathological function of the endocannabinoid system, and to assess finally if further endocannabinoid‐based medicines with advantages over other drugs are to be developed in the future.

Note added in proof Another breakthrough in endocannabinoid research has recently been achieved with the cloning of the NAPE‐PLD for AEA biosynthesis (Okamoto et al., J. Biol. Chem., in press). The enzyme is a member of the zinc metallo‐hydrolase family.