Depolarization-induced suppression of inhibition (DSI) is a prevailing form of endocannabinoid signalling. However, several discrepancies have arisen regarding the roles played by the two major brain endocannabinoids, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide, in mediating DSI. Here we studied endocannabinoid signalling in the prefrontal cortex (PFC), where several components of the endocannabinoid system have been identified, but endocannabinoid signalling remains largely unexplored. In voltage clamp recordings from mouse PFC pyramidal neurons, depolarizing steps significantly suppressed IPSCs induced by application of the cholinergic agonist carbachol. DSI in PFC neurons was abolished by extra- or intracellular application of tetrahydrolipstatin (THL), an inhibitor of the <em>2</em>-AG synthesis enzyme diacylglycerol lipase (DAGL). Moreover, DSI was enhanced by inhibiting <em>2</em>-AG degradation, but was unaffected by inhibiting anandamide degradation. THL, however, may affect other enzymes of lipid metabolism and does not selectively target the α (DAGLα) or β (DAGLβ) isoforms of DAGL. Therefore, we studied DSI in the PFC of DAGLα(-/-) and DAGLβ(-/-) mice generated via insertional mutagenesis by gene-trapping with retroviral vectors. Gene trapping strongly reduced DAGLα or DAGLβ mRNA levels in a locus-specific manner. In DAGLα(-/-) mice cortical levels of <em>2</em>-AG were significantly decreased and DSI was completely abolished, whereas DAGLβ deficiency did not alter cortical <em>2</em>-AG levels or DSI. Importantly, cortical levels of anandamide were not significantly affected in DAGLα(-/-) or DAGLβ(-/-) mice. The chronic decrease of <em>2</em>-AG levels in DAGLα(-/-) mice did not globally alter inhibitory transmission or the response of cannabinoid-sensitive synapses to cannabinoid receptor stimulation, although it altered some intrinsic membrane properties. Finally, we found that repetitive action potential firing of PFC pyramidal neurons suppressed synaptic inhibition in a DAGLα-dependent manner. These results show that DSI is a prominent form of endocannabinoid signalling in PFC circuits. Moreover, the close agreement between our pharmacological and genetic studies indicates that <em>2</em>-AG synthesized by postsynaptic DAGLα mediates DSI in PFC neurons.

The understanding of the pharmacology surrounding the cannabinergic system has seen many advances since the discovery of the CB1 receptor in the mammalian brain and the CB<em>2</em> receptor in the periphery. Among these advances is the discovery of the endogenous ligands arachidonoylethanolamide (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> amide (<em>2</em>-AG), which are selective agonists for the CB1 and CB<em>2</em> receptors, respectively. These endogenous neuromodulators involved in the cannabinergic system are thought to be produced on demand and are metabolized by the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAG lipase). Recently, we characterized a reuptake system that facilitates the transport of anandamide across the cell membrane and subsequently developed selective inhibitors of this transport, which have been found to have therapeutic potential as analgesic and peripheral vasodilators. The cannabinergic proteins currently being explored, which include the CB1 and CB<em>2</em> receptors, FAAH and the anandamide transporter, are excellent targets for the development of therapeutically useful drugs for a range of conditions including pain, loss of appetite, immunosuppression, peripheral vascular disease and motor disorders. As cannabinoid research has progressed, various potent and selective cannabimimetic ligands, targeting these four cannabinoid proteins, have been designed and synthesized. Many of these ligands serve as important molecular probes, providing structural information regarding the binding sites of the cannabinergic proteins, as well as pharmacological tools, which have been playing pivotal roles in research aimed at understanding the biochemical and physiological aspects of the endocannabinoid system. This review will focus on some of the current cannabinergic ligands and probes and their pharmacological and therapeutic potential.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is an endogenous cannabinoid that binds to CB1 and CB<em>2</em> cannabinoid receptors, inducing cannabimimetic effects. However, the cannabimimetic effects of <em>2</em>-AG are weak in vivo due to its rapid enzymatic hydrolysis. The enzymatic hydrolysis of <em>2</em>-AG has been proposed to mainly occur by monoglyceride lipase (monoacylglycerol lipase). Fatty acid amide hydrolase (FAAH), the enzyme responsible for the hydrolysis of N-arachidonoylethanolamide (AEA), is also able to hydrolyse <em>2</em>-AG. In the present study, we investigated the hydrolysis of endocannabinoids in rat cerebellar membranes and observed that enzymatic activity towards <em>2</em>-AG was 50-fold higher than that towards AEA. Furthermore, various inhibitors for <em>2</em>-AG hydrolase activity were studied in rat cerebellar membranes. <em>2</em>-AG hydrolysis was inhibited by methyl arachidonylfluorophosphonate, hexadecylsulphonyl fluoride and phenylmethylsulphonyl fluoride with ic(50) values of <em>2</em>.<em>2</em> nM, <em>2</em>41 nM and 155 microM, respectively. Potent FAAH inhibitors, such as OL-53 and URB597, did not inhibit the hydrolysis of <em>2</em>-AG, suggesting that <em>2</em>-AG is inactivated in rat cerebellar membranes by an enzyme distinct of FAAH. The observation that the hydrolysis of 1(3)-AG and <em>2</em>-AG occurred at equal rates supports the role of MGL in <em>2</em>-AG inactivation. This enzyme assay provides a useful method for future inhibition studies of <em>2</em>-AG degrading enzyme(s) in brain membrane preparation having considerably higher MGL-like activity when compared to FAAH activity.

Enzymes for the biosynthesis and degradation of the endocannabinoid <em>2</em>-arachidonoyl glycerol (<em>2</em>-AG) have been cloned and are the sn-1-selective-diacylglycerol lipases alpha and beta (DAGLalpha and beta) and the monoacylglycerol lipase (MAGL), respectively. Here, we used membranes from COS cells over-expressing recombinant human DAGLalpha to screen new synthetic substances as DAGLalpha inhibitors, and cytosolic fractions from wild-type COS cells to look for MAGL inhibitors. DAGLalpha and MAGL activities were assessed by using sn-1-[14C]-oleoyl-<em>2</em>-arachidonoyl-glycerol and <em>2</em>-[3H]-<em>arachidonoylglycerol</em> as substrates, respectively. We screened known compounds as well as new phosphonate derivatives of oleic acid and fluoro-phosphinoyl esters of different length. Apart from the general lipase inhibitor tetrahydrolipstatin (orlistat) (IC50 approximately 60 nM), the most potent inhibitors of DAGLalpha were O-3640 [octadec-9-enoic acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester] (IC50 = 500 nM), and O-3841 [octadec-9-enoic acid 1-methoxymethyl-<em>2</em>-(fluoro-methyl-phosphinoyloxy)-ethyl ester] (IC50 = 160 nM). Apart from being almost inactive on MAGL, these two compounds showed high selectivity over rat liver triacylglycerol lipase, rat N-acylphosphatidyl-ethanolamine-selective phospholipase D (involved in anandamide biosynthesis), rat fatty acid amide hydrolase and human recombinant cannabinoid CB1 and CB<em>2</em> receptors. Methylarachidonoyl-fluorophosphonate and the novel compound UP-101 [O-ethyl-O-p-nitro-phenyl oleylphosphonate] inhibited both DAGLalpha and MAGL with similar potencies (IC50 = 0.8-0.1 and 3.7-3.<em>2</em> microM, respectively). Thus, we report the first potent and specific inhibitors of the biosynthesis of <em>2</em>-AG that may be used as pharmacological tools to investigate the biological role of this endocannabinoid.

Cyclooxygenase-<em>2</em> (COX-<em>2</em>) is an enzyme that plays a key role in inflammatory processes. Classically, this enzyme is upregulated in inflammatory situations and is responsible for the generation of prostaglandins (PGs) from arachidonic acid (AA). One lesser-known property of COX-<em>2</em> is its ability to metabolize the endocannabinoids, N-arachidonoylethanolamine (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Endocannabinoid metabolism by COX-<em>2</em> is not merely a means to terminate their actions. On the contrary, it generates PG analogs, namely PG-glycerol esters (PG-G) for <em>2</em>-AG and PG-ethanolamides (PG-EA or prostamides) for AEA. Although the formation of these COX-<em>2</em>-derived metabolites of the endocannabinoids has been known for a while, their biological effects remain to be fully elucidated. Recently, several studies have focused on the role of these PG-G or PG-EA in vivo. In this review we take a closer look at the literature concerning these novel bioactive lipids and their role in inflammation.

1. Two endocannabinoids, arachidonoyl ethanolamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) bind and activate G-protein-coupled cannabinoid receptors, but limited data exist on their relative ability to activate G-proteins. <em>2</em>. Here we assess agonist potency and efficacy of various cannabinoids, including <em>2</em>-AG, HU-310 (<em>2</em>-arachidonoyl glyceryl ether, a third putative endocannabinoid), HU-313 (another ether analogue of <em>2</em>-AG), AEA, R-methanandamide (an enzymatically stable analogue of AEA), and CP-55,940 at rat brain CB(1) receptors using agonist-stimulated [(35)S]-GTPgammaS binding to cerebellar membranes and whole brain sections. Degradation of endocannabinoids under experimental conditions was monitored by HPLC. 3. To enhance efficacy differences, agonist dose-response curves were generated using increasing GDP concentrations. At 10(-6) M GDP, all compounds, except HU-313, produced full agonists responses approximately <em>2</em>.5 fold over basal. The superior efficacy of <em>2</em>-AG over all other compounds became evident by increasing GDP (10(-5) and 10(-4) M). 4. In membrane incubations, <em>2</em>-AG was degraded by 85% whereas AEA and HU-310 were stable. Pretreatment of membranes with phenylmethylsulphonyl fluoride inhibited <em>2</em>-AG degradation, resulting in <em>2</em> fold increase in agonist potency. Such pretreatment had no effect on AEA potency. 5. Responses in brain sections were otherwise consistent with membrane binding data, but <em>2</em>-AG evoked only a weak signal in brain sections, apparently due to more extensive degradation. 6. These data establish that even under conditions of substantial degradation, <em>2</em>-AG is a full efficacy agonist, clearly more potent than AEA, in mediating CB(1) receptor-dependent G-protein activity in native membranes.

Chronic stress is the primary environmental risk factor for the development and exacerbation of affective disorders, thus understanding the neuroadaptations that occur in response to stress is a critical step in the development of novel therapeutics for depressive and anxiety disorders. Brain endocannabinoid (eCB) signaling is known to modulate emotional behavior and stress responses, and levels of the eCB <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are elevated in response to chronic homotypic stress exposure. However, the role of <em>2</em>-AG in the synaptic and behavioral adaptations to chronic stress is poorly understood. Here, we show that stress-induced development of anxiety-like behavior is paralleled by a transient appearance of low-frequency stimulation-induced, <em>2</em>-AG-mediated long-term depression at GABAergic synapses in the basolateral amygdala, a key region involved in motivation, affective regulation, and emotional learning. This enhancement of <em>2</em>-AG signaling is mediated, in part, via downregulation of the primary <em>2</em>-AG-degrading enzyme monoacylglycerol lipase (MAGL). Acute in vivo inhibition of MAGL had little effect on anxiety-related behaviors. However, chronic stress-induced anxiety-like behavior and emergence of long-term depression of GABAergic transmission was prevented by chronic MAGL inhibition, likely via an occlusive mechanism. These data indicate that chronic stress reversibly gates eCB synaptic plasticity at inhibitory synapses in the amygdala, and in vivo augmentation of <em>2</em>-AG levels prevents both behavioral and synaptic adaptations to chronic stress.

The novel endogenous cannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) was rapidly inactivated by intact rat basophilic leukaemia (RBL-<em>2</em>H3) and mouse neuroblastoma (N18TG<em>2</em>) cells through diffusion/hydrolysis/reacylation processes. The hydrolysis of <em>2</em>-AG was inhibited by typical esterase inhibitors and by more specific blockers of 'fatty acid amide hydrolase' (FAAH), the enzyme catalysing the hydrolysis of the other 'endocannabinoid', anandamide (AEA). No evidence for a facilitated-diffusion process was found. A <em>2</em>-AG-hydrolysing activity was detected in homogenates from both cell lines, with the highest levels in membrane fractions. It exhibited an optimal pH at 10, and recognized both <em>2</em>- and 1(3)- isomers of mono<em>arachidonoylglycerol</em> with similar efficiencies. The apparent Km and Vmax values for -3H-<em>2</em>-AG hydrolysis were 91 microM and <em>2</em>9 microM and <em>2</em>.4 and 1.8 nmol.min-1.mg of protein-1 respectively in N18TG<em>2</em> and RBL-<em>2</em>H3 cells. [3H]<em>2</em>-AG hydrolysis was inhibited by Cu<em>2</em>+, Zn<em>2</em>+ and p-hydroxymercuribenzoate, and by <em>2</em>- or 1(3)-monolinoleoyl- and -linolenoyl-glycerols, but not by the oleoyl, palmitoyl and myristoyl congeners. Purified fractions from solubilized membrane proteins catalysed, at pH 9.5, the hydrolysis of <em>2</em>-AG as well as AEA. Accordingly, AEA as well as FAAH inhibitors, including arachidonoyltrifluoromethyl ketone (ATFMK), blocked [3H]<em>2</em>-AG hydrolysis by N18TG<em>2</em> and RBL-<em>2</em>H3 membranes, whereas <em>2</em>-AG inhibited [14C]AEA hydrolysis. FAAH blockade by ATFMK preserved from inactivation the <em>2</em>-AG synthesized de novo by intact N18TG<em>2</em> cells stimulated with ionomycin. These data suggest that FAAH may be one of the enzymes deputed to the physiological inactivation of <em>2</em>-AG, and create intriguing possibilities for the cross-regulation of <em>2</em>-AG and AEA levels.

The endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> are predominantly regulated by the respective catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Inhibition of these enzymes elevates endocannabinoid levels and attenuates neuropathic pain. In the present study, CB₁ and CB₂ receptor-deficient mice were subjected to chronic constriction injury (CCI) of the sciatic nerve to examine the relative contribution of each receptor for the anti-allodynic effects of the FAAH inhibitor, PF-3845, and the MAGL inhibitor, JZL184. CCI caused marked hypersensitivity to mechanical and cold stimuli, which was not altered by deletion of either the CB₁ or CB₂ receptor, but was attenuated by gabapentin, as well as by each enzyme inhibitor. Whereas PF-3845 lacked anti-allodynic efficacy in both knockout lines, JZL184 did not produce anti-allodynic effects in CB₁ (-/-) mice, but retained its anti-allodynic effects in CB₂ (-/-) mice. These data indicate that FAAH and MAGL inhibitors reduce nerve injury-related hyperalgesic states through distinct cannabinoid receptor mechanisms of action. In conclusion, although endogenous cannabinoids do not appear to play a tonic role in long-term expression of neuropathic pain states, both FAAH and MAGL represent potential therapeutic targets for the development of pharmacological agents to treat chronic pain resulting from nerve injury.

CONCLUSIONS

This article presents data addressing the cannabinoid receptor mechanisms underlying the anti-allodynic actions of endocannabinoid catabolic enzyme inhibitors in the mouse sciatic nerve ligation model. Fatty acid amide hydrolase and monoacylglycerol lipase inhibitors reduced allodynia through distinct cannabinoid receptor mechanisms. These enzymes offer potential targets to treat neuropathic pain.

Acute or chronic alterations in energy status alter the balance between excitatory and inhibitory synaptic transmission and associated synaptic plasticity to allow for the adaptation of energy metabolism to new homeostatic requirements. The impact of such changes on endocannabinoid and cannabinoid receptor type 1 (CB1)-mediated modulation of synaptic transmission and strength is not known, despite the fact that this signaling system is an important target for the development of new drugs against obesity. We investigated whether CB1-expressing excitatory vs. inhibitory inputs to orexin-A-containing neurons in the lateral hypothalamus are altered in obesity and how this modifies endocannabinoid control of these neurons. In lean mice, these inputs are mostly excitatory. By confocal and ultrastructural microscopic analyses, we observed that in leptin-knockout (ob/ob) obese mice, and in mice with diet-induced obesity, orexinergic neurons receive predominantly inhibitory CB1-expressing inputs and overexpress the biosynthetic enzyme for the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>, which retrogradely inhibits synaptic transmission at CB1-expressing axon terminals. Patch-clamp recordings also showed increased CB1-sensitive inhibitory innervation of orexinergic neurons in ob/ob mice. These alterations are reversed by leptin administration, partly through activation of the mammalian target of rapamycin pathway in neuropeptide-Y-ergic neurons of the arcuate nucleus, and are accompanied by CB1-mediated enhancement of orexinergic innervation of target brain areas. We propose that enhanced inhibitory control of orexin-A neurons, and their CB1-mediated disinhibition, are a consequence of leptin signaling impairment in the arcuate nucleus. We also provide initial evidence of the participation of this phenomenon in hyperphagia and hormonal dysregulation in obesity.

Abstinence symptoms in cannabis-dependent individuals are believed to contribute to the maintenance of regular marijuana use. However, there are currently no medications approved by the FDA to treat cannabis-related disorders. The only treatment currently shown consistently to alleviate cannabinoid withdrawal in both animals and humans is substitution therapy using the psychoactive constituent of marijuana, Delta(9)-tetrahydrocannabinol (THC). However, new genetic and pharmacological tools are available to increase endocannabinoid levels by targeting fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the enzymes responsible for the degradation of the endogenous cannabinoid ligands anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, respectively. In the present study, we investigated whether increasing endogenous cannabinoids levels, through the use of FAAH (-/-) mice as well as the FAAH inhibitor URB597 or the MAGL inhibitor JZL184, would reduce the intensity of withdrawal signs precipitated by the CB(1) receptor antagonist rimonabant in THC-dependent mice. Strikingly, acute administration of either URB597 or JZL184 significantly attenuated rimonabant-precipitated withdrawal signs in THC-dependent mice. In contrast, FAAH (-/-) mice showed identical withdrawal responses as wild-type mice under a variety of conditions, suggesting that the absence of this enzyme across the development of dependence and during rimonabant challenge does not affect withdrawal responses. Of importance, subchronic administration of URB597 did not lead to cannabinoid dependence and neither URB597 nor JZL184 impaired rotarod motor coordination. These results support the concept of targeting endocannabinoid metabolizing enzymes as a promising treatment for cannabis withdrawal.

The role of endocannabinoid signaling in the response of the brain to injury is tantalizing but not clear. In this study, transient middle cerebral artery occlusion (MCAo) was used to produce ischemia/reperfusion injury. Brain content of N-arachidonoylethanolamine (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> were determined during MCAo. Whole brain AEA content was significantly increased after 30, 60 and 1<em>2</em>0 min MCAo compared with sham-operated brain. The increase in AEA was localized to the ischemic hemisphere after 30 min MCAo, but at 60 and 1<em>2</em>0 min, was also increased in the contralateral hemisphere. <em>2</em>-<em>Arachidonoylglycerol</em> content was unaffected by MCAo. In a second set of studies, injury was assessed <em>2</em>4 h after <em>2</em> h MCAo. Rats administered a single dose (3 mg/kg) of the cannabinoid receptor type 1 (CB1) receptor antagonist SR141716 prior to MCAo exhibited a 50% reduction in infarct volume and a 40% improvement in neurological function compared with vehicle control. A second CB1 receptor antagonist, LY3<em>2</em>0135 (6 mg/kg), also significantly improved neurological function. The CB1 receptor agonist, WIN 55<em>2</em>1<em>2</em>-<em>2</em> (0.1-1 mg/kg) did not affect either infarct volume or neurological score.

Transient increases in nucleus accumbens (NAc) dopamine concentration are observed when animals are presented with motivationally salient stimuli and are theorized to energize reward seeking. They arise from high-frequency firing of dopamine neurons in the ventral tegmental area (VTA), which also results in the release of endocannabinoids from dopamine cell bodies. In this context, endocannabinoids are thought to regulate reward seeking by modulating dopamine signaling, although a direct link has never been demonstrated. To test this, we pharmacologically manipulated endocannabinoid neurotransmission in the VTA while measuring transient changes in dopamine concentration in the NAc during reward seeking. Disrupting endocannabinoid signaling dramatically reduced, whereas augmenting levels of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>AG) increased, cue-evoked dopamine concentrations and reward seeking. These data suggest that <em>2</em>AG in the VTA regulates reward seeking by sculpting ethologically relevant patterns of dopamine release during reward-directed behavior.

OBJECTIVE

The cannabinoid receptor type <em>2</em> (CB<em>2</em>) has protective effects in chronic degenerative diseases. Our aim was to assess the potential relevance of the CB<em>2</em> receptor in both human and experimental diabetic nephropathy (DN).

METHODS

CB<em>2</em> expression was studied in kidney biopsies from patients with advanced DN, in early experimental diabetes, and in cultured podocytes. Levels of endocannabinoids and related enzymes were measured in the renal cortex from diabetic mice. To assess the functional role of CB<em>2</em>, streptozotocin-induced diabetic mice were treated for 14 weeks with AM1<em>2</em>41, a selective CB<em>2</em> agonist. In these animals, we studied albuminuria, renal function, expression of podocyte proteins (nephrin and zonula occludens-1), and markers of both fibrosis (fibronectin and transforming growth factor-β1) and inflammation (monocyte chemoattractant protein-1 [MCP-1], CC chemokine receptor <em>2</em> [CCR<em>2</em>], and monocyte markers). CB<em>2</em> signaling was assessed in cultured podocytes.

RESULTS

Podocytes express the CB<em>2</em> receptor both in vitro and in vivo. CB<em>2</em> was downregulated in kidney biopsies from patients with advanced DN, and renal levels of the CB<em>2</em> ligand <em>2</em>-<em>arachidonoylglycerol</em> were reduced in diabetic mice, suggesting impaired CB<em>2</em> regulation. In experimental diabetes, AM1<em>2</em>41 ameliorated albuminuria, podocyte protein downregulation, and glomerular monocyte infiltration, without affecting early markers of fibrosis. In addition, AM1<em>2</em>41 reduced CCR<em>2</em> expression in both renal cortex and cultured podocytes, suggesting that CB<em>2</em> activation may interfere with the deleterious effects of MCP-1 signaling.

CONCLUSIONS

The CB<em>2</em> receptor is expressed by podocytes, and in experimental diabetes, CB<em>2</em> activation ameliorates both albuminuria and podocyte protein loss, suggesting a protective effect of signaling through CB<em>2</em> in DN.

The endocannabinoid system consists of G protein-coupled cannabinoid CB(1) and CB(<em>2</em>) receptors, of endogenous compounds known as endocannabinoids that can target these receptors, of enzymes that catalyse endocannabinoid biosynthesis and metabolism, and of processes responsible for the cellular uptake of some endocannabinoids. This review presents in vitro evidence that most or all of the following 13 compounds are probably orthosteric endocannabinoids since they have all been detected in mammalian tissues in one or more investigation, and all been found to bind to cannabinoid receptors, probably to an orthosteric site: anandamide, <em>2</em>-<em>arachidonoylglycerol</em>, noladin ether, dihomo-γ-linolenoylethanolamide, virodhamine, oleamide, docosahexaenoylethanolamide, eicosapentaenoylethanolamide, sphingosine, docosatetraenoylethanolamide, N-arachidonoyldopamine, N-oleoyldopamine and haemopressin. In addition, this review describes in vitro findings that suggest that the first eight of these compounds can activate CB(1) and sometimes also CB(<em>2</em>) receptors and that another two of these compounds are CB(1) receptor antagonists (sphingosine) or antagonists/inverse agonists (haemopressin). Evidence for the existence of at least three allosteric endocannabinoids is also presented. These endogenous compounds appear to target allosteric sites on cannabinoid receptors in vitro, either as negative allosteric modulators of the CB1 receptor (pepcan-1<em>2</em> and pregnenolone) or as positive allosteric modulators of this receptor (lipoxin A(4)) or of the CB(<em>2</em>) receptor (pepcan-1<em>2</em>). Also discussed are current in vitro data that indicate the extent to which some established or putative orthosteric endocannabinoids seem to target non-cannabinoid receptors and ion channels, particularly at concentrations at which they have been found to interact with CB(1) or CB(<em>2</em>) receptors.

CB(1)- and CB(<em>2</em>)-type cannabinoid receptors mediate effects of the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide in mammals. In canonical endocannabinoid-mediated synaptic plasticity, <em>2</em>-AG is generated postsynaptically by diacylglycerol lipase alpha and acts via presynaptic CB(1)-type cannabinoid receptors to inhibit neurotransmitter release. Electrophysiological studies on lampreys indicate that this retrograde signalling mechanism occurs throughout the vertebrates, whereas system-level studies point to conserved roles for endocannabinoid signalling in neural mechanisms of learning and control of locomotor activity and feeding. CB(1)/CB(<em>2</em>)-type receptors originated in a common ancestor of extant chordates, and in the sea squirt Ciona intestinalis a CB(1)/CB(<em>2</em>)-type receptor is targeted to axons, indicative of an ancient role for cannabinoid receptors as axonal regulators of neuronal signalling. Although CB(1)/CB(<em>2</em>)-type receptors are unique to chordates, enzymes involved in biosynthesis/inactivation of endocannabinoids occur throughout the animal kingdom. Accordingly, non-CB(1)/CB(<em>2</em>)-mediated mechanisms of endocannabinoid signalling have been postulated. For example, there is evidence that <em>2</em>-AG mediates retrograde signalling at synapses in the nervous system of the leech Hirudo medicinalis by activating presynaptic transient receptor potential vanilloid-type ion channels. Thus, postsynaptic synthesis of <em>2</em>-AG or anandamide may be a phylogenetically widespread phenomenon, and a variety of proteins may have evolved as presynaptic (or postsynaptic) receptors for endocannabinoids.

The endogenous cannabinoid signalling system, composed of endogenous cannabinoids, cannabinoid receptors and the enzymes that synthesize and degrade the endogenous cannabinoids, is much more complex than initially conceptualized. <em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is the most abundant endocannabinoid and plays a major role in CNS development and synaptic plasticity. Over the past decade, many key players in <em>2</em>-AG synthesis and degradation have been identified and characterized. Most <em>2</em>-AG is synthesized from membrane phospholipids via sequential activation of a phospholipase Cβ and a diacylglycerol lipase, although other pathways may contribute in specialized settings. <em>2</em>-AG breakdown is more complicated with at least eight different enzymes participating. These enzymes can either degrade <em>2</em>-AG into its components, arachidonic acid and glycerol, or transform <em>2</em>-AG into highly bioactive signal molecules. The implications of the precise temporal and spatial control of the expression and function of these pleiotropic metabolizing enzymes have only recently come to be appreciated. In this review, we will focus on the primary organization of the synthetic and degradative pathways of <em>2</em>-AG and then discuss more recent findings and their implications, with an eye towards the biological and therapeutic implications of manipulating <em>2</em>-AG synthesis and metabolism.

BACKGROUND

This article is part of a themed section on Cannabinoids <em>2</em>013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.<em>2</em>014.171.issue-6.

Alcoholism can result in fatty liver that can progress to steatohepatitis, cirrhosis, and liver cancer. Mice fed alcohol develop fatty liver through endocannabinoid activation of hepatic CB(1) cannabinoid receptors (CB(1)R), which increases lipogenesis and decreases fatty acid oxidation. Chronic alcohol feeding also up-regulates CB(1)R in hepatocytes in vivo, which could be replicated in vitro by co-culturing control hepatocytes with hepatic stellate cells (HSC) isolated from ethanol-fed mice, implicating HSC-derived mediator(s) in the regulation of hepatic CB(1)R (Jeong, W. I., Osei-Hyiaman, D., Park, O., Liu, J., Bátkai, S., Mukhopadhyay, P., Horiguchi, N., Harvey-White, J., Marsicano, G., Lutz, B., Gao, B., and Kunos, G. (<em>2</em>008) Cell Metab. 7, <em>2</em><em>2</em>7-<em>2</em>35). HSC being a rich source of retinoic acid (RA), we tested whether RA and its receptors may regulate CB(1)R expression in cultured mouse hepatocytes. Incubation of hepatocytes with RA or RA receptor (RAR) agonists increased CB(1)R mRNA and protein, the most efficacious being the RARgamma agonist CD437 and the pan-RAR agonist TTNPB. The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) also increased hepatic CB(1)R expression, which was mediated indirectly via RA, because it was absent in hepatocytes from mice lacking retinaldehyde dehydrogenase 1, the enzyme catalyzing the generation of RA from retinaldehyde. The binding of RARgamma to the CB(1)R gene 5' upstream domain in hepatocytes treated with RAR agonists or <em>2</em>-AG was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift and antibody supershift assays. Finally, TTNPB-induced CB(1)R expression was attenuated by small interfering RNA knockdown of RARgamma in hepatocytes. We conclude that RARgamma regulates CB(1)R expression and is thus involved in the control of hepatic fat metabolism by endocannabinoids.

The G-protein coupled receptors for Δ⁹-tetrahydrocannabinol, the major psychoactive principle of marijuana, are known as cannabinoid receptors of type 1 (CB₁) and <em>2</em> (CB₂) and play important functions in degenerative and inflammatory disorders of the central nervous system. Whilst CB₁ receptors are mostly expressed in neurons, where they regulate neurotransmitter release and synaptic strength, CB₂ receptors are found mostly in glial cells and microglia, which become activated and over-express these receptors during disorders such as Alzheimer's disease, multiple sclerosis, amyotropic lateral sclerosis, Parkinson's disease, and Huntington's chorea. The neuromodulatory actions at CB₁ receptors by endogenous agonists ('endocannabinoids'), of which anandamide and <em>2</em>-<em>arachidonoylglycerol</em> are the two most studied representatives, allows them to counteract the neurochemical unbalances arising during these disorders. In contrast, the immunomodulatory effects of these lipophilic mediators at CB₂ receptors regulate the activity and function of glia and microglia. Indeed, the level of expression of CB₁ and CB₂ receptors or of enzymes controlling endocannabinoid levels, and hence the concentrations of endocannabinoids, undergo time- and brain region-specific changes during neurodegenerative and neuroinflammatory disorders, with the initial attempt to counteract excitotoxicity and inflammation. Here we discuss this plasticity of the endocannabinoid system during the aforementioned central nervous system disorders, as well as its dysregulation, both of which have opened the way to the use of either direct and indirect activators or blockers of CB₁ and CB₂ receptors for the treatment of the symptoms or progression of these diseases.

Intraluminal administration of the endocannabinoids N-arachidonoyl-ethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) causes inflammation similar to that caused by Clostridium difficile toxin A in the rat ileum. The effects of anandamide and <em>2</em>-AG were significantly inhibited by pretreatment with the specific capsaicin receptor (vanilloid receptor subtype 1; VR1) antagonist capsazepine. Pretreatment with the CB1 and CB<em>2</em> cannabinoid receptor antagonists N-piperidino-5-(4-chlorophenyl)-1-(<em>2</em>,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716) and N-[1S)-endo-1,3,3-trimethylbicyclo[<em>2</em>.<em>2</em>.1]heptan-<em>2</em>-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR1445<em>2</em>8) did not affect the responses to anandamide. It has previously been shown that intraluminal toxin A stimulates substance P (SP) release from primary sensory neurons and that pretreatment with SP receptor [neurokinin (NK)-1 receptor] antagonists inhibits the inflammatory effects of toxin A. Anandamide stimulated SP release and this was blocked by capsazepine pretreatment. Also, pretreatment with the specific NK-1 receptor antagonist (<em>2</em>S,3S)-3-([3,5-bis[trifluoromethyl)phenyl]methoxy)-<em>2</em>-phenylpiperidine (L-733,060) significantly inhibited the inflammatory effects of both toxin A and anandamide. Toxin A increased tissue concentrations of anandamide and <em>2</em>-AG in the ileum, and these effects were enhanced after pretreatment with inhibitors of fatty acid amide hydrolase, a major endocannabinoid-degrading enzyme. The toxin A-stimulated release of anandamide but not <em>2</em>-AG was selective over their congeners. These results demonstrate that the endocannabinoids anandamide and <em>2</em>-AG stimulate intestinal primary sensory neurons via the capsaicin VR1 receptor to release SP, resulting in enteritis, and that endocannabinoids may mediate the inflammatory effects of toxin A.

Observational studies in humans suggest that exposure to marijuana and other cannabis-derived drugs produces a wide range of subjective effects on mood tone and emotionality. These observations have their counterpart in animal studies, showing that cannabinoid agonists strongly affect emotional reactivity in directions that vary depending on dose and context. Based on these evidence, the activation of central CB(1) receptor has emerged as potential target for the development of antianxiety and antidepressant therapies. However, the variable effects of exogenous cannabinoid agonists have gradually shifted the interest to the alternative approach of amplifying the effects of endogenous cannabinoids (EC), namely anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), by preventing their deactivation. The enzyme fatty acid amide hydrolase (FAAH) has been the target of intense research efforts aimed at developing potent and selective inhibitors that might prolong AEA actions in vivo. Among the inhibitors developed, the compound URB597 was found to potently inhibit FAAH activity in vivo and cause brain AEA levels to increase. Interestingly, the enhanced AEA tone produced by URB597 does not result in the behavioral effects typical of a direct-acting cannabinoid agonist. Though URB597 does not elicit a full-fledged cannabinoid profile of behavioral responses, it does elicit marked anxiolytic-like and antidepressant-like effects in rats and mice. Such effects involve the downstream activation of CB(1) receptors, since they are attenuated by the CB(1) antagonist SR141716 (rimonabant). Parallel to FAAH inhibition, similar results can also be observed by pharmacologically blocking the AEA transport system, which is responsible of the intracellular uptake of AEA from the synaptic cleft. The reason why FAAH inhibition approach produces a smaller set of cannabimimetic effects might depend on the mechanism of EC synthesis and release upon neuronal activation and on the target selectivity of the drug. The mechanism of EC release is commonly referred to as "on request", since they are not synthesized and stored in synaptic vesicles, such as classical neurotransmitters, but are synthesized from membrane precursors and immediately released in the synaptic cleft following neuronal activation. The neural stimulation in specific brain areas, for example, those involved in the regulation of mood tone and/or emotional reactivity, would result in an increased EC tone in these same areas, but not necessarily in others. Therefore, inhibition of AEA metabolism activity could amplify CB(1) activation mainly where AEA release is higher. Furthermore, the inhibition of FAAH causes an accumulation of AEA but not <em>2</em>-AG, which, being <em>2</em>00-fold more abundant than AEA in the brain, might differently modulate CB(1)-mediated behavioral responses. The evidence outlined above supports the hypothesis that the EC system plays an important role in anxiety and mood disorders and suggests that modulation of FAAH activity might be a pharmacological target for novel anxiolytic and antidepressant therapies.

Investigations of the pathways involved in the metabolism of endocannabinoids have grown exponentially in recent years following the discovery of cannabinoid receptors (CB) and their endogenous ligands, such as anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). The in vivo biosynthesis of AEA has been shown to occur through several pathways mediated by N-acylphosphatidylethanolamide-phospholipase D (NAPE-PLD), a secretory PLA(<em>2</em>) and PLC. <em>2</em>-AG, a second endocannabinoid is generated through the action of selective enzymes such as phosphatidic acid phsophohydrolase, diacylglycerol lipase (DAGL), phosphoinositide-specific PLC (PI-PLC) and lyso-PLC. A putative membrane transporter or facilitated diffusion is involved in the cellular uptake or release of endocannabinoids. AEA is metabolized by fatty acid amidohydrolase (FAAH) and <em>2</em>-AG is metabolized by both FAAH and monoacylglycerol lipase (MAGL). The author presents an integrative overview of current research on the enzymes involved in the metabolism of endocannabinoids and discusses possible therapeutic interventions for various diseases, including addiction.

Functional interactions between glucocorticoids and the endocannabinoid system have been repeatedly documented; yet, to date, no studies have demonstrated in vivo that glucocorticoid hormones regulate endocannabinoid signaling. We demonstrate that systemic administration of the glucocorticoid corticosterone (3 and 10 mg/kg) resulted in an increase in the tissue content of the endocannabinoid N-arachidonylethanolamine (AEA) within several limbic structures (amygdala, hippocampus, hypothalamus), but not the prefrontal cortex, of male rats. Tissue AEA content was increased at 10min and returned to control 1h post-corticosterone administration. The other primary endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em>, was found to be elevated by corticosterone exclusively within the hypothalamus. The rapidity of the change suggests that glucocorticoids act through a non-genomic pathway. Tissue contents of two other N-acylethanolamines, palmitoylethanolamide and oleolyethanolamide, were not affected by corticosterone treatment, suggesting that the mechanism of regulation is neither fatty acid amide nor N-acylphosphatidylethanolamine phospholipase D. These data provide in vivo support for non-genomic steroid effects in mammals and suggest that AEA is a mediator of these effects.

It is not yet clear if the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N-arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits <em>2</em>-AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]<em>2</em>-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 +/- <em>2</em>.0 and 15.3 +/- 3.1 microM, Bmax 1.70 +/- 0.30 and 0.<em>2</em>4 +/- 0.04 nmol.min-1.mg protein-1, respectively). Despite these similar Km values, <em>2</em>-AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 +/- 3.9 microM) significantly higher than those required to either <em>2</em>-AG or AEA to inhibit [3H]<em>2</em>-AG uptake (Ki = 18.9 +/- 1.8 and <em>2</em>0.5 +/- 3.<em>2</em> microM, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]<em>2</em>-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]<em>2</em>-AG alone; (b) the uptake of [14C]AEA and [3H]<em>2</em>-AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 +/- 0.7 and 10.<em>2</em> +/- 1.7 microM, respectively) and linvanil (Ki = 9.5 +/- 0.7 and 6.4 +/- 1.<em>2</em> microM, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]<em>2</em>-AG, thus suggesting that <em>2</em>-AG action can be regulated through NO release; (d) AEA and <em>2</em>-AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.