Local environmental cues are indispensable for axonal growth and guidance during brain circuit formation. Here, we combine genetic and pharmacological tools, as well as systems neuroanatomy in human fetuses and mouse models, to study the role of endocannabinoid and Slit/Robo signalling in axonal growth. We show that excess <em>2</em>-<em>arachidonoylglycerol</em>, an endocannabinoid affecting directional axonal growth, triggers corpus callosum enlargement due to the errant CB1 cannabinoid receptor-containing corticofugal axon spreading. This phenotype mechanistically relies on the premature differentiation and end-feet proliferation of CB<em>2</em>R-expressing oligodendrocytes. We further show the dependence of both axonal Robo1 positioning and oligodendroglial Slit<em>2</em> production on cell-type-specific cannabinoid receptor activation. Accordingly, Robo1 and/or Slit<em>2</em> manipulation limits endocannabinoid modulation of axon guidance. We conclude that endocannabinoids can configure focal Slit<em>2</em>/Robo1 signalling to modulate directional axonal growth, which may provide a basis for understanding impaired brain wiring associated with metabolic deficits and prenatal drug exposure.

The plant Cannabis sativa contains cannabinoids represented by Δ9-tetrahydrocannabinol, which exert psychoactivity and immunomodulation through cannabinoid CB1 and CB<em>2</em> receptors, respectively, in animal tissues. Arachidonoylethanolamide (also referred to as anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are well known as two major endogenous agonists of these receptors (termed "endocannabinoids") and show various cannabimimetic bioactivities. However, only <em>2</em>-AG is a full agonist for CB1 and CB<em>2</em> and mediates retrograde signals at the synapse, strongly suggesting that <em>2</em>-AG is physiologically more important than anandamide. The metabolic pathways of these two endocannabinoids are completely different. <em>2</em>-AG is mostly produced from inositol phospholipids via diacylglycerol by phospholipase C and diacylglycerol lipase and then degraded by monoacylglycerol lipase. On the other hand, anandamide is concomitantly produced with larger amounts of other N-acylethanolamines via N-acyl-phosphatidylethanolamines (NAPEs). Although this pathway consists of calcium-dependent N-acyltransferase and NAPE-hydrolyzing phospholipase D, recent studies revealed the involvement of several new enzymes. Quantitatively major N-acylethanolamines include palmitoylethanolamide and oleoylethanolamide, which do not bind to cannabinoid receptors but exert anti-inflammatory, analgesic, and anorexic effects through receptors such as peroxisome proliferator-activated receptor α. The biosynthesis of these non-endocannabinoid N-acylethanolamines rather than anandamide may be the primary significance of this pathway. Here, we provide an overview of the biological activities and metabolisms of endocannabinoids (<em>2</em>-AG and anandamide) and non-endocannabinoid N-acylethanolamines.

Monoacylglycerol lipase (MAGL) is part of the endocannabinoid and the prostaglandin signaling system. MAGL degrades the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) into glycerol and arachidonic acid. MAGL-induced arachidonic acid is the primary source for prostaglandin synthesis in the brain. <em>2</em>-AG mainly induces neuroprotective and anti-inflammatory effects, whereas prostaglandins are related to pro-inflammatory effects inducing neurotoxicity. Therefore, inhibition of MAGL represents a promising target for neurological diseases characterized by inflammation. However, as <em>2</em>-AG is an agonist for the cannabinoid receptor 1 (CB1), inhibition of MAGL might be associated with unwanted cannabimimetic effects. Here, we show that oral administration of KML<em>2</em>9, a highly selective inhibitor of MAGL, induced large and dose-dependent changes in <em>2</em>-AG levels in vivo in brain and spinal cord of mice. Of note, MAGL inhibition by KML<em>2</em>9 induced a decrease in prostaglandin levels in brain and most peripheral tissues but not in the spinal cord. MAGL expression was highest in fat, liver and brain, whereas the cytosolic phospholipase A<em>2</em> (cPLA<em>2</em>), a further enzyme responsible for arachidonic acid production, was highly expressed in spinal cord, muscle and spleen. In addition, high doses (10 mg/kg) of KML<em>2</em>9 induced some cannabimimetic effects in vivo in the tetrad test, including hypothermia, analgesia and hypomotility without induction of cataleptic behavior. In summary, inhibition of MAGL by KML<em>2</em>9 represents a promising strategy for targeting the cannabinoid and prostaglandin system of the brain with only a moderate induction of cannabimimetic effects.

Endocannabinoids (eCBs) are endogenous lipids that bind principally type-1 and type-<em>2</em> cannabinoid (CB(1) and CB(<em>2</em>)) receptors. N-Arachidonoylethanolamine (AEA, anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are the best characterized eCBs that are released from membrane phospholipid precursors through multiple biosynthetic pathways. Together with their receptors and metabolic enzymes, eCBs form the so-called "eCB system". The later has been involved in a wide variety of actions, including modulation of basal ganglia function. Consistently, both eCB levels and CB(1) receptor expression are high in several basal ganglia regions, and more specifically in the striatum and in its target projection areas. In these regions, the eCB system establishes a close functional interaction with dopaminergic neurotransmission, supporting a relevant role for eCBs in the control of voluntary movements. Accordingly, compelling experimental and clinical evidence suggests that a profound rearrangement of the eCB system in the basal ganglia follows dopamine depletion, as it occurs in Parkinson's disease (PD). In this article, we provide a brief survey of the evidence that the eCB system changes in both animal models of, and patients suffering from, PD. A striking convergence of findings is observed between both rodent and primate models and PD patients, indicating that the eCB system undergoes dynamic, adaptive changes, aimed at restoring an apparent homeostasis within the basal ganglia network.

OBJECTIVE

Capsaicin, an agonist of transient receptor potential vanilloid 1 (TRPV1) channels, is pro-nociceptive in the periphery but is anti-nociceptive when administered into the ventrolateral periaqueductal gray (vlPAG), a midbrain region for initiating descending pain inhibition. Here, we investigated how activation of TRPV1 channels in the vlPAG leads to anti-nociception.

METHODS

We examined synaptic transmission and neuronal activity using whole-cell recordings in vlPAG slices in vitro and hot-plate nociceptive responses in rats after drug microinjection into the vlPAG in vivo.

RESULTS

Capsaicin (1-10 µM) depressed evoked GABAergic inhibitory postsynaptic currents (eIPSCs) in vlPAG slices presynaptically, while increasing miniature excitatory PSC frequency. Capsaicin-induced eIPSC depression was antagonized by cannabinoid CB₁ and metabotropic glutamate (mGlu₅) receptor antagonists, and prevented by inhibiting diacylglycerol lipase (DAGL), which converts DAG into <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), an endocannabinoid. Capsaicin induced membrane depolarization in <em>2</em>/3 neurons recorded but, overall, increased neuronal firings by increasing evoked postsynaptic potentials. Intra-vlPAG capsaicin reduced hot-plate responses in rats, effects blocked by CB₁ and mGlu receptor antagonists. Effects of capsaicin were antagonized by SB 366791, a TRPV1 channel antagonist.

CONCLUSIONS

Capsaicin activated TRPV1s on glutamatergic terminals to release glutamate which activated postsynaptic mGlu₅ receptors, yielding <em>2</em>-AG from DAG by DAGL hydrolysis. <em>2</em>-AG induces retrograde inhibition (disinhibition) of GABA release via presynaptic CB₁ receptors. This disinhibition in the vlPAG leads to anti-nociception by activating the descending pain inhibitory pathway. This is a novel TRPV1 channel-mediated anti-nociceptive mechanism in the brain and a new interaction between vanilloid and endocannabinoid systems.

OBJECTIVE

Cerebrovascular smooth muscle cells express the CB1 cannabinoid receptor and CB1 agonists produce vasodilatation of the middle cerebral artery (MCA). The thromboxane A<em>2</em> mimetic, U-46619, increased the content of the endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the MCA and <em>2</em>-AG moderated the vasoconstriction produced by U46619 in this tissue. The purposes of this study were to examine the extent to which <em>2</em>-AG is catabolized by cerebral arteries and to determine whether blockade of <em>2</em>-AG inactivation potentiates its feedback inhibition of U-44619-mediated vasoconstriction.

METHODS

The diameters of isolated, perfused MCA from male rats were measured using videomicroscopy.

RESULTS

Exogenous <em>2</em>-AG produces a CB1 receptor-dependent and concentration-related increase in the diameter of MCA constricted with 5-HT. The E (max) for <em>2</em>-AG dilation is increased 4-fold in the presence of the metabolic inhibitors 3-(decylthio)-1,1,1-trifluropropan-<em>2</em>-one (DETFP), URB754 and URB597. To examine the role of catabolism in the effects of endogenous <em>2</em>-AG, vasoconstriction induced by U-46619 was studied. DETFP and URB754, but not the fatty acid amide hydrolase inhibitor, URB597, significantly increased the EC(50) for U-46619. These data support a physiological role for endocannabinoid feedback inhibition in the effects of U-46619 and indicate that endogenously produced <em>2</em>-AG is also efficiently catabolized within the MCA.

CONCLUSIONS

MCA express mechanisms for the efficient inactivation of <em>2</em>-AG, providing further support for an endocannabinoid feedback mechanism that opposes thromboxane-mediated vasoconstriction. These data suggest that potentiation of endogenously produced <em>2</em>-AG could be a novel therapeutic approach to the treatment of thrombotic stroke.

Fatty acid amide hydrolase (FAAH) is an intracellular serine hydrolase, which catalyzes the hydrolysis of the endocannabinoid N-arachidonoylethanolamide to arachidonic acid and ethanolamine. FAAH also hydrolyzes another endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). However, <em>2</em>-AG has been assumed to be hydrolyzed mainly by monoacylglycerol lipase (MAGL) or a MAGL-like enzyme. Inhibition of FAAH or MAGL activity might lead to beneficial effects in many physiological disorders such as pain, inflammation, and anxiety due to increased endocannabinoid-induced activation of cannabinoid receptors CB1 and CB<em>2</em>. In the present study, a total of 34 novel compounds were designed, synthesized, characterized, and tested against FAAH and MAGL-like enzyme activity. Altogether, 16 compounds were found to inhibit FAAH with half-maximal inhibition concentrations (IC50) between <em>2</em>8 and 380 nM. All the active compounds belong to the structural family of carbamates. Compounds 14 and 18 were found to be the most potent FAAH inhibitors, which may serve as lead structures for novel FAAH inhibitors.

Endocannabinoids (eCBs) inhibit neurotransmitter release throughout the central nervous system. Using the Ceratomandibularis muscle from the lizard Anolis carolinensis we asked whether eCBs play a similar role at the vertebrate neuromuscular junction. We report here that the CB(1) cannabinoid receptor is concentrated on motor terminals and that eCBs mediate the inhibition of neurotransmitter release induced by the activation of M(3) muscarinic acetylcholine (ACh) receptors. N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(<em>2</em>,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, a CB(1) antagonist, prevents muscarine from inhibiting release and arachidonylcyclopropylamide (ACPA), a CB(1) receptor agonist, mimics M(3) activation and occludes the effect of muscarine. As for its mechanism of action, ACPA reduces the action-potential-evoked calcium transient in the nerve terminal and this decrease is more than sufficient to account for the observed inhibition of neurotransmitter release. Similar to muscarine, the inhibition of synaptic transmission by ACPA requires nitric oxide, acting via the synthesis of cGMP and the activation of cGMP-dependent protein kinase. <em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is responsible for the majority of the effects of eCB as inhibitors of phospholipase C and diacylglycerol lipase, two enzymes responsible for synthesis of <em>2</em>-AG, significantly limit muscarine-induced inhibition of neurotransmitter release. Lastly, the injection of (5Z,8Z,11Z,14Z)-N-(4-hydroxy-<em>2</em>-methylphenyl)-5,8,11,14-eicosatetraenamide (an inhibitor of eCB transport) into the muscle prevents muscarine, but not ACPA, from inhibiting ACh release. These results collectively lead to a model of the vertebrate neuromuscular junction whereby <em>2</em>-AG mediates the muscarine-induced inhibition of ACh release. To demonstrate the physiological relevance of this model we show that the CB(1) antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(<em>2</em>,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide prevents synaptic inhibition induced by <em>2</em>0 min of 1-Hz stimulation.

The effect of WIN55<em>2</em>1<em>2</em>-<em>2</em>, a cannabinoid receptor agonist, on acute inflammation of mouse ear was investigated. We found that topical application of WIN55<em>2</em>1<em>2</em>-<em>2</em> suppressed ear swelling induced by 1<em>2</em>-O-tetradecanoylphorbol 13-acetate or <em>2</em>-<em>arachidonoylglycerol</em>. Similar inhibition was observed with CP55940, another cannabinoid receptor agonist, and HU-308, a cannabinoid CB(<em>2</em>) receptor-selective agonist. WIN55<em>2</em>1<em>2</em>-<em>2</em> also suppressed the infiltration of leukocytes induced by 1<em>2</em>-O-tetradecanoylphorbol 13-acetate. On the other hand, WIN55<em>2</em>1<em>2</em>-3, an inactive enantiomer of WIN55<em>2</em>1<em>2</em>-<em>2</em>, exerted only small effects on inflammation. Notably, SR1445<em>2</em>8, a cannabinoid CB(<em>2</em>) receptor antagonist, also suppressed inflammatory reactions in mouse ear. Thus, both the cannabinoid CB(<em>2</em>) receptor agonist and antagonist are capable of reducing inflammatory reactions. We then investigated the mechanism underlying WIN55<em>2</em>1<em>2</em>-<em>2</em>-induced suppression of inflammation using cultured cells. We found that the addition of WIN55<em>2</em>1<em>2</em>-<em>2</em> together with <em>2</em>-<em>arachidonoylglycerol</em> blocked <em>2</em>-<em>arachidonoylglycerol</em>-induced migration of human promyelocytic leukemia HL-60 cells that had been differentiated into macrophage-like cells. The restoration of <em>2</em>-<em>arachidonoylglycerol</em>-desensitized cells and WIN55<em>2</em>1<em>2</em>-<em>2</em>-desensitized cells from an anergic condition was examined next. We found that <em>2</em>-<em>arachidonoylglycerol</em>-treated cells rapidly recovered the capacity to respond to <em>2</em>-<em>arachidonoylglycerol</em>. On the other hand, the anergic condition toward <em>2</em>-<em>arachidonoylglycerol</em> continued for a longer period after pretreatment with WIN55<em>2</em>1<em>2</em>-<em>2</em>. These results suggest that the anti-inflammatory activity of WIN55<em>2</em>1<em>2</em>-<em>2</em> is attributable, at least in part, to interference with the actions of the endogenous ligand, <em>2</em>-<em>arachidonoylglycerol</em>.

We investigated whether <em>2</em>-<em>arachidonoylglycerol</em>, an endogenous cannabinoid receptor ligand, is involved in acetylcholine- and calcium ionophore A<em>2</em>3187-induced relaxations in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) and indomethacin, which is considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF). In rabbit mesenteric arterial rings pre-constricted with noradrenaline, <em>2</em>-<em>arachidonoylglycerol</em> caused concentration-dependent relaxation. The <em>2</em>-<em>arachidonoylglycerol</em>-induced relaxations were not affected by endothelium removal. N-piperidino-5-(4-chlorophenyl)-1-(<em>2</em>,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) and 1-(<em>2</em>,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morholinyl-1H-pyrazole-3-carboxamide (AM<em>2</em>81), cannabinoid CB(1) receptor antagonists, significantly attenuated <em>2</em>-<em>arachidonoylglycerol</em>-induced relaxation and the acetylcholine-induced relaxation only slightly, but not the calcium ionophore A<em>2</em>3187-induced relaxation. On the other hand, charybdotoxin plus apamin, K(+) channel blockers, significantly attenuated acetylcholine and calcium ionohore A<em>2</em>3187-induced relaxations but not <em>2</em>-<em>arachidonoylglycerol</em>-induced relaxations. These results suggest that <em>2</em>-<em>arachidonoylglycerol</em> can cause relaxations via cannabinoid CB(1) receptors, but is not involved in EDHF-mediated relaxations.

OBJECTIVE

The vasorelaxant effect of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) has been well characterised in animals. <em>2</em>-AG is present in human vascular cells and is up-regulated in cardiovascular pathophysiology. However, the acute vascular actions of <em>2</em>-AG have not been explored in humans.

METHODS

Mesenteric arteries were obtained from patients receiving colorectal surgery and mounted on a myograph. Arteries were contracted and <em>2</em>-AG concentration-response curves were carried out. Mechanisms of action were characterised pharmacologically. Post hoc analysis was carried out to assess the effects of cardiovascular disease/risk factors on <em>2</em>-AG responses.

RESULTS

<em>2</em>-AG caused vasorelaxation of human mesenteric arteries, independent of cannabinoid receptor or transient receptor potential vanilloid-1 activation, the endothelium, nitric oxide or metabolism via monoacyglycerol lipase or fatty acid amide hydrolase. <em>2</em>-AG-induced vasorelaxation was reduced in the presence of indomethacin and flurbiprofen, suggesting a role for cyclooxygenase metabolism <em>2</em>-AG. Responses to <em>2</em>-AG were also reduced in the presence of Cay10441, L-16198<em>2</em> and potentiated in the presence of AH6809, suggesting that metabolism of <em>2</em>-AG produces both vasorelaxant and vasoconstrictor prostanoids. Finally, <em>2</em>-AG-induced vasorelaxation was dependent on potassium efflux and the presence of extracellular calcium.

CONCLUSIONS

We have shown for the first time that <em>2</em>-AG causes vasorelaxation of human mesenteric arteries. Vasorelaxation is dependent on COX metabolism, activation of prostanoid receptors (EP4 & IP) and ion channel modulation. <em>2</em>-AG responses are blunted in patients with cardiovascular risk factors.

Bilateral olfactory bulbectomy (OBX) in rodents produces behavioral and neurochemical changes associated clinically with depression and schizophrenia. Most notably, OBX induces hyperlocomotion in response to the stress of exposure to a novel environment. We examined the role of the endocannabinoid system in regulating this locomotor response in OBX and sham-operated rats. In our study, OBX-induced hyperactivity was restricted to the first 3 min of the open field test, demonstrating the presence of novelty (0-3 min) and habituation (3-30 min) phases of the open field locomotor response. Levels of the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide were decreased in the ventral striatum, a brain region deafferented by OBX, whereas cannabinoid receptor densities were unaltered. In sham-operated rats, <em>2</em>-AG levels in the ventral striatum were negatively correlated with distance traveled during the novelty phase. Thus, low levels of <em>2</em>-AG are reflected in a hyperactive open field response. This correlation was not observed in OBX rats. Conversely, <em>2</em>-AG levels in endocannabinoid-compromised OBX rats correlated with distance traveled during the habituation phase. In OBX rats, pharmacological blockade of cannabinoid CB(1) receptors with either AM<em>2</em>51 (1 mg kg(-1) i.p.) or rimonabant (1 mg kg(-1) i.p.) increased distance traveled during the habituation phase. Thus, blockade of endocannabinoid signaling impairs habituation of the hyperlocomotor response in OBX, but not sham-operated, rats. By contrast, in sham-operated rats, effects of CB(1) antagonism were restricted to the novelty phase. These findings suggest that dysregulation in the endocannabinoid system, and <em>2</em>-AG in particular, is implicated in the hyperactive locomotor response induced by OBX. Our studies suggest that drugs that enhance <em>2</em>-AG signaling, such as <em>2</em>-AG degradation inhibitors, might be useful in human brain disorders modeled by OBX.

OBJECTIVE

Endocannabinoids are synthesized from lipid precursors at the plasma membranes of virtually all cell types, including cardiac myocytes. Endocannabinoids can modulate neuronal and vascular ion channels through receptor-independent actions; however, their effects on cardiac K(+) channels are unknown. This study was undertaken to determine the receptor-independent effects of endocannabinoids such as anandamide (N-arachidonoylethanolamine, AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and endocannabinoid-related compounds such as N-palmitoylethanolamine (PEA), N-oleoylethanolamine (OEA), the endogenous lipid lysophosphatidylinositol (LPI), and the fatty acids from which some of these compounds are endogenously synthesized, on human cardiac Kv1.5 channels, which generate the ultrarapid delayed rectifier current (I(Kur)).

RESULTS

hKv1.5 currents (I(hKv1.5)) were recorded in mouse fibroblasts (Ltk(-) cells) by using the whole-cell patch-clamp technique. Most of these compounds inhibited I(hKv1.5) in a concentration-dependent manner, the potency being determined by the number of C atoms in the fatty acyl chain. Indeed, AEA and <em>2</em>-AG, which are arachidonic acid (<em>2</em>0:4) derivatives, exhibited the highest potency (IC(50) approximately 0.9-<em>2</em>.5 microM), whereas PEA, a palmitic acid (PA-16:0) derivative, exhibited the lowest potency. The inhibition was independent of cannabinoid receptor engagement and of changes in the order and microviscosity of the membrane. Furthermore, blockade induced by AEA and <em>2</em>-AG was abolished upon mutation of the R487 residue, which determines the external tetraethylammonium sensitivity and is located in the external entryway of the pore. AEA significantly prolonged the duration of action potentials (APs) recorded in mouse left atria.

CONCLUSIONS

These results indicate that endocannabinoids block human cardiac Kv1.5 channels by interacting with an extracellular binding site, a mechanism by which these compounds regulate atrial AP shape.

Recent studies have highlighted the importance of paracrine growth factors as mediators of pro-angiogenic effects by endothelial progenitor cells (EPCs), but little is known about the release of lipid-based factors like endocannabinoids by EPCs. In the current study, the release of the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> by distinct human EPC sub-types was measured using HPLC/tandem mass-spectrometry. Anandamide release was highest by adult blood colony-forming EPCs at baseline and they also demonstrated increased <em>2</em>-<em>arachidonoylglycerol</em> release with TNF-alpha stimulation. Treatment of mature endothelial cells with endocannabinoids significantly reduced the induction of the pro-inflammatory adhesion molecule CD106 (VCAM-1) by TNF-alpha.

Since the discovery of the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the early 1990s, the endocannabinoid system has been implicated in a wide array of physiological processes, such as control of food intake and energy balance, fertility and obesity. As the importance of this system becomes apparent, there is a tremendous need for robust, sensitive and efficient analytical methodology for the examination of the endocannabinoids, their congeners and putative metabolites. This review will summarize quantitative analytical methodology as reported in the literature from 199<em>2</em> to present for the analysis of endocannabinoids and related compounds.

Regulation of Ca(<em>2</em>+) homeostasis plays a critical role in oligodendrocyte function and survival. Cannabinoid CB(1) and CB(<em>2</em>) receptors have been shown to regulate Ca(<em>2</em>+) levels and/or K(+) currents in a variety of cell types. In this study we investigated the effect of cannabinoid compounds on the Ca(<em>2</em>+) influx elicited in cultured oligodendrocytes by transient membrane depolarization with an elevated extracellular K(+) concentration (50 mM). The CB(1) receptor agonist arachidonoyl-chloro-ethanolamide (ACEA) elicited a concentration-dependent inhibition of depolarization-evoked Ca(<em>2</em>+) transients in oligodendroglial somata with a maximal effect (94+/-3)% and an EC(50) of 1.3+/-0.03 microM. This activity was mimicked by the CB(1)/CB(<em>2</em>) agonist CP55,940, as well as by the endocannabinoids N-arachidonoyl-ethanolamine (anandamide, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), whereas the CB(<em>2</em>) receptor selective agonist JWH133 was ineffective. The CB(1) receptor antagonist AM<em>2</em>51 (1 microM) also reduced the Ca(<em>2</em>+) response evoked by high extracellular K(+) and did not prevent the inhibition elicited by ACEA (3 microM). Nevertheless, the ability of ACEA and AEA to reduce depolarization-evoked Ca(<em>2</em>+) transients was significantly reduced in oligodendrocytes from CB(1) receptor knockout mice, as well as by pretreatment with pertussis toxin. Bath application of the inwardly rectifying K(+) channels (Kir channels) blockers BaCl(<em>2</em>) (300 microM) and CsCl(<em>2</em>) (1 mM) reduced the size of voltage-induced Ca(<em>2</em>+) influx and partially prevented the inhibitory effect of ACEA. Our results indicate that cannabinoids inhibit depolarization-evoked Ca(<em>2</em>+) transients in oligodendrocytes via CB(1) receptor-independent and -dependent mechanisms that involve the activation of PTX-sensitive G(i/o) proteins and the blockade of Kir channels.

Cyclooxygenase-<em>2</em> (COX-<em>2</em>) oxygenates arachidonic acid (AA) and the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-<em>2</em> acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of <em>2</em>-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments <em>2</em>-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-<em>2</em> complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-<em>2</em> increases <em>2</em>-AG oxygenation by increasing kcat and preventing inhibitory binding of <em>2</em>-AG. AM-8138 restored the activity of COX-<em>2</em> mutants that exhibited very poor <em>2</em>-AG oxygenating activity and increased the activity of COX-1 toward <em>2</em>-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-<em>2</em>-dependent <em>2</em>-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or <em>2</em>-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced <em>2</em>-AG oxygenation in intact RAW<em>2</em>64.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.

The endocannabinoid system can be manipulated pharmacologically in a variety of ways, including directly acting agonists and inverse agonists, and indirectly acting compounds which affect the synthesis, cellular accumulation and metabolism of the two main endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>. In this overview, the most commonly used compounds are discussed, primarily with respect to their targets of action and to their selectivities vis a vis "off targets". For direct acting compounds such as cannabinoid receptor agonists, it is suggested that the use of several compounds with different chemical structures at relevant doses or concentrations is likely to minimise the risk of misinterpreting an "off target" effect as being an action mediated by cannabinoid receptors. For indirectly acting compounds, the same reasoning applies, and in the case of compounds affecting the accumulation of anandamide, it is important to recognize that the molecular target of these compounds is far from clear. Nonetheless, judicious use of the array of pharmacological tools currently available, and combination of these tools with RNA interference techniques and the use of genetically modified animals, provides a powerful approach with which to characterize the endocannabinoid system in the body.

Studies with the monoacylglycerol lipase blocker JZL184 have suggested that enhanced <em>2</em>-<em>arachidonoylglycerol</em> signaling suppresses locomotion, lowers body temperature, and decreases anxiety. Although the neurochemical effects of JZL184 develop within 30 min, its behavioral and autonomic effects have been studied much later. To clarify temporal dynamics, we studied the effects of intraperitoneal injections of JZL184 in mice on home-cage locomotion and body temperature for 1<em>2</em>0 min using in-vivo biotelemetry. We also studied the effects of 4, 8, and 16 mg/kg JZL184 in the open field and elevated plus maze at various time points. In the home cage, JZL184 blunted injection-induced body temperature increases but exerted no long-term effects. Vehicle injections increased the duration of rapid movements whereas the duration of motionless periods was decreased, a pattern also abolished by JZL184. Although the highest dose exerted a mild long-term effect on the relative duration of motionless periods, JZL184 seemed to have phasic rather than tonic effects in the home cage. By contrast, open field and plus maze behavior was affected 80 and 1<em>2</em>0 min but not 40 min after treatments, which may indicate tonic rather than phasic effects in these tests. Our findings confirm earlier reports of a mild anxiolytic effect of JZL184, but surprisingly, the compound markedly and dose dependently increased locomotion in the open field in both CD1 and C57BL/6J mice. These findings are difficult to reconcile at present, but suggest that the effects of monoacylglycerol lipase inhibition are more complex than previously believed and may depend strongly on as yet unidentified factors such as environmental conditions, the time of testing, species/strains, etc.

Free arachidonic acid is functionally interlinked with different lipid signaling networks including those involving prostanoid pathways, the endocannabinoid system, N-acylethanolamines, as well as steroids. A sensitive and specific LC-MS/MS method for the quantification of arachidonic acid, prostaglandin E<em>2</em>, thromboxane B<em>2</em>, anandamide, <em>2</em>-<em>arachidonoylglycerol</em>, noladin ether, lineoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, steroyl ethanolamide, aldosterone, cortisol, dehydroepiandrosterone, progesterone, and testosterone in human plasma was developed and validated. Analytes were extracted using acetonitrile precipitation followed by solid phase extraction. Separations were performed by UFLC using a C18 column and analyzed on a triple quadrupole MS with electron spray ionization. Analytes were run first in negative mode and, subsequently, in positive mode in two independent LC-MS/MS runs. For each analyte, two MRM transitions were collected in order to confirm identity. All analytes showed good linearity over the investigated concentration range (r>0.98). Validated LLOQs ranged from 0.1 to 190ng/mL and LODs ranged from 0.04 to 1<em>2</em>.3ng/mL. Our data show that this LC-MS/MS method is suitable for the quantification of a diverse set of bioactive lipids in plasma from human donors (n=3<em>2</em>). The determined plasma levels are in agreement with the literature, thus providing a versatile method to explore pathophysiological processes in which changes of these lipids are implicated.

In addition to binding intracellular fatty acids, fatty-acid-binding proteins (FABPs) have recently been reported to also transport the endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), arachidonic acid derivatives that function as neurotransmitters and mediate a diverse set of physiological and psychological processes. To understand how the endocannabinoids bind to FABPs, the crystal structures of FABP5 in complex with AEA, <em>2</em>-AG and the inhibitor BMS-309403 were determined. These ligands are shown to interact primarily with the substrate-binding pocket via hydrophobic interactions as well as a common hydrogen bond to the Tyr131 residue. This work advances our understanding of FABP5-endocannabinoid interactions and may be useful for future efforts in the development of small-molecule inhibitors to raise endocannabinoid levels.

OBJECTIVE

We have shown the beneficial effects of cannabinoids in a murine model of hepatic encephalopathy following thioacetamide and now report their effects on the liver injury.

METHODS

Fulminant hepatic failure (FHF) was induced by administration of <em>2</em>00 mg/kg thioacetamide to wild-type (WT) and CB<em>2</em> Knockout (KO) mice. Twenty-four hours later, mice were injected with <em>2</em>-<em>arachidonoylglycerol</em> (CB1, CB<em>2</em>, and TRPV1 agonist), HU308 (CB<em>2</em> agonist), SR141716 A (CB1 receptor blocker), SR141716 A+<em>2</em>-AG, and SR1445<em>2</em>8 (CB<em>2</em> receptor blocker), capsaicin and capsazepine (TRPV1 agonist and antagonist receptors). Mice were sacrificed <em>2</em> days after thioacetamide administration (day 3) and liver biochemistry and histopathology as well as evaluation of <em>2</em>-<em>arachidonoylglycerol</em> levels were performed on liver tissue.

RESULTS

Liver histopathology undertaken 48 h after thioacetamide showed evidence of necrosis and inflammation. SR141716 A, HU308, and <em>2</em>-<em>arachidonoylglycerol</em> reduced inflammation and promoted regeneration 1 day after their administration. Liver enzymes increased after thioacetamide administration and were reversed after SR141716 A and <em>2</em>-<em>arachidonoylglycerol</em> administered alone or combined, HU-308, but not SR1445<em>2</em>8. Thus, the beneficial effects mediated through CB<em>2</em> receptors. However, CB<em>2</em> KO mice still modulated liver function via the TRPV1 receptors. Capsaicin improved both liver pathology and function in WT thioacetamide-treated mice, while capsazepine impaired it.

CONCLUSIONS

The similar pattern found between the effect of cannabinoids and their antagonists on brain and liver indicated that the therapeutic effect might be directed by the improvement in both organs through CB<em>2</em> receptors and/or TRPV1 receptors. Modulation of these systems may have therapeutic potential.

Early-life inflammation has been shown to exert profound effects on brain development and behavior, including altered emotional behavior, stress responsivity and neurochemical/neuropeptide receptor expression and function. The current study extends this research by examining the impact of inflammation, triggered with the bacterial compound lipopolysaccharide (LPS) on postnatal day (P) 14, on social behavior during adolescence. We investigated the role that the endocannabinoid (eCB) system plays in sociability after early-life LPS. To test this, multiple cohorts of Sprague Dawley rats were injected with LPS on P14. In adolescence, rats were subjected to behavioral testing in a reciprocal social interaction paradigm as well as the open field. We quantified eCB levels in the amygdala of P14 and adolescent animals (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) as well as adolescent amygdaloid cannabinoid receptor 1 (CB1) binding site density and the hydrolytic activity of the enzyme fatty acid amide hydrolase (FAAH), which metabolizes the eCB anandamide. Additionally, we examined the impact of FAAH inhibition on alterations in social behavior. Our results indicate that P14 LPS decreases adolescent social behavior (play and social non-play) in males and females at P40. This behavioral alteration is accompanied by decreased CB1 binding, increased anandamide levels and increased FAAH activity. Oral administration of the FAAH inhibitor PF-04457845 (1mg/kg) prior to the social interaction task normalizes LPS-induced alterations in social behavior, while not affecting social behavior in the control group. Infusion of 10ng PF-04457845 into the basolateral amygdala normalized social behavior in LPS injected females. These data suggest that alterations in eCB signaling following postnatal inflammation contribute to impairments in social behavior during adolescence and that inhibition of FAAH could be a novel target for disorders involving social deficits such as social anxiety disorders or autism.

The endocannabinoid, N-arachidonoylethanolamine (AEA), is degraded by the enzyme fatty acid amide hydrolase (FAAH). This study examined whether the FAAH inhibitor, URB597, increases retinal ganglion cell (RGC) survival following optic nerve axotomy in young and aged animals. URB597 alone, or together with either a CB1 or CB<em>2</em> receptor antagonist, was administered daily for 1 or <em>2</em> weeks post-axotomy. Histological assessment of retinas indicated that URB597 increased RGC survival in young retina at 1 and <em>2</em> weeks post-axotomy. The increase in RGC survival at <em>2</em> weeks was accompanied by a reduction in phagocytic microglia. The CB1 antagonist, AM<em>2</em>81, but not the CB<em>2</em> antagonist, AM630, ablated URB597-mediated RGC neuroprotection. CB1 or CB<em>2</em> antagonism increased phagocytic microglia in URB597 and vehicle-treated animals. In aged animals, URB597 increased RGC survival at 1 week, but not at <em>2</em> weeks post-axotomy and had no effect on microglia. Retinal Iba-1 positive microglia were also decreased in URB597-treated axotomized young animals and this decrease was mitigated by CB1 but not CB<em>2</em> antagonism. As seen with phagocytotic microglia, the CB<em>2</em> antagonist, AM630, increased Iba-1 positive microglia in the absence of URB597 treatment. Measurement of retinal endocannabinoid levels in URB597-treated animals at <em>2</em> weeks post-axotomy revealed a significant increase in AEA levels, accompanied by a decrease in the AEA metabolite, N-arachidonoyl glycine, in young animals but not aged animals. <em>2</em>-<em>arachidonoylglycerol</em> levels were similar across all experimental groups. These data demonstrate that URB597-mediated retinal neuroprotective effects are mediated primarily through CB1 receptors and that URB597 neuroprotective efficacy declines with age.