Restraint stress exposures evoke progressively larger increases in <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in limbic brain regions as the number of repetitions increases. The Porsolt swim test usually involves two swim exposures separated by <em>2</em>4 h, and we asked whether the <em>2</em>-AG response differed between the first and second exposures.

METHODS

Four groups of male C57/Bl6N mice were studied: control; exposed to a single 6 min swim and killed immediately; exposed to a single 6 min swim and killed <em>2</em>4 h later; and exposed to two swims, separated by <em>2</em>4 h, and killed after the second swim. Outcomes were swim behavior, serum corticosterone, and <em>2</em>-AG and <em>2</em>-oleoylglycerol (<em>2</em>-OG) contents in amygdala, hippocampus, and prefrontal cortex.

RESULTS

Mean <em>2</em>-AG contents were not significantly different among the four treatment groups in any brain region and did not correlate with immobility in either forced swim exposure. However, <em>2</em>-AG contents in all three brain regions only of the mice exposed to two swims were significantly, positively correlated with serum corticosterone concentrations measured at the same time. <em>2</em>-OG is present in brain and exhibits a striking regional heterogeneity in control mice. <em>2</em>-OG concentrations in prefrontal cortex were significantly reduced in the mice killed on the second day compared with the mice killed on the first day. As the target of <em>2</em>-OG in brain is not known, the significance of these observations await further studies.

CONCLUSIONS

Although prior exposure to swim stress does not alter brain <em>2</em>-AG contents upon re-exposure, <em>2</em>-AG concentrations in brain become significantly correlated with the hypothalamic-pituitary-adrenal (HPA) axis response to stress when prior exposure to the stress has occurred. These data suggest that even a single exposure to a short period of intense stress sensitizes the <em>2</em>-AG response to re-exposure to that situation and are consistent with a role for endocannabinoid signaling in modulating stress responses.

Endocannabinoids are small signaling lipids, with <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) implicated in modulating axonal growth and synaptic plasticity. The concept of short-range extracellular signaling by endocannabinoids is supported by the lack of trans-synaptic <em>2</em>-AG signaling in mice lacking sn-1-diacylglycerol lipases (DAGLs), synthesizing <em>2</em>-AG. Nevertheless, how far endocannabinoids can spread extracellularly to evoke physiological responses at CB₁ cannabinoid receptors (CB₁Rs) remains poorly understood. Here, we first show that cholinergic innervation of CA1 pyramidal cells of the hippocampus is sensitive to the genetic disruption of <em>2</em>-AG signaling in DAGLα null mice. Next, we exploit a hybrid COS-7-cholinergic neuron co-culture system to demonstrate that heterologous DAGLα overexpression spherically excludes cholinergic growth cones from <em>2</em>-AG-rich extracellular environments, and minimizes cell-cell contact in vitro. CB₁R-mediated exclusion responses lasted 3 days, indicating sustained spherical <em>2</em>-AG availability. Overall, these data suggest that extracellular <em>2</em>-AG concentrations can be sufficient to activate CB₁Rs along discrete spherical boundaries to modulate neuronal responsiveness.

The endogenous cannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide (AEA) play vital roles during nervous system development. The degradation of <em>2</em>-AG and AEA is mediated by monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), respectively. These enzymes are inhibited following developmental chlorpyrifos (CPF) exposure. To investigate whether this inhibition is persistent or whether accumulation of endocannabinoids in the brain occurs, 10-day-old rat pups were orally exposed daily for 7 days to either corn oil or increasing dosages of CPF (1, <em>2</em>.5, or 5mg/kg), and forebrains were collected at 4, 1<em>2</em>, <em>2</em>4, and 48h following the last administration. All dosages inhibited cholinesterase (ChE), FAAH, and MAGL, and elevated AEA and <em>2</em>-AG levels with the greatest effect occurring at 1<em>2</em>h with ChE, FAAH, AEA, and <em>2</em>-AG and at 4h with MAGL. With the high dosage, return to control levels occurred with <em>2</em>-AG (48h) only. With the medium dosage, return to control levels occurred with MAGL, <em>2</em>-AG, and AEA (48h) but not with ChE or FAAH. With the low dosage, return to control levels occurred with MAGL (1<em>2</em>h), ChE and <em>2</em>-AG (<em>2</em>4h), and AEA (48h) but not with FAAH. With the lowest dosage, peak inhibition of FAAH (5<em>2</em>%) is greater than that of ChE (<em>2</em>4%) and that level of FAAH inhibition is sufficient to induce a persistent pattern of elevated AEA. It is possible that this pattern of elevation could alter the appropriate development of neuronal brain circuits.

The diacylglycerol lipases (DAGLalpha and DAGLbeta) synthesize <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), a full agonist at cannabinoid receptors. Dynamic regulation of DAGL expression underpins its role in axonal growth and guidance during development, retrograde synaptic signalling at mature synapses, and maintenance of adult neurogenesis. We show here that DAGLalpha expression is dramatically down-regulated when neural stem (NS) cells are differentiated toward a gamma-aminobutyric acidergic neuronal phenotype. To understand how DAGLalpha expression might be controlled, we sought to identify the core promoter region and regulatory elements within it. The core promoter was identified and shown to contain both an enhancer and a suppressor region. Deletion analysis identified two elements, including a GC-box, that specifically promote expression in NS cells. Bioinformatic analysis identified three candidate transcription factors that might regulate DAGLalpha expression in NS cells by binding to the GC box; these were specificity protein 1 (Sp1), early growth response element 1 (EGR1), and zinc finger DNA-binding protein 89 (ZBP-89). However, Sp1 was the only factor that could bind to the GC-box. A specific mutation within the GC-box that inhibited Sp1 binding reduced DAGLalpha promoter activity in NS cells. Likewise, a dominant negative Sp1 was shown to bind to the GC-box and to suppress DAGLalpha promoter activity specifically in NS cells. Finally, like DAGLalpha, Sp1 was down-regulated during neuronal differentiation. A full characterization of the DAGLalpha promoter will help to elucidate the upstream pathways that regulate DAGLalpha expression in NS cells and their progeny.

Inhibitory neurons in the thalamic reticular nucleus (TRN) play a critical role in controlling information transfer between thalamus and neocortex. GABAergic synapses formed by TRN neurons contact both thalamic relay cells and neurons within TRN. These two types of synapses are thought to have distinct roles for the generation of thalamic network activity, but their selective regulation is poorly understood. In many areas throughout the brain, retrograde signaling mediated by endocannabinoids acts to dynamically regulate synaptic strength over both short and long time scales. However, retrograde signaling has never been demonstrated in the thalamus. Here, we show that depolarization-induced suppression of inhibition (DSI) is prominent at inhibitory synapses interconnecting TRN neurons. DSI is completely abolished in the presence of a cannabinoid receptor 1 (CB1R) antagonist and in mice lacking CB1Rs. DSI is prevented by DAG lipase inhibitors and prolonged by blocking the <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) degradation enzyme monoacylglycerol lipase, indicating that it is mediated by the release of <em>2</em>-AG from TRN neurons. By contrast, DSI is not observed at TRN synapses targeting thalamic relay neurons. A combination of pharmacological and immunohistochemical data indicate that the differences in endocannabinoid signaling at the two synapses are mediated by a synapse-specific targeting of CB1Rs, as well as differences in endocannabinoid release between the two target neurons. Together, our results show that endocannabinoids control transmitter release at specific thalamic synapses, and could dynamically regulate sensory information processing and thalamus-mediated synchronous oscillations.

A growing body of evidence implicates endogenous cannabinoids as modulators of the mesolimbic dopamine system and motivated behavior. Paradoxically, the reinforcing effects of Δ(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, have been difficult to detect in preclinical rodent models. In this study, we investigated the impact of THC and inhibitors of the endocannabinoid hydrolytic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on operant responding for electrical stimulation of the medial forebrain bundle [intracranial self-stimulation (ICSS)], which is known to activate the mesolimbic dopamine system. These drugs were also tested in assays of operant responding for food reinforcement and spontaneous locomotor activity. THC and the MAGL inhibitor JZL184 (4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) attenuated operant responding for ICSS and food, and also reduced spontaneous locomotor activity. In contrast, the FAAH inhibitor PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-<em>2</em>-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) was largely without effect in these assays. Consistent with previous studies showing that combined inhibition of FAAH and MAGL produces a substantially greater cannabimimetic profile than single enzyme inhibition, the dual FAAH-MAGL inhibitor SA-57 (4-[<em>2</em>-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid <em>2</em>-(methylamino)-<em>2</em>-oxoethyl ester) produced a similar magnitude of ICSS depression as that produced by THC. ICSS attenuation by JZL184 was associated with increased brain levels of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), whereas peak effects of SA-57 were associated with increased levels of both N-arachidonoylethanolamine (anandamide) and <em>2</em>-AG. The cannabinoid receptor type 1 receptor antagonist rimonabant, but not the cannabinoid receptor type <em>2</em> receptor antagonist SR1445<em>2</em>8, blocked the attenuating effects of THC, JZL184, and SA-57 on ICSS. Thus, THC, MAGL inhibition, and dual FAAH-MAGL inhibition not only reduce ICSS, but also decrease other reinforced and nonreinforced behaviors.

The need for new pain therapies that provide greater relief without unwanted side-effects drives the search for new drug targets. The identification of endogenous lipid ligands for the two known cannabinoid receptors (CB(1) and CB(<em>2</em>)) has led to numerous studies investigating the role of these endocannabinoids in pain processes. The two most widely studied endocannabinoids are anandamide (AEA; arachidonoyl ethanolamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), but there are also a number of structurally related endogenous lipid signaling molecules that are agonists at cannabinoid and noncannabinoid receptors. These lipid signaling molecules are not stored in synaptic vesicles, but are synthesized and released on-demand and act locally, as they are rapidly inactivated. This suggests that there may be therapeutic potential in modulating levels of these ligands to only have effects in active neural pathways, thereby reducing the potential for side-effects that result from widespread systemic cannabinoid receptor activation. One approach to modulate the levels and duration of action of these lipid signaling molecules is to target the enzymes responsible for their hydrolysis. The two main enzymes responsible for hydrolysis of these lipid signaling molecules are fatty acid amide hydrolase (FAAH) and monoacylglyceride lipase (MGL). This article will discuss the role of the endocannabinoid system in the modulation of pain and will review the current understanding of the properties of the hydrolytic enzymes and the recent advances in developing inhibitors for these targets, with particular relevance to the treatment of pain.

Endocannabinoids are lipid transmitters binding and activating the cannabinoid receptors. Both cannabinoid receptors and endocannabinoids, such as <em>2</em>-<em>arachidonoylglycerol</em> and anandamide, have been shown to control numerous physiological and pathological processes, including in the central nervous system. Thus regulating endocannabinoid levels in-vivo represents an interesting therapeutic perspective in several CNS-related diseases. To date four enzymes - Fatty Acid Amide Hydrolase (FAAH), N-Acylethanolamine-hydrolyzing Acid Amidase (NAAA), Monoacylglycerol Lipase (MAGL), α/β-Hydrolase Domain 6 (ABHD6) - were shown to control endocannabinoid levels in tissues or in intact cells. While the searches for NAAA and ABHD6 inhibitors are still in their beginning, a growing number of selective and potent inhibitors are now available to inhibit FAAH and MAGL activities. Here, based on the literature and patent literature, we review the compounds of the different chemical families that have been developed to inhibit these enzymes, with a special emphasis on FAAH and MAGL inhibitors.

The cannabinoid field is currently an active research area. Anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are the most characterized endogenous cannabinoids (also known as endocannabinoids). These neuromodulators have been implicated in various physiologically relevant phenomena, including mood (Witkin et al., <em>2</em>005), the immune response (Ashton, <em>2</em>007), appetite (Kirkham and Tucci, <em>2</em>006), reproduction (Wang et al., <em>2</em>006), spasticity (Pertwee, <em>2</em>00<em>2</em>), and pain (Hohmann and Suplita, <em>2</em>006). Pharmacological manipulation of AEA and <em>2</em>-AG signaling should prove to have significant therapeutic applications in disorders linked to endocannabinoid signaling. One way to alter endocannabinoid signaling is to regulate the events responsible for termination of the endocannabinoid signal-cellular uptake and metabolism. However, to pharmacologically exploit AEA and/or <em>2</em>-AG signaling in this way, we must first gain a better understanding of the proteins and mechanisms governing these processes. This review serves as an introduction to the endocannabinoid system with an emphasis on the proteins and events responsible for the termination of AEA and <em>2</em>-AG signaling.

BACKGROUND

The endocannabinoid system includes G-protein-coupled cannabinoid receptors, the endocannabinoids N-arachidonoylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em>, and multiple enzymes involved in the biosynthesis and degradation of endocannabinoids, including the anandamide metabolizing enzyme fatty acid amide hydrolase. Endocannabinoids play an important role in the physiologic control of sleep, pain processing, and emesis. The authors therefore investigated the effects of general anesthesia on the endocannabinoid system in humans.

METHODS

The authors measured whole blood levels of anandamide in 1<em>2</em> patients after induction of general anesthesia with etomidate (an agent shown to have no effect on anandamide levels) and maintenance of anesthesia with the volatile agent sevoflurane as well as in 1<em>2</em> patients undergoing total intravenous anesthesia with propofol, a known inhibitor of fatty acid amide hydrolase in the mouse brain. Anandamide levels were measured using high-performance liquid chromatography-tandem mass spectrometry at four time points (before and at 10, <em>2</em>0, 30, and 40 min after induction of anesthesia).

RESULTS

Patients of the sevoflurane group showed a significant decline in anandamide levels from induction of anesthesia to 40 min after induction, whereas anandamide levels in patients of the propofol group remained unchanged (type III sum of squares = 17<em>2</em>5.66, F = 16<em>2</em>.60, P < 0.001, repeated-measures analysis of variance).

CONCLUSIONS

General anesthesia influences the endocannabinoid system in a drug-dependent way, which may explain side effects of general anesthetics such as psychomimetic and antiemetic properties of propofol and the high incidence of postoperative nausea and vomiting after volatile anesthetics. These findings suggest new targets for anesthetic drug development.

Endocannabinoids play essential roles in synaptic plasticity; thus, their dysfunction often causes impairments in memory or cognition. However, it is not well understood whether deficits in the endocannabinoid system account for the cognitive symptoms of schizophrenia. Here, we show that endocannabinoid-mediated synaptic regulation is impaired by the prolonged elevation of neuregulin-1, the abnormality of which is a hallmark in many patients with schizophrenia. When rat hippocampal slices were chronically treated with neuregulin-1, the degradation of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), one of the major endocannabinoids, was enhanced due to the increased expression of its degradative enzyme, monoacylglycerol lipase. As a result, the time course of depolarization-induced <em>2</em>-AG signaling was shortened, and the magnitude of <em>2</em>-AG-dependent long-term depression of inhibitory synapses was reduced. Our study reveals that an alteration in the signaling of <em>2</em>-AG contributes to hippocampal synaptic dysfunction in a hyper-neuregulin-1 condition and thus provides novel insights into potential schizophrenic therapeutics that target the endocannabinoid system.

<em>2</em>-<em>Arachidonoylglycerol</em> was found to inhibit the depolarization-induced increase in [Ca<em>2</em>+]i in NG108-15 cells differentiated with prostaglandin E1 and theophylline in a dose-dependent manner. Such an effect appears to be rather specific to polyunsaturated fatty acid-containing monoacylglycerols such as <em>2</em>-<em>arachidonoylglycerol</em>. Neither <em>2</em>-palmitoylglycerol nor free arachidonic acid exhibited appreciable inhibitory activity. These observations raise the possibility that <em>2</em>-<em>arachidonoylglycerol</em> attenuates the increase in [Ca<em>2</em>+]i, thereby modulating several neural functions in this type of cell.

<em>2</em>-<em>Arachidonoylglycerol</em> is oxygenated by cyclooxygenase-<em>2</em> to form prostaglandin glyceryl esters. Previous work in this laboratory has suggested that PGE(<em>2</em>)-G activates a novel G protein-coupled receptor in a murine macrophage-like cell line, RAW <em>2</em>64.7. To probe the structural determinants for the putative receptor in RAW <em>2</em>64.7 cells, a panel of 10 analogs was tested for their ability to increase intracellular calcium. These analogs included PGE(<em>2</em>)- and PGF(<em>2</em>alpha)-ethanolamide, 4 PGE(<em>2</em>) glyceryl ester analogs, and 4 PGF(<em>2</em>alpha) glyceryl ester analogs. The glyceryl ester analogs differed in the positioning of the hydroxyl groups in the glycerol moiety and the type of linker (ester, amide, or thioester) of the prostaglandin to the glycerol moiety. Compounds were also evaluated in a human non-small cell lung cancer cell line (H1819). The glycerol moiety was required for the calcium response. All glyceryl ester analogs but not ethanolamides caused a concentration-dependent increase in calcium levels in both RAW <em>2</em>64.7 and H1819 cells. An amide or ester linkage was preferable to a thioester linkage. The EC(50) values did not significantly change when the positioning of the hydroxyls was varied. This calcium response induced by the glyceryl ester analogs appears to be independent of the putative hydrolysis products, PGE(<em>2</em>) and PGF(<em>2</em>alpha), and appears to represent a novel signaling pathway.

Sustained exposure to stress or corticosteroids is known to cause changes in brain endocannabinoid (eCB) signaling, such that tissue contents of the eCBs N-arachidonylethanolamine (AEA) are generally reduced while <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) levels increase. These changes in eCB signaling are important for many of the aspects of chronic stress, such as anxiety, reward sensitivity and stress adaptation, yet the mechanisms mediating these changes are not fully understood. We have recently found that the stress-related neuropeptide corticotropin-releasing hormone (CRH), acting through the CRH type 1 receptor (CRHR1), can reduce AEA content by increasing its hydrolysis by the enzyme fatty acid amide hydrolase (FAAH) as well as increase <em>2</em>-AG contents. As extra-hypothalamic CRH is upregulated by chronic corticosteroid or stress exposure, we hypothesized that increased CRH signaling through CRHR1 contributes to the effects of chronic corticosteroid exposure on the eCB system within the amygdala and prefrontal cortex. Male rats were exposed to 7 days of systemic corticosterone capsules, with or without concurrent exposure to a CRHR1 antagonist, after which we examined eCB content. Consistent with previous studies in the amygdala, sustained corticosterone exposure increases CRH mRNA in the prefrontal cortex. As was shown previously, FAAH activity was increased and AEA contents were reduced within the amygdala and prefrontal cortex following chronic corticosterone exposure. Chronic corticosterone exposure also elevated <em>2</em>-AG content in the prefrontal cortex but not the amygdala. These corticosteroid-driven changes were all blocked by systemic CRHR1 antagonism. Consistent with these data indicating sustained increases in CRH signaling can mediate the effects of chronic elevations in corticosteroids, CRH overexpressing mice also exhibited increased FAAH-mediated AEA hydrolysis in the amygdala and prefrontal cortex compared to wild type. CRH overexpression increased <em>2</em>-AG content in the amygdala, but not the prefrontal cortex. These data indicate that chronic elevations in CRH signaling, as is seen following exposure to chronic elevations in corticosterone or stress, drive persistent changes in eCB function. As reductions in AEA signaling mediate the effects of CRH and chronic stress on anxiety, these data provide a mechanism linking these processes.

Endocannabinoids (ECs), such as anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), modulate a number of physiological processes, including pain, appetite and emotional state. Levels of ECs are tightly controlled by enzymatic biosynthesis and degradation in vivo. However, there is limited knowledge about the enzymes that terminate signaling of the major brain EC, <em>2</em>-AG. Identification and quantification of <em>2</em>-AG, 1-AG and arachidonic acid (AA) is important for studying the enzymatic hydrolysis of <em>2</em>-AG. We have developed a sensitive and specific quantification method for simultaneous determination of <em>2</em>-AG, 1-AG and AA from mouse brain and adipose tissues by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a simple brain sample preparation method. The separations were carried out based on reversed phase chromatography. Optimization of electrospray ionization conditions established the limits of detection (S/N = 3) at 50, <em>2</em>5 and 65 fmol for <em>2</em>-AG, 1-AG and AA, respectively. The methods were selective, precise (%R.S.D. < 10%) and sensitive over a range of 0.0<em>2</em>-<em>2</em>0, 0.01-10 and 0.05-50 ng/mg tissue for <em>2</em>-AG, 1-AG and AA, respectively. The quantification method was validated with consideration of the matrix effects and the mass spectrometry (MS) responses of the analytes and the deuterium labeled internal standard (IS). The developed methods were applied to study the hydrolysis of <em>2</em>-AG from mouse brain extracts containing membrane bound monoacylglycerol lipase (MAGL), and to measure the basal levels of <em>2</em>-AG, 1-AG and AA in mouse brain and adipose tissues.

Diacylglycerol lipase α is the key enzyme in the formation of the most prevalent endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> in the brain. In this study we identified the catalytic triad of diacylglycerol lipase α, consisting of serine 47<em>2</em>, aspartate 5<em>2</em>4 and histidine 650. A truncated version of diacylglycerol lipase α, spanning residues 1-687 retains complete catalytic activity suggesting that the C-terminal domain is not required for catalysis. We also report the discovery and the characterization of fluorogenic and chromogenic substrates for diacylglycerol lipase α. Assays performed with these substrates demonstrate equipotent inhibition of diacylglycerol lipase α by tetrahydrolipastatin and RHC-<em>2</em>0867 as compared to reactions performed with the native diacylglycerol substrate. Thus, confirming the utility of assays using these substrates for identification and kinetic characterization of inhibitors from pharmaceutical collections.

Endocannabinoids are amides and esters of long chain fatty acids that can modulate ion channels through both receptor-dependent and receptor-independent effects. Nowadays, their effects on cardiac K(+) channels are unknown even when they can be synthesized within the heart. We have analyzed the direct effects of endocannabinoids, such as anandamide (AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), the endogenous lipid lysophosphatidylinositol, and cannabinoid analogues such as palmitoylethanolamide (PEA), and oleoylethanolamide, as well as the fatty acids from which they are endogenously synthesized, on human cardiac Kv4.3 channels, which generate the transient outward K(+) current (I(to1)). Currents were recorded in Chinese hamster ovary cells, which do not express cannabinoid receptors, by using the whole-cell patch-clamp. All these compounds inhibited I(Kv4.3) in a concentration-dependent manner, AEA and <em>2</em>-AG being the most potent (IC(50) approximately 0.3-0.4 microM), while PEA was the least potent. The potency of block increased as the complexity and the number of C atoms in the fatty acyl chain increased. The effects were not mediated by modifications in the lipid order and microviscosity of the membrane and were independent of the presence of MiRP<em>2</em> or DPP6 subunits in the channel complex. Indeed, effects produced by AEA were reproduced in human atrial I(to1) recorded in isolated myocytes. Moreover, AEA effects were exclusively apparent when it was applied to the external surface of the cell membrane. These results indicate that at low micromolar concentrations the endocannabinoids AEA and <em>2</em>-AG directly block human cardiac Kv4.3 channels, which represent a novel molecular target for these compounds.

In regenerative medicine, platelet by-products containing factors physiologically involved in wound healing, have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects. Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide, <em>2</em>-<em>arachidonoylglycerol</em>, palmitoylethanolamide, and oleoylethanolamide. Investigation of the activity of PRP on the keratinocyte cell line NCTC<em>2</em>544 in physiological and inflammatory conditions showed that, under inflammatory conditions, PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue. Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated a potent antinociceptive effect against both early and late nocifensive responses. This effect was observed following intrapaw injection of (1) total PRP; (<em>2</em>) lipids extracted from PRP; and (3) an endocannabinoid-enriched lipid fraction of PRP. In all conditions, antagonists of endocannabinoid CB1 and CB<em>2</em> receptors, injected in the paw, abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action. In conclusion, we showed that PRP and PRP lipid extract exert a potent antinociceptive activity linked, at least in part, to their endocannabinoids and related compound content, and to their capability of elevating the levels of these lipid mediators in cells.

Complementary genetic and pharmacological approaches to inhibit monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), the primary hydrolytic enzymes of the respective endogenous cannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and N-arachidonoylethanolamine, enable the exploration of potential therapeutic applications and physiologic roles of these enzymes. Complete and simultaneous inhibition of both FAAH and MAGL produces greatly enhanced cannabimimetic responses, including increased antinociception, and other cannabimimetic effects, far beyond those seen with inhibition of either enzyme alone. While cannabinoid receptor type 1 (CB1) function is maintained following chronic FAAH inactivation, prolonged excessive elevation of brain <em>2</em>-AG levels, via MAGL inhibition, elicits both behavioral and molecular signs of cannabinoid tolerance and dependence. Here, we evaluated the consequences of a high dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate; 40 mg/kg] given acutely or for 6 days in FAAH(-/-) and (+/+) mice. While acute administration of JZL184 to FAAH(-/-) mice enhanced the magnitude of a subset of cannabimimetic responses, repeated JZL184 treatment led to tolerance to its antinociceptive effects, cross-tolerance to the pharmacological effects of Δ(9)-tetrahydrocannabinol, decreases in CB1 receptor agonist-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding, and dependence as indicated by rimonabant-precipitated withdrawal behaviors, regardless of genotype. Together, these data suggest that simultaneous elevation of both endocannabinoids elicits enhanced cannabimimetic activity but MAGL inhibition drives CB1 receptor functional tolerance and cannabinoid dependence.

Parkinson's disease (PD) is a common chronic neurodegenerative disorder, usually of idiopathic origin. Symptoms including tremor, bradykinesia, rigidity and postural instability are caused by the progressive loss of dopaminergic neurons in the nigrostriatal region of the brain. Symptomatic therapies are available but no treatment slows or prevents the loss of neurons. Neuroinflammation has been implicated in its pathogenesis. To this end, the present study utilises the 1-methyl-4-phenyl-1,<em>2</em>,3,6-tetrahydropyridine (MPTP) neurotoxin to reproduce the pattern of cell death evident in PD patients. Herein, the role of a potential regulator of an immune response, the endocannabinoid system (ECS), is investigated. The most prevalent endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) (3 and 5mg/kg), was added exogenously and its enzymatic degradation inhibited to provide protection against MPTP-induced cell death. Furthermore, the addition of DFU (<em>2</em>5mg/kg), a selective inhibitor of inflammatory mediator cyclooxygenase-<em>2</em> (COX-<em>2</em>), potentiated these effects. Levels of <em>2</em>-AG were shown to be upregulated in a time- and region-specific manner following MPTP administration, indicating that the ECS represents a natural defence mechanism against inflammation, potentiation of which could provide therapeutic benefits. The results expand the current understanding of the role that this signalling system has and its potential influence in PD.

The mechanisms by which cannabinoids alter coronary vascular tone and cardiac performance are controversial. We investigated the effects of various cannabinoids in spontaneously beating Langendorff-perfused rat hearts. Bolus injections of anandamide (0.1-1 micromol) caused no change in coronary flow (CF) or left ventricular systolic pressure (LVSP). In hearts preperfused with vasopressin to induce vasoconstrictor tone, anandamide or the selective CB1 receptor agonist ACEA (1-100 nmol) dose-dependently increased CF by up to <em>2</em>67% and LVSP by <em>2</em>0 mm Hg. The metabolically stable endocannabinoid derivatives, R-methanandamide and noladin ether, displayed similar effects. In contrast, Delta-THC (10-100 nmol), the major psychoactive ingredient of cannabis, strongly decreased CF and LVSP. The CB<em>2</em> receptor agonist JWH-133 (10-100 nmol) elicited vasodilator and positive inotropic effects only at higher doses. The CB1 antagonists SR141716A and AM-<em>2</em>51 as well as the potassium channel inhibitors tetraethylammonium and iberiotoxin blocked the anandamide-induced increases in CF and LVSP, whereas the CB<em>2</em> antagonist SR1445<em>2</em>8 and the putative "CB3 antagonist" O-1918 did not have an inhibitory effect. Immunohistochemistry revealed the presence of cardiac CB1 but no CB<em>2</em> receptors. Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> were detected in heart tissue. However, combined application of fatty acid amidohydrolase inhibitors and the transport inhibitor AM-404 to augment tissue levels of endocannabinoids was without effect on CF or LVSP. We conclude that in the rat isolated heart with reestablished vasoconstrictor tone, cannabinoids including anandamide elicit coronary vasodilation and a secondary increase in contractility via CB1 receptors and potassium channels.

Exogenous application of neural progenitor cells (NPCs) has successful implications in treating brain disorders, and research is beginning to identify ways to mimic this exogenous application by activating endogenous stem cell compartments. The recent discovery of a functional endocannabinoid system in murine NPCs (mNPCs) represents one potential therapeutic means to influence endogenous stem cell compartments. High levels of the endogenous cannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) persist during CNS inflammation and infection. The goal of this study was to assess the influence of AEA on mNPCs to identify how the endocannabinoid system influences mNPCs in vitro, a potential model to investigate effects of endocannabinoids on endogenous stem cell compartments. Our results show that AEA affects mNPC cell fate determination. Initial glial differentiation was observed, followed by induction of neuronal differentiation with AEA treatment. Cell survival and apoptosis was not affected by AEA. These effects were coupled by an increased phosphorylation of cAMP-responsive element (CRE) binding protein (CREB).

Growing evidence supports the pivotal role played by oxidative stress in tissue injury development, thus resulting in several pathologies including cardiovascular, renal, neuropsychiatric, and neurodegenerative disorders, all characterized by an altered oxidative status. Reactive oxygen and nitrogen species and lipid peroxidation-derived reactive aldehydes including acrolein, malondialdehyde, and 4-hydroxy-<em>2</em>-nonenal, among others, are the main responsible for cellular and tissue damages occurring in redox-dependent processes. In this scenario, a link between the endocannabinoid system (ECS) and redox homeostasis impairment appears to be crucial. Anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, the best characterized endocannabinoids, are able to modulate the activity of several antioxidant enzymes through targeting the cannabinoid receptors type 1 and <em>2</em> as well as additional receptors such as the transient receptor potential vanilloid 1, the peroxisome proliferator-activated receptor alpha, and the orphan G protein-coupled receptors 18 and 55. Moreover, the endocannabinoids lipid analogues N-acylethanolamines showed to protect cell damage and death from reactive aldehydes-induced oxidative stress by restoring the intracellular oxidants-antioxidants balance. In this review, we will provide a better understanding of the main mechanisms triggered by the cross-talk between the oxidative stress and the ECS, focusing also on the enzymatic and non-enzymatic antioxidants as scavengers of reactive aldehydes and their toxic bioactive adducts.

Treatment of intact human neuroblastoma CHP100 cells with anandamide (arachidonoylethanolamide, AEA) or <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) inhibits intracellular fatty acid amide hydrolase (FAAH). This effect was not associated with covalent modifications of FAAH, since specific inhibitors of farnesyltransferase, kinases, phosphatases, glycosyltransferase or nitric oxide synthase were ineffective. Electrophoretic analysis of (33)P-labelled proteins, Western blot with anti-phosphotyrosine antibodies, and glycan analysis of cellular proteins confirmed the absence of covalent modifications of FAAH. The inhibition by AEA was paralleled by an increased arachidonate release, which was not observed upon treatment of cells with linoleoylethanolamide, palmitoylethanolamide, or oleoylethanolamide. Moreover, cell treatment with AEA or <em>2</em>-AG increased the activity of cyclooxygenase and 5-lipoxygenase, and the hydro(pero)xides generated from arachidonate by lipoxygenase were shown to inhibit FAAH, with inhibition constants in the low micromolar range. Consistently, inhibitors of 5-lipoxygenase, but not those of cyclooxygenase, significantly counteracted the inhibition of FAAH by AEA or <em>2</em>-AG.