Finding of endocannabinoids in human eye tissues: implications for glaucoma

Cannabinoid CB(1) receptors are involved in ocular physiology and may regulate intraocular pressure (IOP). However, endocannabinoid levels in human ocular tissues of cornea, iris, ciliary body, retina, and choroid from normal and glaucomatous donors have not been investigated. Anandamide (N-arachidonoylethanolamine; AEA), 2-arachidonoylglycerol (2-AG), and the anandamide congener, palmitoylethanolamide (PEA), were detected in all the human tissues examined. In eyes from patients with glaucoma, significantly decreased 2-AG and PEA levels were detected in the ciliary body, an important tissue in the regulation of IOP. The findings suggest that these endogenous compounds may have a role in this disease, particularly with respect to regulation of IOP.     

Changes in endocannabinoid and palmitoylethanolamide levels in eye tissues of patients with diabetic retinopathy and age-related macular degeneration

Cannabinoid receptors and the endocannabinoids (anandamide (N-arachidonoylethanolamine--AEA) and 2-arachidonoylglycerol (2-AG)), as well as the AEA congener, palmitoylethanolamide (PEA), are involved in ocular physiology. We measured endocannabinoid and PEA levels by isotope-dilution liquid chromatography-mass spectrometric analysis in post-mortem eye tissues of patients with diabetic retinopathy (DR) or age-related macular degeneration (AMD). In eyes with DR, significantly enhanced levels of AEA were found in the retina ( approximately 1.8-fold), ciliary body ( approximately 1.5-fold) and, to a lesser extent, cornea ( approximately 1.3-fold). Surprisingly, 2-AG levels were significantly higher ( approximately 3-fold) only in the iris, whereas PEA levels only slightly increased ( approximately 1.3-fold) in the ciliary body. In eyes with AMD, significantly enhanced levels of AEA were found in the choroid ( approximately 1.3-fold), ciliary body ( approximately 1.4-fold) and cornea ( approximately 1.4-fold), whereas in the retina only a trend towards an increase ( approximately 1.5-fold) was observed. The tissue- and disease-selective nature of the changes observed suggests that the compounds analyzed here may play different roles in the control of eye function under different pathological conditions.     

Endocannabinoids control spasticity in a multiple sclerosis model.

Spasticity is a complicating sign in multiple sclerosis that also develops in a model of chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice. In areas associated with nerve damage, increased levels of the endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and of the AEA congener, palmitoylethanolamide (PEA), were detected here, whereas comparable levels of these compounds were found in normal and non-spastic CREAE mice. While exogenously administered endocannabinoids and PEA ameliorate spasticity, selective inhibitors of endocannabinoid re-uptake and hydrolysis-probably through the enhancement of endogenous levels of AEA, and, possibly, 2-arachidonoyl glycerol-significantly ameliorated spasticity to an extent comparable with that observed previously with potent cannabinoid receptor agonists. These studies provide definitive evidence for the tonic control of spasticity by the endocannabinoid system and open new horizons to therapy of multiple sclerosis, and other neuromuscular diseases, based on agents modulating endocannabinoid levels and action, which exhibit little psychotropic activity.     

A Progressive Loss of phosphoSer138-Profilin Aligns with Symptomatic Course in the R6/2 Mouse Model of Huntington’s Disease: Possible Sex-Dependent Signaling

The R6/2 transgenic mouse model of Huntington’s disease (HD) carries several copies of exon1 of the huntingtin gene that contains a highly pathogenic 120 CAG-repeat expansion. We used kinome analysis to screen for kinase activity patterns in neural tissues from wildtype (WT) and R6/2 mice at a pre-symptomatic (e.g., embryonic) and symptomatic (e.g., between 3 and 10 weeks postnatal) time points. We identified changes in several signaling cascades, for example, the Akt/FoxO3/CDK2, mTOR/ULK1, and RAF/MEK/CREB pathways. We also identified the Rho-Rac GTPase cascade that contributes to cytoskeleton organization through modulation of the actin-binding proteins, cofilin and profilin. Immunoblotting revealed higher levels of phosphoSer138-profilin in embryonic R6/2 mouse samples (cf. WT mice) that diminish progressively and significantly over the postnatal, symptomatic course of the disease. We detected sex- and genotype-dependent patterns in the phosphorylation of actin-regulators such a ROCK2, PAK, LIMK1, cofilin, and SSH1L, yet none of these aligned consistently with the changing levels of phosphoSer138-profilin. This could be reflecting an imbalance in the sequential influences these regulators are known to exert on actin signaling. The translational potential of these observations was inferred from preliminary observations of changes in LIMK-cofilin signaling and loss of neurite integrity in neural stem cells derived from an HD patient (versus a healthy control). Our observations suggest that a pre-symptomatic, neurodevelopmental onset of change in the phosphorylation of Ser138-profilin, potentially downstream of distinct signaling changes in male and female mice, could be contributing to cytoskeletal phenotypes in the R6/2 mouse model of HD pathology.

     

Lipopolysaccharide induces anandamide synthesis in macrophages via CD14/MAPK/phosphoinositide 3-kinase/NF-kappaB independently of platelet-activating factor

Macrophage-derived endocannabinoids have been implicated in endotoxin (lipopolysaccharide (LPS))-induced hypotension, but the endocannabinoid involved and the mechanism of its regulation by LPS are unknown. In RAW264.7 mouse macrophages, LPS (10 ng/ml) increases anandamide (AEA) levels >10-fold via CD14-, NF-kappaB-, and p44/42-dependent, platelet-activating factor-independent activation of the AEA biosynthetic enzymes, N-acyltransferase and phospholipase D. LPS also induces the AEA-degrading enzyme fatty acid amidohydrolase (FAAH), and inhibition of FAAH activity potentiates, whereas actinomycin D or cycloheximide blocks the LPS-induced increase in AEA levels and N-acyltransferase and phospholipase D activities. In contrast, cellular levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are unaffected by LPS but increased by platelet-activating factor. LPS similarly induces AEA, but not 2-AG, in mouse peritoneal macrophages where basal AEA levels are higher, and the LPS-stimulated increase in AEA is potentiated in cells from FAAH-/- as compared with FAAH+/+ mice. Intravenous administration of 107 LPS-treated mouse macrophages to anesthetized rats elicits hypotension, which is much greater in response to FAAH-/- than FAAH+/+ cells and is susceptible to inhibition by SR141716, a cannabinoid CB1 receptor antagonist. We conclude that AEA and 2-AG synthesis are differentially regulated in macrophages, and AEA rather than 2-AG is a major contributor to LPS-induced hypotension.     

Release of Endocannabinoids into the Cerebrospinal Fluid during the Induction of the Trigemino-Hypoglossal Reflex in Rats

The endocannabinoid system (ECS) plays an important role in pain processing and modulation. Since the specific effects of endocannabinoids within the orofacial area are largely unknown, we aimed to determine whether an increase in the endocannabinoid concentration in the cerebrospinal fluid (CSF) caused by the peripheral administration of the FAAH inhibitor URB597 and tooth pulp stimulation would affect the transmission of impulses between the sensory and motor centers localized in the vicinity of the third and fourth cerebral ventricles. The study objectives were evaluated on rats using a method that allowed the recording of the amplitude of evoked tongue jerks (ETJ) in response to noxious tooth pulp stimulation and URB597 treatment. The amplitude of ETJ was a measure of the effect of endocannabinoids on the neural structures. The concentrations of the endocannabinoids tested (AEA and 2-AG) were determined in the CSF, along with the expression of the cannabinoid receptors (CB1 and CB2) in the tissues of the mesencephalon, thalamus, and hypothalamus. We demonstrated that anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), was significantly increased in the CSF after treatment with a FAAH inhibitor, while tooth pulp stimulation had no effect on the AEA and 2-AG concentrations in the CSF. We also found positive correlations between the CSF AEA concentration and cannabinoid receptor type 1 (CB1R) expression in the brain, and between 2-AG and cannabinoid receptor type 2 (CB2R), and negative correlations between the CSF concentration of AEA and brain CB2R expression, and between 2-AG and CB1R. Our study shows that endogenous AEA, which diffuses through the cerebroventricular ependyma into CSF and exerts a modulatory effect mediated by CB1Rs, alters the properties of neurons in the trigeminal sensory nuclei, interneurons, and motoneurons of the hypoglossal nerve. In addition, our findings may be consistent with the emerging concept that AEA and 2-AG have different regulatory mechanisms because they are involved differently in orofacial pain. We also suggest that FAAH inhibition may offer a therapeutic approach to the treatment of orofacial pain.     

Sex-dependent effects of neonatal maternal deprivation on endocannabinoid levels in the adipose tissue: influence of diet

Maternal deprivation (MD) during neonatal life has diverse long-term effects, including modification of metabolism. We have previously reported that MD modifies the metabolic response to high-fat diet (HFD) intake, with this response being different between males and females, while previous studies indicate that in mice with HFD-induced obesity, endocannabinoid (EC) levels are markedly altered in various brown and white adipose tissue depots. Here, we analyzed the effects of MD (24 h at postnatal day 9), alone or in combination with a HFD from weaning until the end of the experiment in Wistar rats of both sexes. Brown and white perirenal and subcutaneous adipose tissues were collected and the levels of anandamide (AEA), 2-arachidonoylglycerol (2-AG), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) were determined. In males, MD increased the content of OEA in brown and 2-AG in subcutaneous adipose tissues, while in females the content of 2-AG was increased in perirenal fat. Moreover, in females, MD decreased AEA and OEA levels in perirenal and subcutaneous adipose tissues, respectively. HFD decreased the content of 2-AG in brown fat of both sexes and OEA in brown and subcutaneous adipose tissue of control females. In contrast, in subcutaneous fat, HFD increased AEA levels in MD males and OEA levels in control and MD males. The present results show for the first time that MD and HFD induce sex-dependent effects on the main ECs, AEA, and 2-AG, and of AEA-related mediators, OEA and PEA, in the rat brown and white (visceral and subcutaneous) adipose tissues.     

Cannabimimetic fatty acid derivatives in cancer and inflammation

Evidence for the role of the cannabimimetic fatty acid derivatives (CFADs), i.e. anandamide (arachidonoylethanolamide, AEA), 2-arachidonoylglycerol (2-AG) and palmitoylethanolamide (PEA), in the control of inflammation and of the proliferation of tumor cells is reviewed here. The biosynthesis of AEA, PEA, or 2-AG can be induced by stimulation with either Ca(2+) ionophores, lipopolysaccharide, or platelet activating factor in macrophages, and by ionomycin or antigen challenge in rat basophilic leukemia (RBL-2H3) cells (a widely used model for mast cells). These cells also inactivate CFADs through re-uptake and/or hydrolysis and/or esterification processes. AEA and PEA modulate cytokine and/or arachidonate release from macrophages in vitro, regulate serotonin secretion from RBL-2H3 cells, and are analgesic in some animal models of inflammatory pain. However, the involvement of endogenous CFADs and cannabinoid CB(1) and CB(2) receptors in these effects is still controversial. In human breast and prostate cancer cells, AEA and 2-AG, but not PEA, potently inhibit prolactin and/or nerve growth factor (NGF)-induced cell proliferation. Vanillyl-derivatives of anandamide, such as olvanil and arvanil, exhibit even higher anti-proliferative activity. These effects are due to suppression of the levels of the 100 kDa prolactin receptor or of the high affinity NGF receptors (trk), are mediated by CB(1)-like cannabinoid receptors, and are enhanced by other CFADs. Inhibition of adenylyl cyclase and activation of mitogen-activated protein kinase underlie the anti-mitogenic actions of AEA. The possibility that CFADs act as local inhibitors of the proliferation of human breast cancer is discussed here.     

Changes in endocannabinoid and palmitoylethanolamide levels in eye tissues of patients with diabetic retinopathy and age-related macular degeneration

Cannabinoid receptors and the endocannabinoids (anandamide (N-arachidonoylethanolamine—AEA) and 2-arachidonoylglycerol (2-AG)), as well as the AEA congener, palmitoylethanolamide (PEA), are involved in ocular physiology. We measured endocannabinoid and PEA levels by isotope-dilution liquid chromatography-mass spectrometric analysis in post-mortem eye tissues of patients with diabetic retinopathy (DR) or age-related macular degeneration (AMD). In eyes with DR, significantly enhanced levels of AEA were found in the retina (∼1.8-fold), ciliary body (∼1.5-fold) and, to a lesser extent, cornea (∼1.3-fold). Surprisingly, 2-AG levels were significantly higher (∼3-fold) only in the iris, whereas PEA levels only slightly increased (∼1.3-fold) in the ciliary body. In eyes with AMD, significantly enhanced levels of AEA were found in the choroid (∼1.3-fold), ciliary body (∼1.4-fold) and cornea (∼1.4-fold), whereas in the retina only a trend towards an increase (∼1.5-fold) was observed. The tissue- and disease-selective nature of the changes observed suggests that the compounds analyzed here may play different roles in the control of eye function under different pathological conditions.     

Dynamic changes to the endocannabinoid system in models of chronic pain

The analgesic effects of cannabinoid ligands, mediated by CB1 receptors are well established. However, the side-effect profile of CB1 receptor ligands has necessitated the search for alternative cannabinoid-based approaches to analgesia. Herein, we review the current literature describing the impact of chronic pain states on the key components of the endocannabinoid receptor system, in terms of regionally restricted changes in receptor expression and levels of key metabolic enzymes that influence the local levels of the endocannabinoids. The evidence that spinal CB2 receptors have a novel role in the modulation of nociceptive processing in models of neuropathic pain, as well as in models of cancer pain and arthritis is discussed. Recent advances in our understanding of the spinal location of the key enzymes that regulate the levels of the endocannabinoid 2-AG are discussed alongside the outcomes of recent studies of the effects of inhibiting the catabolism of 2-AG in models of pain. The complexities of the enzymes capable of metabolizing both anandamide (AEA) and 2-AG have become increasingly apparent. More recently, it has come to light that some of the metabolites of AEA and 2-AG generated by cyclooxygenase-2, lipoxygenases and cytochrome P450 are biologically active and can either exacerbate or inhibit nociceptive signalling.     

The role of dopamine in motor symptoms in the R6/2 transgenic mouse model of Huntington's disease

In both Huntington's disease (HD) patients and genetic mouse models of HD, there is a pre-symptomatic loss of dopamine (DA) receptors, suggesting that dysfunctional dopaminergic neurotransmission may be involved in early HD presentation. However, the role of DA in HD symptoms is not fully understood. In this study, we examined the possibility that dysfunctional dopaminergic neurotransmission contributes to the progressive decline in motor function of a transgenic mouse model of HD (R6/2 line). We found that R6/2 mice display an age-dependent abnormal behavioural response to (+)-methamphetamine (METH) and a dose-dependent increase in sensitivity to METH toxicity compared with wild-type (WT) mice. R6/2 mice also showed an attenuated response to cocaine, indicating that DA release may be compromised. Striatal DA levels were reduced in R6/2 mice by 9 weeks of age. Replacement of DA by chronic treatment with laevodopa (L-DOPA, administered as Sinemet) caused short-term improvements in activity and rearing behaviour, and abolished abnormal spontaneous hindlimb grooming. However, long-term treatment with L-DOPA had deleterious effects on survival and rotarod performance of R6/2 mice. These results suggest that dysfunctional DA neurotransmission contributes to phenotype development in R6/2 mice and thus also may be important in symptom progression in HD.     

The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB₁and CB₂receptors in the mouse hippocampus

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.     

Quantitative profiling of endocannabinoids and related compounds in rat brain using liquid chromatography-tandem electrospray ionization mass spectrometry

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous identification and quantification of eight endocannabinoid (EC) or related "entourage" compounds in rat brain tissue. Analytes were extracted and purified from rat brain tissue using an ethyl acetate/hexane solvent extraction, followed by a solid phase extraction (SPE) protocol. Chromatographic separation was achieved using a gradient elution, with a mobile phase of acetonitrile, formic acid, and ammonium acetate, at pH 3.6. A Thermo Hypersil C8 HyPurity Advance column (100x2.1 mm i.d., 3 microm) was used with a flow rate of 0.3 ml/min). Anandamide (AEA), 2-arachidonyl glycerol (2-AG), 2-arachidonylglyceryl ether (noladin ether), O-arachidonyl ethanolamide (virodhamine), 2-linoleoyl glycerol (2-LG), arachidonyl glycine, oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA) were quantified by positive ion tandem electrospray ionization mass spectrometry. Internal standards were deuterated AEA, deuterated 2-AG, and heptadecanoyl ethanolamide (HEA). Linearity was proven over the range of 25 fmol to 250 pmol, with a limit of detection of 25 fmol on column for all analytes except 2-AG, noladin ether, and 2-LG (250 fmol). This corresponded to a limit of quantification in biological tissue of 10 pmol/g for all analytes except 2-AG (100 pmol/g). Intra- and interday precision in biological tissue was routinely approximately 20% or lower, and accuracy was between 65% and 155%. This method was used to quantitatively profile regional differences in nine discrete rat brain regions for AEA, 2-AG, 2-LG, OEA, PEA, noladin ether, virodhamine, and arachidonyl glycine.     

Hind limb suspension and long-chain omega-3 PUFA increase mRNA endocannabinoid system levels in skeletal muscle

Muscle disuse has numerous physiological consequences that end up with significant catabolic metabolism and ultimately tissue atrophy. What is not known is how muscle atrophy affects the endocannabinoid (EC) system. Arachidonic acid (AA) is the substrate for anandamide (AEA) and 2-arachidonylgycerol (2-AG), which act as agonists for cannabinoid receptors CB1 and CB2 found in muscle. Diets with n-3 polyunsaturated fatty acids (PUFA) have been shown to reduce tissue levels of AA, AEA and 2-AG. Therefore, we hypothesized that hind limb suspension (HS)-induced muscle atrophy and intake of n-3 PUFA will change mRNA levels of the EC system. Mice were randomized and assigned to a moderate n-3 PUFA [11.7 g/kg eicosapentaenoic acid (EPA)+docosahexaenoic acid (DHA)], high n-3 PUFA (17.6 g/kg EPA+DHA) or control diets for 12 days and then subjected to HS or continued weight bearing (WB) for 14 days. HS resulted in body weight, epididymal fat pad and quadriceps muscle loss compared to WB. Compared to WB, HS had greater mRNA levels of AEA and 2-AG synthesis enzymes and CB2 in the atrophied quadriceps muscle. The high n-3 PUFA diet resulted in greater mRNA levels of EC synthesis enzymes, and CB1 and CB2. The higher mRNA levels for EC with HS and dietary n-3 PUFA suggest that muscle disuse and diet induce changes in the EC system to sensitize muscle in response to metabolic and physiological consequences of atrophy.     

Endocannabinoids Are Expressed in Bone Marrow Stromal Niches and Play a Role in Interactions of Hematopoietic Stem and Progenitor Cells with the Bone Marrow Microenvironment

Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB₁ and CB₂. The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB₁ receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10⁷ cells), and 2-AG (75.2 ng/10⁷ cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10⁷ cells) and 2-AG (98.8 ng/10⁷ cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB₁ agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1^−/− showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)^−/− mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1^−/−, as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.     

The endocannabinoid system in neurodegeneration

Endocannabinoids are bioactive lipids, that comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the best studied endocannabinoids, and act as agonists of cannabinoid receptors. Thus, AEA and 2-AG mimic several pharmacological effects of the exogenous cannabinoid delta9-tetrahydrocannabinol, the psychoactive principle of hashish and marijuana. It is known that the activity of endocannabinoids at their receptors is limited by cellular uptake through specific membrane transporters, followed by intracellular degradation by a fatty acid amide hydrolase (for AEA and partly 2-AG) or by a monoacylglycerol lipase (for 2-AG). Together with AEA, 2-AG and congeners, the proteins that bind, transport and metabolize these lipids form the "endocannabinoid system". This new system will be briefly presented in this review, in order to put in a better perspective the role of the endocannabinoid pathway in neurodegenerative disorders, like Parkinson's disease, Huntington's disease, and multiple sclerosis. In addition, the potential exploitation of antagonists of endocannabinoid receptors, or of inhibitors of endocannabinoid metabolism, as next-generation therapeutics will be discussed.     

Lipidomics profile of a NAPE-PLD KO mouse provides evidence of a broader role of this enzyme in lipid metabolism in the brain

A leading hypothesis of N-acyl ethanolamine (NAE) biosynthesis, including the endogenous cannabinoid anandamide (AEA), is that it depends on hydrolysis of N-acyl-phosphatidylethanolamines (NAPE) by a NAPE-specific phospholipase D (NAPE-PLD). Thus, deletion of NAPE-PLD should attenuate NAE levels. Previous analyses of two different NAPE-PLD knockout (KO) strains produced contradictory data on the importance of NAPE-PLD to AEA biosynthesis. Here, we examine this hypothesis with a strain of NAPE-PLD KO mice whose lipidome is uncharacterized. Using HPLC/MS/MS, over 70 lipids, including the AEA metabolite, N-arachidonoyl glycine (NAGly), the endocannabinoid 2-arachidonyl glycerol (2-AG) and prostaglandins (PGE₂ and PGF_2α), and over 60 lipoamines were analyzed in 8 brain regions of KO and wild-type (WT) mice.Lipidomics analysis of this third NAPE-PLD KO strain shows a broad range of lipids that were differentially affected by lipid species and brain region. Importantly, all 6 NAEs measured were significantly reduced, though the magnitude of the effect varied by fatty acid saturation length and brain region. 2-AG levels were only impacted in the brainstem, where levels were significantly increased in KO mice. Correspondingly, levels of arachidonic acid were significantly decreased exclusively in brainstem. NAGly levels were significantly increased in 4 brain regions and levels of PGE₂ increased in 6 of 8 brain regions in KO mice. These data indicate that deletion of NAPE-PLD has far broader effects on the lipidome than previously recognized. Therefore, behavioral characteristics of suppressing NAPE-PLD activity may be due to a myriad of effects on lipids and not simply due to reduced AEA biosynthesis.     

Effect of lipid rafts on Cb2 receptor signaling and 2-arachidonoyl-glycerol metabolism in human immune cells

Recently, we have shown that treatment of rat C6 glioma cells with the raft disruptor methyl-beta-cyclodextrin (MCD) doubles the binding of anandamide (AEA) to type-1 cannabinoid receptors (CB1R), followed by CB1R-dependent signaling via adenylate cyclase and p42/p44 MAPK activity. In the present study, we investigated whether type-2 cannabinoid receptors (CB2R), widely expressed in immune cells, also are modulated by MCD. We show that treatment of human DAUDI leukemia cells with MCD does not affect AEA binding to CB2R, and that receptor activation triggers similar [35S]guanosine-5'-O-(3-thiotriphosphate) binding in MCD-treated and control cells, similar adenylate cyclase and MAPK activity, and similar MAPK-dependent protection against apoptosis. The other AEA-binding receptor transient receptor potential channel vanilloid receptor subunit 1, the AEA synthetase N-acyl-phosphatidylethanolamine-phospholipase D, and the AEA hydrolase fatty acid amide hydrolase were not affected by MCD, whereas the AEA membrane transporter was inhibited (approximately 55%) compared with controls. Furthermore, neither diacylglycerol lipase nor monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoylglycerol, were affected by MCD in DAUDI or C6 cells, whereas the transport of 2-arachidonoylglycerol was reduced to approximately 50%. Instead, membrane cholesterol enrichment almost doubled the uptake of AEA and 2-arachidonoylglycerol in both cell types. Finally, transfection experiments with human U937 immune cells, and the use of primary cells expressing CB1R or CB2R, ruled out that the cellular environment could account per se for the different modulation of CB receptor subtypes by MCD. In conclusion, the present data demonstrate that lipid rafts control CB1R, but not CB2R, and endocannabinoid transport in immune and neuronal cells.     

Cannabinoid receptor antagonists and fatty acids alter endocannabinoid system gene expression and COX activity

Cyclooxygenase (COX) possesses substrate affinity for the endocannabinoids (EC) anandamide (AEA) and 2-arachidonylglycerol (2-AG). We hypothesized that selective antagonism/activation of the cannabinoid receptors will increase COX activity and the availability of EC as substrates will lead to higher COX activity. Since the relationship between EC signaling of the endocannabinoid system (ECS) and the COX pathway in muscle has not been investigated, we examined agonist, antagonists and polyunsaturated fatty acid effects on ECS genes in myoblasts. At 50% confluency, C2C12 myoblasts were pretreated with 5 μM of the cannabinoid receptor (CB)2 inverse agonist AM630 for 2 h and one with both AM630 and 1 μM of the CB1 antagonist NESS0327. Cell cultures pretreated with AM630 were then administered with 25 μM of either arachidonic acid (20:4n6), eicosapentaenoate (EPA) (20:5n3), docosahexaenoate (DHA) (22:6n3), AEA or bovine serum albumin (vehicle control) for 24 h. Quantitative polymerase chain reaction analyses were performed looking at ECS and prostaglandin genes. Total COX activity and COX-1 protein were greater in the AM630+AEA-treated cells compared to all other cell cultures. The mRNA for the AEA synthesis enzyme N-acyl phosphatidylethanolamine phospholipase D and the 2-AG synthesis enzymes diacylglycerol lipase (DAGL)α and DAGLβ were higher in AM630+EPA-treated cells compared to the other groups. The mRNA levels of CB1 and CB2 were both highest in the AM630+EPA group. The mRNA for interleukin-6 and tumor necrosis factor-α was higher with AEA but lower with DHA and docosahexaenoyl ethanolamide (DHEA), supporting previous findings that the EC AEA supports activation of the COX system. These findings suggest that COX activity and protein levels are influenced by the ECS, specifically by the ligand AEA for CB1 and by inverse agonism of CB2.     

The endocannabinoid 2-arachidonoylglycerol (2-AG) and metabolizing enzymes during rat fetoplacental development: A role in uterine remodelling

The main endocannabinoids (EC) identified in mammalian tissues are N-arachidonoylethanolamide (AEA, anandamide), and 2-arachidonoylglycerol (2-AG). AEA levels are critical in pregnancy, especially during implantation, decidualization, and placental development. As 2-AG functions in pregnancy are still largely undefined, we hypothesized that it may also have a role during fetoplacental development. We showed that 2-AG is not only present in the rat mesometrial decidua and plasma during fetoplacental development, but that both 2-AG synthesizing (diacylglycerol lipase) and degradation (monoacylglycerol lipase) enzymes are expressed by decidual cells. While lower concentrations of 2-AG induced apoptosis of rat primary decidual cells, via the CB1 receptor, higher concentrations induced a dramatic effect on cell morphology, cell viability and lactate dehydrogenase release, triggered through a mechanism independent of CB1. This study provides evidences that 2-AG fluctuation in maternal tissues throughout normal pregnancy is primarily regulated by its metabolizing enzymes. Together, these data supports the hypothesis that a deregulation of the endocannabinoid system through aberrant cannabinoid signalling may impact normal uterine remodelling process and consequently normal pregnancy.