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.     

Effect of polyunsaturated fatty acids on endocannabinoid and N-acyl-ethanolamine levels in mouse adipocytes

The tissue concentrations of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoyl-ethanolamine (anandamide), are altered in the adipose tissue of mice fed a high fat diet. We have investigated here the effect on endocannabinoid levels of incubation of mouse 3T3-F442A adipocytes with several free polyunstaurated fatty acids (PUFAs), including linolenic acid (LA), alpha-linolenic acid (ALA), arachidonic acid (AA) and docosahexaenoic acid (DHA), as well as oleic acid (OA) and palmitic acid (PA). By using mass spectrometric methods, we quantified the levels of endocannabinoids, of two anandamide congeners, N-palmitoyl-ethanolamine (PEA) and N-oleoyl-ethanolamine (OEA), and of fatty acids esterified in triacylglycerols or phospholipids, which act as 2-AG and/or N-acyl-ethanolamine precursors. Incubation with AA strongly elevated 2-AG levels and the amounts of AA esterified in triacylglycerols and on glycerol carbon atom 2 (sn-2), but not 1 (sn-1), in phospholipids. Incubation with DHA decreased 2-AG and anandamide levels and the amounts of AA esterified on both the sn-2 and sn-1 position of phospholipids, but not on triacylglycerols. PEA levels augmented following incubation of adipocytes with OA and PA, with no corresponding changes in phospholipids and triacylglycerols. We suggest that dietary PUFAs might modulate the levels of adipocyte phospholipids that act as endocannabinoid precursors.     

Eicosapentaenoic acid decreases expression of anandamide synthesis enzyme and cannabinoid receptor 2 in osteoblast-like cells

Anandamide (AEA) is an endogenous agonist for the cannabinoid receptor 2 (CB2) which is expressed in osteoblasts. Arachidonic acid (AA) is the precursor for AEA and dietary n-3 polyunsaturated fatty acids (PUFA) are known to reduce the concentrations of AA in tissues and cells. Therefore, we hypothesized that n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which reduce AA in cells, could lower AEA in osteoblasts by altering enzyme expression of the endocannabinoid (EC) system. MC3T3-E1 osteoblast-like cells were grown for 6, 10, 15, 20, 25 or 30 days in osteogenic medium. Osteoblasts were treated with 10 μM of AA, EPA, DHA, oleic acid (OA) or EPA+DHA (5 μM each) for 72 h prior to their collection for measurement of mRNA and alkaline phosphatase (ALP) activity. Compared to vehicle control, osteoblasts treated with AA had higher levels of AA and n-6 PUFA while those treated with EPA and DHA had lower n-6 but higher n-3 PUFA. Independent of the fatty acid treatments, osteoblasts matured normally as evidenced by ALP activity. N-acyl phosphatidylethanolamine-selective phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH) and CB2 mRNA expression were higher at 20 days compared to 10 days. NAPE-PLD and CB2 mRNA was lower in osteoblasts treated with EPA compared to all other groups. Thus, mRNA expression for NAPE-PLD, FAAH, and CB2 increased during osteoblast maturation and EPA reduced mRNA for NAPE-PLD and CB2 receptor. In conclusion, EPA lowered mRNA levels for proteins of the EC system and mRNA for AEA synthesis/degradation is reported in osteoblasts.     

Hydrolysis of prostaglandin glycerol esters by the endocannabinoid-hydrolyzing enzymes, monoacylglycerol lipase and fatty acid amide hydrolase

Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variety of prostaglandin-EA's and prostaglandin-G's nearly as diverse as those derived from arachidonic acid. Thus, COX-2 may regulate endocannabinoid levels in neurons during retrograde signaling or produce novel endocannabinoid metabolites for receptor activation. Endocannabinoid-metabolizing enzymes are important regulators of their action, so we tested whether PG-G levels may be regulated by monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH). We found that PG-Gs are poor substrates for purified MGL and FAAH compared to 2-AG and/or AEA. Determination of substrate specificity demonstrates a 30-100- and 150-200-fold preference of MGL and FAAH for 2-AG over PG-Gs, respectively. The substrate specificity of AEA compared to those of PG-Gs was approximately 200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH.     

Anandamide and 2-arachidonoylglycerol inhibit fatty acid amide hydrolase by activating the lipoxygenase pathway of the arachidonate cascade

Treatment of intact human neuroblastoma CHP100 cells with anandamide (arachidonoylethanolamide, AEA) or 2-arachidonoylglycerol (2-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 2-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 2-AG.     

Critical enzymes involved in endocannabinoid metabolism

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

Human adipose tissue binds and metabolizes the endocannabinoids anandamide and 2-arachidonoylglycerol

Endocannabinoids are a group of biologically active endogenous lipids that have recently emerged as important mediators in energy balance control. The two best studied endocannabinoids, anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the endogenous ligands of the central and peripheral cannabinoid receptors. Furthermore, AEA binds to the transient receptor potential vanilloid type-1 (TRPV1), a capsaicin-sensitive, non-selective cation channel. The synthesis of these endocannabinoids is catalyzed by the N-acylphosphatidylethanolamine-selective phospholipase D (NAPE-PLD) and the sn-1-selective diacylglycerol lipase (DAGL), whereas their degradation is accomplished by the fatty acid amide hydrolase (FAAH) and the monoglyceride lipase (MGL), respectively. We investigated the presence of a functional endocannabinoid system in human adipose tissue from seven healthy subjects. Subcutaneous abdominal adipose tissue underwent biochemical and molecular biology analyses, aimed at testing the expression of this system and its functional activity. AEA and 2-AG levels were detected and quantified by HPLC. Real time PCR analyzed the expression of the endocannabinoid system and immunofluorescence assays showed the distribution of its components in the adipose tissue. Furthermore, binding assay for the cannabinoid and vanilloid receptors and activity assay for each metabolic enzyme of the endocannabinoid system gave clear evidence of a fully operating system. The data presented herein show for the first time that the human adipose tissue is able to bind AEA and 2-AG and that it is endowed with the biochemical machinery to metabolize endocannabinoids.     

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.     

Biochemistry and pharmacology of the endocannabinoids arachidonylethanolamide and 2-arachidonylglycerol

The purpose of this review is to discuss the cellular synthesis and inactivation of two putative endogenous ligands of the cannabinoid receptor, N-arachidonylethanolamine (AEA) and 2-arachidonylglycerol (2-AG). Both ligands are synthesized by neurons and brain tissue in response to increased intracellular calcium concentrations. Both ligands are substrates for fatty acid amide hydrolase (FAAH). Both AEA and 2-AG bind to the neuronal form of the cannabinoid receptor (CB1). AEA binds the receptor with moderate affinity and has the characteristics of a partial agonist, whereas, 2-AG binds with low affinity but exhibits full efficacy. Two possible physiological roles of the endocannabinoids and the CB1 receptor are discussed: the regulation of gestation and the regulation of gastrointestinal motility.     

Dietary Linoleic Acid Elevates Endogenous 2-AG and Anandamide and Induces Obesity

Suppressing hyperactive endocannabinoid tone is a critical target for reducing obesity. The backbone of both endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) is the ω-6 fatty acid arachidonic acid (AA). Here we posited that excessive dietary intake of linoleic acid (LA), the precursor of AA, would induce endocannabinoid hyperactivity and promote obesity. LA was isolated as an independent variable to reflect the dietary increase in LA from 1 percent of energy (en%) to 8 en% occurring in the United States during the 20th century. Mice were fed diets containing 1 en% LA, 8 en% LA, and 8 en% LA + 1 en% eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) in medium-fat diets (35 en% fat) and high-fat diets (60 en%) for 14 weeks from weaning. Increasing LA from 1 en% to 8 en% elevated AA-phospholipids (PL) in liver and erythrocytes, tripled 2-AG + 1-AG and AEA associated with increased food intake, feed efficiency, and adiposity in mice. Reducing AA-PL by adding 1 en% long-chain ω-3 fats to 8 en% LA diets resulted in metabolic patterns resembling 1 en% LA diets. Selectively reducing LA to 1 en% reversed the obesogenic properties of a 60 en% fat diet. These animal diets modeled 20th century increases of human LA consumption, changes that closely correlate with increasing prevalence rates of obesity. In summary, dietary LA increased tissue AA, and subsequently elevated 2-AG + 1-AG and AEA resulting in the development of diet-induced obesity. The adipogenic effect of LA can be prevented by consuming sufficient EPA and DHA to reduce the AA-PL pool and normalize endocannabinoid tone.     

Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are valuable commodities that provide important human health benefits. We report the transgenic production of significant amounts of AA and EPA in Brassica juncea seeds via a stepwise metabolic engineering strategy. Using a series of transformations with increasing numbers of transgenes, we demonstrate the incremental production of VLCPUFAs, achieving AA levels of up to 25% and EPA levels of up to 15% of total seed fatty acids. Both fatty acids were almost exclusively found in triacylglycerols, with AA located preferentially at sn-2 and sn-3 positions and EPA distributed almost equally at all three positions. Moreover, we reconstituted the DHA biosynthetic pathway in plant seeds, demonstrating the practical feasibility of large-scale production of this important omega-3 fatty acid in oilseed crops.     

Methylation and acetylation of 15-hydroxyanandamide modulate its interaction with the endocannabinoid system

The biological activity of endocannabinoids like anandamide (AEA) and 2-arachidonoylglycerol (2-AG) is subjected in vivo to a “metabolic control”, exerted mainly by catabolic enzymes. AEA is inactivated by fatty acid amide hydrolase (FAAH), that is inhibited competitively by hydroxyanandamides (HAEAs) generated from AEA by lipoxygenase activity. Among these derivatives, 15-HAEA has been shown to be an effective (K_i ∼0.6 μM) FAAH inhibitor, that blocks also type-1 cannabinoid receptor (CB1R) but not other components of the “endocannabinoid system (ECS)”, like the AEA transporter (AMT) or CB2R. Here, we extended the study of the effect of 15-HAEA on the AEA synthetase (NAPE-PLD) and the AEA-binding vanilloid receptor (TRPV1), showing that 15-HAEA activates the former (up to ∼140% of controls) and inhibits the latter protein (down to ∼70%). We also show that 15-HAEA halves the synthesis of 2-AG and almost doubles the transport of this compound across the membrane. In addition, we synthesized methyl and acetyl derivatives of 15-HAEA (15-MeOAEA and 15-AcOAEA, respectively), in order to check their ability to modulate FAAH and the other ECS elements. In fact, methylation and acetylation are common biochemical reactions in the cellular environment. We show that 15-MeOAEA, unlike 15-AcOAEA, is still a powerful competitive inhibitor of FAAH (K_i ∼0.7 μM), and that both derivatives have negligible interactions with the other proteins of ECS. Therefore, 15-MeOAEA is a FAAH inhibitor more selective than 15-HAEA. Further molecular dynamics analysis gave clues to the molecular requirements for the interaction of 15-HAEA and 15-MeOAEA with FAAH.     

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.     

Inhibition of Endocannabinoid Metabolism by the Metabolites of Ibuprofen and Flurbiprofen

Background

In addition to their effects upon prostaglandin synthesis, the non-steroidal anti-inflammatory drugs ibuprofen and flurbiprofen inhibit the metabolism of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) by cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH), respectively. Here, we investigated whether these effects upon endocannabinoid metabolism are shared by the main metabolites of ibuprofen and flurbiprofen.

Methodology/Principal Findings

COX activities were measured via changes in oxygen consumption due to oxygenation of arachidonic acid (for COX-1) and arachidonic acid and 2-AG (for COX-2). FAAH activity was quantified by measuring hydrolysis of tritium labelled AEA in rat brain homogenates. The ability of ibuprofen and flurbiprofen to inhibit COX-2-catalysed oxygenation of 2-AG at lower concentrations than the oxygenation of arachidonic acid was seen with 4′-hydroxyflurbiprofen and possibly also 3′-hydroxyibuprofen, albeit at lower potencies than the parent compounds. All ibuprofen and flurbiprofen metabolites retained the ability to inhibit FAAH in a pH-dependent manner, although the potency was lower than seen with the parent compounds.

Conclusions/Significance

It is concluded that the primary metabolites of ibuprofen and flurbiprofen retain some of the properties of the parent compound with respect to inhibition of endocannabinoid metabolism. However, these effects are unlikely to contribute to the actions of the parent compounds in vivo.     

Docosahexaenoic Acid Is a Major n-3 Polyunsaturated Fatty Acid in Bovine Retinal Microvessels

Abstract: The aim of this study was to purify microvessels from bovine retina and also to cultivate bovine retinal endothelial cells (BRECs) or intramural pericytes, to determine their fatty acid composition. Microvessels were obtained after Dounce homogenization of the retina followed by centrifugation on albumin cushion and finally microvessels in the pellet were trapped on a 100-µm nylon filter. Contamination of microvessel preparations by neuronal tissue, assessed after both microscopic examination and western blotting with a monoclonal antibody raised against rhodopsin, was minor. In the entire bovine retina, docosahexaenoic acid (DHA) represented 23.3% of the total fatty acids and there was about three times less arachidonic acid (AA) (8.2%) than DHA. In contrast, DHA and AA levels were almost equivalent in the retinal microvessels with ∼10% of total fatty acids. When compared with intact microvessels, the DHA proportion of confluent monolayers of both BRECs or pericytes in primary cultures dropped to ∼2% of the total fatty acids, whereas AA was unchanged. Culture medium supplementation with unesterified DHA (10 µ M ) restored the DHA proportion of BRECs close to the microvascular value at the expense of linoleic acid without affecting AA very much. In contrast, DHA supplementation in pericytes increased the DHA proportion of these cells at the expense of AA. In conclusion, DHA of intact microvessels represented 10% of the total fatty acids, which was close to the AA proportion. Mild DHA supplementation of BRECs or pericytes in primary cultures restored their DHA proportion to the original microvessel value. This high percentage of polyunsaturated fatty acids in retinal microvessels should allow us to test the hypothesis that oxidation products derived from these fatty acids may be involved in the pathogenic process leading to diabetic retinopathy.     

A new perspective on cannabinoid signalling: complementary localization of fatty acid amide hydrolase and the CB1 receptor in rat brain.

CB1-type cannabinoid receptors in the brain mediate effects of the drug cannabis. Anandamide and sn-2 arachidonylglycerol (2-AG) are putative endogenous ligands for CB1 receptors, but it is not known which cells in the brain produce these molecules. Recently, an enzyme which catalyses hydrolysis of anandamide and 2-AG, known as fatty acid amide hydrolase (FAAH), was identified in mammals. Here we have analysed the distribution of FAAH in rat brain and compared its cellular localization with CB1-type cannabinoid receptors using immunocytochemistry. High concentrations of FAAH activity were detected in the cerebellum, hippocampus and neocortex, regions of the rat brain which are enriched with cannabinoid receptors. Immunocytochemical analysis of these brain regions revealed a complementary pattern of FAAH and CB1 expression with CB1 immunoreactivity occurring in fibres surrounding FAAH-immunoreactive cell bodies and/or dendrites. In the cerebellum, FAAH was expressed in the cell bodies of Purkinje cells and CB1 was expressed in the axons of granule cells and basket cells, neurons which are presynaptic to Purkinje cells. The close correspondence in the distribution of FAAH and CB1 in rat brain and the complementary pattern of FAAH and CB1 expression at the cellular level provides important new evidence that FAAH may participate in cannabinoid signalling mechanisms of the brain.     

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.     

Comparison of anandamide transport in FAAH wild-type and knockout neurons: evidence for contributions by both FAAH and the CB1 receptor to anandamide uptake

The cellular inactivation of the endogenous cannabinoid (endocannabinoid) anandamide (AEA) represents a controversial and intensely investigated subject. This process has been proposed to involve two proteins, a transporter that promotes the cellular uptake of AEA and fatty acid amide hydrolase (FAAH), which hydrolyzes AEA to arachidonic acid. However, whereas the role of FAAH in AEA metabolism is well-characterized, the identity of the putative AEA transporter remains enigmatic. Indeed, the indirect pharmacological evidence used to support the existence of an AEA transporter has been suggested also to be compatible with a model in which AEA uptake is driven by simple diffusion coupled to FAAH metabolism. Here, we have directly addressed the contribution of FAAH to AEA uptake by examining this process in neuronal preparations from FAAH(-/-) mice and in the presence of the uptake inhibitor UCM707. The results of these studies reveal that (i) care should be taken to avoid the presence of artifacts when studying the cellular uptake of lipophilic molecules like AEA, (ii) FAAH significantly contributes to AEA uptake, especially with longer incubation times, and (iii) a UCM707-sensitive protein(s) distinct from FAAH also participates in AEA uptake. Interestingly, the FAAH-independent component of AEA transport was significantly reduced by pretreatment of neurons with the cannabinoid receptor 1 (CB1) antagonist SR141716A. Collectively, these results indicate that the protein-dependent uptake of AEA is largely mediated by known constituents of the endocannabinoid system (FAAH and the CB1 receptor), although a partial contribution of an additional UCM707-sensitive protein is also suggested.     

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.     

A novel phosphatidylcholine which contains pentadecanoic acid at sn-1 and docosahexaenoic acid at sn-2 in Schizochytrium sp. F26-b

Docosahexaenoic acid (DHA, 22:6n-3)-containing phospholipids are a ubiquitous component of the central nervous system and retina, however their physiological and pharmacological functions have not been fully elucidated. Here, we report a novel DHA-containing phosphatidylcholine (PC) in a marine single cell eukaryote, Schizochytrium sp. F26-b. Interestingly, 31.8% of all the fatty acid in F26-b is DHA, which is incorporated into triacylglycerols and various phospholipids. In phospholipids, DHA was found to make up about 50% of total fatty acid. To identify phospholipid species containing DHA, the fraction of phospholipids from strain F26-b was subjected to normal phase high-performance liquid chromatography (HPLC). It was found that DHA was incorporated into PC, lyso-PC, phosphatidylethanolamine, and phosphatidylinositol. The major DHA-containing phospholipid was PC in which 32.5% of the fatty acid was DHA. The structure of PC was analyzed further by phospholipase A2 treatment, fast atom bombardment mass spectrometry, and 1H- and 13C-NMR after purification of the PC with reverse phase HPLC. Collectively, it was clarified that the major PC contains pentadecanoic acid (C15:0) at sn-1 and DHA at sn-2; the systematic name of this novel PC is therefore "1-pentadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine."