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.     

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.     

Cannabinoid drugs and enhancement of endocannabinoid responses: strategies for a wide array of disease states

The endogenous cannabinoid system has revealed potential avenues to treat many disease states. Medicinal indications of cannabinoid drugs including compounds that result in enhanced endocannabinoid responses (EER) have expanded markedly in recent years. The wide range of indications covers chemotherapy complications, tumor growth, addiction, pain, multiple sclerosis, glaucoma, inflammation, eating disorders, age-related neurodegenerative disorders, as well as epileptic seizures, traumatic brain injury, cerebral ischemia, and other excitotoxic insults. Indeed, a great effort has led to the discovery of agents that selectively activate the cannabinoid system or that enhance the endogenous pathways of cannabinergic signaling. The endocannabinoid system is comprised of three primary components: (i) cannabinoid receptors, (ii) endocannabinoid transport system, and (iii) hydrolysis enzymes that break down the endogenous ligands. Two known endocannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), are lipid molecules that are greatly elevated in response to a variety of pathological events. This increase in endocannabinoid levels is suggested to be part of an on-demand compensatory response. Furthermore, activation of signaling pathways mediated by the endogenous cannabinoid system promotes repair and cell survival. Similar cell maintenance effects are elicited by EER through inhibitors of the endocannabinoid deactivation processes (i.e., internalization and hydrolysis). The therapeutic potential of the endocannabinoid system has yet to be fully determined, and the number of medical maladies that may be treated will likely continue to grow. This review will underline studies that demonstrate medicinal applications for agents that influence the endocannabinoid system.     

The endocannabinoid 2-arachidonoyl-glycerol activates human neutrophils: critical role of its hydrolysis and de novo leukotriene B4 biosynthesis

Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B(4) biosynthesis, or LTB(4) receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB(4) biosynthesis, and an autocrine activation loop involving LTB(4) receptor 1.     

Endocannabinoid structure-activity relationships for interaction at the cannabinoid receptors

Anandamide (N -arachidonoylethanolamine) was the first ligand to be identified as an endogenous ligand of the G-protein coupled cannabinoid CB1 receptor. Subsequently, two other fatty acid ethanolamides, N -homo- gamma -linolenylethanolamine and N -7,10,13,16-docosatetraenylethanolamine were identified as endogenous cannabinoid ligands. A fatty acid ester, 2-arachidonoylglycerol (2-AG), and a fatty acid ether, 2-arachidonyl glyceryl ether also have been isolated and shown to be endogenous cannabinoid ligands. Recent studies have postulated the existence of carrier-mediated anandamide transport that is essential for termination of the biological effects of anandamide. A membrane bound amidohydrolase (fatty acid amide hydrolase, FAAH), located intracellularly, hydrolyzes and inactivates anandamide and other endogenous cannabinoids such as 2-AG. 2-AG has also been proposed to be an endogenous CB2 ligand. Structure-activity relationships (SARs) for endocannabinoid interaction with the CB receptors are currently emerging in the literature. This review considers cannabinoid receptor SAR developed to date for the endocannabinoids with emphasis upon the conformational implications for endocannabinoid recognition at the cannabinoid receptors.     

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 endocannabinoid system,anandamide and the regulation of mammalian cell apoptosis

Endocannabinoids are a new class of lipid mediators, which include amides, esters and ethers of long-chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the main endogenous agonists of cannabinoid receptors able to mimic several pharmacological effects of Delta-9-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. The pathways leading to the synthesis and release of AEA and 2-AG from neuronal and non-neuronal cells are still rather uncertain. Instead, it is known that the activity of AEA is limited by cellular uptake through a specific membrane transporter, followed by intracellular degradation by a fatty acid amide hydrolase. Together with AEA and congeners these proteins form the 'endocannabinoid system'. Here, the involvement of AEA in apoptosis and the underlying signal transduction pathways will be reviewed, along with the metabolic routes and the molecular targets of this endocannabinoid. Also, recent findings on the apoptotic potential of AEA for neuronal cell differentiation and brain development will be discussed.     

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.     

Enhancement of endocannabinoid signaling by fatty acid amide hydrolase inhibition: A neuroprotective therapeutic modality

AimsThis review posits that fatty acid amide hydrolase (FAAH) inhibition has therapeutic potential against neuropathological states including traumatic brain injury; Alzheimer's, Huntington's, and Parkinson's diseases; and stroke.Main methodsThis proposition is supported by data from numerous in vitro and in vivo experiments establishing metabolic and pharmacological contexts for the neuroprotective role of the endogenous cannabinoid (“endocannabinoid”) system and selective FAAH inhibitors.Key findingsThe systems biology of endocannabinoid signaling involves two main cannabinoid receptors, the principal endocannabinoid lipid mediators N-arachidonoylethanolamine (“anandamide”) (AEA) and 2-arachidonoyl glycerol (2-AG), related metabolites, and the proteins involved in endocannabinoid biosynthesis, biotransformation, and transit. The endocannabinoid system is capable of activating distinct signaling pathways on-demand in response to pathogenic events or stimuli, thereby enhancing cell survival and promoting tissue repair. Accumulating data suggest that endocannabinoid system modulation at discrete targets is a promising pharmacotherapeutic strategy for treating various medical conditions. In particular, neuronal injury activates cannabinoid signaling in the central nervous system as an intrinsic neuroprotective response. Indirect potentiation of this salutary response through pharmacological inhibition of FAAH, an endocannabinoid-deactivating enzyme, and consequent activation of signaling pathways downstream from cannabinoid receptors have been shown to promote neuronal maintenance and function.SignificanceThis therapeutic modality has the potential to offer site- and event-specific neuroprotection under conditions where endocannabinoids are being produced as part of a physiological protective mechanism. In contrast, direct application of cannabinoid receptor agonists to the central nervous system may activate CB receptors indiscriminately and invite unwanted psychotrophic effects.     

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.     

Brain regional distribution of endocannabinoids: implications for their biosynthesis and biological function

The amounts, in nine different rat brain regions, of the two endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoylglycerol (2-AG), and of the putative AEA precursor N-arachidonoyl-phosphatidylethanolamine (NArPE), were determined by isotope-dilution gas chromatography-mass spectrometry and compared to the number of cannabinoid binding sites in each region. The distribution of NArPE, reported here for the first time, exhibited a good correlation with that of AEA, the former metabolite being 3-13 times more abundant than the endocannabinoid in all regions. The highest amounts of both metabolites (up to 358.5 and 87 pmol/g wet weight tissue, respectively) were found in the brainstem and striatum, and the lowest in the diencephalon, cortex, and cerebellum. These data support the hypothesis that, in the brain, AEA is a metabolic product of NArPE and may reach levels compatible with its proposed neuromodulatory function. The brain distribution of 2-AG, also described in this study for the first time, was found to correlate with that of AEA with levels ranging from 2.0 to 14.0 nmol/g (in the diencephalon and brainstem, respectively). The distribution of the endocannabinoids did not match exactly with that of cannabinoid binding sites, suggesting either that these compounds are not necessarily produced near their molecular targets, or that they play functional roles additional to the activation of cannabinoid receptors. Regional differences in the ligand/receptor ratios may also lead to predict corresponding differences in the efficiency of receptor activation, as shown by previous studies.     

Endocannabinoids: Friends and foes of reproduction

Endocannabinoids are fatty acid amides like anandamide (AEA), and monoacylglycerols like 2-arachidonoylglycerol, that bind to cannabinoid, vanilloid and peroxisome proliferator-activated receptors. Their biological actions are controlled through not yet fully characterized cellular mechanisms. These compounds, together with their related enzymes, that include key proteins for the synthesis and degradation of endocannabinoids, cannabinoid and non-cannabinoid receptors, and purported membrane transporter(s), form the “endocannabinoid system (ECS)”. In the past few years AEA and related ECS elements have emerged as essential players in various aspects of human reproduction, both for males and females. Here, the key features of the ECS and the potential of its components to direct human fertility towards a positive or negative end will be reviewed. In particular, the involvement of AEA and related ECS elements in regulating embryo oviductal transport, blastocyst implantation and placental development (in females), and sperm survival, motility, capacitation and acrosome reaction (in males) will be addressed, as well as the role of endocannabinoids in sperm–oviduct interactions. Additionally, the possibility that blood AEA and its hydrolase FAAH may represent reliable diagnostic markers of natural and assisted reproduction in humans will be discussed, along with the therapeutic exploitation of ECS-oriented drugs as useful fertility enhancers.     

Opposing actions of endocannabinoids on cholangiocarcinoma growth is via the differential activation of Notch signaling

The endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2-AG) have opposing effects on cholangiocarcinoma growth. Implicated in cancer, Notch signaling requires the γ-secretase complex for activation. The aims of this study were to determine if the opposing effects of endocannabinoids depend on the differential activation of the Notch receptors and to demonstrate that the differential activation of these receptors are due to presenilin 1 containing- and presenilin 2 containing-γ-secretase complexes. Mz-ChA-1 cells were treated with AEA or 2-AG. Notch receptor expression, activation, and nuclear translocation were determined. Specific roles for Notch 1 and 2 on cannabinoid-induced effects were determined by transient transfection of Notch 1 or 2 shRNA vectors before stimulation with AEA or 2-AG. Expression of presenilin 1 and 2 was determined after AEA or 2-AG treatment, and the involvement of presenilin 1 and 2 in the cannabinoid-induced effects was demonstrated in cell lines with low presenilin 1 or 2 expression. Antiproliferative effects of AEA required increased Notch 1 mRNA, activation, and nuclear translocation, whereas the growth-promoting effects induced by 2-AG required increased Notch 2 mRNA expression, activation, and nuclear translocation. AEA increased presenilin 1 expression and recruitment into the γ-secretase complex, whereas 2-AG increased expression and recruitment of presenilin 2. The development of novel therapeutic strategies aimed at modulating the endocannabinoid system or mimicking the mode of action of AEA on Notch signaling pathways would prove beneficial for cholangiocarcinoma management.     

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.     

Anandamide and 2-arachidonoylglycerol: Pharmacological Properties, Functional Features, and Emerging Specificities of the Two Major Endocannabinoids

Since the discovery of endocannabinoids and their receptors, two major members of the endocannabinoid family, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), have been regarded almost as twin brothers. Pharmacological properties were initially considered to be similar, as these molecules were believed mutually exchangeable and almost indistinguishable in the regulation of synaptic functions, such as long- and short-term synaptic plasticity, and in behavioral aspects, such as learning and memory, reward and addiction, antinociception, and anxiety. In recent years, however, endocannabinoid signaling specificity began to emerge, in particular, due to the production of genetically engineered mice lacking key enzymes in endocannabinoid synthesis or degradation, together with the development of selective inhibitors of AEA or 2-AG catabolic enzymes. Evidence now suggests that AEA and 2-AG possess specific pharmacological properties, are engaged in different forms of synaptic plasticity, and take part in different behavioral functions. In this review, we provide an overview on similarities and specificities of the two endocannabinoids in the CNS and on the unresolved questions concerning their role in synaptic signaling.     

Endocannabinoids,hormone-cytokine networks and human fertility

Anandamide (N -arachidonoylethanolamine, AEA) is a major endocannabinoid, shown to impair mouse pregnancy and embryo development and to induce apoptosis in blastocysts. Here, we review the roles of AEA, of the AEA-binding cannabinoid (CB) receptors, of the selective AEA membrane transporter (AMT), and of the AEA-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), in human gestation. In particular, we discuss the interplay between the endocannabinoid system and the hormone-cytokine array involved in the control of human pregnancy, showing that the endocannabinoids take part in the immunological adaptation occurring during early pregnancy. In this line, we discuss the critical role of FAAH in human peripheral lymphocytes, showing that the expression of this enzyme is regulated by progesterone, Th1 and Th2 cytokines, which also regulate fertility. Moreover, we show that AEA and the other endocannabinoid, 2-arachidonoylglycerol, inhibit the release of the fertility-promoting cytokine leukemia inhibitory factor from human lymphocytes. Taken together, low FAAH and consistently high blood levels of AEA, but not CB receptors or AMT, can be early (<8 weeks of gestation) markers of spontaneous abortion, potentially useful as diagnostic tools for large-scale, routine monitoring of gestation in humans.     

The evolution and comparative neurobiology of endocannabinoid signalling

CB₁- and CB₂-type cannabinoid receptors mediate effects of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide in mammals. In canonical endocannabinoid-mediated synaptic plasticity, 2-AG is generated postsynaptically by diacylglycerol lipase alpha and acts via presynaptic CB₁-type cannabinoid receptors to inhibit neurotransmitter release. Electrophysiological studies on lampreys indicate that this retrograde signalling mechanism occurs throughout the vertebrates, whereas system-level studies point to conserved roles for endocannabinoid signalling in neural mechanisms of learning and control of locomotor activity and feeding. CB₁/CB₂-type receptors originated in a common ancestor of extant chordates, and in the sea squirt Ciona intestinalis a CB₁/CB₂-type receptor is targeted to axons, indicative of an ancient role for cannabinoid receptors as axonal regulators of neuronal signalling. Although CB₁/CB₂-type receptors are unique to chordates, enzymes involved in biosynthesis/inactivation of endocannabinoids occur throughout the animal kingdom. Accordingly, non-CB₁/CB₂-mediated mechanisms of endocannabinoid signalling have been postulated. For example, there is evidence that 2-AG mediates retrograde signalling at synapses in the nervous system of the leech Hirudo medicinalis by activating presynaptic transient receptor potential vanilloid-type ion channels. Thus, postsynaptic synthesis of 2-AG or anandamide may be a phylogenetically widespread phenomenon, and a variety of proteins may have evolved as presynaptic (or postsynaptic) receptors for endocannabinoids.     

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.     

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.