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.     

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.     

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.     

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.     

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.     

Biosynthesis and degradation of the endocannabinoid 2-arachidonoylglycerol

2-Arachidonoylglycerol (2-AG) is a monoacylglycerol (MAG) molecule containing an esterified arachidonic acid chain at sn-2 position of the glycerol backbone. Together with structurally similar N-arachidonoylethanolamine (anandamide), 2-AG has been extensively studied as an endogenous ligand of cannabinoid receptors (an endocannabinoid) in brain and other mammalian tissues. Accumulating evidence demonstrates that the endocannabinoid system, including the central-type cannabinoid receptor CB1 and 2-AG, is responsible for synaptic retrograde signaling in the central nervous system. As 2-AG is rapidly formed from membrane phospholipids on cellular stimuli and degraded to arachidonic acid and glycerol, the enzymes catalyzing its biosynthesis and degradation are believed to play crucial roles in the regulation of its tissue levels. The major biosynthetic pathway appears to consist of sequential hydrolyses of inositol phospholipids via diacylglycerol (DAG) by β-type phospholipase C and DAG lipase, while MAG lipase is a principal enzyme in the degradation. In this short review, we will briefly outline rapid advances in enzymological research on the biosynthetic and degradative pathways of 2-AG.     

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.     

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.     

Characterization of biotin-anandamide, a novel tool for the visualization of anandamide accumulation

Anandamide (N-arachidonoylethanolamide; AEA) acts as an endogenous agonist of both cannabinoid and vanilloid receptors. During the last two decades, its metabolic pathways and biological activity have been investigated extensively and relatively well characterized. In contrast, at present, the effective nature and mechanism of AEA transport remain controversial and still unsolved issues. Here, we report the characterization of a biotinylated analog of AEA (b-AEA) that has the same lipophilicity of the parent compound. In addition, by means of biochemical assays and fluorescence microscopy, we show that b-AEA is accumulated inside the cells in a way superimposable on that of AEA. Conversely, b-AEA does not interact or interfere with the other components of the endocannabinoid system, such as type-1 and type-2 cannabinoid receptors, vanilloid receptor, AEA synthetase (N-acylphosphatidylethanolamine-hydrolyzing phospholipase D), or AEA hydrolase (fatty acid amide hydrolase). Together, our data suggest that b-AEA could be a very useful probe for visualizing the accumulation and intracellular distribution of this endocannabinoid.     

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.     

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.     

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.     

Analysis of endocannabinoids by Ag+ coordination tandem mass spectrometry

The neutral arachidonic acid derivatives N-arachidonylethanolamide (anandamide or AEA) and 2-arachidonylglycerol (2-AG) have been identified as endogenous ligands for the cannabinoid receptors. Quantitation of these endocannabinoids from various tissues has been shown to be essential in the elucidation of cannabinoid-mediated processes in vivo. Here, we describe a novel method for the detection and quantitation of AEA and 2-AG from mammalian tissue. We exploit the ability of silver cation to bind to the polyunsaturated arachidonate backbone of both molecules to form the charged species [M+Ag](+). These complexes are amenable to liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, resulting in the simple and specific quantitation of AEA and 2-AG. The limits of detection of 2-AG and AEA are 13 and 14fmol, respectively, on-column. This method provides an alternative to existing methods, which employ derivation and/or selected ion monitoring (when mass spectrometric detection is used), and may facilitate the understanding of the physiological roles of this new class of compounds.     

Regulation of neural progenitor cell fate by anandamide

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

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.     

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 uptake by cells of 2-arachidonoylglycerol, an endogenous agonist of cannabinoid receptors.

It is not yet clear if the endocannabinoid 2-arachidonoylglycerol (2-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N-arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2-AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]2-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 +/- 2.0 and 15.3 +/- 3.1 microM, Bmax 1.70 +/- 0.30 and 0.24 +/- 0.04 nmol.min-1.mg protein-1, respectively). Despite these similar Km values, 2-AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 +/- 3.9 microM) significantly higher than those required to either 2-AG or AEA to inhibit [3H]2-AG uptake (Ki = 18.9 +/- 1.8 and 20.5 +/- 3.2 microM, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2-AG alone; (b) the uptake of [14C]AEA and [3H]2-AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 +/- 0.7 and 10.2 +/- 1.7 microM, respectively) and linvanil (Ki = 9.5 +/- 0.7 and 6.4 +/- 1.2 microM, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]2-AG, thus suggesting that 2-AG action can be regulated through NO release; (d) AEA and 2-AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.     

The endocannabinoid system in human keratinocytes. Evidence that anandamide inhibits epidermal differentiation through CB1 receptor-dependent inhibition of protein kinase C,activation protein-1, and transglutaminase

Anandamide (AEA), a prominent member of the endogenous ligands of cannabinoid receptors (endocannabinoids), is known to affect several functions of brain and peripheral tissues. A potential role for AEA in skin pathophysiology has been proposed, yet its molecular basis remains unknown. Here we report unprecedented evidence that spontaneously immortalized human keratinocytes (HaCaT) and normal human epidermal keratinocytes (NHEK) have the biochemical machinery to bind and metabolize AEA, i.e. a functional type-1 cannabinoid receptor (CB1R), a selective AEA membrane transporter (AMT), an AEA-degrading fatty acid amide hydrolase (FAAH), and an AEA-synthesizing phospholipase D (PLD). We show that, unlike CB1R and PLD, the activity of AMT and the activity and expression of FAAH increase while the endogenous levels of AEA decrease in HaCaT and NHEK cells induced to differentiate in vitro by 12-O-tetradecanoylphorbol 13-acetate (TPA) plus calcium. We also show that exogenous AEA inhibits the formation of cornified envelopes, a hallmark of keratinocyte differentiation, in HaCaT and NHEK cells treated with TPA plus calcium, through a CB1R-dependent reduction of transglutaminase and protein kinase C activity. Moreover, transient expression in HaCaT cells of the chloramphenicol acetyltransferase reporter gene under control of the loricrin promoter, which contained a wild-type or mutated activating protein-1 (AP-1) site, showed that AEA inhibited AP-1 in a CB1R-dependent manner. Taken together, these data demonstrate that human keratinocytes partake in the peripheral endocannabinoid system and show a novel signaling mechanism of CB1 receptors, which may have important implications in epidermal differentiation and skin development.     

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.