Cannabinoid receptors and the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> have been suggested to regulate food intake in several animal phyla. Orthologs of the mammalian cannabinoid CB(1) and CB(<em>2</em>) receptors have been identified in fish. We investigated the presence of this endocannabinoid system in the brain of the goldfish Carassius auratus and its role in food consumption. CB(1)-like immunoreactivity was distributed throughout the goldfish brain. The prosencephalon showed strong CB(1)-like immunoreactivity in the telencephalon and the inferior lobes of the posterior hypothalamus. Endocannabinoids were detected in all brain regions of C. auratus and an anandamide-hydrolysing enzymatic activity with features similar to those of mammalian fatty acid amide hydrolase was found. Food deprivation for <em>2</em>4 h was accompanied by a significant increase of anandamide, but not <em>2</em>-<em>arachidonoylglycerol</em>, levels only in the telencephalon. Anandamide caused a dose-dependent effect on food intake within <em>2</em> h of intraperitoneal administration to satiated fish and significantly enhanced or reduced food intake at low (1 pg/g body weight) or intermediate (10 pg/g) doses, respectively, the highest dose tested (100 pg/g) being inactive. We suggest that endocannabinoids might variously contribute to adaptive responses to food shortage in fish.

Lipodystrophy is a major disease involving severe alterations of adipose tissue distribution and metabolism. Mutations in genes encoding the nuclear envelope protein lamin A or its processing enzyme, the metalloproteinase Zmpste<em>2</em>4, cause diverse human progeroid syndromes that are commonly characterized by a selective loss of adipose tissue. Similarly to humans, mice deficient in Zmpste<em>2</em>4 accumulate prelamin A and display phenotypic features of accelerated aging, including lipodystrophy. Herein, we report the proteome and phosphoproteome of adipose tissue as well as serum metabolome in lipodystrophy by using Zmpste<em>2</em>4(-/-) mice as experimental model. We show that Zmpste<em>2</em>4 deficiency enhanced lipolysis, fatty acid biogenesis and β-oxidation as well as decreased fatty acid re-esterification, thus pointing to an increased partitioning of fatty acid toward β-oxidation and away from storage that likely underlies the observed size reduction of Zmpste<em>2</em>4-null adipocytes. Besides the mitochondrial proteins related to lipid metabolism, other protein networks related to mitochondrial function, including those involved in tricarboxylic acid cycle and oxidative phosphorylation, were up-regulated in Zmpste<em>2</em>4(-/-) mice. These results, together with the observation of an increased mitochondrial response to oxidative stress, support the relationship between defective prelamin A processing and mitochondrial dysfunction and highlight the relevance of oxidative damage in lipoatrophy and aging. We also show that absence of Zmpste<em>2</em>4 profoundly alters the processing of the cytoskeletal protein vimentin and identify a novel protein dysregulated in lipodystrophy, High-Mobility Group Box-1 Protein. Finally, we found several lipid derivates with important roles in energy balance, such as Lysophosphatidylcholine or <em>2</em>-<em>arachidonoylglycerol</em>, to be dysregulated in Zmpste<em>2</em>4(-/-) serum. Together, our findings in Zmpste<em>2</em>4(-/-) mice may be useful to unveil the mechanisms underlying adipose tissue dysfunction and its overall contribution to body homeostasis in progeria and other lipodystrophy syndromes as well as to develop novel strategies to prevent or ameliorate these diseases.

The amygdala is a sexually dimorphic brain region critical for the regulation of social, cognitive, and emotional behaviors, but both the nature and the source of sex differences in the amygdala are largely unknown. We have identified a unique sex difference in the developing rat medial amygdala (MeA) that is regulated by cannabinoids. Newborn females had higher rates of cell proliferation than males. Treatment of neonates with the cannabinoid receptor agonist, WIN 55,<em>2</em>1<em>2</em>-<em>2</em> (WIN), reduced cell proliferation in females to that of males and a wide range of WIN doses had no effect on cell proliferation in males. The effect of WIN on cell proliferation in the MeA was prevented by coinfusions of a CB<em>2</em> but not CB1 receptor antagonist. Females had higher amygdala content of the endocannabinoid degradation enzymes, fatty acid amid hydrolase, and monoacylglycerol lipase than males, and lower amounts of the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> and N-arachidonylethanolamide (anandamide). Inhibition of the degradation of <em>2</em>-<em>arachidonoylglycerol</em> in females occluded the sex difference in cell proliferation. Analyses of cell fate revealed that females had significantly more newly generated glial cells but not more newly generated neurons than males, and treatment with WIN significantly decreased glial cell genesis in females but not males. Finally, early exposure to cannabinoids masculinized juvenile play behavior in females but did not alter this behavior in males. Collectively, our findings suggest that sex differences in endocannabinoids mediate a sex difference in glial cell genesis in the developing MeA that impacts sex-specific behaviors in adolescence.

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

Endocannabinoids are natural lipids able to bind to cannabinoid and vanilloid receptors. Their biological actions at the central and peripheral level are under the tight control of the proteins responsible for their synthesis, transport and degradation. In the last few years, several reports have pointed out these lipid mediators as critical signals, together with sex hormones and cytokines, in various aspects of animal and human reproduction. The identification of anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in reproductive cells and tissues of invertebrates, vertebrates and mammals highlights the key role played by these endogenous compounds along the evolutionary axis. Here, we review the main actions of endocannabinoids on female and male reproductive events, and discuss the interplay between them, steroid hormones and cytokines in regulating fertility. In addition, we discuss the involvement of endocannabinoid signalling in ensuring a correct chromatin remodeling, and hence a good DNA quality, in sperm cells.

Endocannabinoids (eCBs) are important endogenous lipid mediators in synaptic transmission and plasticity and are oxygenated by cyclooxygenase-<em>2</em> (COX-<em>2</em>) to form new types of prostaglandins. However, little is known about whether COX-<em>2</em> oxidative metabolism of eCBs and their metabolites alter synaptic signaling. Here we demonstrate that increased COX-<em>2</em> expression significantly enhances basal synaptic transmission and augments long-term potentiation (LTP) in the mouse hippocampus. This augmentation was inhibited in the presence of a selective COX-<em>2</em> inhibitor or with deletion of the COX-<em>2</em> gene. The CB(1) receptor-mediated depolarization-induced suppression of inhibition (DSI) was diminished when COX-<em>2</em> expression was increased either with lipopolysaccharide (LPS) stimulation or transgenic neuronal over-expression of COX-<em>2</em>. Conversely, DSI was potentiated when COX-<em>2</em> activity was pharmacologically or genetically inhibited. Interestingly, COX-<em>2</em> oxidative metabolites of eCBs elevated LTP, an effect opposite to that of their parent molecules <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and arachidonoyl ethanolamide (AEA). In addition, the ERK/MAPK and IP(3) pathways were found to mediate PGE(<em>2</em>)-G-induced enhancement of LTP. Our results indicate that COX-<em>2</em> oxidative metabolism of eCBs is an important signaling pathway in modulation of synaptic transmission and plasticity.

The endocannabinoid system has recently emerged as a vital component of the stress response and is an appealing target for the treatment of mood and anxiety disorders. Additionally, corticolimbic endocannabinoid signaling is important for stress-induced regulation of emotional behavior. However, the mechanism by which this occurs remains elusive. Combining biochemical and behavioral analyses within the forced swim test, we examined whether stress-induced regulation of endocannabinoid signaling in the medial prefrontal cortex contributes to behavioral responses to stress, and whether these responses are dependent on serotonergic neurotransmission. Forced swim stress produced a rapid and pronounced reduction in medial prefrontal anandamide content, but had no effect on <em>2</em>-<em>arachidonoylglycerol</em> content within this region. Local administration of the anandamide hydrolysis inhibitor URB597 (0.01μg) into the ventromedial region of the prefrontal cortex decreased passive coping responses and increased active behavioral strategies, a phenomenon which was blocked by local antagonism of the CB(1) receptor. Furthermore, local inhibition of anandamide hydrolysis within the medial PFC increased the firing rate of serotonergic neurons within the dorsal raphe, suggesting that prefrontal cortical endocannabinoid signaling may modulate stress coping behaviors through a regulation of serotonergic neurotransmission. Accordingly, serotonin depletion prevented the ability of inhibition of anandamide hydrolysis within the medial PFC to promote active stress coping responses. Collectively, these data argue that stress-induced changes in endocannabinoid signaling within the medial PFC modulate stress-coping behaviors through a regulation of serotonergic neurotransmission and provide a neuroanatomical framework by which we may understand the mechanisms subserving the antidepressant potential of the endocannabinoid system.

Recent studies have demonstrated a loss of cannabinoid CB1 receptors in the basal ganglia in Huntington's disease (HD), but there are no data on endocannabinoid levels in this disease. In the present study, we have addressed this question by using rats with bilateral intrastriatal injections of 3-nitropropionic acid (3-NP), a toxin that, through the selective damage of striatal GABAergic efferent neurons, produces a useful model of HD. Twelve days after the lesion, 3-NP-lesioned rats exhibited motor disturbances, characterized by an ambulatory hyperactivity accompanied by a loss of guided activities. Analysis of GABA contents in the basal ganglia showed a trend towards a reduction compatible with motor hyperactivity. In addition, CB1 receptor binding and, to a greater extent, CB1 receptor activation of GTP-binding proteins, were also reduced in the basal ganglia. These changes were paralleled by a decrease of the contents of the two endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, in the striatum, and by an increase, particularly of anandamide, in the ventral mesencephalon where the substantia nigra is located. Both CB1 receptors and endocannabinoid levels were not altered in the cerebral cortex, an area not affected by the lesion. In summary, behavioral and biochemical changes observed in rats intrastriatally lesioned with 3-NP were similar to those occurring in the brain of HD patients. As expected, a loss of CB1 receptor function was evident in the basal ganglia of these rats and this was accompanied by different changes in endocannabinoid levels.

The balance between detrimental, pro-aging, often stochastic processes and counteracting homeostatic mechanisms largely determines the progression of aging. There is substantial evidence suggesting that the endocannabinoid system (ECS) is part of the latter system because it modulates the physiological processes underlying aging. The activity of the ECS declines during aging, as CB1 receptor expression and coupling to G proteins are reduced in the brain tissues of older animals and the levels of the major endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are lower. However, a direct link between endocannabinoid tone and aging symptoms has not been demonstrated. Here we show that a low dose of Δ9-tetrahydrocannabinol (THC) reversed the age-related decline in cognitive performance of mice aged 1<em>2</em> and 18 months. This behavioral effect was accompanied by enhanced expression of synaptic marker proteins and increased hippocampal spine density. THC treatment restored hippocampal gene transcription patterns such that the expression profiles of THC-treated mice aged 1<em>2</em> months closely resembled those of THC-free animals aged <em>2</em> months. The transcriptional effects of THC were critically dependent on glutamatergic CB1 receptors and histone acetylation, as their inhibition blocked the beneficial effects of THC. Thus, restoration of CB1 signaling in old individuals could be an effective strategy to treat age-related cognitive impairments.

Two endogenous ligands for cannabinoid CB1 receptors, anandamide (N-arachidonoylethanolamine) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), have been identified and characterized. <em>2</em>-AG is the most prevalent endogenous cannabinoid ligand in the brain, and electrophysiological studies suggest <em>2</em>-AG, rather than anandamide, is the true natural ligand for cannabinoid receptors and the key endocannabinoid involved in retrograde signaling in the brain. Here, we evaluated intravenously administered <em>2</em>-AG for reinforcing effects in nonhuman primates. Squirrel monkeys that previously self-administered anandamide or nicotine under a fixed-ratio schedule with a 60 s timeout after each injection had their self-administration behavior extinguished by vehicle substitution and were then given the opportunity to self-administer <em>2</em>-AG. Intravenous <em>2</em>-AG was a very effective reinforcer of drug-taking behavior, maintaining higher numbers of self-administered injections per session and higher rates of responding than vehicle across a wide range of doses. To assess involvement of CB1 receptors in the reinforcing effects of <em>2</em>-AG, we pretreated monkeys with the cannabinoid CB(1) receptor inverse agonist/antagonist rimonabant [N-piperidino-5-(4-chlorophenyl)-1-(<em>2</em>,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide]. Rimonabant produced persistent blockade of <em>2</em>-AG self-administration without affecting responding maintained by food under similar conditions. Thus, <em>2</em>-AG was actively self-administered by monkeys with or without a history of cannabinoid self-administration, and the reinforcing effects of <em>2</em>-AG were mediated by CB1 receptors. Self-administration of <em>2</em>-AG by squirrel monkeys provides a valuable procedure for studying abuse liability of medications that interfere with <em>2</em>-AG signaling within the brain and for investigating mechanisms involved in the reinforcing effects of endocannabinoids.

Endocannabinoids, defined in 1995 as endogenous agonists of cannabinoid receptors, their anabolic and catabolic pathways, and the enzymes involved in these pathways (the "endocannabinoid enzymes"), are the subject of this review. A general strategy seems to apply to the regulation of the levels of the two major endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Five endocannabinoid enzymes have been cloned to date: two are responsible for the biosynthesis and degradation of anandamide, the NAPE-selective phospholipase D and the fatty acid amide hydrolase, respectively; the other three catalyse the biosynthesis and degradation of <em>2</em>-AG, the sn-1-selective diacylglycerol lipases alpha and beta and the monoacylglycerol lipase, respectively. The major features of these five proteins, their relative weight in determining endocannabinoid levels, and the possible targeting of some of them for therapeutic purpose, as well as the possibility of the existence of alternative anabolic and catabolic pathways are discussed.

We employed in vivo microdialysis to characterize the effect of an ethanol challenge injection on endocannabinoid levels in the nucleus accumbens of ethanol-naïve and chronic ethanol-treated rats. Ethanol (0.75 and <em>2</em> g/kg, i.p.) dose-dependently increased dialysate <em>2</em>-<em>arachidonoylglycerol</em> (to a maximum 157 +/- <em>2</em>0% of baseline) and decreased anandamide (to a minimum 5<em>2</em> +/- 9% of baseline) in ethanol-naïve rats. The endocannabinoid clearance inhibitor N-(4-hydrophenyl) arachidonoylamide (AM404; 3 mg/kg) potentiated ethanol effects on <em>2</em>-<em>arachidonoylglycerol</em> levels but did not alter ethanol-induced decreases in anandamide. AM404 alone did not alter dialysate levels of either endocannabinoid. Then, we characterized the effect of ethanol challenge on nucleus accumbens endocannabinoid levels in rats previously maintained on an ethanol-containing liquid diet. Ethanol challenge produced a greater and more prolonged increase in <em>2</em>-<em>arachidonoylglycerol</em> (to a maximum 394 +/- 135% of baseline) in ethanol-experienced than in ethanol-naïve rats. The profile in ethanol-experienced rats was similar to that produced by AM404 pre-treatment in ethanol-naïve rats. AM404 in ethanol-experienced rats led to a further enhancement in the <em>2</em>-<em>arachidonoylglycerol</em> response to ethanol challenge (to a maximum 704 +/- 174% of baseline). Our findings demonstrate that ethanol-induced increases in nucleus accumbens <em>2</em>-<em>arachidonoylglycerol</em> are potentiated in animals with a history of ethanol consumption.

Upon the identification of anandamide (AEA) in the porcine brain, numerous studies contributed to the current state of knowledge regarding all elements that form the "endocannabinoid system (ECS)."How this complex system of receptors, ligands, and enzymes is integrated in helping to regulate fundamental processes at level of central nervous and peripheral systems and how its regulation and dysregulation might counteract disturbances of such functions, is nowadays still under investigation. However, the most recent advances on the physiological distribution and functional role of ECS allowed the progress of various research tools aimed at the therapeutic exploitation of endocannabinoid (eCB) signaling, as well as the development of novel drugs with pharmacological advantages. Here, we shall briefly overview the metabolic and signal transduction pathways of the main eCBs representatives, AEA, and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and we will discuss the therapeutic potential of new ECS-oriented drugs.

OBJECTIVE

Palmitoylethanolamide (PEA) is an endogenous congener of anandamide and potentiates its actions at cannabinoid CB1 and CB<em>2</em> receptors, and at transient receptor potential vanilloid type-1 (TRPV1) channels. The other endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), was recently suggested to act as a TRPV1 channel agonist. We investigated if PEA enhanced levels of <em>2</em>-AG in vitro or in vivo and <em>2</em>-AG activity at TRPV1 channels.

METHODS

Endogenous lipid levels were measured by LC-MS in (i) human keratinocytes incubated with PEA (10-<em>2</em>0 μM, 40 min, 6 and <em>2</em>4 h, 37°C); (ii) the blood of spontaneously Ascaris suum hypersensitive beagle dogs given a single oral dose of ultramicronized PEA (30 mg·kg(-1), 1, <em>2</em>, 4 and 8 h from administration); (iii) the blood of healthy volunteers given a single oral dose of micronized PEA (300 mg, <em>2</em>, 4 and 6 h from administration). Effects of <em>2</em>-AG at TRPV1 channels were assessed by measuring intracellular Ca(<em>2</em>+) in HEK-<em>2</em>93 cells over-expressing human TRPV1 channels.

RESULTS

PEA elevated <em>2</em>-AG levels in keratinocytes (∼3-fold) and in human and canine plasma (∼<em>2</em> and ∼<em>2</em>0-fold respectively). <em>2</em>-AG dose-dependently raised intracellular Ca(<em>2</em>+) in HEK-<em>2</em>93-TRPV1 cells in a TRPV1-dependent manner and desensitized the cells to capsaicin. PEA only slightly enhanced <em>2</em>-AG activation of TRPV1 channels, but significantly increased <em>2</em>-AG-induced TRPV1 desensitization to capsaicin (IC50 from 0.75 ± 0.04 to 0.45 ± 0.0<em>2</em> μM, with PEA <em>2</em> μM).

CONCLUSIONS

These observations may explain why several effects of PEA are attenuated by cannabinoid receptor or TRPV1 channel antagonists.

BACKGROUND

This article is part of a themed section on Endocannabinoids. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.7/issuetoc.

Acute administration of Δ(9)-tetrahydrocannabinol (THC) or exposure to marijuana smoke impairs short-term spatial memory in water maze tasks through a CB(1) receptor mechanism of action. N-Arachidonoylethanolamine (anandamide; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are endogenous cannabinoids that are predominantly metabolized by the respective enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Although the MAGL inhibitor JZL184 enhances short-term synaptic plasticity, it has yet to be evaluated in the Morris water maze. Previous research demonstrated that simultaneous, complete blockade of FAAH and MAGL produces full blown THC-like effects. Thus, in the following studies we tested whether dual blockade of FAAH and MAGL would impair learning in a repeated acquisition Morris water maze task. Mice treated with the dual FAAH/MAGL inhibitor JZL195 (<em>2</em>0 mg/kg) as well as JZL184-treated FAAH -/- mice displayed robust deficits in Morris water maze performance that were similar in magnitude to THC-treated mice. While <em>2</em>0 or 40 mg/kg impaired water maze performance in FAAH -/- mice, only the high dose of JZL184 disrupted performance in FAAH +/+ mice. The memory impairing effects of JZL184 were blocked by the CB(1) receptor antagonist rimonabant. Neither JZL184 nor JZL195 impaired performance in a cued version of the water maze task, arguing against the notion that sensorimotor or motivational deficits accounted for the impaired acquisition performance. JZL184 increased <em>2</em>-AG levels in the hippocampus, prefrontal cortex, and cerebellum to a similar degree in FAAH -/- and +/+ mice. FAAH -/- mice, regardless of drug treatment, possessed elevated AEA levels in each brain region assessed. The results of this study reveal that concomitant increases in AEA and <em>2</em>-AG disrupt short-term spatial memory performance in a manner similar to that of THC.

The endocannabinoid signalling system in mammals comprises several molecular components, including cannabinoid receptors (e.g. CB1, CB<em>2</em>), putative endogenous ligands for these receptors [e.g. anandamide, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)] and enzymes involved in the biosynthesis and inactivation of anandamide (e.g. NAPE-PLD, FAAH) and <em>2</em>-AG (e.g. DAG lipase, MGL). In this review we examine the occurrence of these molecules in non-mammalian organisms (in particular, animals and plants) by surveying published data and by basic local alignment search tool (BLAST) analysis of the GenBank database and of genomic sequence data from several vertebrate and invertebrate species. We conclude that the ability of cells to synthesise molecules that are categorised as "endocannabinoids" in mammals is an evolutionarily ancient phenomenon that may date back to the unicellular common ancestor of animals and plants. However, exploitation of these molecules for intercellular signalling may have occurred independently in different lineages during the evolution of the eukaryotes. The CB1- and CB<em>2</em>-type receptors that mediate effects of endocannabinoids in mammals occur throughout the vertebrates, and an orthologue of vertebrate cannabinoid receptors was recently identified in the deuterostomian invertebrate Ciona intestinalis (CiCBR). However, orthologues of the vertebrate cannabinoid receptors are not found in protostomian invertebrates (e.g. Drosophila, Caenorhabditis elegans). Therefore, it is likely that a CB1/CB<em>2</em>-type cannabinoid receptor originated in a deuterostomian invertebrate. This phylogenetic information provides a basis for exploitation of selected non-mammalian organisms as model systems for research on endocannabinoid signalling.

Anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) were the first endocannabinoids to be characterized, that bind two G protein-coupled receptors, CB1 and CB<em>2</em>. AEA synthesized by multiple pathways, including NAPE-specific phospholipase D (NAPE-PLD) and degraded by the fatty acid amide hydrolase (FAAH). AEA levels are critical in regulating embryo development and the "window" of implantation. We examined the expression of nape-pld mRNA, CB1 and FAAH in human placenta hypothesizing that their altered signaling may contribute to spontaneous miscarriage. First trimester placentas from women with spontaneous miscarriage (group 1) were matched with placentas from women who underwent termination (group <em>2</em>). Nape-pld expression was analyzed by RT-PCR; CB1 and FAAH expression by Western blot and immunohistochemistry. Nape-pld mRNA expression was higher in group <em>2</em> than in group 1. Western blot analysis revealed higher CB1 expression and lower or absent FAAH in group 1 than in group <em>2</em>. Immunohistochemistry confirmed CB1 and FAAH signals in group 1 and group <em>2</em> placentas, respectively. Human placenta contains the enzymes to synthesize AEA. Moreover, placental tissue represents a target for endocannabinoids whose activity may regulate pregnancy outcome. In particular, very low or absent FAAH and high CB1 levels correspond with spontaneous miscarriage.

The actions of cannabis are mediated by receptors that are part of an endogenous cannabinoid system. The endocannabinoid system (ECS) consists of the naturally occurring ligands N-arachidonoylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), their biosynthetic and degradative enzymes, and the cannabinoid (CB) receptors CB1 and CB<em>2</em>. The ECS is a widely distributed transmitter system that controls gut functions peripherally and centrally. It is an important physiologic regulator of gastrointestinal motility. Polymorphisms in the gene encoding CB1 (CNR1) have been associated with some forms of irritable bowel syndrome. The ECS is involved in the control of nausea and vomiting and visceral sensation. The homeostatic role of the ECS also extends to the control of intestinal inflammation. We review the mechanisms by which the ECS links stress and visceral pain. CB1 in sensory ganglia controls visceral sensation, and transcription of CNR1 is modified through epigenetic processes under conditions of chronic stress. These processes might link stress with abdominal pain. The ECS is also involved centrally in the manifestation of stress, and endocannabinoid signaling reduces the activity of hypothalamic-pituitary-adrenal pathways via actions in specific brain regions, notably the prefrontal cortex, amygdala, and hypothalamus. Agents that modulate the ECS are in early stages of development for treatment of gastrointestinal diseases. Increasing our understanding of the ECS will greatly advance our knowledge of interactions between the brain and gut and could lead to new treatments for gastrointestinal disorders.

Endocannabinoids (endogenous ligands of cannabinoid receptors) such as anandamide (N-arachidonoylethanolamine) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are inactivated upon enzymatic hydrolysis. Recent progress in the enzymological and molecular biological studies on the 'endocannabinoid hydrolases' is reviewed in this article. Anandamide is hydrolyzed to arachidonic acid and ethanolamine by a membrane-bound amidase generally referred to as fatty acid amide hydrolase (FAAH). This enzyme has a broad substrate specificity, hydrolyzing oleamide (an endogenous sleep-inducing factor) and <em>2</em>-AG as well as anandamide. cDNA cloning revealed that FAAH is composed of 579 amino acids and belongs to the amidase signature family. A serine residue functioning as a catalytic nucleophile and several other catalytically important residues were identified in its primary structure. Furthermore, recent generation and analysis of the FAAH gene-deficient mice demonstrated the central role of this enzyme in the metabolism of anandamide. Alternatively, an amidase, which is distinct from FAAH but also hydrolyzing anandamide and other N-acylethanolamines at acidic pH, was identified in human megakaryoblastic cells and rat organs such as lung and spleen. As for the <em>2</em>-AG hydrolysis, in addition to the known monoacylglycerol lipase, other esterases and FAAH may be involved.

BACKGROUND

Marijuana, the most used recreational drug, has been shown to have adverse effects on human reproduction. Endogenous cannabinoids (also called endocannabinoids) bind to the same receptors as those of Δ(9)-tetrahydrocannabinol (THC), the psychoactive component of Cannabis sativa. The most extensively studied endocannabinoids are anandamide (N-arachidonoylethanolamine, AEA) and <em>2</em>-<em>arachidonoylglycerol</em>. The endocannabinoids, their congeners and the cannabinoid receptors, together with the metabolic enzymes and putative transporters form the endocannabinoid system (ECS). In this review, we summarize current knowledge about the relationships of ECS, sex steroid hormones and cytokines in female fertility, and underline the importance of this endocannabinoid-hormone-cytokine network.

METHODS

Pubmed and the Web of Science databases were searched for studies published since 1985, looking into the ECS, sex hormones, type-1/<em>2</em> T-helper (Th1/Th<em>2</em>) cytokines, leukaemia inhibitory factor, leptin and reproduction.

RESULTS

The ECS plays a pivotal role in human reproduction. The enzymes involved in the synthesis and degradation of endocannabinoids normalize levels of AEA for successful implantation. The AEA degrading enzyme (fatty acid amide hydrolase) activity as well as AEA content in blood may potentially be used for the monitoring of early pregnancies. Progesterone and oestrogen are involved in the maintenance of endocannabinoid levels. The ECS plays an important role in the immune regulation of human fertility.

CONCLUSIONS

The available studies suggest that tight control of the endocannabinoid-hormone-cytokine network is required for successful implantation and early pregnancy maintenance. This hormone-cytokine network is a key element at the maternal-foetal interface, and any defect in such a network may result in foetal loss.

A great need exists for the development of new medications to treat pain resulting from various disease states and types of injury. Given that the endogenous cannabinoid (that is, endocannabinoid) system modulates neuronal and immune cell function, both of which play key roles in pain, therapeutics targeting this system hold promise as novel analgesics. Potential therapeutic targets include the cannabinoid receptors, type 1 and <em>2</em>, as well as biosynthetic and catabolic enzymes of the endocannabinoids N-arachidonoylethanolamine and <em>2</em>-<em>arachidonoylglycerol</em>. Notably, cannabinoid receptor agonists as well as inhibitors of endocannabinoid-regulating enzymes fatty acid amide hydrolase and monoacylglycerol lipase produce reliable antinociceptive effects, and offer opioid-sparing antinociceptive effects in myriad preclinical inflammatory and neuropathic pain models. Emerging clinical studies show that 'medicinal' cannabis or cannabinoid-based medications relieve pain in human diseases such as cancer, multiple sclerosis, and fibromyalgia. However, clinical data have yet to demonstrate the analgesic efficacy of inhibitors of endocannabinoid-regulating enzymes. Likewise, the question of whether pharmacotherapies aimed at the endocannabinoid system promote opioid-sparing effects in the treatment of pain reflects an important area of research. Here we examine the preclinical and clinical evidence of various endocannabinoid system targets as potential therapeutic strategies for inflammatory and neuropathic pain conditions.

1. Chronic alcohol exposure modifies endocannabinoid levels in different brain regions, while pharmacological targeting of the endocannabinoid system has been reported to influence ethanol intake in laboratory animals. <em>2</em>. The present study was aimed at evaluating the pattern of changes of endocannabinoids and their receptors, with emphasis on reward-related brain areas, in Wistar rats subjected to consecutive phases of alcoholization, alcohol deprivation (abstinence), and voluntary consumption of alcohol (relapse). 3. We observed that, in the limbic forebrain, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) contents increased after 7 days of alcoholization, then to dramatically decrease after 48 h of alcohol deprivation and, in the case of <em>2</em>-AG, to further decrease when rats were allowed to relapse to alcohol consumption. By contrast, in the midbrain, there was a marked reduction in AEA, but not <em>2</em>-AG, content, after alcoholization. This decrease was not affected during alcohol abstinence, but both AEA and <em>2</em>-AG contents were then significantly reduced when rats were allowed to relapse to alcohol consumption. 4. Based on these data, we examined whether pharmacological activation/blockade of endocannabinoid transmission might influence ethanol intake in rats allowed to relapse to alcohol consumption after subsequent periods of alcoholization and alcohol deprivation. 5. Treatment with either Delta(9)-tetrahydrocannabinol or CP55,940, two cannabinoid agonists, reduced both total liquid and ethanol intake but did not affect ethanol preference. Treatment with SR141716, a selective cannabinoid CB(1) receptor antagonist, also produced a significant reduction in both total liquid and ethanol intake without affecting ethanol preference. Accordingly, none of these effects on ethanol intake were accompanied by changes in dopamine and GABA in limbic structures. 6. In summary, the levels of endocannabinoids underwent significant changes in reward-related areas during alcoholization, alcohol deprivation, and relapse, showing the lowest values in this latter phase. Treatment with cannabinoid agonists or a selective CB(1) receptor antagonist resulted in a reduction of ethanol intake by rats allowed to relapse to alcohol consumption after periods of alcoholization and alcohol deprivation, but these effects did not appear to be due to changes in neurobiological substrates currently involved in alcohol reinforcement/relapse.

Stearoylethanolamide (SEA) is present in human, rat and mouse brain in amounts comparable with those of the endocannabinoid anandamide (arachidonoylethanolamide; AEA). Yet, the biological activity of SEA has never been investigated. We synthesized unlabelled and radiolabelled SEA to investigate its binding, degradation and biological activity in rat C6 glioma cells. We report that SEA binds to a specific site distinct from known cannabinoid or vanilloid receptors, and that AEA and capsazepine partly (approx. 50%) antagonized this binding. Treatment of C6 cells with SEA inhibits cellular nitric oxide synthase and does not affect adenylate cyclase, whereas treatment with cannabinoid type 1 agonist <em>2</em>-<em>arachidonoylglycerol</em> activates the former enzyme and inhibits the latter. C6 cells also have a specific SEA membrane transporter, which is inhibited by NO, and a fatty acid amide hydrolase capable of cleaving SEA. In these cells, SEA shows pro-apoptotic activity, due to elevation of intracellular calcium, activation of the arachidonate cascade and mitochondrial uncoupling. NO further enhances SEA-induced apoptosis. Moreover, the cannabinoid type 1 receptor-mediated decrease in cAMP induced by AEA in C6 cells is potentiated by SEA, suggesting that this compound also has an 'entourage' effect. Taken together, this study shows that SEA is an endocannabinoid-like compound which binds to and is transported by new components of the endocannabinoid system. It seems noteworthy that degradation and pro-apoptotic activity of SEA are regulated by NO in a way opposite to that reported for AEA.

The endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> are removed from the extracellular space by a process of cellular uptake followed by metabolism. Although the enzymes responsible for endocannabinoid metabolism have been well characterised, the processes involved in uptake have been the subject of much controversy. Recent studies, however, have identified intracellular transport proteins (fatty acid binding proteins 5 and 7, heat shock protein 70, albumin, and fatty acid amide hydrolase-like AEA transporter protein) that shuttle AEA from the plasma membrane to its metabolic enzymes. Proteins such as the fatty acid amide hydrolase-like anandamide transporter protein may be useful targets for novel therapeutic strategies aimed at potentiating AEA signalling. In this article I review the current state of the art of endocannabinoid uptake.