The Endogenous Cannabinoid and the Nitricoxidergic Systems in the Modulation of Stress Responses.

The effects on stress-induced analgesia (SIA) from endogenous cannabinoid system (ECS) and nitric oxide (NO) interaction after 1 h of restraint stress were evaluated in male Wistar rats. The animals were subjected to 1 h of restraint and then injected with different combinations of cannabinoid receptor type 1 agonist anandamide (AEA) or antagonist AM251 along with an NO donor, NO precursor, or inhibitor of NO synthase. Nociception was evaluated using paw pressure (PP) or hot plate (HP) tests. AEA was administered immediately after the end of restraint-SIA (r-SIA). Administration of NO precursor reversed the pronociceptive effect of the CB1 agonist on r-SIA. Both the CB1 antagonist and the NOS inhibitor neutralized the pro-analgesic effect of L-arginine (L-arg). Administration of an NO donor, instead, increased r-SIA. Our experiments confirmed that the endogenous cannabinoid and the NO-ergic systems interact in the modulation of r-SIA. This interaction probably implies NO as a second messenger of the ECS.

Differential effect of interferon-alpha treatment on AEA and 2-AG levels.

The endocannabinoid (eCB) system is one of the key players in immunoregulation, and reduced activity of the eCB system has been linked with depressive-like behaviours in animal studies and depression in clinical samples. There is a well-established link between immune activation and depression, such as following the administration of the pro-inflammatory cytokine, interferon-α (IFN-α), used to treat hepatitis C viral (HCV) infection. However, the role of peripheral endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), following immunotherapy with IFN-α and in IFN-α -induced depression, have not been examined yet. In this study, we investigated whether circulating AEA and 2-AG were modified by treatment with IFN-α and whether they were involved in the development of IFN-α-induced depression. We also explored whether circulating eCBs were associated with peripheral cytokines during and after IFN-α treatment. We measured serum concentrations of AEA and 2-AG using High Performance Liquid Chromatography with Tandem Mass Spectrometry, and serum concentrations of cytokines using Meso Scale Discovery electrochemiluminescence V-PLEX assay, in 70 patients with HCV infection and 41 healthy subjects. We assessed HCV patients at baseline, IFN-α-treatment weeks (TW) 4 and 24, end of treatment (END) and at six months follow-up (FU). We assessed depression using M.I.N.I. International Neuropsychiatric Interview. We found a different pattern of change in peripheral AEA and 2-AG during and after IFN-α treatment. Whilst 2-AG increased earlier in immunotherapy (TW4), remained elevated throughout treatment, and reduced at six months follow-up (FU), AEA increased later in treatment (TW24) and remained elevated six months post-treatment. We also found that baseline levels of AEA were lower in HCV patients compared with healthy controls, whereas there were no differences in 2-AG levels. Interestingly, AEA, but not 2-AG, was significantly, negatively correlated with interleukin (IL)-2 and IL-17a at six months follow-up. We did not find any difference in both eCBs between patients with and without IFN-α-induced depression, at any time point. Our findings suggest that AEA and 2-AG are involved in different stages of immunoregulation following IFN-α treatment, where AEA might be involved in chronic inflammation. Lack of association between peripheral eCBs and IFN-α-induced depression suggests that different biological mechanisms may underpin inflammation-induced depression compared with classic "psychiatric" depression, or that any changes in the eCB system in depression may not be captured by peripheral AEA and 2-AG.

Design and synthesis of cyanamides as potent and selective N-acylethanolamine acid amidase inhibitors.

N-acylethanolamine acid amidase (NAAA) inhibition represents an exciting novel approach to treat inflammation and pain. NAAA is a cysteine amidase which preferentially hydrolyzes the endogenous biolipids palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). PEA is an endogenous agonist of the nuclear peroxisome proliferator-activated receptor-α (PPAR-α), which is a key regulator of inflammation and pain. Thus, blocking the degradation of PEA with NAAA inhibitors results in augmentation of the PEA/PPAR-α signaling pathway and regulation of inflammatory and pain processes. We have prepared a new series of NAAA inhibitors exploring the azetidine-nitrile (cyanamide) pharmacophore that led to the discovery of highly potent and selective compounds. Key analogs demonstrated single-digit nanomolar potency for hNAAA and showed >100-fold selectivity against serine hydrolases FAAH, MGL and ABHD6, and cysteine protease cathepsin K. Additionally, we have identified potent and selective dual NAAA-FAAH inhibitors to investigate a potential synergism between two distinct anti-inflammatory molecular pathways, the PEA/PPAR-α anti-inflammatory signaling pathway,1-4 and the cannabinoid receptors CB1 and CB2 pathways which are known for their antiinflammatory and antinociceptive properties.5-8 Our ligand design strategy followed a traditional structure-activity relationship (SAR) approach and was supported by molecular modeling studies of reported X-ray structures of hNAAA. Several inhibitors were evaluated in stability assays and demonstrated very good plasma stability (t1/2 > 2 h; human and rodents). The disclosed cyanamides represent promising new pharmacological tools to investigate the potential role of NAAA inhibitors and dual NAAA-FAAH inhibitors as therapeutic agents for the treatment of inflammation and pain.

Alterations in Rat Accumbens Dopamine, Endocannabinoids and GABA Content During WIN55,212-2 Treatment: The Role of Ghrelin.

The endocannabinoid/CB1R system as well as the central ghrelin signalling with its growth hormone secretagogoue receptors (GHS-R1A) are importantly involved in food intake and reward/reinforcement processing and show distinct overlaps in distribution within the relevant brain regions including the hypothalamus (food intake), the ventral tegmental area (VTA) and the nucleus accumbens (NAC) (reward/reinforcement). The significant mutual interaction between these systems in food intake has been documented; however, the possible role of ghrelin/GHS-R1A in the cannabinoid reinforcement effects and addiction remain unclear. Therefore, the principal aim of the present study was to investigate whether pretreatment with GHS-R1A antagonist/JMV2959 could reduce the CB1R agonist/WIN55,212-2-induced dopamine efflux in the nucleus accumbens shell (NACSh), which is considered a crucial trigger impulse of the addiction process. The synthetic aminoalklylindol cannabinoid WIN55,212-2 administration into the posterior VTA induced significant accumbens dopamine release, which was significantly reduced by the 3 mg/kg i.p. JMV2959 pretreatment. Simultaneously, the cannabinoid-increased accumbens dopamine metabolic turnover was significantly augmented by the JMV2959 pretreament. The intracerebral WIN55,212-2 administration also increased the endocannabinoid arachidonoylethanolamide/anandamide and the 2-arachidonoylglycerol/2-AG extracellular levels in the NACSh, which was moderately but significantly attenuated by the JMV2959 pretreatment. Moreover, the cannabinoid-induced decrease in accumbens γ-aminobutyric acid/gamma-aminobutyric acid levels was reversed by the JMV2959 pretreatment. The behavioural study in the LABORAS cage showed that 3 mg/kg JMV2959 pretreatment also significantly reduced the systemic WIN55,212-2-induced behavioural stimulation. Our results demonstrate that the ghrelin/GHS-R1A system significantly participates in the rewarding/reinforcing effects of the cannabinoid/CB1 agonist that are involved in cannabinoid addiction processing.

Synthesis and evaluation of potent and selective MGL inhibitors as a glaucoma treatment.

Monoacylglycerol lipase (MGL) inhibition provides a potential treatment approach to glaucoma through the regulation of ocular 2-arachidonoylglycerol (2-AG) levels and the activation of CB1 receptors. Herein, we report the discovery of new series of carbamates as highly potent and selective MGL inhibitors. The new inhibitors showed potent nanomolar inhibitory activity against recombinant human and purified rat MGL, were selective (>1000-fold) against serine hydrolases FAAH and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Protein-based 1H NMR experiments indicated that inhibitor 2 rapidly formed a covalent adduct with MGL with a residence time of about 6 h. This interconversion process "intrinsic reversibility" was exploited by modifications of the ligand's size (length and bulkiness) to generate analogs with "tunable' adduct residence time (τ). Inhibitor 2 was evaluated in a normotensive murine model for assessing intraocular pressure (IOP), which could lead to glaucoma, a major cause of blindness. Inhibitor 2 was found to decrease ocular pressure by ∼4.5 mmHg in a sustained manner for at least 12 h after a single ocular application, underscoring the potential for topically-administered MGL inhibitors as a novel therapeutic target for the treatment of glaucoma.

Masturbation to Orgasm Stimulates the Release of the Endocannabinoid 2-Arachidonoylglycerol in Humans.

BACKGROUND: Endocannabinoids are critical for rewarding behaviors such as eating, physical exercise, and social interaction. The role of endocannabinoids in mammalian sexual behavior has been suggested because of the influence of cannabinoid receptor agonists and antagonists on rodent sexual activity. However, the involvement of endocannabinoids in human sexual behavior has not been studied. AIM: To investigate plasma endocannabinoid levels before and after masturbation in healthy male and female volunteers. OUTCOMES: Plasma levels of the endocannabinoids 2-arachidonoylglycerol (2-AG), anandamide, the endocannabinoid-like lipids oleoyl ethanolamide and palmitoyl ethanolamide, arachidonic acid, and cortisol before and after masturbation to orgasm. METHODS: In study 1, endocannabinoid and cortisol levels were measured before and after masturbation to orgasm. In study 2, masturbation to orgasm was compared with a control condition using a single-blinded, randomized, 2-session crossover design. RESULTS: In study 1, masturbation to orgasm significantly increased plasma levels of the endocannabinoid 2-AG, whereas anandamide, oleoyl ethanolamide, palmitoyl ethanolamide, arachidonic acid, and cortisol levels were not altered. In study 2, only masturbation to orgasm, not the control condition, led to a significant increase in 2-AG levels. Interestingly, we also found a significant increase of oleoyl ethanolamide after masturbation to orgasm in study 2. CLINICAL TRANSLATION: Endocannabinoids might play an important role in the sexual response cycle, leading to possible implications for the understanding and treatment of sexual dysfunctions. STRENGTHS AND LIMITATIONS: We found an increase of 2-AG through masturbation to orgasm in 2 studies including a single-blinded randomized design. The exact role of endocannabinoid release as part of the sexual response cycle and the biological significance of the finding should be studied further. Cannabis and other drug use and the attainment of orgasm were self-reported in the present study. CONCLUSION: Our data indicate that the endocannabinoid 2-AG is involved in the human sexual response cycle and we hypothesize that 2-AG release plays a role in the rewarding consequences of sexual arousal and orgasm. Fuss J, Bindila L, Wiedemann K, et al. Masturbation to Orgasm Stimulates the Release of the Endocannabinoid 2-Arachidonoylglycerol in Humans. J Sex Med 2017;14:1372-1379.

Unraveling the Endocannabinoid System: Exploring Its Therapeutic Potential in Autism Spectrum Disorder.

The salient features of autism spectrum disorder (ASD) encompass persistent difficulties in social communication, as well as the presence of restricted and repetitive facets of behavior, hobbies, or pursuits, which are often accompanied with cognitive limitations. Over the past few decades, a sizable number of studies have been conducted to enhance our understanding of the pathophysiology of ASD. Preclinical rat models have proven to be extremely valuable in simulating and analyzing the roles of a wide range of established environmental and genetic factors. Recent research has also demonstrated the significant involvement of the endocannabinoid system (ECS) in the pathogenesis of several neuropsychiatric diseases, including ASD. In fact, the ECS has the potential to regulate a multitude of metabolic and cellular pathways associated with autism, including the immune system. Moreover, the ECS has emerged as a promising target for intervention with high predictive validity. Particularly noteworthy are resent preclinical studies in rodents, which describe the onset of ASD-like symptoms after various genetic or pharmacological interventions targeting the ECS, providing encouraging evidence for further exploration in this area.

A neuroplasticity hypothesis of chronic stress in the basolateral amygdala.

Chronic stress plays a role in the etiology of several affective and anxiety-related disorders. Despite this, its mechanistic effects on the brain are still unclear. Of particular interest is the effect of chronic stress on the amygdala, which plays a key role in the regulation of emotional responses and memory consolidation. This review proposes a neuroplasticity model for the effects of chronic stress in this region, emphasizing the roles of glutamate and BDNF signaling. This model provides a review of recent discoveries of the effects of chronic stress in the amygdala and reveals pathways for future research.

Cell-autonomous and differential endocannabinoid signaling impacts the development of presynaptic retinal ganglion cell axon connectivity in vivo.

Cannabis exposure during gestation evokes significant molecular modifications to neurodevelopmental programs leading to neurophysiological and behavioral abnormalities in humans. The main neuronal receptor for Δ9-tetrahydrocannabinol (THC) is the type-1 cannabinoid receptor CB1R, one of the most abundant G-protein-coupled receptors in the nervous system. While THC is the major psychoactive phytocannabinoid, endocannabinoids (eCBs) are the endogenous ligands of CB1R and are known to act as retrograde messengers to modulate synaptic plasticity at different time scales in the adult brain. Accumulating evidence indicates that eCB signaling through activation of CB1R plays a central role in neural development. During development, most CB1R localized to axons of projection neurons, and in mice eCB signaling impacts axon fasciculation. Understanding of eCB-mediated structural plasticity during development, however, requires the identification of the precise spatial and temporal dynamics of CB1R-mediated modifications at the level of individual neurons in the intact brain. Here, the cell-autonomous role of CB1R and the effects of CB1R-mediated eCB signaling were investigated using targeted single-cell knockdown and pharmacologic treatments in Xenopus. We imaged axonal arbors of retinal ganglion cells (RGCs) in real time following downregulation of CB1R via morpholino (MO) knockdown. We also analyzed RGC axons with altered eCB signaling following treatment with URB597, a selective inhibitor of the enzyme that degrades Anandamide (AEA), or JZL184, an inhibitor of the enzyme that blocks 2-Arachidonoylglycerol (2-AG) hydrolysis, at two distinct stages of retinotectal development. Our results demonstrate that CB1R knockdown impacts RGC axon branching at their target and that differential 2-AG and AEA-mediated eCB signaling contributes to presynaptic structural connectivity at the time that axons terminate and when retinotectal synaptic connections are made. Altering CB1R levels through CB1R MO knockdown similarly impacted dendritic morphology of tectal neurons, thus supporting both pre- and postsynaptic cell-autonomous roles for CB1R-mediated eCB signaling.

Activity-Dependent Modulation of Tonic GABA Currents by Endocannabinoids in Hirudo verbana.

Endocannabinoids are lipid neuromodulators that are synthesized on demand and primarily signal in a retrograde manner to elicit depression of excitatory and inhibitory synapses. Despite the considerable interest in their potential analgesic effects, there is evidence that endocannabinoids can have both pro-nociceptive and anti-nociceptive effects. The mechanisms contributing to the opposing effects of endocannabinoids in nociception need to be better understood before cannabinoid-based therapies can be effectively utilized to treat pain. Using the medicinal leech, Hirudo verbana, this work investigates whether endocannabinoids modulate tonic inhibition onto non-nociceptive afferents. In voltage clamp recordings, we analyzed changes in the tonic inhibition in pressure-sensitive (P) cells following pre-treatment with endocannabinoids, 2-arachidonoylglycerol (2-AG) or anandamide (AEA). We also tested whether high frequency stimulation (HFS) of nociceptive (N) cells could also modulate tonic inhibition. Both endocannabinoid application and N cell HFS depressed tonic inhibition in the P cell. Depression of tonic inhibition by N cell HFS was blocked by SB 366791 (a TRPV1 inhibitor). SB 366791 also prevented 2-AG-and AEA-induced depression of tonic inhibition. HFS-induced depression was not blocked by tetrahydrolipstatin (THL), which prevents 2-AG synthesis, nor AM 251 (a CB1 receptor inverse agonist). These results illustrate a novel activity-dependent modulation of tonic GABA currents that is mediated by endocannabinoid signaling and is likely to play an important role in sensitization of non-nociceptive afferent pathways.

2-AG and anandamide enhance hippocampal long-term potentiation via suppression of inhibition.

It is widely accepted that exogenous cannabinoids can impair short-term memory and cognition in humans and other animals. This is likely related to the inhibition of long-term potentiation (LTP), a form of synaptic plasticity, by the global and sustained activation of CB1 cannabinoid receptors in the presence of exogenous agonists. Conversely, the temporally and spatially restricted release of endogenous cannabinoid (eCB) ligands may enhance synaptic plasticity in a synapse-specific manner. We examined the role of eCB signaling in LTP by recording field excitatory postsynaptic potentials (fEPSPs) in the CA1 stratum radiatum in hippocampal slices from juvenile mice. LTP was induced either electrically, by theta burst stimulation (TBS), or pharmacologically, by treatment for 15 min with a solution designed to increase intracellular cAMP (chem-LTP). A stable and long-lasting potentiation in fEPSP slope following TBS was significantly reduced by blocking cannabinoid receptor activation with CB1 receptor antagonists. Chem-LTP caused a sustained 2-fold increase in fEPSP slope and was also blocked by CB1 receptor antagonists. TBS-LTP was partially reduced by inhibiting the synthesis of the endogenous ligands 2-arachidonylglycerol (2-AG) and anandamide. A similar effect was observed with chem-LTP. Blocking inhibitory synapses completely prevented the effect of CB1 receptor antagonists or inhibition of eCB synthesis on TBS-LTP and chem-LTP. These results indicate that simultaneous activation of CB1 receptors by 2-AG and anandamide enhances TBS-induced and pharmacologically-induced LTP, and this effect is mediated by the suppression of inhibition at GABAergic synapses.

Sex differences in endocannabinoids during 3 years of Mediterranean diet intervention: Association with insulin resistance and weight loss in a population with metabolic syndrome.

Background: Excess circulating endocannabinoids (eCBs) and imbalanced N-acylethanolamines (NAEs) related eCBs abundance could influence dietary weight loss success. We aimed to examine sex differences in the impact of a 3-years Mediterranean diet (MedDiet) intervention on circulating eCBs, NAEs and their precursor fatty acids, and to analyze the interplay between changes in eCBs or NAEs ratios, insulin resistance and the achievement of clinically meaningful weight reductions. Methods: Prospective cohort study in a subsample of N = 105 participants (54.3% women; 65.6 ± 4.6 years) with overweight or obesity and metabolic syndrome that underwent a 3-years MedDiet intervention (PREDIMED-Plus study). Plasma eCBs and NAEs, including 2-arachidonoylglycerol (2-AG), anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), fatty acids, diet, glycemic homeostasis (including the assessment of insulin resistance-HOMA-IR), and cardiovascular risk markers were monitored (at 0-6-12-36 months). Results: Mediterranean diet adherence increased in both sexes and remained high during the 3 years of follow-up. Reductions in body weight, glycemic and cardiovascular parameters were larger in men than in women. Women presented higher concentrations of NAEs than men throughout the study. In both sexes, AEA and other NAEs (including OEA, and PEA) decreased after 6 months (for AEA: -4.9%), whereas the ratio OEA/AEA increased after 1 year (+5.8%). Changes in 2-AG (-3.9%) and the ratio OEA/PEA (+8.2%) persisted over the 3 years of follow-up. In women, 6-months changes in AEA (OR = 0.65) and the ratio OEA/AEA (OR = 3.28) were associated with the achievement of 8% weight reductions and correlated with HOMA-IR changes (r = 0.29 and r = -0.34). In men, OEA/PEA changes were associated with 8% weight reductions (OR = 2.62) and correlated with HOMA-IR changes (r = -0.32). Conclusion: A 3-years MedDiet intervention modulated plasma concentrations of eCBs and NAEs. Changes in AEA and in the relative abundance of NAEs were associated with clinically meaningful weight reductions. However, marked sex differences were identified in eCBs and NAEs, as well as in the efficacy of the intervention in terms of glycemic and cardiovascular parameters, which could be related to post-menopause alterations in glucose metabolism. These findings support a sex-balanced research strategy for a better understanding of the mechanisms underlying the regulation of body weight loss.

The role of endocannabinoid pathway in the neuropathology of Alzheimer's disease: Can the inhibitors of MAGL and FAAH prove to be potential therapeutic targets against the cognitive impairment associated with Alzheimer's disease?

Alzheimer's disease is a neurodegenerative disease characterized by progressive decline of cognitive function in combination with neuronal death. Current approved treatment target single dysregulated pathway instead of multiple mechanism, resulting in lack of efficacy in slowing down disease progression. The proclivity of endocannabinoid system to exert neuroprotective action and mitigate symptoms of neurodegeneration condition has received substantial interest. Growing evidence suggest the endocannabinoids (eCB) system, viz. anadamide (AEA) and arachidonoyl glycerol (2-AG), as potential therapeutic targets with the ability to modify Alzheimer's pathology by targeting the inflammatory, neurodegenerative and cognitive aspects of the disease. In order to modulate endocannabinoid system, number of agents have been reported amongst which are inhibitors of the monoacylglycerol (MAGL) and fatty acid amide hydrolase (FAAH), the enzymes that hydrolyses 2-AG and AEA respectively. However, little is known regarding the exact mechanistic signalling and their effects on pathophysiology and cognitive decline associated with Alzheimer's disease. Both MAGL and FAAH inhibitors possess fascinating properties that may offer a multi-faceted approach for the treatment of Alzheimer's disease such as potential to protect neurons from deleterious effect of amyloid-ß, reducing phosphorylation of tau, reducing amyloid-ß induced oxidative stress, stimulating neurotrophin to support brain intrinsic repair mechanism etc. Based on empirical evidence, MAGL and FAAH inhibitors might have potential for therapeutic efficacy against cognitive impairment associated with Alzheimer's disease. The aim of this review is to summarize the experimental studies demonstrating the polyvalent properties of MAGL or FAAH inhibitor compounds for the treatment of Alzheimer's disease, and also effect of these on learning and types of memories, which together encourage to study these compounds over other therapeutics targets. Further research in this direction would enhance the molecular mechanisms and development of applicable interventions for the treatment of Alzheimer's disease, which nevertheless stay as the primary unmet need.

Genetic Blockade of NAAA Cell-specifically Regulates Fatty Acid Ethanolamides (FAEs) Metabolism and Inflammatory Responses.

N-Acylethanolamine acid amidase (NAAA) is a lysosomal enzyme responsible for the hydrolysis of fatty acid ethanolamides (FAEs). However, the role of NAAA in FAEs metabolism and regulation of pain and inflammation remains mostly unknown. Here, we generated NAAA-deficient (NAAA-/-) mice using CRISPR-Cas9 technique, and found that deletion of NAAA increased PEA and AEA levels in bone marrow (BM) and macrophages, and elevated AEA levels in lungs. Unexpectedly, genetic blockade of NAAA caused moderately effective anti-inflammatory effects in lipopolysaccharides (LPS)-induced acute lung injury (ALI), and poor analgesic effects in carrageenan-induced hyperalgesia and sciatic nerve injury (SNI)-induced mechanical allodynia. These data contrasted with acute (single dose) or chronic NAAA inhibition by F96, which produced marked anti-inflammation and analgesia in these models. BM chimera experiments indicated that these phenotypes were associated with the absence of NAAA in non-BM cells, whereas deletion of NAAA in BM or BM-derived cells in rodent models resulted in potent analgesic and anti-inflammatory phenotypes. When combined, current study suggested that genetic blockade of NAAA regulated FAEs metabolism and inflammatory responses in a cell-specifical manner.

Potassium channels as molecular targets of endocannabinoids.

Endocannabinoids are a group of endogenous mediators derived from membrane lipids, which are implicated in a wide variety of physiological functions such as blood pressure regulation, immunity, pain, memory, reward, perception, reproduction, and sleep. N-Arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG) represent two major endocannabinoids in the human body and they exert many of their cellular and organ system effects by activating the Gi/o protein-coupled, cannabinoid type 1 (CB1) and type 2 (CB2) receptors. However, not all effects of cannabinoids are ascribable to their interaction with CB1 and CB2 receptors; indeed, macromolecules like other types of receptors, ion channels, transcription factors, enzymes, transporters, and cellular structure have been suggested to mediate the functional effects of cannabinoids. Among the proposed molecular targets of endocannabinoids, potassium channels constitute an intriguing group, because these channels not only are crucial in shaping action potentials and controlling the membrane potential and cell excitability, thereby regulating a wide array of physiological processes, but also serve as potential therapeutic targets for the treatment of cancer and metabolic, neurological and cardiovascular disorders. This review sought to survey evidence pertaining to the CB1 and CB2 receptor-independent actions of endocannabinoids on ion channels, with an emphasis on AEA and potassium channels. To better understand the functional roles as well as potential medicinal uses of cannabinoids in human health and disease, further mechanistic studies to delineate interactions between various types of cannabinoids and ion channels, including members in the potassium channel superfamily, are warranted.

Endocannabinoid Anandamide Attenuates Acute Respiratory Distress Syndrome through Modulation of Microbiome in the Gut-Lung Axis.

Acute respiratory distress syndrome (ARDS) is a serious lung condition characterized by severe hypoxemia leading to limitations of oxygen needed for lung function. In this study, we investigated the effect of anandamide (AEA), an endogenous cannabinoid, on Staphylococcal enterotoxin B (SEB)-mediated ARDS in female mice. Single-cell RNA sequencing data showed that the lung epithelial cells from AEA-treated mice showed increased levels of antimicrobial peptides (AMPs) and tight junction proteins. MiSeq sequencing data on 16S RNA and LEfSe analysis demonstrated that SEB caused significant alterations in the microbiota, with increases in pathogenic bacteria in both the lungs and the gut, while treatment with AEA reversed this effect and induced beneficial bacteria. AEA treatment suppressed inflammation both in the lungs as well as gut-associated mesenteric lymph nodes (MLNs). AEA triggered several bacterial species that produced increased levels of short-chain fatty acids (SCFAs), including butyrate. Furthermore, administration of butyrate alone could attenuate SEB-mediated ARDS. Taken together, our data indicate that AEA treatment attenuates SEB-mediated ARDS by suppressing inflammation and preventing dysbiosis, both in the lungs and the gut, through the induction of AMPs, tight junction proteins, and SCFAs that stabilize the gut-lung microbial axis driving immune homeostasis.

The Endocannabinoid Anandamide Attenuates Acute Respiratory Distress Syndrome by Downregulating miRNA that Target Inflammatory Pathways.

Acute respiratory distress syndrome (ARDS) is defined as a type of respiratory failure that is caused by a variety of insults such as pneumonia, sepsis, trauma and certain viral infections. In this study, we investigated the effect of an endocannabinoid, anandamide (AEA), on ARDS induced in the mouse by Staphylococcus Enterotoxin B (SEB). Administration of a single intranasal dose of SEB in mice and treated with exogenous AEA at a dose of 40 mg/kg body weight led to the amelioration of ARDS in mice. Clinically, plethysmography results indicated that there was an improvement in lung function after AEA treatment accompanied by a decrease of inflammatory cell infiltrate. There was also a significant decrease in pro-inflammatory cytokines IL-2, TNF-α, and IFN-γ, and immune cells including CD4+ T cells, CD8+ T cells, Vß8+ T cells, and NK+ T cells in the lungs. Concurrently, an increase in anti-inflammatory phenotypes such as CD11b + Gr1+ Myeloid-derived Suppressor Cells (MDSCs), CD4 + FOXP3 + Tregs, and CD4+IL10 + cells was observed in the lungs. Microarray data showed that AEA treatment in ARDS mice significantly altered numerous miRNA including downregulation of miRNA-23a-3p, which caused an upregulation of arginase (ARG1), which encodes for arginase, a marker for MDSCs, as well as TGF-ß2, which induces Tregs. AEA also caused down-regulation of miRNA-34a-5p which led to induction of FoxP3, a master regulator of Tregs. Transfection of T cells using miRNA-23a-3p or miRNA-34a-5p mimics and inhibitors confirmed that these miRNAs targeted ARG1, TGFß2 and FoxP3. In conclusion, the data obtained from this study suggests that endocannabinoids such as AEA can attenuate ARDS induced by SEB by suppressing inflammation through down-regulation of key miRNA that regulate immunosuppressive pathways involving the induction of MDSCs and Tregs.

Familial abnormalities of endocannabinoid signaling in schizophrenia.

OBJECTIVES: Epidemiological and experimental evidence suggests that the endocannabinoid system plays a pathophysiological role in schizophrenia. This is reflected by elevated cerebrospinal levels of the endocannabinoid anandamide in schizophrenia and its initial prodromal states. METHODS: We analyzed plasma concentrations of anandamide, 2-arachidonoyl-sn-glycerol, palmitoylethanolamide and oleoylethanolamide from 25 twin pairs discordant for schizophrenia, six discordant for bipolar disorder and eight healthy twin pairs to determine hereditary traits. RESULTS: Twin pairs discordant for schizophrenia or bipolar disorder had significantly higher levels of anandamide and palmitoylethanolamide compared to healthy twins (both P < 0.002). Non-affected twins discordant for schizophrenia, who developed a psychotic disorder within 5 years follow-up showed lower anandamide (P = 0.042) and 2-arachidonoyl-sn-glycerol levels (P = 0.049) than twins who remained healthy. CONCLUSIONS: We suggest that the protective upregulation of endocannabinoid signalling reflects either a hereditary trait or mirrors a modulating response to genetically influenced cerebral function involving, e.g., other neurotransmitters or energy metabolism.

Oleoyl glycine: interference with the aversive effects of acute naloxone-precipitated MWD, but not morphine reward, in male Sprague-Dawley rats.

RATIONALE: Oleoyl glycine (OlGly), a recently discovered fatty acid amide that is structurally similar to N- acylethanolamines, which include the endocannabinoid, anandamide (AEA), as well as endogenous peroxisome proliferator-activated receptor alpha (PPARα) agonists oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), has been shown to interfere with nicotine reward and dependence in mice. OBJECTIVES AND METHODS: Behavioral and molecular techniques were used to investigate the ability of OlGly to interfere with the affective properties of morphine and morphine withdrawal (MWD) in male Sprague-Dawley rats. RESULTS: Synthetic OlGly (1-30 mg/kg, intraperitoneal [ip]) produced neither a place preference nor aversion on its own; however, at doses of 1 and 5 mg/kg, ip, it blocked the aversive effects of MWD in a place aversion paradigm. This effect was reversed by the cannabinoid 1 (CB1) receptor antagonist, AM251 (1 mg/kg, ip), but not the PPARα antagonist, MK886 (1 mg/kg, ip). OlGly (5 or 30 mg/kg, ip) did not interfere with a morphine-induced place preference or reinstatement of a previously extinguished morphine-induced place preference. Ex vivo analysis of tissue (nucleus accumbens, amygdala, prefrontal cortex, and interoceptive insular cortex) collected from rats experiencing naloxone-precipitated MWD revealed that OlGly was selectively elevated in the nucleus accumbens. MWD did not modify levels of the endocannabinoids 2-AG and AEA, nor those of the PPARα ligands, OEA and PEA, in any region evaluated. CONCLUSION: Here, we show that OlGly interferes with the aversive properties of acute naloxone-precipitated morphine withdrawal in rats. These results suggest that OlGly may reduce the impact of MWD and may possess efficacy in treating opiate withdrawal.

Endocannabinoids and endocannabinoid-like compounds modulate hypoxia-induced permeability in CaCo-2 cells via CB1, TRPV1, and PPARα.

BACKGROUND AND PURPOSE: We have previously reported that endocannabinoids modulate permeability in Caco-2 cells under inflammatory conditions and hypothesised in the present study that endocannabinoids could also modulate permeability in ischemia/reperfusion. EXPERIMENTAL APPROACH: Caco-2 cells were grown on cell culture inserts to confluence. Trans-epithelial electrical resistance (TEER) was used to measure permeability. To generate hypoxia (0% O2), a GasPak™ EZ anaerobe pouch system was used. Endocannabinoids were applied to the apical or basolateral membrane in the presence or absence of receptor antagonists. KEY RESULTS: Complete hypoxia decreased TEER (increased permeability) by ~35% after 4 h (recoverable) and ~50% after 6 h (non-recoverable). When applied either pre- or post-hypoxia, apical application of N-arachidonoyl-dopamine (NADA, via TRPV1), oleamide (OA, via TRPV1) and oleoylethanolamine (OEA, via TRPV1) inhibited the increase in permeability. Apical administration of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) worsened the permeability effect of hypoxia (both via CB1). Basolateral application of NADA (via TRPV1), OA (via CB1 and TRPV1), noladin ether (NE, via PPARα), and palmitoylethanolamine (PEA, via PPARα) restored permeability after 4 h hypoxia, whereas OEA increased permeability (via PPARα). After 6 h hypoxia, where permeability does not recover, only basolateral application PEA sustainably decreased permeability, and NE decreased permeability. CONCLUSIONS AND IMPLICATIONS: A variety of endocannabinoids and endocannabinoid-like compounds modulate Caco-2 permeability in hypoxia/reoxygenation, which involves multiple targets, depending on whether the compounds are applied to the basolateral or apical membrane. CB1 antagonism and TRPV1 or PPARα agonism may represent novel therapeutic targets against several intestinal disorders associated with increased permeability.