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2-Monoacylglycerol (2-MAG) is one of the digestion products of dietary lipids. We recently demonstrated that a 2-MAG, 2-arachidonoyl glycerol (2-AG) potently stimulated cholecystokinin (CCK) secretion via cannabinoid receptor 1 (CB1) in a murine CCK-producing cell line, STC-1. CCK plays a crucial role in suppressing postprandial gastric emptying. To examine the effect of 2-AG on gastric emptying, we performed acetaminophen and phenol red recovery tests under oral or intraperitoneal administration of 2-AG in mice. Orally administered 2-AG (25 mg/kg) suppressed the gastric emptying rate in mice, as determined by the acetaminophen absorption test and phenol red recovery test. Intraperitoneal administration of a cholecystokinin A receptor antagonist (0.5 mg/kg) attenuated the gastric inhibitory emptying effect. In addition, both oral (10 mg/kg) and intraperitoneal (0.5 mg/kg) administration of a CB1 antagonist counteracted the 2-AG-induced gastric inhibitory effect. Furthermore, intraperitoneal 2-AG (25 mg/kg) suppressed gastric emptying. These results indicate that 2-AG exhibits an inhibitory effect on gastric emptying in mice, possibly mediated by stimulating both CCK secretion via CB1 expressed in CCK-producing cells and acting on CB1 expressed in the peripheral nerves. Our findings provide novel insights into the 2-MAG-sensing mechanism in enteroendocrine cells and the physiological role of 2-MAG.

Cholecystokinin (CCK) is a peptide hormone secreted from enteroendocrine cells and regulates the exocrine pancreas, gastric motility, and appetite. Dietary triacylglycerols are hydrolyzed to fatty acids (FA) and 2-monoacylglycerols (2-MAG) in the small intestine. Although it is well known that FA stimulate CCK secretion, whether 2-MAG have the CCK-releasing activity remains unclear. We examined the CCK-releasing activity of four commercially available 2-MAG in a murine CCK-producing cell line, STC-1, and the molecular mechanism underlying 2-MAG-induced CCK secretion. CCK released from the cells was measured using ELISA. Among four 2-MAG (2-palmitoyl, 2-oleoyl, 2-linoleoyl, and 2-arachidonoyl monoacylglycerols) examined, 2-arachidonoyl glycerol (2-AG) potently stimulated CCK secretion in a dose-dependent manner. Structurally related compounds, such as 2-arachidonoyl glycerol ether and 1-arachidonoyl glycerol, did not stimulate CCK secretion. Both arachidonic acid and 2-AG stimulated CCK secretion at 100 mu M, but only 2-AG did at 50 mu M. 2-AG-induced CCK secretion but not arachidonic acid-induced CCK secretion was attenuated by treatment with a cannabinoid receptor 1 (CB1) antagonist. These results indicate that a specific 2-MAG, 2-AG, directly stimulates CCK secretion via CB1.

Emerging evidence indicates that the endogenous cannabinoid system modulates the behavioral and physiological effects of nicotine. Fatty acid-binding proteins (FABPs) are among the primary intracellular trafficking mechanisms of endogenous cannabinoids, such as anandamide. To this end, changes in FABP expression may similarly impact the behavioral manifestations associated with nicotine, particularly its addictive properties. FABP5+/+ and FABP5−/− mice were tested for nicotine-conditioned place preference (CPP) at two different doses (0.1 or 0.5 mg/kg). The nicotine-paired chamber was assigned as their least preferred chamber during preconditioning. Following 8 days of conditioning, the mice were injected with either nicotine or saline. The mice were allowed to access to all the chambers on the test day, and their times spent in the drug chamber on the preconditioning versus the test days were used to examine the drug preference score. The CPP results showed that the FABP5−/− mice displayed a higher place preference for 0.1 mg/kg nicotine than the FABP5+/+ mice, while no CPP difference was observed for 0.5 mg/kg nicotine between the genotypes. In conclusion, FABP5 plays an important role in regulating nicotine place preference. Further research is warranted to identify the precise mechanisms. The results suggest that dysregulated cannabinoid signaling may impact nicotine-seeking behavior.

Corneal endothelial cells are crucially involved in maintaining the transparency of the human cornea. Severe losses in corneal endothelial cell density (ECD) can lead to corneal edema, gradual loss of vision, and at worst blindness. Cell degeneration processes and apoptosis are implicated with Ca2+. Thus, phenotypic characteristics of these cells are controlled by Ca2+-dependent cellular mechanisms. Transient receptor potential channels (TRPs) are substantially involved in the regulation of intracellular calcium concentration ([Ca2+]i) whereby TRP vanilloid type 1 (TRPV1) (capsaicin receptor) modulates apoptosis. Studies have shown that G protein-coupled receptors (GPCRs) such as cannabinoid receptor type 1 (CB1) are expressed in human corneal epithelial cells (HCEPs). Further studies about nerve growth factor (NGF) revealed that exogenous NGF can stimulate the proliferation of HCEP. In this study, the functional expression of TRPV1, CB1, and NGF in human corneal endothelial cells (HCECs) was investigated. [Ca2+]i was measured by fluorescence calcium imaging and whole-cell currents were measured by the planar patch-clamp technique. The human HCEC line (HCEC-12) was used as an established in vitro cell model and a TRPV1 transduced HCEC-12 (TRPV1-HCEC-12) was used as a heterologous cell expression system. Extracellular application of 10 µM capsaicin (CAP) led to an increase of the fluorescence ratio (f340/f380), which is proportional to [Ca2+]i, and an increase of whole-cell currents. Both effects were inhibited by the TRPV1 antagonist capsazepine (CPZ) (10 µM). The CB1 agonist WIN 55,212-2 (10 µM), increased [Ca2+]i and whole-cell currents, which could be blocked with the CB1 antagonist AM251 (10 µM). The CAP (10 µM) induced Ca2+ rise was inhibited below the Ca2+ baseline if the HCEC-12 cells were preincubated with 10 µM WIN 55,212-2, whereas 10 µM AM251 only slightly influenced this effect. The CAP-induced Ca2+ increase was at considerably higher levels in TRPV1 transduced-HCEC-12 than in non-transduced HCEC-12 (controls). The WIN 55,212-2-induced Ca2+ response pattern was changed if the cells were preincubated with CAP or CPZ (both 10 µM). Finally, NGF (50 ng/ml) increased [Ca2+]i, which was suppressed by CPZ. Taken together, the functional expression of CB1 and TRPV1 was first shown in normal HCEC-12 as well as in TRPV1-HCEC-12, in which the effects were at higher levels confirming the successful overexpression of TRPV1 in HCEC-12. Furthermore, there is a crosstalk between TRPV1 and CB1 as well as NGF indicating a complex Ca2+ regulation in HCEC-12. The results contribute to a better understanding of Ca2+ regulatory mechanisms and may be useful for the development of novel strategies to improve the preservation of corneal grafts for keratoplasty., Korneale Endothelzellen sind maßgeblich an der Aufrechterhaltung der Transparenz der humanen Hornhaut involviert. Geringe Endothelzelldichte (ECD) kann zu kornealem Ödem, allmählichen Sehverlust und schlimmstenfalls zur Erblindung führen. Daran sind degenerative Prozesse und Apoptose mit Ca2+ beteiligt. Von daher sind die Eigenschaften dieser Zellen von Ca2+ abhängigen zellulären Mechanismen geprägt. In der Regulation der intrazellulären Ca2+ Konzentration ([Ca2+]i) sind „transient receptor potential“ (TRP) Kanäle substanziell beteiligt wobei der TRP Vanilloid 1 (TRPV1) bekannt als Capsaicinrezeptor Apoptose moduliert. Studien haben gezeigt, dass G-Protein gekoppelte Rezeptoren wie der Cannabinoid Rezeptor 1 (CB1) in humanen Hornhautepithelzellen (HCEPs) exprimiert werden. Studien zum Nervenwachstumsfaktor (NGF) ergaben, dass exogenes NGF die Proliferation von kornealen Endothel- und Epithelzellen von Kaninchen und Menschen stimuliert. In dieser Arbeit wurde die funktionelle Expression von TRPV1, CB1 und NGF in humanen kornealen Endothelzellen (HCEC) untersucht. [Ca2+]i wurde mit Hilfe von Fluoreszenz Calcium Imaging und Ganzzellströme wurden mittels planarer Patch-Clamp Technik gemessen. Die HCEC-12 Zelllinie wurde als etabliertes in vitro Zellmodell sowie eine TRPV1-transduzierte HCEC-12 Zelllinie als heterologes Zellsystem verwendet. Extrazelluläre Applikation von 10 µM Capsaicin (CAP) führte sowohl zu Erhöhung des Fluoreszenzratios (f340/f380), das proportional zu [Ca2+]i ist, also auch zu Erhöhung der Ganzzellströme. Beide Effekte konnten mit dem TRPV1 Kanalblocker Capsazepin (CPZ) (10 µM) unterdrückt werden. Der CB1 Agonist WIN 55,212-2 (10 µM) erhöhte [Ca2+]i und Ganzzellströme, welche mit dem CB1 Antagonisten AM251 (10 µM) geblockt werden konnten. Der CAP-induzierte Ca2+ Anstieg konnten in Zellen, die mit 10 µM WIN 55,212-2 vorinkubiert wurden, bis unter die Ca2+ Basislinie unterdrückt werden, während 10 µM AM251 diesen Effekt eher geringfügig beeinflusste. Der CAP-induzierte Ca2+ Anstieg war in TRPV1-HCEC-12 erwartungsgemäß wesentlich stärker als in nicht-transduzierten HCEC-12 (Kontrollen). Das WIN 55,212-2-induzierte Ca2+ Antwortmuster änderte sich in Zellen, die mit CAP oder CPZ behandelt wurden (10 µM). Schließlich erhöhte auch NGF (50 ng/ml) [Ca2+]i, welche ebenfalls durch CPZ unterdrückt werden konnte. Zusammenfassend konnte erstmalig die funktionale Expression von CB1 und TRPV1 in normalen HCEC-12 sowie TRPV1-transduzierten HCEC-12 gezeigt werden, wobei die Effekte in den transduzierten Zellen stärker waren und damit die erfolgreiche Transduktion von TRPV1 in HCEC-12 bestätigte. Darüber hinaus besteht ein Crosstalk zwischen TRPV1 und CB1 sowie NGF, welcher auf eine komplexe Ca2+ Regulation in HCEC-12 hindeutet. Die Ergebnisse tragen zu einem besseren Verständnis der Ca2+ Regulationsmechanismen bei und können für die Entwicklung neuartiger Strategien für die Verbesserung der Erhaltung von Hornhauttransplantaten für die Keratoplastik beitragen.

Cannabinoid receptor 1 (CB1), abundantly expressed in the CNS, is a promising target for the pharmacological treatment of pathological pain conditions due to its function as an inhibitor of neurotransmitter release from presynaptic neurons. Recently, the SGIP1 protein has been found to interact with the CB1 receptor and participates in the modulation of nociception. However, whether SGIP1 modulates spinal CB1 receptor signaling at the spinal cord level is unknown. To answer this question, we used the patch-clamp method in the superficial spinal cord dorsal horn neurons of wild-type (WT) and knockout (KO) SGIP1 mice to measure spontaneous (s) and miniature (m) excitatory postsynaptic currents (EPSCs). Results of naive mice and mice with carrageenan-induced peripheral inflammation were compared. The results show that the efficacy of the CB1 receptor agonist WIN 55,212-2 at the first spinal nociceptive synapse is identical in naive mice for both SGIP1 WT and KO phenotypes. The control frequencies of both groups of neurons did not differ in naïve conditions or the peripheral inflammation model. On the contrary, the WIN 55,212-2 application was more effective in SGIP1 KO mice during peripheral inflammation. This study further addressed how CB1 receptor activation affects spinal inhibitory synaptic...

Specific and selective anti-CB1 antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB1 receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB1 receptor as the best choice for specific end-use purposes. Specifically, an antibody against a long fragment of the extracellular amino tail of CB1 receptor (but not one against a short sequence of the extreme amino-terminus) detected strong surface staining when applied to live cells, whereas two different antibodies against an identical fragment of the extreme carboxy-terminus of CB1 receptor (but not one against an upstream peptide) showed acceptable performance on all platforms, although they behaved differently in immunohistochemical assays depending on the tissue fixation procedure used and showed different specificity in Western blot assays, which made each of them particularly suitable for one of those techniques. Our results provide a framework to interpret past and future results derived from the use of different anti-CB1 antibodies in the context of current knowledge about the CB1 receptor at the molecular level, and highlight the need for an adequate validation for specific purposes, not only before antibodies are placed on the market, but also before the decision to discontinue them is made.

Electroacupuncture (EA) pretreatment induces cerebral ischemic tolerance; however, the mechanism remains poorly understood. This study aimed to determine the participation of peroxisome proliferato...

Over the last decade, new psychoactive substances (NPS) have continuously been the focus of the international society since their emergence on the illicit drug market. NPS can be classified into six groups including; synthetic cannabinoid receptor agonists (SCRAs), stimulants, opioids, dissociatives, sedatives/hypnotics, and classic hallucinogens with psychoactive effects. These are sold as "herbal incense," "bath salts," "legal highs," and "research chemicals". They can be synthesized easily with slight changes in the chemical moieties of known psychoactive substances. NPS are sold worldwide via on- and off-line markets without proper scientific evaluation regarding their safety or harmfulness. Abuse of NPS poses a serious public health issue, and systematic studies on their adverse effects are lacking. Therefore, it would be meaningful to collect currently available data in order to understand NPS and to establish viable solutions to cope with the various health issues related to them. In this article, we reviewed the general pharmacological characteristics, recent findings, and adverse effects of representative NPS; SCRAs. SCRAs are known as the most commonly abused NPS. Most SCRAs, cannabinoid receptor 1 and cannabinoid receptor 2 agonists, are often associated with severe toxicities, including cardiotoxicity, immunotoxicity, and even death, unlike natural cannabinoid Δ9-Tetrahydrocannabinol.

Background: Mental disorders linked with dysfunction in the temporal cortex, such as anxiety and depression, can increase the morbidity and mortality of people living with HIV (PLWHA). Expressions of both nucleobindin 1 (NUCB1) and cannabinoid receptor 1 (CNR1) in the neurons have been found to alter in patients with depressive disorder, but whether it is involved in the development of depression in the context of HIV infection is unknown. Objectives To investigate the effects of NUCB1 on depressive disorder among PLWHA and preliminarily explore the underlying molecular mechanisms. Methods: Individuals who were newly HIV diagnosed were assessed on the Hospital Anxiety and Depression scale (HADS). Then SHIV-infected rhesus monkeys were used to investigate the possible involvement of the NUCB1 and the CNR1 protein in depression-like behavior. Results: The prevalence rate of depression among PLWHA was 27.33% (41/150). The mechanism results showing elevated NUCB1 levels in cerebrospinal fluid from HIV-infected patients suffering from depression were confirmed compared to those of HIV-infected patients. Moreover, the immunohistochemical analysis indicated the expression of NUCB1 in the temporal cortex neurons of SHIV-infected monkeys was higher than that of the healthy control. Conversely, CNR1 expression was down-regulated at protein levels. Conclusions: Depression symptoms are common among PLWHA and associate with NUCB1 expression increases, and NUCB1 may be a potential target for depression.

l-Theanine is a characteristic amino acid in tea with various effects including antioxidant and anti-inflammatory effects. Previously, most studies had reported that l-theanine regulates the immune function in vivo by inhibiting the expression of the inflammatory factors, but how l-theanine regulates the inflammatory factors' pathway is not known. In this study, we innovatively found the binding target of l-theanine in vivo-cannabinoid receptor 1, and demonstrated that l-theanine regulated the immune function and glutamine metabolism by competitively binding cannabinoid receptor 1. Mechanistically, l-theanine competitively binds cannabinoid receptor 1, leading to inhibition of cannabinoid receptor 1 activity, and regulates glutamine metabolism and immune function in normal and E44813-stressed rats. In normal rats, l-theanine inhibits ERK1/2 phosphorylation through Gβy by antagonizing cannabinoid receptor 1, thus affecting GS expression. From the point of view of immune signaling, after LTA antagonizes the activity of cannabinoid receptor 1, it relieves the inhibition of cannabinoid receptor 1 on COX-2 expression, downregulates Pdcd4 expression and NFκB, and ultimately enhances the expression of the anti-inflammatory factor IL-10. In E44813-stressed rats, l-theanine promotes the nuclear translocation of p-ERK1/2 by inhibiting the activity of cannabinoid receptor 1, and finally acts on GS. At the same time, it decreases the expression of the pro-inflammatory factor TNF-α and increases the expression of the anti-inflammatory factor IL-10 in stressed rats through the COX2-Pdcd4-NFκB-IL10 and TNFα pathways. In summary, these results demonstrate that l-theanine regulates glutamine metabolism and immune function by competitively binding to cannabinoid receptor 1.

Xuelian Zhang,1 Haiqing Zhu,2 Xiaoyan Xing,1 Chunyu Zhang3 1Department of Endocrinology, China-Japan Friendship Hospital, Beijing 100029, People’s Republic of China; 2Department of Endocrinology, Emergency General Hospital, Beijing 100028, People’s Republic of China; 3Department of Statistical Teaching and Research, China-Japan Friendship Hospital, Beijing 100029, People’s Republic of ChinaCorrespondence: Xiaoyan XingDepartment of Endocrinology, China-Japan Friendship Hospital, Chaoyang District, Beijing 100029, People’s Republic of ChinaEmail 3382648198@qq.comBackground: The cannabinoid receptor 1 (CNR1) gene polymorphism is reportedly associated with components of metabolic syndrome and coronary artery diseases in patients with type 2 diabetes mellitus (T2DM). We investigated whether the common variant rs10493353 polymorphism is associated with diabetic nephropathy (DN) in T2DM patients.Patients and Methods: T2DM patients with DN were enrolled as a case group, and patients with only T2DM as a control group. Demographic data and biochemical parameters were collected. The polymerase chain reaction-based restriction fragment length polymorphism technique was used for genotyping. The odds ratio and 90% confidence interval were calculated to assess the association between genotypes and the risk of DN.Results: In total, 320 T2DM patients and 320 DN patients were enrolled. Compared with T2DM patients, the DN patients have a significantly larger body mass index (BMI), longer duration of disease, and higher proportions of smokers, drinkers, and hypertension. The risk of DN was significantly decreased by genotypes AA (OR=0.39, 95% CI=0.23– 0.67) and GA (OR=0.53, 95% CI=0.37– 0.75) vs GG (codominant model), GA/AA vs GG (OR=0.49, 95% CI=0.35– 0.67; dominant model), AA vs GG/GA (OR=0.47, 95% CI=0.28– 0.80; recessive model), and the A allele (OR=0.52, 95% CI=0.40– 0.68; allele model). Multiple logistic regressions still show significant levels. Negative interactions were found between gene and clinical parameters, including drinking, smoking, BMI, and hypertension.Conclusion: The A allele of CNR1 gene rs10493353 may be a protective factor for DN in T2DM patients. The risk factors of DN can affect the protective role of A allele in the progression of DN.Keywords: diabetes mellitus, diabetic nephropathy, gene polymorphism, cannabinoid receptor 1

Xuelian Zhang,1 Haiqing Zhu,2 Xiaoyan Xing,1 Chunyu Zhang3 1Department of Endocrinology, China-Japan Friendship Hospital, Beijing 100029, People’s Republic of China; 2Department of Endocrinology, Emergency General Hospital, Beijing 100028, People’s Republic of China; 3Department of Statistical Teaching and Research, China-Japan Friendship Hospital, Beijing 100029, People’s Republic of ChinaCorrespondence: Xiaoyan XingDepartment of Endocrinology, China-Japan Friendship Hospital, Chaoyang District, Beijing 100029, People’s Republic of ChinaEmail 3382648198@qq.comBackground: The cannabinoid receptor 1 (CNR1) gene polymorphism is reportedly associated with components of metabolic syndrome and coronary artery diseases in patients with type 2 diabetes mellitus (T2DM). We investigated whether the common variant rs10493353 polymorphism is associated with diabetic nephropathy (DN) in T2DM patients.Patients and Methods: T2DM patients with DN were enrolled as a case group, and patients with only T2DM as a control group. Demographic data and biochemical parameters were collected. The polymerase chain reaction-based restriction fragment length polymorphism technique was used for genotyping. The odds ratio and 90% confidence interval were calculated to assess the association between genotypes and the risk of DN.Results: In total, 320 T2DM patients and 320 DN patients were enrolled. Compared with T2DM patients, the DN patients have a significantly larger body mass index (BMI), longer duration of disease, and higher proportions of smokers, drinkers, and hypertension. The risk of DN was significantly decreased by genotypes AA (OR=0.39, 95% CI=0.23– 0.67) and GA (OR=0.53, 95% CI=0.37– 0.75) vs GG (codominant model), GA/AA vs GG (OR=0.49, 95% CI=0.35– 0.67; dominant model), AA vs GG/GA (OR=0.47, 95% CI=0.28– 0.80; recessive model), and the A allele (OR=0.52, 95% CI=0.40– 0.68; allele model). Multiple logistic regressions still show significant levels. Negative interactions were found between gene and clinical parameters, including drinking, smoking, BMI, and hypertension.Conclusion: The A allele of CNR1 gene rs10493353 may be a protective factor for DN in T2DM patients. The risk factors of DN can affect the protective role of A allele in the progression of DN.Keywords: diabetes mellitus, diabetic nephropathy, gene polymorphism, cannabinoid receptor 1

Background: Cannabinoid receptor 1 has its crystallographic structure available in complex with agonists and inverse agonists, which paved the way to establish an understanding of the structural basis of interactions with ligands. Dipyrone is a prodrug with analgesic capabilities and is widely used in some countries. Recently some evidence of a dipyrone metabolite acting over the Cannabinoid Receptor 1has been shown. Objective: Our goal here is to explore the dipyrone metabolite 4-aminoantipyrine as a Cannabinoid Receptor 1 agonist, reviewing dipyrone characteristics, and investigating the structural basis for its interaction with the Cannabinoid Receptor 1. Method: We reviewed here recent functional studies related to the dipyrone metabolite focusing on its action as a Cannabinoid Receptor 1 agonist. We also analyzed protein-ligand interactions for this complex obtained through docking simulations against the crystallographic structure of the Cannabinoid Receptor 1. Results: Analysis of the crystallographic structure and docking simulations revealed that most of the interactions present in the docked pose were also present in the crystallographic structure of Cannabinoid Receptor 1 and agonist. Conclusion: Analysis of the complex of 4-aminoantipyrine and Cannabinoid Receptor 1 revealed the pivotal role played by residues Phe 170, Phe 174, Phe 177, Phe 189, Leu 193, Val 196, and Phe 379, besides the conserved hydrogen bond at Ser 383. The mechanistic analysis and the present computational study suggest that the dipyrone metabolite 4-aminoantipyrine interacts with the Cannabinoid Receptor 1.

Synthetic cannabinoid (SC) use has persisted in the United States despite schedule-1 placement under the Synthetic Drug Abuse Prevention Act of 2012 [1]. Analysis of the National Poison Data System indicates that hospitalisations caused by SC use increased significantly between 2010 and 2015 [2]. Moreover, there is a trend of the increasing use of such compounds among adolescents [3]. SCs are often 30–100-fold more potent than Δ9-tetrahydrocannabinol (THC), the major psychoactive ingredient of cannabis, in activating Cannabinoid receptor 1 (CB1R). Users are attracted to SCs because of the cheaper, novel and stronger highs such substances offer compared to cannabis, and because the compounds are not screened for in typical drug tests [1, 2]. Among those hospitalised for SC use, some patients exhibited respiratory failure [4–7], pulmonary infiltrates [5, 7], alveolar damage or haemorrhage [5–7] and histopathologic features similar to organising pneumonia [4–6]. The mechanism by which SCs damage pulmonary tissue has yet to be elucidated – whether by SC binding at CB1R, CB2R or another receptor, and what downstream effects such binding elicits. Solving this conundrum is the first step in optimising treatment for patients presenting with SC-related respiratory distress., Synthetic cannabinoids (SCs) induce a pro-inflammatory condition by activating cannabinoid receptor 1 (CB1R) in the lungs of mice, which raises a potential therapeutic use of CB1R antagonists in SC-induced lung disease resulting in hospitalisation https://bit.ly/31bWw4Q

Nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3) has been regarded as an important initiator or promoter in multiple inflammatory diseases. However, the relationship between cannabinoid receptor 1 (CB1) and macrophage NLRP3 inflammasome and the corresponding molecular mechanism in liver inflammation remain unclear. Mouse liver injury models were induced by carbon tetrachloride (CCl4) or methionine-choline-deficient and high fat (MCDHF) diet. Human liver tissues were obtained from patients with different chronic liver diseases. CB1 expression was increased in liver tissue and macrophages of CCl4- and MCDHF-treated mice, positively correlated with NLRP3. CB1 agonist ACEA (Arachiodonyl-2’-Chloroethylamide) promoted NLRP3 expression and NLRP3 inflammasome activation in macrophages. CB1 blockade with its antagonist AM281 reduced NLRP3 expression, inflammasome activation, and liver inflammation in CCl4- and MCDHF-treated mice. MicroRNA-30b-5p (miR-30b-5p), screened by the intersection of bioinformatics databases and downregulated miRNAs in injured liver, negatively correlated with NLRP3 in mouse and human liver. miR-30b-5p was involved in CB1-mediated activation of NLRP3 inflammasome in macrophages by directly targeting NLRP3. Importantly, administration of miR-30b-5p agomir targeted NLRP3 and attenuated liver inflammation in the injured liver. Altogether, CB1/miR-30b-5p axis modulates NLRP3 expression and NLPR3 inflammasome activation in macrophages during liver inflammation, which provides a potential target for liver disease., Graphical Abstract, In this study, the critical role of CB1 was explored in macrophage NLRP3 inflammasome, and miR-30b-5p was identified as a negative regulator of NLRP3 expression and NLRP3 inflammasome activation in macrophages during liver inflammation. The findings highlight the potential of CB1/miR-30b-5p/NLRP3 axis as a therapeutic target for liver disease.

Cannabinoid receptor 1 (CB1R) controls various physiological and pathological conditions, including memory, motivation, and inflammation, and is thus an interesting target for positron emission tomography (PET). Herein, we report a ruthenium-mediated radiolabeling synthesis and preclinical evaluation of a new CB1R specific radiotracer, [18F]FPATPP. [18F]FPATPP was produced with 16.7 ± 5.7% decay-corrected radiochemical yield and >95 GBq/μmol molar activity. The tracer showed high stability, low defluorination, and high specific binding to CB1Rs in mouse brain.

This article belongs to the Special Issue Seeking New Antidepressant Agents., The anxiolytic and antidepressant properties of cannabidiol (CBD) have been evaluated in several studies. However, the molecular mechanisms involved in these actions remain unclear. A total of 130 male mice were used. CBD’s ability to modulate emotional disturbances (anxiety and depressive-like behaviors) was evaluated at different doses in wild-type (CD1; 10, 20 and 30 mg/kg; i.p.) and knockout (CB1KO, CB2KO; GPR55KO; 20 mg/kg) mice. Moreover, CBD effects (20 mg/kg; i.p.) were evaluated in mice previously treated with the CB1r-antagonist SR141716A (2mg/kg; i.p.). Relative gene expression analyses of Cnr1 and Cnr2, Gpr55 and GABA(A)α2 and γ2 receptor subunits were performed in the amygdala (AMY) and hippocampus (HIPP) of CD1 mice. CBD (10 and 20 mg/kg) showed anxiolytic and antidepressant actions in CD1 mice, being more effective at 20 mg/kg. Its administration did not induce anxiolytic actions in CB1KO mice, contrary to CB2KO and GPR55KO. In all of them, the lack of cannabinoid receptors did not modify the antidepressant activity of CBD. Interestingly, the administration of the CB1r antagonist SR141716A blocked the anxiolytic-like activity of CBD. Real-time PCR studies revealed a significant reduction in Cnr1 and GABA(A)α2 and γ2 gene expression in the HIPP and AMY of CD1 mice treated with CBD. Opposite changes were observed in the Cnr2. Indeed, Gpr55 was increased in the AMY and reduced in the HIPP. CB1r appears to play a relevant role in modulating the anxiolytic actions of CBD. Moreover, this study revealed that CBD also modified the gene expression of GABA(A) subunits α2 and γ2 and CB1r, CB2r and GPR55, in a dose- and brain-region-dependent manner, supporting a multimodal mechanism of action for CBD., This work was supported by “Instituto de Salud Carlos III” (RD. PI18/00576, Spanish Ministerio de Ciencia e Inovación) and RD21/0009/0008 “Red de Investigación en Atención Primaria de Adicciones”) to J.M.

International audience; Cannabinoid receptor 1 (CB1R), a G protein-coupled receptor, plays a fundamental role in synaptic plasticity. Abnormal activity and deregulation of CB1R signaling result in a broad spectrum of pathological conditions. CB1R signaling is regulated by receptor desensitization including phosphorylation of residues within the intracellular C terminus by G protein-coupled receptor kinases (GRKs) that may lead to endocytosis. Furthermore, CB1R signaling is regulated by the protein Src homology 3-domain growth factor receptor-bound 2-like (SGIP1) that hinders receptor internalization, while enhancing CB1R association with β-arrestin. It has been postulated that phosphorylation of two clusters of serine/threonine residues, 425 SMGDS429 and 460 TMSVSTDTS468 , within the CB1R C-tail controls dynamics of the association between receptor and its interaction partners involved in desensitization. Several molecular determinants of these events are still not well understood. We hypothesized that the dynamics of these interactions are modulated by SGIP1. Using a panel of CB1Rs mutated in the aforementioned serine and threonine residues, together with an array of Bioluminescence energy transfer-based (BRET) sensors, we discovered that GRK3 forms complexes with Gβγ subunits of G proteins that largely independent of GRK3's interaction with CB1R. Furthermore, CB1R interacts only with activated GRK3. Interestingly, phosphorylation of two specific residues on CB1R triggers GRK3 dissociation from the desensitized receptor. SGIP1 increases the association of GRK3 with Gβγ subunits of G proteins, and with CB1R. Altogether, our data suggest that the CB1R signalosome complex is dynamically controlled by sequential phosphorylation of the receptor C-tail and is also modified by SGIP1.

The endocannabinoid system regulates appetite and energy expenditure and inhibitors of cannabinoid receptor 1 (CB-1) induce weight loss with improvement in components of the metabolic syndrome. While CB-1 blockage in brain is responsible for weight loss, many of the metabolic benefits associated with CB-1 blockade have been attributed to inhibition of CB-1 signaling in the periphery. As a result, there has been interest in developing a peripherally restricted CB-1 inhibitor for the treatment of nonalcoholic fatty liver disease (NAFLD) that would lack the unwanted centrally mediated side effects. Here, we produced mice that lacked CB-1 in hepatocytes or stellate cells to determine if CB-1 signaling contributes to the development of NAFLD or liver fibrosis. Deletion of CB-1 in hepatocytes did not alter the development of NAFLD in mice fed a high-sucrose diet (HSD) or a high-fat diet (HFD). Similarly, deletion of CB-1 specifically in stellate cells also did not prevent the development of NAFLD in mice fed the HFD, nor did it protect mice from carbon tetrachloride–induced fibrosis. Combined, these studies do not support a direct role for hepatocyte or stellate cell CB-1 signaling in the development of NAFLD or liver fibrosis.

The binding of a series of small organic molecules, acting as agonists of the cannabinoid receptor CB1, was investigated by means of three methods of computational chemistry. Binding modes were predicted by means of molecular docking, and binding free energy was estimated via docking, molecular-mechanics Poisson-Boltzmann surface area method, and multistate Bennett acceptance ratio. No evident correlation was observed for the molecules between the experimental characteristics of affinity and three computed binding free energy estimates. The reasons for the discrepancy were discussed.

2-Arachidonoylglycerol (2-AG) is acting as a full agonist of cannabinoid receptor 1 and 2. Direct manipulation of 2-AG levels is a challenging task. The amphiphilic properties and the instability of 2-AG in aqueous media complicate its use as a drug-like molecule. Additionally, inhibition of the protein machinery that regulates 2-AG levels may also affect other monoacylglycerols. Therefore, we developed a novel method to elevate 2-AG levels with a flash of light. The resulting tool is a photoactivatable "caged" 2-arachidonoylglycerol (cg2-AG) allowing for the rapid photorelease of the signaling lipid in live cells. We characterized the mechanism of uncaging and the effect of 2-AG on the regulation of the β-cell signaling network. After uncaging of 2-AG, we monitored calcium levels, CB1-GIRK channel coupling, and CB1-mediated inhibition of adenylate cyclase and protein kinase A activity.

Mammalian ω3- and ω6-PUFAs are synthesized from essential fatty acids (EFAs) or supplied by the diet. PUFAs are constitutive elements of membrane architecture and precursors of lipid signaling molecules. EFAs and long-chain (LC)-PUFAs are precursors in the synthesis of endocannabinoid ligands of Gi/o protein-coupled cannabinoid receptor (CB)1 and CB2 in the endocannabinoid system, which critically regulate energy homeostasis as the metabolic signaling system in hypothalamic neuronal circuits and behavioral parameters. We utilized the auxotrophic fatty acid desaturase 2-deficient (fads2−/−) mouse, deficient in LC-PUFA synthesis, to follow the age-dependent dynamics of the PUFA pattern in the CNS-phospholipidome in unbiased dietary studies of three cohorts on sustained LC-PUFA-free ω6-arachidonic acid- and DHA-supplemented diets and their impact on the precursor pool of CB1 ligands. We discovered the transformation of eicosa-all cis-5,11,14-trienoic acid, uncommon in mammalian lipidomes, into two novel endocannabinoids, 20:35,11,14-ethanolamide and 2-20:35,11,14-glycerol. Their function as ligands of CB1 has been characterized in HEK293 cells. Labeling experiments excluded Δ8-desaturase activity and proved the position specificity of FADS2. The fads2−/− mutant might serve as an unbiased model in vivo in the development of novel CB1 agonists and antagonists.

Objective To explore the expression pattern of cannabinoid receptor 1 (CBR1) in reproductive toxicity induced by dibutyl phthalate (DBP) exposure in male rats, and investigate the underlying mechanism. Methods Sixty healthy male Sprague-Dawley rats were randomly divided into 4 groups (15 animals in each group), that is, control group, and low-, medium- and high-dose groups, exposure to 100, 250 and 500 mg/kg DBP, respectively, by intragastric injection daily for 28 consecutive days. Computer-aided sperm analysis system (CASA) was used to detect sperm vitality, concentration, and rate of deformity. HE staining was employed to observe the changes in testicular tissue. The serum levels of testosterone (T), estradiol (E2), luteinizing hormone (LH), follicle stimulating hormone (FSH) and sex hormone binding protein (SHBG) were measured by ELISA. The expression of p38, ERK, AKT and CBR1 at mRNA and protein levels was measured by RT-PCR and Western blotting. Results Compared with the control rats, the sperm motility and total sperm motility were reduced in low- (20.1±9.2 and 73.6±3.2), middle- (22.3±6.2 and 40.7±24.4) and high-dose (21.0±14.2 and 41.2±4.6) exposure groups (P < 0.05). Sperm concentration was significantly lower in the high-dose group (152.0±67.0, P < 0.05). Compared with the control group, serum T levels were significantly lower in the middle- (19.65±2.65) and high-dose groups (17.40±3.50, both P < 0.05). The serum level of E2 was significantly decreased in each exposure group (2.09±0.08, 2.11±0.77, 2.05±0.02) when compared to the control group. So were the serum LH levels (7.80±1.42, 7.68±2.28, 6.43±0.90) (all P < 0.05). While the serum level of FSH was all significantly increased in each exposure group (2.88±0.37, 2.89±0.29, 3.37±0.30), when compared to the control group (P < 0.05). In the testis tissue, the expression of CBR1 was increased at mRNA and protein levels than that of the control group, and the expression of p-p38 and p-ERK was significantly decreased than the control group (P < 0.05). Conclusion DBP exposure results in impairment of testicular tissue structure and decrease of sperm motility in male rats, which may be due to the up-regulation of CBR1 and the subsequent inhibition of ERK/p38MAPK phosphorylation.

INTRODUCTION: The endocannabinoid system is an important regulator of various physiological processes. Preclinical and clinical studies indicate that attenuation of the endocannabinoid system via antagonism of the type 1 cannabinoid receptor (CB1) is an excellent strategy to treat obesity, metabolic syndrome and associated disorders. However, centrally acting antagonists of CB1 also produce adverse effects like depression and anxiety. Current efforts are geared towards discovery and optimization of antagonists and modulators of CB1 that have limited brain penetration. AREAS COVERED: Several recent publications and patent applications support the development of peripherally acting CB1 receptor antagonists and modulators. In this review, recent patents and applications (2015 – 2018) are summarized and discussed. EXPERT OPINION: Approximately 30 new inventions have been reported since 2015, along with 3 recent commercial deals, highlighting the importance of this class of therapeutics. Taken together, peripherally acting CB1 receptor antagonists and modulators are an emerging class of drugs for metabolic syndrome, non-alcoholic steatohepatitis (NASH) and other important disorders where this receptor has been implicated.

Cannabinoid receptors have been identified as potential targets for analgesia from studies on animal physiology and behavior, and from human clinical trials. Here, we sought to improve translational understanding of the mechanisms of cannabinoid-mediated peripheral analgesia. Human lumbar dorsal root ganglia were rapidly recovered from organ donors to perform physiological and anatomical investigations into the potential for cannabinoids to mediate analgesia at the level of the peripheral nervous system. Anatomical characterization of in situ gene expression and immunoreactivity showed that 61 and 53% of human sensory neurons express the CB1 gene and receptor, respectively. Calcium influx evoked by the algogen capsaicin was measured by Fura-2AM in dissociated human sensory neurons pre-exposed to the inflammatory mediator prostaglandin E2 (PGE2) alone or together with CB13 (1 μM), a cannabinoid agonist with limited blood–brain barrier permeability. Both a higher proportion of neurons and a greater magnitude of response to capsaicin were observed after exposure to CB13, indicating cannabinoid-mediated sensitization. In contrast, membrane properties measured by patch-clamp electrophysiology demonstrated that CB13 suppressed excitability and reduced action potential discharge in PGE2-pre-incubated sensory neurons, suggesting the suppression of sensitization. This bidirectional modulation of sensory neuron activity suggests that cannabinoids may suppress overall membrane excitability while simultaneously enhancing responsivity to TRPV1-mediated stimuli. We conclude that peripherally restricted cannabinoids may have both pro- and anti-nociceptive effects in human sensory neurons.

Cannabinoid receptors typically include type 1 (CB1) and type 2 (CB2), and they have attracted extensive attention in the central nervous system (CNS) and immune system. Due to more in-depth studies in recent years, it has been found that the typical CB1 and CB2 receptors confer functional importance far beyond the CNS and immune system. In particular, many works have reported the critical involvement of the CB1 and CB2 receptors in myocardial injuries. Both pharmacological and genetic approaches have been used for studying CB1 and CB2 functions in these studies, revealing that the brother receptors have many basic differences and sometimes antagonistic functions in a variety of myocardial injuries, despite some sequence or location identity they share. Herein, we introduce the general differences of CB1 and CB2 cannabinoid receptors, and summarize the functional rivalries between the two brother receptors in the setting of myocardial injuries. We point out the importance of individual receptor-based modulation, instead of dual receptor modulators, when treating myocardial injuries.

Genetic markers of the endocannabinoid system have been linked to a variety of addiction-related behaviors that extend beyond cannabis use. In the current study we investigate the relationship between endocannabinoid (eCB) genetic markers and alcohol use disorder (AUD) in European adolescents (14–18 years old) followed in the IMAGEN study (n = 2,051) and explore replication in a cohort of North American adolescents from Canadian Saguenay Youth Study (SYS) (n = 772). Case-control status is represented by a score of more than 7 on the Alcohol Use Disorder Identification Test (AUDIT). First a set-based test method was used to examine if a relationship between the eCB system and AUDIT case/control status exists at the gene level. Using only SNPs that are both independent and significantly associated to case-control status, we perform Fisher's exact test to determine SNP level odds ratios in relation to case-control status and then perform logistic regressions as post-hoc analysis, while considering various covariates. Generalized multifactor dimensionality reduction (GMDR) was used to analyze the most robust SNP×SNP interaction of the five eCB genes with positive AUDIT screen. While no gene-sets were significantly associated to AUDIT scores after correction for multiple tests, in the case/control analysis, 7 SNPs were significantly associated with AUDIT scores of > 7 (p < 0.05; ORCNR1 (pcorrected =0.042, OR = 0.73) and rs507961 in MGLL (pcorrected = 0.043, OR = 0.78). Logistic regression showed that both rs9353525 (CNR1) and rs507961 (MGLL) remained significantly associated with positive AUDIT screens (p < 0.01; OR < 1) after correction for multiple covariables and interaction of covariable × SNP. This result was not replicated in the SYS cohort. The GMDR model revealed a significant three-SNP interaction (p = 0.006) involving rs484061 (MGLL), rs4963307 (DAGLA), and rs7766029 (CNR1) predicted case-control status, after correcting for multiple covariables in the IMAGEN sample. A binomial logistic regression of the combination of these three SNPs by phenotype in the SYS cohort showed a result in the same direction as seen in the IMAGEN cohort (BETA = 0.501, p = 0.06). While preliminary, the present study suggests that the eCB system may play a role in the development of AUD in adolescents.

The endocannabinoid system (ECS) is involved in a variety of brain functions, mainly through the activation of the type-1 cannabinoid receptors (CB1R). CB1R are highly expressed throughout the brain at different structural, cellular and subcellular locations and its activity and expression levels have a direct impact in synaptic activity and behavior. In the last few decades, astrocytes have arisen as active players of brain physiology through their participation in the tripartite synapse and through their metabolic interaction with neurons. Here, we discuss some of the mechanisms by which astroglial CB1R at different subcellular locations, regulate astrocyte calcium signals and have an impact on gliotransmission and metabolic regulation. In addition, we discuss evidence pointing at astrocytes as potential important sources of endocannabinoid synthesis and release. Thus, we summarize recent findings that add further complexity and establish that the ECS is a fundamental effector of astrocyte functions in the brain. This article is part of the special issue on ‘Cannabinoids'.

Pain is a crucial component of the body's innate defenses, which helps us to respond to the damage that is threatening or imminent. If the pain persists even after the injury has healed, or arises for no apparent reason, it itself becomes harmful. Nociception begins with the detection of a noxious stimulus that irritates free nerve endings on the peripheral projections of spinal ganglion neurons. If the stimulus induces depolarization of the cell and an action potential forms, information of the stimulus is conducted by thinly myelinated Aδ fibers, or unmyelinated C fibers to the spinal cord dorsal horn. Here, the first synapses of sensory pathways are located, which allow the transmission of nociception to secondary afferent neurons, and these further direct the information to the higher centers of the CNS. Synapses in the dorsal horn are key to modulating nociceptive signaling, in which the endocannabinoid system, including endogenous cannabinoids and their receptors, plays a significant role. However, under pathological conditions such as the development of neuropathic pain or neuroinflammation, changes in the expression and function of agonists and receptors of the endocannabinoid system occur. These changes are of great importance in the onset and persistence of pathological pain. The study of...

CB1 receptors mediate most central nervous system effects ascribed to the phytocannabinoid Δ-9-tetrahydrocannabinol (THC). CB1 receptors are guanine nucleotide-binding regulatory protein-coupled receptors that primarily activate Gi/o proteins, resulting in the inhibition of adenylyl cyclase. CB1 receptors are expressed mainly on the membranes of presynaptic terminals. The effects of CB1 receptor activation consist of release inhibition of a variety of neurotransmitters. Once activated, CB1 receptors undergo inactivation and internalization. CB1 receptors are located throughout the central nervous system with reported low density in the medullary nucleus.

Molecular mechanism of Cannabinoid receptor 1 regulation by SGIP1 Abstract Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1 (SGIP1) has been identified as an interacting partner of cannabinoid receptor 1 (CB1R). Their protein-protein interaction was confirmed by co-immunoprecipitation. SGIP1 hinders the internalization of activated CB1R and modulates its signaling in HEK293 cells. Employing whole-cell patch-clamp electrophysiology, we have shown that SGIP1 affects CB1R signaling in autaptic hippocampal neurons. Using a battery of behavioral tests in SGIP1 constitutive knock-out (SGIP1-/- ) and WT mice, we investigated the consequences of SGIP1 deletion on behavior regulated by the endocannabinoid system. In SGIP1-/- mice, exploratory levels, working memory and sensorimotor gating were unaltered. SGIP1-/- mice showed decreased anxiety-like and depressive-like behaviors. Fear extinction to tone was enhanced in SGIP1-/- females. Several cannabinoid tetrad behaviors were altered in the absence of SGIP1. SGIP1-/- males exhibited abnormal THC withdrawal behaviors. SGIP1 deletion also reduced acute nociception, and SGIP1-/- mice were more sensitive to antinociceptive effects of CB1R agonists and morphine. CB1R-SGIP1 interaction results in profound modification of CB1R...

In this thesis, we have studied the potential of the zebrafish larval model in studying the ECS, as a complementary model to the existing rodent models. More specifically, we have looked at the role of the ECS in regulating locomotion and anxiety, and its interaction with the hypothalamic-pituitary-interrenal (HPI) axis, or stress axis. This study has provided us with an interesting animal model which allows for pharmacological screening of Cnr1 agonists, and their involvement in the CNS, as shown by a change in locomotion, anxiety-like behavior and HPI axis activity. The zebrafish larval model can be used as a complementary model to the existing rodent animal models, to study the ECS. The zebrafish larval model brings several interesting features, such as optical transparency and possibilities for high-throughput screening. Furthermore, a complete ECS is present, there is lack of endogenous activity, allowing for exogenous compound screening, and zebrafish data is generally in line with rodent literature. Since the ECS is involved in many diseases, more research of this system may result in the discovery of novel drugs and drug targets.

The endocannabinoid system (ECS) is known to modulate not only food intake but also pain, especially via the cannabinoid type 1 receptor (CB1R) expressed throughout the central nervous system and the peripheral tissues. Our previous study demonstrated that fasting produces an analgesic effect in adult male mice, which is reversed by intraperitoneal (i.p.) administration of CB1R antagonist (SR 141716). In the present study, we further examined the effect of CB1R expressed in the peripheral tissues. In the formalin-induced inflammatory pain model, i.p. administration of peripherally restricted CB1R antagonist (AM 6545) reversed fasting-induced analgesia. However, intraplantar administration of SR 141716 did not affect fasting-induced analgesia. Furthermore, mRNA expression of CB1R did not change in the formalin model by fasting in the dorsal root ganglia. The formalin-induced c-Fos expression at the spinal cord level was not affected by fasting, and in vivo recording from the superficial dorsal horn of the lumbar spinal cord revealed that fasting did not affect formalin-induced neural activity, which indicates minimal involvement of the spinal cord in fasting-induced analgesia. Finally, when we performed subdiaphragmatic vagotomy to block the hunger signal from the gastrointestinal (GI) system, AM 6545 did not affect fasting-induced analgesia, but SR 141716 still reversed fasting-induced analgesia. Taken together, our results suggest that both peripheral and central CB1Rs contribute to fasting-induced analgesic effects and the CB1Rs in the GI system which transmit fasting signals to the brain, rather than those in the peripheral sensory neurons, may contribute to fasting-induced analgesic effects.

In small hibernators, global downregulation of the endocannabinoid system (ECS), which is involved in modulating neuronal signaling, feeding behavior, energy metabolism, and circannual rhythms, has been reported to possibly drive physiological adaptation to the hibernating state. In hibernating brown bears (Ursus arctos), we hypothesized that beyond an overall suppression of the ECS, seasonal shift in endocannabinoids compounds could be linked to bear’s peculiar features that include hibernation without arousal episodes and capacity to react to external disturbance. We explored circulating lipids in serum and the ECS in plasma and metabolically active tissues in free-ranging subadult Scandinavian brown bears when both active and hibernating. In winter bear serum, in addition to a 2-fold increase in total fatty acid concentration, we found significant changes in relative proportions of circulating fatty acids, such as a 2-fold increase in docosahexaenoic acid C22:6 n-3 and a decrease in arachidonic acid C20:4 n-6. In adipose and muscle tissues of hibernating bears, we found significant lower concentrations of 2-arachidonoylglycerol (2-AG), a major ligand of cannabinoid receptors 1 (CB1) and 2 (CB2). Lower mRNA level for genes encoding CB1 and CB2 were also found in winter muscle and adipose tissue, respectively. The observed reduction in ECS tone may promote fatty acid mobilization from body fat stores, and favor carbohydrate metabolism in skeletal muscle of hibernating bears. Additionally, high circulating level of the endocannabinoid-like compound N-oleoylethanolamide (OEA) in winter could favor lipolysis and fatty acid oxidation in peripheral tissues. We also speculated on a role of OEA in the conservation of an anorexigenic signal and in the maintenance of torpor during hibernation, while sustaining the capacity of bears to sense stimuli from the environment.

The aim of this work was to determine whether Capsaicin may exert a vascular regulation through the activation of CB1 and/or CB2 receptors causing vasorelaxation in the rat aorta. Our results show the location of TRPV1 mainly in the endothelial and smooth muscle cells membrane. Nevertheless, Capsaicin caused vasorelaxation of this artery through a mechanism independent of TRPV1, since the specific antagonists Capsazepine and SB-366791 did not block the effect of Capsaicin. Because the significant expression of CB1 and CB2 receptors has been previously reported in the rat aorta, we used antagonists for these two receptors prior to the addition of Capsaicin. In these experiments, we found that the inhibition of CB1 using AM281, decreases the vasorelaxant effect caused by Capsaicin. On the other hand, the vasorelaxant effect is not altered in the presence of the CB2 receptor antagonist AM630. Furthermore, a partial decrease of the effect of Capsaicin was also seen when L-type calcium channels are blocked. A complete block of Capsaicin-induced vasorelaxation was achieved using a combination of Verapamil and AM281. In accordance to our results, Capsaicin-induced vasorelaxation of the rat aorta is neither dependent of TRPV1 or CB2 receptors, but rather it is strongly suggested that a tandem mechanism between inactivation of L-type calcium channels and the direct activation of CB1 receptors is involved. These findings are supported by CB1 docking simulation which predicted a binding site on CB1 receptors for Capsaicin.

G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB1), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB1 signaling. Furthermore, we found that CB1 hotspot periodicity increased upon CB1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB1. Our results suggest that CB1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy when being activated.

Introduction and Objective: Org27569 is a prototypical allosteric modulator of the cannabinoid receptor 1 (CB1). It belongs to the indole-2-carboxamide scaffold and has been intensively investigated in pharmacology and in structure–activity relationship (SAR) studies. Although azaindoles are rare in natural products and differ only by the presence of an extra ring nitrogen, they were demonstrated as valuable bioisosteres in many pharmacologically important molecules. To extend the SAR investigation of the indole-2-carboxamide class of CB1 allosteric modulators, azaindole (pyrrolopyridine) rings were used to replace the indole ring of Org27569 analogs to explore the potential of azaindole-2-carboxamides as CB1 allosteric modulators. Using 6- and 7-azaindole in lieu of the indole moiety within this class of CB1 allosteric modulators indeed improved the aqueous solubility. Materials and Methods: We synthesized 6- and 7-azaindole-2-carboxamides and their indole-2-carboxamide counterparts. The molecules were evaluated by [3H]CP55,940 binding and [35S]GTPγS binding assays for their allosteric modulation of the CB1 receptor. Results: The 7-azaindole-2-carboxamides lost the ability to bind to the CB1 receptor. The 6-azaindole-2-carboxamides (e.g., 3c and 3d) showed markedly reduced binding affinities to the CB1 receptor in comparison with their indole-2-carboxamide counterparts. However, they behaved similarly as indole-2-carboxamides in potentiating the orthosteric agonist binding and inhibiting the orthosteric agonist-induced G-protein coupling. The results indicated that some azaindole scaffolds (e.g., 6-azaindole) are worth further exploration, whereas the 7-azaindole ring is not a viable bioisostere of the indole ring in the Org27569 class of CB1 allosteric modulators.

Contradictory findings on the role of the type 1 cannabinoid receptor (CB1R) during the pathogenesis of Alzheimer’s disease (AD) have been reported. Here, we evaluated the CB1R brain profile in an AD mouse model using longitudinal positron emission tomography with an inverse agonist for CB1R, [18F]FMPEP-d2. APP/PS1-21 and wild-type (n 1⁄4 8 in each group) mice were repeatedly imaged between 6 to 15 months of age, accompanied by brain autoradiography, western blot, and CB1R immunohistochemistry with additional mice. [18F]FMPEP-d2 positron emission tomography demonstrated lower (p < 0.05) binding ratios in the parietotemporal cortex and hippocampus of APP/PS1-21 mice compared with age-matched wild-type mice. Western blot demonstrated no differences between APP/PS1-21 and wild-type mice in the CB1R abundance, whereas significantly lower (p < 0.05) receptor expression was observed in male than female mice. The results provide the first demonstration that [18F]FMPEP-d2 is a promising imaging tool for AD research in terms of CB1R availability, but not expression. This finding may further facilitate the development of novel therapeutic approaches based on endocannabinoid regulation.

Background: Cerebral evoked potentials (CEP) induced by colorectal distension (CRD) in conscious rats provides a novel method in studies of visceral sensitivity. The aim of this study was to explore the pharmacological effect on CEP of compounds known to reduce the visceromotor response to CRD. Methods: Epidural electrodes were chronically implanted in eight female Sprague-Dawley rats. Evoked potentials were elicited by colorectal rapid balloon distensions (100 ms, 80 mmHg) and the effect of WIN55 (cannabinoid CB receptor agonist), clonidine (adrenergic α 2 receptor agonist), MPEP (mGluR5 receptor antagonist), pregabalin (ligand of α 2δ subunits in voltage-gated calcium channels) and baclofen (GABA-B receptor agonist) on amplitudes and latency of CEP were determined. Results: WIN55 (0.1 μmol kg −1), clonidine (0.05 μmol kg −1), MPEP (10 μmol kg −1) and pregabalin (200 μmol kg −1) caused a significant, p < 0.05, reduction of the N2 to P2 peak-to-peak amplitude by 23 ± 8%, 25 ± 8%, 39 ± 5%, and 47 ± 6% respectively. Baclofen (9 μmol kg −1) induced a prolongation of the N2 peak latency of 18 ± 4% but had no significant effect on the amplitudes. Conclusion: The obtained results suggest that MPEP, WIN55, clonidine, and pregabalin reduce visceral nociceptive input to the brain, whereas the lack of effect of baclofen on CRD evoked CEP amplitudes suggest that the effect on VMR is not due to a direct analgesic effect. Brain responses to colorectal distension provide a useful tool to evaluate pharmacological effects in rats and may serve as a valuable preclinical model for understanding pharmacological mechanisms related to visceral sensitivity.

The endocannabinoid signalling system was discovered because receptors in this system are the targets of compounds present in psychotropic preparations of Cannabis sativa. The search for new therapeutics that target endocannabinoid signalling is both challenging and potentially rewarding, as endocannabinoids are implicated in numerous physiological and pathological processes. Hundreds of mediators chemically related to the endocannabinoids, often with similar metabolic pathways but different targets, have complicated the development of inhibitors of endocannabinoid metabolic enzymes but have also stimulated the rational design of multi-target drugs. Meanwhile, drugs based on botanical cannabinoids have come to the clinical forefront, synthetic agonists designed to bind cannabinoid receptor 1 with very high affinity have become a societal threat and the gut microbiome has been found to signal in part through the endocannabinoid network. The current development of drugs that alter endocannabinoid signalling and how this complex system could be pharmacologically manipulated in the future are described in this Opinion article.

Objectives To explore the antihyperglycaemic and antidiabetic effects and to determine the acute toxicity of 5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (ENP-9). Methods The antihyperglycaemic effect of ENP-9 (50 mg/kg) was determined by oral glucose tolerance test (OGTT). Also, the acute (16, 50 and 160 mg/kg) and subacute (50 mg/kg/day for 10 days) antidiabetic effects of ENP-9 were determined. After subacute treatment, blood samples were analysed to determine glucose and lipid profiles. Also, an acute toxicity determination of ENP-9 was conducted followed the OECD recommendation. Molecular docking was performed using AutoDock 4.2.6 at human cannabinoid receptor 1 (PDB code 5TGZ). Key findings Acute Administration of ENP-9 showed significant antidiabetic effect and decreased the maximum OGTT peak, compared to the control group (P < 0.05). Moreover, the 10 days treatment induced a decrease in plasma glucose levels, being significant at the end of the experiments (P < 0.05); however, triacylglycerols and cholesterol were not modified. Finally, LD50 of ENP-9 was estimated to be greater than 2000 mg/kg. Molecular docking suggests that ENP-9 may act as rimonabant does. Conclusions ENP-9 showed significant antihyperglycaemic and antidiabetic properties and also was demonstrated to be safety in the studied doses, which might allow future studies for its potential development as antidiabetic agent.

Many malignant cancers, including breast cancer, have a propensity to invade bones, leading to excruciating bone pain. Opioids are the primary analgesics used to alleviate this cancer-induced bone pain (CIBP) but are associated with numerous severe side effects, including enhanced bone degradation, which significantly impairs patients’ quality of life. In contrast, agonists activating only peripheral CB1 receptors (CB1Rs) have been shown to effectively alleviate multiple chronic pain conditions with limited side effects, yet no studies have evaluated their role(s) in CIBP. Here, we demonstrate for the first time that a peripherally selective CB1R agonist can effectively suppress CIBP. Our studies using a syngeneic murine model of CIBP show that both acute and sustained administration of a peripherally restricted CB1R agonist, 4-{2-[−(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3-yl]ethyl} morpholine (PrNMI), significantly alleviated spontaneous pain behaviors in the animals. This analgesic effect by PrNMI can be reversed by a systemic administration but not spinal injection of SR141716, a selective CB1R antagonist. Additionally, the cancer-induced bone loss in the animals was not exacerbated by a repeated administration of PrNMI. Furthermore, catalepsy and hypothermia, the common side effects induced by cannabinoids, were measured at the supra-therapeutic doses of PrNMI tested. PrNMI induced mild sedation, yet no anxiety nor a decrease in limb movements were detected. Overall, our studies demonstrate that CIBP can be effectively managed by using a peripherally restricted CB1R agonist, PrNMI, without inducing dose-limiting central side effects. Thus, targeting peripheral CB1Rs could be an alternative therapeutic strategy for the treatment of CIBP.

Re-investigation of the methanolic extract of Ficus pandurata Hance (Moraceae) fruits, afforded four new ceramides namely, panduramides A-D (1-3 and 5), along with newbouldiamide (4). Their structures were determined on the basis of various spectroscopic and chemical methods, as well as comparison with literature. The isolated compounds were evaluated for their antimicrobial, antimalarial, anti-leishmanial, and cytotoxic activities. In addition, their radioligand displacement affinity on opioid and cannabinoid receptors was assessed. Compound 1 exhibited good affinity towards cannabinoid receptor 1 (CB1) with displacement value of 69.9%.

In the last decades, cannabinoid receptor 2 (CB2R) has continued to receive attention as a key therapeutic target in neuroprotection. Indeed, several findings highlight the neuroprotective effects of CB2R through suppression of both neuronal excitability and reactive microglia. Additionally, CB2R seems to be a more promising target than cannabinoid receptor 1 (CB1R) thanks to the lack of central side effects, its lower expression levels in the central nervous system (CNS), and its inducibility, since its expression enhances quickly in the brain following pathological conditions. This review aims to provide a thorough overview of the main natural and synthetic selective CB2R modulators, their chemical classification and their potential therapeutic usefulness in neuroprotection, a crucial aspect for the treatment of neurodegenerative diseases.

Cannabinoid pharmacology has been intensely studied because of cannabis' pervasive medicinal and non-medicinal uses as well as for the therapeutic potential of cannabinoid-based drugs for the treatment of pain, anxiety, substance abuse, obesity, cancer and neurodegenerative disorders. The identification of allosteric modulators of the cannabinoid receptor 1 (CB1) has given a new direction to the development of cannabinoid-based therapeutics due to the many advantages offered by targeting allosteric site(s). Allosteric receptor modulators hold potential to develop subtype-specific and pathway-specific therapeutics. Here we briefly discuss the first-generation of allosteric modulators of CB1 receptor, their structure-activity relationships, signaling pathways and the allosteric binding site(s) on the CB1 receptor. This article is part of the Special Issue entitled "A New Dawn in Cannabinoid Neurobiology".

Cannabinoid receptor 1 gene (CNR1) variants have been related to affective information processing and, in particular, to stress release. Here, we aimed to examine whether the endocannabinoid system via CNR1 signaling modulates affective working memory, the memory system that transiently maintains and manipulates emotionally charged material. We focused on rs2180619 (A > G) polymorphism and examined genotype data collected from 231 healthy females. Analyses showed how a general positivity bias in working memory (i.e., better memory for positive words) emerged as task strings lengthened only in carriers of the major allele (AA/AG). Differently, GG carriers showed better memory for affective items in general (i.e., positive and negative words). These findings are some of the first to directly highlight the role of variant on promoter of the CNR1 gene in affective working memory and to evidence a differentiation among CNR1 genotypes in terms of larger difficulties in disengaging from negative stimuli in GG carriers.