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β-Caryophyllene (BCP), a bicyclic sesquiterpene that is a component of the essential oils of various spice and food plants, has been described as a selective CB2 cannabinoid receptor agonist. In the present study, the effect of BCP on angiogenesis was investigated. It was found that conditioned media (CM) from BCP-treated hypoxic A549 lung cancer cells exhibited a concentration-dependent inhibitory effect on human umbilical vein endothelial cell (HUVEC) tube formation induced by CM from vehicle-treated hypoxic A549 cells. There was an associated concentration-dependent decrease in the proangiogenic factor vascular endothelial growth factor (VEGF) in the CM, with both BCP inhibitory effects (tube formation, VEGF secretion) being CB2 receptor-dependent. A reduction of the transcription factor hypoxia-inducible factor 1α (HIF-1α) was furthermore detected. The antiangiogenic and VEGF-lowering properties of BCP were confirmed when CM from another lung cancer cell line, H358, were tested. When directly exposed to HUVECs, BCP showed no significant effect on tube formation, but at 10 µM, impaired VEGF receptor 2 (VEGFR2) phosphorylation triggered by recombinant VEGF in a CB2 receptor-independent manner. In summary, BCP has a dual antiangiogenic effect on HUVECs, manifested in the inhibition of tube formation through modulation of the tumor cell secretome and additionally in the inhibition of VEGF-induced VEGFR2 activation. Because the CB2 agonist has no psychoactive properties, BCP should continue to be evaluated preclinically for further antitumor effects. [ABSTRACT FROM AUTHOR]

β‐caryophyllene (BCP) is a cannabinoid receptor CB2 agonist plant‐derived terpenoid found in different essential oil plants, including rosemary, black pepper, copaiba and cannabis. It has GRAS (generally recognized as safe) status and is approved by the FDA (Food and Drug Administration) for food use. BCP displays agonist activity on the CB2 receptor and is a potential therapeutic target in several neuropsychiatric disorders, including anxiety and drug addiction. Unlike CB1 receptors, activation of the CB2 receptors is devoid of psychotomimetic and addictive properties. In this regard, this study aimed to evaluate the effects of BCP on incentive salience ("wanting") performance and motivational properties elicited by sweetened palatable foods in female Swiss mice. After 9 days of training for incentive salience performance for a sweet reward (hazelnut cream with chocolate), food‐restricted mice received a systemic injection of BCP (50 and 100 mg/kg) before testing over 3 days. Moreover, independent groups of female mice were tested on sweet reward‐induced conditioned place preference (CPP) for 22 consecutive days. To evaluate BCP effects on the expression of seeking behaviour for sweetened food, mice received a single intraperitoneal injection of BCP (50 mg/kg) 30 min before testing on the CPP task. BCP significantly decreased the incentive performance for a sweet reward compared with the control group in a CB2 receptor‐dependent manner. Also, BCP suppressed the expression of sweet reward‐CPP. Altogether, these preclinical data demonstrate the potential role of BCP in treating disorders associated with food addiction‐like behaviour. [ABSTRACT FROM AUTHOR]

Genetic and environmental factors are implicated in the etiology of neuropsychiatric diseases. Inbred mouse strains, including the 129S1/SvImJ (S1), constitute important models to study the influence of genetic factors in these conditions. S1 mice displayed anxiogenic-like behavior, impaired fear extinction, and increased prepulse inhibition (PPI) of startle reflex compared to C57BL/6J (BL6) mice. Given the role played by the endocannabinoid system (ECS) in these responses, we evaluated the expression of the ECS components in different brain regions in S1 mice. Gene expression levels of the cannabinoid type-1 and type-2 receptors (CB1R and CB2R) and the endocannabinoid metabolizing enzymes varied depending on the brain region evaluated. Notably, CB2R expression markedly increased in the amygdala, prefrontal cortex and hippocampus in S1 mice. Moreover, CB2R blockade with SR144528 partially rescued the anxiogenic phenotype in S1 mice, while CB2R activation with JWH133 potentiated the deficits in fear extinction and the PPI of startle reflex in this mouse strain. These data suggest that CB2R is involved in the behavioral alterations observed in S1 mice and underline the importance of this cannabinoid receptor subtype in the regulation of certain central nervous system disorders. [ABSTRACT FROM AUTHOR]

Genetic and environmental factors are implicated in the etiology of neuropsychiatric diseases. Inbred mouse strains, including the 129S1/SvImJ (S1), constitute important models to study the influence of genetic factors in these conditions. S1 mice displayed anxiogenic-like behavior, impaired fear extinction, and increased prepulse inhibition (PPI) of startle reflex compared to C57BL/6J (BL6) mice. Given the role played by the endocannabinoid system (ECS) in these responses, we evaluated the expression of the ECS components in different brain regions in S1 mice. Gene expression levels of the cannabinoid type-1 and type-2 receptors (CB1R and CB2R) and the endocannabinoid metabolizing enzymes varied depending on the brain region evaluated. Notably, CB2R expression markedly increased in the amygdala, prefrontal cortex and hippocampus in S1 mice. Moreover, CB2R blockade with SR144528 partially rescued the anxiogenic phenotype in S1 mice, while CB2R activation with JWH133 potentiated the deficits in fear extinction and the PPI of startle reflex in this mouse strain. These data suggest that CB2R is involved in the behavioral alterations observed in S1 mice and underline the importance of this cannabinoid receptor subtype in the regulation of certain central nervous system disorders.

Background: We have previously reported that the endocannabinoid receptor inverse agonist AM630 is a potent inhibitor of isocitrade dehydrogenase-1 wild-type glioblastoma (GBM) core tumour cell proliferation. To uncover the mechanism behind the anti-tumour effects we have performed a transcriptional analysis of AM630 activity both in the tumour core cells (U87) and the invasive margin cells (GIN-8), the latter representing a better proxy of post-surgical residual disease. Results: The core and invasive margin cells exhibited markedly different gene expression profiles and only the core cells had high expression of a potential AM630 target, the CB1 receptor. Both cell types had moderate expression of the HTR2B serotonin receptor, a reported AM630 target. We found that the AM630 driven transcriptional response was substantially higher in the central cells than in the invasive margin cells, with the former driving the up regulation of immune response and the down regulation of cell cycle and metastatic pathways and correlating with transcriptional responses driven by established anti-neoplastics as well as serotonin receptor antagonists. Conclusion: Our results highlight the different gene sets involved in the core and invasive margin cell lines derived from GBM and an associated marked difference in responsiveness to AM630. Our findings identify AM630 as an anti-neoplastic drug in the context of the core cells, showing a high correlation with the activity of known antiproliferative drugs. However, we reveal a key set of similarities between the two cell lines that may inform therapeutic intervention. [ABSTRACT FROM AUTHOR]

β-arrestins are partners of the G protein-coupled receptors (GPCRs), regulating their intracellular trafficking and signaling. Development of biased GPCR agonists, selectively targeting either G protein or β-arrestin pathways, are in the focus of interest due to their therapeutic potential in different pathological conditions. The CB2 cannabinoid receptor (CB2R) is a GPCR involved in various functions in the periphery and the central nervous system. Two common occurring variants of CB2R, harboring Q63R or L133I missense mutations, have been implicated in the development of a diverse set of disorders. To evaluate the effect of these mutations, we characterized the binding profile of these mutant CB2 receptors to G proteins and β-arrestin2. Although their ability to inhibit cAMP signaling was similar, the Q63R mutant had increased, whereas the L133I mutant receptor had decreased β-arrestin2 binding. In line with these observations, the variants also had altered intracellular trafficking. Our results show that two common variants of the CB2 receptor have biased signaling properties, which may contribute to the pathogenesis of the associated disorders and may offer CB2R as a target for further development of biased receptor activation strategies. [ABSTRACT FROM AUTHOR]

Aims Myocardial infarction (MI) leads to an enhanced release of endocannabinoids and a massive accumulation of neutrophils and monocytes within the ischaemic myocardium. These myeloid cells originate from haematopoietic precursors in the bone marrow and are rapidly mobilized in response to MI. We aimed to determine whether endocannabinoid signalling is involved in myeloid cell mobilization and cardiac recruitment after ischaemia onset. Methods and results Intravenous administration of endocannabinoid 2-arachidonoylglycerol (2-AG) into wild type (WT) C57BL6 mice induced a rapid increase of blood neutrophil and monocyte counts as measured by flow cytometry. This effect was blunted when using cannabinoid receptor 2 knockout mice. In response to MI induced in WT mice, the lipidomic analysis revealed significantly elevated plasma and cardiac levels of the endocannabinoid 2-AG 24 h after infarction, but no changes in anandamide, palmitoylethanolamide, and oleoylethanolamide. This was a consequence of an increased expression of 2-AG synthesizing enzyme diacylglycerol lipase and a decrease of metabolizing enzyme monoacylglycerol lipase (MAGL) in infarcted hearts, as determined by quantitative RT–PCR analysis. The opposite mRNA expression pattern was observed in bone marrow. Pharmacological blockade of MAGL with JZL184 and thus increased systemic 2-AG levels in WT mice subjected to MI resulted in elevated cardiac CXCL1, CXCL2, and MMP9 protein levels as well as higher cardiac neutrophil and monocyte counts 24 h after infarction compared with vehicle-treated mice. Increased post-MI inflammation in these mice led to an increased infarct size, an impaired ventricular scar formation assessed by histology and a worsened cardiac function in echocardiography evaluations up to 21 days. Likewise, JZL184-administration in a myocardial ischaemia-reperfusion model increased cardiac myeloid cell recruitment and resulted in a larger fibrotic scar size. Conclusion These findings suggest that changes in endocannabinoid gradients due to altered tissue levels contribute to myeloid cell recruitment from the bone marrow to the infarcted heart, with crucial consequences on cardiac healing and function. [ABSTRACT FROM AUTHOR]

Emerging evidence suggests a complex interplay between the endocannabinoid system, omega-3 fatty acids and the immune system in the promotion of brain self-repair. However, it is unknown if all omega-3 fatty acids elicit similar effects on adult neurogenesis and if such effects are mediated or regulated by interactions with the endocannabinoid system. This study investigated the effects of DHA and EPA on neural stem cell (NSC) fate and the role of the endocannabinoid signalling pathways in these effects. EPA, but not DHA, significantly increased proliferation of NSCs compared to controls, an effect associated with enhanced levels of the endocannabinoid 2-arachidonylglycerol (2-AG) and p-p38 MAPK, effects attenuated by pre-treatment with CB1 (AM251) or CB2 (AM630) receptor antagonists. Furthermore, in NSCs derived from IL-1β deficient mice, EPA significantly decreased proliferation and p-p38 MAPK levels compared to controls, suggesting a key role for IL-1β signalling in the effects observed. Although DHA similarly increased 2-AG levels in wild-type NSCs, there was no concomitant increase in proliferation or p-p38 MAPK activity. In addition, in NSCs from IL-1β deficient mice, DHA significantly increased proliferation without effects on p-P38 MAPK, suggesting effects of DHA are mediated via alternative signalling pathways. These results provide crucial new insights into the divergent effects of EPA and DHA in regulating NSC proliferation and the pathways involved, and highlight the therapeutic potential of their interplay with endocannabinoid signalling in brain repair. [ABSTRACT FROM AUTHOR]

In the brain, CB1 cannabinoid receptors primarily mediate the effects of cannabinoids, but CB2 cannabinoid receptors (CB2Rs) have recently been discovered in the nervous system and also implicated in neuromodulatory roles. To understand the mechanisms of CB2R functions in the brain, it is essential to localize CB2Rs, but the types of cells expressing CB2Rs have been controversial. Unequivocal localization of CB2Rs in the brain has been impeded in part by the low expression levels of CB2Rs and poor specificity of detection methods. Here, we used an ultrasensitive and specific in situ hybridization method called the RNAscope to determine the spatial pattern of CB2R mRNA expression in the mouse hippocampus. CB2R mRNAs were mostly expressed in a subset of excitatory and inhibitory neurons in the CA1, CA3 and dentate gyrus areas, but rarely in microglia. CB2R knock-out mice were used as a negative control. Using the quantitative real-time polymerase chain reaction, we also found that the temporal pattern of CB2R mRNA expression was stable during postnatal development. Consistent with previous reports, the immunological detection of CB2Rs was not reliable, implying extremely low levels of the protein expression and/or insufficient specificity of the current anti-CB2R antibodies. Our findings of the expression patterns of CB2R mRNAs may help determine the cell types involved in, and hence the mechanisms of, the CB2R-mediated neuromodulation. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Several studies provided evidence that the endocannabinoid system (ECS) is involved in psychiatric diseases, like major depression, schizophrenia and bipolar disorder (BD), mainly focusing on CB1 cannabinoid receptor, and FAAH, the fatty acid amide hydrolase involved in endocannabinoid metabolism. In this study we investigated the possible association of BD with three missense SNPs, of the gene CNR2, encoding for CB2 cannabinoid receptor. Methods: The possible association between BD and three CNR2 missense SNPs, namely rs2501432 (315A>G; Arg63Gln), rs41311993 (524C>A; Leu133Ile) and rs2229579 (1073C>T; Tyr316His), was investigated through a case–control study. Eighty patients and one hundred and sixty healthy subjects were recruited. Allele Specific Oligonucleotide (ASO)-PCR and restriction fragment length polymorphism (RFLP) methods were used for genotyping. Results: A statistically significant association was found between BD and the CNR2 524C>A; Leu133Ile (P(χ 2)=0.001; OR=4.74; 95% C.I.=2.52–10.50) while no statistically significant difference between BD and control group was observed for the other two SNPs. Conclusion: Though further investigations are necessary to confirm this data, our results suggest that CB2 cannabinoid receptor may play a role in BD. [Copyright &y& Elsevier]

CB2 is a Gi protein-coupled receptor activated by endo- and phytocannabinoids, thus inhibiting stimulated adenylyl cyclase activity. CB2 is expressed in bone cells and Cb2 null mice show a marked age-related bone loss. CB2-specific agonists both attenuate and rescue ovariectomy-induced bone loss. Activation of CB2 stimulates osteoblast proliferation and bone marrow derived colony-forming units osteoblastic. Here we show that selective and nonselective CB2 agonists are mitogenic in MC3T3 E1 and newborn mouse calvarial osteoblastic cultures. The CB2 mitogenic signaling depends critically on the stimulation of Erk1/2 phosphorylation and de novo synthesis of MAP kinase-activated protein kinase 2 ( Mapkapk2) mRNA and protein. Further downstream, CB2 activation enhances CREB transcriptional activity and cyclin D1 mRNA expression. The CB2-induced stimulation of CREB and cyclin D1 is inhibitable by pertussis toxin, the MEK-Erk1/2 inhibitors PD098059 and U0126, and Mapkapk2 siRNA. These data demonstrate that in osteoblasts CB2 targets a Gi protein-cyclin D1 mitogenic axis. Erk1/2 phosphorylation and Mapkapk2 protein synthesis are critical intermediates in this axis. © 2011 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Abstract: In contrast to the numerous reports on the pharmacological effects of Δ9-tetrahydrocannabinol (THC), the pharmacological activity of another substituent of Cannabis sativa, cannabichromene (CBC) remains comparatively unknown. In the present study, we investigated whether CBC elicits cannabinoid activity in the tetrad assay, which consists of the following four endpoints: hypomotility, antinociception, catalepsy, and hypothermia. Because cannabinoids are well documented to possess anti-inflammatory properties, we examined CBC, THC, and combination of both phytocannabinoids in the lipopolysaccharide (LPS) paw edema assay. CBC elicited activity in the tetrad that was not blocked by the CB1 receptor antagonist, rimonabant. Moreover, a behaviorally inactive dose of THC augmented the effects of CBC in the tetrad that was associated with an increase in THC brain concentrations. Both CBC and THC elicited dose-dependent anti-inflammatory effects in the LPS-induced paw edema model. The CB2 receptor, SR144528 blocked the anti-edematous actions of THC, but not those produced by CBC. Isobolographic analysis revealed that the anti-edematous effects of these cannabinoids in combination were additive. Although CBC produced pharmacological effects, unlike THC, its underlying mechanism of action did not involve CB1 or CB2 receptors. In addition, there was evidence of a possible pharmacokinetic component in which CBC dose-dependently increased THC brain levels following an i.v. injection of 0.3mg/kg THC. In conclusion, CBC produced a subset of behavioral activity in the tetrad assay and reduced LPS-induced paw edema through a noncannabinoid receptor mechanism of action. These effects were augmented when CBC and THC were co-administered. [Copyright &y& Elsevier]

Abstract: Preventive treatment with cannabinoid agonists has been reported to reduce the infarct size in a mouse model of myocardial ischemia/reperfusion. Here we investigated the possible cardioprotective effect of selective CB2 cannabinoid receptor activation during ischemia. We performed left coronary artery ligature in C57Bl/6 mice for 30 min, followed by 24 h of reperfusion. Five minutes before reperfusion, mice received intraperitoneal injection of the CB2 selective agonist JWH-133 (20 mg/kg) or vehicle. Infarct size was assessed histologically and by cardiac troponin I (cTnI) ELISA. Immunohistochemical analysis of leukocyte infiltration, oxidative stress in situ quantification, real-time RT-PCR analysis of inflammatory mediators as well as western blots for kinase phosphorylation was also performed. In addition, we studied chemotaxis and integrin expression of human neutrophils in vitro. JWH-133 significantly reduced the infarct size (I/area at risk: 19.27%±1.91) as compared to vehicle-treated mice (31.77%±2.7). This was associated with a reduction of oxidative stress and neutrophil infiltration in the infarcted myocardium, whereas activation of ERK 1/2 and STAT-3 was increased. Preinjection of PI3K inhibitor LY294002, MEK 1/2 inhibitor U0126 and JAK-2 inhibitor AG-490 partially abrogated the JWH-133 mediated infarct size reduction. No changes in cardiac CXCL1, CXCL2, CCL3, TNF-α, and ICAM-1 expression levels were found. Furthermore, JWH-133 inhibited the TNF-α induced chemotaxis and integrin CD18/CD11b (Mac-1) upregulation on human neutrophils. Our data suggest that JWH-133 administration during ischemia reduces the infarct size in a mouse model of myocardial ischemia/reperfusion through a direct cardioprotective activity on cardiomyocytes and neutrophils. [Copyright &y& Elsevier]

The endogenous cannabinoid N-arachidonoyl ethanolamine (anandamide; AEA) produces most of its pharmacological effects by binding and activating CB1 and CB2 cannabinoid receptors within the CNS and periphery. However, the actions of AEA are short lived because of its rapid catabolism by fatty acid amide hydrolase (FAAH). Indeed, FAAH knockout mice as well as animals treated with FAAH inhibitors are severely impaired in their ability to hydrolyze AEA as well as a variety of noncannabinoid lipid signaling molecules and consequently possess greatly elevated levels of these endogenous ligands. In this mini review, we describe recent research that has investigated the functional consequences of inhibiting this enzyme in a wide range of animal models of inflammatory and neuropathic pain states. FAAH-compromised animals reliably display antinociceptive and anti-inflammatory phenotypes with a similar efficacy as direct-acting cannabinoid receptor agonists, such as Δ9-tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa. Importantly, FAAH blockade does not elicit any apparent psychomimetic effects associated with THC or produce reinforcing effects that are predictive of human drug abuse. The beneficial effects caused by FAAH blockade in these models are predominantly mediated through the activation of CB1 and/or CB2 receptors, though noncannabinoid mechanisms of actions can also play contributory or even primary roles. Collectively, the current body of scientific literature suggests that activating the endogenous cannabinoid system by targeting FAAH is a promising strategy to treat pain and inflammation but lacks untoward side effects typically associated with Cannabis sativa. [ABSTRACT FROM AUTHOR]

Abstract: Formerly considered as an exclusively peripheral receptor, it is now accepted that CB2 cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB2 receptors in the brain need to be clarified. The aim of our work was to study the μ-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB2 receptor antagonist SR144528 in brainstem of mice deficient in either CB1 or CB2 receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB2 cannabinoid antagonist SR144528, suggesting a CB2 receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [35S]GTPγS binding assay to analyze the capability of μ-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB1 wild-type and CB1 knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB1 wild-type and CB1 knockout mice. In vitro addition of 1μM SR144528 caused a decrease in the maximal stimulation of DAMGO in [35S]GTPγS binding assays in CB2 wild-type brainstem membranes whereas no significant changes were observed in CB2 receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB2 cannabinoid receptors. [Copyright &y& Elsevier]

Nerve injuries often lead to neuropathic pain syndrome. The mechanisms contributing to this syndrome involve local inflammatory responses, activation of glia cells, and changes in the plasticity of neuronal nociceptive pathways. Cannabinoid CB2 receptors contribute to the local containment of neuropathic pain by modulating glial activation in response to nerve injury. Thus, neuropathic pain spreads in mice lacking CB2 receptors beyond the site of nerve injury. To further investigate the mechanisms leading to the enhanced manifestation of neuropathic pain, we have established expression profiles of spinal cord tissues from wild-type and CB2-deficient mice after nerve injury. An enhanced interferon-γ (IFN-γ) response was revealed in the absence of CB2 signaling. Immunofluorescence stainings demonstrated an IFN-γ production by astrocytes and neurons ispilateral to the nerve injury in wild-type animals. In contrast, CB2-deficient mice showed neuronal and astrocytic IFN-γ immunoreactivity also in the contralateral region, thus matching the pattern of nociceptive hypersensitivity in these animals. Experiments in BV-2 microglia cells revealed that transcriptional changes induced by IFN-γ in two key elements for neuropathic pain development, iNOS (inducible nitric oxide synthase) and CCR2, are modulated by CB2 receptor signaling. The most direct support for a functional involvement of IFN-γ as a mediator of CB2 signaling was obtained with a double knock-out mouse strain deficient in CB2 receptors and IFN-γ. These animals no longer show the enhanced manifestations of neuropathic pain observed in CB2 knock-outs. These data clearly demonstrate that the CB2 receptor-mediated control of neuropathic pain is IFN-γ dependent. [ABSTRACT FROM AUTHOR]

Neuropathic pain is a clinical manifestation of nerve injury difficult to treat even with potent analgesic compounds. Here, we used different lines of genetically modified mice to clarify the role played by CB2 cannabinoid receptors in the regulation of the central immune responses leading to the development of neuropathic pain. CB2 knock-out mice and wild-type littermates were exposed to sciatic nerve injury, and both genotypes developed a similar hyperalgesia and allodynia in the ipsilateral paw. Most strikingly, knock-outs also developed a contralateral mirror image pain, associated with an enhanced microglial and astrocytic expression in the contralateral spinal horn. In agreement, hyperalgesia, allodynia, and microglial and astrocytic activation induced by sciatic nerve injury were attenuated in transgenic mice overexpressing CB2 receptors. These results demonstrate the crucial role of CB2 cannabinoid receptor in modulating glial activation in response to nerve injury. The enhanced manifestations of neuropathic pain were replicated in irradiated wild-type mice reconstituted with bone marrow cells from CB2 knock-outs, thus demonstrating the implication of the CB2 receptor expressed in hematopoietic cells in the development of neuropathic pain at the spinal cord. [ABSTRACT FROM AUTHOR]

The functional expression of neuronal CB2 cannabinoid receptors (CB2-Rs) in the brain has been controversial. We and others have now demonstrated that CB2-Rs are expressed in neurons and glial cells in the brain. However, the subcellular localization of these receptors has not been characterized. In this study we used immunohistochemical electron microscopy to determine the subcellular distribution of CB2-Rs in two brain regions. Brain sections from the CA1 hippocampal area and substantia nigra were immunostained for CB2-Rs and analyzed by electron microscopy. In each region immunoperoxidase labeling for CB2-Rs was detected in neurons as well as in glial and endothelial cells. In neuronal cells, CB2-R immunoreactivity was observed in somata and large and medium-sized dendrites. In the soma, the CB2-R labeling was mainly associated with the rough endoplasmic reticulum and Golgi apparatus, suggesting its endogenous synthesis. In the dendrites, the CB2-R labeling was observed in the cytoplasm and was associated with the plasma membrane near the area of synaptic contact with axon terminals, indicating a postsynaptic distribution of these receptors. In CB2-Rs in immunoreactive glial and endothelial cells, the labeling was also found to be associated with the plasma membrane. In the substantia nigra, some unmyelinated axons were immunoreactive for CB2-Rs, but we rarely found CB2-R-labeled axon terminals. These results extend our previous detection of postsynaptic cortical CB2-Rs and provide additional ultrastructural evidence that CB2-Rs are mainly postsynaptic in the CA1 area of the hippocampus and substantia nigra. The functional implication of pre- and/or postsynaptic localization of CB2-Rs remains to be determined. [ABSTRACT FROM AUTHOR]

The psychoactive cannabinoids from Cannabis sativa L. and the arachidonic acid-derived endocannabinoids are nonselective natural ligands for cannabinoid receptor type 1 (CB1) and CB2 receptors. Although the CB1 receptor is responsible for the psychomodulatory effects, activation of the CB2 receptor is a potential therapeutic strategy for the treatment of inflammation, pain, atherosclerosis, and osteoporosis. Here, we report that the widespread plant volatile (E)-β-caryophyllene [(E)-BCP] selectively binds to the CB2 receptor (K1 = 155 ± 4 nM) and that it is a functional CB2 agonist. Intriguingly, (E)-BCP is a common constituent of the essential oils of numerous spice and food plants and a major component in Cannabis. Molecular docking simulations have identified a putative binding site of (E)-BCP in the CB2 receptor, showing ligand π-π stacking interactions with residues F117 and W258. Upon binding to the CB2 receptor, (E)-BCP inhibits adenylate cylcase, leads to intracellular calcium transients and weakly activates the mitogen-activated kinases Erk1/2 and p38 in primary human monocytes. (E)-BCP (500 nM) inhibits lipopolysaccharide (LPS)-induced proinflammatory cytokine expression in peripheral blood and attenuates LPS-stimulated Erk1/2 and JNK1/2 phosphorylation in monocytes. Furthermore, peroral (E)-BCP at 5 mg/kg strongly reduces the carrageenan-induced inflammatory response in wild-type mice but not in mice lacking CB2 receptors, providing evidence that this natural product exerts cannabimimetic effects in vivo. These results identify (E)-BCP as a functional nonpsychoactive CB2 receptor ligand in foodstuff and as a macrocyclic antiinflammatory cannabinoid in Cannabis. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss, paralysis and death within 2–5 years of diagnosis. Currently, no effective pharmacological agents exist for the treatment of this devastating disease. Neuroinflammation may accelerate the progression of ALS. Cannabinoids produce anti-inflammatory actions via cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), and delay the progression of neuroinflammatory diseases. Additionally, CB2 receptors, which normally exist primarily in the periphery, are dramatically up-regulated in inflamed neural tissues associated with CNS disorders. In G93A-SOD1 mutant mice, the most well-characterized animal model of ALS, endogenous cannabinoids are elevated in spinal cords of symptomatic mice. Furthermore, treatment with non-selective cannabinoid partial agonists prior to, or upon, symptom appearance minimally delays disease onset and prolongs survival through undefined mechanisms. We demonstrate that mRNA, receptor binding and function of CB2, but not CB1, receptors are dramatically and selectively up-regulated in spinal cords of G93A-SOD1 mice in a temporal pattern paralleling disease progression. More importantly, daily injections of the selective CB2 agonist AM-1241, initiated at symptom onset, increase the survival interval after disease onset by 56%. Therefore, CB2 agonists may slow motor neuron degeneration and preserve motor function, and represent a novel therapeutic modality for treatment of ALS. [ABSTRACT FROM AUTHOR]

Abstract: CB2 cannabinoid receptors exist in immune cells including macrophages. Affinity-purified antibodies against the CB2 receptor identified a 45 kDa protein in rat brain, human tonsil and rat and mouse microglia, but not mouse N18TG2 neuroblastoma cells. Intracerebroventricular lipopolysaccharide (LPS) increased immunoreactive CB2 receptors in brain membranes detected by Western blot. LPS increased immunodetectable CB2 receptors in cultured RAW 264.7 macrophages, and this was partially attenuated by cyclohexamide or the protein kinase A and C inhibitors H8 and bis-indolylmaleimide. Forskolin or dibutyryl cyclic AMP increased CB2 receptor immunoreactivity, suggesting the involvement of the cyclic AMP–protein kinase A–Cyclic AMP response element pathway in the regulation of CB2 receptor levels. [Copyright &y& Elsevier]

For over a decade, until recently, it was thought that marijuana acts by activating brain-type cannabinoid receptors called CB1, and that a second type called CB2 cannabinoid receptor was found only in peripheral tissues. Neuronal CB2 receptors in the brain had been controversial. We reported the discovery and functional presence of CB2 cannabinoid receptors in the mammalian brain that may be involved in depression and drug abuse and this was supported by reports of identification of neuronal CB2 receptors that are involved in emesis. RT-PCR, immunoblotting, hippocampal cultures, immunohistochemistry, transmission electron microscopy, and stereotaxic techniques with behavioral assays were used to determine the functional expression of CB2 cannabinoid receptors in the rat brain and mouse brain exposed to chronic mild stress or treated with abused drugs. RT-PCR analyses supported the expression of brain CB2 receptor transcripts at levels much lower than those of CB1 receptors. In situ hybridization revealed CB2 mRNA in cerebellar neurons of wild-type but not of CB2 knockout mice. Abundant CB2 receptor immunoreactivity (iCB2) in neuronal and glial processes was detected in the brain. The effect of direct CB2 antisense oligonucleotide injection into the brain and treatment with JWH015 in motor function and plus-maze tests also demonstrated the functional presence of CB2 cannabinoid receptors in the central nervous system. In humans, there was a high incidence of Q63R polymorphism in the CB2 gene in Japanese alcoholics and depressed subjects. Contrary to the prevailing view that CB2 cannabinoid receptors are restricted to peripheral tissues and predominantly in immune cells, we demonstrated that CB2 cannabinoid receptors and their gene transcripts are widely distributed in the brain. This multifocal expression of iCB2 in the brain suggests that CB2 receptors may play broader roles than previously anticipated and may therefore be exploited as new targets in the treatment of depression and substance abuse. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Two well-characterized cannabinoid receptors (CBrs), CB1 and CB2, mediate the effects of cannabinoids and marijuana use, with functional evidence for other CBrs. CB1 receptors are expressed primarily in brain and peripheral tissues. For over a decade several laboratories were unable to detect CB2 receptors in brain and were known to be intensely expressed in peripheral and immune tissues and have traditionally been referred to as peripheral CB2 CBrs. We have reported the discovery and functional presence of CB2 cannabinoid receptors in mammalian brain that may be involved in depression and drug abuse and this was supported by reports of identification of neuronal CB2 receptors that are involved in emesis. We used RT-PCR, immunoblotting, hippocampal cultures, immunohistochemistry, transmission electron microscopy, and stereotaxic techniques with behavioral assays to determine the functional expression of CB2 CBrs in rat brain and mice brain exposed to chronic mild stress (CMS) or those treated with abused drugs. RT-PCR analyses supported the expression of brain CB2 receptor transcripts at levels much lower than those of CB1 receptors. In situ hybridization revealed CB2 mRNA in cerebellar neurons of wild-type but not of CB2 knockout mice. Abundant CB2 receptor immunoreactivity (iCB2) in neuronal and glial processes was detected in brain and CB2 expression was detected in neuron-specific enolase (NSE) positive hippocampal cell cultures. The effect of direct CB2 antisense oligonucleotide injection into the brain and treatment with JWH015 in motor function and plus-maze tests also demonstrated the functional presence of CB2 cannabinoid receptors in the central nervous system (CNS). Thus, contrary to the prevailing view that CB2 CBrs are restricted to peripheral tissues and predominantly in immune cells, we demonstrated that CB2 CBrs and their gene transcripts are widely distributed in the brain. This multifocal expression of CB2 immunoreactivity in brain suggests that CB2 receptors may play broader roles in the brain than previously anticipated and may be exploited as new targets in the treatment of depression and substance abuse. [ABSTRACT FROM AUTHOR]

Abstract: Brain expression of CB2 cannabinoid receptors has been much less well established and characterized in comparison to the expression of brain CB1 receptors. Since CB2 receptors are intensely expressed in peripheral and immune tissues, expression in brain microglia has been anticipated. Nevertheless, we now describe expression of CB2-receptor-like immunoreactivity in brain in neuronal patterns that support broader CNS roles for this receptor. Two anti-CB2 affinity purified polyclonal antibodies were raised in rabbits immunized with peptide conjugates that corresponded to amino acids 1–33 and 20–33. Western blot analyses revealed specific bands that were identified using these sera and were absent when the sera were preadsorbed with 8.3 μg/ml of the immunizing peptides. These studies, and initial RT-PCR analyses of brain CB1 and CB2 mRNAs, also support the expression of brain CB2 receptor transcripts at levels much lower than those of CB1 receptors. CB2 cannabinoid receptor mRNA was clearly expressed in the cerebellum of wild type but not in CB2 knockout mice. CB2 immunostaining was detected in the interpolar part of spinal 5th nucleus of wild type but not in CB2 knockout mice, using a mouse C-terminal CB2 receptor antibody. Immunohistochemical analyses revealed abundant immunostaining for CB2 receptors in apparent neuronal and glial processes in a number of rat brain areas. Cerebellar Purkinje cells and hippocampal pyramidal cells revealed substantial immunoreactivity that was absent when sections were stained with preadsorbed sera. CB2 immunoreactivity was also observed in olfactory tubercle, islands of Calleja, cerebral cortex, striatum, thalamic nuclei, hippocampus, amygdala, substantia nigra, periaqueductal gray, paratrochlear nucleus, paralemniscal nucleus, red nucleus, pontine nuclei, inferior colliculus and the parvocellular portion of the medial vestibular nucleus. In-vitro, CB2 immunoreactivity was also present in hippocampal cell cultures. The multifocal expression of CB2 immunoreactivity in glial and neuronal patterns in a number of brain regions suggests reevaluation of the possible roles that CB2 receptors may play in the brain. [Copyright &y& Elsevier]

Abstract: The aim of the present study was to investigate the effects of cannabinoid agonists on established inflammatory hyperalgesia. We have compared the effects of pre-administration versus post-administration of a potent non-selective cannabinoid agonist HU210 and a selective CB2 receptor agonist JWH-133 on hindpaw weight bearing and paw oedema in the carrageenan model of inflammatory hyperalgesia. For comparative purposes we also determined the effects of the μ-opioid receptor agonist morphine and the COX2 inhibitor rofecoxib in this model. At 3h following intraplantar injection of carrageenan (2%, 100μl) there was a significant (P<0.001) reduction in weight bearing on the ipsilateral hindpaw, compared to vehicle treated rats and a concomitant increase in ipsilateral hindpaw volume (P<0.001), compared to vehicle treated rats. Systemic administration of HU210 (10μg/kg) and JWH-133 (10mg/kg) at 3h following injection of carrageenan, significantly attenuated decreases in ipsilateral hindpaw weight bearing (P<0.05 for both) and paw volume (P<0.001 for both). Pre-administration of HU210 and JWH-133 had similar effects on weight bearing in this model. Pre-administered HU210 also significantly decreased carrageenan-induced changes in paw volume (P<0.001), this was not the case for JWH-133. Effects of post-administered HU210 and JWH-133 on ipsilateral hindpaw weight bearing and paw volume were comparable to the effect of systemic post-administration of morphine and rofecoxib (3mg/kg for both). In summary, both HU210 and JWH-133 attenuated established inflammatory hypersensitivity and swelling, suggesting that cannabinoid-based drugs have clinical potential for the treatment of established inflammatory pain responses. [Copyright &y& Elsevier]

[Display omitted] As a continuation of earlier work on classical cannabinoids bearing bulky side chains we report here the design, synthesis, and biological evaluation of 3′-functionalized oxa-adamantyl cannabinoids as a novel class of cannabinergic ligands. Key synthetic steps involve nucleophilic addition/transannular cyclization of aryllithium to epoxyketone in the presence of cerium chloride and stereoselective construction of the tricyclic cannabinoid nucleus. The synthesis of the oxa-adamantyl cannabinoids is convenient, and amenable to scale up allowing the preparation of these analogs in sufficient quantities for detailed in vitro evaluation. The novel oxa-adamantyl cannabinoids reported here were found to be high affinity ligands for the CB1 and CB2 cannabinoid receptors. In the cyclase assay these compounds were found to behave as potent and efficacious CB1 receptor agonists. Isothiocyanate analog AM10504 is capable of irreversibly labeling both the CB1 and CB2 receptors. [ABSTRACT FROM AUTHOR]

The endocannabinoid system, which spans the central and peripheral nervous systems and regulates many biologic processes, is an important target for probe discovery and medications development. Whereas the earliest endocannabinoid receptor probes were derivatives of the non-selective phytocannabinoids isolated from Cannabis species, modern drug discovery techniques have expanded the definitions of what constitutes a CB1R or CB2R cannabinoid receptor ligand. This review highlights recent advances in synthetic cannabinoid receptor chemistry and pharmacology. We provide examples of new CB1R- and CB2R-selective probes, and discuss rational approaches to the design of peripherally-restricted agents. We also describe structural classes of positive- and negative allosteric modulators (PAMs and NAMs) of CB1R and CB2R. Finally, we introduce new opportunities for cannabinoid receptor probe development that have emerged in recent years, including biased agonists that may lead to medications lacking adverse effects. • Aminoalkylindoles are synthetically simple CB1R and CB2R ligands. • New CB1R probes were discovered by "scaffold-hopping." • CB2R-selective agonists and antagonists with high selectivity are now available. • New scaffolds of PAMs and NAMs are needed for CB1R and CB2R. • Peripherally restricted and biased agonists offer new opportunities for therapeutic investigation. [ABSTRACT FROM AUTHOR]

We previously reported the 2-oxopyridine-3-carboxamide derivative EC21a as the first small synthetic CB2R positive allosteric modulator which displayed antinociceptive activity in vivo in an experimental mouse model of neuropathic pain. Herein, we extended the structure-activity relationships of EC21a through structural modifications regarding the p -fluoro benzyl moiety at position 1 and the amide group in position 3 of the central core. The characterization in vitro was assessed through radioligand binding experiments and functional assays (GTPγS, cAMP, βarrestin2). Among the new compounds, the derivatives A1 (SV-10a) and A5 (SB-13a) characterized respectively by fluorine atom or by chlorine atom in ortho position of the benzylic group at position 1 and by a cycloheptane-carboxamide at position 3 of the central core, showed positive allosteric behavior on CB2R. They enhanced the efficacy of CP55,940 in 35S]GTPγS assay, and modulated CP55,940-dependent βarrestin2 recruitment and cAMP inhibition. The obtained results extend our knowledge of the structural requirements for interaction with the allosteric site of CB2R. Image 1 • We synthesized 3-substituted 1,2-dihydropyridine-2-oxo derivatives. • Derivatives A1 and A5 were shown to be CB2R PAMs. • Their behaviour as PAMs was assessed through radioligand binding and functional assays. • A1 and A5 enhanced the efficacy of CP55,940 in 35S]GTPγS assay. • A1 and A5 modulated CP55,940-dependent βarrestin2 recruitment and cAMP inhibition. [ABSTRACT FROM AUTHOR]

β-arrestins are partners of the G protein-coupled receptors (GPCRs), regulating their intracellular trafficking and signaling. Development of biased GPCR agonists, selectively targeting either G protein or β-arrestin pathways, are in the focus of interest due to their therapeutic potential in different pathological conditions. The CB 2 cannabinoid receptor (CB 2 R) is a GPCR involved in various functions in the periphery and the central nervous system. Two common occurring variants of CB 2 R, harboring Q63R or L133I missense mutations, have been implicated in the development of a diverse set of disorders. To evaluate the effect of these mutations, we characterized the binding profile of these mutant CB 2 receptors to G proteins and β-arrestin2. Although their ability to inhibit cAMP signaling was similar, the Q63R mutant had increased, whereas the L133I mutant receptor had decreased β-arrestin2 binding. In line with these observations, the variants also had altered intracellular trafficking. Our results show that two common variants of the CB 2 receptor have biased signaling properties, which may contribute to the pathogenesis of the associated disorders and may offer CB 2 R as a target for further development of biased receptor activation strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Turu, Soltész-Katona, Tóth, Juhász, Cserző, Misák, Balla, Caron and Hunyady.)

Cannabinoid receptor type II, or CB 2 , is an integral membrane protein that belongs to a large class of G-protein-coupled receptors (GPCR)s. CB 2 is a part of the endocannabinoid system, which plays an important role in the regulation of immune response, inflammation, and pain. Information about the structure and function of CB 2 is essential for the development of specific ligands targeting this receptor. We present here a methodology for recombinant expression of CB 2 and its stable isotope labeling, purification, and reconstitution into liposomes, in preparation for its characterization by nuclear magnetic resonance (NMR). Correctly folded, functional CB 2 labeled with [ 13 C, 15 N]tryptophan or uniformly labeled with 13 C and 15 N is expressed in a medium of defined composition, under controlled aeration, pH, and temperature conditions. The receptor is purified by affinity chromatography and reconstituted into lipid bilayers in the form of proteoliposomes suitable for analysis by NMR spectroscopy. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

During life, new neurons are continually added to hippocampal circuitry, with evidence suggesting that these adult-born neurons are functionally linked to cognition and emotion. The mammalian brain contains actively dividing neural stem cells in discrete regions, including the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus. Once mature, these neurons integrate into neuronal networks, forming synaptic connections with interneurons, mossy cells and CA3 pyramidal cells Linked Article This article is a commentary on Avraham et al., pp. 468-479 of volume 171 issue 2. To view this paper visit [ABSTRACT FROM AUTHOR]