Evidence that the plant cannabinoid cannabigerol is a highly potent α2-adrenoceptor agonist and moderately potent 5HT1A receptor antagonist

Background and purpose:

Cannabis is the source of at least seventy phytocannabinoids. The pharmacology of most of these has been little investigated, three notable exceptions being Δ⁹-tetrahydrocannabinol, cannabidiol and Δ⁹-tetrahydrocannabivarin. This investigation addressed the question of whether the little-studied phytocannabinoid, cannabigerol, can activate or block any G protein-coupled receptor.

Experimental approach:

The [³⁵S]GTPγS binding assay, performed with mouse brain membranes, was used to test the ability of cannabigerol to produce G protein-coupled receptor activation or blockade. Its ability to displace [³H]CP55940 from mouse CB₁ and human CB₂ cannabinoid receptors and to inhibit electrically evoked contractions of the mouse isolated vas deferens was also investigated.

Key results:

In the brain membrane experiments, cannabigerol behaved as a potent α₂-adrenoceptor agonist (EC₅₀= 0.2 nM) and antagonized the 5-HT_1A receptor agonist, R-(+)-8-hydroxy-2-(di-n-propylamino)tetralin (apparent K_B= 51.9 nM). At 10 µM, it also behaved as a CB₁ receptor competitive antagonist. Additionally, cannabigerol inhibited evoked contractions of the vas deferens in a manner that appeared to be α₂-adrenoceptor-mediated (EC₅₀= 72.8 nM) and displayed significant affinity for mouse CB₁ and human CB₂ receptors.

Conclusions and implications:

This investigation has provided the first evidence that cannabigerol can activate α₂-adrenoceptors, bind to cannabinoid CB₁ and CB₂ receptors and block CB₁ and 5-HT_1A receptors. It will now be important to investigate why cannabigerol produced signs of agonism more potently in the [³⁵S]GTPγS binding assay than in the vas deferens and also whether it can inhibit noradrenaline uptake in this isolated tissue and in the brain.     

Palmitoylethanolamide,a naturally occurring lipid,is an orally effective intestinal anti-inflammatory agent

BACKGROUND AND PURPOSE

Palmitoylethanolamide (PEA) acts via several targets, including cannabinoid CB₁ and CB₂ receptors, transient receptor potential vanilloid type-1 (TRPV1) ion channels, peroxisome proliferator-activated receptor alpha (PPAR α) and orphan G protein-coupled receptor 55 (GRR55), all involved in the control of intestinal inflammation. Here, we investigated the effect of PEA in a murine model of colitis.

EXPERIMENTAL APPROACH

Colitis was induced in mice by intracolonic administration of dinitrobenzenesulfonic acid (DNBS). Inflammation was assessed by evaluating inflammatory markers/parameters and by histology; intestinal permeability by a fluorescent method; colonic cell proliferation by immunohistochemistry; PEA and endocannabinoid levels by liquid chromatography mass spectrometry; receptor and enzyme mRNA expression by quantitative RT-PCR.

KEY RESULTS

DNBS administration caused inflammatory damage, increased colonic levels of PEA and endocannabinoids, down-regulation of mRNA for TRPV1 and GPR55 but no changes in mRNA for CB₁, CB₂ and PPARα. Exogenous PEA (i.p. and/or p.o., 1 mg·kg⁻¹) attenuated inflammation and intestinal permeability, stimulated colonic cell proliferation, and increased colonic TRPV1 and CB₁ receptor expression. The anti-inflammatory effect of PEA was attenuated or abolished by CB₂ receptor, GPR55 or PPARα antagonists and further increased by the TRPV1 antagonist capsazepine.

CONCLUSIONS AND IMPLICATIONS

PEA improves murine experimental colitis, the effect being mediated by CB₂ receptors, GPR55 and PPARα, and modulated by TRPV1 channels.     

Activation of peroxisome proliferator-activated receptor γ in brain inhibits inflammatory pain, dorsal horn expression of Fos, and local edema

Systemic administration of thiazolidinediones reduces peripheral inflammation in vivo, presumably by acting at peroxisome proliferator-activated receptor γ (PPARγ) in peripheral tissues. Based on a rapidly growing body of literature indicating the CNS as a functional target of PPARγ actions, we postulated that brain PPARγ modulates peripheral edema and the processing of inflammatory pain signals in the dorsal horn of the spinal cord. To test this in the plantar carrageenan model of inflammatory pain, we measured paw edema, heat hyperalgesia, and dorsal horn expression of the immediate-early gene c-fos after intracerebroventricular (ICV) administration of PPARγ ligands or vehicle. We found that ICV rosiglitazone (0.5-50 μg) or 15d-PGJ₂ (50-200 μg), but not vehicle, dose-dependently reduced paw thickness, paw volume and behavioral withdrawal responses to noxious heat. These anti-inflammatory and anti-hyperalgesia effects result from direct actions in the brain and not diffusion to other sites, because intraperitoneal and intrathecal administration of rosiglitazone (50 μg) and 15d-PGJ₂ (200 μg) had no effect. PPARγ agonists changed neither overt behavior nor motor coordination, indicating that non-specific behavioral effects do not contribute to PPAR ligand-induced anti-hyperalgesia. ICV administration of structurally dissimilar PPARγ antagonists (either GW9662 or BADGE) reversed the anti-inflammatory and anti-hyperalgesic actions of both rosiglitazone and 15d-PGJ₂. To evaluate the effects of PPARγ agonists on a classic marker of noxious stimulus-evoked gene expression, we quantified Fos protein expression in the dorsal horn. The number of carrageenan-induced Fos-like immunoreactive profiles was less in rosiglitazone-treated rats as compared to vehicle controls. We conclude that pharmacological activation of PPARγ in the brain rapidly inhibits local edema and the spinal transmission of noxious inflammatory signals.     

PPARγ disease gene network and identification of therapeutic targets for prostate cancer

Peroxisome proliferator-activated receptor (PPAR) belongs to the nuclear hormone receptor superfamily. Recently published reports demonstrate the importance of a direct repeat 2 (DR2) as a PPARγ-responsive element in addition to the canonical direct repeat 1 (DR1) Peroxisome proliferator response elements (PPREs). However, a comprehensive and systematic approach to constructing de novo disease-specific gene networks for PPARγ is lacking, especially one that includes PPARγ target genes containing either DR1 or DR2 site within their promoter region. Here, we computationally identified 1154 PPARγ direct target genes and constructed the PPARγ disease gene network, which revealed 138 PPARγ target genes that are associated with 65 unique diseases. The network shows that PPARγ target genes are highly associated with cancer and neurological diseases. Thirty-eight PPARγ direct target genes were found to be involved in prostate cancer and two key (hub) PPARγ direct target genes, PRKCZ and PGK1, were experimentally validated to be repressed upon PPARγ activation by its natural ligand, 15d-PGJ₂ in three prostrate cancer cell lines. We proposed that PRKCZ and PGK1 could be novel therapeutic targets for prostate cancer. These investigations would not only aid in understanding the molecular mechanisms by which PPARγ regulates disease targets but would also lead to the identification of novel PPARγ gene targets.     

The plant cannabinoid ��9-tetrahydrocannabivarin can decrease signs of inflammation and inflammatory pain in mice

Background and purpose:

The phytocannabinoid, Δ⁹-tetrahydrocannabivarin (THCV), can block cannabinoid CB₁ receptors. This investigation explored its ability to activate CB₂ receptors, there being evidence that combined CB₂ activation/CB₁ blockade would ameliorate certain disorders.

Experimental approach:

We tested the ability of THCV to activate CB₂ receptors by determining whether: (i) it inhibited forskolin-stimulated cyclic AMP production by Chinese hamster ovary (CHO) cells transfected with human CB₂ (hCB₂) receptors; (ii) it stimulated [³⁵S]GTPγS binding to hCB₂ CHO cell and mouse spleen membranes; (iii) it attenuated signs of inflammation/hyperalgesia induced in mouse hind paws by intraplantar injection of carrageenan or formalin; and (iv) any such anti-inflammatory or anti-hyperalgesic effects were blocked by a CB₁ or CB₂ receptor antagonist.

Key results:

THCV inhibited cyclic AMP production by hCB₂ CHO cells (EC₅₀= 38 nM), but not by hCB₁ or untransfected CHO cells or by hCB₂ CHO cells pre-incubated with pertussis toxin (100 ng·mL⁻¹) and stimulated [³⁵S]GTPγS binding to hCB₂ CHO and mouse spleen membranes. THCV (0.3 or 1 mg·kg⁻¹ i.p.) decreased carrageenan-induced oedema in a manner that seemed to be CB₂ receptor-mediated and suppressed carrageenan-induced hyperalgesia. THCV (i.p.) also decreased pain behaviour in phase 2 of the formalin test at 1 mg·kg⁻¹, and in both phases of this test at 5 mg·kg⁻¹; these effects of THCV appeared to be CB₁ and CB₂ receptor mediated.

Conclusions and implications:

THCV can activate CB₂ receptors in vitro and decrease signs of inflammation and inflammatory pain in mice partly via CB₁ and/or CB₂ receptor activation.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x     

A CB₁/CB₂ receptor agonist, WIN 55,212-2, exerts its therapeutic effect in a viral autoimmune model of multiple sclerosis by restoring self-tolerance to myelin

Infection of mice with Theiler's murine encephalomyelitis virus (TMEV) leads to the development of TMEV-induced demyelinating disease (TMEV-IDD), an autoimmune, demyelinating and neurodegenerative pathology that serves as a model of multiple sclerosis. Activation of endogenous CB₁/CB₂ cannabinoid receptors inhibits inflammation and improves the clinical status of TMEV-IDD animals. In the present study, mice with established TMEV-IDD were treated with the CB₁/CB₂ receptor agonist WIN 55,212-2 (WIN), which restored self-tolerance to a myelin self-antigen while ameliorating the disease in a long-term manner. Accordingly, disruption of self-tolerance with cyclophosphamide provoked chronic relapse. Furthermore, transfer of splenocytes from WIN-treated TMEV-IDD mice to TMEV-infected mice at disease onset prevented the autoimmune inflammatory response and motor impairment. The therapeutic effect of WIN correlated with a decrease in the activation of CD4⁺CD25⁺Foxp3^? T cells and an increase in regulatory CD4⁺CD25⁺Foxp3⁺ T cells in the CNS, along with alterations in the cytokine and chemokine milieu. These findings demonstrate for the first time that the suppression of autoimmune responses to myelin antigens underlies the therapeutic effect of CB₁/CB₂ cannabinoid agonists in the treatment of multiple sclerosis.     

A Cannabinoid CB1 Receptor-Positive Allosteric Modulator Reduces Neuropathic Pain in the Mouse with No Psychoactive Effects

The CB₁ receptor represents a promising target for the treatment of several disorders including pain-related disease states. However, therapeutic applications of Δ⁹-tetrahydrocannabinol and other CB₁ orthosteric receptor agonists remain limited because of psychoactive side effects. Positive allosteric modulators (PAMs) offer an alternative approach to enhance CB₁ receptor function for therapeutic gain with the promise of reduced side effects. Here we describe the development of the novel synthetic CB₁ PAM, 6-methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011), which augments the in vitro and in vivo pharmacological actions of the CB₁ orthosteric agonists CP55,940 and N-arachidonoylethanolamine (AEA). ZCZ011 potentiated binding of [³H]CP55,940 to the CB₁ receptor as well as enhancing AEA-stimulated [³⁵S]GTPγS binding in mouse brain membranes and β-arrestin recruitment and ERK phosphorylation in hCB₁ cells. In the whole animal, ZCZ011 is brain penetrant, increased the potency of these orthosteric agonists in mouse behavioral assays indicative of cannabimimetic activity, including antinociception, hypothermia, catalepsy, locomotor activity, and in the drug discrimination paradigm. Administration of ZCZ011 alone was devoid of activity in these assays and did not produce a conditioned place preference or aversion, but elicited CB₁ receptor-mediated antinociceptive effects in the chronic constriction nerve injury model of neuropathic pain and carrageenan model of inflammatory pain. These data suggest that ZCZ011 acts as a CB₁ PAM and provide the first proof of principle that CB₁ PAMs offer a promising strategy to treat neuropathic and inflammatory pain with minimal or no cannabimimetic side effects.     

Characterization of cannabinoid receptor ligands in tissues natively expressing cannabinoid CB2 receptors

Background and Purpose

Although cannabinoid CB₂ receptor ligands have been widely characterized in recombinant systems in vitro, little pharmacological characterization has been performed in tissues natively expressing CB₂ receptors. The aim of this study was to compare the pharmacology of CB₂ receptor ligands in tissue natively expressing CB₂ receptors (human, rat and mouse spleen) and hCB₂-transfected CHO cells.

Experimental Approach

We tested the ability of well-known cannabinoid CB₂ receptor ligands to stimulate or inhibit [³⁵S]GTPγS binding to mouse, rat and human spleen membranes and to hCB₂-transfected CHO cell membranes. cAMP assays were also performed in hCB₂-CHO cells.

Key Results

The data presented demonstrate that: (i) CP 55,940, WIN 55,212-2 and JWH 133 behave as CB₂ receptor full agonists both in spleen and hCB₂-CHO cells, in both [³⁵S]GTPγS and cAMP assays; (ii) JWH 015 behaves as a low-efficacy agonist in spleen as well as in hCB₂-CHO cells when tested in the [³⁵S]GTPγS assay, while it displays full agonism when tested in the cAMP assay using hCB₂-CHO cells; (iii) (R)-AM 1241 and GW 405833 behave as agonists in the [³⁵S]GTPγS assay using spleen, instead it behaves as a low-efficacy inverse agonist in hCB₂-CHO cells; and (iv) SR 144528, AM 630 and JTE 907 behave as CB₂ receptor inverse agonists in all the tissues.

Conclusion and Implications

Our results demonstrate that CB₂ receptor ligands can display differential pharmacology when assays are conducted in tissues that natively express CB₂ receptors and imply that conclusions from recombinant CB₂ receptors should be treated with caution.     

Inhibition of COX-2-mediated eicosanoid production plays a major role in the anti-inflammatory effects of the endocannabinoid N-docosahexaenoylethanolamine (DHEA) in macrophages

BACKGROUND AND PURPOSE

N-docosahexaenoylethanolamine (DHEA) is the ethanolamine conjugate of the long-chain polyunsaturated n-3 fatty acid docosahexaenoic (DHA; 22: 6n-3). Its concentration in animal tissues and human plasma increases when diets rich in fish or krill oil are consumed. DHEA displays anti-inflammatory properties in vitro and was found to be released during an inflammatory response in mice. Here, we further examine possible targets involved in the immune-modulating effects of DHEA.

EXPERIMENTAL APPROACH

Antagonists for cannabinoid (CB)₁ and CB₂ receptors and PPAR_γ were used to explore effects of DHEA on NO release by LPS-stimulated RAW264.7 cells. The possible involvement of CB₂ receptors was studied by comparing effects in LPS-stimulated peritoneal macrophages obtained from CB₂^−/− and CB₂^+/+ mice. Effects on NF-κB activation were determined using a reporter cell line. To study DHEA effects on COX-2 and lipoxygenase activity, 21 different eicosanoids produced by LPS-stimulated RAW264.7 cells were quantified by LC-MS/MS. Finally, effects on mRNA expression profiles were analysed using gene arrays followed by Ingenuity® Pathways Analysis.

KEY RESULTS

CB₁ and CB₂ receptors or PPARs were not involved in the effects of DHEA on NO release. NF-κB and IFN-β, key elements of the myeloid differentiation primary response protein D88 (MyD88)-dependent and MyD88-independent pathways were not decreased. By contrast, DHEA significantly reduced levels of several COX-2-derived eicosanoids. Gene expression analysis provided support for an effect on COX–2-mediated pathways.

CONCLUSIONS AND IMPLICATIONS

Our findings suggest that the anti-inflammatory effects of DHEA in macrophages predominantly take place via inhibition of eicosanoids produced through COX-2.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids 2013 published in volume 171 issue 6. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2014.171.issue-6/issuetoc     

Very low density lipoprotein receptor promotes adipocyte differentiation and mediates the proadipogenic effect of peroxisome proliferator-activated receptor gamma agonists

Very low density lipoprotein receptor (VLDLR) is a member of the low density receptor family, expressed mostly in adipose tissue, heart, and skeletal muscles. VLDLR binds apolipoprotein-E-triglyceride-rich lipoproteins and plays a key role in lipid metabolism. In adipocytes, VLDLR expression increases with differentiation but it is not known whether it plays a role in the adipogenesis. Here we report that VLDLR expression in 3T3-L1 adipocytes is upregulated by PPARγ agonist 15-deoxy-delta^12,14-prostaglandin J₂ (15d-PGJ₂) in dose- and time-dependant manners. Knockdown of peroxisome proliferator-activated receptor-γ (PPARγ) with siRNA abolished pioglitazone- and 15d-PGJ₂-induced VLDLR expression and simultaneously reduced VLDL uptake in adipocytes. In addition, PPARγ-agonist treatment of control mouse adipocytes (vldlr^+/+) enhanced adipogenesis and VLDL uptake concurrently with the induction of VLDLR expression. However, vldlr deficiency (vldlr^−/−) significantly blunted the proadipogenic effects of PPARγ agonists. Sequence analysis revealed the presence of a putative PPARγ responsive sequence (PPRE) within the vldlr promoter, which is responsive to natural (15d-PGJ₂) and synthetic (pioglitazone) PPARγ agonists. Reporter gene assays using serial deletion of the 5′-flanking region showed that this putative PPRE site induced promoter transactivation, while a site-targeted mutation abolished transactivation. Moreover, electrophoresis mobility shift assay (EMSA) and chromatic immunoprecipitation (ChIP) assays showed the specific binding of PPARγ to the PPRE sequence.Together, these results support a crucial function for VLDLR in adipocyte differentiation and mediation of the proadipogenic effect of PPARγ.     

Very low density lipoprotein receptor promotes adipocyte differentiation and mediates the proadipogenic effect of peroxisome proliferator-activated receptor gamma agonists

Very low density lipoprotein receptor (VLDLR) is a member of the low density receptor family, expressed mostly in adipose tissue, heart, and skeletal muscles. VLDLR binds apolipoprotein-E-triglyceride-rich lipoproteins and plays a key role in lipid metabolism. In adipocytes, VLDLR expression increases with differentiation but it is not known whether it plays a role in the adipogenesis. Here we report that VLDLR expression in 3T3-L1 adipocytes is upregulated by PPARγ agonist 15-deoxy-delta^12,14-prostaglandin J₂ (15d-PGJ₂) in dose- and time-dependant manners. Knockdown of peroxisome proliferator-activated receptor-γ (PPARγ) with siRNA abolished pioglitazone- and 15d-PGJ₂-induced VLDLR expression and simultaneously reduced VLDL uptake in adipocytes. In addition, PPARγ-agonist treatment of control mouse adipocytes (vldlr^+/+) enhanced adipogenesis and VLDL uptake concurrently with the induction of VLDLR expression. However, vldlr deficiency (vldlr^−/−) significantly blunted the proadipogenic effects of PPARγ agonists. Sequence analysis revealed the presence of a putative PPARγ responsive sequence (PPRE) within the vldlr promoter, which is responsive to natural (15d-PGJ₂) and synthetic (pioglitazone) PPARγ agonists. Reporter gene assays using serial deletion of the 5′-flanking region showed that this putative PPRE site induced promoter transactivation, while a site-targeted mutation abolished transactivation. Moreover, electrophoresis mobility shift assay (EMSA) and chromatic immunoprecipitation (ChIP) assays showed the specific binding of PPARγ to the PPRE sequence.Together, these results support a crucial function for VLDLR in adipocyte differentiation and mediation of the proadipogenic effect of PPARγ.     

Cannabidiol for neurodegenerative disorders: important new clinical applications for this phytocannabinoid?

Cannabidiol (CBD) is a phytocannabinoid with therapeutic properties for numerous disorders exerted through molecular mechanisms that are yet to be completely identified. CBD acts in some experimental models as an anti-inflammatory, anticonvulsant, anti-oxidant, anti-emetic, anxiolytic and antipsychotic agent, and is therefore a potential medicine for the treatment of neuroinflammation, epilepsy, oxidative injury, vomiting and nausea, anxiety and schizophrenia, respectively. The neuroprotective potential of CBD, based on the combination of its anti-inflammatory and anti-oxidant properties, is of particular interest and is presently under intense preclinical research in numerous neurodegenerative disorders. In fact, CBD combined with Δ⁹-tetrahydrocannabinol is already under clinical evaluation in patients with Huntington''s disease to determine its potential as a disease-modifying therapy. The neuroprotective properties of CBD do not appear to be exerted by the activation of key targets within the endocannabinoid system for plant-derived cannabinoids like Δ⁹-tetrahydrocannabinol, i.e. CB₁ and CB₂ receptors, as CBD has negligible activity at these cannabinoid receptors, although certain activity at the CB₂ receptor has been documented in specific pathological conditions (i.e. damage of immature brain). Within the endocannabinoid system, CBD has been shown to have an inhibitory effect on the inactivation of endocannabinoids (i.e. inhibition of FAAH enzyme), thereby enhancing the action of these endogenous molecules on cannabinoid receptors, which is also noted in certain pathological conditions. CBD acts not only through the endocannabinoid system, but also causes direct or indirect activation of metabotropic receptors for serotonin or adenosine, and can target nuclear receptors of the PPAR family and also ion channels.     

Chronic blockade of cannabinoid CB2 receptors induces anxiolytic-like actions associated with alterations in GABA(A) receptors

BACKGROUND AND PURPOSE

The aim of this study was to explore the effects of CB₂ receptor agonist and antagonist in the regulation of anxiety-like behaviours.

EXPERIMENTAL APPROACHES

Effects of acute and chronic treatment with the CB₂ receptor agonist JWH133 and CB₂ receptor antagonist AM630 were evaluated in the light-dark box (LDB) and elevated plus maze (EPM) tests in Swiss ICR mice. CB₂ receptor, GABA_Aα₂ and GABA_Aγ₂ gene and protein expression in the cortex and amygdala of mice chronically treated with JWH133 or AM630 were examined by RT-PCR and Western blot. Effects of chronic AM630 treatment were evaluated in spontaneously anxious DBA/2 mice in LDB.

KEY RESULTS

Acute JWH133 treatment failed to produce any effect. Acute AM630 treatment increased anxiety and was blocked by pre-treatment with JWH133. Chronic JWH133 treatment increased anxiety-like behaviour whereas chronic AM630 treatment was anxiolytic in LDB and EPM tests. Chronic AM630 treatment increased gene and reduced protein expression of CB₂ receptors, GABA_Aα₂ and GABA_Aγ₂ in cortex and amygdala. Chronic JWH133 treatment resulted in opposite gene and protein alterations. In addition, chronic AM630 administration decreased the anxiety of DBA/2 mice in the LDB test.

CONCLUSIONS AND IMPLICATIONS

The opposing behavioural and molecular changes observed after chronic treatment with AM630 or JWH133 support the key role of CB₂ receptors in the regulation of anxiety. Indeed, the efficacy of AM630 in reducing the anxiety of the spontaneously anxious DBA/2 strain of mice strengthens the potential of the CB₂ receptor as a new target in the treatment of anxiety-related disorders.     

Regulation of Cell Surface CB<Subscript>2</Subscript> Receptor during Human B Cell Activation and Differentiation

Cannabinoid receptor type 2 (CB₂) is the primary receptor pathway mediating the immunologic consequences of cannabinoids. We recently reported that human peripheral blood B cells express CB₂ on both the extracellular membrane and at intracellular sites, where-as monocytes and T cells only express intracellular CB₂. To better understand the pattern of CB₂ expression by human B cells, we examined CD20⁺ B cells from three tissue sources. Both surface and intracellular expression were present and uniform in cord blood B cells, where all cells exhibited a naïve mature phenotype (IgD⁺/CD38^Dim). While naïve mature and quiescent memory B cells (IgD^?/CD38^?) from tonsils and peripheral blood exhibited a similar pattern, tonsillar activated B cells (IgD^?/CD38⁺) expressed little to no surface CB₂. We hypothesized that regulation of the surface CB₂ receptor may occur during B cell activation. Consistent with this, a B cell lymphoma cell line known to exhibit an activated phenotype (SUDHL-4) was found to lack cell surface CB₂ but express intracellular CB₂. Furthermore, in vitro activation of human cord blood resulted in a down-regulation of surface CB₂ on those B cells acquiring the activated phenotype but not on those retaining IgD expression. Using a CB₂ expressing cell line (293 T/CB₂-GFP), confocal microscopy confirmed the presence of both cell surface expression and multifocal intracellular expression, the latter of which co-localized with endoplasmic reticulum but not with mitochondria, lysosomes, or nucleus. Our findings suggest a dynamic multi-compartment expression pattern for CB₂ in B cells that is specifically modulated during the course of B cell activation.     

15-Deoxy-Δ-prostaglandin J2 inhibits fibrogenic response in human hepatoma cells

Liver fibrosis can be induced by environmental chemicals or toxicants, and finally stimulates fibrogenic cytokines expression, such as transforming growth factor-β (TGF-β) and its downstream mediator connective tissue growth factor (CTGF). 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) is a metabolite of arachidonic acid, can act as a peroxisome proliferator-activated receptor γ (PPARγ) ligand, and function as either anti-inflammatory or inflammatory agents in different cell types. In this study, CTGF was detected in three human hepatoma cell lines, Hep3B, HepG2, and Huh-7, and it was up-regulated by TGF-β. 15d-PGJ₂ significantly inhibited TGF-β-induced CTGF protein and mRNA expressions, and promoter activity in hepatoma cells. 15d-PGJ₂ suppressed TGF-β-induced Smad2 phosphorylation, however enhancing the phosphorylation of ERK, c-Jun N-terminal kinase (JNK), and p38 in TGF-β-treated Hep3B cells. Other PPAR ligands like the PPARγ agonist, troglitazone; the PPARα agonist, Wy-14643, and bezafibrate were also able to inhibit TGF-β-induced CTGF. The results suggest that 15d-PGJ₂ inhibits TGF-β-induced CTGF expression by inhibiting the phosphorylation of Smad2, which is independent of PPAR, and 15d-PGJ₂ might also act through a PPAR-dependent mechanism in human hepatoma cells. 15d-PGJ₂ might have a beneficent effect on prevention of liver fibrosis induced by environmental toxicants.     

A new cannabinoid CB2 receptor agonist HU-910 attenuates oxidative stress, inflammation and cell death associated with hepatic ischaemia/reperfusion injury

BACKGROUND AND PURPOSE

Cannabinoid CB₂ receptor activation has been reported to attenuate myocardial, cerebral and hepatic ischaemia-reperfusion (I/R) injury.

EXPERIMENTAL APPROACH

We have investigated the effects of a novel CB₂ receptor agonist ((1S,4R)-2-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-7,7-dimethylbicyclo[2.2.1]hept-2-en-1-yl)methanol (HU-910) on liver injury induced by 1 h of ischaemia followed by 2, 6 or 24 h of reperfusion, using a well-established mouse model of segmental hepatic I/R.

KEY RESULTS

Displacement of [³H]CP55940 by HU-910 from specific binding sites in CHO cell membranes transfected with human CB₂ or CB₁ receptors (hCB_1/2) yielded K_i values of 6 nM and 1.4 µM respectively. HU-910 inhibited forskolin-stimulated cyclic AMP production by hCB₂ CHO cells (EC₅₀= 162 nM) and yielded EC₅₀ of 26.4 nM in [³⁵S]GTPγS binding assays using hCB₂ expressing CHO membranes. HU-910 given before ischaemia significantly attenuated levels of I/R-induced hepatic pro-inflammatory chemokines (CCL3 and CXCL2), TNF-α, inter-cellular adhesion molecule-1, neutrophil infiltration, oxidative stress and cell death. Some of the beneficial effect of HU-910 also persisted when given at the beginning of the reperfusion or 1 h after the ischaemic episode. Furthermore, HU-910 attenuated the bacterial endotoxin-triggered TNF-α production in isolated Kupffer cells and expression of adhesion molecules in primary human liver sinusoidal endothelial cells stimulated with TNF-α. Pretreatment with a CB₂ receptor antagonist attenuated the protective effects of HU-910, while pretreatment with a CB₁ antagonist tended to enhance them.

CONCLUSION AND IMPLICATIONS

HU-910 is a potent CB₂ receptor agonist which may exert protective effects in various diseases associated with inflammation and tissue injury.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7