Central antinociception induced by μ-opioid receptor agonist morphine,but not δ- or κ-, is mediated by cannabinoid CB1 receptor

Background and purpose:

It has been demonstrated that cannabinoids evoke the release of endogenous opioids to produce antinociception; however, no information exists regarding the participation of cannabinoids in the antinociceptive mechanisms of opioids. The aim of the present study was to determine whether endocannabinoids are involved in central antinociception induced by activation of µ-, δ- and κ-opioid receptors.

Experimental approach:

Nociceptive threshold to thermal stimulation was measured according to the tail-flick test in Swiss mice. Morphine (5 µg), SNC80 (4 µg), bremazocine (4 µg), AM251 (2 and 4 µg), AM630 (2 and 4 µg) and MAFP (0.1 and 0.4 µg) were administered by the intracerebroventricular route.

Key results:

The CB₁-selective cannabinoid receptor antagonist AM251 completely reversed the central antinociception induced by morphine in a dose-dependent manner. In contrast, the CB₂-selective cannabinoid receptor antagonist AM630 did not antagonize this effect. Additionally, the administration of the anandamide amidase inhibitor, MAFP, significantly enhanced the antinociception induced by morphine. In contrast, the antinociceptive effects of δ- and κ-opioid receptor agonists were not affected by the cannabinoid antagonists. The antagonists alone caused no hyperalgesic or antinociceptive effects.

Conclusions and implications:

The results provide evidence for the involvement of cannabinoid CB₁ receptors in the central antinociception induced by activation of µ-opioid receptors by the agonist morphine. The release of endocannabinoids appears not to be involved in central antinociception induced by activation of κ- and δ-opioid receptors.     

Peripheral interactions between cannabinoid and opioid systems contribute to the antinociceptive effect of crotalphine

Background and Purpose

Crotalphine is an antinociceptive peptide that, despite its opioid-like activity, does not induce some of the characteristic side effects of opioids, and its amino acid sequence has no homology to any known opioid peptide. Here, we evaluated the involvement of the peripheral cannabinoid system in the crotalphine effect and its interaction with the opioid system.

Experimental Approach

Hyperalgesia was evaluated using the rat paw pressure test. Involvement of the cannabinoid system was determined using a selective cannabinoid receptor antagonist. Cannabinoid and opioid receptor activation were evaluated in paw slices by immunofluorescence assays using conformation state-sensitive antibodies. The release of endogenous opioid peptides from skin tissue was measured using a commercial enzyme immunoassay (EIA).

Key Results

Both p.o. (0.008–1.0 μg·kg⁻¹) and intraplantar (0.0006 μg per paw) administration of crotalphine induced antinociception in PGE₂-induced hyperalgesia. Antinociception by p.o. crotalphine (1 μg·kg⁻¹) was blocked by AM630 (50 μg per paw), a CB₂ receptor antagonist, and by antiserum anti-dynorphin A (1 μg per paw). Immunoassay studies confirmed that crotalphine increased the activation of both κ-opioid (51.7%) and CB₂ (28.5%) receptors in paw tissue. The local release of dynorphin A from paw skin was confirmed by in vitro EIA and blocked by AM630.

Conclusions and Implications

Crotalphine-induced antinociception involves peripheral CB₂ cannabinoid receptors and local release of dynorphin A, which is dependent on CB₂ receptor activation. These results enhance our understanding of the mechanisms involved in the peripheral effect of crotalphine, as well as the interaction between the opioid and cannabinoid systems.     

Rimonabant inhibits TNF-α-induced endothelial IL-6 secretion via CB1 receptor and cAMP-dependent protein kinase pathway

Aim:

To investigate whether rimonabant, a cannabinoid receptor antagonist, had inhibitory effects on inflammatory reactions in human umbilical vein endothelial cells (HUVEC).

Methods:

TNF-α-induced IL-6 production was measured by ELISA and effects on related signaling pathways were investigated by immunoblot analysis. Cellular cAMP level was measured using kinase-coupled luciferase reaction.

Results:

Rimonabant at 1 and 10 μmol/L significantly inhibited TNF-α-induced IL-6 production when added 15, 30 and 60 minutes before TNF-α treatment. Rimonabant also inhibited TNF-α-induced phosphorylation of IκB kinase (IKK) α/β and IκB-α degradation. ACEA, a cannabinoid receptor subtype 1 (CB1) agonist, added before rimonabant abolished the former effects of rimonabant. H-89, an inhibitor of cAMP-dependent protein kinase (PKA), abolished the inhibitory effects of rimonabant on TNF-α induced IL-6 production. Rimonabant also increased the phosphorylation of PKA regulatory subunit II (PKA-RII), implying the essential role of PKA activation in the inhibitory effects of rimonabant. Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin did not abolish the inhibitory effects of rimonabant on TNF-α induced IL-6 production.

Conclusion:

Rimonabant had anti-inflammatory effects on endothelial cells and inhibited TNF-α-induced IKKα/β phosphorylation, IκB-α degradation and IL-6 production in HUVEC. This effect was related to CB1 antagonism and PKA activation.     

Physical and functional interaction between CB1 cannabinoid receptors and ��2-adrenoceptors

Background and purpose:

The CB₁ cannabinoid receptor and the β₂-adrenoceptor are G protein-coupled receptors (GPCRs) co-expressed in many tissues. The present study examined physical and functional interactions between these receptors in a heterologous expression system and in primary human ocular cells.

Experimental approach:

Physical interactions between CB₁ receptors and β₂-adrenoceptors were assessed using bioluminescence resonance energy transfer (BRET). Functional interactions between these receptors were evaluated by examining receptor trafficking, as well as extracellular signal-regulated kinase (ERK) and cyclic AMP response element binding protein (CREB) signalling.

Key results:

Physical interactions between CB₁ receptors and β₂-adrenoceptors were demonstrated using BRET. In human embryonic kidney (HEK) 293H cells, co-expression of β₂-adrenoceptors tempered the constitutive activity and increased cell surface expression of CB₁ receptors. Co-expression altered the signalling properties of CB₁receptors, resulting in increased Gα_i-dependent ERK phosphorylation, but decreased non-Gα_i-mediated CREB phosphorylation. The CB₁ receptor inverse agonist AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) attenuated β₂-adrenoceptor-pERK signalling in cells expressing both receptors, while the CB₁ receptor neutral antagonist O-2050 ((6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) did not. The actions of AM251 and O-2050 were further examined in primary human trabecular meshwork (HTM) cells, which are ocular cells endogenously co-expressing CB₁ receptors and β₂-adrenoceptors. In HTM cells, as in HEK 293H cells, AM251 but not O-2050, altered the β₂-adrenoceptor–pERK response.

Conclusion and implications:

A complex interaction was demonstrated between CB₁ receptors and β₂-adrenoceptors in HEK 293H cells. As similar functional interactions were also observed in HTM cells, such interactions may affect the pharmacology of these receptors in tissues where they are endogenously co-expressed.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     

Virodhamine relaxes the human pulmonary artery through the endothelial cannabinoid receptor and indirectly through a COX product

Background and purpose:

The endocannabinoid virodhamine is a partial agonist at the cannabinoid CB₁ receptor and a full agonist at the CB₂ receptor, and relaxes rat mesenteric arteries through endothelial cannabinoid receptors. Its concentration in the periphery exceeds that of the endocannabinoid anandamide. Here, we examined the influence of virodhamine on the human pulmonary artery.

Experimental approach:

Isolated human pulmonary arteries were obtained during resections for lung carcinoma. Vasorelaxant effects of virodhamine were examined on endothelium-intact vessels precontracted with 5-HT or KCl.

Key results:

Virodhamine, unlike WIN 55,212-2, relaxed 5-HT-precontracted vessels concentration dependently. The effect of virodhamine was reduced by endothelium denudation, two antagonists of the endothelial cannabinoid receptor, cannabidiol and O-1918, and a high concentration of the CB₁ receptor antagonist rimonabant (5 μM), but only slightly attenuated by the NOS inhibitor L-NAME and not affected by a lower concentration of rimonabant (100 nM) or by the CB₂ and vanilloid receptor antagonists SR 144528 and capsazepine, respectively. The COX inhibitor indomethacin and the fatty acid amide hydrolase inhibitor URB597 and combined administration of selective blockers of small (apamin) and intermediate and large (charybdotoxin) conductance Ca²⁺-activated K⁺ channels attenuated virodhamine-induced relaxation. The vasorelaxant potency of virodhamine was lower in KCl- than in 5-HT-precontracted preparations.

Conclusions and implications:

Virodhamine relaxes the human pulmonary artery through the putative endothelial cannabinoid receptor and indirectly through a COX-derived vasorelaxant prostanoid formed from the virodhamine metabolite, arachidonic acid. One or both of these mechanisms may stimulate vasorelaxant Ca²⁺-activated K⁺ channels.     

Cannabinoids and bone: endocannabinoids modulate human osteoclast function in vitro

BACKGROUND AND PURPOSE

Both CB₁ and CB₂ cannabinoid receptors have been shown to play a role in bone metabolism. Crucially, previous studies have focussed on the effects of cannabinoid ligands in murine bone cells. This study aimed to investigate the effects of cannabinoids on human bone cells in vitro.

EXPERIMENTAL APPROACH

Quantitative RT-PCR was used to determine expression of cannabinoid receptors and liquid chromatography-electrospray ionization tandem mass spectrometry was used to determine the presence of endocannabinoids in human bone cells. The effect of cannabinoids on human osteoclast formation, polarization and resorption was determined by assessing the number of cells expressing α_vβ₃ or with F-actin rings, or measurement of resorption area.

KEY RESULTS

Human osteoclasts express both CB₁ and CB₂ receptors. CB₂ expression was significantly higher in human monocytes compared to differentiated osteoclasts. Furthermore, the differentiation of human osteoclasts from monocytes was associated with a reduction in 2-AG levels and an increase in anandamide (AEA) levels. Treatment of osteoclasts with LPS significantly increased levels of AEA. Nanomolar concentrations of AEA and the synthetic agonists CP 55 940 and JWH015 stimulated human osteoclast polarization and resorption; these effects were attenuated in the presence of CB₁ and/or CB₂ antagonists.

CONCLUSIONS AND IMPLICATIONS

Low concentrations of cannabinoids activate human osteoclasts in vitro. There is a dynamic regulation of the expression of the CB₂ receptor and the production of the endocannabinoids during the differentiation of human bone cells. These data suggest that small molecules modulating the endocannabinoid system could be important therapeutics in human bone disease.

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     

Kaempferol acts through mitogen-activated protein kinases and protein kinase B/AKT to elicit protection in a model of neuroinflammation in BV2 microglial cells

BACKGROUND AND PURPOSE

Kaempferol, a dietary flavonoid and phyto-oestrogen, is known to have anti-inflammatory properties. Microglial activation has been implicated in various neurodegenerative diseases. Anti-inflammatory effects of kaempferol and the underlying mechanisms were investigated by using LPS-stimulated microglial BV2 cells.

EXPERIMENTAL APPROACH

Cell viability was measured using MTT and neutral red assays. elisa, Western blot, immunocytochemistry and electrophoretic mobility-shift assay were used to analyse NO, PGE₂, TNF-α and IL-1β production, inducible NOS (iNOS), COX-2 expression and the involvement of signalling pathways such as toll-like receptor-4 (TLR4), MAPK cascades, PKB (AKT) and NF-κB. Accumulation of reaction oxygen species (ROS) was measured by nitroblue tetrazolium and 2′7′-dichlorofluorescein diacetate assay. Matrix metalloproteinase activity was investigated by zymography and immunoblot assay. Phagocytotic activity was assessed by use of latex beads.

KEY RESULTS

Kaempferol significantly attenuated LPS-induced NO, PGE₂, TNF-α, IL-1β and ROS production and phagocytosis in a concentration-dependent manner. Kaempferol suppressed the expression of iNOS, COX-2, MMP-3 and blocked the TLR4 activation. Moreover, kaempferol inhibited LPS-induced NF-κB activation and p38 MAPK, JNK and AKT phosphorylation.

CONCLUSION AND IMPLICATIONS

Kaempferol was able to reduce LPS-induced inflammatory mediators through the down-regulation of TLR4, NF-κB, p38 MAPK, JNK and AKT suggesting that kaempferol has therapeutic potential for the treatment of neuroinflammatory diseases.     

Inflammatory muscle pain is dependent on the activation of kinin B₁ and B₂ receptors and intracellular kinase pathways

BACKGROUND AND PURPOSE

B₁ and B₂ kinin receptors are involved in pain transmission but they may have different roles in the muscle pain induced by intense exercise or inflammation. We investigated the contribution of each of these receptors, and the intracellular pathways involved, in the initial development and maintenance of the muscle pain associated with inflammation-induced tissue damage.

EXPERIMENTAL APPROACH

Mechanical hyperalgesia was measured using the Randall–Selitto apparatus after injecting 5% formalin solution into the gastrocnemius muscle in mice treated with selective antagonists for B₁ or B₂ receptors. The expression of kinin receptors and cytokines and the activation of intracellular kinases were monitored by real-time PCR and immunohistochemistry.

KEY RESULTS

The i.m. injection of formalin induced an overexpression of B₁ and B₂ receptors. This overexpression was associated with the mechanical hyperalgesia induced by formalin because treatment with B₁ receptor antagonists (des-Arg⁹[Leu⁸]-BK, DALBK, and SSR240612) or B₂ receptor antagonists (HOE 140 and {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657) prevented the hyperalgesia. Formalin increased myeloperoxidase activity, and up-regulated TNF-α, IL-1β and IL-6 in gastrocnemius. Myeloperoxidase activity and TNF-α mRNA expression were inhibited by either DALBK or HOE 140, whereas IL-6 was inhibited only by HOE 140. The hyperalgesia induced by i.m. formalin was dependent on the activation of intracellular MAPKs p38, JNK and PKC.

CONCLUSIONS AND IMPLICATIONS

Inflammatory muscle pain involves a cascade of events that is dependent on the activation of PKC, p38 and JNK, and the synthesis of IL-1β, TNF-α and IL-6 associated with the up-regulation of both B₁ and B₂ kinin receptors.     

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.     

Potential anxiolytic- and antidepressant-like effects of salvinorin A,the main active ingredient of Salvia divinorum,in rodents

Background and purpose:

Drugs targeting brain κ-opioid receptors produce profound alterations in mood. In the present study we investigated the possible anxiolytic- and antidepressant-like effects of the κ-opioid receptor agonist salvinorin A, the main active ingredient of Salvia divinorum, in rats and mice.

Experimental approach:

Experiments were performed on male Sprague-Dawley rats or male Albino Swiss mice. The anxiolytic-like effects were tested by using the elevated plus maze, in rats. The antidepressant-like effect was estimated through the forced swim (rats) and the tail suspension (mice) test. κ-Opioid receptor involvement was investigated pretreating animals with the κ-opioid receptor antagonist, nor-binaltorphimine (1 or 10 mg·kg⁻¹), while direct or indirect activity at CB₁ cannabinoid receptors was evaluated with the CB₁ cannabinoid receptor antagonist, N-(piperidin-1-yl) -5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251, 0.5 or 3 mg·kg⁻¹), binding to striatal membranes of naïve rats and assay of fatty acid amide hydrolase in prefrontal cortex, hippocampus and amygdala.

Key results:

Salvinorin A, given s.c. (0.001–1000 µg·kg⁻¹), exhibited both anxiolytic- and antidepressant-like effects that were prevented by nor-binaltorphimine or AM251 (0.5 or 3 mg·kg⁻¹). Salvinorin A reduced fatty acid amide hydrolase activity in amygdala but had very weak affinity for cannabinoid CB₁ receptors.

Conclusions and implications:

The anxiolytic- and antidepressant-like effects of Salvinorin A are mediated by both κ-opioid and endocannabinoid systems and may partly explain the subjective symptoms reported by recreational users of S. divinorum.     

Differential effect of opioid and cannabinoid receptor blockade on heroin-seeking reinstatement and cannabinoid substitution in heroin-abstinent rats

BACKGROUND AND PURPOSE

Opioids and cannabinoids interact in drug addiction and relapse. We investigated the effect of the opioid receptor antagonist naloxone and/or the cannabinoid CB₁ receptor antagonist rimonabant on cannabinoid-induced reinstatement of heroin seeking and on cannabinoid substitution in heroin-abstinent rats.

EXPERIMENTAL APPROACH

Rats were trained to self-administer heroin (30 µg·kg⁻¹ per infusion) under a fixed-ratio 1 reinforcement schedule. After extinction of self-administration (SA) behaviour, we confirmed the effect of naloxone (0.1–1 mg·kg⁻¹) and rimonabant (0.3–3 mg·kg⁻¹) on the reinstatement of heroin seeking induced by priming with the CB₁ receptor agonist WIN55,212-2 (WIN, 0.15–0.3 mg·kg⁻¹). Then, in a parallel set of heroin-trained rats, we evaluated whether WIN (12.5 µg·kg⁻¹ per infusion) SA substituted for heroin SA after different periods of extinction. In groups of rats in which substitution occurred, we studied the effect of both antagonists on cannabinoid intake.

KEY RESULTS

Cannabinoid-induced reinstatement of heroin seeking was significantly attenuated by naloxone (1 mg·kg⁻¹) and rimonabant (3 mg·kg⁻¹) and fully blocked by co-administration of sub-threshold doses of the two antagonists. Moreover, contrary to immediate (1 day) or delayed (90 days) drug substitution, rats readily self-administered WIN when access was given after 7, 14 or 21 days of extinction from heroin, and showed a response rate that was positively correlated with the extinction period. In these animals, cannabinoid intake was increased by naloxone (1 mg·kg⁻¹) and decreased by rimonabant (3 mg·kg⁻¹).

CONCLUSIONS AND IMPLICATIONS

Our findings extend previous research on the crosstalk between cannabinoid and opioid receptors in relapse mechanisms, which suggests a differential role in heroin-seeking reinstatement and cannabinoid substitution in heroin-abstinent rats.

LINKED ARTICLES

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

The antinociceptive triterpene β-amyrin inhibits 2-arachidonoylglycerol (2-AG) hydrolysis without directly targeting cannabinoid receptors

BACKGROUND AND PURPOSE

Pharmacological activation of cannabinoid CB₁ and CB₂ receptors is a therapeutic strategy to treat chronic and inflammatory pain. It was recently reported that a mixture of natural triterpenes α- and β-amyrin bound selectively to CB₁ receptors with a subnanomolar K_i value (133 pM). Orally administered α/β-amyrin inhibited inflammatory and persistent neuropathic pain in mice through both CB₁ and CB₂ receptors. Here, we investigated effects of amyrins on the major components of the endocannabinoid system.

EXPERIMENTAL APPROACH

We measured CB receptor binding interactions of α- and β-amyrin in validated binding assays using hCB₁ and hCB₂ transfected CHO-K1 cells. Effects on endocannabinoid transport in U937 cells and breakdown using homogenates of BV2 cells and pig brain, as well as purified enzymes, were also studied.

KEY RESULTS

There was no binding of either α- or β-amyrin to hCB receptors in our assays (K_i > 10 µM). The triterpene β-amyrin potently inhibited 2-arachidonoyl glycerol (2-AG) hydrolysis in pig brain homogenates, but not that of anandamide. Although β-amyrin only weakly inhibited purified human monoacylglycerol lipase (MAGL), it also inhibited α,β-hydrolases and more potently inhibited 2-AG breakdown than α-amyrin and the MAGL inhibitor pristimerin in BV2 cell and pig brain homogenates.

CONCLUSIONS AND IMPLICATIONS

We propose that β-amyrin exerts its analgesic and anti-inflammatory pharmacological effects via indirect cannabimimetic mechanisms by inhibiting the degradation of the endocannabinoid 2-AG without interacting directly with CB receptors. Triterpenoids appear to offer a very broad and largely unexplored scaffold for inhibitors of the enzymic degradation of 2-AG.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8     

Inhibitory effect of salvinorin A, from Salvia divinorum, on ileitis-induced hypermotility: cross-talk between kappa-opioid and cannabinoid CB(1) receptors

Background and purpose:

Salvinorin A, the active component of the hallucinogenic herb Salvia divinorum, inhibits intestinal motility through activation of κ-opioid receptors (KORs). However, this compound may have target(s) other than the KORs in the inflamed gut. Because intestinal inflammation upregulates cannabinoid receptors and endogenous cannabinoids, in the present study we investigated the possible involvement of the endogenous cannabinoid system in salvinorin A-induced delay in motility in the inflamed gut.

Experimental approach:

Motility in vivo was measured by evaluating the distribution of a fluorescent marker along the small intestine; intestinal inflammation was induced by the irritant croton oil; direct or indirect activity at cannabinoid receptors was evaluated by means of binding, enzymic and cellular uptake assays.

Key results:

Salvinorin A as well as the KOR agonist U-50488 reduced motility in croton oil treated mice. The inhibitory effect of both salvinorin A and U-50488 was counteracted by the KOR antagonist nor-binaltorphimine and by the cannabinoid CB₁ receptor antagonist rimonabant. Rimonabant, however, did not counteract the inhibitory effect of salvinorin A on motility in control mice. Binding experiments showed very weak affinity of salvinorin A for cannabinoid CB₁ and CB₂ and no inhibitory effect on 2-arachidonoylglycerol and anandamide hydrolysis and cellular uptake.

Conclusions and implications:

The inhibitory effect of salvinorin A on motility reveals a functional interaction between cannabinoid CB₁ receptors and KORs in the inflamed—but not in the normal—gut in vivo.     

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.     

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     

MDA7: a novel selective agonist for CB2 receptors that prevents allodynia in rat neuropathic pain models

Background and purpose:

There is growing interest in using cannabinoid type 2 (CB₂) receptor agonists for the treatment of neuropathic pain. In this report, we describe the pharmacological characteristics of MDA7 (1-[(3-benzyl-3-methyl-2,3-dihydro-1-benzofuran-6-yl)carbonyl]piperidine), a novel CB₂ receptor agonist.

Experimental approach:

We characterized the pharmacological profile of MDA7 by using radioligand-binding assays and in vitro functional assays at human cannabinoid type 1 (CB₁) and CB₂ receptors. In vitro functional assays were performed at rat CB₁ and CB₂ receptors. The effects of MDA7 in reversing neuropathic pain were assessed in spinal nerve ligation and paclitaxel-induced neuropathy models in rats.

Key results:

MDA7 exhibited selectivity and agonist affinity at human and rat CB₂ receptors. MDA7 treatment attenuated tactile allodynia produced by spinal nerve ligation or by paclitaxel in a dose-related manner. These effects were selectively antagonized by a CB₂ receptor antagonist but not by CB₁ or opioid receptor antagonists. MDA7 did not affect rat locomotor activity.

Conclusion and implications:

MDA7, a novel selective CB₂ agonist, was effective in suppressing neuropathic nociception in two rat models without affecting locomotor behaviour. These results confirm the potential for CB₂ agonists in the treatment of neuropathic pain.     

��-Opioid receptors stimulate GLUT1-mediated glucose uptake through Src- and IGF-1 receptor-dependent activation of PI3-kinase signalling in CHO cells

BACKGROUND AND PURPOSE

Although opioids have been reported to affect glucose homeostasis, relatively little is known on the role of δ-opioid receptors. We have investigated the regulation of glucose transport by human δ-opioid receptors expressed in Chinese hamster ovary cells.

EXPERIMENTAL APPROACH

The uptake of [³H]-2-deoxy-D-glucose and 3-O-[methyl-[³H]]-D-glucose in response to δ-opioid receptor ligands and the expression of GLUT1, GLUT3 and GLUT4 glucose transporters were examined. Moreover, the effects of intracellular signal transduction inhibitors on δ-opioid receptor-regulated [³H]-2-deoxy-D-glucose uptake and protein phosphorylation were investigated.

KEY RESULTS

Activation of δ-opioid receptors rapidly stimulated [³H]-2-deoxy-D-glucose and 3-O-[methyl-[³H]]-D-glucose uptakes, which were blocked by the GLUT inhibitors cytochalasin B and phloretin. The stimulation of [³H]-2-deoxy-D-glucose uptake that occurred without a change in plasma membrane GLUT1 – required the coupling to G_i/G_o proteins – was independent of cAMP and extracellular signal-regulated protein kinases, and was suppressed by blockade of Src and insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinases. Inhibition of phosphatidylinositol 3-kinase (PI3K) by wortmannin or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and by PI3Kα, but not γ, isoform-selective inhibitors greatly reduced the δ-opioid receptor stimulation of glucose uptake. Moreover, the response was attenuated by overexpressing a dominant-negative kinase-deficient Akt form and by chemical inhibition of Akt. Stimulation of δ-opioid receptors increased protein kinase Cζ/λ (PKCζ/λ) phosphorylation and a selective PKCζ/λ inhibitor slightly reduced opioid stimulation of glucose uptake.

CONCLUSIONS AND IMPLICATIONS

δ-Opioid receptors stimulated glucose transport probably by enhancing GLUT1 intrinsic activity through a signalling cascade involving G_i/G_o, Src, IGF-1R, PI3Kα, Akt and, to a minor extent, PKCζ/λ. This effect may contribute to the opioid regulation of glucose homeostasis in physio-pathological conditions.     

The fatty acid amide hydrolase (FAAH) inhibitor PF-3845 acts in the nervous system to reverse LPS-induced tactile allodynia in mice

BACKGROUND AND PURPOSE

Inflammatory pain presents a problem of clinical relevance and often elicits allodynia, a condition in which non-noxious stimuli are perceived as painful. One potential target to treat inflammatory pain is the endogenous cannabinoid (endocannabinoid) system, which is comprised of CB₁ and CB₂ cannabinoid receptors and several endogenous ligands, including anandamide (AEA). Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without the psychomimetic side effects associated with Δ⁹-tetrahydrocannabinol (THC).

EXPERIMENTAL APPROACH

Allodynia was induced by intraplantar injection of LPS. Complementary genetic and pharmacological approaches were used to determine the strategy of blocking FAAH to reverse LPS-induced allodynia. Endocannabinoid levels were quantified using mass spectroscopy analyses.

KEY RESULTS

FAAH (−/−) mice or wild-type mice treated with FAAH inhibitors (URB597, OL-135 and PF-3845) displayed an anti-allodynic phenotype. Furthermore, i.p. PF-3845 increased AEA levels in the brain and spinal cord. Additionally, intraplantar PF-3845 produced a partial reduction in allodynia. However, the anti-allodynic phenotype was absent in mice expressing FAAH exclusively in the nervous system under a neural specific enolase promoter, implicating the involvement of neuronal fatty acid amides (FAAs). The anti-allodynic effects of FAAH-compromised mice required activation of both CB₁ and CB₂ receptors, but other potential targets of FAA substrates (i.e. µ-opioid, TRPV1 and PPARα receptors) had no apparent role.

CONCLUSIONS AND IMPLICATIONS

AEA is the primary FAAH substrate reducing LPS-induced tactile allodynia. Blockade of neuronal FAAH reverses allodynia through the activation of both cannabinoid receptors and represents a promising target to treat inflammatory pain.

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     

Cannabinoids mediate opposing effects on inflammation-induced intestinal permeability

BACKGROUND AND PURPOSE

Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion and intestinal motility. The ability to modulate intestinal permeability in inflammation may be important in therapy aimed at maintaining epithelial barrier integrity. The aim of the present study was to determine whether cannabinoids modulate the increased permeability associated with inflammation in vitro.

EXPERIMENTAL APPROACH

Confluent Caco-2 cell monolayers were treated for 24 h with IFNγ and TNFα (10 ng·mL⁻¹). Monolayer permeability was measured using transepithelial electrical resistance and flux measurements. Cannabinoids were applied either apically or basolaterally after inflammation was established. Potential mechanisms of action were investigated using antagonists for CB₁, CB₂, TRPV1, PPARγ and PPARα. A role for the endocannabinoid system was established using inhibitors of the synthesis and degradation of endocannabinoids.

KEY RESULTS

Δ⁹-Tetrahydrocannabinol (THC) and cannabidiol accelerated the recovery from cytokine-induced increased permeability; an effect sensitive to CB₁ receptor antagonism. Anandamide and 2-arachidonylglycerol further increased permeability in the presence of cytokines; this effect was also sensitive to CB₁ antagonism. No role for the CB₂ receptor was identified in these studies. Co-application of THC, cannabidiol or a CB₁ antagonist with the cytokines ameliorated their effect on permeability. Inhibiting the breakdown of endocannabinoids worsened, whereas inhibiting the synthesis of endocannabinoids attenuated, the increased permeability associated with inflammation.

CONCLUSIONS AND IMPLICATIONS

These findings suggest that locally produced endocannabinoids, acting via CB₁ receptors play a role in mediating changes in permeability with inflammation, and that phytocannabinoids have therapeutic potential for reversing the disordered intestinal permeability associated with inflammation.

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     

Glycyrrhizin attenuates rat ischemic spinal cord injury by suppressing inflammatory cytokines and HMGB1

Aim:

To investigate the neuroprotective effect of glycyrrhizin (Gly) against the ischemic injury of rat spinal cord and the possible role of the nuclear protein high-mobility group box 1 (HMGB1) in the process.

Methods:

Male Sprague-Dawley rats were subjected to 45 min aortic occlusion to induce transient lumbar spinal cord ischemia. The motor functions of the animals were assessed according to the modified Tarlov scale. The animals were sacrificed 72 h after reperfusion and the lumbar spinal cord segment (L2–L4) was taken out for histopathological examination and Western blotting analysis. Serum inflammatory cytokine and HMGB1 levels were analyzed using ELISA.

Results:

Gly (6 mg/kg) administered intravenously 30 min before inducing the transient lumbar spinal cord ischemia significantly improved the hind-limb motor function scores, and reduced the number of apoptotic neurons, which was accompanied by reduced levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the plasma and injured spinal cord. Moreover, the serum HMGB1 level correlated well with the serum TNF-α, IL-1β and IL-6 levels during the time period of reperfusion.

Conclusion:

The results suggest that Gly can attenuate the transient spinal cord ischemic injury in rats via reducing inflammatory cytokines and inhibiting the release of HMGB1.