Inhibition of monoacylglycerol lipase attenuates vomiting in Suncus murinus and 2-arachidonoyl glycerol attenuates nausea in rats

BACKGROUND AND PURPOSE

To evaluate the role of 2-arachidonoyl glycerol (2AG) in the regulation of nausea and vomiting using animal models of vomiting and of nausea-like behaviour (conditioned gaping).

EXPERIMENTAL APPROACH

Vomiting was assessed in shrews (Suncus murinus), pretreated with JZL184, a selective monoacylglycerol lipase (MAGL) inhibitor which elevates endogenous 2AG levels, 1 h before administering the emetogenic compound, LiCl. Regulation of nausea-like behaviour in rats by exogenous 2AG or its metabolite arachidonic acid (AA) was assessed, using the conditioned gaping model. The role of cannabinoid CB₁ receptors, CB₂ receptors and cyclooxygenase (COX) inhibition in suppression of vomiting or nausea-like behaviour was assessed.

KEY RESULTS

JZL184 dose-dependently suppressed vomiting in shrews, an effect prevented by pretreatment with the CB₁ receptor inverse agonist/antagonist, AM251. In shrew brain tissue, JZL184 inhibited MAGL activity in vivo. In rats, 2AG suppressed LiCl-induced conditioned gaping but this effect was not prevented by AM251 or the CB₂ receptor antagonist, AM630. Instead, the COX inhibitor, indomethacin, prevented suppression of conditioned gaping by 2AG or AA. However, when rats were pretreated with a high dose of JZL184 (40 mg·kg⁻¹), suppression of gaping by 2AG was partially reversed by AM251. Suppression of conditioned gaping was not due to interference with learning because the same dose of 2AG did not modify the strength of conditioned freezing to a shock-paired tone.

CONCLUSIONS AND IMPLICATIONS

Our results suggest that manipulations that elevate 2AG may have anti-emetic or anti-nausea potential.

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     

Effects of palmitoylation of Cys(415) in helix 8 of the CB(1) cannabinoid receptor on membrane localization and signalling

BACKGROUND AND PURPOSE

The CB₁ cannabinoid receptor is regulated by its association with membrane microdomains such as lipid rafts. Here, we investigated the role of palmitoylation of the CB₁ receptor by analysing the functional consequences of site-specific mutation of Cys⁴¹⁵, the likely site of palmitoylation at the end of helix 8, in terms of membrane association, raft targeting and signalling.

EXPERIMENTAL APPROACH

The palmitoylation state of CB₁ receptors in rat forebrain was assessed by depalmitoylation/repalmitoylation experiments. Cys⁴¹⁵ was replaced with alanine by site-directed mutagenesis. Green fluorescence protein chimeras of both wild-type and mutant receptors were transiently expressed and functionally characterized in SH-SY5Y cells and HEK-293 cells by means of confocal microscopy, cytofluorimetry and competitive binding assays. Confocal fluorescence recovery after photobleaching was used to assess receptor membrane dynamics, whereas signalling activity was assessed by [³⁵S]GTPγS, cAMP and co-immunoprecipitation assays.

KEY RESULTS

Endogenous CB₁ receptors in rat brain were palmitoylated. Mutation of Cys⁴¹⁵ prevented the palmitoylation of the receptor in transfected cells and reduced its recruitment to plasma membrane and lipid rafts; it also increased protein diffusional mobility. The same mutation markedly reduced the functional coupling of CB₁ receptors with G-proteins and adenylyl cyclase, whereas depalmitoylation abolished receptor association with a specific subset of G-proteins.

CONCLUSIONS AND IMPLICATIONS

CB₁ receptors were post-translationally modified by palmitoylation. Mutation of Cys⁴¹⁵ provides a receptor that is functionally impaired in terms of membrane targeting and signalling.

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     

Differential signalling in human cannabinoid CB1 receptors and their splice variants in autaptic hippocampal neurones

BACKGROUND AND PURPOSE

Cannabinoids such as Δ⁹- tetrahydrocannabinol, the major psychoactive component of marijuana and hashish, primarily act via cannabinoid CB₁ and CB₂ receptors to produce characteristic behavioural effects in humans. Due to the tractability of rodent models for electrophysiological and behavioural studies, most of the studies of cannabinoid receptor action have used rodent cannabinoid receptors. While CB₁ receptors are relatively well-conserved among mammals, human CB₁ (hCB₁) differs from rCB₁ and mCB₁ receptors at 13 residues, which may result in differential signalling. In addition, two hCB₁ splice variants (hCB_1a and hCB_1b) have been reported, diverging in their amino-termini relative to hCB₁ receptors. In this study, we have examined hCB₁ signalling in neurones.

EXPERIMENTAL APPROACH

hCB₁, hCB_1a hCB_1b or rCB₁ receptors were expressed in autaptic cultured hippocampal neurones from CB₁^−/− mice. Such cells express a complete endogenous cannabinoid signalling system. Electrophysiological techniques were used to assess CB₁ receptor-mediated signalling.

KEY RESULTS

Expressed in autaptic hippocampal neurones cultured from CB₁^−/− mice, hCB₁, hCB_1a and hCB_1b signal differentially from one another and from rodent CB₁ receptors. Specifically, hCB₁ receptors inhibit synaptic transmission less effectively than rCB₁ receptors.

CONCLUSIONS AND IMPLICATIONS

Our results suggest that cannabinoid receptor signalling in humans is quantitatively very different from that in rodents. As the problems of marijuana and hashish abuse occur in humans, our results highlight the importance of studying hCB₁ receptors. They also suggest further study of the distribution and function of hCB₁ receptor splice variants, given their differential signalling and potential impact on human health.

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 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     

So what do we call GPR18 now?

The further characterization of the orphan GPCR GPR18 conducted by McHugh et al. in this issue of the British Journal of Pharmacology has generated a pharmacological profile that raises some interesting questions about the nomenclature of this receptor and may also prompt some questions about the pharmacological definition of the classical cannabinoid receptors, CB₁ and CB₂.

LINKED ARTICLES

This article is a commentary on McHugh et al., pp. 2414–2424 of this issue and is part of a themed section on Cannabinoids in Biology and Medicine. To view McHugh et al. visit http://dx.doi.org/10.1111/j.1476-5381.2011.01497.x. 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     

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     

Cannabinoid CB1 receptors transactivate multiple receptor tyrosine kinases and regulate serine/threonine kinases to activate ERK in neuronal cells

BACKGROUND AND PURPOSE

Signalling networks that regulate the progression of cannabinoid CB₁ receptor-mediated extracellular signal-regulated kinase (ERK) activation in neurons are poorly understood. We investigated the cellular mechanisms involved in CB₁ receptor-stimulated ERK phosphorylation in a neuronal cell model.

EXPERIMENTAL APPROACH

Murine N18TG2 neuronal cells were used to analyse the effect of specific protein kinase and phosphatase inhibitors on CB₁ receptor-stimulated ERK phosphorylation. The LI-COR In Cell Western assay and immunoblotting were used to measure ERK phosphorylation.

KEY RESULTS

The time-course of CB₁ receptor-stimulated ERK activation occurs in three phases that are regulated by distinct cellular mechanisms in N18TG2 cells. Phase I (0–5 min) maximal ERK phosphorylation is mediated by CB₁ receptor-stimulated ligand-independent transactivation of multiple receptor tyrosine kinases (RTKs). Phase I requires G_i/oβγ subunit-stimulated phosphatidylinositol 3-kinase activation and Src kinase activation and is modulated by inhibition of cAMP-activated protein kinase A (PKA) levels. Src kinase activation is regulated by the protein tyrosine phosphatases 1B and Shp1. The Phase II (5–10 min) rapid decline in ERK phosphorylation involves PKA inhibition and serine/threonine phosphatase PP1/PP2A activation. The Phase III (>10 min) plateau in ERK phosphorylation is mediated by CB₁ receptor-stimulated, ligand-independent, transactivation of multiple RTKs.

CONCLUSIONS AND IMPLICATIONS

The complex expression of CB₁ receptor-stimulated ERK activation provides cellular selectivity, modulation of sensitivity to agonists, and coincidence detection with RTK signalling. RTK and PKA pathways may provide routes to novel CB₁-based therapeutic interventions in the treatment of addictive disorders or neurodegenerative diseases.

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     

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.     

Δ9-Tetrahydrocannabinol (Δ9-THC) attenuates mouse sperm motility and male fecundity

BACKGROUND AND PURPOSE

Numerous studies have shown that N-arachidonoylethanolamine (AEA) can inhibit sperm motility and function but the ability of cannabinoids to inhibit sperm motility is not well understood. We investigated the effects of WIN 55,212-2, a CB₁ cannabinoid receptor agonist, and Δ⁹-tetrahydracannabinol (Δ⁹-THC) on the ATP levels and motility of murine sperm in vitro. In addition, the effects of acute administration of Δ⁹-THC on male fecundity were determined.

EXPERIMENTAL APPROACH

Effects of Δ⁹-THC on basal sperm kinematics were determined using computer-assisted sperm analysis (CASA). Stop-motion imaging was performed to measure sperm beat frequency. The effect of Δ⁹-THC on sperm ATP was determined using a luciferase assay. Male fertility was determined by evaluating the size of litters sired by Δ⁹-THC-treated males.

KEY RESULTS

Pretreatment of sperm for 15 min with 1 µM Δ⁹-THC reduced their basal motility and attenuated the ability of bicarbonate to stimulate flagellar beat frequency. Treatment with 5 µM WIN 55,212-2 or 10 µM Δ⁹-THC for 30 min reduced sperm ATP levels. In sperm lacking CB₁ receptors this inhibitory effect of WIN 55,212-2 on ATP was attenuated whereas that of Δ⁹-THC persisted. Administration of 50 mg·kg⁻¹Δ⁹-THC to male mice just before mating caused a 20% decrease in embryonic litter size.

CONCLUSIONS AND IMPLICATIONS

Δ⁹-THC inhibits both basal and bicarbonate-stimulated sperm motility in vitro and reduces male fertility in vivo. High concentrations of WIN 55,212-2 or Δ⁹-THC inhibit ATP production in sperm; this effect of WIN 55,212-2 is CB₁ receptor-dependent whereas that of Δ⁹-THC is not.

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     

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     

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     

The endocannabinoid system in the rat dorsolateral periaqueductal grey mediates fear-conditioned analgesia and controls fear expression in the presence of nociceptive tone

BACKGROUND AND PURPOSE

Endocannabinoids in the midbrain periaqueductal grey (PAG) modulate nociception and unconditioned stress-induced analgesia; however, their role in fear-conditioned analgesia (FCA) has not been examined. The present study examined the role of the endocannabinoid system in the dorsolateral (dl) PAG in formalin-evoked nociceptive behaviour, conditioned fear and FCA in rats.

EXPERIMENTAL APPROACH

Rats received intra-dlPAG administration of the CB₁ receptor antagonist/inverse agonist rimonabant, or vehicle, before re-exposure to a context paired 24 h previously with foot shock. Formalin-evoked nociceptive behaviour and fear-related behaviours (freezing and 22 kHz ultrasonic vocalization) were assessed. In a separate cohort, levels of endocannabinoids [2-arachidonoyl glycerol (2-AG) and N-arachidonoyl ethanolamide (anandamide; AEA)] and the related N-acylethanolamines (NAEs) [N-palmitoyl ethanolamide (PEA) and N-oleoyl ethanolamide (OEA)] were measured in dlPAG tissue following re-exposure to conditioned context in the presence or absence of formalin-evoked nociceptive tone.

KEY RESULTS

Re-exposure of rats to the context previously associated with foot shock resulted in FCA. Intra-dlPAG administration of rimonabant significantly attenuated FCA and fear-related behaviours expressed in the presence of nociceptive tone. Conditioned fear without formalin-evoked nociceptive tone was associated with increased levels of 2-AG, AEA, PEA and OEA in the dlPAG. FCA was specifically associated with an increase in AEA levels in the dlPAG.

CONCLUSIONS AND IMPLICATIONS

Conditioned fear to context mobilises endocannabinoids and NAEs in the dlPAG. These data support a role for endocannabinoids in the dlPAG in mediating the potent suppression of pain responding which occurs during exposure to conditioned aversive contexts.

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     

Δ8-Tetrahydrocannabivarin prevents hepatic ischaemia/reperfusion injury by decreasing oxidative stress and inflammatory responses through cannabinoid CB2 receptors

BACKGROUND AND PURPOSE

Activation of cannabinoid CB₂ receptors protects against various forms of ischaemia-reperfusion (I/R) injury. Δ⁸-Tetrahydrocannabivarin (Δ⁸-THCV) is a synthetic analogue of the plant cannabinoid Δ⁹-tetrahydrocannabivarin, which exhibits anti-inflammatory effects in rodents involving activation of CB₂ receptors. Here, we assessed effects of Δ⁸-THCV and its metabolite 11-OH-Δ⁸-THCV on CB₂ receptors and against hepatic I/R injury.

EXPERIMENTAL APPROACH

Effects in vitro were measured with human CB₂ receptors expressed in CHO cells. Hepatic I/R injury was assessed in mice with 1h ischaemia and 2, 6 or 24h reperfusion in vivo.

KEY RESULTS

Displacement of [³H]CP55940 by Δ⁸-THCV or 11-OH-Δ⁸-THCV from specific binding sites in CHO cell membranes transfected with human CB₂ receptors (hCB₂) yielded K_i values of 68.4 and 59.95 nM respectively. Δ⁸-THCV or 11-OH-Δ⁸-THCV inhibited forskolin-stimulated cAMP production by hCB₂ CHO cells (EC₅₀= 12.95 and 14.3 nM respectively). Δ⁸-THCV, given before induction of I/R, attenuated hepatic injury (measured by serum alanine aminotransferase and aspartate aminotransferase levels), decreased tissue protein carbonyl adducts, 4-hydroxy-2-nonenal, the chemokines CCL3 and CXCL2,TNF-α, intercellular adhesion molecule 1 (CD54) mRNA levels, tissue neutrophil infiltration, caspase 3/7 activity and DNA fragmentation. Protective effects of Δ⁸-THCV against liver damage were still present when the compound was given at the beginning of reperfusion. Pretreatment with a CB₂ receptor antagonist attenuated the protective effects of Δ⁸-THCV, while a CB₁ antagonist tended to enhance it.

CONCLUSIONS AND IMPLICATIONS

Δ⁸-THCV activated CB₂ receptors in vitro, and decreased tissue injury and inflammation in vivo, associated with I/R partly via CB₂ receptor activation.

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     

The cannabinoid CB1 receptor antagonists rimonabant (SR141716) and AM251 directly potentiate GABA(A) receptors

BACKGROUND AND PURPOSE

Rimonabant (SR141716) and the structurally related AM251 are widely used in pharmacological experiments as selective cannabinoid receptor CB₁ antagonists / inverse agonists. Concentrations of 0.5–10 µM are usually applied in in vitro experiments. We intended to show that these drugs did not act at GABA_A receptors but found a significant positive allosteric modulation instead.

EXPERIMENTAL APPROACH

Recombinant GABA_A receptors were expressed in Xenopus oocytes. Receptors were exposed to AM251 or rimonabant in the absence and presence of GABA. Standard electrophysiological techniques were used to monitor the elicited ionic currents.

KEY RESULTS

AM251 dose-dependently potentiated responses to 0.5 µM GABA at the recombinant α₁β₂γ₂ GABA_A receptor with an EC₅₀ below 1 µM and a maximal potentiation of about eightfold. The Hill coefficient indicated that more than one binding site for AM251 was located in this receptor. Rimonabant had a lower affinity, but a fourfold higher efficacy. AM251 potentiated also currents mediated by α₁β₂, α_xβ₂γ₂ (x = 2,3,5,6), α₁β₃γ₂ and α₄β₂δ GABA_A receptors, but not those mediated by α₁β₁γ₂. Interestingly, the CB₁ receptor antagonists {"type":"entrez-nucleotide","attrs":{"text":"LY320135","term_id":"1257555575","term_text":"LY320135"}}LY320135 and O-2050 did not significantly affect α₁β₂γ₂ GABA_A receptor-mediated currents at concentrations of 1 µM.

CONCLUSIONS AND IMPLICATIONS

This study identified rimonabant and AM251 as positive allosteric modulators of GABA_A receptors. Thus, potential GABAergic effects of commonly used concentrations of these compounds should be considered in in vitro experiments, especially at extrasynaptic sites where GABA concentrations are low.

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     

Compartmentalization of endocannabinoids into lipid rafts in a microglial cell line devoid of caveolin-1

BACKGROUND AND PURPOSE

N-acyl ethanolamines (NAEs) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids and along with related lipids are synthesized on demand from membrane phospholipids. Here, we have studied the compartmentalization of NAEs and 2-AG into lipid raft fractions isolated from the caveolin-1-lacking microglial cell line BV-2, following vehicle or cannabidiol (CBD) treatment. Results were compared with those from the caveolin-1-positive F-11 cell line.

EXPERIMENTAL APPROACH

BV-2 cells were incubated with CBD or vehicle. Cells were fractionated using a detergent-free continuous OptiPrep density gradient. Lipids in fractions were quantified using HPLC/MS/MS. Proteins were measured using Western blot.

KEY RESULTS

BV-2 cells were devoid of caveolin-1. Lipid rafts were isolated from BV-2 cells as confirmed by co-localization with flotillin-1 and sphingomyelin. Small amounts of cannabinoid CB₁ receptors were found in lipid raft fractions. After incubation with CBD, levels and distribution in lipid rafts of 2-AG, N-arachidonoyl ethanolamine (AEA), and N-oleoyl ethanolamine (OEA) were not changed. Conversely, the levels of the saturated N-stearoyl ethanolamine (SEA) and N-palmitoyl ethanolamine (PEA) were elevated in lipid raft fractions. In whole cells with growth medium, CBD treatment increased AEA and OEA time-dependently, while levels of 2-AG, PEA and SEA did not change.

CONCLUSIONS AND IMPLICATIONS

Whereas levels of 2-AG were not affected by CBD treatment, the distribution and levels of NAEs showed significant changes. Among the NAEs, the degree of acyl chain saturation predicted the compartmentalization after CBD treatment suggesting a shift in cell signalling activity.

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     

Symptom-relieving and neuroprotective effects of the phytocannabinoid Δ⁹-THCV in animal models of Parkinson's disease

BACKGROUND AND PURPOSE

Previous findings have indicated that a cannabinoid, such as Δ⁹-THCV, which has antioxidant properties and the ability to activate CB₂ receptors but to block CB₁, might be a promising therapy for alleviating symptoms and delaying neurodegeneration in Parkinson''s disease (PD).

EXPERIMENTAL APPROACH

The ability of Δ⁹-THCV to reduce motor inhibition and provide neuroprotection was investigated in rats lesioned with 6-hydroxydopamine and in mice lesioned with lipopolysaccharide (LPS).

KEY RESULTS

Acute administration of Δ⁹-THCV attenuated the motor inhibition caused by 6-hydroxydopamine, presumably through changes in glutamatergic transmission. Moreover, chronic administration of Δ⁹-THCV attenuated the loss of tyrosine hydroxylase–positive neurones caused by 6-hydroxydopamine in the substantia nigra, through an effect related to its antioxidant properties (it was reproduced by cannabidiol -enriched botanical extract). In addition, CB₂ receptor–deficient mice responded to 6-hydroxydopamine in a similar manner to wild-type animals, and CB₂ receptors were poorly up-regulated in the rat substantia nigra in response to 6-hydroxydopamine. By contrast, the substantia nigra of mice that had been injected with LPS exhibited a greater up-regulation of CB₂ receptors. In these animals, Δ⁹-THCV also caused preservation of tyrosine hydroxylase–positive neurones. This effect probably involved CB₂ receptors as it was also elicited by the selective CB₂ receptor agonist, HU-308, and CB₂ receptor–deficient mice were more vulnerable to LPS lesions.

CONCLUSIONS AND IMPLICATIONS

Given its antioxidant properties and its ability to activate CB₂ but to block CB₁ receptors, Δ⁹-THCV has a promising pharmacological profile for delaying disease progression in PD and also for ameliorating parkinsonian symptoms.

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     

Imaging calcium signals in vivo: a powerful tool in physiology and pharmacology

The design and engineering of organic fluorescent Ca²⁺ indicators approximately 30 years ago opened the door for imaging cellular Ca²⁺ signals with a high degree of temporal and spatial resolution. Over this time, Ca²⁺ imaging has revolutionized our approaches for tissue-level spatiotemporal analysis of functional organization and has matured into a powerful tool for in situ imaging of cellular activity in the living animal. In vivo Ca²⁺ imaging with temporal resolution at the millisecond range and spatial resolution at micrometer range has been achieved through novel designs of Ca²⁺ sensors, development of modern microscopes and powerful imaging techniques such as two-photon microscopy. Imaging Ca²⁺ signals in ensembles of cells within tissue in 3D allows for analysis of integrated cellular function, which, in the case of the brain, enables recording activity patterns in local circuits. The recent development of miniaturized compact, fibre-optic-based, mechanically flexible microendoscopes capable of two-photon microscopy opens the door for imaging activity in awake, behaving animals. This development is poised to open a new chapter in physiological experiments and for pharmacological approaches in the development of novel therapies.

LINKED ARTICLES

This article is part of a themed section on Imaging. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2011.163.issue-8BJP has previously published an Imaging in Pharmacology themed section, edited by A Davenport and C Daly. To view this section visit http://dx.doi.org/10.1111/bph.2010.159.issue-4     

Δ(9) -Tetrahydrocannabinol and N-arachidonyl glycine are full agonists at GPR18 receptors and induce migration in human endometrial HEC-1B cells

BACKGROUND AND PURPOSE

Endometriosis is a disorder in which the endometrium forms growths outside the uterus and is associated with chronic pain. Recent evidence suggests that endometrial motility plays a role in the aetiology of endometriosis. The endocannabinoid system regulates cellular migration. Given the growing involvement of the endocannabinoids in reproduction, we investigated the role of the endocannabinoid system in migration of endometrial cells.

EXPERIMENTAL APPROACH

Migration of the human endometrial HEC-1B cells was assayed. Standard PCR techniques were used to determine the presence of the GPCR, GPR18, in HEC-1B cells, and p44/42 MAPK was assayed in stably transfected HEK293-GPR18 cells to determine receptor specificity for known cannabinoid agonists and antagonists. N-arachidonoyl ethanolamine (AEA) metabolism was measured, using HPLC/MS/MS for lipid analysis.

KEY RESULTS

AEA, Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and N-arachidonoyl glycine (NAGly) induce migration of HEC-1B cells through cannabinoid CB₁ receptor-independent mechanisms. MAPK activation in HEK293-GPR18 cells revealed novel pharmacology for known CB₁ and CB₂ receptor ligands at GPR18 receptors, including Δ⁹-THC, which activates MAPK at nanomolar concentrations, whereas WIN 55212-2, CP55940, JWH-133 and JWH-015, and arachidonyl-1-hydroxy-2-propylamide (R1-methanandamide) had no effect. Moreover, HEC-1B migration and MAPK activation by NAGly and Δ⁹-THC were antagonized by Pertussis toxin, AM251 and cannabidiol.

CONCLUSIONS AND IMPLICATIONS

An understanding of the function and regulation of GPR18 and its molecular interactions with endogenous ligands, and how phytocannabinoids play a role with GPR18 signalling is vital if we are to comprehensively assess the function of the cannabinoid signalling system in human health and disease.

LINKED ARTICLES

This article is commented on by Alexander, pp. 2411–2413 of this issue and is part of a themed section on Cannabinoids in Biology and Medicine. To view Alexander visit http://dx.doi.org/10.1111/j.1476-5381.2011.01731.x. 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     

The monoacylglycerol lipase inhibitor JZL184 attenuates LPS-induced increases in cytokine expression in the rat frontal cortex and plasma: differential mechanisms of action

Background and purpose

JZL184 is a selective inhibitor of monoacylglycerol lipase (MAGL), the enzyme that preferentially catabolizes the endocannabinoid 2-arachidonoyl glycerol (2-AG). Here, we have studied the effects of JZL184 on inflammatory cytokines in the brain and plasma following an acute immune challenge and the underlying receptor and molecular mechanisms involved.

Experimental approach

JZL184 and/or the CB₁ receptor antagonist, AM251 or the CB₂receptor antagonist, AM630 were administered to rats 30 min before lipopolysaccharide (LPS). 2 h later cytokine expression and levels, MAGL activity, 2-AG, arachidonic acid and prostaglandin levels were measured in the frontal cortex, plasma and spleen.

Key results

JZL184 attenuated LPS-induced increases in IL-1β, IL-6, TNF-α and IL-10 but not the expression of the inhibitor of NFkB (IκBα) in rat frontal cortex. AM251 attenuated JZL184-induced decreases in frontal cortical IL-1β expression. Although arachidonic acid levels in the frontal cortex were reduced in JZL184-treated rats, MAGL activity, 2-AG, PGE₂ and PGD₂ were unchanged. In comparison, MAGL activity was inhibited and 2-AG levels enhanced in the spleen following JZL184. In plasma, LPS-induced increases in TNF-α and IL-10 levels were attenuated by JZL184, an effect partially blocked by AM251. In addition, AM630 blocked LPS-induced increases in plasma IL-1β in the presence, but not absence, of JZL184.

Conclusion and implications

Inhibition of peripheral MAGL in rats by JZL184 suppressed LPS-induced circulating cytokines that in turn may modulate central cytokine expression. The data provide further evidence for the endocannabinoid system as a therapeutic target in treatment of central and peripheral inflammatory disorders.

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.2013.169.issue-4 & http://dx.doi.org/10.1111/bph.2012.167.issue-8