Capsaicin in the periaqueductal gray induces analgesia via metabotropic glutamate receptor-mediated endocannabinoid retrograde disinhibition

BACKGROUND AND PURPOSE

Capsaicin, an agonist of transient receptor potential vanilloid 1 (TRPV1) channels, is pro-nociceptive in the periphery but is anti-nociceptive when administered into the ventrolateral periaqueductal gray (vlPAG), a midbrain region for initiating descending pain inhibition. Here, we investigated how activation of TRPV1 channels in the vlPAG leads to anti-nociception.

EXPERIMENTAL APPROACH

We examined synaptic transmission and neuronal activity using whole-cell recordings in vlPAG slices in vitro and hot-plate nociceptive responses in rats after drug microinjection into the vlPAG in vivo.

KEY RESULTS

Capsaicin (1–10 µM) depressed evoked GABAergic inhibitory postsynaptic currents (eIPSCs) in vlPAG slices presynaptically, while increasing miniature excitatory PSC frequency. Capsaicin-induced eIPSC depression was antagonized by cannabinoid CB₁ and metabotropic glutamate (mGlu₅) receptor antagonists, and prevented by inhibiting diacylglycerol lipase (DAGL), which converts DAG into 2-arachidonoylglycerol (2-AG), an endocannabinoid. Capsaicin induced membrane depolarization in 2/3 neurons recorded but, overall, increased neuronal firings by increasing evoked postsynaptic potentials. Intra-vlPAG capsaicin reduced hot-plate responses in rats, effects blocked by CB₁ and mGlu receptor antagonists. Effects of capsaicin were antagonized by SB 366791, a TRPV1 channel antagonist.

CONCLUSIONS AND IMPLICATIONS

Capsaicin activated TRPV1s on glutamatergic terminals to release glutamate which activated postsynaptic mGlu₅ receptors, yielding 2-AG from DAG by DAGL hydrolysis. 2-AG induces retrograde inhibition (disinhibition) of GABA release via presynaptic CB₁ receptors. This disinhibition in the vlPAG leads to anti-nociception by activating the descending pain inhibitory pathway. This is a novel TRPV1 channel-mediated anti-nociceptive mechanism in the brain and a new interaction between vanilloid and endocannabinoid systems.     

Cannabidiol improves brain and liver function in a fulminant hepatic failure-induced model of hepatic encephalopathy in mice

BACKGROUND AND PURPOSE

Hepatic encephalopathy is a neuropsychiatric disorder of complex pathogenesis caused by acute or chronic liver failure. We investigated the effects of cannabidiol, a non-psychoactive constituent of Cannabis sativa with anti-inflammatory properties that activates the 5-hydroxytryptamine receptor 5-HT_1A, on brain and liver functions in a model of hepatic encephalopathy associated with fulminant hepatic failure induced in mice by thioacetamide.

EXPERIMENTAL APPROACH

Female Sabra mice were injected with either saline or thioacetamide and were treated with either vehicle or cannabidiol. Neurological and motor functions were evaluated 2 and 3 days, respectively, after induction of hepatic failure, after which brains and livers were removed for histopathological analysis and blood was drawn for analysis of plasma liver enzymes. In a separate group of animals, cognitive function was tested after 8 days and brain 5-HT levels were measured 12 days after induction of hepatic failure.

KEY RESULTS

Neurological and cognitive functions were severely impaired in thioacetamide-treated mice and were restored by cannabidiol. Similarly, decreased motor activity in thioacetamide-treated mice was partially restored by cannabidiol. Increased plasma levels of ammonia, bilirubin and liver enzymes, as well as enhanced 5-HT levels in thioacetamide-treated mice were normalized following cannabidiol administration. Likewise, astrogliosis in the brains of thioacetamide-treated mice was moderated after cannabidiol treatment.

CONCLUSIONS AND IMPLICATIONS

Cannabidiol restores liver function, normalizes 5-HT levels and improves brain pathology in accordance with normalization of brain function. Therefore, the effects of cannabidiol may result from a combination of its actions in the liver and 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.     

Novel selective cannabinoid CB(1) receptor antagonist MJ08 with potent in vivo bioactivity and inverse agonistic effects

Aim:

To characterize the biological profiles of MJ08, a novel selective CB₁ receptor antagonist.

Methods:

Radioligand binding assays were performed using rat brain and spleen membrane preparations. CB₁ and CB₂ receptor redistribution and intracellular Ca²⁺ ([Ca²⁺]_i) assays were performed with IN CELL Analyzer. Inverse agonism was studied using intracellular cAMP assays, and in guinea-pig ileum and mouse vas deferens smooth muscle preparations. In vivo pharmacologic profile was assessed in diet-induced obesity (DIO) mice.

Results:

In radioligand binding assay, MJ08 selectively antagonized CB₁ receptor (IC₅₀=99.9 nmol/L). In EGFP-CB₁_U2OS cells, its IC₅₀ value against CB₁ receptor activation was 30.23 nmol/L (SR141716A: 32.16 nmol/L). WIN 55,212-2 (1 μmol/L) increased [Ca²⁺]_i in the primary cultured hippocampal neuronal cells and decreased cAMP accumulation in CHO-hCB₁ cells. MJ08 (10 nmol/L–10 μmol/L) blocked both the WIN 55,212-2-induced effects. Furthermore, MJ08 reversed the inhibition of electrically evoked twitches of mouse vas deferens by WIN 55,212-2 (pA₂=10.29±1.05). MJ08 and SR141716A both showed an inverse agonism activity by markedly promoting the contraction force and frequency of guinea pig ileum muscle. MJ08 significantly increased the cAMP level in CHO-hCB₁ cells with an EC₅₀ value of 78.6 nmol/L, which was lower than the EC₅₀ value for SR141716A (159.2 nmol/L). Besides the more potent pharmacological effects of cannabinoid CB₁ receptor antagonism in DIO mice, such as reducing food intake, decreasing body weight, and ameliorating dyslipidemia, MJ08 (10 mg/kg) unexpectedly raised the fasted blood glucose in vivo.

Conclusion:

MJ08 is a novel, potent and selective CB₁ receptor antagonist/inverse agonist with potent bioactive responses in vitro and in vivo that may be useful for disclosure the versatile nature of CB₁ receptors.     

Hypotensive effect of anandamide through the activation of CB<Subscript>1</Subscript> and VR<Subscript>1</Subscript> spinal receptors in urethane-anesthetized rats

This study examined the effect of intrathecal (i.t.) injection of the endocannabinoid anandamide in urethane-anesthetized rats. The tip of the i.t. cannula was positioned at the T₁₂–L₁ level of the spinal cord. Either anandamide or its metabolically stable analogue methanandamide (25 to 100 nmol) produced dose-dependent decreases in the blood pressure that persisted at least for up to 30 min. The hypotensive responses to 100 nmol anandamide and to 100 nmol methanandamide were –17.7±1.6 mmHg (n=5) and –17.9±2.0 mmHg (n=4), respectively. Hypotensive effects were also obtained with the CB₁ cannabinoid receptor agonist WIN 55212-2 (20 nmol; i.t.) as well as with the vanilloid VR₁ receptor agonist capsaicin (3 nmol; i.t.). Nicotinic ganglionic blockade with hexamethonium bromide [10 mg/kg; intravenous(i.v.)] abolished the responses to both anandamide and capsaicin. The i.t. administration of the CB₁ receptor antagonist, 20 nmol SR 141716A, as well as the VR₁ receptor antagonist, 20 nmol capsazepine, prevented almost completely the hypotensive responses to both anandamide and methanandamide. SR 141716A prevented the hypotension caused by WIN 55212-2 but did not modify the response to the vanilloid receptor agonist capsaicin. On the contrary, capsazepine antagonized the hypotension caused by capsaicin but failed to affect the decrease in blood pressure caused by the CB1 cannabinoid receptor agonist WIN 55212-2. These results suggest that anandamide could modulate the blood pressure through the activation of cannabinoid CB₁ receptors and vanilloid VR₁ receptors localized at the spinal cord.     

Spinal and peripheral analgesic effects of the CB2 cannabinoid receptor agonist AM1241 in two models of bone cancer-induced pain

Background and purpose:

The activation of CB₂ receptors induces analgesia in experimental models of chronic pain. The present experiments were designed to study whether the activation of peripheral or spinal CB₂ receptors relieves thermal hyperalgesia and mechanical allodynia in two models of bone cancer pain.

Experimental approach:

NCTC 2472 osteosarcoma or B16-F10 melanoma cells were intratibially inoculated to C3H/He and C57BL/6 mice. Thermal hyperalgesia was assessed by the unilateral hot plate test and mechanical allodynia by the von Frey test. AM1241 (CB₂ receptor agonist), AM251 (CB₁ receptor antagonist), SR144528 (CB₂ receptor antagonist) and naloxone were used. CB₂ receptor expression was measured by Western blot.

Key results:

AM1241 (0.3–10 mg·kg⁻¹) abolished thermal hyperalgesia and mechanical allodynia in both tumour models. The antihyperalgesic effect was antagonized by subcutaneous, intrathecal or peri-tumour administration of SR144528. In contrast, the antiallodynic effect was inhibited by systemic or intrathecal, but not peri-tumour, injection of SR144528. The effects of AM1241 were unchanged by AM251 but were prevented by naloxone. No change in CB₂ receptor expression was found in spinal cord or dorsal root ganglia.

Conclusions and implications:

Spinal CB₂ receptors are involved in the antiallodynic effect induced by AM1241 in two neoplastic models while peripheral and spinal receptors participate in the antihyperalgesic effects. Both effects were mediated by endogenous opiates. The use of drugs that activate CB₂ receptors could be a useful strategy to counteract bone cancer-induced pain symptoms.     

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.     

Rimonabant (SR141716) induces metabolism and acquisition of fertilizing ability in human sperm

Background and purpose:

The endocannabinoid system and the cannabinoid CB₁ receptor have been identified in human sperm, and it is well known that endocannabinoids have pronounced adverse effects on male and female reproduction. In order to elucidate further the pathophysiological role of the endocannabinoid system in male fertility, we investigated the activity of the CB₁ receptor antagonist rimonabant (SR141716) on the fertilizing ability of human sperm.

Experimental approach:

We evaluated in vitro the effects of rimonabant on motility, survival, capacitation, acrosin activity and metabolism of human sperm. Particularly, capacitation was studied by using three different approaches: intracellular free Ca²⁺ content assay, cholesterol efflux assay and protein tyrosine phosphorylation analysis.

Key results:

Rimonabant significantly increased sperm motility and viability through the induction of pAkt and pBcl2, key proteins of cell survival and metabolism, and it induced acrosome reaction and capacitation as well. Rimonabant reduced the triglyceride content of sperm, while enhancing lipase and acyl-CoA dehydrogenase activities, implying an overall lipolytic action in these cells. Rimonabant also affected sperm glucose metabolism by decreasing phosphorylation of glycogen synthase kinase 3 and increasing glucose-6-phosphate dehydrogenase activity, suggesting a role in inducing sperm energy expenditure. Intriguingly, agonism at the CB₁ receptor, with an anandamide analogue or a selective inhibitor of fatty acid amide hydrolase, produced opposing effects on human sperm functions.

Conclusions and implications:

Our data suggest that blockade of the CB₁ receptor by rimonabant induces the acquisition of fertilizing ability and stimulates energy expenditure in human sperm.     

Inhibitory activity of the novel CB2 receptor agonist, GW833972A, on guinea-pig and human sensory nerve function in the airways

Background and purpose:

Sensory nerves regulate central and local reflexes such as airway plasma protein leakage, bronchoconstriction and cough. Sensory nerve activity may be enhanced during inflammation such that these protective effects become exacerbated and deleterious. Cannabinoids are known to inhibit airway sensory nerve function. However, there is still controversy surrounding which receptor is involved in eliciting these effects.

Experimental approach:

We have adopted a pharmacological approach, including using a novel, more selective CB₂ receptor agonist, GW 833972A (1000-fold selective CB₂/CB₁), and receptor selective antagonists to investigate the inhibitory activity of cannabinoids on sensory nerve activity in vitro and in vivo in guinea-pig models of cough and plasma extravasation.

Key results:

GW 833972A inhibited capsaicin-induced depolarization of the human and guinea-pig and prostaglandin E₂ (PGE₂) and hypertonic saline-induced depolarization of the guinea-pig isolated vagus nerve in vitro. GW 833972A also inhibited citric acid-induced cough but not plasma extravasation in the guinea-pig and this effect was blocked by a CB₂ receptor antagonist.

Conclusions and implications:

This confirms and extends previous studies highlighting the role of CB₂ receptors in the modulation of sensory nerve activity elicited both by the exogenous ligands capsaicin and hypertonic saline but also by endogenous modulators such as PGE₂ and low pH stimuli. These data establish the CB₂ receptor as an interesting target for the treatment of chronic cough.     

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.     

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.     

Effect of Δ9-tetrahydrocannabinol,a cannabinoid receptor agonist,on the triggering of transient lower oesophageal sphincter relaxations in dogs and humans

Background and purpose

Transient lower oesophageal sphincter relaxations (TLESRs) are the main mechanism underlying gastro-oesophageal reflux and are a potential pharmacological treatment target. We evaluated the effect of the CB₁/CB₂ receptor agonist Δ⁹-tetrahydrocannabinol (Δ⁹-THC) on TLESRs in dogs. Based on these findings, the effect of Δ⁹-THC was studied in healthy volunteers.

Experimental approach

In dogs, manometry was used to evaluate the effect of Δ⁹-THC in the presence and absence of the CB₁ receptor antagonist SR141716A on TLESRs induced by gastric distension. Secondly, the effect of 10 and 20 mg Δ⁹-THC was studied in 18 healthy volunteers in a placebo-controlled study. Manometry was performed before and for 3 h after meal ingestion on three occasions.

Key results

In dogs, Δ⁹-THC dose-dependently inhibited TLESRs and reduced acid reflux rate. SR141716A significantly reversed the effects of Δ⁹-THC on TLESRs. Similarly, in healthy volunteers, Δ⁹-THC significantly reduced the number of TLESRs and caused a non-significant reduction of acid reflux episodes in the first postprandial hour. In addition, lower oesophageal sphincter pressure and swallowing were significantly reduced by Δ⁹-THC. After intake of 20 mg, half of the subjects experienced nausea and vomiting leading to premature termination of the study. Other side-effects were hypotension, tachycardia and central effects.

Conclusions and implications

Δ⁹-THC significantly inhibited the increase in meal-induced TLESRs and reduced spontaneous swallowing in both dogs and humans. In humans, Δ⁹-THC significantly reduced basal lower oesophageal sphincter pressure. These findings confirm previous observations in dogs and indicate that cannabinoid receptors are also involved in the triggering of TLESRs in humans.     

The endocannabinoid system in renal cells: regulation of Na+ transport by CB1 receptors through distinct cell signalling pathways

Background and Purpose

The function of the endocannabinoid system (ECS) in renal tissue is not completely understood. Kidney function is closely related to ion reabsorption in the proximal tubule, the nephron segment responsible for the re-absorption of 70–80% of the filtrate. We studied the effect of compounds modulating the activity of cannabinoid (CB) receptors on the active re-absorption of Na⁺ in LLC-PK1 cells.

Experimental Approach

Changes in Na⁺/K⁺-ATPase activity were assessed after treatment with WIN55,212-2 (WIN), a non-selective lipid agonist, and haemopressin (HP), an inverse peptide agonist at CB₁ receptors. Pharmacological tools were used to investigate the signalling pathways involved in the modulation of Na⁺ transport.

Key Results

In addition to CB₁ and CB₂ receptors and TRPV1 channels, the mRNAs encoding for enzymes of the ECS were also expressed in LLC-PK1. WIN (10⁻⁷ M) and HP (10⁻⁶ M) altered Na⁺ re-absorption in LLC-PK1 in a dual manner. They both acutely (after 1 min) increased Na⁺/K⁺-ATPase activity in a TRPV1 antagonist-sensitive way. WIN''s stimulating effect persisted for 30 min, and this effect was partially blocked by a CB₁ antagonist or a PKC inhibitor. In contrast, HP inhibited Na⁺/K⁺-ATPase after 30 min incubation, and this effect was attenuated by a CB₁ antagonist or a PKA inhibitor.

Conclusion and Implications

The ECS is expressed in LLC-PK1 cells. Both CB₁ receptors and TRPV1 channels regulate Na⁺/K⁺-ATPase activity in these cells, and are modulated by lipid and peptide CB₁ receptor ligands, which act via different signalling pathways.     

Tetrahydrocannabinolic acid reduces nausea-induced conditioned gaping in rats and vomiting in Suncus murinus

BACKGROUND AND PURPOSE

We evaluated the anti-emetic and anti-nausea properties of the acid precursor of Δ⁹-tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), and determined its mechanism of action in these animal models.

EXPERIMENTAL APPROACH

We investigated the effect of THCA on lithium chloride- (LiCl) induced conditioned gaping (nausea-induced behaviour) to a flavour, and context (a model of anticipatory nausea) in rats, and on LiCl-induced vomiting in Suncus murinus. Furthermore, we investigated THCA''s ability to induce hypothermia and suppress locomotion [rodent tasks to assess cannabinoid₁ (CB₁) receptor agonist-like activity], and measured plasma and brain THCA and THC levels. We also determined whether THCA''s effect could be blocked by pretreatment with SR141716 (SR, a CB₁ receptor antagonist).

KEY RESULTS

In rats, THCA (0.05 and/or 0.5 mg·kg⁻¹) suppressed LiCl-induced conditioned gaping to a flavour and context; the latter effect blocked by the CB₁ receptor antagonist, SR, but not by the 5-hydroxytryptamine-1A receptor antagonist, WAY100635. In S. murinus, THCA (0.05 and 0.5 mg·kg⁻¹) reduced LiCl-induced vomiting, an effect that was reversed with SR. A comparatively low dose of THC (0.05 mg·kg⁻¹) did not suppress conditioned gaping to a LiCl-paired flavour or context. THCA did not induce hypothermia or reduce locomotion, indicating non-CB₁ agonist-like effects. THCA, but not THC was detected in plasma samples.

CONCLUSIONS AND IMPLICATIONS

THCA potently reduced conditioned gaping in rats and vomiting in S. murinus, effects that were blocked by SR. These data suggest that THCA may be a more potent alternative to THC in the treatment of nausea and vomiting.     

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     

AM630 behaves as a protean ligand at the human cannabinoid CB2 receptor

BACKGROUND AND PURPOSE

We have investigated how pre-incubating hCB₂ CHO cells with the CB₂ receptor antagonists/inverse agonists, AM630 and SR144528, affects how these and other ligands target hCB₂ receptors in these cells or their membranes.

EXPERIMENTAL APPROACH

We tested the ability of AM630, SR144528 and of the CB₁/CB₂ receptor agonists, CP55940 and R-(+)-WIN55212, to modulate forskolin-stimulated cAMP production in hCB₂ CHO cells or [³⁵S]-GTPγS binding to membranes prepared from these cells, or to displace [³H]-CP55940 from whole cells and membranes. Assays were also performed with the CB₂ receptor partial agonist, Δ⁹-tetrahydrocannabivarin. Some cells were pre-incubated with AM630 or SR144528 and then washed extensively.

KEY RESULTS

AM630 behaved as a low-potency neutral competitive antagonist in AM630-pre-incubated cells, a low-potency agonist in SR144528-pre-incubated cells, and a much higher-potency inverse agonist/antagonist in vehicle-pre-incubated cells. AM630 pre-incubation (i) reduced the inverse efficacy of SR144528 without abolishing it; (ii) increased the efficacy of Δ⁹-tetrahydrocannabivarin; and (iii) did not affect the potency with which AM630 displaced [³H]-CP55940 from whole cells or its inverse agonist potency and efficacy in the [³⁵S]-GTPγS membrane assay.

CONCLUSIONS AND IMPLICATIONS

These results suggest that AM630 is a protean ligand that can target a constitutively active form of the hCB₂ receptor (R*) with low affinity to produce agonism or neutral antagonism and a constitutively inactive form of this receptor (R) with much higher affinity to produce inverse agonism, and that the constitutive activity of whole cells is decreased less by pre-incubation with AM630 than with the higher-efficacy inverse agonist, SR144528.

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     

Effect of the CB1 receptor antagonists rimonabant and AM251 on the firing rate of dorsal raphe nucleus neurons in rat brain slices

Background and purpose:

Previous studies have suggested a regulation of 5-hydroxytryptamine (5-HT) neurons by the endocannabinoid system. The aim of our work was to examine the effect of two CB₁ receptor antagonists, SR141716A (rimonabant, Sanofi-Synthélabo Recherche, Montpellier, France) and N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251, Tocris Cookson, Bristol, UK), on the firing rate of dorsal raphe nucleus (DRN) neurons.

Experimental approach:

Single-unit extracellular recordings were performed to study the effect of CB₁ receptor antagonists in slices of the DRN from rat brain.

Key results:

Rimonabant (1 µM) and AM251 (1 µM) decreased the firing rate of about 50% of all the recorded DRN 5-HT cells. The GABA_Areceptor antagonist picrotoxin (20 µM) (Sigma) prevented and also reversed the inhibitory effect of rimonabant (1 µM) and AM251 (1 µM), suggesting that CB₁ receptors regulate 5-HT neurons through the GABAergic system. However, the CB₁/CB₂ receptor agonist R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)-methyl]pyrrolol[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate salt (10 µM) (WIN55212-2, Sigma, St. Louis, MO, USA) failed to change the firing activity of non-5-HT (presumably GABAergic) neurons in the DRN. The endocannabinoid N-(2-hydroxyethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (anandamide, Tocris Cookson) (10 µM) also inhibited the firing activity of a number of 5-HT neurons, but this inhibition was not blocked by rimonabant (1 µM) or AM251 (1 µM), and the stable analogue R-(+) N-(2-hydroxy-1methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (methanandamide, Tocris Cookson) (10 µM) did not mimic this effect. The selective CB₁ receptor agonist arachidonoyl-2-chloroethylamide (ACEA) (1 µM) only slightly increased the firing rate of DRN 5-HT cells.

Conclusions and implications:

These results suggest a tonic/constitutive regulation of DRN 5-HT neurons by the endocannabinoid system, which may occur through a CB₁ receptor-mediated inhibition of the GABAergic system. The inhibitory effect of anandamide may be mediated through a CB₁ receptor-independent mechanism.     

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.     

Endocannabinoid modulation of hyperaemia evoked by physiologically relevant stimuli in the rat primary somatosensory cortex

Background and purpose:

In vitro studies demonstrate that cannabinoid CB₁ receptors subserve activity-dependent suppression of inhibition in the neocortex. To examine this mechanism in vivo, we assessed the effects of local changes in CB₁ receptor activity on somatosensory cortex neuronal activation by whisker movement in rats.

Experimental approach:

Laser Doppler flowmetry and c-Fos immunohistochemistry were used to measure changes in local blood flow and neuronal activation, respectively. All drugs were applied directly to the cranium above the whisker barrel fields of the primary somatosensory cortex.

Key results:

The CB₁ receptor agonist WIN55212-2 potentiated the hyperaemia induced by whisker movement and this potentiation was occluded by bicuculline. The CB₁ receptor antagonists, rimonabant and AM251, inhibited hyperaemic responses to whisker movement; indicating that activation of endogenous CB₁ receptors increased during whisker movement. Whisker movement-induced expression of c-Fos protein in neurons of the whisker barrel cortex was inhibited by rimonabant. Movement of the whiskers increased the 2-arachidonoylglycerol content in the contralateral, compared to the ipsilateral, sensory cortex.

Conclusions and implications:

These results support the hypothesis that endocannabinoid signalling is recruited during physiologically relevant activation of the sensory cortex. These data support the hypothesis that the primary effect of CB₁ receptor activation within the activated whisker barrel cortex is to inhibit GABA release, resulting in disinhibition of neuronal activation. These studies provide physiological data involving endocannabinoid signalling in activity-dependent regulation of neuronal activation and provide a mechanistic basis for the effects of cannabis use on sensory processing in humans.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     

Cannabinoid-1 receptor activation induces reactive oxygen species-dependent and -independent mitogen-activated protein kinase activation and cell death in human coronary artery endothelial cells

Background and purpose:

Impaired endothelial activity and/or cell death play a critical role in the development of vascular dysfunction associated with congestive heart failure, diabetic complications, hypertension, coronary artery disease and atherosclerosis. Increasing evidence suggests that cannabinoid 1 (CB₁) receptor inhibition is beneficial in atherosclerosis and cardiovascular inflammation both in experimental models, as well as in humans. Here, we investigated the effects of CB₁ receptor activation with the endocannabinoid anandamide (AEA) or synthetic agonist HU210 on cell death and interrelated signal transduction pathways in human primary coronary artery endothelial cells (HCAECs).

Experimental approach:

Cell death, CB₁ receptor expression, reactive oxygen species (ROS) generation and activation of signal transduction pathways in HCAECs were determined by flow cytometry and molecular biology tools.

Key results:

In HCAECs expressing CB₁ receptors (demonstrated by Western immunoblot and flow cytometry) AEA (5–15 µM) or HU210 (30–1000 nM) triggered concentration- and time-dependent activation of p38 and c-Jun NH₂-terminal protein kinase (JNK)–mitogen-activated protein kinases (MAPKs), cell death and ROS generation. The AEA- or HU210-induced cell death and MAPK activation were attenuated by CB₁ antagonists [SR141716 (rimonabant) and AM281], inhibitors of p38 and JNK–MAPKs or the antioxidant N-acetylcysteine. N-acetylcysteine alone prevented AEA- or HU210-induced ROS generation, but only partially attenuated MAPK activation and cell death. In contrast, in combination with CB₁ antagonists, N-acetylcysteine completely prevented these effects.

Conclusions and implications:

CB₁ receptor activation in endothelial cells may amplify the ROS–MAPK activation–cell death pathway in pathological conditions when the endocannabinoid synthetic or metabolic pathways are dysregulated by excessive inflammation and/or oxidative/nitrosative stress, thereby contributing to the development of endothelial dysfunction and pathophysiology of multiple cardiovascular diseases.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