Endocannabinoids modulate human blood–brain barrier permeability in vitro

Background and Purpose

Endocannabinoids alter permeability at various epithelial barriers, and cannabinoid receptors and endocannabinoid levels are elevated by stroke, with potential neuroprotective effects. We therefore explored the role of endocannabinoids in modulating blood–brain barrier (BBB) permeability in normal conditions and in an ischaemia/reperfusion model.

Experimental Approach

Human brain microvascular endothelial cell and astrocyte co-cultures modelled the BBB. Ischaemia was modelled by oxygen-glucose deprivation (OGD) and permeability was measured by transepithelial electrical resistance. Endocannabinoids or endocannabinoid-like compounds were assessed for their ability to modulate baseline permeability or OGD-induced hyperpermeability. Target sites of action were investigated using receptor antagonists and subsequently identified with real-time PCR.

Key Results

Anandamide (10 μM) and oleoylethanolamide (OEA, 10 μM) decreased BBB permeability (i.e. increased resistance). This was mediated by cannabinoid CB₂ receptors, transient receptor potential vanilloid 1 (TRPV1) channels, calcitonin gene-regulated peptide (CGRP) receptor (anandamide only) and PPARα (OEA only). Application of OEA, palmitoylethanolamide (both PPARα mediated) or virodhamine (all 10 μM) decreased the OGD-induced increase in permeability during reperfusion. 2-Arachidonoyl glycerol, noladin ether and oleamide did not affect BBB permeability in normal or OGD conditions. N-arachidonoyl-dopamine increased permeability through a cytotoxic mechanism. PPARα and γ, CB₁ receptors, TRPV1 channels and CGRP receptors were expressed in both cell types, but mRNA for CB₂ receptors was only present in astrocytes.

Conclusion and Implication

The endocannabinoids may play an important modulatory role in normal BBB physiology, and also afford protection to the BBB during ischaemic stroke, through a number of target sites.     

Fluorescent ligand binding reveals heterogeneous distribution of adrenoceptors and ‘cannabinoid-like’ receptors in small arteries

Background and purpose:

Pharmacological analysis of synergism or functional antagonism between different receptors commonly assumes that interacting receptors are located in the same cells. We have now investigated the distribution of α-adrenoceptors, β-adrenoceptors and cannabinoid-like (GPR55) receptors in the mouse arteries.

Experimental approach:

Fluorescence intensity from vascular tissue incubated with fluorescent ligands (α₁-adrenoceptor ligand, BODIPY-FL-prazosin, QAPB; β-adrenoceptor ligand, TMR-{"type":"entrez-protein","attrs":{"text":"CGP12177","term_id":"877152897","term_text":"CGP12177"}}CGP12177; fluorescent angiotensin II; a novel diarylpyrazole cannabinoid ligand (Tocrifluor 1117, T1117) was measured with confocal microscopy. Small mesenteric and tail arteries of wild-type and α_1B/D-adrenoceptor-KO mice were used.

Key results:

T1117, a fluorescent form of the cannabinoid CB₁ receptor antagonist AM251, was a ligand for GPR55, with low affinity for CB₁ receptors. In mesenteric arterial smooth muscle cells, α_1A-adrenoceptors were predominantly located in different cells from those with β-adrenoceptors, angiotensin receptors or cannabinoid-like (GPR55) receptors. Cells with β-adrenoceptors predominated at arterial branches. Endothelial cells expressed β-adrenoceptors, α-adrenoceptors and cannabinoid-like receptors. Only endothelial α-adrenoceptors appeared in clusters. Adventitia was a rich source of G protein-coupled receptors (GPCRs), particularly fibroblasts and nerve tracts, where Schwann cells bound α-adrenoceptor, β-adrenoceptor and CB-receptor ligands, with a mix of separate receptor locations and co-localization.

Conclusions and implications:

Within each cell type, each GPCR had a distinctive heterogeneous distribution with limited co-localization, providing a guide to the possibilities for functional synergism, and suggesting a new paradigm for synergism in which interactions may be either between cells or involve converging intracellular signalling processes.This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x     

Evaluation of the insulin releasing and antihyperglycaemic activities of GPR55 lipid agonists using clonal beta-cells,isolated pancreatic islets and mice

Background and Purpose

G-protein coupled receptor (GPR)55 is a novel lipid sensing receptor activated by both cannabinoid endogenous ligands (endocannabinoids) and other non-cannabinoid lipid transmitters. This study assessed the effects of various GPR55 agonists on glucose homeostasis.

Experimental Approach

Insulin secretion and changes in intracellular Ca²⁺ and cAMP in response to glucose and a range of GPR55 agonists [endogenous ligands (OEA, PEA), chemically synthetic cannabidiol (CBD) analogues (Abn-CBD, 0–1602), an analogue of rimonabant (AM-251) and antagonist (CBD)] were investigated in clonal BRIN-BD11 cells and mouse pancreatic islets. Cytotoxicity was assessed by LDH release, cellular localization by double-staining immunohistochemistry and in vivo effects assessed in mice.

Key Results

The most potent and selective GPR55 agonist was the synthetic CBD analogue, Abn-CBD (pEC₅₀ 10.33), maximum stimulation of 67% at 10⁻⁴ mol·L⁻¹ (P < 0.001) in BRIN-BD11 cells. AM-251 (pEC₅₀ 7.0), OEA (pEC₅₀ 7.0), 0–1602 (pEC₅₀ 7.3) and PEA (pEC₅₀ 6.0) stimulated insulin secretion. Results were corroborated by islet studies, with no cytotoxic effects. Concentration-dependent insulin secretion by GPR55 agonists was glucose-sensitive and accompanied by elevations of [Ca²⁺]_i (P < 0.01–P < 0.001) and cAMP (P < 0.05–P < 0.01). GPR55 agonists exhibited insulinotropic and glucose lowering activity in vivo. GPR55 was expressed on BRIN-BD11 cells and confined to islet beta cells with no distribution on alpha cells.

Conclusion and Implications

These results demonstrate GPR55 is distributed in pancreatic beta cells and is a strong activator of insulin secretion, with glucose-lowering effects in vivo. Development of agents agonizing the GPR55 receptor may have therapeutic potential in the treatment of type 2 diabetes.     

Interaction between cannabinoid CB1 receptors and endogenous ATP in the control of spontaneous mechanical activity in mouse ileum

Background and purpose:

Although it is well accepted that cannabinoids modulate intestinal motility by reducing cholinergic neurotransmission mediated by CB₁ receptors, it is not known whether the endocannabinoids are involved in more complex circuits and if they interact with other systems. The aim of the present study was to examine possible interactions between cannabinoid CB₁ receptors and purines in the control of spontaneous contractility of longitudinal muscle in mouse ileum.

Experimental approach:

The mechanical activity of longitudinally oriented ileal segments from mice was recorded as isometric contractions.

Key results:

The selective CB₁ receptor agonist, N-(2-chloroethyl)5,8,11,14-eicosaetraenamide (ACEA) reduced, concentration dependently, spontaneous contractions in mouse ileum. This effect was almost abolished by tetrodotoxin (TTX) or atropine. Inhibition by ACEA was not affected by theophylline (P1 receptor antagonist) or by P2Y receptor desensitization with adenosine 5′[β-thio]diphosphate trilithium salt, but was significantly reversed by pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (P2 receptor antagonist), by P2X receptor desensitization with α,β-methyleneadenosine 5′-triphosphate lithium salt (α,β-MeATP) or by 8,8′-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino) bis(1,3,5-naphthalenetrisulphonic acid)] (P2X receptor antagonist). Contractile responses to α,β-MeATP (P2X receptor agonist) were virtually abolished by TTX or atropine, suggesting that they were mediated by acetylcholine released from neurones, and significantly reduced by ACEA.

Conclusion and implications:

In mouse ileum, activation of CB₁ receptors, apart from reducing acetylcholine release from cholinergic nerves, was able to modulate negatively, endogenous purinergic effects, mediated by P2X receptors, on cholinergic neurons. Our study provides evidence for a role of cannabinoids in the modulation of interneuronal purinergic transmission.     

Brain RVD-haemopressin,a haemoglobin-derived peptide,inhibits bombesin-induced central activation of adrenomedullary outflow in the rat

BACKGROUND AND PURPOSE

Haemopressin and RVD-haemopressin, derived from the haemoglobin α-chain, are bioactive peptides found in brain and are ligands for cannabinoid CB₁ receptors. Activation of brain CB₁ receptors inhibited the secretion of adrenal catecholamines (noradrenaline and adrenaline) induced by i.c.v. bombesin in the rat. Here, we investigated the effects of two haemoglobin-derived peptides on this bombesin-induced response

EXPERIMENTAL APPROACH

Anaesthetised male Wistar rats were pretreated with either haemoglobin-derived peptide, given i.c.v., 30 min before i.c.v. bombesin and plasma catecholamines were subsequently measured electrochemically after HPLC. Direct effects of bombesin on secretion of adrenal catecholamines were examined using bovine adrenal chromaffin cells. Furthermore, activation of haemoglobin α-positive spinally projecting neurons in the rat hypothalamic paraventricular nucleus (PVN, a regulatory centre of central adrenomedullary outflow) after i.c.v. bombesin was assessed by immunohistochemical techniques.

KEY RESULTS

Bombesin given i.c.v. dose-dependently elevated plasma catecholamines whereas incubation with bombesin had no effect on spontaneous and nicotine-induced secretion of catecholamines from chromaffin cells. The bombesin-induced increase in catecholamines was inhibited by pretreatment with i.c.v. RVD-haemopressin (CB₁ receptor agonist) but not after pretreatment with haemopressin (CB₁ receptor inverse agonist). Bombesin activated haemoglobin α-positive spinally projecting neurons in the PVN.

CONCLUSIONS AND IMPLICATIONS

The haemoglobin-derived peptide RVD-haemopressin in the brain plays an inhibitory role in bombesin-induced activation of central adrenomedullary outflow via brain CB₁ receptors in the rat. These findings provide basic information for the therapeutic use of haemoglobin-derived peptides in the modulation of central adrenomedullary outflow.     

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.     

Pioglitazone ameliorates memory deficits in streptozotocin-induced diabetic mice by reducing brain β-amyloid through PPARγ activation

Aim:

To examine the effects of pioglitazone, a PPARγ agonist, on memory performance and brain amyloidogenesis in streptozotocin (STZ)-induced diabetic mice.

Methods:

ICR male mice were injected with STZ (150 mg/kg, iv) to induce experimental diabetes. Pioglitazone (9 and 18 mg·kg^-1·d^-1, po) was administered for 6 weeks. Passive avoidance and Morris water maze (MWM) tests were used to evaluate cognitive function. The blood glucose and serum insulin levels were detected using the glucose oxidase method and an ELISA assay, respectively. β-amyloid (Aβ), β-amyloid precursor protein (APP), β-amyloid precursor protein cleaving enzyme 1 (BACE1), NF-κB p65, the receptor for advanced glycation end products (RAGE) and PPARγ in the brains were analyzed using Western blotting assays.

Results:

The STZ-induced diabetic mice characterized by hyperglycemia and hypoinsulinemia performed poorly in both the passive avoidance and MWM tests, accompanied by increased Aβ_1–40/Aβ_1–42, APP, BACE1, NF-κB p65 and RAGE levels and decreased PPARγ level in the hippocampus and cortex. Chronic pioglitazone treatment significantly ameliorated the memory deficits and amyloidogenesis of STZ-induced diabetic mice, and suppressed expression of APP, BACE1, RAGE and NF-κB p65, and activated PPARγ in the hippocampus and cortex. However, pioglitazone did not significantly affect blood glucose and insulin levels.

Conclusion:

Pioglitazone ameliorates memory deficits in STZ-induced diabetic mice by reducing brain Aβ level via activation of PPARγ, which is independent of its effects on blood glucose and insulin levels. The results suggest that pioglitazone may be used for treating the cognitive dysfunction in type 1 diabetes mellitus.     

Endocannabinoid-like N-arachidonoyl serine is a novel pro-angiogenic mediator

BACKGROUND AND PURPOSE

N-arachidonoyl serine (ARA-S) is a recently identified endocannabinoid-like lipid with weak affinity for the fully characterized cannabinoid receptors (CB₁ and CB₂) and the transient receptor potential vanilloid receptor 1 (TRPV-1). ARA-S induces vasodilatation and shows vasoprotective potential via activation of key signalling pathways in endothelial cells. Based on these findings, the effect of ARA-S on endothelial functions was further studied.

EXPERIMENTAL APPROACH

Primary human dermal microvascular endothelial cells (HMVEC) were used to investigate effects of ARA-S (0–10 µM) on certain endothelial functions, using cell proliferation, migration and wound repair models in vitro, and angiogenesis assays in vitro and ex vivo. Selective CB receptor antagonists and specific siRNAs were deployed to block individual CB receptors.

KEY RESULTS

We found that ARA-S stimulated angiogenesis and endothelial wound healing through induction of vascular endothelial growth factor C and its cognate receptor expression in primary HMVEC. Moreover, knock-down of G protein-coupled receptor 55 (GPR55) partly inhibited ARA-S-induced signal transduction and endothelial functions.

CONCLUSIONS AND IMPLICATIONS

Our results indicate that ARA-S is a pro-angiogenic factor in addition to a vessel dilator. The GPR55 receptor may serve as one target of ARA-S.     

Cannabinoid CB(2) receptor attenuates morphine-induced inflammatory responses in activated microglial cells

BACKGROUND AND PURPOSE

Among several pharmacological properties, analgesia is the most common feature shared by either opioid or cannabinoid systems. Cannabinoids and opioids are distinct drug classes that have been historically used separately or in combination to treat different pain states. In the present study, we characterized the signal transduction pathways mediated by cannabinoid CB₂ and µ-opioid receptors in quiescent and LPS-stimulated murine microglial cells.

EXPERIMENTAL APPROACH

We examined the effects of µ-opioid and CB₂ receptor stimulation on phosphorylation of MAPKs and Akt and on IL-1β, TNF-α, IL-6 and NO production in primary mouse microglial cells.

KEY RESULTS

Morphine enhanced release of the proinflammatory cytokines, IL-1β, TNF-α, IL-6, and of NO via µ-opioid receptor in activated microglial cells. In contrast, CB₂ receptor stimulation attenuated morphine-induced microglial proinflammatory mediator increases, interfering with morphine action by acting on the Akt-ERK1/2 signalling pathway.

CONCLUSIONS AND IMPLICATIONS

Because glial activation opposes opioid analgesia and enhances opioid tolerance and dependence, we suggest that CB₂ receptors, by inhibiting microglial activity, may be potential targets to increase clinical efficacy of opioids.     

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     

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.     

Tobacco smoke affects expression of peroxisome proliferator-activated receptor-γ in monocyte/macrophages of patients with coronary heart disease

Background and purpose:

Tobacco smoke represents a relevant risk factor for coronary heart disease (CHD). Although peroxisome proliferator-activated receptor (PPAR)γ activation reduces inflammation and atherosclerosis, expression of PPARγ in cells and its modulation by smoking are poorly investigated. We previously reported that monocyte/macrophages from healthy smokers exhibited an enhanced constitutive expression of PPARγ. Here, we evaluated PPARγ expression and basal cytokine release in monocytes and monocyte-derived macrophages (MDMs) from 85 CHD patients, classified by their smoking habit (smokers, non-smokers and ex-smokers), and assessed the role of PPARγ ligands in this context.

Experimental approach:

PPARγ protein was detected by Western blot and semi-quantified by PPARγ/β-actin ratio; cytokine release was measured by elisa and nuclear factor-kappaB (NF-κB) translocation by electrophoretic mobility shift assays.

Key results:

As compared to the other groups, MDMs from smoker CHD patients exhibited a reduced PPARγ/β-actin ratio and an increased spontaneous release of tumour necrosis factor-α (TNF-α) and interleukin-6, but with no major variations in monocytes. In cells from selected CHD patients, rosiglitazone inhibited TNF-α release and NF-κB translocation induced by phorbol-12-myristate 13-acetate. The selective PPARγ antagonist GW9662 reversed these effects, with some variations related to smoking habit.

Conclusions and implications:

In CHD patients, exposure to tobacco smoke profoundly affected PPARγ expression, and this was related to levels of secretion of pro-inflammatory cytokines. MDMs from CHD smokers showed the lowest PPARγ expression and released more inflammatory cytokines. Moreover, rosiglitazone''s ability to inhibit cytokine release and its reversal by GW9662 clearly indicated PPARγ involvement in these changes in CHD patients.     

β-Amyloid exacerbates inflammation in astrocytes lacking fatty acid amide hydrolase through a mechanism involving PPAR-α, PPAR-γ and TRPV1, but not CB₁ or CB₂ receptors

BACKGROUND AND PURPOSE

The endocannabinoid system may regulate glial cell functions and their responses to pathological stimuli, specifically, Alzheimer''s disease. One experimental approach is the enhancement of endocannabinoid tone by blocking the activity of degradative enzymes, such as fatty acid amide hydrolase (FAAH).

EXPERIMENTAL APPROACH

We examined the role of FAAH in the response of astrocytes to the pathologic form of β-amyloid (Aβ). Astrocytes from wild-type mice (WT) and from mice lacking FAAH (FAAH-KO) were incubated with Aβ for 8, 24 and 48 h, and their inflammatory responses were quantified by elisa, western-blotting and real-time quantitative-PCR.

KEY RESULTS

FAAH-KO astrocytes were significantly more responsive to Aβ than WT astrocytes, as shown by the higher production of pro-inflammatory cytokines. Expression of COX-2, inducible NOS and TNF-α was also increased in Aβ-exposed KO astrocytes compared with that in WTs. These effects were accompanied by a differential pattern of activation of signalling cascades involved in mediating inflammatory responses, such as ERK1/2, p38MAPK and NFκ B. PPAR-α and PPAR-γ as well as transient receptor potential vanilloid-1 (TRPV1), but not cannabinoid CB₁ or CB₂ receptors, mediate some of the differential changes observed in Aβ-exposed FAAH-KO astrocytes. The pharmacological blockade of FAAH did not render astrocytes more sensitive to Aβ. In contrast, exogenous addition of several acylethanolamides (anandamide, palmitoylethanolamide and oleoylethanolamide) induced an antiinflammatory response.

CONCLUSIONS

The genetic deletion of FAAH in astrocytes exacerbated their inflammatory phenotype against Aβ in a process involving PPAR-α, PPAR-γ and TRPV1 receptors.     

Urethane,but not pentobarbitone,attenuates presynaptic receptor function in rats: a contribution to the choice of anaesthetic

Background and purpose:

We examined whether cannabinoid CB₁ and histamine H₃ receptors resemble α₂-adrenoceptors in that their presynaptically mediated cardiovascular effects are less marked in urethane- than in pentobarbitone-anaesthetized pithed rats.

Experimental approach:

Effects of the cannabinoid agonist CP-55,940 and the H₃ receptor agonist imetit on electrically induced tachycardic and vasopressor responses, respectively, was compared in pithed rats anaesthetized with urethane or pentobarbitone. The affinity of urethane for the three receptors was measured by radioligand binding studies in rat brain cortex membranes and its potency assessed in superfused mouse tissues preincubated with ³H-noradrenaline.

Key results:

The neurogenic tachycardic response was less markedly inhibited by CP-55,940 in urethane- than in pentobarbitone-anaesthetized pithed rats. Imetit inhibited the neurogenic vasopressor response after pentobarbitone but not after urethane. The catecholamine-induced tachycardic and vasopressor response did not differ between rats anaesthetized with either compound. Urethane 10 mM (plasma concentration reached under anaesthesia) did not affect binding to CB₁ or H₃ receptors and α₂ adrenoceptors, nor did it alter the inhibitory effect of agonists at the three receptors on electrically evoked ³H-noradrenaline release.

Conclusions and implications:

Urethane, but not pentobarbitone, abolished the H₃ receptor-mediated vascular response in pithed rats and attenuated the CB₁ receptor-mediated cardiac response much more than pentobarbitone. The weaker effects of CB₁, H₃ and α₂ receptor agonists cannot be explained by antagonism by urethane at the three receptors in vitro. Pentobarbitone, but not urethane, is suitable as an anaesthetic for investigations of inhibitory presynaptic receptor function in pithed and anaesthetized rats.     

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

Background and purpose:

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

Experimental approach:

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

Key results:

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

Conclusions and implications:

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