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PubMed2000

Modulation of peristalsis by cannabinoid CB(1) ligands in the isolated guinea-pig ileum.

The effect of cannabinoid drugs on peristalsis in the guinea-pig ileum was studied. Peristalsis was induced by delivering fluid into the oral end of an isolated intestinal segment. Longitudinal muscle reflex contraction, threshold pressure and threshold volume to trigger peristalsis, compliance of the intestinal wall during the preparatory phase (a reflection of the resistance of the wall to distension) and maximal ejection pressure during the emptying phase of peristalsis were measured. The cannabinoid agonists WIN 55,212-2 (0.3 - 300 nM) and CP55,940 (0.3 - 300 nM) significantly decreased longitudinal muscle reflex contraction, compliance and maximal ejection pressure, while increased threshold pressure and volume to elicit peristalsis. These effects were not modified by the opioid antagonist naloxone (1 microM) and by the alpha-adrenoceptor antagonist phentolamine (1 microM). The inhibitory effect of both WIN 55,212-2 and CP55,940 on intestinal peristalsis was antagonized by the cannabinoid CB(1) receptor antagonist SR141716A (0.1 microM), but not by the cannabinoid CB(2) receptor antagonist SR144528 (0.1 microM). In absence of other drugs, the CB(1) receptor antagonists SR141716A (0.01 - 1 microM) and AM281 (0.01 - 1 microM) slightly (approximatively 20%) but significantly increased maximal ejection pressure during the empty phase of peristalsis without modifying longitudinal muscle reflex contraction, threshold pressure, threshold volume to trigger peristalsis and compliance. It is concluded that activation of CB(1) receptors reduces peristalsis efficiency in the isolated guinea-pig, and that the emptying phase of peristalsis could be tonically inhibited by the endogenous cannabinoid system.

Animals↗
PubMed2000

O-1057, a potent water-soluble cannabinoid receptor agonist with antinociceptive properties.

Cannabinoids have low water solubility, necessitating the use of a solubilizing agent. In this paper we investigated whether a novel water-soluble cannabinoid, 3-(5'-cyano-1', 1'-dimethylpentyl)-1-(4-N-morpholinobutyryloxy)-Delta(8)- tetrahydroca nnabinol hydrochloride (O-1057), would interact with cannabinoid receptors when water or saline were used as the only vehicle. O-1057 displaced [(3)H]-CP55940 from specific binding sites on Chinese hamster ovary (CHO) cell membranes expressing CB(1) or CB(2) cannabinoid receptors, with pK(i) values of 8.36 and 7.95 respectively. It also displaced [(3)H]-CP55940 from specific binding sites on rat brain membranes (pK(i) = 7.86). O-1057 inhibited forskolin-stimulated cyclic AMP production by both CB(1)- and CB(2)-transfected CHO cells (pEC(50) = 9.16 and 9.72 respectively), its potency matching that of CP55940 and exceeding that of Delta(9)-tetrahydrocannabinol. In the mouse isolated vas deferens, O-1057 inhibited electrically-evoked contractions with pEC(50) and E(max) values of 9.73 and 76.84% respectively. It was antagonized by 100 nM SR141716A, the pK(B) of SR141716A against O-1057 (8.90) approximating to that against CP55940 (8.97). O-1057 also behaved as a CB(1) receptor agonist in vivo, reducing mouse spontaneous activity and rectal temperature when injected intravenously and inducing antinociception in the mouse tail flick test when given intravenously (ED(50) = 0.02 mg kg(-1)), intrathecally, intracerebroventricularly or by gavage. In all these assays, O-1057 was more potent than Delta(9)-tetrahydrocannabinol and, at 0.1 mg kg(-1) i.v., was antagonized by SR141716A (3 mg kg(-1) i.v.). These data demonstrate the ability of the water-soluble cannabinoid, O-1057, to act as a potent agonist at CB(1) and CB(2) receptors and warrant investigation of the clinical potential of O-1057 as an analgesic.

Analgesics↗
PubMed2000

Characterization of the effects of cannabinoids on guinea-pig tracheal smooth muscle tone: role in the modulation of acetylcholine release from parasympathetic nerves.

We investigated the ability of the cannabinoid agonists CP55,940 (CB(1)/CB(2)) and anandamide (endogenous cannabinoid) to modulate electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. We assessed whether modulation of transmitter release translated to an impact on functional responses by investigating the effect of these agents on contractile responses evoked by EFS and ACh. Furthermore, we evaluated the ability of these compounds to elicit bronchodilation in pre-contracted guinea-pig tracheal strips. CP55,940 and anandamide significantly inhibited EFS-evoked ACh release (maximal inhibition of 35.1+/-2.9% and 33.4+/-6.4% at 1 microM, P<0.05, respectively). The CB(1) receptor antagonist SR 141716A (1 microM), had no effect on ACh release and failed to reverse the inhibitory effect of CP55,940 (1 microM). Paradoxically, CP55,940 had no significant effect on EFS-evoked cholinergic contractile responses. Furthermore, CP55,940 did not relax pre-contracted tracheal strips or affect contractile responses to exogenous ACh. This lack of activity on smooth muscle tone is consistent with the fact that no detectable specific binding of [(3)H] CP55,940 was found in tracheal homogenates. These data suggest that cannabinoid agonists inhibit ACh release from cholinergic nerve terminals via activation of CB(2) receptors but that this inhibitory action does not impact on functional responses such as cholinergic contraction.

Acetylcholine↗
PubMed2001

Separation of cannabinoid receptor affinity and efficacy in delta-8-tetrahydrocannabinol side-chain analogues.

1. The activities of a number of side-chain analogues of delta-8-tetrahydrocannabinol (Delta(8)-THC) in rat cerebellar membrane preparations were tested. 2. The affinities of each compound for the CB(1) receptor were compared by their respective abilities to displace [(3)H]-SR141716A and their efficacies compared by stimulation of [(35)S]-GTPgammaS binding. 3. It was found that the affinities varied from 0.19+/-0.03 nM for 3-norpentyl-3-[6'-cyano,1',1'-dimethyl]hexyl-Delta(8)-THC to 395+/-66.3 nM for 5'-[N-(4-chlorophenyl)]-1',1'-dimethyl-carboxamido-Delta(8)-THC. 4. The efficacies of these compounds varied greatly, ranging from the very low efficacy exhibited to acetylenic compounds such as 1'-heptyn-Delta(8)-THC and 4'-octyn-Delta(8)-THC to higher efficacy compounds such as 5'-(4-cyanophenoxy)-1',1'-dimethyl-Delta(8)-THC and 5'-[N-(4-aminosulphonylphenyl)]-1',1' dimethyl-carboxamido Delta(8)-THC. All agonist activities were antagonized by the CB(1)-selective antagonist SR141716A. 5. It was found that a ligand's CB(1) affinity and efficacy are differentially altered by modifications in the side-chain. Decreasing the flexibility of the side-chain reduced efficacy but largely did not alter affinity. Additionally, the positioning of electrostatic moieties, such as cyano groups, within the side-chain also has contrasting effects on these two properties. 6. In summary, this report details the characterization of a number of novel Delta(8)-THC analogues in rat cerebellar membranes. It provides the first detailed pharmacological analysis of how the inclusion of electrostatic moieties in the side-chain and also how alteration of the side-chain's flexibility may differentially affect a CB(1) cannabinoid receptor ligand's affinity and efficacy.

Animals↗
PubMed2001

Pharmacological analysis of cannabinoid receptor activity in the rat vas deferens.

1. The interaction between the cannabinoid agonists, WIN 55,212-2 or CP 55,940 with the CB(1) receptor-selective antagonists, SR141716A or LY320135 was investigated using the rat electrically-stimulated vas deferens bioassay. 2. Tissues were stimulated by single-field pulses (150 V, 0.5 ms) delivered every 30 mins. In the presence of nifedipine (3 microM), agonists elicited a concentration-dependent inhibition of the contractile response, with pEC(50) values of 7.93 and 6.84 for WIN 55,212-2 and CP 55,940, respectively. 3. SR141716A and LY320135 caused parallel dextral displacements of the agonist concentration-response curves. However, the shift of the agonist curves by either antagonist was accompanied by a concentration-dependent enhancement of basal (agonist-independent) tissue contraction. 4. Addition of the amidase inhibitor, phenylmethylsulphonylfluoride (200 microM), resulted in a significant reduction of the basal twitch response, an effect consistent with the presence of tonic receptor activation mediated by the endogenous cannabinoid, anandamide. 5. In light of these findings, we propose a theoretical model of competitive agonist-antagonist interaction in the presence of endogenous agonist tone that was used to derive an optimized analytical approach for the determination of antagonist potency estimates under conditions of tonic receptor activation. 6. This approach yielded pK(B) estimates for SR141716A and LY320135 that were in good agreement with their activity at cannabinoid CB(1) receptors. 7. It is concluded that the rat vas deferens contains prejunctional cannabinoid CB(1) receptors that are under tonic activation from endogenous substances; under these conditions our analytical approach is preferable to the standard methods for the determination of antagonist potency.

Animals↗
PubMed2001

Differential effects of anandamide on acetylcholine release in the guinea-pig ileum mediated via vanilloid and non-CB1 cannabinoid receptors.

1. The effects of anandamide on [3H]-acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [3H]-choline. 2. Anandamide increased both basal [3H]-acetylcholine release (pEC(50) 6.3) and muscle tone (pEC(50) 6.3). The concentration-response curves for anandamide were shifted to the right by 1 microM capsazepine (pK(B) 7.5 and 7.6), and by the combined blockade of NK1 and NK3 tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 microM). The CB1 and CB2 receptor antagonists, SR141716A (1 microM) and SR144528 (30 nM), did not modify the facilitatory effects of anandamide. 3. Anandamide inhibited the electrically-evoked release of [3H]-acetylcholine (pEC(50) 5.8) and contractions (pEC(50) 5.2). The contractile response to the muscarinic agonist methacholine was not significantly affected by 10 microM anandamide. 4. The inhibitory effects of anandamide were not changed by either capsazepine (1 microM), SR144528 (30 nM) or CP99994 plus SR142801 (each 0.1 microM). SR141716A (1 microM) produced rightward shifts in the inhibitory concentration-response curves for anandamide yielding pK(B) values of 6.6 and 6.2. 5. CP55940 inhibited the evoked [3H]-acetylcholine release and contractions, and SR141716A (0.1 microM) shifted the concentration-response curves of CP55940 to the right with pK(B) values of 8.4 and 8.9. 6. The experiments confirm the existence of release-inhibitory CB1 receptors on cholinergic myenteric neurones. We conclude that anandamide inhibits the evoked acetylcholine release via stimulation of a receptor that is different from the CB1 and CB2 receptor. Furthermore, anandamide increases basal acetylcholine release via stimulation of vanilloid receptors located at primary afferent fibres.

Acetylcholine↗
PubMed2001

A possible role of lipoxygenase in the activation of vanilloid receptors by anandamide in the guinea-pig bronchus.

1. In the absence of indomethacin, anandamide did not contract the guinea-pig bronchus at concentrations up to 100 microM. In the presence of indomethacin (10 microM), anandamide induced concentration-related contractions with a pEC(50) value of 5.18+/-0.11. It was significantly less potent than capsaicin (pEC(50) 7.01+/-0.1). The anandamide uptake inhibitor AM404, produced only a 14.1+/-3.22% contraction at 100 microM. All experiments were conducted in the presence of PMSF (20 microM). 2. The vanilloid receptor antagonist, capsazepine (10 microM), significantly attenuated the contractile effect of anandamide, the response to 100 microM anandamide being 40.53+/-7.04% in the presence of vehicle and 1.57+/-8.93% in the presence of 10 microM capsazepine. The contractile actions of anandamide and AM404 were markedly enhanced by the peptidase inhibitor thiorphan. 3. The log concentration-response curve of anandamide was unaltered by the CB1 receptor antagonist, SR141716A. The pEC(50) values for anandamide were 4.88+/-0.08 and 5.17+/-0.19 in the presence of vehicle and SR141716A (1 microM) respectively. 4. The lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA) and 5,8,11 eicosatriynoic acid (ETI) reduced the effect of 100 microM anandamide from 34.7+/-1.9% (vehicle) to 7.7+/-5% (ETYA, 10 microM) and from 41.85+/-4.25% (n=6) (vehicle) to 10.31+/-3.54 (n=6) (ETI, 20 microM). Neither inhibitor significantly affected contraction of the tissue by substance P. 5. This study provides evidence that anandamide acts on vanilloid receptors in the guinea-pig isolated bronchus. These data raise the possibility that the contractile action of anandamide may be due, at least in part, to lipoxygenase metabolites of this fatty acid amide that are vanilloid receptor agonists.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed2001

Cannabinoid CB1-receptor mediated regulation of gastrointestinal motility in mice in a model of intestinal inflammation.

1. We have studied the effect of cannabinoid agonists (CP 55,940 and cannabinol) on intestinal motility in a model of intestinal inflammation (induced by oral croton oil in mice) and measured cannabinoid receptor expression, endocannabinoids (anandamide and 2-arachidonylglycerol) and anandamide amidohydrolase activity both in physiological and pathophysiological states. 2. CP 55,940 (0.03 - 10 nmol mouse(-1)) and cannabinol (10 - 3000 nmol mouse(-1)) were more active in delaying intestinal motility in croton oil-treated mice than in control mice. These inhibitory effects were counteracted by the selective cannabinoid CB(1) receptor antagonist SR141716A (16 nmol mouse(-1)). SR141716A (1 - 300 nmol mouse(-1)), administered alone, increased intestinal motility to the same extent in both control and croton oil-treated mice. 3. Croton oil-induced intestinal inflammation was associated with an increased expression of CB(1) receptor, an unprecedented example of up-regulation of cannabinoid receptors during inflammation. 4. High levels of anandamide and 2-arachidonylglycerol were detected in the small intestine, although no differences were observed between control and croton oil-treated mice; by contrast anandamide amidohydrolase activity increased 2 fold in the inflamed small intestine. 5. It is concluded that inflammation of the gut increases the potency of cannabinoid agonists possibly by 'up-regulating' CB(1) receptor expression; in addition, endocannabinoids, whose turnover is increased in inflamed gut, might tonically inhibit intestinal motility.

Analgesics↗
PubMed2001

Effects of cannabinoids on adrenaline release from adrenal medullary cells.

1. The objective of the present study was to analyse the peripheral effects of cannabinoids on adrenaline release from adrenal chromaffin cells. 2. In pithed rabbits with electrically stimulated sympathetic outflow, intravenous injection of the cannabinoid receptor agonists WIN55212-2 and CP55940 (5, 50 and 500 microg x kg(-1)) markedly lowered the plasma adrenaline concentration. The effect of WIN55212-2 was attenuated by the selective CB1 cannabinoid receptor antagonist SR141716A (500 microg x kg(-1)). WIN55212-3 (same doses as WIN55212-2), the enantiomer of WIN55212-2 lacking affinity for cannabinoid receptors, had no effect on the plasma adrenaline concentration. 3. In rabbit isolated adrenal glands, the release of adrenaline elicited by electrical stimulation was measured by fast cyclic voltammetry. Electrically-evoked adrenaline release was inhibited by WIN55212-2 (0.3, 1, 3 and 10 microM) and this effect was antagonized by SR141716A (1 microM). The non-cholinergic component of adrenaline release observed after blockade of nicotinic (by hexamethonium 100 microM) and muscarinic (by atropine 0.5 microM) acetylcholine receptors was not depressed by WIN55212-2. WIN55212-3 (10 microM) had no effect on adrenaline release. 4. No detectable specific CB1 receptor binding and mRNA expression were found in rabbit adrenal glands with autoradiography and in situ hybridization. 5. The results show that cannabinoids inhibit adrenaline secretion in rabbit isolated adrenal glands; the likely mechanism is a presynaptic CB1 receptor-mediated inhibition of acetylcholine release from preganglionic sympathetic neurons. The inhibition of adrenaline secretion in adrenal glands most probably accounts for the decrease in the plasma adrenaline concentration observed after cannabinoid administration in pithed rabbits.

Adrenal Glands↗
PubMed2002

Role of the endocannabinoid system in MDMA intracerebral self-administration in rats.

I.c.v. self-administration of MDMA (0.01-2 micro g per infusion), alone and in combination with CP 55,940 (0.4 micro g infusion(-1)), was studied on an operant responding procedure. On the basis of individual preference for one of two levers, developed during training, rats were allowed to self-administer vehicle from the preferred lever and MDMA from the other. Pressings on the MDMA associated-lever, except for the maximal unit dose, progressively increased. The combination of CP 55,940 with MDMA (1 micro g infusion(-1)) reduced the number of drug-associated lever pressings compared to the single drugs. Pre-treatment with SR 141716A (0.5 mg kg(-1) i.p.), 15 min before each daily session, significantly increased MDMA self-administration. These findings suggest that MDMA self-administration is under endogenous tonic control by the endocannabinoid system.

Animals↗
PubMed2003

Inhibition of guinea-pig and human sensory nerve activity and the cough reflex in guinea-pigs by cannabinoid (CB2) receptor activation.

1. There is considerable interest in novel therapies for cough, since currently used agents such as codeine have limited beneficial value due to the associated side effects. Sensory nerves in the airways mediate the cough reflex via activation of C-fibres and RARs. Evidence suggests that cannabinoids may inhibit sensory nerve-mediated responses. 2. We have investigated the inhibitory actions of cannabinoids on sensory nerve depolarisation mediated by capsaicin, hypertonic saline and PGE2 on isolated guinea-pig and human vagus nerve preparations, and the cough reflex in conscious guinea-pigs. 3. The non-selective cannabinoid (CB) receptor agonist, CP 55940, and the selective CB2 agonist, JWH 133 inhibited sensory nerve depolarisations of the guinea-pig vagus nerve induced by hypertonic saline, capsaicin and PGE2. These responses were abolished by the CB2 receptor antagonist SR144528, and unaffected by the CB1 antagonist SR141716A. Similarly, JWH 133 inhibited capsaicin-evoked nerve depolarisations in the human vagus nerve, and was prevented by SR144528. 4. Using a guinea-pig in vivo model of cough, JWH 133 (10 mg kg-1, i.p., 20 min) significantly reduced citric acid-induced cough in conscious guinea pigs compared to those treated with the vehicle control. 5. These data show that activation of the CB2 receptor subtype inhibits sensory nerve activation of guinea-pig and human vagus nerve, and the cough reflex in guinea-pigs, suggesting that the development of CB2 agonists, devoid of CB1-mediated central effects, will provide a new and safe antitussive treatment for chronic cough.

Animals↗
PubMed2003

Effects of acute treatment with paroxetine, citalopram and venlafaxine in vivo on noradrenaline and serotonin outflow: a microdialysis study in Swiss mice.

1. This study investigated whether a single administration of a range of doses (1, 4 and 8 mg kg-1, i.p.) of paroxetine, citalopram or venlafaxine may simultaneously increase extracellular levels of 5-HT ([5-HT]ext) and noradrenaline ([NA]ext) by using in vivo microdialysis in the frontal cortex (FCx) of awake, freely moving Swiss mice. 2. In vivo, paroxetine induced similar increases in cortical [5-HT]ext at the three doses tested, and induced a statistically significant increase in cortical [NA]ext at 4 and 8 mg x kg-1. Citalopram increased neither [5-HT]ext nor [NA]ext at the lowest dose, but increased both neurotransmitter levels at 4 and 8 mg x kg-1. At these doses, citalopram induced greater increases in cortical [5-HT]ext than in [NA]ext. Venlafaxine increased [5-HT]ext and [NA]ext to about 400 and 140% of the respective basal values at 8 mg kg-1. 3. Citalopram and paroxetine have the highest potency to increase cortical [5-HT]ext and [NA]ext, respectively. In addition, the rank of order of efficacy of these antidepressant drugs to increase [5-HT]ext in vivo in the FCx of mice was as follows: venlafaxine>citalopram>paroxetine, while the efficacy to increase cortical [NA]ext in mice of paroxetine and citalopram is similar, and greater than that of venlafaxine. 4. In conclusion, extracellular levels of cortical [NA]ext increase with the highest doses of the very selective SSRI citalopram, as well as with the very potent SSRI paroxetine. Surprisingly, the SNRI venlafaxine increased cortical [5-HT]ext to a greater extent rather than [NA]ext in the range of doses studied in mice.

Animals↗
PubMed2004

Cannabinoid CB1 receptor-mediated modulation of evoked dopamine release and of adenylyl cyclase activity in the human neocortex.

1. The present study investigated the binding characteristics of various ligands to cannabinoid CB(1) receptors in human neocortex and amygdala. In addition, the functionality of CB(1) receptors in the human neocortex was assessed by examining the effects of CB(1) receptor ligands on evoked [(3)H]-dopamine (DA) release in superfused brain slices and on synaptosomal cAMP accumulation. 2. Saturation-binding assays in human neocortical and amygdala synaptosomes using a radiolabelled cannabinoid receptor agonist ([(3)H]-CP55.940) revealed pK(d) values of 8.96 and 8.63, respectively. The numbers of binding sites (B(max)) were 3.99 and 2.67 pmol (mg protein)(-1), respectively. 3. Various cannabinoid receptor ligands inhibited [(3)H]-CP55.940 binding with rank order potencies corresponding to those of previous studies in animal tissues. 4. Electrically evoked [(3)H]-DA release from human neocortical slices was inhibited by CP55.940 (IC(50) 6.76 nm, I(max) 65%) and strongly enhanced by the cannabinoid receptor antagonist AM251. However, [(3)H]-DA release was not influenced in rat neocortex. In human tissue, the estimated endocannabinoid concentration in the biophase of the release-modulating CB(1) receptors was 1.07 nm, expressed in CP55.940 units. 5. K(+)-evoked [(3)H]-DA release in the presence of tetrodotoxin (TTX) was strongly inhibited by CP55.940 in humans, but not in rats. 6. In human tissue, CP55.940 inhibited forskolin-stimulated cAMP accumulation (IC(50) 20.89 nm, I(max) 35%). AM251 blocked this effect and per se increased forskolin-stimulated cAMP accumulation by approximately 20%. 7. In conclusion, cannabinoids modulate [(3)H]-DA release and adenylyl cyclase activity in the human neocortex. CB(1) receptors are located on dopaminergic nerve terminals and seem to be tonically activated by endocannabinoids.

Adenylyl Cyclases↗
PubMed2005

Synergistic and additive interactions of the cannabinoid agonist CP55,940 with mu opioid receptor and alpha2-adrenoceptor agonists in acute pain models in mice.

1. Cannabinoid receptor agonists elicit analgesic effects in acute and chronic pain states via spinal and supraspinal pathways. We investigated whether the combination of a cannabinoid agonist with other classes of antinociceptive drugs exerted supra-additive (synergistic) or additive effects in acute pain models in mice. 2. The interactions between the cannabinoid agonist CP55,940, alpha2-adrenoceptor agonist dexmedetomidine and mu-opioid receptor agonist morphine were evaluated by isobolographic analysis of antinociception in hot plate (55 degrees C) and tail flick assays in conscious male Swiss mice. Drug interactions were examined by administering fixed-ratio combinations of agonists (s.c.) in 1:1, 3:1 and 1:3 ratios of their respective ED50 fractions. 3. CP55,940, dexmedetomidine and morphine all caused dose-dependent antinociception. In the hot plate and tail flick assays, ED50 values (mg kg(-1)) were CP55,940 1.13 and 0.51, dexmedetomidine 0.066 and 0.023, and morphine 29.4 and 11.3, respectively. Synergistic interactions existed between CP55,940 and dexmedetomidine in the hot plate assay, and CP55,940 and morphine in both assays. Additive interactions were found for CP55,940 and dexmedetomidine in the tail flick assay, and dexmedetomidine and morphine in both assays. 4. Thus, an alpha2-adrenoceptor agonist or mu opioid receptor agonist when combined with a cannabinoid receptor agonist showed significant synergy in antinociception in the hot plate test. However, for the tail flick nociceptive response to heat, only cannabinoid and mu opioid receptor antinociceptive synergy was demonstrated. If these results translate to humans, then prudent selection of dose and receptor-specific agonists may allow an improved therapeutic separation from unwanted side effects.

Acute Disease↗
PubMed2005

The effects of Delta9-tetrahydrocannabinol in rat mesenteric vasculature, and its interactions with the endocannabinoid anandamide.

1 Delta9-tetrahydrocannabinol (THC) produces varying effects in mesenteric arteries: vasorelaxation (third-order branches, G3), modest vasorelaxation (G2), no effect (G1) and vasoconstriction (the superior mesenteric artery, G0). 2 In G3, vasorelaxation to THC was inhibited by pertussis toxin, but was unaffected by the CB1 receptor antagonist, AM251 (1 microM), incubation with the TRPV1 receptor agonist capsaicin (10 microM, 1 h), the TRPV1 receptor antagonist capsazepine (10 microM) or de-endothelialisation. 3 In G3, vasorelaxation to THC was inhibited by high K+ buffer, and by the following K+ channel inhibitors: charybdotoxin (100 nM), apamin (500 nM) and barium chloride (30 microM), but not by 4-aminopyridine, glibenclamide or tertiapin. 4 In G3, THC (10 and 100 microM) inhibited the contractile response to Ca2+ in a Ca2+-free, high potassium buffer, indicating that THC blocks Ca2+ influx. 5 In G0, the vasoconstrictor responses to THC were inhibited by de-endothelialisation and SR141716A (100 nM), but not by the endothelin (ET(A)) receptor antagonist FR139317 (1 microM).THC (1 and 10 microM) antagonised vasorelaxation to anandamide in G3 but not G0. THC did not antagonise the noncannabinoid verapamil, capsaicin or the CB1 receptor agonist CP55,940. 6 THC (10 and 100 microM) inhibited endothelium-derived relaxing factor (EDHF)-mediated responses to carbachol in a manner similar to the gap junction inhibitor 18alpha-glycyrrhetinic acid. 7 These data show that THC causes vasorelaxation through activation of K+ channels and inhibition of Ca2+ channels, and this involves non-CB1, non-TRPV1 but G-protein-coupled receptors. In G0, THC does not cause relaxation and at high concentrations causes contractions. Importantly, THC antagonises the effects of anandamide, possibly through inhibition of EDHF activity.

Animals↗
PubMed2005

Identification of WIN55212-3 as a competitive neutral antagonist of the human cannabinoid CB2 receptor.

1. Several G protein-coupled receptors (GPCRs), including cannabinoid CB(1) and CB(2) receptors, show constitutive activity under heterologous expression. Such a tonic response is generated in the absence of an activating ligand, and can be inhibited by inverse agonists. Neutral antagonists, however, are silent at such receptors, but can reverse both agonist and inverse agonist responses. To date, no neutral antagonist for the CB(2) receptor has been reported. 2. Here, by monitoring receptor-dependent G protein activation, we demonstrate that WIN55212-3 acts as a neutral antagonist at the human CB(2) (hCB(2)) receptor. WIN55212-3 alone, at concentrations </=10(-4) M, behaved as a silent ligand exhibiting no agonist or inverse agonist activity. However, WIN55212-3 competitively antagonized cannabinoid agonist CP-55,940-stimulated responses (pA(2) 6.1). Importantly, the inverse agonism evoked by SR144528 in hCB(2) was dose-dependently reversed by WIN55212-3 (pEC(50) 5.3+/-0.2), indicating true neutral antagonist behavior. 3. Furthermore, WIN55212-3 also antagonized CB(1) receptor signaling in a competitive manner (pA(2) 5.6), but behaved as a partial inverse agonist (pIC(50) 5.5+/-0.1) at the constitutively active human CB(1). 4. Additionally, WIN55212-3 antagonized signaling of the human melatonin MT(1) receptor, with modest activity at the human muscarinic M4 receptor, but it was inactive towards several other GPCRs. 5. These data identify WIN55212-3 as a true neutral hCB(2) receptor antagonist. WIN55212-3 offers a valuable tool for further characterization of ligand activities at the CB(2) receptor and may serve as a lead compound in further efforts to develop more potent and selective neutral CB(2) receptor antagonists.

Animals↗
PubMed2007

Control of spasticity in a multiple sclerosis model is mediated by CB1, not CB2, cannabinoid receptors.

BACKGROUND AND PURPOSE: There is increasing evidence to suggest that cannabis can ameliorate muscle-spasticity in multiple sclerosis, as was objectively shown in experimental autoimmune encephalomyelitis models. The purpose of this study was to investigate further the involvement of CB1 and CB2)cannabinoid receptors in the control of experimental spasticity. EXPERIMENTAL APPROACH: Spasticity was induced in wildtype and CB1-deficient mice following the development of relapsing, experimental autoimmune encephalomyelitis. Spastic-hindlimb stiffness was measured by the resistance to flexion against a strain gauge following the administration of CB1 and CB2 agonists. KEY RESULTS: As previously suggested, some CB2-selective agonists (RWJ400065) could inhibit spasticity. Importantly, however, the anti-spastic activity of RWJ400065 and the therapeutic effect of non-selective CB1/CB2 agonists (R(+)WIN55,212-2 and CP55, 940) was lost in spastic, CB1-deficit mice. CONCLUSIONS AND IMPLICATIONS: The CB1 receptor controls spasticity and cross-reactivity to this receptor appears to account for the therapeutic action of some CB2 agonists. As cannabinoid-induced psychoactivity is also mediated by the CB1 receptor, it will be difficult to truly dissociate the therapeutic effects from the well-known, adverse effects of cannabinoids when using cannabis as a medicine. The lack of knowledge on the true diversity of the cannabinoid system coupled with the lack of total specificity of current cannabinoid reagents makes interpretation of in vivo results difficult, if using a purely pharmacological approach. Gene knockout technology provides an important tool in target validation and indicates that the CB1 receptor is the main cannabinoid target for an anti-spastic effect.

Animals↗
PubMed2000

Gaucher disease: expression and characterization of mild and severe acid beta-glucosidase mutations in Portuguese type 1 patients.

Type 1 Gaucher disease (GD), the most prevalent lysosomal storage disease, results from the deficient activity of acid beta-glucosidase. Molecular analysis of 12 unrelated Portuguese patients with type 1 GD identified three novel acid beta-glucosidase mutations (F109V, W184R and R395P), as well as three previously reported, but uncharacterized, lesions (R359Q, G377S and N396T). The type 1 probands were either heteroallelic for the well-characterized common lesion, N370S, and the F109V, W184R, R359Q or N396T lesions or homoallelic for the G377S or N396T mutations. Expression of the W184R, R359Q and R395P mutations revealed very low specific activities based on cross-reacting immunologic material (CRIM SAs of 0.0004, 0.016 and 0.045, respectively), consistent with their being found only in type 1 patients who had a neuroprotective N370S allele. In contrast, the F109V, G377S and N396T alleles had significant acid beta-glucosidase activity (CRIM specific activities of 0.15, 0.17, 0.14, respectively), in agreement with their being mild type 1 alleles. Thus, these studies identified additional acid beta-glucosidase mutations in the Portuguese population and demonstrated that the G377S and N396T mutations were neuroprotective, consistent with the mild clinical phenotypes of the type 1 patients who were homoallelic for the G377S and N396T lesions.

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