The endogenous cannabinoid agonist, anandamide stimulates sensory nerves in guinea-pig airways

The endogenous cannabinoid agonist, anandamide produced a modest contractile response in guinea-pig isolated bronchus compared with the vanilloid receptor agonist capsaicin. The contractile response to both anandamide and capsaicin was inhibited by the vanilloid receptor antagonist, capsazepine. Furthermore, the NK(2)-selective antagonist, SR48968 but not the NK(1)-selective antagonist, SR140333 inhibited contractile responses to anandamide. The contractile response to anandamide was abolished in tissues desensitized by capsaicin. However, anandamide failed to cross-desensitize the contractile response to capsaicin. The contractile response to anandamide was not significantly altered in the presence of the CB(1) receptor antagonist, SR141716A, nor the amidase inhibitor, phenylmethylsulphonyl fluoride (PMSF) but was significantly increased in the presence of the neutral endopeptidase inhibitor, thiorphan. The cannabinoid agonist, CP55,940 failed to significantly attenuate the excitatory non-adrenergic non-cholinergic (eNANC) response in guinea-pig airways. In contrast, the ORL(1) receptor agonist, nociceptin, significantly inhibited this response. The results demonstrate that anandamide induces a modest contractile response in guinea-pig isolated bronchus that is dependent upon the activation of vanilloid receptors on airway sensory nerves. However, cannabinoid receptors do not appear to play a role in this regard, nor in regulating the release of neuropeptides from airway sensory nerves under physiological conditions.     

Anandamide and methanandamide induce both vanilloid VR1- and cannabinoid CB1 receptor-mediated changes in heart rate and blood pressure in anaesthetized rats

In anaesthetized rats activation of vanilloid receptors on sensory vagal nerves elicits rapid bradycardia and hypotension (Bezold-Jarisch reflex). Recent in vitro experiments revealed that the endogenous cannabinoid ligand anandamide acts as an agonist at the vanilloid VRI receptors. The present study was aimed at examining whether vanilloid VR1 receptors are involved in the cardiovascular effects of anandamide in the anaesthetized rat. Intravenous injection of anandamide, its stable analogue methanandamide and the vanilloid receptor agonist capsaicin produced a dose-dependent immediate and short-lasting decrease in heart rate and blood pressure with the following rank order of potencies: capsaicin > methanandamide > anandamide. This bradycardia was dose-dependently diminished by the selective vanilloid receptor antagonist capsazepine (0.3-3 micromol/kg) and the nonselective inhibitor of these receptors, ruthenium red (1-10 micromol/kg). Both antagonists reduced or tended to reduce the hypotension stimulated by the agonists. Following this bradycardia and hypotension (presumably evoked by the Bezold-Jarisch reflex; phase I), capsaicin, anandamide and methanandamide led to a brief vasopressor effect (phase II). Subsequently both anandamides, but not capsaicin, induced a more prolonged decrease in blood pressure (phase III). Capsazepine and ruthenium red (at doses up to 3 tmol/kg and 10 micromol/kg, respectively) failed to affect these changes in blood pressure. The cannabinoid CB1 receptor antagonist SR 141716 at 3 micromol/kg abolished the prolonged decrease in blood pressure (phase III) induced by anandamide and methanandamide, but had no effect on the reflex bradycardia and hypotension (phase I) and on the subsequent vasopressor effect (phase II) evoked by capsaicin, anandamide and methanandamide. In conclusion, the endogenous cannabinoid receptor agonist anandamide and its stable analogue methanandamide induce reflex bradycardia and hypotension (phase I) by activating the vanilloid VRI receptor. Whereas the mechanism underlying the brief vasopressor effect (phase II) is unknown, the prolonged hypotension (phase III) results from stimulation of the cannabinoid CB1 receptor.     

Anandamide induces cardiovascular and respiratory reflexes via vasosensory nerves in the anaesthetized rat

1. We tested the hypothesis that sensory nerves innervating blood vessels play a role in the local and systemic regulation of the cardiovascular and respiratory (CVR) systems. We measured CVR reflexes evoked by administration of anandamide (86 - 863 nmoles) and capsaicin (0.3 - 10 nmoles) into the hindlimb vasculature of anaesthetized rats. 2. Anandamide and capsaicin each caused a rapid dose-dependent reflex fall in blood pressure and an increase in ventilation when injected intra-arterially into the hindlimb. 3. Action of both agonists at the vanilloid receptor (VR1) on perivascular sensory nerves was investigated using capsazepine (1 mg kg(-1) i.a.) a competitive VR1 antagonist, ruthenium red (1 mg kg(-1) i.a.), a non-competitive antagonist at VR1, or a desensitizing dose of capsaicin (200 nmoles i.a.). The cannabinoid receptor antagonist SR141716 (1 mg kg(-1) i.a.) was used to determine agonist activity at the CB(1) receptor. 4. Capsazepine, ruthenium red, or acute VR1 desensitization by capsaicin-pretreatment, markedly attenuated the reflex CVR responses evoked by anandamide and capsaicin (P< 0.05; paired Student's t-test). Blockade of CB(1) had no significant effect on the responses to anandamide. 5. Local sectioning of the femoral and sciatic nerves attenuated CVR responses to anandamide and capsaicin (P< 0.05). Vagotomy or carotid sinus sectioning had no significant effect on anandamide- or capsaicin-induced responses. 6. These data demonstrate that both the endogenous cannabinoid, anandamide, and the vanilloid, capsaicin, evoke CVR reflexes when injected intra-arterially into the rat hindlimb. These responses appear to be mediated reflexly via VR1 located on sensory nerve endings within the hindlimb vasculature.     

Neurobehavioral activity in mice of N-vanillyl-arachidonyl-amide

We studied the cannabimimetic properties of N-vanillyl-arachidonoyl-amide (arvanil), a potential agonist of cannabinoid CB(1) and capsaicin VR(1) receptors, and an inhibitor of the facilitated transport of the endocannabinoid anandamide. Arvanil and anandamide exhibited similar affinities for the cannabinoid CB(1) receptor, but arvanil was less efficacious in inducing cannabinoid CB(1) receptor-mediated GTPgammaS binding. The K(i) of arvanil for the vanilloid VR(1) receptor was 0.28 microM. Administered i.v. to mice, arvanil was 100 times more potent than anandamide in producing hypothermia, analgesia, catalepsy and inhibiting spontaneous activity. These effects were not attenuated by the cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chloro-phenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.HCl (SR141716A). Arvanil (i.t. administration) induced analgesia in the tail-flick test that was not blocked by either SR141716A or the vanilloid VR(1) antagonist capsazepine. Conversely, capsaicin was less potent as an analgesic (ED(50) 180 ng/mouse, i.t.) and its effects attenuated by capsazepine. The analgesic effect of anandamide (i.t.) was also unaffected by SR141716A but was 750-fold less potent (ED(50) 20.5 microg/mouse) than capsaicin. These data indicate that the neurobehavioral effects exerted by arvanil are not due to activation of cannabinoid CB(1) or vanilloid VR(1) receptors.     

The effect of cannabinoids on capsaicin-evoked calcitonin gene-related peptide (CGRP) release from the isolated paw skin of diabetic and non-diabetic rats

Sensory neural dysfunction is common in patients with peripheral neuropathy, a major complication of diabetes mellitus. In animal models of inflammatory and neuropathic pain cannabinoids potently attenuate pain behaviour, cannabinoid (CB) receptors located on nociceptive primary afferent neurones being important in their anti-hyperalgesic actions. A key measure of sensory neurone function is stimulus-evoked neuropeptide release. We investigated the effect of cannabinoid on capsaicin-evoked release of calcitonin gene-related peptide (CGRP) from the rat paw skin in vitro, comparing non-diabetic and streptozotocin-induced diabetic animals. Diabetes caused a greater than two-fold increase in basal and capsaicin-evoked CGRP release. The synthetic CB(1)/CB(2) receptor agonist, CP55940 (100 nM), inhibited capsaicin-evoked CGRP release in both non-diabetic (30.92+/-7.69%, P<0.05) and diabetic animals (37.82+/-9.85%, P<0.05). The CB(1) receptor antagonist SR141716A (100 nM), but not the CB(2) receptor antagonist SR144528 (100 nM), significantly attenuated the inhibitory action of CP55940. The endogenous cannabinoid, anandamide (100 nM) inhibited capsaicin-evoked CGRP release in non-diabetic animals (28.88+/-7.12%, P<0.05) but neither the CB(1) nor the CB(2) receptor antagonist attenuated this action of anandamide. Anandamide (100 nM) did not significantly inhibit capsaicin-evoked CGRP release from the paw skin of diabetic animals, but it did produce a small stimulation of CGRP release at high concentrations (10 microM). These data suggest that peripheral CB(1) receptors mediate inhibition of capsaicin-evoked neuropeptide release from the paw skin of both non-diabetic and diabetic animals. However, pathological changes in the diabetic animals appear to preclude the non-CB(1) receptor mediated inhibitory action of the endogenous cannabinoid, anandamide.     

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.     

Mechanisms of anandamide-induced vasorelaxation in rat isolated coronary arteries

1. The cannabinoid arachidonyl ethanolamide (anandamide) caused concentration-dependent relaxation of 5-HT-precontracted, myograph-mounted, segments of rat left anterior descending coronary artery. 2. This relaxation was endothelium-independent, unaffected by the fatty acid amide hydrolase inhibitor, arachidonyl trifluoromethyl ketone (10 microM), and mimicked by the non-hydrolysable anandamide derivative, methanandamide. 3. Relaxations to anandamide were attenuated by the cannabinoid receptor antagonist, SR 141716A (3 microM), but unaffected by AM 251 (1 microM) and AM 630 (1 microM), more selective antagonists of cannabinoid CB(1) and CB(2) receptors respectively. Palmitoylethanolamide, a selective CB(2) receptor agonist, did not relax precontracted coronary arteries. 4. Anandamide relaxations were not affected by inhibition of sensory nerve transmission with capsaicin (10 microM) or blockade of vanilloid VR1 receptors with capsazepine (5 microM). Nevertheless capsaicin relaxed coronary arteries in a concentration-dependent and capsazepine-sensitive manner, confirming functional sensory nerves were present. In contrast, capsazepine and capsaicin did inhibit anandamide relaxations in methoxamine-precontracted rat small mesenteric arteries. 5. Relaxations to anandamide were inhibited by TEA (1 mM) or iberiotoxin (50 nM), blockers of large conductance, Ca(2+)-activated K(+) channels (BK(Ca)). Gap junction inhibition with 18alpha-glycyrrhetinic acid (100 microM) did not affect anandamide relaxations. 6. This study shows anandamide relaxes the rat coronary artery by a novel mechanism. Anandamide-induced relaxations do not involve the endothelium, degradation into active metabolites, or activation of cannabinoid CB(1) or CB(2) receptors, but may involve activation of BK(Ca). Vanilloid receptor activation also has no role in the effects of anandamide in coronary arteries, even though functional sensory nerves are present.     

Arvanil-induced inhibition of spasticity and persistent pain: evidence for therapeutic sites of action different from the vanilloid VR1 receptor and cannabinoid CB(1)/CB(2) receptors

Activation of cannabinoid receptors causes inhibition of spasticity, in a mouse model of multiple sclerosis, and of persistent pain, in the rat formalin test. The endocannabinoid anandamide inhibits spasticity and persistent pain. It not only binds to cannabinoid receptors but is also a full agonist at vanilloid receptors of type 1 (VR1). We found here that vanilloid VR1 receptor agonists (capsaicin and N-N'-(3-methoxy-4-aminoethoxy-benzyl)-(4-tert-butyl-benzyl)-urea [SDZ-249-665]) exhibit a small, albeit significant, inhibition of spasticity that can be attenuated by the vanilloid VR1 receptor antagonist, capsazepine. Arvanil, a structural "hybrid" between capsaicin and anandamide, was a potent inhibitor of spasticity at doses (e.g. 0.01 mg/kg i.v.) where capsaicin and cannabinoid CB(1) receptor agonists were ineffective. The anti-spastic effect of arvanil was unchanged in cannabinoid CB(1) receptor gene-deficient mice or in wildtype mice in the presence of both cannabinoid and vanilloid receptor antagonists. Likewise, arvanil (0.1-0.25 mg/kg) exhibited a potent analgesic effect in the formalin test, which was not reversed by cannabinoid and vanilloid receptor antagonists. These findings suggest that activation by arvanil of sites of action different from cannabinoid CB(1)/CB(2) receptors and vanilloid VR1 receptors leads to anti-spastic/analgesic effects that might be exploited therapeutically.     

Intraplantar injection of anandamide inhibits mechanically-evoked responses of spinal neurones via activation of CB2 receptors in anaesthetised rats

Anti-nociceptive effects of the endocannabinoid anandamide are well established. Anandamide has, however, also been shown to activate pro-nociceptive vanilloid 1 (VR1) receptors present on primary afferent nociceptors. The aim of the present study was to determine the effect of intraplantar injection of anandamide on dorsal spinal neuronal responses in control rats and rats with hindpaw carrageenan-induced inflammation. Effects of intraplantar administration of anandamide (50 microg in 50 microl) on peripheral mechanically-evoked responses of spinal neurones were studied in halothane-anaesthetised rats in vivo. Responses of spinal neurones to mechanical punctate stimulation (von Frey filaments, 8-80 g) of the peripheral receptive field were similar in non-inflamed rats and rats with hindpaw carrageenan-induced inflammation. Intraplantar injection of anandamide, but not vehicle, significantly (P<0.05) inhibited innocuous and noxious mechanically-evoked responses of spinal neurones in rats with hindpaw inflammation, but not in non-inflamed rats. Co-administration of the cannabinoid (2) (CB(2)) receptor antagonist, SR144528 (10 microg in 50 microl), but not the cannabinoid (1) (CB(1)) receptor antagonist, SR141716A (10 microg in 50 microl), significantly blocked inhibitory effects of anandamide on peripheral evoked neuronal responses in rats with hindpaw inflammation. This study demonstrates inhibitory effects of exogenous anandamide on mechanically-evoked responses under inflammatory conditions in vivo, which are mediated by peripheral CB(2) receptors.     

Vasorelaxant effects of oleamide in rat small mesenteric artery indicate action at a novel cannabinoid receptor

Oleamide (cis-9-octadecenoamide) exhibits some cannabimimetic responses despite its low affinities at the currently known cannabinoid receptors. Here we have investigated whether or not it is a vasorelaxant in rat small mesenteric arteries. Oleamide elicited vasorelaxation (EC50=1.2+/-0.2 microM, Rmax=99.1+/-3.9%, n=8) which was reduced by endothelial removal. Nitric oxide synthase inhibition reduced the response (EC50=5.3+/-1.6 microM, Rmax=59.2+/-7.7%, n=7; P<0.01) as did blockade of Ca2+-sensitive K+ channels (KCa) with apamin plus charybdotoxin (both 50 nM) (EC50=2.1+/-0.2 microM, Rmax=58.4+/-1.9%, n=5; P<0.05). Desensitisation of vanilloid receptors with capsaicin (10 microM for 30 min) shifted the oleamide concentration-response curve approximately 30-fold to the right (n=7; P<0.01). Pertussis toxin (400 ng ml-1 for 2 h) caused a two-fold shift in the response curve (EC50=2.2+/-0.4 microM, Rmax=66.8+/-4.5%, n=6; P<0.01). Rimonabant (CB1 cannabinoid receptor antagonist; SR141716A; 3 microM) significantly inhibited relaxation induced by oleamide (EC50=3.5+/-0.3 microM, Rmax=75.1+/-1.9%; n=8; P<0.05). In contrast, neither the more selective CB1 receptor antagonist, AM251 (1 microM), nor the CB2 antagonist, SR144528 (1 microM), had significant effects. O-1918 (10 microM), a putative antagonist at a novel endothelial cannabinoid receptor (abnormal-cannabidiol site), markedly reduced the relaxation to oleamide (n=7; P<0.01).It is concluded that oleamide responses in the rat isolated small mesenteric artery are partly dependent on the presence of the endothelium, activation of Ca2+-sensitive K+ channels (KC)) and involve capsaicin-sensitive sensory nerves. Oleamide may share a receptor (sensitive to rimonabant and O-1918, and coupled to KC) and Gi/o) with anandamide in this vessel. This might be distinct from both of the known cannabinoid receptors and the novel abnormal-cannabidiol site.     

Arachidonic acid release and prostaglandin F(2alpha) formation induced by anandamide and capsaicin in PC12 cells

Anandamide, an endogenous agonist of cannabinoid receptors, activates various signal transduction pathways. Anandamide also activates vanilloid VR(1) receptor, which was a nonselective cation channel with high Ca(2+) permeability and had sensitivity to capsaicin, a pungent principle in hot pepper. The effects of anandamide and capsaicin on arachidonic acid metabolism in neuronal cells have not been well established. We examined the effects of anandamide and capsaicin on arachidonic acid release in rat pheochromocytoma PC12 cells. Both agents stimulated [3H]arachidonic acid release in a concentration-dependent manner from the prelabeled PC12 cells even in the absence of extracellular CaCl(2). The effect of anandamide was neither mimicked by an agonist nor inhibited by an antagonist for cannabinoid receptors. The effects of anandamide and capsaicin were inhibited by phospholipase A(2) inhibitors, but not by an antagonist for vanilloid VR(1) receptor. In PC12 cells preincubated with anandamide or capsaicin, [3H]arachidonic acid release was marked and both agents were no more effective. Co-addition of anandamide or capsaicin synergistically enhanced [3H]arachidonic acid release by mastoparan in the absence of CaCl(2). Anandamide stimulated prostaglandin F(2alpha) formation. These findings suggest that anandamide and capsaicin stimulated arachidonic acid metabolism in cannabinoid receptors- and vanilloid VR(1) receptor-independent manner in PC12 cells. The possible mechanisms are also discussed.     

Effect of vanilloid drugs on gastrointestinal transit in mice

1. We have studied the effect of capsaicin, piperine and anandamide, drugs which activate vanilloid receptors and capsazepine, a vanilloid receptor antagonist, on upper gastrointestinal motility in mice. 2. Piperine (0.5 - 20 mg kg(-1) i.p.) and anandamide (0.5 - 20 mg kg(-1) i.p.), dose-dependently delayed gastrointestinal motility, while capsaicin (up to 3 mg kg(-1) i.p.) was without effect. Capsazepine (15 mg kg(-1) i.p.) neither per se affected gastrointestinal motility nor did it counteract the inhibitory effect of both piperine (10 mg kg(-1)) and anandamide (10 mg kg(-1)). 3. A per se non effective dose of SR141716A (0.3 mg kg(-1) i.p.), a cannabinoid CB(1) receptor antagonist, counteracted the inhibitory effect of anandamide (10 mg kg(-1)) but not of piperine (10 mg kg(-1)). By contrast, the inhibitory effect of piperine (10 mg kg(-1)) but not of anandamide (10 mg kg(-1)) was strongly attenuated in capsaicin (75 mg kg(-1) in total, s.c.)-treated mice. 4. Pretreatment of mice with N(G)-nitro-L-arginine methyl ester (25 mg kg(-1) i.p.), yohimbine (1 mg kg(-1), i.p.), naloxone (2 mg kg(-1) i.p.), or hexamethonium (1 mg kg(-1) i.p.) did not modify the inhibitory effect of both piperine (10 mg kg(-1)) and anandamide (10 mg kg(-1)). 5. The present study indicates that the vanilloid ligands anandamide and piperine, but not capsaicin, can reduce upper gastrointestinal motility. The effect of piperine involves capsaicin-sensitive neurones, but not vanilloid receptors, while the effect of anandamide involves cannabinoid CB(1), but not vanilloid 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.     

Endothelium-independent relaxation to cannabinoids in rat-isolated mesenteric artery and role of Ca2+ influx

(1) Three cannabinoid receptor agonists, anandamide (CB(1) receptor-selective) and the aminoalkyl-indoles, JWH 015(2-methyl-1-propyl-1H-indol-3-yl)-1-napthalenylmethanone; (CB(2) receptor-selective), R-(+)-WIN 55,212-2 (R-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolol[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone; slightly CB(2) receptor-selective), as well as the enantiomer S-(-)-WIN 55,212-3(S-(-)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolol[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone; inactive at cannabinoid receptors), induced endothelium-independent relaxation of methoxamine-precontracted isolated small mesenteric artery of rat. KCL (60 mM) precontraction did not affect relaxation to the aminoalkylindoles, but reduced that to anandamide. (2) SR14176A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; 3 micro M; CB(1) receptor antagonist) inhibited relaxation only to JWH 015 and anandamide. Neither AM 251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; CB(1) antagonist) nor SR 144528 (N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide; CB(2) antagonist; both at 3 micro M) affected any of the relaxations. (3) Vanilloid receptor desensitisation with capsaicin reduced anandamide relaxation; addition of SR 141716A (3 micro M) then caused further inhibition. SR 141716A did not affect capsaicin-induced relaxation. (4) The aminoalkylindoles inhibited CaCl(2)-induced contractions in methoxamine-stimulated vessels previously depleted of intracellular Ca(2+). These inhibitory effects were greatly reduced or abolished in ionomycin-(a calcium ionophore) contracted vessels. Anandamide also caused vanilloid receptor-independent, SR 141716A- (3 micro M) insensitive, inhibition of CaCl(2) contractions. (5) In conclusion, the aminoalkylindoles JWH 015, R-(+)-WIN 55,212-2 and S-(-)-WIN 55,212-3 relax rat small mesenteric artery mainly by inhibiting Ca(2+) influx into vascular smooth muscle. Anandamide causes vasorelaxation by activating vanilloid receptors, but may also inhibit Ca(2+) entry. Relaxation to JWH 015 and anandamide was sensitive to SR 141716A, but there is no other evidence for the involvement of CB(1) or CB(2) receptors in responses to these compounds.     

Contractile response to a cannabimimetic eicosanoid, 2-arachidonoylglycerol,of longitudinal smooth muscle from the guinea-pig distal colon in vitro

The effect of 2-arachidonoylglycerol, a cannabimimetic eicosanoid, was studied on mucosa-free longitudinal muscle strips isolated from the guinea-pig distal colon. In the presence of indomethacin (3 microM) and N(G)-nitro-L-arginine (100 microM), 2-arachidonoylglycerol (10 nM-10 microM) produced concentration-dependent and tetrodotoxin (1 microM)-sensitive contractions of the longitudinal muscle strips. The contractions were markedly attenuated in the presence of atropine (0.2 microM), and partially by hexamethonium (100 microM) pretreatment. The response to 2-arachidonoylglycerol was mimicked with N-arachidonoylethanolamine (anandamide, 0.1-30 microM), another cannabimimetic eicosanoid, but the cannabinoid CB(1)/CB(2) receptor agonist, R-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2) (0.1-10 microM), and the vanilloid receptor agonist, (all Z)-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM 404) (10-30 microM), were without effect. The cannabinoid CB(1) receptor antagonist, N-piperidino-5-(4-chlorophenyl)-l-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) (1 microM), the cannabinoid CB(2) receptor antagonist, [N-[1S]-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-l-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) (1 microM), and the vanilloid receptor antagonist, capsazepine (10 microM), did not shift the concentration-response curve for 2-arachidonoylglycerol to the right. The contractile action of 2-arachidonoylglycerol was also partially attenuated in the presence of nordihydroguaiaretic acid (10 microM), a lipoxygenase inhibitor. These results indicate that 2-arachidonoylglycerol produces contraction of longitudinal muscle of the guinea-pig distal colon via mainly stimulation of myenteric cholinergic neurones, and that neither cannabinoid CB(1)/CB(2) receptors nor vanilloid receptors contributed to the response. The present results suggest the possibility that lipoxygenase metabolites may also contribute, at least in part, to the contractile action of 2-arachidonoylglycerol.     

Characterisation of the vasorelaxant properties of the novel endocannabinoid N-arachidonoyl-dopamine (NADA)

1. We have investigated the vascular effects of N-arachidonoyl-dopamine (NADA), a novel endocannabinoid/vanilloid. NADA caused vasorelaxant effects comparable to those of anandamide in small mesenteric vessels (G3), the superior mesenteric artery (G0) and in the aorta. 2. In G3, addition of N(G)-nitro-l-arginine methyl ester (300 microm) or the dopamine (D(1)) receptor antagonist (SCH23390, 1 microm) did not affect responses to NADA. In the presence of 60 mm KCl, after de-endothelialisation, or after K(+) channel inhibition with charybdotoxin (100 nm) and apamin (500 nm), relaxant responses to NADA were inhibited. 3. In G3, pretreatment with the vanilloid receptor (VR) agonist capsaicin (10 microm) or the VR antagonist capsazepine (10 microm) reduced vasorelaxation to NADA. 4. In G3, application of the CB(1) antagonist SR141716A at 1 microm but not 100 nm reduced the potency of NADA. Another CB(1) antagonist, AM251 (100 nm and 1 microm), did not affect vasorelaxation to NADA. After endothelial denudation, SR141716A (1 microm) did not reduce the responses further. A combination of capsaicin and SR141716A (1 microm) reduced vasorelaxation to NADA further than with capsaicin pretreatment alone. The novel endothelial cannabinoid (CB) receptor antagonist O-1918 opposed vasorelaxation to NADA in G3. 5. In the superior mesenteric artery (G0), vasorelaxation to NADA was not dependent on an intact endothelium and was not sensitive to O-1918, but was sensitive to capsaicin and SR141716A or AM251 (both 100 nm). 6. The results of the present study demonstrate for the first time that NADA is a potent vasorelaxant. In G3, the effects of NADA are mediated by stimulation of the VR and the novel endothelial CB receptor, while in G0, vasorelaxation is mediated through VR(1) and CB(1) receptors.     

Anandamide induces cough in conscious guinea-pigs through VR1 receptors

1. Endogenous neuronal lipid mediator anandamide, which can be synthesized in the lung, is a ligand of both cannabinoid (CB) and vanilloid receptors (VR). The tussigenic effect of anandamide has not been studied. The current study was designed to test the direct tussigenic effect of anandamide in conscious guinea-pigs, and its effect on VR1 receptor function in isolated primary guinea-pig nodose ganglia neurons. 2. Anandamide (0.3-3 mg.ml(-1)), when given by aerosol, induced cough in conscious guinea-pigs in a concentration dependent manner. When guinea-pigs were pretreated with capsazepine, a VR1 antagonist, the anandamide-induced cough was significantly inhibited. Pretreatment with CB1 (SR 141716A) and CB2 (SR 144528) antagonists had no effect on anandamide-induced cough. These results indicate that anandamide-induced cough is mediated through the activation of VR1 receptors. 3. Anandamide (10-100 micro M) increased intracellular Ca(2+) concentration estimated by Fluo-4 fluorescence change in isolated guinea-pig nodose ganglia cells. The anandamide-induced Ca(2+) response was inhibited by two different VR1 antagonists: capsazepine (1 micro M) and iodo-resiniferatoxin (I-RTX, 0.1 micro M), indicating that anandamide-induced Ca(2+) response was through VR1 channel activation. In contrast, the CB1 (SR 141716A, 1 micro M) and CB2 (SR 144528, 0.1 micro M) receptor antagonists had no effect on Ca(2+) response to anandamide. 4. In conclusion, these results provide evidence that anandamide activates native vanilloid receptors in isolated guinea-pig nodose ganglia cells and induces cough through activation of VR1 receptors.     

Hypolocomotor effects in rats of capsaicin and two long chain capsaicin homologues

Capsaicin and its analogue N-arachidonoyl-vanillyl-amine (arvanil) are agonists of vanilloid VR₁ receptors, and suppress spontaneous activity in mice through an unknown mechanism. Here, we tested in rats the effect on motor behavior of: (1) capsaicin; (2) N-linoleoyl-vanillyl-amine (livanil) and N--linolenoyl-vanillyl-amine (linvanil), which, unlike arvanil, have very little affinity for cannabinoid CB₁ receptors; and (3) the endocannabinoid anandamide (N-arachidonoyl-ethanolamine), which is a full agonist at both cannabinoid CB₁ and vanilloid VR₁ receptors. All compounds, administered i.p., dose-dependently (0.1–10 mg/kg) inhibited ambulation and stereotypic behavior and increased inactivity in the open field test. The rank of potency observed in vivo (livanil>capsaicin>linvanil>anandamide) bore little resemblance with the relative potencies in a functional assay for rat vanilloid VR₁ receptors (livanil=linvanil>capsaicin>anandamide) and even less with the relative affinities in rat CB₁ receptor binding assays (anandamide>livanil>linvanil>capsaicin). The vanilloid VR₁ receptor antagonist capsazepine (10 mg/kg, i.p.) blocked the effect of capsaicin but not of livanil or anandamide, whereas the CB₁ receptor antagonist (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.HCl (SR141716A, 3 mg/kg, i.p.) antagonized the actions of the CB₁ receptor agonist Δ⁹-tetrahydrocannabinol, but not of livanil, anandamide or capsaicin. Anandamide occluded the effects of livanil on locomotion, possibly suggestive of a common mechanism of action for the two compounds. Finally, stimulation with capsaicin of cells expressing rat vanilloid VR₁ receptors led to anandamide formation. These data suggest that motor behavior can be suppressed by the activation of: (1) vanilloid receptors, possibly via the intermediacy of anandamide; or (2) capsazepine- and SR141716A-insensitive sites of action for anandamide, livanil and linvanil, possibly the same that were previously suggested to mediate arvanil hypokinetic effects in mice.     

The differential contractile responses to capsaicin and anandamide in muscle strips isolated from the rat urinary bladder

The contractile responses to capsaicin and anandamide, exogenous and endogenous agonists for transient receptor potential vanilloid receptor 1 (TRPV1), respectively, were investigated in muscle strips isolated from the rat urinary bladder. Capsaicin and anandamide produced concentration-dependent contractions of the muscle strips. The contractile response induced by capsaicin disappeared within approximately 20 min. In contrast, anandamide produced contractile responses lasting at least for 30 min. Capsaicin produced additive contractile responses in anandamide-treated muscle strips. The contractile response to anandamide was attenuated, but not abolished in strips desensitized by capsaicin. The response to capsaicin was abolished in the presence of a TRPV1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-chlorphyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC), but not altered in the presence of either tetrodotoxin, atropine or indomethacin. In the presence of SR140333, a tachykinin NK₁ receptor antagonist or SR48968, an NK₂ receptor antagonist, the response to capsaicin was attenuated. The response to anandamide was partially attenuated in the presence of ONO8130, a prostanoid EP₁ receptor antagonist, URB597, a fatty-acid amide hydrolase inhibitor, BCTC, SR140333 or SR48968, and almost completely abolished by indomethacin. Neither tetrodotoxin, atropine, a cannabinoid CB₁ receptor antagonist, AM251, nor a cannabinoid CB₂ receptor antagonist, AM630, had any effect on the response to anandamide. These results indicate that capsaicin produces muscle contractions by stimulating the TRPV1 receptor, followed by release of neuropeptides that can activate tachykinin NK₁ and/or NK₂ receptors in the bladder and that the contractile response to anandamide is mediated at least in part by activation of prostanoid EP₁ receptors due to production of prostaglandins in addition to TRPV1 receptor activation.     

Cannabinoid modulation of peripheral autonomic and sensory neurotransmission

Cannabinoids are cell membrane-derived signalling molecules that are released from nerves, blood cells and endothelial cells, and have diverse biological effects. They act at two distinct types of G-protein-coupled receptors, cannabinoid CB(1) and CB(2) receptors. Cannabinoid CB(1) receptors are highly localised in the central nervous system and are also found in some peripheral tissues, and cannabinoid CB(2) receptors are found outside the central nervous system, in particular in association with immune tissues. Novel actions of cannabinoids at non-CB(1) non-CB(2) cannabinoid-like receptors and vanilloid VR1 receptors have also recently been described. There is growing evidence that, among other roles, cannabinoids can act at prejunctional sites to modulate peripheral autonomic and sensory neurotransmission, and the present article is aimed at providing an overview of this. Inhibitory cannabinoid CB(1) receptors are expressed on the peripheral terminals of autonomic and sensory nerves. The role of cannabinoid receptor ligands in modulation of sensory neurotransmission is complex, as certain of these (anandamide, an "endocannabinoid", and N-arachidonoyl-dopamine, an "endovanilloid") also activate vanilloid VR1 receptors (coexpressed with cannabinoid CB(1) receptors), which excites sensory nerves and causes a release of sensory neurotransmitter. The fact that the activities of anandamide and N-arachidonoyl-dopamine span two distinct receptor families raises important questions about cannabinoid/vanilloid nomenclature, and as both compounds are structurally related to the archetypal vanilloid capsaicin, all three are arguably members of the same family of signalling molecules. Anandamide is released from nerves, but unlike classical neurotransmitters, it is not stored in and released from nerve vesicles, but is released on demand from the nerve cell membrane. In the central nervous system, cannabinoids function as retrograde signalling molecules, inhibiting via presynaptic cannabinoid CB(1) receptors the release of classical transmitter following release from the postsynaptic cell. At the neuroeffector junction, it is more likely that cannabinoids are released from prejunctional sites, as the neuroeffector junction is wide in some peripheral tissues and cannabinoids are rapidly taken up and inactivated. Understanding the actions of cannabinoids as modulators of peripheral neurotransmission is relevant to a variety of biological systems and possibly their disorders.