Anandamide is a partial agonist at native vanilloid receptors in acutely isolated mouse trigeminal sensory neurons

1. The endogenous fatty acid anandamide (AEA) is a partial agonist at cannabinoid CB1 receptors and has been reported to be a full agonist at the recombinant vanilloid receptor, VR1. 2. Whole cell voltage clamp techniques were used to examine the efficacy of AEA and related analogues methanandamide and N-(4-hydroxyphenyl)-arachidonylamide (AM404) at native VR1 receptors in acutely isolated mouse trigeminal neurons. 3. Superfusion of the VR1 agonist capsaicin onto small trigeminal neurons voltage clamped at +40 mV produced outward currents in most cells, with a pEC(50) of 6.3+/-0.1 (maximum currents at 10-30 micro M). 4. AEA produced outward currents with a pEC(50) of 5.6+/-0.1. Maximal AEA currents (30-100 micro M) were 38+/-2% of the capsaicin maximum. AEA currents were blocked by the VR1 antagonist capsazepine (30 micro M), but unaffected by the CB1 antagonist SR141716A (1 micro M). 5. Methanandamide and AM404 were less potent than AEA at activating VR1. Methanandamide (100 micro M) produced currents 37+/-6% of the capsaicin maximum, the highest concentration of AM404 tested (100 micro M) produced currents that were 55+/-9% of the capsaicin maximum. 6. Capsazepine abolished the currents produced by AM404 (100 micro M) and strongly attenuated (>70%) those produced by methanandamide (100 micro M). 7. Co-superfusion of AEA (30 micro M, methanandamide (100 micro M) or AM404 (100 micro M) with capsaicin (3 micro M) resulted in a significant reduction of the capsaicin current. 8. These data indicate that AEA, methanandamide and AM404 activate native VR1 receptors, but that all three compounds are partial agonists when compared with capsaicin.     

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.     

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.     

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.     

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.     

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 acts as a vasodilator of dural blood vessels in vivo by activating TRPV1 receptors

Migraine pathophysiology is believed to involve the release of neuropeptides via the activation of trigeminal afferents that innervate the cranial vasculature. Anandamide, the endogenous ligand to the cannabinoid receptor, is able to inhibit neurogenic dural vasodilatation, calcitonin gene-related peptide (CGRP)-induced and nitric oxide-induced dural vessel dilation in the intravital microscopy model. In an in vitro setting anandamide is also able to activate the vanilloid type 1 (TRPV1) receptor and cause vasodilation, via the release of CGRP. In this study we used intravital microscopy to study whether anandamide behaves as a TRPV1 receptor agonist in the trigeminovascular system. We examined if anandamide-induced dural vasodilation involves CGRP release that can be reversed by the CGRP receptor antagonist, CGRP(8-37), and whether like capsaicin the anandamide effect could be reversed by the TRPV1 receptor antagonist, capsazepine. Anandamide 1 (19+/-9%, n=12), 3 (29+/-5%, n=37), 5 (74+/-7%, n=13) and 10 mg kg(-1) (89+/-18%, n=6) was able to cause a dose-dependent increase in dural vessel diameter. Capsazepine (3 mg kg(-1), t(5)=6.2, P<0.05) and CGRP(8-37) (300 micrograms kg(-1), t(6)=11.1, P<0.05) attenuated the anandamide-induced dural vessel dilation when compared to control (Student's paired t-test). AM251 (3 mg kg(-1)), a cannabinoid type 1 (CB(1)) receptor antagonist, was unable to reverse this anandamide-induced dilation. The study demonstrates that anandamide acts as a TRPV1 receptor agonist in the trigeminovascular system, activating TRPV1 receptors that promote CGRP release and cause vasodilation independent of any action at the CB(1) receptor. Anandamide has been shown previously to inhibit trigeminovascular neurons and prevent vasodilation, through an action at CB(1) receptors.     

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.     

AM404, an inhibitor of anandamide reuptake decreases Fos-immunoreactivity in the spinal cord of neuropathic rats after non-noxious stimulation

Cannabinoids like anandamide are involved in pain transmission. In this study we evaluated the effects of administrating N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404), an inhibitor of anandamide reuptake and monitoring the expression of c-fos, a marker of activated neurons in an experimental model of neuropathic pain (sciatic nerve tying). Fos expression was monitored 14 days after tying of sciatic nerve and 2 h after non-noxious stimulation. We showed that non-noxious stimulation increased Fos-positivity in the dorsal superficial laminae of the lumbar spinal cord of tied animals but not in the control animals. AM404 significantly reduced Fos induction in tied animals. Co-administration of cannabinoid CB1 receptor, cannabinoid CB2 receptor and transient receptor potential vanilloid type 1 (TRPV-1) antagonists reduced the effect of AM404 and this reduction was higher using cannabinoid CB1 receptor antagonist. These results suggest that AM404 could be a useful drug to reduce neuropathic pain and that cannabinoid CB1 receptor, cannabinoid CB2 receptor and vanilloid TRPV-1 receptor are involved.     

Structure-activity relationship for the endogenous cannabinoid, anandamide, and certain of its analogues at vanilloid receptors in transfected cells and vas deferens

1. This study was directed at exploring the structure-activity relationship for anandamide and certain of its analogues at the rat VR1 receptor in transfected cells and at investigating the relative extent to which anandamide interacts with CB(1) and vanilloid receptors in the mouse vas deferens. 2. pK(i) values for displacement of [(3)H]-resiniferatoxin from membranes of rVR1 transfected CHO cells were significantly less for anandamide (5.78) than for its structural analogues N-(4-hydroxyphenyl)-arachidonylamide (AM404; 6.18) and N-(3-methoxy-4-hydroxy)benzyl-arachidonylamide (arvanil; 6.77). 3. pEC(50) values for stimulating (45)Ca(2+) uptake into rVR1 transfected CHO cells were significantly less for anandamide (5.80) than for AM404 (6.32) or arvanil (9.29). Arvanil was also significantly more potent than capsaicin (pEC(50)=7.37), a compound with the same substituted benzyl polar head group as arvanil. 4. In the mouse vas deferens, resiniferatoxin was 218 times more potent than capsaicin as an inhibitor of electrically-evoked contractions. Both drugs were antagonized to a similar extent by capsazepine (pK(B)=6.93 and 7.18 respectively) but were not antagonized by SR141716A (1 microM). Anandamide was less susceptible than capsaicin to antagonism by capsazepine (pK(B)=6.02) and less susceptible to antagonism by SR141716A (pK(B)=8.66) than methanandamide (pK(B)=9.56). WIN55212 was antagonized by SR141716A (pK(B)=9.02) but not by capsazepine (10 microM). 5. In conclusion, anandamide and certain of its analogues have affinity and efficacy at the rat VR1 receptor. In the mouse vas deferens, which seems to express vanilloid and CB(1) receptors, both receptor types appear to contribute to anandamide-induced inhibition of evoked contractions.     

The endogenous cannabinoid anandamide activates vanilloid receptors in the rat hippocampal slice

We have previously reported that the synthetic cannabinoid receptor agonist WIN55,212-2 causes a selective reduction in paired-pulse depression of population spikes in the CA1 region of the rat hippocampal slice. This effect is consistent with the observation that activation of cannabinoid receptors inhibits GABA release in the hippocampus. We have now investigated the actions of the putative endogenous cannabinoids 2-arachidonoyl-glycerol (2-AG) and anandamide in this system. 2-AG mimicked the effect of WIN55,212-2 by selectively reducing paired-pulse depression at concentrations of 1–30 μM. In contrast, anandamide caused a selective increase in paired-pulse depression at concentrations of 1–30 μM. This effect was mimicked by the vanilloid receptor agonists capsaicin and resiniferatoxin, and blocked by the vanilloid receptor antagonist capsazepine, but not by the cannabinoid receptor antagonist AM281. These results are the first to demonstrate a clear functional vanilloid receptor-mediated effect in the hippocampus, and further, that anandamide but not 2-AG acts at these receptors to increase paired-pulse depression of population spikes.     

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.     

Sex-linked differences in the vasorelaxant effects of anandamide in vascular mesenteric beds: role of oestrogens

Anandamide (0.01 to 10 microM) caused greater concentration-dependent reductions of the contractile-induced responses to noradrenaline in female than in male mesenteric vascular beds isolated from adult Sprague-Dawley rats. Greater relaxant responses in females were also induced by the vanilloid TRPV1 receptor agonist capsaicin (0.01 to 10 microM), whereas no sex differences were observed for the relaxations caused by either acetylcholine or sodium nitroprusside. The effect of anandamide in either sex was reduced by the vanilloid TRPV1 receptor antagonist capsazepine but not by the cannabinoid CB1 receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716A). In males, the anandamide-induced relaxations were potentiated by in vitro exposure during 5 min to 0.5 microM 17beta-oestradiol and unmodified by the protein synthesis inhibitor cycloheximide. The vasorelaxant effects of anandamide in female rats were decreased by ovariectomy. This decrease was prevented by in vivo treatment with 17beta-oestradiol-3-benzoate (450 microg/kg i.m., once a week during 3 weeks) and counteracted by in vitro exposure to oestrogen. In vivo treatment with 17beta-oestradiol also potentiated anandamide-induced responses in males. In conclusion, this study shows an oestrogen-dependent sensitivity to the vanilloid TRPV1 receptor-mediated vasorelaxant effects of anandamide in the mesenteric vasculature of Sprague-Dawley rats, that could be mediated by both genomic and non-genomic mechanisms.     

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.     

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.     

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.     

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.     

Role of TRPV1 and cannabinoid CB1 receptors in AM 404-evoked hypothermia in rats

AM 404 inhibits endocannabinoid uptake and enhances the cannabinoid CB(1)-mediated effects of endogenous cannabinoids. Accumulating evidence also suggests that AM 404 acts at sites other than the endocannabinoid system. One site is the transient receptor potential vanilloid 1 cation channel (TRPV1). A useful endpoint for discriminating between TRPV1- or CB(1)-mediated effects of AM 404 is hypothermia. This is because TRPV1 or CB(1) receptor activation produces a significant hypothermia in rats. The present study investigated the effects of AM 404 (1, 5, 10 and 20 mg/kg, i.p.) on body temperature in rats and the involvement of TRPV1 and CB(1) receptors in the effects of AM 404. Doses of 10 and 20 mg/kg of AM 404 produced significant hypothermia. Pre-treatment with capsazepine (30 mg/kg, i.p.) blocked the hypothermia caused by 10 and 20 mg/kg of AM 404. Pre-treatment with SB 366791 (2 mg/kg, i.p.), a new TRPV1 antagonist, also abolished the hypothermia evoked by AM 404 (20 mg/kg, i.p.). In contrast, pre-treatment with SR 141716A (Rimonabant), a CB(1) antagonist, or AA-5-HT, a fatty acid amide hydrolase (FAAH) blocker, did not affect AM 404-evoked hypothermia. The present data demonstrate that AM 404 evokes a significant hypothermia in rats that is dependent on TRPV1 receptor activation.     

Long-term inhibition of nitric oxide synthase potentiates effects of anandamide in the rat mesenteric bed

In rat isolated mesenteric beds, anandamide induced a concentration-dependent reduction (0.01-50 microM) of the contractile responses elicited by bolus administration of noradrenaline. The anandamide-induced reductions of noradrenaline responses were unmodified by the in vitro exposure to the nitric oxide synthase (NOS) inhibitor, 100 microM L-N(G)-nitro-L-arginine methyl ester (L-NAME), whereas they were significantly potentiated after the long-term in vivo administration of L-NAME (70 mg/kg/day during 4 weeks). Responses to anandamide were not potentiated and even reduced in mesenteric beds from rats made hypertensive by aortic coarctation. In mesenteric beds isolated from either untreated or in vivo L-NAME treated rats, concentration-response curves to anandamide were significantly attenuated by the non-selective K+ channel blocker tetraethylammonium (TEA) but were not modified by either endothelium removal, or the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) or the cannabinoid receptor antagonists 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl] (4-methoxyphenyl) methanone (AM630) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281). On the other hand, the vanilloid receptor agonist (E)-N-[4-hydroxy-3-methoxyphenyl)methyl]-8-methyl-6-nonenamide (capsaicin) induced a concentration-dependent inhibition of noradrenaline-induced vasoconstriction, and the vanilloid receptor antagonist N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide (capsazepine) caused a significant reduction of anandamide-induced responses in mesenteric beds isolated from both control and chronic L-NAME treated rats. The non-metabolizable analogue of anandamide, methanandamide, produced higher reductions of noradrenaline responses than anandamide in mesenteric beds isolated from controls but not from the L-NAME treated rats. Moreover, in mesenteric beds from untreated but not from L-NAME treated rats, the effects of anandamide were significantly potentiated by the inhibitor of endocannabinoid degradation, 200 microM phenylmethylsulphonyl fluoride (PMSF), and by the inhibitor of anandamide uptake, 5 microM (all Z)-N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM404). It is concluded that long-term inhibition of NOS potentiates anandamide-induced relaxations probably through changes in either endocannabinoid metabolism or uptake. A possible compensatory role for endocannabinoids in vascular function in situations in which nitric oxide (NO) synthesis is long-term impaired arises from the present results.     

Characterisation using FLIPR of human vanilloid VR1 receptor pharmacology

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.