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.     

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.     

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.     

Cannabidiol lacks the vanilloid VR1-mediated vasorespiratory effects of capsaicin and anandamide in anaesthetised rats

The results of vasorespiratory studies in rats anaesthetised with pentobarbital show that (+/-) cannabidiol, a cannabinoid that lacks psychotropic actions and is inactive at cannabinoid (CB) receptors, does not affect respiration or blood pressure when injected (1-2000 microg; 3.2-6360 nmol i.a.). Cannabidiol in doses up to 2 mg (6360 nmol) i.a. or i.v. did not affect the fall in mean blood pressure or the increase in ventilation (respiratory minute volume) caused by capsaicin and high doses of anandamide, responses that are mediated by activation of vanilloid VR1 (TRPV1) receptors in this species. Similar results were obtained with (-) cannabidiol (30-100 microg i.a.; 95-318 nmol). It has previously been shown using human embryonic kidney (HEK) cells over-expressing vanilloid human VR1 (hVR1) receptors that cannabidiol is a full agonist at vanilloid VR1 receptors in vitro. However, in the intact rat cannabidiol lacked vanilloid VR1 receptor agonist effects. We conclude that there are substantial functional differences between human and rat vanilloid VR1 receptors with respect to the actions of cannabidiol as an agonist at vanilloid VR1 receptors. Studies in vivo show that cannabidiol lacks any significant effect on mean blood pressure or respiratory minute volume when injected i.a. or i.v., and that this cannabinoid does not modulate the vanilloid VR1 receptor-mediated cardiovascular and ventilatory changes reflexly evoked by capsaicin or anandamide in rats anaesthetised with pentobarbital.     

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.     

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.     

Capsaicin-like effects of N-arachidonoyl-dopamine in the isolated guinea pig bronchi and urinary bladder

A capsaicin-like endogenous ligand of vanilloid (VR1) receptors, N-arachidonoyl-dopamine, was recently identified in bovine and rat nervous tissue, and found to be almost as potent as capsaicin, and 5-10-fold more potent than anandamide, on these receptors, both in isolated cells and in vivo. Here we have investigated if N-arachidonoyl-dopamine also exerts other capsaicin-like effects at VR1 receptors in some isolated organ preparations. N-arachidonoyl-dopamine exerted a potent contractile response of guinea pig isolated bronchi (EC50=12.6 +/- 1.7 microM, Emax=69.2 +/- 2.4% of carbachol Emax), which was blocked by pre-treatment with capsaicin or with the VR1 antagonist capsazepine, as well as by a combination of tachykinin NK1 and NK2 receptor antagonists. In this assay, N-arachidonoyl-dopamine was less and more potent and/or efficacious than capsaicin (EC50=40.0 nM; Emax=93.5%) and anandamide (EC50=15.2 microM, Emax=38.0%), respectively. Unlike capsaicin and anandamide, forskolin or ethanol did not enhance N-arachidonoyl-dopamine effect in this preparation, whereas epithelial denudation resulted in a 2.5-fold increase in potency without affecting the efficacy. N-arachidonoyl-dopamine also contracted the isolated guinea pig urinary bladder, although in this preparation, as well as in the isolated rat urinary bladder, the potency (EC50=3.7 +/- 0.3 and 19.9 +/- 0.1 microM) and/or efficacy (Emax=12.0 +/- 0.1% and 20.7 +/- 0.7% of carbachol Emax) of the compound were significantly lower than those of both capsaicin and anandamide. These data suggest that the extent to which exogenous N-arachidonoyl-dopamine activates VR1 receptor in isolated organs is largely dependent on pharmacodynamics and bioavailability.     

Cloning and functional characterization of the guinea pig vanilloid receptor 1

We have cloned a guinea pig Vanilloid receptor 1 (VR1) from a dorsal root ganglion cDNA library and expressed it in CHO cells. The receptor has been functionally characterized by measuring changes in intracellular calcium produced by capsaicin, low pH and noxious heat.Capsaicin produced a concentration-dependent increase in intracellular calcium in guinea pig VR1-CHO cells with an estimated EC(50) of 0.17 +/- 0.0065 micro M, similar to that previously reported for rat and human VR1. Olvanil and resiniferatoxin were also effective agonists (EC(50) values of 0.0087 +/- 0.0035 micro M and 0.067 +/- 0.014 micro M, respectively), but 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) and anandamide showed little agonist activity up to 10 micro M. As with human and rat VR1, guinea pig VR1 was also activated by pH below 6.0 and by noxious heat (>42 degrees C). Capsazepine acted as an antagonist of capsaicin responses in guinea pig VR1-CHO cells (IC(50) of 0.324 +/- 0.041 micro M ), as seen at rat VR1. However, in contrast to its lack of activity against pH and heat responses at rat VR1, capsazepine was an effective antagonist of these responses at guinea pig VR1. Capsazepine displayed an IC(50) of 0.355 +/- 25 micro M against pH 5.5, and provided complete blockade of heat responses at 1 micro M. Thus, capsazepine can significantly inhibit calcium influx due to heat and pH 5.5 at guinea pig VR1 and human VR1 but is inactive against these activators at rat VR1.     

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.     

Activation of vanilloid receptor 1 by resiniferatoxin mobilizes calcium from inositol 1,4,5-trisphosphate-sensitive stores

1 Capsaicin and resiniferatoxin (RTX) stimulate Ca2+ influx by activating vanilloid receptor 1 (VR1), a ligand-gated Ca2+ channel on sensory neurones. We investigated whether VR1 activation could also trigger Ca2+ mobilization from intracellular Ca2+ stores. 2 Human VR1-transfected HEK293 cells (hVR1-HEK293) were loaded with Fluo-3 or a mixture of Fluo-4 and Fura Red and imaged on a fluorometric imaging plate reader (FLIPR) and confocal microscope respectively. 3 In Ca2+ -free media, RTX caused a transient elevation in intracellular free Ca2+ concentration in hVR1-HEK293 cells (pEC(50) 6.45+/-0.05) but not in wild type cells. Capsaicin (100 microM) did not cause Ca2+ mobilization under these conditions. 4 RTX-mediated Ca2+ mobilization was inhibited by the VR1 receptor antagonist capsazepine (pIC(50) 5.84+/-0.04), the Ca2+ pump inhibitor thapsigargin (pIC(50) 7.77+/-0.04), the phospholipase C inhibitor U-73122 (pIC(50) 5.35+/-0.05) and by depletion of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores by pretreatment with the acetylcholine-receptor agonist carbachol (20 microM, 2 min). These data suggest that RTX causes Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in hVR1-HEK293 cells. 5 In the presence of extracellular Ca2+, both capsaicin-mediated and RTX-mediated Ca2+ rises were attenuated by U-73122 (10 microM, 30 min) and thapsigargin (1 microM, 30 min). We conclude that VR1 is able to couple to Ca2+ mobilization by a Ca2+ dependent mechanism, mediated by capsaicin and RTX, and a Ca2+ independent mechanism mediated by RTX alone.     

Albaconol from the mushroom Albatrellus confluens induces contraction and desensitization in guinea pig trachea

The contraction and desensitization induced by albaconol and the influence of capsazepine, capsaicin and extracellular Ca2+ were investigated to see whether the actions were mediated via a specific VR receptor in guinea pig trachea spiral strips in vitro. Both albaconol and capsaicin were contractors of tracheal smooth muscle, but albaconol was not so potent as capsaicin, with -log (M) EC50 values of 4.23 +/- 0.18 (n = 10) and 7.33 +/- 0.21 (n = 10) respectively. 2.5 microM capsazepine competitively antagonized the contractile response to albaconol and capsaicin. Albaconol increased the contraction induced by a low dose of capsaicin (10(-10) to 10(-9) M), but non-competitively antagonized the contraction induced by a high dose of capsaicin (10(-8) to 10(-3) M). Either albaconol (1 or 100 mM) or capsaicin (3 or 10 microM) was able to desensitize the isolated guinea pig bronchi to subsequent addition of albaconol. Capsazepine (5.0 microM) significantly prevented the desensitization induced by either albaconol (1 or 100mM) or capsaicin (3 or 10 microM). Extracellular Ca2+ was essential for albaconol to induce excitation, but it did not affect albaconol- or capsaicin-induced desensitization. In summary, the results from the present study suggest that albaconol induces contraction and desensitization of guinea pig trachea in vitro as a partial agonist for VR.     

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.     

The anandamide transport inhibitor AM404 activates vanilloid receptors

The possibility that the anandamide transport inhibitor N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM404), structurally similar to the vanilloid receptor agonists anandamide and capsaicin, may also activate vanilloid receptors and cause vasodilation was examined. AM404 evoked concentration-dependent relaxations in segments of rat isolated hepatic artery contracted with phenylephrine. Relaxations were abolished in preparations pre-treated with capsaicin. The calcitonin-gene related peptide (CGRP) receptor antagonist CGRP-(8-37) also abolished relaxations. The vanilloid receptor antagonist capsazepine inhibited vasodilation by AM404 and blocked AM404-induced currents in patch-clamp experiments on Xenopus oocytes expressing the vanilloid subtype 1 receptor (VR1). In conclusion, AM404 activates native and cloned vanilloid receptors.     

Effects of piperine, the pungent component of black pepper, at the human vanilloid receptor (TRPV1)

1. We have characterised the effects of piperine, a pungent alkaloid found in black pepper, on the human vanilloid receptor TRPV1 using whole-cell patch-clamp electrophysiology. 2. Piperine produced a clear agonist activity at the human TRPV1 receptor yielding rapidly activating whole-cell currents that were antagonised by the competitive TRPV1 antagonist capsazepine and the non-competitive TRPV1 blocker ruthenium red. 3. The current-voltage relationship of piperine-activated currents showed pronounced outward rectification (25+/-4-fold between -70 and +70 mV) and a reversal potential of 0.0+/-0.4 mV, which was indistinguishable from that of the prototypical TRPV1 agonist capsaicin. 4. Although piperine was a less potent agonist (EC50=37.9+/-1.9 microM) than capsaicin (EC50=0.29+/-0.05 microM), it demonstrated a much greater efficacy (approximately two-fold) at TRPV1. 5. This difference in efficacy did not appear to be related to the proton-mediated regulation of the receptor since a similar degree of potentiation was observed for responses evoked by piperine (230+/-20%, n=11) or capsaicin (284+/-32%, n=8) upon acidification to pH 6.5. 6. The effects of piperine upon receptor desensitisation were also unable to explain this effect since piperine resulted in more pronounced macroscopic desensitisation (t(1/2)=9.9+/-0.7 s) than capsaicin (t(1/2)>20 s) and also caused greater tachyphylaxis in response to repetitive agonist applications. 7. Overall, our data suggest that the effects of piperine at human TRPV1 are similar to those of capsaicin except for its propensity to induce greater receptor desensitisation and, rather remarkably, exhibit a greater efficacy than capsaicin itself. These results may provide insight into the TRPV1-mediated effects of piperine on gastrointestinal function.     

Pharmacological differences between the human and rat vanilloid receptor 1 (VR1)

Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells. Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with similar EC(50) values. Capsaicin had a lower potency on both channels, when measured electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90+/-0.20 microM; human=1.90+/-0.30 microM: CHO cells: rat=0.20+/-0.06 microM; human=0.19+/-0.08 microM). In CHO cell lines co-expressing either rat or human VR1 and the calcium sensitive, luminescent protein, aequorin, the EC(50) values for capsaicin-induced responses were similar in both cell lines (rat=0.35+/-0.06 microM, human=0.53+/-0.03 microM). The threshold for activation by acidic solutions was lower for human VR1 channels than that for rat VR1 (EC(50) pH 5.49+/-0.04 and pH 5.78+/-0.09, respectively). The threshold for heat activation was identical (42 degrees C) for rat and human VR1. PPAHV was an agonist at rat VR1 (EC(50) between 3 and 10 microM) but was virtually inactive at the human VR1 (EC(50)>10 microM). Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of human VR1 than rat VR1. Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also more effective at inhibiting the noxious heat response of human than of rat VR1.     

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.     

Anandamide transport inhibitor AM404 and structurally related compounds inhibit synaptic transmission between rat hippocampal neurons in culture independent of cannabinoid CB1 receptors

N-(hydroxyphenyl)-arachidonamide (AM404) is an inhibitor of endocannabinoid transport. We examined the effects of AM404 on glutamatergic synaptic transmission using network-driven increases in intracellular Ca2+ concentration ([Ca2+] spikes) as an assay. At a concentration of 1 microM AM404 inhibited [Ca2+]i spiking by 73+/-8%. The cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), the vanilloid VR1 receptor antagonist capsazepine (CPZ), and treatment with pertussis toxin failed to block AM404-mediated inhibition. AM404 (3 microM) inhibited action-potential-evoked Ca2+ influx by 58+/-3% but failed to affect calcium influx evoked by depolarization with 30 mM K+, suggesting that the inhibition of electrically evoked [Ca2+]i increases and that [Ca2+]i spiking was due to inhibition of Na+ channels. Palmitoylethanolamide (PMEA), capsaicin (CAP) and (5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (VDM11), compounds structurally similar to AM404, inhibited [Ca2+]i spiking by 34+/-10%, 42+/-18% and 67+/-12%, respectively. Thus, AM404 and related compounds inhibit depolarization-induced Ca2+ influx independent of cannabinoid receptors, suggesting caution when using these agents as pharmacological probes to study synaptic transmission.     

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.     

Iodo-resiniferatoxin, a new potent vanilloid receptor antagonist

The highly potent vanilloid receptor (VR) agonist resiniferatoxin has been radiolabeled with 125I, and the pharmacology to the cloned rodent VR, VR1, and the endogenous VR in rat spinal cord membranes has been characterized. [125I]RTX binding to human embryonic kidney 293 cells expressing VR1 was reversible and with high affinity (Kd = 4.3 nM) in an apparent monophasic manner. In rat spinal cord membranes, [125I]RTX bound with a similar high affinity (Kd = 4.2 nM) to a limited number of binding sites (Bmax = 51 +/- 8 fmol/mg of protein). The pharmacology of recombinant rodent VR1 and the endogenous rat VR1 was indistinguishable when measuring displacement of [125I]RTX binding (i.e., the following rank order of affinity was observed: RTX > I-RTX > olvanil > capsaicin > capsazepine). Capsaicin and RTX induced large nondesensitizing currents in Xenopus laevis oocytes expressing VR1 (EC50 values were 1300 nM and 0.2 nM, respectively), whereas I-RTX induced no current per se at concentrations up to 10 microM. However, I-RTX completely blocked capsaicin-induced currents (IC50 = 3.9 nM). In vivo, I-RTX effectively blocked the pain responses elicited by capsaicin (ED50 = 16 ng/mouse, intrathecally). The present study showed that I-RTX is at least 40-fold more potent than the previously known VR antagonist, capsazepine. Thus, I-RTX as well as its radiolabeled form, should be highly useful for further exploring the physiological roles of VRs in the brain and periphery.     

Evidence of a novel site mediating anandamide-induced negative inotropic and coronary vasodilatator responses in rat isolated hearts

1. Cannabinoids are known to cause coronary vasodilatation and reduce left ventricular developed pressure (LVDP) in isolated hearts although the identity of the receptor(s) mediating these responses is unknown. Our objective was to pharmacologically characterize cannabinoid receptors mediating cardiac responses to the endocannabinoid, anandamide. 2. Dose-response curves for coronary perfusion pressure (CPP) and LVDP were constructed to anandamide, R-(+)-methanandamide, palmitoylethanolamide (PEA) and JWH015 in isolated Langendorff-perfused rat hearts. Anandamide dose-response curves were also constructed in the presence of antagonists selective for CB(1), CB(2) or VR(1) receptors. 3. Anandamide and methanadamide significantly reduced CPP and LVDP but the selective CB(2) receptor agonists, PEA and JWH015 had no significant effect, compared with equivalent vehicle doses. 4. Single bolus additions of the selective CB(1)-receptor agonist, ACEA (5 nmol), decreased LVDP and CPP. When combined with JWH015 (5 nmol) these responses were not augmented. 5. Anandamide-mediated reductions in CPP were significantly blocked by the selective CB(1) receptor antagonists SR 141716A (1 microM) and AM251 (1 microM) and the selective CB(2) receptor antagonist SR 144528 (1 microM) but not by another selective CB(2) receptor antagonist AM630 (10 microM) nor the vanilloid VR(1) receptor antagonist capsazepine (10 microM). 6. SR 141716A, AM281 and SR 144528 significantly blocked negative inotropic responses to anandamide that were not significantly affected by AM251, AM630 and capsazepine. 7. One or more novel sites mediate negative inotropic and coronary vasodilatatory responses to anandamide. These sites can be distinguished from classical CB(1) and CB(2) receptors, as responses are sensitive to both SR 141716A and SR 144528.