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.     

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.     

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.     

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.     

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

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

Evidence that the plant cannabinoid Delta9-tetrahydrocannabivarin is a cannabinoid CB1 and CB2 receptor antagonist

Delta9-tetrahydrocannabivarin (THCV) displaced [(3)H]CP55940 from specific binding sites on mouse brain and CHO-hCB(2) cell membranes (K(i)=75.4 and 62.8 nM, respectively).THCV (1 microM) also antagonized CP55940-induced stimulation of [(35)S]GTPgammaS binding to these membranes (apparent K(B)=93.1 and 10.1 nM, respectively).In the mouse vas deferens, the ability of Delta9-tetrahydrocannabinol (THC) to inhibit electrically evoked contractions was antagonized by THCV, its apparent K(B)-value (96.7 nM) approximating the apparent K(B)-values for its antagonism of CP55940- and R-(+)-WIN55212-induced stimulation of [(35)S]GTPgammaS binding to mouse brain membranes. THCV also antagonized R-(+)-WIN55212, anandamide, methanandamide and CP55940 in the vas deferens, but with lower apparent K(B)-values (1.5, 1.2, 4.6 and 10.3 nM, respectively).THCV (100 nM) did not oppose clonidine, capsaicin or (-)-7-hydroxy-cannabidiol-dimethylheptyl-induced inhibition of electrically evoked contractions of the vas deferens.Contractile responses of the vas deferens to phenylephrine hydrochloride or beta,gamma-methylene-ATP were not reduced by 1microM THCV or R-(+)-WIN55212, suggesting that THCV interacts with R-(+)-WIN55212 at prejunctional sites.At 32 microM, THCV did reduce contractile responses to phenylephrine hydrochloride and beta,gamma-methylene-ATP, and above 3 microM it inhibited electrically evoked contractions of the vas deferens in an SR141716A-independent manner.In conclusion, THCV behaves as a competitive CB(1) and CB(2) receptor antagonist. In the vas deferens, it antagonized several cannabinoids more potently than THC and was also more potent against CP55940 and R-(+)-WIN55212 in this tissue than in brain membranes. The bases of these agonist- and tissue-dependent effects remain to be established.     

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.     

Pharmacological characterisation of cannabinoid CB(1) receptors in the rat and mouse

The role of cannabinoid CB(1) receptors in sympathetic neurotransmission was characterised in nerve-mediated responses of isolated right atria, vasa deferentia and small mesenteric resistance arteries using the cannabinoid CB(1) receptor agonists Delta(9)-tetrahydrocannabinol, CP 55,940 and anandamide and the cannabinoid CB(1)-selective antagonist SR 141716A. In the mouse vas deferens, the twitch response was completely inhibited by each of the putative cannabinoid receptor agonists with pIC(50) values of CP 55,940, 9.2+/-0.1; Delta(9)-tetrahydrocannabinol, 8.4+/-0.1; anandamide, 7.1+/-0.1. SR 141716A 10-100 nM was a competitive antagonist of all three agonists with a pK(B) value of 8.4-8.6, consistent with an interaction at the cannabinoid CB(1) receptor. In the rat vas deferens CP 55,940 (0.01-10 microM) inhibited the contractions to a significant extent (88.5+/-0.5% at 10 microM; pIC(50) of 7.1+/-0.1) while Delta(9)-tetrahydrocannabinol and anandamide (both up to 10 microM) were inactive. CP 55,940 exhibited low potency in rat compared with mouse vas deferens and the rat concentration-response curve was not competitively antagonised by SR 141716A (100 nM) or SR 144528 (10 nM-10 microM), suggesting an interaction at a receptor(s) distinct from cannabinoid CB(1) or CB(2). Sympathetic nerve-induced tachycardia in rat and mouse atria, and rat mesenteric artery smooth muscle contractile responses to perivascular nerve stimulation, were not inhibited by Delta(9)-tetrahydrocannabinol, CP 55,940 or anandamide up to 1 microM. These data indicate that cannabinoid CB(1) receptor activation inhibits sympathetic neurotransmission only in the mouse vas deferens and thus point to species and regional differences in cannabinoid CB(1) receptor involvement in pre-synaptic inhibition of sympathetic neurotransmission and CP 55,940 may have inhibitory actions in rat vas deferens unrelated to cannabinoid receptor activity.     

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.     

High affinity antagonists of the vanilloid receptor

The vanilloid receptor VR1 has attracted great interest as a sensory transducer for capsaicin, protons, and heat, and as a therapeutic target. Here we characterize two novel VR1 antagonists, KJM429 [N-(4-tert-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea] and JYL1421 [N-(4-tert-butylbenzyl)-N'-[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea], with enhanced activity compared with capsazepine on rat VR1 expressed in Chinese hamster ovary (CHO) cells. JYL1421, the more potent of the two novel antagonists, inhibited [(3)H]resiniferatoxin binding to rVR1 with an affinity of 53.5 +/- 6.5 nM and antagonized capsaicin-induced calcium uptake with an EC(50) of 9.2 +/- 1.6 nM, reflecting 25- and 60-fold greater potencies than capsazepine. Both JYL1421 and KJM429 antagonized RTX as well as capsaicin and their mechanism was competitive. The responses to JYL1421 and KJM429 differed for calcium uptake by rVR1 induced by heat or pH. JYL1421 antagonized the response to both pH 6.0 and 5.5, whereas KJM429 antagonized at pH 6.0 but was an agonist at lower pH (<5.5). For heat, JYL1421 fully antagonized and KJM429 partially antagonized. Capsazepine showed only weak antagonism for both pH and heat. Responses of rVR1 to different activators could thus be differentially affected by different ligands. In cultured dorsal root ganglion neurons, JYL1421 and KJM429 likewise behaved as antagonists for capsaicin, confirming that the antagonism is not limited to heterologous expression systems. Finally, JYL1421 and KJM429 had little or no effect on ATP-induced calcium uptake in CHO cells lacking rVR1, unlike capsazepine. We conclude that JYL1421 is a competitive antagonist of rVR1, blocking response to all three of the agonists (capsaicin, heat, and protons) with enhanced potency relative to capsazepine.     

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.     

Characterization of the anandamide induced depolarization of guinea-pig isolated vagus nerve

1. There is considerable interest in elucidating potential endogenously derived agonists of the vanilloid receptor and the role of anandamide in this regard has received considerable attention. In the present study, we have used an electrophysiological technique to investigate the mechanism of activation of vanilloid receptors in an isolated vagal preparation. 2. Both capsaicin and anandamide depolarized de-sheathed whole vagal nerve preparations that was antagonized by the VR1 antagonist, capsazepine (P<0.05) whilst this response was unaltered by the cannabinoid (CB1) selective antagonist SR141716A or the CB2 selective antagonist, SR144528, thereby ruling out a role for cannabinoid receptors in this response. 3. The PKC activator, phorbol-12-myristate-13-acetate (PMA) augmented depolarization to both anandamide and capsaicin and this response was significantly inhibited with the PKC inhibitor, bisindolylmaleimide (BIM) (P<0.05). 4. The role of lipoxygenase products in the depolarization to anandamide was investigated in the presence of the lipoxygenase inhibitor, 5,8,11-Eicosatriynoic acid (ETI). Depolarization to anandamide and arachidonic acid was significantly inhibited in the presence of ET1 (P<0.05). However, in the absence of calcium depolarization to anandamide was not inhibited by ETI. 5. Using confocal microscopy we have demonstrated the presence of vanilloid receptors on both neuropeptide containing nerves and nerves that did not stain for sensory neuropeptides. 6. These results demonstrate that anandamide evokes depolarization of guinea-pig vagus nerve, following activation of vanilloid receptors, a component of which involves the generation of lipoxygenase products. Furthermore, these receptors are distributed in both neuropeptide and non-neuropeptide containing nerves.     

Pharmacological Characterization of the Vanilloid Receptor in the Rat Isolated Vas Deferens

The present study set out to further characterize the vanilloid receptor in the rat isolated vas deferens. In this preparation, both capsaicin and resiniferatoxin (RTX) evoked a concentration-dependent inhibition in the amplitude of electrically-evoked contractions with pEC50 values of 7.62 ± 0.03 and 12.2 ± 0.21 respectively. Responses to capsaicin were fast in onset and faded rapidly over a 30-min exposure period, whereas those to RTX were slow in onset and well maintained, an observation believed to reflect pharmacokinetic differences in the rate of penetration to the vanilloid receptor. Responses to both agonists showed mutual cross-desensitization and were antagonized by both the vanilloid-receptor antagonist capsazepine and the ion-channel blocker ruthenium red. The capsaicin analogue, olvanil failed to either mimic or antagonize capsaicin-evoked responses in the rat isolated vas deferens, an effect at variance with previous observations in other tissues. The reason for these differences is unclear, but the possibility of multiple classes of receptor cannot at this stage be ruled out.     

Temperature-dependent activation of recombinant rat vanilloid VR1 receptors expressed in HEK293 cells by capsaicin and anandamide.

Capsaicin activates vanilloid (VR1) receptors found on sensory neurons. These ligand-gated ion channels are also sensitive to low pH, elevated temperature and the endocannabinoid, anandamide. In this study, we have measured capsaicin- and anandamide-induced elevations in intracellular calcium concentrations ([Ca(2+)](i)) in fura-2 loaded HEK293 cells stably expressing the rat VR1 receptor at 22, 37 and 50 degrees C. Both capsaicin and anandamide produced a concentration-dependent elevation in [Ca(2+)](i) at all temperatures. pEC(50) values were 7.74 and 5.69 at 22 degrees C and 6.90 and 5.15 at 37 degrees C for capsaicin and anandamide, respectively. At 50 degrees C, the pEC(50) value for capsaicin was 6.36 but the response to anandamide did not saturate. Responses to both agonists were sensitive to ruthenium red and capsazepine at all temperatures. This temperature-dependent reduction in potency may result from desensitization.     

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 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 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.     

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.     

The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1)

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.     

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.