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 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.     

Possible mechanisms of cannabinoid-induced antinociception in the spinal cord.

Anandamide is an endogenous ligand at both the inhibitory cannabinoid CB(1) receptor and the excitatory vanilloid receptor 1 (VR1). The CB(1) receptor and vanilloid VR1 receptor are expressed in about 50% and 40% of dorsal root ganglion neurons, respectively. While all vanilloid VR1 receptor-expressing cells belong to the calcitonin gene-related peptide-containing and isolectin B4-binding sub-populations of nociceptive primary sensory neurons, about 80% of the cannabinoid CB(1) receptor-expressing cells belong to those sub-populations. Furthermore, all vanilloid VR1 receptor-expressing cells co-express the cannabinoid CB(1) receptor. In agreement with these findings, neonatal capsaicin treatment that induces degeneration of capsaicin-sensitive, vanilloid VR1 receptor-expressing, thin, unmyelinated, nociceptive primary afferent fibres significantly reduced the cannabinoid CB(1) receptor immunostaining in the superficial spinal dorsal horn. Synthetic cannabinoid CB(1) receptor agonists, which do not have affinity at the vanilloid VR1 receptor, and low concentrations of anandamide both reduce the frequency of miniature excitatory postsynaptic currents and electrical stimulation-evoked or capsaicin-induced excitatory postsynaptic currents in substantia gelatinosa cells in the spinal cord without any effect on their amplitude. These effects are blocked by selective cannabinoid CB(1) receptor antagonists. Furthermore, the paired-pulse ratio is increased while the postsynaptic response of substantia gelatinosa neurons induced by alpha-amino-3-hydroxy-5-methylisoxasole-propionic acid (AMPA) in the presence of tetrodotoxin is unchanged following cannabinoid CB(1) receptor activation. These results strongly suggest that the cannabinoid CB(1) receptor is expressed presynaptically and that the activation of these receptors by synthetic cannabinoid CB(1) receptor agonists or low concentration of anandamide results in inhibition of transmitter release from nociceptive primary sensory neurons. High concentrations of anandamide, on the other hand, increase the frequency of miniature excitatory postsynaptic currents recorded from substantia gelatinosa neurons. This increase is blocked by ruthenium red, suggesting that this effect is mediated through the vanilloid VR1 receptor. Thus, anandamide at high concentrations can activate the VR1 and produce an opposite, excitatory effect to its inhibitory action produced at low concentrations through cannabinoid CB(1) receptor activation. This "dual", concentration-dependent effect of anandamide could be an important presynaptic modulatory mechanism in the spinal nociceptive system.     

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.     

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

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

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Vanilloid receptor ligands: hopes and realities for the future

Neurons possessing C-fibers transmit nociceptive information into the central nervous system and participate in various reflex responses. These neurons carry receptors that bind capsaicin, recently identified as the vanilloid VR1 receptor. Excitation of these cells by capsaicin is followed by a lasting refractory state, termed desensitisation, in which the neurons fail to respond to a variety of noxious stimuli. Desensitisation to capsaicin has a clear therapeutic potential in relieving neuropathic pain and ameliorating urinary bladder overactivity, just to cite 2 important examples. Vanilloids may also be beneficial in the treatment of benign prostate hyperplasia (BPH). Since the majority of elderly patients have neuropathic pain co-existent with urinary incontinence and/or BPH, a drug that ameliorates pain and improves urinary symptoms at the same time promises to be of great clinical value in geriatric medicine. In fact, capsaicin has already been shown to have a role in the treatment of conditions that can arise in the elderly, including herpes zoster-related neuropathic pain, diabetic neuropathy, postmastectomy pain, uraemic itching associated with renal failure, and urinary incontinence. The potent VR1 agonist resiniferatoxin, now in phase II clinical trials, appears to be superior to capsaicin in terms of its tolerability profile. Recent discoveries enhance the therapeutic potential of vanilloids. The recognition that VR1 also functions as a principal receptor for protons and eicosanoids implies that VR1 antagonists may be of value in the treatment of inflammatory hyperalgesia and pain. Animal experimentation has already lent support to this assumption. The discovery of VR1-expressing cells in the brain as well as in non-neural tissues such as the kidney and urothelium places VR1 in a much broader perspective than peripheral pain perception, and is hoped to identify further, yet unsuspected, indications for vanilloid therapy. The realisation that VR1 and cannabinoid CB1 receptors have overlapping ligand recognition properties may also have far-reaching implications for vanilloid therapy. In fact, arvanil, a combined agonist of VR1 and CB1 receptors, has already proved to be a powerful analgesic drug in the mouse. From academic molecular biology laboratories to industrial drug discovery centres to the clinics, there is a steady flow of new data, forcing us to constantly revise the ways we are thinking about vanilloid receptor ligands and their hopes and realities for the future. This review covers the most promising current trends in vanilloid research with special emphasis on geriatric medicine.     

Pharmacological and molecular characterization of a dorsal root ganglion cell line expressing cannabinoid CB(1) and CB(2) receptors

The behavioral effects evoked by cannabinoids are primarily mediated by the CB(1) and CB(2) cannabinoid receptor subtypes. In vitro pharmacology of cannabinoid receptors has been elucidated using recombinant expression systems expressing either CB(1) or CB(2) receptors, with limited characterization in native cell lines endogenously expressing both CB(1) and CB(2) receptors. In the current study, we report the molecular and pharmacological characterization of the F-11 cell line, a hybridoma of rat dorsal root ganglion neurons and mouse neuroblastoma (N18TG2) cells, reported to endogenously express both cannabinoid receptors. The present study revealed that both receptors are of mouse origin in F-11 cells, and describes the relative gene expression levels between the two receptors. Pharmacological characterization of the F-11 cell line using cannabinoid agonists and antagonists indicated that the functional responses to these cannabinoid ligands are mainly mediated by CB(1) receptors. The non-selective cannabinoid ligands CP 55,940 and WIN 55212-2 are potent agonists and their efficacies in adenylate cyclase and MAPK assays are inhibited by the CB(1) selective antagonist SR141716A (SR1), but not by the CB(2) selective antagonist SR144528 (SR2). The endocannabinoid ligand 2AG, although not active in adenylate cyclase assays, was a potent activator of MAPK signaling in F-11 cells. The analysis of CB(1) and CB(2) receptor gene expression and the characterization of cannabinoid receptor pharmacology in the F-11 cell line demonstrate that it can be used as a tool for interrogating the endogenous signal transduction of cannabinoid receptor subtypes.     

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.     

Cannabinoid modulation of peripheral autonomic and sensory neurotransmission

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

Mechanisms underlying tissue selectivity of anandamide and other vanilloid receptor agonists

Anandamide acts as a full vanilloid receptor agonist in many bioassay systems, but it is a weak activator of primary afferents in the airways. To address this discrepancy, we compared the effect of different vanilloid receptor agonists in isolated airways and mesenteric arteries of guinea pig using preparations containing different phenotypes of the capsaicin-sensitive sensory nerve. We found that anandamide is a powerful vasodilator of mesenteric arteries but a weak constrictor of main bronchi. These effects of anandamide are mediated by vanilloid receptors on primary afferents and do not involve cannabinoid receptors. Anandamide also contracts isolated lung strips, an effect caused by the hydrolysis of anandamide and subsequent formation of cyclooxygenase products. Although capsaicin is equally potent in bronchi and mesenteric arteries, anandamide, resiniferatoxin, and particularly olvanil are significantly less potent in bronchi. Competition experiments with the vanilloid receptor antagonist capsazepine did not provide evidence of vanilloid receptor heterogeneity. Arachidonoyl-5-methoxytryptamine (VDM13), an inhibitor of the anandamide membrane transporter, attenuates responses to olvanil and anandamide, but not capsaicin and resiniferatoxin, in mesenteric arteries. VDM13 did not affect responses to these agonists in bronchi, suggesting that the anandamide membrane transporter is absent in this phenotype of the sensory nerve. Computer simulations using an operational model of agonism were consistent, with differences in intrinsic efficacy and receptor content being responsible for the remaining differences in agonist potency between the tissues. This study describes differences between vanilloid receptor agonists regarding tissue selectivity and provides a conceptual framework for developing tissue-selective vanilloid receptor agonists devoid of bronchoconstrictor activity.     

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.     

Non-cannabinoid CB1, non-cannabinoid CB2 antinociceptive effects of several novel compounds in the PPQ stretch test in mice

The analgesic and anti-hyperalgesic effects of cannabinoid- and vanilloid-like compounds, plus the fatty acid amide hydrolase (FAAH) inhibitor Cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597), and acetaminophen, were evaluated in the phenyl-p-quinone (PPQ) pain model, using different routes of administration in combination with opioid and cannabinoid receptor antagonists. All the compounds tested produced analgesic effects. Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and (R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide ((R)-methanandamide) were active by three routes of administration: i.p., s.c. and, p.o. Delta(9)-THC produced ED(50)s of 2.2 mg/kg (0.3-15.6) i.p., 9 mg/kg (4.3-18.9) s.c., and 6.4 mg/kg (5.5-7.6) p.o. Similarly, (R)-methanandamide yielded ED(50)s of 2.9 mg/kg (1-8) i.p., 11 mg/kg (7-17) s.c., and 11 mg/kg (0.9-134) p.o. N-vanillyl-arachidonyl-amide (arvanil) was active by two routes, producing ED(50)s of 4.7 mg/kg (3.0-7.4) s.c. and 0.06 mg/kg (0.02-0.2) i.p. Palmitoylethanolamide, URB597, and acetaminophen were active i.p., resulting in ED(50)s of 3.7 mg/kg (3.2-4.2), 22.9 mg/kg (11.1-47.2), and 160 mg/kg (63-405), respectively. None of the cannabinoid or opioid receptor antagonists tested blocked the compounds evaluated, with two exceptions: the antinociceptive effects of Delta(9)-THC and URB597 were completely blocked by SR141716A, a cannabinoid CB(1) receptor antagonist. Western immunoassays performed using three opioid receptor antibodies, a cannabinoid CB(1) receptor antibody and a transient receptor potential vanilloid type 1(TRPV(1)) receptor antibody, yielded no change in receptor protein levels after short-term arvanil, (R)-methanandamide or Delta(9)-THC administration. These data suggest that all the compounds tested, except Delta(9)-THC and URB597, produced analgesia via a non-cannabinoid CB(1), non-cannabinoid CB(2) pain pathway not yet identified.     

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.