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.     

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.     

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.     

Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin.

1. Capsazepine is a synthetic analogue of the sensory neurone excitotoxin, capsaicin. The present study shows the capsazepine acts as a competitive antagonist of capsaicin. 2. Capsazepine (10 microM) reversibly reduced or abolished the current response to capsaicin (500 nM) of voltage-clamped dorsal root ganglion (DRG) neurones from rats. In contrast, the responses to 50 microM gamma-aminobutyric acid (GABA) and 5 microM adenosine 5'-triphosphate (ATP) were unaffected. 3. The effects of capsazepine were examined quantitatively with radioactive ion flux experiments. Capsazepine inhibited the capsaicin (500 nM)-induced 45Ca2+ uptake in cultures of rat DRG neurones with an IC50 of 420 +/- 46 nM (mean +/- s.e.mean, n = 6). The 45Ca2+ uptake evoked by resiniferatoxin (RTX), a potent capsaicin-like agonist was also inhibited. (Log concentration)-effect curves for RTX (0.3 nM-1 microM) were shifted in a competitive manner by capsazepine. The Schild plot of the data had a slope of 1.08 +/- 0.15 (s.e.) and gave an apparent Kd estimate for capsazepine of 220 nM (95% confidence limits, 57-400 nM). 4. Capsazepine also inhibited the capsaicin- and RTX-evoked efflux of 86Rb+ from cultured DRG neurones. The inhibition appeared to be competitive and Schild plots yielded apparent Kd estimates of 148 nM (95% confidence limits, 30-332 nM) with capsaicin as the agonist and 107 nM (95% confidence limits, 49-162 nM) with RTX as agonist. 5. A similar competitive inhibition by capsazepine was seen for capsaicin-induced [14C]-guanidinium efflux from segments of adult rat vagus nerves (apparent Kd = 690 nM; 95% confidence limits, 63 nM-1.45 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxicity of capsaicin in monkey kidney cells: lack of antagonistic effects of capsazepine and Ruthenium red

Capsaicin is a natural product of Capsicum peppers, excitatory effects of which have been shown to be mediated by the recently cloned vanilloid receptor 1 (VR1). Since previous studies have shown that capsaicin inhibits protein synthesis, experiments were performed to investigate whether this effect is mediated by VR1 receptor on cultured monkey kidney cells (Vero cells). The capsaicin uptake was assessed in cellular homogenate and in medium by high-performance liquid chromatography (HPLC) separation and quantification on C18 reverse-phase column and fluorescence detection. Toxic effects were assessed by incorporation of [³H]L-leucine into cellular proteins in the presence of capsazepine, the VR1 vanilloid receptor antagonist and Ruthenium red or tyrosine or calcium. Capsazepine (1 to 256 μM) did not modify the uptake rate of capsaicin for incubation times up to 24 h and did not antagonize capsaicin-induced protein synthesis inhibition. It rather inhibited protein synthesis per se from 100 to 256 μM. Ruthenium red which blocks mitochondrial calcium uptake, inhibited protein synthesis and did not antagonise or increase synergistically the effects of capsaicin. Interestingly in a medium deprived of calcium and supplemented by calcium chloride (10–50 μM) the protein synthesis inhibition induced by capsaicin is antagonised somehow. There was no prevention of capsaicin diffusion into the cells. Tyrosine, which seems to be the best preventive agent of capsaicin inhibitory effects, prevents its metabolism but not its diffusion. Capsaicin might enter cells by diffusion and interfere with protein synthesis machinery by competition with tyrosine which in turn prevents the metabolism of capsaicin. The results of the present study suggest that cell responses to capsaicin may be transduced through at least two molecular pathways, one involving VR1, since the receptor antagonist capsazepine fails to prevent the inhibitory effect of capsaicin in Vero cells of renal origin. Received: 27 August 1999 / Accepted: 3 November 1999     

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.     

Relaxant effect of capsazepine in the isolated rat ileum

We have evaluated the effect of the vanilloid receptor agonists resiniferatoxin (RTX), capsaicin and piperine and of the vanilloid receptor antagonist capsazepine on the resting tone in the isolated rat ileum. Capsazepine (10(-8)-3 x 10(-5) M) produced a concentration-related relaxation (8 +/-3%-49 +/-3%) of the rat ileum. By contrast RTX (up to 10(-8) M), capsaicin (up to 10(-6) M) and piperine (up to 10(-5) M) were without effect. Pre-treatment with capsaicin [either in vivo (50 mg/kg s.c.) or in vitro (10(-6) M)] did not modify the inhibitory effect of capsazepine. The L-type Ca2+ channel antagonist nifedipine (10(-6) M), but not the N-type Ca2+ channel antagonist omega-conotoxin GVIA (3 x 10(-8) M) nor the Na+ channel blocker tetrodotoxin (3 x 10(-7) M), counteracted the inhibitory effect of capsazepine. The NK1 receptor antagonist SR 140333 (10(-7) M), the NK2 receptor antagonist SR 48968 (10(-6) M), the NK3 receptor antagonist SR 142801 (10(-7) M), atropine (10(-6) M), hexamethonium (10(-4) M), phentolamine (10(-6) M) plus propranolol (10(-6) M), N(G)-nitro- L-arginine methyl ester ( L-NAME 3 x 10(-4) M), apamin (10(-7) M), methysergide (10(-6) M), the calcitonin gene-related peptide (CGRP) antagonist hCGRP 8-37 (1.5 x 10(-6) M), the VIP antagonist hGRF 1-29 (10(-5) M) did not modify the inhibitory effect of capsazepine. Capsazepine (2.5-40 mg/kg) also decreased upper gastrointestinal transit in vivo. It is concluded that the vanilloid antagonist capsazepine has a direct relaxing effect on rat intestinal smooth muscle which could involve L-type calcium channels. We found no evidence to suggest that capsazepine is antagonizing an endogenous vanilloid.     

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.     

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.     

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.     

Respiratory action of capsaicin microinjected into the nucleus of the solitary tract: involvement of vanilloid and tachykinin receptors.

1. The respiratory response to microinjection of capsaicin into the commissural nucleus of the solitary tract (cNTS) of urethane-anaesthetized rats was investigated in the absence and presence of the competitive vanilloid (capsaicin) antagonist, capsazepine, and selective tachykinin NK1, NK2 and NK3 antagonists (RP 67580, SR 48968 and SR 142801, respectively). 2. Microinjection of capsaicin reduced respiratory frequency but not tidal volume (VT), leading to an overall reduction in minute ventilation (VE). The effect was dose-dependent between 0.5 and 2 nmol capsaicin. Doses greater than 2 nmol produced apnoea. Tachyphylaxis was observed following repeated injection of capsaicin (1 nmol, 30 min apart). 3. Capsazepine (1 nmol) had no effect on frequency or VT when injected alone but completely blocked the respiratory response to capsaicin (1 nmol). 4. RP 67580 (1 but not 5 nmol) alone depressed frequency and VT slightly. Moreover, RP 67580 appeared to potentiate the bradypnoeic effect of capsaicin. In contrast, SR 48968 and SR 142801 (1 and 5 nmol) alone had no significant effect on respiration. However, both agents significantly attenuated the reduction in frequency produced by capsaicin. 5. In conclusion, microinjection of capsaicin into the cNTS decreases overall ventilation, primarily by reducing frequency. The action of capsaicin appears from the data to be mediated by vanilloid receptors since it is blocked by the competitive vanilloid antagonist capsazepine and is subject to tachyphylaxis. However, since NK2 (SR 48968) and NK3 (SR 142801) receptor antagonists block the actions of capsaicin, we propose that capsaicin acts also by releasing tachykinins from central afferent terminals in the cNTS.     

Capsaicin-, Resiniferatoxin-, and Lactic Acid-Evoked Vascular Effects in the Pig Nasal Mucosa in vivo with Reference to Characterization of the Vanilloid Receptor

Abstract: Nasal cavity volume, mucosal and superficial skin blood flow as well as renal splenic vascular effects of capsaicin, resiniferatoxin and lactic acid were investigated, using a novel in vivo pig model. The present results show that locally intraarterially injected capsaicin, resiniferatoxin and lactic acid evoke similar vasodilatory responses, although with different duration, in the nasal mucosa and superficial skin as well as an increase in heart rate and mean arterial blood pressure. Nasal vascular responses evoked by capsaicin, resiniferatoxin and lactic acid were unaffected by the cyclooxygenase inhibitor diclofenac. Moreover, chlorisondamine did not alter the nasal vasodilatory responses evoked by capsaicin and lactic acid. However, chlorisondamine abolished sympathetic reflex-mediated vasoconstrictor effects of capsaicin in the spleen and kidney. Lactic acid-evoked vasodilation in the nasal mucosa and skin was inhibited by the 8-37 fragment of calcitonin gene-related peptide, a calcitonin gene-related peptide-receptor antagonist. Lactic acid-evoked vasoconstriction in the spleen and kidney was reduced but not abolished by chlorisondamine, suggesting that the effects of lactic acid are not exclusively reflex-mediated. Capsazepine did not inhibit the vasodilatation in the nasal mucosa evoked by capsaicin and lactic acid. [³H]Resiniferatoxin bound to pig nasal mucosa membranes with an affinity of 134 pM in a non-cooperative fashion; this binding behaviour contrasted to the apparent positive cooperativity (a Hill coefficient of 2.2) of specific resiniferatoxin binding to pig spinal cord preparations. Specific [³H]resiniferatoxin binding to nasal mucosa membranes was fully inhibited by capsaicin (K_i = 5 μM) and lactic acid (IC₅₀ at pH 5.0) but not by capsazepine (up to 10 μM), in accord with the physiological findings. Capsazepine, by contrast, displaced [³H]resiniferatoxin from spinal vanilloid receptors with an affinity of 3 μM. These findings show the presence of vanilloid receptors in the pig nasal mucosa and suggest heterogeneity in the properties of vanilloid receptors in the pig. Furthermore, lactic acid evokes vascular effects similar to those of capsaicin and resiniferatoxin, possibly via interaction of protons and/or proton-generated substances at vanilloid receptors with a subsequent release of calcitonin gene-related peptide.     

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.     

NEUTROPHILS IN LUNG INFLAMMATION: Which Reactive Oxygen Species are Being Measured?

The oxidative burst in circulating polymorphonuclear leukocytes (PMN) plays a fundamental role in pulmonary defense and injury. Flow cytometric techniques have been developed for quantitation of oxidative burst activity at the single cell level using 2',7'dichlorofluorescin (DCFH). However, the specific reactive oxidant species being measured using this method are not clearly defined. Isolated human PMN were loaded with DCFH diacetate, stimulated with phorbol myristate acetate (PMA) in the presence or absence of specific reagents, and analyzed using flow cytometry. Addition of PMA resulted in a 90-fold increase in the fluorescence intensity of DCFH-loaded neutrophils (p &lt; .01). Inhibition of NADPH oxidase activity using a calmodulin antagonist (W-13) decreased PMA-induced DCFH oxidation by 70% (p &lt; .05). Inhibition of nitric oxide synthase using N^G -monomethyl-L-arginine (NMMA) did not significantly reduce DCFH oxidation, and did not alter the action of W-13. Addition of superoxide dismutase (SOD) had no effect, but catalase, with or without SOD, suppressed DCFH oxidation by 90% (p &lt; .01). These data suggest that hydrogen peroxide, and not NO, is primarily responsible for the PMA-induced oxidation of DCFH in human PMN under these conditions.     

Effect of capsazepine on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) induced by low pH, capsaicin and potassium in rat soleus muscle.

1. We have determined the effect of the competitive antagonist capsazepine at the capsaicin receptor on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from rat isolated soleus muscle induced by capsaicin (1 microM), by superfusion with low pH medium (pH 5) or by KCl (80 mM). 2. Each one of the three stimuli tested produced a marked CGRP-LI release. Total evoked release (fmol g-1) was 482 +/- 69, 169 +/- 20 and 253 +/- 43 for capsiacin, low pH medium and KCL, respectively. 3. Prior application of capsiacin (10 microM for 30 min followed by 30 min of washout) to produce capasaicin desensitization in vitro abolished CGRP-LI release induced by the three stimuli. 4. Capsazepine (1-100 microM, 45 min preincubation) inhibited the evoked CGRP-LI release. Capsaicin-induced release was significantly inhibited by 77, 92 and 96% with 10, 30 and 100 microM capsazepine, respectively. Low pH-induced release was inhibited by 78, 84, 88 and 93% with 3, 10, 30 and 100 microM capsazepine, respectively. KCl-induced release was significantly inhibited by 55 and 93% with 30 and 100 microM (but not with 10 microM) capsazepine, respectively. 5. These findings demonstrate that capsazepine prevents low pH- and capsaicin-induced CGRP-LI release from rat soleus muscle at concentrations which do not affect the release evoked by KCl. These findings imply a relationship between the action of low pH and activation of the capsaicin receptor. At high concentrations, capsazepine produces a nonspecific inhibitory effect on CGRP-LI release from peripheral endings of the capsaicin-sensitive primary afferent neurone.     

A comparison of capsazepine and ruthenium red as capsaicin antagonists in the rat isolated urinary bladder and vas deferens.

1. The ability of capsazepine, a recently developed capsaicin receptor antagonist, to prevent the effects of capsaicin on the rat isolated urinary bladder (contraction) and vas deferens (inhibition of electrically-evoked twitches) was compared to that of ruthenium red, a dye which behaves as a functional antagonist of capsaicin. 2. In the rat bladder, capsazepine (3-30 microM) produced a concentration-dependent rightward shift of the curve to capsaicin without any significant depression of the maximal response to the agonist. By contrast, ruthenium red (10-30 microM) produced a non-competitive type of antagonism, characterized by marked depression of the maximal response attainable. Similar findings were obtained in the rat isolated vas deferens in which capsazepine (10 microM) produced a rightward shift of the curve to capsaicin while ruthenium red (3 microM) depressed the maximal response to the agonist. 3. At the concentrations used to block the effect of capsaicin, neither capsazepine nor ruthenium red affected the contractile response of the rat urinary bladder produced by either neurokinin A or electrical field stimulation or the twitch inhibition produced by rat alpha-calcitonin gene-related peptide (alpha CGRP) in the vas deferens. 4. These findings provide additional evidence that both capsazepine and ruthenium red are valuable tools for exploration of the function of capsaicin-sensitive primary afferent neurones. The antagonism of the action of capsaicin by capsazepine is entirely consistent with the proposed interaction of this substance with a vanilloid receptor located on primary afferents, while the action of ruthenium red apparently involves a more complex, non-competitive antagonism.     

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.     

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.     

Stimulatory effect of centrally injected capsaicin, an agonist of vanilloid receptors, on gastric acid secretion in rats.

Capsaicin, the main pungent ingredient in chilli peppers, acts through specific vanilloid receptors on sensory neurons. The vanilloid receptors have been localized in the brain. We describe here a stimulatory effect of centrally injected capsaicin on gastric acid secretion in urethane-anesthetized rats. Injection of capsaicin (10-30 nmol per rat) into the lateral cerebroventricle markedly stimulated the secretion. Injection of capsazepine (30 nmol) or ruthenium red (30 nmol), antagonists for vanilloid receptors, into the lateral cerebroventricle inhibited the secretion induced by capsaicin, although these antagonists alone significantly stimulated the secretion. Injection of capsaicin into the fourth cerebroventricle also stimulated gastric acid secretion. The effects of centrally injected capsaicin into the lateral and fourth cerebroventricle were mediated via the vagus cholinergic nerve, because the effects were abolished by bilateral vagotomy at the cervical level. The present findings showed that central injection of capsaicin stimulated gastric acid secretion, via vanilloid receptors in the central nervous system (CNS), and through vagus nerve mechanisms in the perfused stomach of urethane-anesthetized rats.     

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.