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

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

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.     

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

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

Selective antagonism of capsaicin by capsazepine: evidence for a spinal receptor site in capsaicin-induced antinociception.

1. Capsazepine has recently been described as a competitive capsaicin antagonist. We have used this compound to test the hypotheses that the in vitro and in vivo effects of capsaicin are due to interactions with a specific receptor. 2. In an in vitro preparation of the neonatal rat spinal cord with functionally connected tail, the activation of nociceptive afferent fibres by the application of capsaicin, bradykinin or noxious heat (48 degrees C) to the tail could be measured by recording a depolarizing response from a spinal ventral root. Application of capsaicin or substance P to the spinal cord also evoked a depolarizing response which was recorded in a ventral root. 3. When capsazepine (50 nM-20 microM) was administered to the tail or spinal cord it did not evoke any measurable response. However on the tail, capsazepine reversibly antagonized (IC50 = 254 +/- 28 nM) the responses to capsaicin but not to heat or bradykinin administered to the same site. Similarly capsazepine administration to the spinal cord antagonized the responses evoked by capsaicin (IC50 = 230 +/- 20 nM) applied to the cord but not responses evoked by substance P on the cord or by noxious heat and capsaicin on the tail. 4. In halothane anaesthetized rats, C-fibre responses evoked by transcutaneous electrical stimulation of the receptive field were recorded from single wide dynamic range neurones located in the spinal dorsal horn. C-fibre evoked discharges were consistently reduced by the systemic administration of capsaicin (20 mumol kg-1, s.c.) and this action of capsaicin was antagonized by capsazepine (100 mumol kg-1) administered by the same route.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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

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

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)     

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.     

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.     

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     

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.     

Capsazepine as a selective antagonist of capsaicin-induced activation of C-fibres in guinea-pig bronchi.

We investigated the action of capsazepine, an antagonist of the actions of capsaicin on sensory neurones, on the contractile responses evoked by capsaicin or by electrical field stimulation (EFS) in guinea-pig bronchi. Capsazepine (10(-5) M) selectively inhibited responses to capsaicin, producing a significant change in EC50 values but not the Hill coefficient (nH), suggesting that capsazepine acts as a competitive antagonist (apparent pKB = 5.12) whereas ruthenium red is a non-competitive antagonist. Capsazepine and ruthenium red were without effect on EFS-induced responses.     

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.     

Expression of vanilloid receptors in rat gastric epithelial cells: role in cellular protection

Vanilloid receptors subtype 1 (VR1), a nonselective cation channel responsive to capsaicin, protons, and noxious heat, has been recently identified in not only neural but also non-neural cells. In the present study, we demonstrated the peripheral expression of VR1 in gastric mucosal epithelial cells and investigated the role of the receptor in cellular protection. The rat gastric mucosal epithelial cell line was used. The expression of VR1 was examined by Western blotting and RT-PCR. Cell damage was induced by immersion in 10% ethanol or acid (pH 4.0) for 30 min, and cell viability was determined by MTT assay. Capsaicin or resiniferatoxin was added 30 min before the challenge with ethanol or acid, while capsazepine or ruthenium red (a VR1 antagonist) was added simultaneously with capsaicin. The distinct expression of VR1 protein and mRNA was detected in rat gastric mucosal epithelial cell line as well as in the rat stomach and spinal cord by Western blotting and RT-PCR, respectively. The cDNA sequence of the PCR product was found to be almost identical to that of the authentic VR1 (99.8%) when the product was subcloned and sequenced. On the other hand, the cell damage induced by ethanol or acid was dose-dependently prevented by pretreatment with capsaicin. The protective effect of capsaicin was mimicked by resiniferatoxin and almost totally abolished by co-addition of capsazepine or ruthenium red. These findings suggest that VR1 is expressed peripherally in gastric mucosal epithelial cells and plays a cellular protective role.     

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.     

Iodo-resiniferatoxin, a new potent vanilloid receptor antagonist

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

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.     

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.     

Modulation of Capsaicin‐Sensitive Nerve Activation by Low pH Solutions in Guinea‐Pig Lung

Abstract: We have studied the stimulation of airways sensory nerves by low pH solutions and concomitantly induced bronchoconstriction. The effect of low pH buffer and lactic acid solutions at the same pH (5 and 6) were compared and the influence of low pH on the capsaicin effect was recorded. We have used the isolated guinea‐pig perfused lung model taking the insufflation pressure as an indicator of bronchial smooth muscle tone while the calcitonin gene‐related peptide‐like immunoreactivity measured in the lung perfusate represented sensory nerves activation. Low pH buffer and lactic acid solution (3 and 4.1 mM) at the same pH of 5 and 6 induced pH‐dependent bronchoconstriction and peptides release which were completely abolished after systemic pretreatment with capsaicin. Both responses were significantly inhibited after Ca²⁺‐free infusion. Capsazepine (10^?6 M), a selective capsaicin antagonist, significantly reduced the calcitonin gene‐related peptide‐like immunoreactivity overflow evoked by all the solutions studied. Diclofenac (10^?5 M), a cyclooxygenase blocker, inhibited pH 5, pH 6 and lactic acid 3 mM (pH 6)‐evoked peptide release, but not lactic acid 4.1 mM (pH 5). The functional response was not significantly modified after diclofenac while only the lactic acid 3 mM response was significantly reduced by capsazepine. There was a synergistic interaction between capsaicin and low pH buffer on calcitonin gene‐related peptide‐like immunoreactivity release and an additive effect on bronchoconstriction. It is concluded that in the isolated perfused guinea‐pig lung, lactic acid and low pH buffer induced calcitonin gene‐related peptide‐like immunoreactivity release and bronchoconstriction by stimulation of capsaicin‐sensitive C fibres via a pathway partly dependent of extracellular Ca²⁺. The mechanism of calcitonin gene‐related peptide‐like immunoreactivity release seems to be the same at pH 6, while differences are evident at pH 5 between low pH buffer and lactic acid. Our results also suggest that proton activity could exert a modulatory role on the capsaicin‐sensitive sensory nerves by a mechanism which remains to be clarified.     

Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology

The vanilloid receptor (VR1) is a ligand-gated ion channel, which plays an important role in nociceptive processing. Therefore, a pharmacological characterization of the recently cloned rat VR1 (rVR1) was undertaken. HEK293 cells stable expressing rVR1 (rVR1-HEK293) were loaded with Fluo-3AM and then incubated at 25 degrees C for 30 min with or without various antagonists or signal transduction modifying agents. Then intracellular calcium concentrations ([Ca(2+)](i)) were monitored using FLIPR, before and after the addition of various agonists. The rank order of potency of agonists (resiniferatoxin (RTX)>capsaicin>olvanil>PPAHV) was as expected, and all were full agonists. The potencies of capsaicin and olvanil, but not RTX or PPAHV, were enhanced at pH 6.4 (pEC(50) values of 7.47+/-0.06, 7.16+/-0.06, 8.19+/-0.06 and 6.02+/-0.03 respectively at pH 7.4 vs 7.71+/-0.05, 7.58+/-0.14, 8.10+/-0.05 and 6.04+/-0.08 at pH 6.4). Capsazepine, isovelleral and ruthenium red all inhibited the capsaicin (100 nM)-induced Ca(2+) response in rVR1-HEK293 cells, with pK(B) values of 7.52+/-0.08, 6.92+/-0.11 and 8.09+/-0.12 respectively (n=6 each). The response to RTX and olvanil were also inhibited by these compounds. None displayed any agonist-like activity. The removal of extracellular Ca(2+) abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 microM) partially (approximately 20%) inhibited, the capsaicin (10 microM)-induced Ca(2+) response. However, tetrodotoxin (3 microM), nimodipine (10 microM), omega-GVIA conotoxin (1 microM), thapsigargin (1 microM), U73122 (3 microM) or H-89 (3 microM) had no effect on the capsaicin (100 nM)-induced response. In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand-gated Ca(2+) channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.