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.     

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.     

Molecular cloning, functional characterization of the porcine transient receptor potential V1 (pTRPV1) and pharmacological comparison with endogenous pTRPV1

In the present study, we cloned a porcine orthologue of transient receptor potential V1 (pTRPV1) and heterologously expressed it in human embryonic kidney (HEK) 293 cells to characterize its pharmacological properties. At the amino acid level, pTRPV1 was highly homologous (83-90%) to other orthologues of TRPV1. The expression of receptors was examined with current and [Ca2+]i responses to capsaicin using whole-cell patch-clamp and fura-2 ratio imaging techniques, respectively, and by immunostaining with an anti-TRPV1 antibody. The receptors were characterized by changes in [Ca2+]i in response to various vanilloid agonists, low pH and heat and by the effects of TRPV1 antagonists on them. The various TRPV1 agonists activated pTRPV1 in a dose-dependent manner in the order of potency of resiniferatoxin (RTX) > olvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), phorbol 12,13-dinonanoate 20-homovanillate (PDNHV). Isovelleral and scutigeral had no effect. Endogenous vanilloids (anandamide > 15 (s)-HPETE > NADA), low pH and noxious heat (>42 degrees C) activated pTRPV1. Comparison of amino acid sequences with various mammalian TRPV1 homologues suggested some novel putative vanilloid recognition sites. TRPV1 antagonists, iodoRTX, ruthenium red and capsazepine suppressed capsaicin-induced responses. Similar to human TRPV1, but not rodent TRPV1, capsazepine was effective in blocking pH- and heat-induced responses. Similar pharmacological profiles were observed in cultured porcine dorsal root ganglion neurons. We discuss putative amino acid residues related to pharmacological differences among mammalian TRPV1 homologues.     

The cloned rat vanilloid receptor VR1 mediates both R-type binding and C-type calcium response in dorsal root ganglion neurons.

[(3)H]Resiniferatoxin (RTX) binding and calcium uptake by rat dorsal root ganglion (DRG) neurons show distinct structure-activity relations, suggestive of independent vanilloid receptor (VR) subtypes. We have now characterized ligand binding to rat VR1 expressed in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells and compared the structure-activity relations with those for calcium mobilization. Human embryonic kidney cells (HEK293/VR1 cells) and Chinese hamster ovary cells transfected with VR1 (CHO/VR1 cells) bound [(3)H]RTX with affinities of 84 and 103 pM, respectively, and positive cooperativity (Hill numbers were 2.1 and 1.8). These parameters are similar to those determined with rat DRG membranes expressing native VRs (a K(d) of 70 pM and a Hill number of 1.7). The typical vanilloid agonists olvanil and capsaicin inhibited [(3)H]RTX binding to HEK293/VR1 cells with K(i) values of 0.4 and 4.0 microM, respectively. The corresponding values in DRG membranes were 0.3 and 2.5 microM. HEK293/VR1 cells and DRG membranes also recognized the novel vanilloids isovelleral and scutigeral with similar K(i) values (18 and 20 microM in HEK293/VR1 cells; 24 and 21 microM in DRGs). The competitive vanilloid receptor antagonist capsazepine inhibited [(3)H]RTX binding to HEK293/VR1 cells with a K(i) value of 6.2 microM and binding to DRG membranes with a K(i) value of 8.6 microM. RTX and capsaicin induced calcium mobilization in HEK293/VR1 cells with EC(50) values of 4.1 and 82 nM, respectively. Thus, the relative potencies of RTX (more potent for binding) and capsaicin (more potent for calcium mobilization) are similar in DRG neurons and cells transfected with VR1. We conclude that VR1 can account for both the ligand binding and calcium uptake observed in rat DRG neurons.     

A non-pungent triprenyl phenol of fungal origin, scutigeral, stimulates rat dorsal root ganglion neurons via interaction at vanilloid receptors

1. A [3H]-resiniferatoxin (RTX) binding assay utilizing rat spinal cord membranes was employed to identify novel vanilloids in a collection of natural products of fungal origin. Of the five active compounds found (scutigeral, acetyl-scutigeral, ovinal, neogrifolin, and methyl-neogrifolin), scutigeral (Ki=19 microM), isolated from the edible mushroom Albatrellus ovinus, was selected for further characterization. 2. Scutigeral induced a dose-dependent 45Ca uptake by rat dorsal root ganglion neurons with an EC50 of 1.6 microM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine (IC50=5.2 microM). 3. [3H]-RTX binding isotherms were shifted by scutigeral (10-80 microM) in a competitive manner. The Schild plot of the data had a slope of 0.8 and gave an apparent Kd estimate for scutigeral of 32 microM. 4. Although in the above assays scutigeral mimicked capsaicin, it was not pungent on the human tongue up to a dose of 100 nmol per tongue, nor did it provoke protective wiping movements in the rat (up to 100 microM) upon intraocular instillation. 5. In accord with being non-pungent, scutigeral (5 microM) did not elicit a measurable inward current in isolated rat dorsal root ganglion neurons under voltage-clamp conditions. It did, however, reduce the proportion of neurons (from 61 to 15%) that responded to a subsequent capsaicin (1 microM) challenge. In these neurons, scutigeral both delayed (from 27 to 72 s) and diminished (from 5.0 to 1.9 nA) the maximal current evoked by capsaicin. 6. In conclusion, scutigeral and its congeners form a new chemical class of vanilloids, the triprenyl phenols. Scutigeral promises to be a novel chemical lead for the development of orally active, non-pungent vanilloids.     

Inhibition of [3H]resiniferatoxin binding to rat dorsal root ganglion membranes as a novel approach in evaluating compounds with capsaicin-like activity

Summary We have recently reported the specific binding of [³H]resiniferatoxin to sensory ganglion membranes; this binding appears to represent the postulated vanilloid (capsaicin) receptor. In the present report, we compare the structure/activity relations for binding to rat dorsal root ganglion membranes and for biological responses in the rat, using a series of vanilloids of the capsaicin (homovanilloyl-decylamide, homovanilloyl-dodecylamide, homovanilloyl-cyclododecylamide, homovanilloyl-hexadecylamide, homovanilloyl-piperidine and nonenoyl-homoveratrylamide) and resiniferatoxin (tinyatoxin, 12-deoxyphorbol 13-phenylacetate 20-homovanillate) classes. We find that all the tested biologically active vanilloids, but not the inactive structure analogs, compete for the [³H]resiniferatoxin binding sites in rat dorsal root ganglion membranes, and we conclude that the [³H]resiniferatoxin binding assay may provide an efficient approach for evaluating such compounds. We also provide evidence that the [3H]resiniferatoxin receptor is likely to recognize vanilloids which are inserted into the membranes; and that the apparent activity of capsaicinoids may be significantly influenced by factors other than equilibrium binding affinities. Send offprint requests to P. M. Blumberg at the above address     

Resiniferatoxin-type phorboid vanilloids display capsaicin-like selectivity at native vanilloid receptors on rat DRG neurons and at the cloned vanilloid receptor VR1.

1 Although the cloned rat vanilloid receptor VR1 appears to account for both receptor binding and calcium uptake, the identification of vanilloids selective for one or the other response is of importance because these ligands may induce distinct patterns of biological activities. 2 Phorbol 12,13-didecanoate 20-homovanillate (PDDHV) evoked 45Ca(2+)-uptake by rat dorsal root ganglion neurons (expressing native vanilloid receptors) in culture with an EC50 of 70 nM but inhibited [3H]-resiniferatoxin (RTX) binding to rat dorsal root ganglion membranes with a much lower potency (Ki>10,000 nM). This difference in potencies represents a more than 100 fold selectivity for capsaicin-type pharmacology. 3 45Ca2+ influx by PDDHV was fully inhibited by the competitive vanilloid receptor antagonist capsazepine, consistent with the calcium uptake occurring via vanilloid receptors. 4 PDDHV induced calcium mobilization in CHO cells transfected with the cloned rat vanilloid receptor VR1 with an EC50 of 125 nM and inhibited [3H]-RTX binding to these cells with an estimated Ki of 10,000 nM. By contrast, PDDHV failed to evoke a measurable calcium response in non-transfected CHO cells, confirming its action through VR1. 5 We conclude that PDDHV is two orders of magnitude more potent for inducing calcium uptake than for inhibiting RTX binding at vanilloid receptors, making this novel vanilloid a ligand selective for capsaicin-type pharmacology. These results emphasize the importance of monitoring multiple endpoints for evaluation of vanilloid receptor structure-activity relations. Furthermore, PDDHV now provides a tool to explore the biological correlates of capsaicin-type vanilloid pharmacology.     

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

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

Activities of prenylphenol derivatives from fruitbodies of Albatrellus spp. on the human and rat vanilloid receptor 1 (VR1) and characterisation of the novel natural product,confluentin

Several prenylphenols from basidiocarps of European and Chinese Albatrellus spp., namely grifolin (1), neogrifolin (2), confluentin (3), scutigeral (4), and albaconol (5) were investigated concerning their activities in test models for vanilloid receptor modulation. The isolation of these compounds from A. confluens and structure elucidation of the novel natural product confluentin (3) are described. The effects of scutigeral and neogrifolin on vanilloid receptors were studied by means of electrophysiological methodology on rat dorsal root ganglion neurons as well as on recombinant cell lines expressing the rat VR1 receptor. Concurrently, the effects of compounds 1-5 on a reporter cell line expressing the human vanilloid receptor VR1 were measured. In contrast to previous studies reported in the literature, the results of these investigations suggest that fungal prenylphenols act as weak antagonists (activity in the microM range), rather than exhibiting agonistic activities.     

Respiratory actions of vanilloid receptor agonists in the nucleus of the solitary tract: comparison of resiniferatoxin with non-pungent agents and anandamide

1. Activation of vanilloid receptors on sensory nerve terminals in the commissural nucleus of the solitary tract (cNTS) of rats with capsaicin, produces respiratory slowing. In this study, we used microinjection techniques employing pungent and non-pungent vanilloids to further characterize vanilloid receptors in the cNTS. 2. Microinjection of the pungent vanilloid, resiniferatoxin (RTX), into the cNTS of urethane-anaesthetized rats, dose-dependently reduced respiratory rate without affecting tidal volume. RTX was 20 fold more potent at slowing respiration ( approximately ED(50), 100 pmol) than capsaicin ( approximately ED(50), 2 nmol). Doses of RTX greater than 100 pmol caused either irregular (dyspnoeic) breathing or terminal apnoea (>250 pmol). The respiratory slowing response to RTX (75 pmol), was dose-dependently attenuated by injecting RTX (but not vehicle) into the same site 60 min earlier. 3. The non-pungent phorbol derivative of RTX, phorbol 12-phenylacetete 13-acetate 20-homovanillate (PPAHV, 0.1-1 nmol), also slowed respiration (ED(50), approximately 1 nmol) and almost abolished response to RTX (75 pmol) injected into the same site 60 min later. 4. In contrast to RTX, PPAHV and capsaicin, the putative endogenous vanilloid receptor agonist, arachidonyl ethanolamide (AEA), and non-pungent capsaicin derivative, olvanil, had no direct effect on respiration. However, both AEA and olvanil dose-dependently reduced the respiratory response to injection of RTX (75 pmol) 60 min later into the same site (EC(50)s, for AEA and olvanil, approximately 2 and 0.2 nmol, respectively). 5. These studies suggest that both pungent and non-pungent vanilloids interact with vanilloid receptors in the cNTS. However, whereas RTX and PPAHV activate and subsequently desensitize vanilloid receptors on sensory nerve terminals in the cNTS, olvanil and AEA fail to activate despite readily desensitizing responses to RTX in this region.     

Noladin ether, a putative endocannabinoid, attenuates sensory neurotransmission in the rat isolated mesenteric arterial bed via a non-CB1/CB2 G(i/o) linked receptor

1 Noladin ether has recently been reported to be an endocannabinoid, with selectivity for the cannabinoid (CB) CB1 receptor. In the present study, we investigated the effects of noladin ether in the rat isolated mesenteric arterial bed, cultured dorsal root ganglia (DRG) cells and human vanilloid (TRPV1)-receptor-expressing HEK293 cells (TRPV1-HEK293 cells). 2 Electrical field stimulation of the mesenteric bed evoked frequency-dependent vasorelaxation due to the action of calcitonin gene-related peptide (CGRP) released from sensory nerves. Noladin ether (0.1-3 microm) attenuated sensory neurogenic relaxation in a concentration-dependent manner. Noladin ether (1 microm) reduced vasorelaxation at a submaximal frequency (8 Hz), from 57.3+/-6.8 to 23.3+/-3.8% (P<0.05, n=4). 3 The inhibitory effects of noladin ether were unaffected by the CB1 antagonists SR141716A and LY320135, and the CB2 antagonist SR144528 (1 microm). 4 Noladin ether had no effect on vasorelaxation elicited by exogenous CGRP or capsaicin. These data suggest that noladin ether is acting at a prejunctional site and no interaction with TRPV1 is involved. 5 In mesenteric beds from pertussis toxin (PTX)-pretreated rats, the inhibitory actions of noladin ether on sensory neurotransmission were abolished, indicating the involvement of G(i/o) protein-coupled receptors. 6 Noladin ether evoked a concentration-dependent increase in intracellular Ca2+ concentration in TRPV1-HEK293 cells at 10 microm (36.5+/-3.2% of maximal capsaicin-induced response), but it was a less potent agonist than both capsaicin and anandamide and at 1 microm it was essentially inactive. Noladin ether (1 microm) had no effect on capsaicin-evoked Ca2+ responses in DRG cells, and produced no response alone, indicating it neither modulates nor acts directly on TRPV1 receptors. 7 These data demonstrate that noladin ether attenuates sensory neurotransmission in rat mesenteric arteries via a non-CB1 non-CB2 PTX-sensitive prejunctional site, independently of TRPV1 receptors.     

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.     

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.     

Yohimbine inhibits firing activities of rat dorsal root ganglion neurons by blocking Na+ channels and vanilloid VR1 receptors

Yohimbine, an indole alkaloid, is a natural alpha(2)-adrenoceptor antagonist and is frequently used to assess the mechanism of a drug's effect on alpha-adrenoceptors. Recently, several studies showed that yohimbine exhibited analgesic effects in in vivo animal models. However, the underlying mechanism is not known. We investigated the effects of yohimbine on Na(+) channels and vanilloid VR1 receptors in dorsal root ganglion cells. We found that yohimbine inhibited tetrodotoxin-sensitive Na(+) channels (Na(V)1.2), the tetrodotoxin-resistant Na(+) channels, including both slow inactivating (Na(V)1.8) and persistent (Na(V)1.9) Na(+) channels, and capsaicin-sensitive vanilloid VR1 receptors. Action potential firing activities of dorsal root ganglion neurons evoked by current injection or capsaicin were eliminated by yohimbine. The blocking effects of yohimbine on nociceptive-related ion channels and firing activities of dorsal root ganglion neurons may underlie the ionic mechanism of yohimbine's analgesic effects observed in in vivo studies.     

Cannabinoid inhibition of the capsaicin-induced calcium response in rat dorsal root ganglion neurones

Cannabinoids have marked inhibitory effects on somatosensory processing, which may arise from actions at both peripheral and central cannabinoid receptors. Here, the effect of a synthetic cannabinoid agonist HU210 on capsaicin-evoked responses in adult rat dorsal root ganglion (DRG) neurones was studied. The vanilloid capsaicin produced a concentration-related increase in intracellular calcium in DRG neurones, which was significantly inhibited by HU210 (1 microM). The cannabinoid CB(1) receptor antagonist SR141716A (1 microM) had no effect alone and did not influence the response to capsaicin but significantly reversed the inhibitory effect of HU210. These data indicate that DRG CB(1) receptors are functional and can inhibit nociceptive responses.     

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.     

Cloning and functional characterization of the guinea pig vanilloid receptor 1

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

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.     

Methoctramine analogues inhibit responses to capsaicin and protons in rat dorsal root ganglion neurons

We have investigated the possibility that vanilloid receptors have a binding site for polyamines and determined the consequences of binding to such a site. Whole-cell and single-channel patch-clamp recordings were used to investigate the effect of the tetraamine, methoctramine, and 16 of its analogues on capsaicin and proton induced responses of foetal rat dorsal root ganglion neurons. All but two methoctramine analogues inhibited responses to 10 microM capsaicin with IC50 values in the range of 2-70 microM at a holding potential of -100 mV. Inhibition was generally non-competitive and voltage-dependent. Methoctramine at 10 microM reduced the single channel mean open time (>3-fold), but also increased the mean closed time (1.7-fold). Sustained responses to pH 5.4 were antagonized by methoctramine with similar potency to capsaicin responses. Similar data were obtained with adult rat dorsal root ganglion neurons. These data indicate that methoctramine analogues bind to vanilloid receptors to inhibit their function.     

Multiple actions of anandamide on neonatal rat cultured sensory neurones

1. We have investigated the effects of the endocannabinoid anandamide (AEA) on neuronal excitability and vanilloid TRPV1 receptors in neonatal rat cultured dorsal root ganglion neurones. 2. Using whole-cell patch-clamp electrophysiology, we found that AEA inhibits high-voltage-activated Ca(2+) currents by 33+/-9% (five out of eight neurones) in the absence of the CB(1) receptor antagonist SR141716A (100 nM) and by 32+/-6% (seven out of 10 neurones) in the presence of SR141716A. 3. Fura-2 fluorescence Ca(2+) imaging revealed that AEA produced distinct effects on Ca(2+) transients produced by depolarisation evoked by 30 mM KCl. In a population of neurones of larger somal area (372+/-20 microM(2)), it significantly enhanced Ca(2+) transients (80.26+/-13.12% at 1 microM), an effect that persists after pertussis toxin pretreatment. In a population of neurones of smaller somal area (279+/-18 microM(2)), AEA significantly inhibits Ca(2+) transients (30.75+/-3.54% at 1 microM), an effect that is abolished by PTX pretreatment. 4. Extracellular application of 100 nM AEA failed to evoke TRPV1 receptor inward currents in seven out of eight neurones that responded to capsaicin (1 microM), with a mean inward current of -0.94+/-0.21 nA. In contrast, intracellular application of 100 nM AEA elicited robust inward currents in approximately 62% of neurones, the mean population response was -0.85+/-0.21 nA. When AEA was applied to the intracellular environment with capsazepine (1 microM), the mean population inward current was -0.01+/-0.01 nA. Under control conditions, mean population current fluctuations of -0.09+/-0.05 nA were observed.