Possible Consequences of Blocking Transient Receptor Potential Vanilloid 1
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The cloning of the first sensory Transient Receptor Potential (TRP) channel, TRPVanilloid 1 (TRPV1) in 1997, initiated a new era of pain research and coincided with the Decade of Pain Control and Research promulgated by the United States Congress. When cloned, TRPV1 channel was shown to be predominantly expressed in nociceptors (C- and A -fibers) and are activated by physical and chemical stimuli. Channel function can be amplified by transcriptional upregulation and posttranslational modification by proinflammatory agents. Indeed, TRPV1 gene disruption confirms that it is involved in transmitting inflammatory thermal hypersensitivity, but not acute thermal or mechanical pain sensitivity. Based on its distribution and functions, TRPV1 is considered as an ideal target for developing small molecule antagonists. Now, there is a growing body of evidence that TRPV1 is expressed in non-sensory neurons and non-neuronal cells. This raises the possibility of unwanted effects that may result from targeting TRPV1. A major consequence of TRPV1 blockade that has come to light in clinical trials following administration of antagonists is hyperthermia. This observation has threatened the abandonment of TRPV1 antagonists, although they are proven to be useful in certain modalities of pain. In this review, we will discuss the expression and functions of TRPV1 in various organ systems and highlight the consequences that might be associated with blocking the receptor.Keywords:
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In this issue of the BJUI, Charrua et al. 1 report on the possible interaction of two members of the vanilloid subfamily of transient receptor potential (TRP) channels in the control of rat urinary bladder function, TRPV1 and TRPV4. TRP channels are a family of cation-selective channels with 28 known mammalian members. Six of them belong to the subfamily of vanilloid receptors (TRPV channels) and fall into four groups, TRPV1/TRPV2, TRPV3, TRPV4, and TRPV5/TRPV6. The physiological and pharmacological interest in these channels results largely from the finding that they can be activated by a plethora of physical and chemical stimuli; accordingly, they have been implicated in sensory function and pathophysiology of many organ systems 2. A breakthrough in our understanding of such channels came with the reporting of TRPV1 and TRPV4 knock-out mice, which also exhibit a bladder phenotype; the role of TRP channels in lower urinary tract function has comprehensively been reviewed recently 3. While the physiological regulation of TRPV1 by endogenous mediators is poorly understood, natural compounds such as capsaicin or resiniferatoxin are acute agonists of TRPV1 channels; however, over time, they desensitise the channel and hence act as inhibitors. These compounds have shown promise in the treatment of detrusor overactivity but also have problems attributed to their initial agonist effects 3. TRPV4 are activated experimentally by hypotonicity induced cell swelling and several chemicals and more physiologically by moderate heat, stretch and shear stress, leading to the proposition that they may functions as a stretch sensor in the bladder. The inhibitory effects of TRPV1 agonists manifest only after prolonged exposure once desensitisation of their agonist effects occurs, and this initial agonistic phase is a source of undesirable effects. Therefore, a search is on for small molecules that have direct antagonist effects. Charrua et al. 1 now report that two small molecule antagonists at TRPV1 and TRPV4, (SB355791 and RN1734, respectively) even in high doses did not affect bladder function in control rats. Intravesical installation of lipopolysaccharide is used to create an animal model of cystitis as it induces inflammation, detrusor overactivity and bladder pain. In this model, a high dose of the TRPV4 inhibitor reduced detrusor overactivity, whereas even the high dose of the TRPV1 inhibitor did not; however, a combination of ineffective doses of both inhibitors markedly decreased bladder reflex activity. On the other hand, each of the two drugs caused partial analgesia, but their combination was not more effective than either drug alone. This indicates an interesting functional interaction between TRPV1 and TRPV4 channels, which is specific for the overactivity vs the pain response. Previously, the Cruz group reported that bladder overactivity induced by nerve growth factor depends on the presence of functionally active TRPV1 4. Taken together this work shines light on networks of multiple mediators and their receptors that cooperate in the regulation of bladder function but previously have mainly been viewed in isolation. Such work may also have therapeutic consequences. As target-saturating concentrations of ligands at any of these receptors may cause relevant adverse effects, targeting multiple such receptors in low doses may open an avenue for a multi-pronged approach, particularly in patients with bladder dysfunction difficult to control with present treatment options. This multiple target, low-dose approach is a therapeutically fascinating idea, but finding the right combination of doses in such a setting is a nightmare for any drug development scientist. Moreover, much of the specific role of such targets in pathophysiology remains to be explored before the present findings can be translated into clinical treatments, and the Charrua et al. study 1 will also help such efforts in other ways. Some of the initial thinking on the function of TRP channels in the control of bladder and other functions has been based on localisation studies with TRP channel antibodies, which may have been flawed. Similar to many other receptor antibodies 5, several of those directed against TRPV1 channels also have been shown to lack target specificity 6, leading to misunderstandings about the location and function of such channels. The validation for other TRPV1 and TRPV4 antibodies presented by Charrua et al. 1 will allow more robust studies in this regard and help to develop more valid understanding of TRP channels in physiology, pathophysiology and as treatment targets. The author currently is an employee of Boehringer Ingelheim.
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Stimulation of the vanilloid receptor‐1 (TRPV1) results in the activation of nociceptive and neurogenic inflammatory responses. Poor specificity and potency of TRPV1 antagonists has, however, limited the clarification of the physiological role of TRPV1. Recently, iodo‐resiniferatoxin (I‐RTX) has been reported to bind as a high affinity antagonist at the native and heterologously expressed rat TRPV1. Here we have studied the ability of I‐RTX to block a series of TRPV1 mediated nociceptive and neurogenic inflammatory responses in different species (including transfected human TRPV1). We have demonstrated that I‐RTX inhibited capsaicin‐induced mobilization of intracellular Ca 2+ in rat trigeminal neurons (IC 50 0.87 n M ) and in HEK293 cells transfected with the human TRPV1 (IC 50 0.071 n M ). Furthermore, I‐RTX significantly inhibited both capsaicin‐induced CGRP release from slices of rat dorsal spinal cord (IC 50 0.27 n M ) and contraction of isolated guinea‐pig and rat urinary bladder (pK B of 10.68 and 9.63, respectively), whilst I‐RTX failed to alter the response to high KCl or SP. Finally, in vivo I‐RTX significantly inhibited acetic acid‐induced writhing in mice (ED 50 0.42 μmol kg −1 ) and plasma extravasation in mouse urinary bladder (ED 50 0.41 μmol kg −1 ). In in vitro and in vivo TRPV1 activated responses I‐RTX was ∼3 log units and ∼20 times more potent than capsazepine, respectively. This high affinity antagonist, I‐RTX, may be an important tool for future studies in pain and neurogenic inflammatory models. British Journal of Pharmacology (2003) 138 , 977–985. doi: 10.1038/sj.bjp.0705110
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Background Transient receptor potential vanilloid subtype 1 (TRPV1) is activated by low pH/ protons and is well known to be involved in hyperalgesia during inflammation. Tumor necrosis factor α (TNF-α), a proinflammatory cytokine, is involved in nociceptive responses causing hyperalgesia through TNF receptor type 1 (TNFR1) activation. Reactive oxygen species (ROS) production is also prominently increased in inflamed tissue. The present study investigated TNFR1 receptors in primary cultured mouse dorsal root ganglion (DRG) neurons after TRPV1 activation and the involvement of ROS. C57BL/6 mice, both TRPV1 knockout and wild type, were used for immunofluorescent and live cell imaging. The L4 and L5 DRGs were dissected bilaterally and cultured overnight. TRPV1 was stimulated with capsaicin or its potent analog, resiniferatoxin. ROS production was measured with live cell imaging and TNFR1 was detected with immunofluorescence in DRG primary cultures. The TRPV1 knockout mice, TRPV1 antagonist, capsazepine, and ROS scavenger, N-tert-Butyl-α-phenylnitrone (PBN), were employed to explore the functional relationship among TRPV1, ROS and TNFR1 in these studies. Results The results demonstrate that TRPV1 activation increases TNFR1 receptors and ROS generation in primary cultures of mouse DRG neurons. Activated increases in TNFR1 receptors and ROS production are absent in TRPV1 deficient mice. The PBN blocks increases in TNFR1 and ROS production induced by capsaicin/resiniferatoxin. Conclusion TRPV1 activation increases TNFR1 in cultured mouse DRG neurons through a ROS signaling pathway, a novel sensitization mechanism in DRG neurons.
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Transient receptor potential vanilloid 1 (TRPV1) is a capsaicin- and heat-gated ion channel required for normal in vivo responses to these painful stimuli. However, growing evidence suggests that TRPV1 also participates in thermoregulation. Therefore, we examined the effects of a selective TRPV1 antagonist, 5-iodoresiniferatoxin (I-RTX), on mouse body temperature. Surprisingly, s.c. administration of I-RTX (0.1–1 μmol/kg) evoked a hypothermic response similar to that evoked by capsaicin (9.8 μmol/kg) in naive wild-type mice, but not in mice pretreated with resiniferatoxin, a potent TRPV1 agonist, or in naive TRPV1-null mice. In response to I-RTX in vitro, HEK293 cells expressing rat TRPV1 exhibited increases in intracellular Ca2+ (biphasic, EC50 = 56.7 nM and 9.9 μM) that depended on Ca2+ influx and outwardly rectifying, capsazepine-sensitive currents that were smaller than those evoked by 1 μM capsaicin. Thus, I-RTX induces TRPV1-dependent hypothermia in vivo and is a partial TRPV1 agonist in vitro.
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Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [3H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met547 and Thr550) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [3H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders. Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [3H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met547 and Thr550) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [3H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders. The receptor for capsaicin (a small vanilloid molecule extracted from “hot” chili peppers), designated vanilloid receptor 1 (also known as VR1 and TRPV1 1The abbreviations used are: TRPV1, transient receptor potential vanilloid type 1; RTX, resiniferatoxin; AEA, arachidonyl ethanolamine; NADA, N-arachidonyldopamine; OLDA, oleoyldopamine; DRG, dorsal root ganglia; PPAHV, 12-phenylacetate 13-acetate 20-homovanillate; BCTC, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide; CHO, Chinese hamster ovary; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid; r/o, rat-rabbit chimera; h/o, human-rabbit chimera; Iodo-RTX, iodo resiniferatoxin; TM, transmembrane domain. (1Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7310) Google Scholar)) has been cloned and shown to be a nonselective cation channel with high permeability to calcium. TRPV1 belongs to a superfamily of ion channels known as transient receptor potential channels (TRPs) several of which appear to be sensors of temperature (2Julius D. Basbaum A.I. Nature. 2001; 413: 203-210Crossref PubMed Scopus (1985) Google Scholar, 3Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 6: 387-396Crossref Scopus (982) Google Scholar). TRPV1 can be activated by exogenous agonists (capsaicin and RTX) and by physical stimuli such as heat (>42 °C) and protons (pH 5). Possible endogenous ligands released during tissue injury have also been suggested, including anandamide (arachidonylethanolamine or AEA) and products of lipoxygenases such as 12-hydroperoxyeicosatetraenoic acid, N-arachidonyldopamine (NADA), and N-oleoyldopamine (OLDA) (4Hwang S.W. Cho H. Kwak J. Lee S.Y. Kang C.J. Jung J. Cho S. Min K.H. Suh Y.G. Kim D. Oh U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6155-6160Crossref PubMed Scopus (976) Google Scholar, 5Olah Z. Karai L. Iadarola M.J. J. Biol. Chem. 2001; 276: 31163-31170Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 6Huang S.M. Bisogno T. Trevisani M. Al-Hayani A. De Petrocellis L. Fezza F. Tognetto M. Petros T.J. Krey J.F. Chu C.J. Miller J.D. Davies S.N. Geppetti P. Walker J.M. Di Marzo V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8400-8405Crossref PubMed Scopus (840) Google Scholar, 7Chu C.J. Huang S.M. De Petrocellis L. Bisogno T. Ewing S.A. Miller J.D. Zipkin R.E. Daddario N. Appendino G. Di Marzo V. Walker J.M. J. Biol. Chem. 2003; 278: 13633-13639Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Ji et al. (8Ji R.R. Samad T.A. Jin S.X. Schmoll R. Woolf C.J. Neuron. 2002; 36: 57-68Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar) reported that TRPV1 is detectable at increased levels after inflammatory injury in rodents and speculated that the increased level of TRPV1 protein combined with the confluence of stimuli present in inflammatory injury states leads to a reduced threshold of activation of nociceptors that express TRPV1, i.e. hyperalgesia. Indeed the converse is true that TRPV1-deficient mice display reduced thermal hypersensitivity following inflammatory tissue injury (9Caterina M.J. Leffler A. Malmberg A.B. Martin W.J. Trafton J. Petersen-Zeitz K.R. Koltzenburg M. Basbaum A.I. Julius D. Science. 2000; 288: 306-313Crossref PubMed Scopus (2991) Google Scholar). Structure-function studies of this channel are in their infancy, but fundamental observations have been reported. Publications of species differences, based upon differential binding of the radiolabeled TRPV1 agonist [3H]RTX to dorsal root ganglia membranes, were recorded even before TRPV1 was cloned (10Szallasi A. Blumberg P.M. Naunyn-Schmiedeberg's Arch. Pharmacol. 1993; 347: 84-91Crossref PubMed Scopus (71) Google Scholar). Of note, rabbits were found to be resistant to the acute toxicity of capsaicin (11Glinsukon T. Stitmunnaithum V. Toskulkao C. Baranawuti T. Tangkrisanavinont V. Toxicon. 1980; 18: 215-220Crossref PubMed Scopus (104) Google Scholar) and were found not to have [3H]RTX binding sites (10Szallasi A. Blumberg P.M. Naunyn-Schmiedeberg's Arch. Pharmacol. 1993; 347: 84-91Crossref PubMed Scopus (71) Google Scholar). These observations have provided the basis for an approach to identify key regions involved in TRPV1 binding and activation by RTX and capsaicin by cloning TRPV1 from capsaicin-sensitive and insensitive species (rat (1Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7310) Google Scholar); human (12Hayes P. Meadows H.J. Gunthorpe M.J. Harries M.H. Duckworth D.M. Cairns W. Harrison D.C. Clarke C.E. Ellington K. Prinjha R.K. Barton A.J. Medhurst A.D. Smith G.D. Topp S. Murdock P. Sanger G.J. Terrett J. Jenkins O. Benham C.D. Randall A.D. Gloger I.S. Davis J.B. Pain. 2000; 88: 205-215Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar); rabbit (13Edenson S. Tamir R. Neering S. Wild K. Yang R. Treanor J. Lile J. Soc. Neurosci. Abstr. 2000; 26: 39.4Google Scholar, 14Qu Y. Klionsky L. Tamir R. Edenson S. Wang J. Lile J. Wild K. Gavva N.R. Treanor J. Biophys. J. 2003; 84 (suppl.): 434Google Scholar); chicken (15Jordt S.E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar); and guinea pig (16Savidge J. Davis C. Shah K. Colley S. Phillips E. Ranasinghe S. Winter J. Kotsonis P. Rang H. McIntyre P. Neuropharmacology. 2002; 43: 450-456Crossref PubMed Scopus (86) Google Scholar)). Rat and human TRPV1 have been pharmacologically characterized proving that capsaicin and RTX are indeed agonists of TRPV1 (capsaicin EC50: 0.05–0.2 μm and RTX EC50: 0.3–11 nm) transiently expressed in HEK293 cells (12Hayes P. Meadows H.J. Gunthorpe M.J. Harries M.H. Duckworth D.M. Cairns W. Harrison D.C. Clarke C.E. Ellington K. Prinjha R.K. Barton A.J. Medhurst A.D. Smith G.D. Topp S. Murdock P. Sanger G.J. Terrett J. Jenkins O. Benham C.D. Randall A.D. Gloger I.S. Davis J.B. Pain. 2000; 88: 205-215Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 17Szallasi A. Blumberg P.M. Annicelli L.L. Krause J.E. Cortright D.N. Mol. Pharmacol. 1999; 56: 581-587Crossref PubMed Scopus (124) Google Scholar, 18McIntyre P. McLatchie L.M. Chambers A. Phillips E. Clarke M. Savidge J. Toms C. Peacock M. Shah K. Winter J. Weerasakera N. Webb M. Rang H.P. Bevan S. James I.F. Br. J. Pharmacol. 2001; 132: 1084-1094Crossref PubMed Scopus (178) Google Scholar, 19Shin J.S. Wang M.H. Hwang S.W. Cho H. Cho S.Y. Kwon M.J. Lee S.Y. Oh U. Neurosci. Lett. 2001; 299: 135-139Crossref PubMed Scopus (31) Google Scholar). Interestingly, these studies have indicated species differences in antagonism, such as the report that capsazepine blocks human but not rat TRPV1 response to low pH (18McIntyre P. McLatchie L.M. Chambers A. Phillips E. Clarke M. Savidge J. Toms C. Peacock M. Shah K. Winter J. Weerasakera N. Webb M. Rang H.P. Bevan S. James I.F. Br. J. Pharmacol. 2001; 132: 1084-1094Crossref PubMed Scopus (178) Google Scholar). Electrophysiological studies using membrane-impermeable analogues of capsaicin (20Jung J. Hwang S.W. Kwak J. Lee S.-Y. Kang C.J. Kim W.B. Kim D. Oh U. J. Neurosci. 1999; 19: 529-538Crossref PubMed Google Scholar) and mutational analysis of extracellular loops (21Jordt S.E. Tominaga M. Julius D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8134-8139Crossref PubMed Scopus (542) Google Scholar, 22Welch J.M. Simon S.A. Reinhart P.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13889-13894Crossref PubMed Scopus (169) Google Scholar) have identified domains that contribute to capsaicin and proton activation, respectively. These studies have demonstrated that capsaicin appears to function from the intracellular side, and protons act on an extracellular site to activate TRPV1. We have previously reported the cloning of rabbit TRPV1 and that it is capsaicin-insensitive but activated by heat (45 °C) and protons (pH 5) in transiently expressed HEK293 cells (13Edenson S. Tamir R. Neering S. Wild K. Yang R. Treanor J. Lile J. Soc. Neurosci. Abstr. 2000; 26: 39.4Google Scholar). Jordt and Julius (15Jordt S.E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar) have more recently shown that heterologously expressed chicken TRPV1 (gTRPV1) is similarly insensitive to activation by capsaicin but sensitive to heat (>42 °C) and proton (pH 4.5) stimuli. Furthermore, Jordt and Julius (15Jordt S.E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar) showed that the TM3/4 region of TRPV1 appeared to be responsible for capsaicin sensitivity. Experiments by other investigators have identified additional residues on the N- and C-terminal domains of TRPV1 that also appear to modify capsaicin sensitivity as well as [3H]RTX binding (23Jung J. Lee S.-Y. Hwang S.W. Cho H. Shin J. Kang Y.S. Kim S. Oh U. J. Biol. Chem. 2002; 277: 44448-44454Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 24Vlachova V. Teisinger J. Suankova K. Lyfenko A. Ettrich R. Vyklicky L. J. Neurosci. 2003; 23: 1340-1350Crossref PubMed Google Scholar). We describe in the present study amino acids in TRPV1 critical for vanilloid sensitivity by utilizing rabbit TRPV1 (Oryctolagus cuniculus, oTRPV1) and selected mutations of the TM3/4 region. We determine, utilizing radioactive calcium (45Ca2+) uptake assays and whole cell patch clamp techniques, the sensitivity of oTRPV1 and mutants to the published activators of TRPV1. Binding of [3H]RTX was used to probe residues important for the high affinity binding of this ligand to TRPV1. Furthermore, we examine the functional sensitivity of oTRPV1 and mutants to capsaicin site antagonists and show that gain of capsaicin sensitivity also confers competitive antagonist action at TRPV1. Last, we present a model of capsaicin and RTX bound to the TM3/4 region of rat TRPV1. Molecular Biology—A cDNA library was made in pSPORT vector from poly(A)+-containing RNA extracted from dorsal root ganglia dissected from New Zealand White rabbits. The library was screened at high stringency (2× SSC, 65 °C) with a rTRPV1 probe (bases 1063–2185, the sequence with GenBank™ accession number AF029310). Several clones were isolated, and the longest full-length clone, designated oTRPV1, was chosen for expression studies. The sequence of this cDNA has been submitted to the GenBank™ (accession number AY487342). Rat-rabbit (r/o) TRPV1 chimera was generated by restriction cloning. ClaI and PmlI restriction sites were introduced into pcDNA3.1-oTRPV1 construct using QuikChange site-directed mutagenesis kit (Stratagene). Ser505-Thr550 fragment was PCR-amplified from rTRPV1 (ClaI and PmlI sites were included in PCR primers) and cloned into oTRPV1 ClaI-PmlI. Point mutations were introduced using the QuikChange kit following the manufacturer's protocol. All constructs were verified by DNA sequencing. In Situ Hybridization—In situ hybridization was carried out using 33P-labeled riboprobes as described previously by Wilcox (25Wilcox J.N. J. Histochem. Cytochem. 1993; 41: 1725-1733Crossref PubMed Scopus (229) Google Scholar). A unique 500-bp PCR fragment of oTRPV1 cDNA was subcloned into the polylinker site of pCR2.1 vector (Invitrogen). Linearized constructs were transcribed with SP6 or T7 RNA polymerase to generate antisense or sense [33P]uridine triphosphate-labeled RNA probes, respectively (Promega SP6/T7 kit). Sections were hybridized overnight at 55 °C with the 33P-labeled antisense or sense riboprobes corresponding to oTRPV1. Sections were examined with dark field and standard illumination (bright field) to allow simultaneous evaluation of tissue morphology and hybridization signal. Transient Transfections—HEK293 cells were maintained in Dulbecco's modified Eagle's medium (supplemented with 10% fetal bovine serum, penicillin, streptomycin, and l-glutamine). Cells were transiently transfected with a cytomegalovirus promoter-based expression vector (pcDNA3.1, Invitrogen) encoding an appropriate TRPV1 receptor by using FuGENE (Roche Applied Science) transfection reagent (75 μl of FuGENE and 45 μg of plasmid per 1.5–1.7 × 107 cells in a 225-cm2 culture flask). After 24 h the large pool of transfected cells was reseeded into Amersham Biosciences Cytostar plates for 45Ca uptake studies, into 3-cm dishes for whole cell patch-clamp recording studies, into clear polystyrene 96-well plates for enzyme-linked immunosorbent assay or immunostaining, or spun down, and pellets were used for [3H]RTX binding assay. Enzyme-linked immunosorbent assay or immunostaining with appropriate anti-VR1 antibodies were used to measure expression levels. Stable Transfections—CHO cells stably expressing rTRPV1, oTRPV1, oTRPV1-I550T, oTRPV1-L547M, or oTRPV1-L547M/I550T were generated by transfection with pcDNA 3.1-expression vector encoding an appropriate TRPV1 cDNA. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal bovine serum, penicillin, streptomycin, l-glutamine, and nonessential amino acids. The CaOPO4 method was used for stable transfections (5 μg of DNA per 2 × 106 cells in 60-mm dishes). 800 μg/ml Geneticin was used as a selection agent. After about 2 weeks of selection single colonies were picked and screened for expression of TRPV1 in a 45Ca2+ uptake assay. Positive clones were expanded and used in all of our studies similarly to HEK293 transients. Functional Assays—The activation of TRPV1 is followed as a function of cellular uptake of radioactive calcium (45Ca2+, ICN). All the 45Ca2+ uptake assays had a final 45Ca2+ at 10 μCi/ml. The assays were as follows: 1) Agonist assay: agonists were incubated with vector or TRPV1 expressing HEK293 cells in 1:1 ratio of F-12 and Hanks' buffered saline solution supplemented with BSA, 0.1 mg/ml, and 1 mm HEPES at pH 7.4 at room temperature for 2 min in the presence of 45Ca2+ prior to compound washout. 2) Capsaicin antagonist assay: compounds were preincubated with vector or TRPV1 expressing HEK293 or CHO cells in Hanks' buffered saline solution supplemented with BSA, 0.1 mg/ml, and 1 mm HEPES at pH 7.4 at room temperature for 2 min prior to addition of 45Ca2+ in F-12 and then left for an additional 2 min prior to compound washout. 3) Proton antagonist assay: compounds were preincubated with vector or TRPV1 expressing HEK293 or CHO cells at room temperature for 2 min prior to addition of 45Ca2+ in 30 mm HEPES/MES buffer (final assay pH 5) and then left for an additional 2 min prior to compound washout. 4) Compound washout and analysis: assay plates were washed two times with phosphate-buffered saline, 0.1 mg/ml BSA using an ELX405 plate washer (Bio-Tek Instruments Inc.) immediately after functional assay. Radioactivity in the 96-well plates was measured using a MicroBeta Jet (Wallac Inc.). Compound activity was then calculated using GraphPad Prism. Maximum 45Ca2+ uptake in agonist dose response was considered as 100% for each agonist in calculating the EC50 values. [3H]RTX Binding Assay—Binding studies with [3H]RTX were carried as described previously with minor modifications (10Szallasi A. Blumberg P.M. Naunyn-Schmiedeberg's Arch. Pharmacol. 1993; 347: 84-91Crossref PubMed Scopus (71) Google Scholar). Binding assay mixtures were set up on ice in glass tubes (Kimble Glass Inc.) and consisted of 200 μl of binding buffer (5 mm KCl, 5.8 mm NaCl, 0.75 mm CaCl2, 2 mm MgCl2, 137 mm sucrose, 10 mm HEPES, pH 7.8), 50 μl of [3H]RTX (different concentrations, 37 Ci/mmol specific activity, PerkinElmer Life Sciences) and 100 μl of cell suspension (0.5–1 × 106/per tube). The assay mix contained BSA at a final concentration of 0.25 mg/ml (Cohn fraction V, Sigma). In each set of experiments, total binding and nonspecific binding were defined in the presence of 3.5 μl of cold RTX (1 μm final concentration). The reaction mixtures were incubated at 37 °C shaking water bath for 1 h (50 rpm). Binding reactions were terminated by chilling the assay mixtures on ice for 5 min. 100 μl of α1-acid glycoprotein (2 mg/ml; Sigma) was added into the binding mix and incubated for an additional 10 min to reduce nonspecific binding. The bound and free ligands were separated by centrifugation in a Beckman 12 Microfuge. The tip of the Microfuge tubes containing the cell pellet was cut off, and the bound radioactivity was determined by scintillation counting (Wallac). Data was analyzed using GraphPad Prism. Electrophysiology—HEK293 cells transiently expressing the TRPV1 channels were maintained at 37 °Cina5%CO2 atmosphere. Whole-cell membrane currents were recorded using the whole cell patch-clamp technique (26Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflugers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15453) Google Scholar). The external calcium-free recording solution contained 140 mm NaCl, 5 mm KCl, 10 mm EGTA, 2 mm MgCl2, 10 mm HEPES, and 10 mm glucose, pH 7.4. Recording micropipettes were filled with an internal recording solution containing 140 mm CsCl, 10 mm EGTA, and 10 mm HEPES, pH 7.2. The micropipettes had resistances ranging from 2 to 4 mÙ and were connected to an AxoPatch 200B patch-clamp amplifier (Axon Instruments Inc.), driven by a desktop computer through a DigiData 1322A digitizer (Axon Instruments Inc.). Liquid junction potentials were manually corrected before establishing the seal. Upon achieving a GÙ seal, the patch was ruptured and whole cell currents were recorded during the application of voltage pulses generated using pClamp version 8.0 software (Axon Instruments Inc.). Currents were filtered at 5 kHz by a low pass 8-pole Bessel filter and acquired at 10 kHz, in episodic mode. All experiments were conducted at room temperature (20–22 °C) by holding the membrane potential at -60 mV. A “sewer-pipe” perfusion system (Rapid Solution Changer model RSC-200, Bio-Logic Science Instrument SA, France) was used to apply solutions directly to the cell under study. Capsaicin and Ruthenium Red were dissolved directly into external recording solution. Recording solutions were adjusted to the desired pH by adding HCl. Data was analyzed using pClamp version 8.0 and Prism version 3.02 (GraphPad). Molecular Modeling—Molecular modeling was carried out using Insight II (2000) software (Accelrys Inc.). Transmembrane helices and connecting segments were modeled using the Biopolymer module of Insight II (2000). RTX and capsaicin structures were generated and minimized using Insight II tools. All studies of mutant TRPV1 function were conducted using transient transfections in HEK293 cells. Transient transfections of HEK293 cells followed by immunohistochemical staining for TRPV1 protein indicated that all TRPV1 cDNAs studied in this report appeared to be expressed. However, this technique did not allow for quantitative analysis of the number of functional channels expressed on the cell surface, and as such all of the data presented here are discussed as relative activity. Interpretation of 45Ca2+ uptake assays utilizing different TRPV1 mutants assumed that all have the same permeability to calcium ions. oTRPV1 gain-of-function mutants were also characterized by stable expression in CHO cells. oTRPV1 Is Less Sensitive to Capsaicin Activation Than Rat TRPV1—To determine if oTRPV1 is indeed less sensitive to vanilloids than other species, oTRPV1 was cloned from a bacterial colony screen of a rabbit DRG cDNA library utilizing hybridization with a radiolabeled 32P-rTRPV1 probe. A cDNA clone (2.4 kb) was identified whose predicted protein sequence had high homology to rTRPV1 (86% identity and 91% similarity) and hTRPV1 (87% identity and 92% similarity; Table I and Fig. 1A). In situ hybridization of rabbit dorsal root ganglia (DRG) sections with a probe generated from the oTRPV1 cDNA revealed strong labeling of cells in the DRG (Fig. 1B) with expression restricted to the small and medium diameter cell bodies consistent with that seen in other species. Studies showed that conditions capable of robustly activating rTRPV1 had a mixed effect on oTRPV1. Whereas oTRPV1 was activated by heat (45 °C) or pH 5 similar to rTRPV1, it was not activated by capsaicin at supra-maximal activation concentrations for rTRPV1 (Fig. 1C). Furthermore, oTRPV1-transfected HEK293 cells did not show any specific [3H]RTX binding, whereas rTRPV1-transfected cells showed specific binding with a KD value of 0.089 ± 0.01 nm.Table IIdentity and homology between TRPV1 from different species (rabbit, oTRPV1; rat, rTRPV1; human, hTRPV1; chicken, gTRPV1; guinea pig, gpTRPV1) For each species identity is shown at the top, and similarity is shown in the bottom row.oTRPV1hTRPV1gpTRPV1gTRPV1%rTRPV18685856591929177oTRPV1878465929077hTRPV184649077gpTRPV16476 Open table in a new tab Agonist sensitivity was also characterized by electrophysiology. Perfusion of voltage-clamped cells transiently expressing TRPV1 with low pH solution elicited an inward current, with amplitudes that increased with lower pH (e.g. Fig. 1D). Average peak currents at pH 5 for rTRPV1 and oTRPV1 were 310.3 ± 85.8 μA/microfarad (n = 5), 281.9 ± 24.6 μA/microfarad (n = 5), respectively. Low pH elicited a much smaller current of 1.02 ± 0.93 pA/picofarad (n = 6 cells) in mock transfected HEK293 cells, which indicated that the proton-activated current in (rat and rabbit) TRPV1-transfected cells was primarily mediated by TRPV1. Large currents were also observed in response to 1 and 10 μm capsaicin in rTRPV1-transfected cells. In contrast, 1 μm capsaicin failed to generate any current in oTRPV1-transfected cells, although 10 μm evoked a small current (Fig. 1D). These experiments confirmed that oTRPV1 was functionally expressed in HEK293 cells and that oTRPV1 was much less sensitive to activation by capsaicin than rTRPV1. However, the small current elicited with 10 μm capsaicin suggested a rudimentary capsaicin-site in oTRPV1. The ability of Ruthenium Red to block oTRPV1 was also tested in patch-clamp studies. Ruthenium Red (10 μm) application 10 s after pH 5 activation blocked 83.4 ± 8.2% of the oTRPV1 current (n = 5 cells; Fig. 1E). These data verified that proton activation of oTRPV1 is sensitive to pore blockade similar to rTRPV1. In summary, sequence similarities to TRPV1s from other species, the activation profile of oTRPV1 by proton and heat, blockade of the proton and heat responses by Ruthenium Red, and the expression pattern of oTRPV1 mRNA in rabbit dorsal root ganglia confirm that oTRPV1 is the rabbit orthologue of TRPV1. The limited sensitivity of oTRPV1 to capsaicin and RTX and lack of detectable [3H]RTX binding agree with published data on the expected properties of oTRPV1 (10Szallasi A. Blumberg P.M. Naunyn-Schmiedeberg's Arch. Pharmacol. 1993; 347: 84-91Crossref PubMed Scopus (71) Google Scholar, 11Glinsukon T. Stitmunnaithum V. Toskulkao C. Baranawuti T. Tangkrisanavinont V. Toxicon. 1980; 18: 215-220Crossref PubMed Scopus (104) Google Scholar). Residue 550 Is an Important Determinant for Vanilloid Sensitivity in oTRPV1—A rat-rabbit chimera (r/o chimera) of TRPV1 was constructed by transfer of transmembrane domains 3 through 4 (amino acids Ser505-Thr550) from rTRPV1 to oTRPV1, because Jordt and Julius (15Jordt S.E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar) previously showed that the TM3/4 region of TRPV1 appears to be responsible for capsaicin sensitivity. Functional analysis of transiently transfected cells by 45Ca2+ uptake showed that the r/o chimera gained sensitivity to vanilloids (EC50 for capsaicin: 0.051 ± 0.029 μm and RTX: 11 ± 5 nm) similar to rTRPV1 (Fig. 2A). Sensitivity of the r/o chimera to capsaicin was also characterized by electrophysiology. Currents evoked by pH 5 and 1 μm capsaicin were similar in the chimera and rTRPV1 (Figs. 1D and 2A, bottom panel). In addition, we also made a human-rabbit chimera (h/o chimera) transferring the Ser505-Thr550 from hTRPV1 to oTRPV1. Similar to r/o chimera, functional analysis showed that h/o chimera gained sensitivity to capsaicin (Fig. 2A), further confirming that the TM3/4 region is responsible for vanilloid sensitivity. Amino acid sequence alignment of the 505–550 region indicated that ten amino acids are different between rat and rabbit TRPV1, and six amino acids are different between human and rabbit TRPV1 (Fig. 2C). To determine which residues within this region were responsible for gain of functional sensitivity to vanilloids in oTRPV1, we mutated the residues that are different in rabbit from both rat and human TRPV1 (A505S, A520S, C534R, T540S, and I550T). Remarkably, changing the single residue at 550 in rabbit to the corresponding residue found in rat and human TRPV1 (I550T) was sufficient to confer gain of function for activation by capsaicin (Fig. 2B). Dose-response curves in 45Ca2+-uptake experiments indicated that the EC50 of capsaicin at the oTRPV1 channel was 14.8 ± 7.9 μm, whereas it was 0.016 ± 0.006 μm for rTRPV1, 0.051 ± 0.029 μm for the r/o chimera (described above), and 0.052 ± 0.034 μm for oTRPV1-I550T. In addition to the above studies using transiently transfected HEK293 cells, we have also verified oTRPV1-I550T sensitivity to vanilloids in stably expressing CHO cells. Vector-transfected HEK293 or parental CHO cells did not show significant 45Ca2+-uptake up to 40 μm capsaicin or 10 μm RTX. Other known TRPV1 agonists (Arvanil, Olvanil, 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), NADA, and OLDA) were inactive up to 40 μm at wild type oTRPV1 but functioned as potent agonists at oTRPV1-I550T (Table II). Changing residues A505S, A520S, C534R, and T540S individually or in various combinations did not cause any changes in response of oTRPV1 to vanilloids (data not shown). Lastly, patch clamp recordings confirmed gain of capsaicin sensitivity in oTRPV1-I550T; currents evoked by pH 5, 1, or 10 μm capsaicin were similar in r/o chimera and oTRPV1-I550T-transfected cells indicating that indeed Thr550 confers vanilloid sensitivity (Fig. 2, A and B).Table IIComparison of agonist activation of TRPV1 and mutants Selected agonists were tested in CHO cells stably expressing rTRPV1, oTRPV1, oTRPV1-L547M, oTRPV1-I550T, or oTRPV1-L547M/I550T. EC50 value for each cell line was determined using Prism software and expressed in micromol
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Resiniferatoxin
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A sensitive response of the nervous system to changes in temperature is of predominant importance for homeotherms to maintain a stable body temperature. A number of temperature-sensitive transient receptor potential (TRP) ion channels have been studied as nociceptors that respond to extreme temperatures and harmful chemicals. Recent findings in the field of pain have established a family of six thermo-TRP channels (TRPA1, TRPM8, TRPV1, TRPV2, TRPV3, and TRPV4) that exhibit sensitivity to increases or decreases in temperature, as well as to chemical substances eliciting the respective hot or cold sensations. In this study, we used behavioral methods to investigate whether mustard oil (allyl isothiocyanate) and capsaicin affect the sensitivity to heat, innocuous and noxious cold, and mechanical stimuli in male rats. The results obtained indicate that TRPA1 and TRPV1 channels are clearly involved in pain reactions, and the TRPA1 agonist allyl isothiocyanate enhances the heat pain sensitivity, possibly by indirectly modulating TRPV1 channels coexpressed in nociceptors with TRPA1. Overall, our data support the role of thermosensitive TRPA1 and TRPV1 channels in pain modulation and show that these two thermoreceptor channels are in a synergistic and/or conditional relationship with noxious heat and cold cutaneous stimulation.
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