The transient receptor potential vanilloid 1: Role in airway inflammation and disease

European Journal of Pharmacology 533 (2006) 207 – 214 www.elsevier.com/locate/ejphar

Review

The transient receptor potential vanilloid 1: Role in airway inflammation and disease Pierangelo Geppetti ⁎, Serena Materazzi, Paola Nicoletti Clinical Pharmacology Unit, Department of Critical Care Medicine and Surgery, University of Florence, Viale Pieraccini, 6, 50139, Florence, Italy Center of Excellence for the Study of Inflammation, University of Ferrara, Via Fossato di Mortara 19, 44100 Ferrara, Italy Accepted 13 December 2005 Available online 7 February 2006

Abstract The transient receptor potential vanilloid 1 (TRPV1) is an excitatory cation channel, rather selectively expressed in a subpopulation of nociceptive, primary sensory neurons that promote neurogenic inflammation via neuropeptide release. TRPV1 is activated by noxious temperature, low extracellular pH and diverse lipid derivatives, and is uniquely sensitive to vanilloid molecules, including capsaicin. TRPV1 expression and sensitivity is highly regulated by diverse G protein-coupled and tyrosine kinase receptors. Other exogenous or endogenous chemical agents, including reactive oxygen species, ethanol and hydrogen sulphide sensitize/activate TRPV1. In the airways, TRPV1 agonists cause cough, bronchoconstriction, microvascular leakage, hyperreactivity and hypersecretion. Patients with asthma and chronic obstructive pulmonary disease are more sensitive to the tussive effect of TRPV1 agonists and TRPV1 activation may contribute to respiratory symptoms caused by acidic media present in the airways during asthma exacerbation, gastroesophageal reflux induced asthma or in other conditions. TRPV1 antagonists may be useful in the treatment of these diseases. © 2005 Elsevier B.V. All rights reserved. Keywords: Transient receptor potential vanilloid 1; Cough; Asthma exacerbation; Acidic media

Contents 1. The transient receptor potential family of channels. . . . . . . . . 2. The transient receptor potential vanilloid 1 . . . . . . . . . . . . . 3. Activation of TRPV1 and neurogenic inflammatory responses . . . 4. Sensitization and regulation of TRPV1 function . . . . . . . . . . 5. Localization and function of TRPV1 in the airways . . . . . . . . 6. Cough and TRPV1 . . . . . . . . . . . . . . . . . . . . . . . . . 7. Putative endogenous TRPV1 agonists and the therapeutic potential 8. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. The transient receptor potential family of channels ⁎ Corresponding author. Clinical Pharmacology Unit, Department of Critical Care Medicine and Surgery, University of Florence, Viale Pieraccini, 6, 50139, Florence, Italy. Tel.: +39 055 4271329; fax: +39 055 4271280. E-mail address: [email protected] (P. Geppetti). 0014-2999/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.12.063

The transient receptor potential (TRP) family of proteins is currently under intense investigation in health and disease because these ion channels have been recognized to sense a vast range of stimuli and because of their wide distribution in different

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tissues and organs. TRPs are putative six-transmembrane proteins that assemble as tetramers to form cation-permeable pores. TRPs have been subdivided in three main subclasses TRPC, TRPM and TRPV (V stands for vanilloid) (Clapham, 2003; Montell et al., 2002). TRPP, TRPML and TRPN are additional and newly proposed subtypes of TRPs. More recently, a novel TRP-like channel, that responds to cold temperature (b 15 °C), has been cloned and termed ANKTM1 or TRPA1 (McKemy et al., 2002; Peier et al., 2002). Uncertainty exists as regard to the precise and multiple roles of TRPs. Their localization in the plasma membranes of neurons or other cells and a large body of evidence collected using a plethora of stimuli indicates that they are sensors of chemical and physical stimuli. TRP channels are the molecules used by mammals and humans to appreciate sweet and bitter tastes, and to discriminate warmth, heat and cold. However, intracellular localization (e.g. in the endoplasmic reticulum (Karai et al., 2004)) and evidence obtained about the cellular regulation of ion flux has suggested a role as modulators of Ca2+ homeostasis (Clapham, 2003; Montell, 1997), downstream to G protein-coupled receptors, most probably via the phospholipase C pathway. However, this otherwise fascinating hypothesis is not supported yet by the identification of a messenger molecule, which directly binds and activates the channel (Clapham, 2003). Phosphatidylinositol-4,5-bisphosphate (PIP2) binding and PIP2 hydrolysis inhibits and activates, respectively, TRPL in the Drosophila (Hardie, 2003) and the mammalian TRPV1 (Chuang et al., 2001; Prescott and Julius, 2003). However, a major role of PIP2 as TRP regulator has been challenged by the observation that constitutive activity of TRPM7 is increased by PIP2 binding and reduced by PIP2 hydrolysis (Runnels et al., 2002). Finally, TRPs have been proposed to regulate the so called capacitance Ca2+ entry or store-operated Ca2+ entry (SOCE). Store-operated Ca2+ entry channels are considered channels that link Ca2+ store depletion with Ca2+ entry. However, final proof that one or more TRPs are the exclusive and selective mechanism that mediate store-operated Ca2+ entry is still lacking (Clapham, 2003). Although there is no evidence for one or more specific and high affinity endogenous ligands for TRPs, a series of lipid derivatives, including arachidonic acid metabolites, have been claimed to gate TRPs. For example the endocannabinoid anandamide and its metabolite, arachidonic acid, activates, directly TRPV1 (Zygmunt et al., 1999) and, via a cytochrome P450 epoxygenase-dependent formation of epoxyeicosatrienoic acids, TRPV4 (Watanabe et al., 2003). 2. The transient receptor potential vanilloid 1 TRPV1, a 426 (in the rat) amino acid protein (Caterina et al., 1997) uniquely sensitive to vanilloid molecules, including capsaicin, the hot principle contained in the plants of the genus Capsicum (Szallasi and Blumberg, 1999), is activated by low extracellular pH (pH 6–5) (Bevan and Geppetti, 1994; Geppetti et al., 1991; Tominaga et al., 1998), and elevated concentrations (in the micromolar range) of the endocannabionid, anandamide (Zygmunt et al., 1999), the lipoxygenase metabolites of arachidonic acid, leukotriene B4 (LTB4) or 12-hydroperoxyei-

cosatetraenoic acids (12-HPETE) (Hwang et al., 2000) and N-arachidonoyl-dopamine (Huang et al., 2002). TRPV1 is also a thermosensor, activated by moderate noxious temperature between 42 and 53 °C (Caterina et al., 1997). TRPV1 together with other TRP channels (TRPA1, TRPM8, TRPV3, TRPV4, and TRPV2) enables mammals to discriminate different temperatures from noxious cold to noxious heat (Clapham, 2003; Montell, 1997; Montell et al., 2002). TRPV1 is highly expressed in a subset of primary sensory neurons of the trigeminal, vagal and dorsal root (DRG) ganglia with C- and A-δ fibers. These neurons have been defined as polymodal nociceptors because of their ability to detect noxious chemical, thermal and high threshold mechanical stimuli. TRPV1 mRNA is also expressed in diverse areas of the central nervous system including the limbic system, striatum, hypothalamus, thalamic nuclei, substantia nigra, reticular formation, locus coeruleus, and cerebellum (Mezey et al., 2000). There is also evidence that TRPV1 mRNA and protein are expressed in nonneuronal cells including, epithelial cells of the urothelium (Birder et al., 2001), keratinocytes (Inoue et al., 2002) and epithelial cells of the palatal rugae (Kido et al., 2003) (see also below). The investigation of the physiological and pathophysiological function, if any, of TRPV1 in non-neuronal cells, as well as in nonsensory neuronal cells may elucidate broader functions than pain perception. However, it should be underlined that there is not clear evidence for such roles yet. TRPV1 gating, excites terminals of primary sensory neurons and causes their depolarization and the initiation of action potentials. Orthodromic propagation of the depolarizing stimulus, contributes to reflex responses, including cough, urinary bladder voiding, peristalsis in the gut and other responses. Antidromic conduction of action potential to collateral nerve fibers, or direct gating of TRPV1 itself, allow Ca2+ influx into the nerve endings, a phenomenon that results in the local release of neuropeptides, including calcitonin gene-related peptide (CGRP) and the tachykinins, substance P (SP) and neurokinin A (NKA). Activation of CGRP receptors and tachykinin (NK1, NK2 and NK3) receptors on effector cells, particularly at the vascular levels, causes a series of inflammatory responses, collectively referred to as neurogenic inflammation (Geppetti and Holzer, 1996). The putative role of TRPV1 as a sensor of noxious temperature and acidic pH justifies the channel enrichment on peripheral terminals of primary sensory neurons. Less clear is the significance of the high TRPV1 expression on central terminals of primary sensory neurons in the dorsal horn of the spinal cord and medulla oblongata, two anatomical sites where noxious temperature or low pH can unlikely be encountered. However, lipid derivates that potentially may be produced in the spinal cord have been shown to stimulate the channel within the dorsal spinal cord (Tognetto et al., 2000), thus suggesting TRPV1 expressed on central terminals of primary sensory neurons may exert an homeostatic role at this level. Whereas TRPV1 is not required for appropriate temperature sensing, its genetic deletion impairs the development of thermal hyperalgesia (Davis et al., 2000). Urinary bladder function was also found altered in TRPV1 knockout mice (Birder et al., 2002). Pharmacological studies with the first

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generation (capsazepine) (Walker et al., 2003), and more recent TRPV1, antagonists (Lee et al., 2003; Pomonis et al., 2003) support the hypothesis that TRPV1 also contributes to mechanical hyperalgesia. 3. Activation of TRPV1 and neurogenic inflammatory responses The term neurogenic inflammation refers to a series of responses mainly present at the vascular level, but that also occur in other tissues and organs with a large variability according to the mammal species under investigation. At the vascular level neurogenic inflammation (in parenthesis the neuropeptide involved) consists of vasodilatation (CGRP), plasma protein extravasation and leukocyte adhesion to the vascular endothelium of postcapillary venules (substance P/neurokinin A) (Geppetti and Holzer, 1996). In non-vascular tissues, neurogenic inflammatory responses include cardiac positive chonotropic effects (CGRP), contraction of the smooth muscle of the iris sphincter (substance P/ neurokinin A), ureter, bladder and urethra (substance P/neurokinin A), relaxation of bladder (CGRP), exocrine gland secretion (substance P/neurokinin A), and other effects. The bronchomotor response in the airways illustrates the marked species-related variation in the effect produced by sensory nerve activation/ tachykinins. Excitation of TRPV1-expressing nerve terminals causes direct bronchoconstriction in the guinea pig (NK2/NK1) and indirect (mainly mediated by epithelial nitric oxide/prostanoids) bronchodilatation in the rat and mouse (NK1). In man, mainly NK2 but also tachykinin NK1 receptors mediate a robust bronchoconstriction (Amadesi et al., 2001). Of particular interest is the ability of tachykinins (NK1) to stimulate seromucous secretion (Geppetti et al., 1993) from bronchial glands, and to excite (NK3) postganglionic cholinergic nerve terminals in the human bronchus (Myers et al., 2005). Neurogenic inflammation markedly contributes to inflammatory responses both at the somatic and visceral levels in different mammal species. In the human skin there is strong evidence that capsaicin or histamine cause a flare response that being blocked by local anesthetics or by repeated application of topical capsaicin (capsaicin desensitization), is mediated by stimulation of terminals of TRPV1-expressing neurons and the subsequent release of neuropeptides. Less clear is, however, whether in man neurogenic inflammation plays a pathophysiological role at the visceral level. There is evidence that CGRP is released by capsaicin from human tissues in vitro (Franco-Cereceda, 1991; Geppetti et al., 1992) and during migraine attacks (Goadsby et al., 1990). A major role of CGRP released from trigeminal perivascular nerve fibers derived from the observation that BIBN 4096BS, a peptoid with high affinity for the CGRP receptor (Doods et al., 2000) that does not cross the blood brain barrier, reduces the pain and other symptoms associated with migraine attacks (Olesen et al., 2004). 4. Sensitization and regulation of TRPV1 function Expression of mRNA/protein and function of TRPV1, as those of other TRP channels, undergo marked plasticity by a

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series of regulatory and inflammatory mediators. TRPV1 plasticity, more than its ‘normal’ expression underlines its possible role in disease. Nerve growth factor (NGF) is required for survival of newborn rat dorsal root ganglia neurons and for expression of the TRPV1-phenotype in adult rat dorsal root ganglia neurons in culture (Bevan and Winter, 1995). NGF via a p38 mitogen-activated protein (MAP) kinase increases TRPV1 protein transportation to the peripheral endings of sensory neurons, a phenomenon associated with an increase in heat hypersensitivity (Ji et al., 2002). Thus, upregulation of TRPV1 could contribute to the proinflammatory role of NGF released from mast cells during asthma exacerbations (Bonini et al., 1996). Protein kinases A and C (PK), and phospholipase A and C metabolites also regulate TRPV1 by diverse mechanisms. The threshold temperature for TRPV1 stimulation is lowered by anandamide through a protein kinase C (PKC)-ε dependent pathway (Premkumar and Ahern, 2000). The major proinflammatory peptide, bradykinin via activation of the B2 receptor sensitizes TRPV1 by diverse intracellular mechanisms, including PKC-ε (Premkumar and Ahern, 2000; Sugiura et al., 2002), displacement of PIP2 from TRPV1 binding (Chuang et al., 2001), and 12- and 5-lipoxygenase metabolites production (Carr et al., 2003; Shin et al., 2002). In vagal afferent C-fibers, bradykinin evokes membrane depolarization and action potential discharge through the additive effects of TRPV1 activation (Lee et al., 2005). Prostaglandins may induce cough (Costello et al., 1985) and one major adverse effect of angiotensin converting enzyme inhibitors is cough (Israili and Hall, 1992). The interesting hypothesis that protein kinase C/protein kinase A-dependent pathways are involved in TRPV1 sensitization that results in a lowered tussive threshold to capsaicin is currently under intense scrutiny. Protease-activated receptor-2 (PAR-2) is stimulated though cleavage of its extracellular tail by proteases such as trypsin and tryptase. PAR-2 is expressed in a large variety of cells, including TRPV1-positive sensory neurons, and PAR-2 stimulation promotes neurogenic inflammation and hyperalgesia (Steinhoff et al., 2000; Vergnolle et al., 2001). A large body of evidence indicates that in the lung PAR-2 activation is associated with inflammatory responses, including exaggeration of allergic reaction (Schmidlin et al., 2002), bronchoconstriction and plasma protein extravasation (Su et al., 2005), all effects mediated, in large part, by a sensory neurogenic mechanism. The recent finding that PAR-2 stimulation upregulates the function of TRPV1 through a PKC-dependent mechanism adds PAR-2 to the list of G protein-coupled receptors that regulating TRPV1 orchestrate the neural components of the inflammatory response in the airways (Amadesi et al., 2004). Sensitization of TRPV1 by PKC and cAMP-dependent protein kinase (PKA) pathways seems to be promiscuously used by different stimuli, including capsaicin, anandamide, heat and protons (Bhave et al., 2002; De Petrocellis et al., 2001; Premkumar and Ahern, 2000; Vellani et al., 2001), but it is not unique to endogenously generated agents. The common notion that exposure to mucosal surfaces or wounds to alcoholic tinctures causes burning pain has remained without an explanation until the observation that ethanol excites TRPV1-expressing rat

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sensory neurons and human embryonic kidney (HEK-293) cells transfected with the human TRPV1, but not wild type HEK-293 cells (Trevisani et al., 2002). TRPV1, usually stimulated at 42 °C, in the presence of ethanol is activated by lower temperatures, as the physiological temperature of 37 °C, because ethanol lowers the threshold temperature for TRPV1 activation by about 8 °C (Trevisani et al., 2002). In the presence of ethanol, effects of TRPV1 agonists, including anandamide and protons, are markedly potentiated (Trevisani et al., 2002). Ethanol-induced asthma is still a poorly understood condition, where a primary role for acetaldehyde has been proposed (Vally and Thompson, 2003). Exposure to ethanol of isolated guinea pig bronchi, and intragastric ethanol in vivo caused bronchoconstriction and bronchial microvascular leakage, through a capsaicin-sensitive, TRPV1-dependent and tachykinin mediated mechanism (Trevisani et al., 2004a). This finding supports the hypothesis that ethanol, by lowering the temperature threshold for TRPV1 activation causes a series of neurogenic proinflammatory responses of relevance for alcoholinduced asthma. 5. Localization and function of TRPV1 in the airways In a large variety of diseases, including migraine, osteoarthrtitis, cystitis and detrusor hyperreflexia, fecal urgency and inflammatory bowel diseases, post-herpetic neuralgia and postmastectomy pain and many others diseases, a role for TRPV1expressing neurons and neurogenic inflammation has been proposed (Geppetti and Holzer, 1996; Geppetti and Trevisani, 2004). Neurogenic inflammation has been proposed also to contribute to asthma (previous reviews have covered this issue, (Barnes, 1986; Bertrand and Geppetti, 1996; Joos and Pauwels, 2001)). Contribution of neurogenic inflammation in chronic obstructive pulmonary disease (COPD), is suggested by the findings that cigarette smoke, the major causative agent of the disease, produces an early inflammatory response completely mediated by sensory neuropeptides (Baluk et al., 1996; Lundberg and Saria, 1983). The hypothesis that TRPV1 contributes to some of the major symptoms of asthma and COPD is corroborated by a series of anatomical, physiological and pathophysiological findings reported below. In the guinea pig TRPV1-positive nerve fibers localized within the epithelium of the trachea and around smooth muscle and blood vessels and within the lower airways, in the vicinity of bronchi and bronchioles, and around alveolar tissue. Although TRPV1 immunoreactive and neuropeptide negative axons were also seen, TRPV1 in the tracheal epithelium mostly co-localized with substance P (Watanabe et al., 2005). Of interest for further discussion is the finding that in the guinea pig no TRPV1 was found localized to airway epithelial cells (Watanabe et al., 2005). In contrast with this immunohistochemical observation, RT-PCR revealed that TRPV1, together with acid sensing ion channel 1a (ASIC1a), and ASIC3 subunits of proton-gated ion channels, are expressed in immortalized human bronchial epithelial cells, normal human bronchial/tracheal epithelial cells, and normal human small airway epithelial cells from the distal airways (Agopyan et al., 2003). TRPV1 seemed to be associated to Ca2+ regulation and apoptosis in

these cells, as apoptotic response and large part of the Ca2+ response caused by exposure of these cells to particulate matter (PM), was inhibited by capsazepine and because particulate matter exposure induced apoptosis in mouse sensory neurons, but not in those pretreated with capsazepine, in the absence of extracellular calcium or in sensory neurons from TRPV1 knockout mice (Agopyan et al., 2004). Thus, the hypothesis was advanced that capsaicin- and acid-sensitive irritant receptors, located on somatosensory cell bodies and their nerve fiber terminals, subserve particulate matter-induced airway inflammation (Veronesi et al., 2000). Neuropeptides (tachykinins and CGRP) released from terminals of TRPV1-expressing neurons have been proposed to contribute to the immune response (van Hagen et al., 1999). However, recent evidence suggests that mouse dendritic cells (DC), a key cell type of the vertebrate immune system, expresses TRPV1 and its activation by capsaicin or heat leads to dendritic cells maturation and draining lymph nodes (Basu and Srivastava, 2005). The intriguing hypothesis that TRPV1 and its putative ligands orchestrate an early immune response is, however, challenged by additional observations that failed to detect the occurrence of a functional TRPV1 in mouse dendritic cells (O'Connell et al., 2005). TRPV1 expression has been recently detected in many other non-neuronal human cells in skin (human mast cells, epidermal keratinocytes) (Stander et al., 2004), and liver (HepG2) cells (Vriens et al., 2004), in prostate epithelial cell lines PC-3 and LNCaP and prostate tissue (Sanchez et al., 2005), and in intra-cytoplasmatic granules matching mitochondrial structures of gastric parietal cells (FaussonePellegrini et al., 2005). It is possible that also in the airways TRPV1 occurs in extraneuronal cells from where it may contribute to homeostasis and inflammation. However, it should be underlined that conclusive evidence that non-neuronal TRPV1 is functional and exerts defined biological roles is still absent (Fig. 1). 6. Cough and TRPV1 Activation of afferent nerve fibers with rapidly adapting receptors (RAR) that conduct action potentials in the A–δ range initiates the cough reflex. RARs are exquisitely sensitive to mechanical perturbation of their receptive fields, but are unaffected by a variety of chemical agents or messengers, including bradykinin, and capsaicin. In contrast, C-fibers are activated by capsaicin and bradykinin, but are much less sensitive to mechanical stimulation (Undem et al., 2002). However, capsaicin and bradykinin substantially reduce the electrical threshold for initiating the cough reflex, and capsazepine prevents the increased cough sensitivity induced by capsaicin (Mazzone et al., in press). A series of experiments with tachykinin receptor antagonists suggested that TRPV1/C-fiber activation sensitizes the cough reflex via central mechanisms (Mazzone et al., in press). A key role played by airway inflammation in the upregulation of the cough reflex is strengthened by the following recent observations: i) inflammation substantially increases the mechanical sensitivity of RAR fibers, and ii) causes a phenotypic

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Fig. 1. Schematic representation of cell types that putatively express functional TRPV1 and the biological functions produced by the channel following exposure to its putative ligands.

switch in neuropeptide innervation of the airways, as RAR neurons synthesize tachykinins and calcitonin gene-related peptide, and finally iii) the proinflammatory peptide, bradykinin, seems to activate C-fiber by stimulating TRPV1 (Undem et al., 2002). In addition, the pivotal function of TRPV1 in the cough response in chronic airway inflammatory disease is underlined by the lower threshold to cough induced by capsaicin (a common tussive stimulus in experimental animals and in man) in patients with asthma, cough variant asthma, and COPD (Doherty et al., 2000; Fujimura et al., 1994; Millqvist, 2000; Wong and Morice, 1999). Although channels known to sense low extracellular pH include ASICs and electrophysiological studies propose that the tussive response to citric acid is mediated by ASICs (Kollarik and Undem, 2002), pharmacological evidence with two TRPV1 antagonists, capsazepine (Lalloo et al., 1995) and iodo-resineferatoxin (Trevisani et al., 2004b) gives robust support to the role of this channel to mediate citric acid-induced cough. Thus, TRPV1 may be considered as a major molecular entity involved in the tussive response in health and disease and its targeting may represent a novel therapeutic strategy in treating cough. Increased expression of TRPV1 has been found in inflammatory diseases of the gut, where its exaggerated expression was associated with the severity of the symptoms (Chan et al., 2003). Similar findings have been obtained in the respiratory tract. Whereas PGP-9.5-positive nerve fibers were not increased in the airway epithelium of patients with chronic cough, a fivefold increase in TRPV1-positive nerve profiles was found in these patients. A significant correlation between capsaicin tussive response and the number of TRPV1-positive nerves was also

found in patients with chronic cough (Groneberg et al., 2004). Expression of TRPV1 has been also found increased in the airway smooth muscle of patients with chronic cough, where it localized in a thapsigargin insensitive compartment (Mitchell et al., 2005). In line with this latter observation, the bronchoconstrictive eicosanoid, 20-hydroxy-eicosatetraenoic acid (20-HETE), a product of cytochrome P-450 (CYP-450) omega-hydroxylase, and capsaicin have been found to produce a capsazepine-sensitive tonic contraction in airway smooth muscle cells. Thus, 20-HETE could be added to the series of putative endogenous TRPV1 agonists, that in the present case contributes to bronchoconstriction by stimulation of non-neuronal TRPV1 channels (Rousseau et al., 2005). 7. Putative endogenous TRPV1 agonists and the therapeutic potential of TRPV1 antagonists in airway disease The current active search for high affinity and selective TRPV1 antagonists has yielded a series of molecules tested in various models of disease that include different types of neuropathic pain and urinary bladder dysfunction. TRPV1 antagonists are under intense investigation in several other animal models of disease. The identification of the endogenous ligand(s) that, during inflammation or injury, activates TRPV1 is of paramount importance to make the channel a valuable therapeutic target. Lipid substances as anandamide (Tucker et al., 2001) or N-arachidonoyl-dopamine (Harrison et al., 2003) have been shown to cause bronchoconstriction in guinea pigs entirely via a TRPV1-dependent pathways. In addition to lipid derivatives, protons seem to play a major role as putative ligands of

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TRPV1 in the airways. The original proposal (Bevan and Geppetti, 1994) that low extracellular pH might activate the capsaicinreceptor was fully confirmed following TRPV1 cloning (Tominaga et al., 1998). Exposure to endogenous and exogenous acids in the airways (acidopnea) evokes cough, bronchoconstriction, airway hyperreactivity, microvascular leakage, and heightened production of mucous, all effects mediated by neurogenic inflammation. The role of acidity to the mechanism of asthma is being increasingly appreciated (Harding, 2003), and now the contribution of inhalation of acidic media because of gastroesophageal reflux disease and following exposure to acid fog, pollution or workplace exposure in asthma seems widely confirmed (Harding, 2003). In addition, a marked decrease in pH in the exhaled breath condensate, that seems to reflect the lining fluid pH of the lower airways, via the activation of TRPV1 and the release of sensory neuropeptides, has been proposed to contribute to the mechanisms of obstructive airway diseases (Hunt et al., 2000; Ricciardolo et al., 2004). In addition to protons, temperature and a large variety of lipids, recent studies have added novel molecules to the stimuli known to produce airways inflammation via TRPV1 activation and neurogenic mechanisms. The odorous and irritant gas, hydrogen sulfide (H2S), has been recently described as an endogenous mediator with diverse biological effects (Li et al., 2005). NaHS, that non-enzymatically generates H2S, increased sensory neuropeptide release in the airways and caused in vivo bronchoconstriction and microvascular leakage in a capsazepinesensitive manner. This novel mechanism may contribute to the irritant action of H2S in the respiratory system, possibly through TRPV1 activation (Trevisani et al., 2005). Electrophysiological results suggest that both the TRPV1 and the purinergic P2X receptors mediate the sensory transduction of reactive oxygen species (ROS), especially H2O2 and OH, by capsaicin-sensitive vagal lung afferent fibers (Ruan et al., 2005). This finding is of particular relevance when considering the role of reactive oxygen species in the mechanism of cigarette smoke induced injury and of COPD. Finally, stimulation of afferent fibers in the upper airways, e.g. in the nose, may modulate the responsiveness of the same type of nerve terminals in the lower airways. The role of NGF to upregulate TRPV1, described previously in isolated neurons (Ji et al., 2002), has received support from recent data in man in vivo. Patients sensitive to scents and chemicals with respiratory symptoms showed a significant increase in NGF in the nasal lavage fluid, a phenomenon associated with an increased tussive response to capsaicin (Millqvist et al., 2005). In another study intranasal capsaicin enhanced the cough response provoked by inhalation of a tussigen in humans (Plevkova et al., 2004). 8. Conclusions Enhancement of the cough response to capsaicin and citric acid is reported in patients suffering from two respiratory illness, asthma and COPD, widely distributed in the general population. Cough by capsaicin and most likely cough by citric acid are mediated by TRPV1 activation. Thus, it is possible that chronic inflammation changes the phenotype of sensory neurons and

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