The Canonical TRPs

The mammalian “canonical” TRPCs are structurally most closely related to Drosophila TRP and include seven proteins (TRPC1-7), although in humans TRPC2 is a pseudogene [17,18,33–35]. All TRPCs are activated by stimulation of G-protein-coupled receptor and receptor tyrosine kinases, commonly downstream of phospholipase C (PLC) activation. These channels play important roles in calcium homeostasis by admitting external Ca^2 + in a receptor- and/or store-operated manner, in the latter case likely in heteromeric complexes with Orai proteins [36].

ASM which controls airway caliber, plays a central role in the pathogenesis of airway hyperresponsiveness in asthma. Increase in [Ca^2 +]_i in ASM causes contraction leading to airway narrowing, and these [Ca^2 +]_i signals are also important for smooth muscle proliferation, hyperplasia, or hypertrophy, the defining characteristics of asthma [37–40]. In addition, ASM can secrete inflammatory mediators, which recruit and activate inflammatory cells, such as mast cells and T-lymphocytes. In turn, inflammatory mediators can alter Ca^2 + homeostasis in ASM and sensitize them to agonists [39]. There is growing evidence for the roles of TRPCs in ASM calcium regulation relevant to these pathogenic events.

The airways are innervated by cholinergic nerve fibers, and the major parasympathetic transmitter acetylcholine, when released, induces excitation and contraction of ASM. Although numerous ion channel mechanisms mediate acetylcholine-induced membrane depolarization, opening of receptor-operated cation channels is one common mechanism present in ASM and other types of visceral smooth muscles [41–43]. Studies of muscarinic receptor and TRPC knockout mice, as well as the intracellular signal transduction pathways, showed that in ileal myocytes both M₂ and M₃ acetylcholine receptor subtypes, which are commonly coexpressed in visceral smooth muscles, converge to activate two main contributors to the muscarinic cation current termed mI_CAT—TRPC4 (which mediates about 80% of mI_CAT) and TRPC6 [44–46]. Although similar studies have not yet been performed on ASM, it should be noted that histamine, which is intimately associated with the pathology of allergy and inflammation of the airways, acting at H₁ receptor activates cation currents in ASM, that require [Ca^2 +]_i rise and simultaneous activation of G_i/o and G_q/11 proteins, but not diacylglycerol formation [43]. These signal transduction pathways are similar to those leading to the activation of mI_CAT by costimulation of M₂ and M₃ receptors [47]. However, I-V relationships of this cation current in ASM differ from mI_CAT I-V curves [43]. It thus appears that receptor-operated cation channels are similar, but not identical, in ASM and gastrointestinal myocytes. Indeed, expression of TRPC4 and -C6, as well as other TRPC channels (TRPC1, -3, -4, and -6) has been described in human ASM [48,49]. In guinea-pig ASM, mRNA encoding TRPC1, -3, -4, -5, and -6 was also detected at levels comparable to the brain, whereas mRNA encoding TRPC2 and TRPC5 was even more abundant [50]. These studies implicate TRPCs as strong candidates for the Ca^2 + influx pathways required for sustained ASM contraction. Indeed, more recently in freshly isolated mouse ASM myocytes, constitutively active single cation channels were described, which could be inhibited by TRPC3, but not TRPC1, antibodies, and siRNAs [51]. Gene silencing experiments revealed that TRPC3 plays an important role in the regulation of the resting membrane potential and [Ca^2 +]_i in ASM, as well as in methacholine-evoked increase in [Ca^2 +]_i [51]. Importantly, in ovalbumin-sensitized mice, a model for airway hyperresponsiveness, ASM cells’ TRPC3 protein expression was increased, accompanied by increased cation channel activity, membrane depolarization, and enhanced contractile responses to methacholine. Although TRPC1 expression was not altered, it was also involved in these cellular responses in asthmatic, but not in normal, ASM [51].

It is interesting to note that similar contributions of TRPC1, -3, and -6 proteins to the formation of native constitutively active, store-, and receptor-activated cation channels have been described in vascular smooth muscles [52]. However, the role of TRPC3 in ASM appears to be more pronounced; for example, TRPC6 gene silencing in guinea-pig primary ASM cells does not affect OAG-induced Ca^2 + signaling [53]. In contrast, TRPC3 gene silencing by siRNA treatment significantly inhibits tumor necrosis factor alpha (TNF-α)- and acetylcholine-induced Ca^2 + influx in cultured human ASM cells [49]. Because TNF-α, the proinflammatory cytokine, contributes to airway hyperresponsiveness by altering ASM [Ca^2 +]_i homeostasis, these results suggest the important role of TRPC3 in inflammatory airway diseases, such as asthma and COPD [54]. In addition, there is evidence that up-regulated TRPC1, which is associated with an enhanced Ca^2 + entry and elevated [Ca^2 +]_i, may play an important role in bronchial constriction and ASM proliferation in asthma [54,55].

Cystic fibrosis (CF), one of the most common severe life-threatening inherited diseases, is caused by an autosomal recessive mutation of the CF transmembrane conductance regulator (CFTR) gene. The most common phenylalanine deletion mutant F508del causes the most severe phenotype—a dehydrated airway surface and impaired mucus clearance due to the lack of chloride secretion and excessive sodium absorption [56]. CFTR regulates various apical epithelial ion channels (e.g., outwardly rectifying Cl⁻ channels, ENaC, K_ATP), but very little is known about its interaction with TRP channels. In aortic endothelial cells isolated from TRPC4^−/− mice functional interaction between TRPC4 and CFTR has been established by showing that, although the expression of CFTR was unchanged, the current was suppressed in TRPC4-deficient cells [57]. It was thus suggested that TRPC4 may provide a scaffold for the formation of functional CFTR channels. Such multiprotein complexes have been recently revealed by coimmunoprecipitation analysis of CFTR, F508del-CFTR, and TRPC6 interactions in human tracheal epithelial cells. Functionally, CFTR down-regulates TRPC6-mediated Ca^2 + influx, but TRPC6 up-regulates CFTR-dependent Cl⁻ transport in airway epithelial cells. Because in CF this functional coupling is lost, TRPC6-mediated Ca^2 + influx is abnormally increased in CF compared with non-CF cells [58].

There is also substantial evidence for the involvement of TRPCs, and TRPC6 in particular, in the regulation of hypoxic pulmonary vasoconstriction, which is responsible for ensuring the physiological ventilation/perfusion matching during acute episodes of local alveolar hypoxia, but with no such role in chronic generalized hypoxia associated with vascular remodeling and pulmonary hypertension [59,60]. TRPC6 may also contribute to the hypersecretion of mucus in chronic bronchitis [61], migration of neutrophils [62], and allergic airway inflammation [63], making it a potential new drug target in asthma and COPD [54]. Compared with WT mice, TRPC6^−/− mice exhibited reduced allergic responses after allergen challenge (e.g., reduced airway eosinophilia and blood IgE levels, decreased levels of Th2 cytokines IL-5 and IL-13), but unaltered lung mucus production [63]. Intriguingly, agonist-induced contractility of tracheal rings was increased in TRPC6-deficient mice, contrary to the expectations based on the previously discussed role of TRPC6 in ASM. However, compensatory up-regulation of TRPC3 in ASM could explain this controversy. TRPC1 also plays an essential role in allergic reactions by negatively regulating TNF-α production by mast cells, as recent comparison of antigen-mediated anaphylaxis in Trpc1^−/− and WT mice has revealed [64].

Macrophages play key roles in the pathophysiology of COPD. Analysis of TRPC expression in human lung macrophages revealed that TRPC6 mRNA expression was significantly elevated in alveolar macrophages from patients with COPD compared with control subjects, whereas there was no difference in the expression levels of TRPC3 and -7 [65]. This increased molecular and functional expression, as confirmed by patch-clamp analysis, was particularly pronounced in small macrophages, and it correlated with COPD diagnosis [65].

The Vanilloid TRPs

This TRP subfamily contains six mammalian members, TRPV1-6. TRPV1-4 are activated by heat, as well as a broad array of chemicals or mechanical stimuli. As already noted, TRPV5 and -6 are highly Ca^2 + permeable and are tightly controlled by [Ca^2 +]_i, which is important for the regulated Ca^2 + uptake in epithelial cells [17,18,34,35,66,67]. There have been many exciting developments regarding the roles of TRPV channels in the respiratory system. These roles often conformed to the expectations based on the knowledge of their diverse expression patterns and activation mechanisms, as well as functional properties of heterologously expressed TRPV channels, but in native cells there have been also many unexpected findings.

Among TRPVs, TRPV1 (capsaicin receptor) remains the most extensively studied channel. It is truly a polymodal sensor, that can be activated by agonists (capsaicin and resiniferatoxin), heat (> 42 °C), protons, endocannabinoid lipids such as anandamide, and eicosanoids [17,18,34,35,66–69]. Furthermore, TRPV1 is sensitized by protein kinase A (PKA), protein kinase C (PKC), Ca^2 +/calmodulin-dependent kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K), and PLC activation (likely in a PIP₂-dependent manner). TRPV1 is widely expressed in neuronal, nociceptive neurons in particular and nonneuronal cells, and thus it is often considered as a focal point of signal transduction and cell communication.

Heterogeneous groups of TRPV1-positive afferent fibers (some colocalized with substance P and CGRP) are found throughout the entire respiratory tract—within and beneath the epithelium, around blood vessels, within ASM and alveoli, and their number increases under allergic inflammatory conditions [70,71]. It is thus not surprising that the majority of studies have focused on TRPV1 roles in sensory nerves and in particular on its role in airway inflammation and sensitization of cough reflex in chronic cough [3,9,11,21,22,24,32,54,72–76]. Indeed, the TRPV1 agonist capsaicin readily provokes cough in human and animal challenge testing. This response is enhanced in asthma and COPD, and it can be inhibited by TRPV1 antagonists, strongly suggesting TRPV1 as a putative molecular target for the development of novel antitussive drugs [21,24,32,75,76]. Endogenous ligands, such as prostaglandin E₂ and bradykinin, are known to sensitize the response to tussive stimuli (via EP3 and B2 receptors, respectively), and they both have been shown to mediate their effects though TRPV1 (and TRPA1) channels [77].

It should be noted that TRPV1 gene polymorphisms can further increase the risk of cough due to irritant exposures, such as cigarette smoke and occupational exposures [27]. In contrast, one genetic variant (SNP) of TRPV1 (TRPV1-I585V) shows decreased channel activity in response to capsaicin and heat, and this loss-of-function is associated with a reduced risk of childhood asthma [78]. Interestingly, comparative analysis of TRPV1- and TRPA1-mediated cough responses in guinea pigs showed higher efficacy of TRPV1 agonists, likely due to higher potency of TRPV1 in the sustained activation of airway C fibers [79].

Our recent study showed that TRPV1 is also functionally expressed in human bronchial epithelial cells, where it can mediate capsaicin- and low pH-induced cation currents [20]. In these cells the TRPV1 agonist capsaicin induced dose-dependent IL-8 release, which could be blocked by the TRPV1 antagonist capsazepine. Moreover, TRPV1 was overexpressed in patients with refractory asthma, suggesting its contribution to airway hypersensitivity in severe asthma [20]. We have also detected molecular expression of TRPV2, -3, -4, and V6 in human bronchial epithelial cells, but their functional and pathophysiological roles in these cells remain to be studied.

In the context of lung inflammatory diseases it is an important finding that TRPV1 activation can lead to lung cell death [80]. Moreover, in animal models of asthma, TRPV1 knockdown attenuated airway hyperresponsiveness, airway inflammation, goblet cell metaplasia, and subepithelial fibrosis induced by IL-13 in BALB/c mice, suggesting that up-regulation of TRPV1 by mediators of allergic inflammation in bronchial epithelia could lead to epithelial injury [81]. TRPV1 was also implicated in a formaldehyde-induced model of asthma in mice [82].

TRPV1 can be selectively activated by some types of PM, such as coal fly ash, the most potent TRPV1 activator [83]. Exposure of human airway and epithelial cells and mouse sensory neurons to PM evoked apoptosis associated with sustained calcium influx through TRPV1 channels, which was completely prevented by capsazepine, the TRPV1 antagonist, or in sensory neurons from TRPV1^−/− mice [84]. In human bronchial epithelial cells, exposure to PM induces [Ca^2 +]_i-dependent production of cytokines IL-1β and IL-8, although the role of TRP channels in this process has not been elucidated [85]. Sepsis-evoked acute lung injury induced by hydrogen sulfide (H₂S) has been shown to enhance neurogenic inflammation, COX-2 and PGE₂ via TRPV1 activation [86]. Consistent with these findings, capsazepine can protect against H₂S-induced lung inflammation [87]. Similarly, sulfur dioxide (SO₂) exposure also causes TRPV1 up-regulation, paralleled by an increased sensitivity of the cough response to capsaicin [88]. Inflammatory cells, such as mast cells, by releasing inflammatory proteases (mast cell tryptase and trypsin) cleave protease-activated receptor 2 (PAR2), which is coexpressed with TRPV1 in small- to medium-diameter neurons and thus sensitize neuronal TRPV1 through PKC [89]. This cascade of events can reduce the temperature threshold for TRPV1 activation from 42 °C to well below body temperature [90]. In addition, endogenous agonists of TRPV1 (endovanilloids) also appear to be involved in lung injury during inflammation [91].

Based on the preceding studies, it is an attractive hypothesis that in neurogenic inflammation enhanced release of pro-tussive inflammatory mediators and inflammatory proteases from nerve endings, epithelial cells, and mast cells could explain TRPV1 sensitization and heightened cough reflex. In this scenario, TRPV1 can function at multiple points, from nerve activation and release of sensory neuropeptides to the secretion of pro-inflammatory cytokines by airway epithelial cells [14,92]. Similar mechanisms acting via up-regulation of neuronal TRPV1 (as well as TRPA1 and TRPM8) channels are also involved in respiratory virus-induced cough hypersensitivity [93] and in bronchoconstriction, although the role of TRPV1 in bronchospasm in humans remains somewhat controversial [26,32]. About two-thirds of the increase in bronchoconstriction induced by hyperventilation with humidified hot air in guinea pigs could be attributed to the cholinergic reflex, likely elicited by the activation of TRPV1-expressing airway afferents, but the remaining bronchoconstriction is caused by other, as yet unidentified, mechanisms [94].

Similar to bronchial epithelial cells, TRPV1 shows increased expression in ASM in patients suffering from chronic cough, but it is present in intracellular compartments, and its exact role in ASM contractility remains unclear [95]. There is recent evidence for overexpression of TRPV1 in asthmatic ASM, whereby TRPV1 channel involved in the regulation of proliferation and apoptosis in asthmatic ASM cells [96]. Interestingly, bronchial hyperresponsiveness was enhanced in TRPV1^−/− mice, whereas inflammation elevated somatostatin concentrations in wild-type (WT), but not in TRPV1^−/− mice. Thus, it is possible that TRPV1 activation can also counteract inflammation via somatostatin release [97].

There is also recent evidence for a TRPV1 role in airway sensory nerves in connection with the action of tiotropium, the long-acting muscarinic receptor antagonist prescribed for its bronchodilator activity in COPD and asthma. However, tiotropium also attenuates cough through its direct inhibition of (i) TRPV1-triggered [Ca^2 +]_i rises, (ii) action potential discharge in airway-specific sensory afferent nerves, and (iii) capsaicin-induced bronchospasm [98].

TRPV1 sensitization in respiratory inflammation is mediated by the PI3K and PKC signaling pathways, causing hypersecretion of mucus and inflammatory cytokines [99]. Nerve growth factor, which itself does not induce a cough response, nevertheless causes a significant increase in the citric acid-induced cough and airway obstruction via TrkA receptor and TRPV1 activation, possibly downstream of PI3K and PLCγ activation [100].

TRPV1 has also been recently implicated in Cl⁻ secretion. Formaldehyde is an environmental pollutant and strong stimulant, which can also be formed endogenously from methylamine. Formaldehyde-induced activation of TRPV1 expressed in the intraepithelial nerve endings stimulated Cl⁻ secretion in rat tracheal epithelium. This secretion was mediated by the release of adrenaline and substance P, and it could be blocked by the CFTR specific inhibitor CFTR_i-172 [101].

Thus, there is substantial evidence implicating TRPV1 in chronic cough, asthma, and COPD, whereas successful use of TRPV1 antagonists for the alleviation of cough and airway hyperresponsiveness in animal models further highlights the promise such drugs hold for treating human respiratory disorders [102,103].

TRPV4 is another member of this subfamily that attracts growing interest in the context of respiratory disorders. TRPV4 channels are widely expressed in neuronal and nonneuronal cells within the respiratory tract, including macrophages and neutrophils, suggesting their relevance to airway inflammation [32]. Jia et al. [104] reported, that TRPV4 is expressed in ASM, where it acts as an osmolarity sensor directly causing hypotonicity-induced [Ca^2 +]_i rise and airway contraction. These findings are relevant to asthma because bronchial fluid in asthmatics can be hypotonic, and inhalation of hypotonic aerosols induces airway narrowing in people with asthma. TRPV4 present in airway macrophages can also be activated by mechanical stimuli. Thus, TRPV4 agonist 4-alpha-phorbol didecanoate (4α-PDD) increased [Ca^2 +]_i and reactive oxygen and nitrogen species in lung WT, but not TRPV4^−/−, macrophages, suggesting a role for TRPV4 in the development of ventilator-induced injury [105]. In addition, activation of TRPV4 by 4α-PDD and 5,6- or 14,15-EET, as well as thapsigargin treatment increased lung endothelial permeability measured by the filtration coefficient. The response to 4α-PDD, but not to thapsigargin, was absent in TRPV4^−/− mice, thus implicating TRPV4 in disruption of the alveolar septal barrier and in acute lung injury [106]. P450-derived epoxyeicosatrienoic acid-dependent regulation of calcium entry via TRPV4 has also been implicated in the endothelial permeability increase in acute lung injury caused by high vascular pressure [107].

Under hypotonic conditions regulatory volume decrease (RVD) occurs in human airway epithelial cells, but RVD is lost in CF airways [108]. Using TRPV4 antisense oligonucleotides treatment, Arniges et al. [109] showed that TRPV4 is uniquely required for the swelling-induced Ca^2 + entry, which is needed for a full RVD in human tracheal epithelial cells. Furthermore, the impaired RVD response in CF airway epithelia is due to the specific lack of TRPV4 activation in response to cell swelling; notably, activation of TRPV4 by 4α-PDD was unimpaired in CF. These data suggest that hypotonic activation of TRPV4 channels depends on CFTR. However, it should be noted that CFTR requirement for TRPV4 activation in airway epithelia is stimulus-dependent because the functional coupling of TRPV4 and large conductance Ca^2 +-activated K⁺ channel in response to high-viscous solutions is preserved in CF [110].

TRPV4 is functionally expressed in ASM cells, and it is likely associated with airflow obstruction in COPD patients. There is genetic evidence that polymorphisms in the TRPV4 gene are associated with COPD [111], but not with asthma [78]. However, McAlexander et al. [112] have provided compelling pharmacological evidence with the use of TRPV4 agonists and antagonists, that, unexpectedly, TRPV4 activation causes human airway constriction entirely due to the production of cysteinyl leukotrienes. The authors propose cysteinyl leukotriene synthesis as a novel mechanism for the involvement of TRPV4 in airway inflammation and obstruction. Although the source of cysteinyl leukotrienes has not been yet elucidated, one possibility is that mast cells in the immediate vicinity to bronchial smooth muscle are involved in leukotriene production [112].

There is also recent evidence that, similar to TRPC6 and other TRPCs, up-regulation of TRPV4 by chronic hypoxia is associated with enhanced myogenic tone, whereas TRPV4 knockout suppresses the development of chronic hypoxic pulmonary hypertension. Specifically, TRPV4 plays an essential role in serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension [113].

The Melastatin TRPs

Eight mammalian members of the TRPM subfamily, TRPM1-8, are structurally and functionally diverse cation channels, which are involved in processes ranging from detection of cold, taste, osmolarity, redox state, and pH to control of Mg^2 + homeostasis and cell proliferation or death [17,18,34,35,66,67]. TRPMs have a TRP box in the C-terminus, similarly to TRPC channels, but their N-terminus lacks the ankyrin repeats found in TRPCs and TRPVs; instead it has a common large TRPM homology domain. Remarkably, three out of eight TRPM members have connections with cancer development. Thus, the founding member melastatin (TRPM1) as well as TRPM5 and TRPM8 were identified by analysis of gene expression in several carcinomas [114].

The roles of these multifunctional cation channels in respiratory disorders are becoming increasingly apparent in connection with the effects produced by air pollution (e.g., oxidative stress, inflammation) and thermal irritants (e.g., cold).

Ozone, particulate matter, nitrogen oxides, and some metals are potent oxidants. In addition, these airborne pollutants can generate reactive oxygen species (ROS), which may also be produced endogenously under hypoxic and ischemia/reperfusion conditions. Oxidative stress through the production of ROS causes damage to all cellular macromolecules, including proteins, lipids, and DNA, thus disrupting normal cell signaling. Oxidative stress plays a role in many diseases, including asthma and COPD.

Various nonselective Ca^2 +-permeable cation channels have been implicated in cell damage or death due to oxidative stress, ischemia, and associated [Ca^2 +]_i overload [115]. TRPM2 is activated by ROS [116–118]. Ca^2 + entry via TRPM2 induces chemokine production in monocytes, followed by inflammatory neutrophil infiltration [119]. Thus, ROS can lead to inflammation and tissue injury with the involvement of TRPM2—a scenario, which is well established in endothelial injury and endothelial hyperpermeability [120–122]. However, in the airways this hypothesis was not confirmed in TRPM2^−/− mice, as no difference in the development of airway inflammation or cell activation between WT and TRPM2^−/− mice could be found, implying that inhibition of TRPM2 activity in COPD would have no anti-inflammatory effect [123]. Moreover, TRPM2 turned out to be protective in endotoxin-induced lung inflammation in mice. Somewhat unexpectedly, in this scenario ROS-induced activation of TRPM2 causes negative feedback inactivation of ROS production in phagocytic cells mediated by the inhibition of the membrane potential-sensitive NADPH oxidase, which prevents lung inflammation [124]. The inhibitory role of TRPM2 in ROS production was also shown in polymorphonuclear leukocytes and macrophages derived from TRPM2^−/− mice [124]. In contrast, deletion of TRPM4, which is a [Ca^2 +]_i-activated channel, results in an enhanced release of histamine, leukotrienes, and tumor necrosis factor from mast cells [125]. Thus, it is attractive to speculate that the balance between the activities of TRPM2 and TRPM4 channels is an important factor in airway inflammation.

Thermal irritants, such as cold air, can provoke bouts of coughing, especially in respiratory virus-induced cough hypersensitivity [75,93]. Other autonomic responses include airway constriction and mucosal secretion, which can trigger asthma attacks [32]. In connection with these well-known cold-induced airway symptoms, the expression and function of the cold- and menthol-receptor TRPM8 in the airways has been examined in several studies, but molecular and functional data remain somewhat contradictory, whereas conclusive evidence for the role of neuronal TRPM8 in these symptoms is still lacking (see [32,126] for recent reviews). The problems of interpreting these data relate to the difficulties with differentiation between TRPA1- and TRPM8-mediated effects and the lack of highly selective ligands. However, as already noted, up-regulation of neuronal TRPM8 clearly contributes to respiratory virus-induced cough hypersensitivity [93].

Epithelial TRPM8 present in the respiratory tract is comparatively better studied. Functional expression of TRPM8 was shown in human lung epithelial cells, whereby its activation causes the release of inflammatory cytokines IL-6 and IL-8 [127]. Interestingly, lung epithelial TRPM8 is represented by its truncated splice variant, localized mainly in the endoplasmic reticulum, which seems similar to prostate cancer epithelial cells [128]. Using immunohistochemistry, RT-PCR, and Western blotting enhanced expression of TRPM8 was found in bronchial epithelial cells in patients with COPD compared to healthy subjects. Functionally, activation of the epithelial TRPM8 isoform causes [Ca^2 +]_i rises, which mediate cold-induced mucus hypersecretion [129]. In addition, TRPM8 expressed in mast cells has been implicated in cold- and menthol-induced histamine release, likely explaining the role of TRPM8 channels in the menthol- and cold-induced allergic responses [130].

TRPM8 immunoreactive nerve fibers are abundant in nasal mucosa, especially around blood vessels, where TRPM8 may mediate neurovascular reflexes. These findings led to the suggestion of a role for TRPM8 in rhinitis [131]. We have recently developed a new method for short-term culture of human nasal epithelial cells (HNEC) for the study of CF pathophysiology [132]. TRPM8 gene transcripts and protein expression were revealed in these cells. Application of the TRPM8 agonist menthol-induced robust membrane current responses, as well as [Ca^2 +]_i rises ablated by the TRPM8 antagonist BCTC [133]. Taken together, these studies show that functional TRPM8 channels are expressed in the upper airway, both in sensory fibers and in airway epithelial cells.

Finally, it should be noted that although TRPM8 is expressed in vascular smooth muscle cells [134,135], its expression in ASM and any direct role in airway constriction remain to be studied.