Usually the nasal mucosa, nasopharynx and larynx are exposed to the highest concentrations of toxicants as these structures represent the first line of defense after inhalation. Typical first symptoms will occur due to mucosal irritation like a tickle of the throat and cough which are both biological alert signs with the aim of initiating an escape reaction from the dangerous environment. Coughing and sneezing are triggered by activation of peripheral sensory nerve endings in the nasal airway lining. These nerve endings can be considered as a first defense mechanism against chemical challenges (Bessac and Jordt 2008, 2010). The purpose of this initial reflex-response is the removal of irritants from the nasal epithelial surface. After activation of nasal trigeminal, glossopharyngeal or vagal chemosensory neurons (type C and small diameter myelinated Aδ fibres) by TIHs, sensory-autonomic pathways elicit parasympathetic reflexes, mediated via cholinergic efferent neurons, resulting in secretions from nasal, lacrimatory and salivary glands in combination with the dilatation of vessels in the nasal mucosa and sinuses (Bessac and Jordt 2008, 2010). Mucosal swelling and leakage are common results leading to narrowing or obstruction of the nasal passage (Baraniuk and Kim 2007). The resulting edema of the larynx and glottis can be life-threatening due to acute obstruction of the upper airways.

When irritants reach the tracheobronchial section, sensory neuronal activity can trigger tracheal and bronchial constriction via contraction of bronchial smooth muscle cells, mucus secretion, and neurogenic inflammation (Fig. 2), events which are considered as typical reactions after exposure to lung irritants (Miller and Chang 2003). Rhonchus is the typical diagnostic finding resulting from mucous secretion and constriction of the bronchial musculature. TIHs can be removed from the respiratory tract by different mechanisms. The airways of the lower respiratory tract are lined with cells that are predominantly ciliated or mucus producing. TIHs are trapped within the mucus layer and are removed by ciliated cells via mucociliary transport. Macrophages, either integrated in the epithelium or macrophages in the alveoli, are capable of phagocytosis and thereby clearing inhaled particles. Macrophages migrate to upper parts of the respiratory tract, where they are trapped in the mucociliar transport and are cleared from the airways. Moreover, activated macrophages release cytokines, chemokines, growth factors, proteolytic enzymes and ROS. These mediators recruit and activate other cells that either participate in tissue regeneration or may contribute to tissue remodeling processes. After exposure to lung irritants, mucociliary clearance may increase when small or moderate concentrations of toxic chemicals are inhaled. However, high concentrations or very aggressive chemical substances may also interfere with the cilia of the respiratory epithelium or influence the epithelial integrity itself. As shown in an in vitro human bronchial co-culture cell culture model, sulfur mustard induced a widening of intercellular spaces and moreover, a loss in cell-matrix adhesion molecules was observed resulting in a decrease of transepithelial resistance (Pohl et al. 2009). Mucin production increased with resultant cessation of ciliar beat.

Chemical substances with little water solubility or high concentrations of TIHs are able to reach deep lung compartments, e.g. the alveoli. The complex damaging processes of TIHs in the alveolar space are depicted in Fig. 3 and include liberation of inflammatory mediators, invasion of inflammatory cells, hypersecretion, and disturbance of cell-cell contacts leading to the destruction of the endothelial-epithelial barrier.

Apparently, type I alveolar epithelial cells (covering 90–95 % of the alveolar surface) appear to be most vulnerable to many lung toxicants probably because of their large surface area. Dysfunctional alveolar type I cells have to be replaced by new type I cells originating from type II alveolar cells. Alternatively, fibroblasts are activated to repair tissue lesions resulting in the formation of connective tissue and scars. Chemicals that affect both alveolar type I and type II cells have to be considered as extremely harmful, as regenerative cells are compromised and tissue damage cannot be adequately repaired. As the blood-air barrier is extremely thin (~0.4–1 μm), toxic substances may penetrate into lung capillaries (especially after damage to alveolar type I cells) and affect the endothelium, which is comparably vulnerable as epithelial cells type I. This damage will result in progressive impairment of the blood-air barrier and permeability will increase. Tight and adherence junctions between cells are loosened, facilitating loss of epithelial resistance. As a result, blood plasma enters from lung capillaries into the lung interstitium. The resulting interstitial edema increases the diffusion distance through the blood-air barrier leading to disturbance of gas exchange. First, edema fluid is evacuated from the lung interstitium by the lymphatic system. Ongoing loss of epithelial and endothelial resistance will increase plasma leakage, overstraining the lymphatic system and resulting in alveolar edema. A pathological cycle is initiated: formation of edema foam, ascending from the alveolar space to the upper parts of the respiratory system, blocking bronchioles and impairing oxygenation. These events then cause increased pulmonary resistance which leads to further leakage into the alveoli. The whole process may result in a toxic lung edema that may be fatal.

Toxic lung edema due to increased alveolar permeability is hard to control, because tissue architecture is affected or even destroyed. Recovery of the integrity of the alveolar epithelial surface is an essential strategy after toxic inhalation injury. Especially, alveolar type I cells are vulnerable and may undergo cell death mediated either by direct TIH effects or indirectly by specific cytokines released from affected cells, residual structural cells or activated macrophages. Various acute inflammatory mediators are expressed in the lung during acute lung injury (e.g. IL-1α, IL-8, TNF-α, C3b, leukotrienes). In parallel, proteases are released from infiltrating neutrophils and mononuclear cells and degrade the extracellular matrix. Resulting debris is subsequently cleared by macrophages (attracted and differentiated from bone marrow and circulatory pools). Next, growth factors (e.g. TGF-ß, CTGF, PDGF) stimulate basal cells (type II alveolar cells) to differentiate into type I cells and other cell types reconstitute connective tissue, vascular tissue, and nerve endings. A careful balance of catalytic and anabolic processes is mandatory for a complete physiological repair. If one of the processes is gaining an upper hand, fibrosis due to enhanced production of collagen and connective tissue or emphysema due to enhanced protease activity may occur (Bonner 2008).

Virtually all pathophysiological processes mentioned above are regulated or modulated by activation of chemosensory molecules in the plasma membrane of cells in the respiratory system. In particular chemosensory TRP channels play a pivotal role in the detection of TIHs, in the control of adaptive responses, and in the initiation of detrimental signaling cascades.

TRP channels represent one of the largest superfamilies of ion channels in the human genome (Clapham et al. 2001; Montell et al. 2002; Nilius and Owsianik 2011; Pedersen et al. 2005). Mammalian TRP channels can be subdivided into six families, i.e. TRPC, TRPM, TRPV, TRPP, TRPML, and TRPA channels as shown in Fig. 4. The common structural features of the members of the TRP superfamily are six transmembrane-spanning domains (TM1-6) and a putative pore region (P) located between transmembrane domains 5 and 6 (see Fig. 5). TRP channels are permeable to cations with striking differences in ion selectivity. Many TRP channels conduct monovalent cations like Na⁺ or K⁺ as well as bivalents like Ca²⁺, Mg²⁺ or bivalent cations of transition metals. Some TRP channels like TRPM4 and TRPM5 show high selectivity for monovalent cations, other channels like TRPV5 or TRPV6 are highly selective for Ca²⁺ (for review: Owsianik et al. 2006).

The archetypal TRP channel, eponymous for the whole super-family, is the product of the trp gene of the fruit fly, Drosophila melanogaster. Cloning of the Drosophila TRP protein was preceded by the characterization of a mutant fruit fly with a visual defect. In this mutant strain, termed transient receptor potential (TRP) (Minke 1977; Montell et al. 1985), exposure to light only led to a short-lived depolarizing current due to an inactivating mutation of the TRP channel, which is an indispensable component of the phototransduction cascade in Drosophila (Hardie and Minke 1992; Montell et al. 1985; Montell and Rubin 1989). In some analogy to Drosophila TRP, many mammalian TRP channels exert sensory functions, although mostly outside of the visual system. Thus, TRP channels like TRPV1 (Caterina et al. 1999; Cesare et al. 1999; Tominaga et al. 1998), TRPV2 (Caterina et al. 1999), TRPV3 (Peier et al. 2002b; Smith et al. 2002; Xu et al. 2002), and TRPV4 (Guler et al. 2002; Watanabe et al. 2002) were reported to be activated by elevated temperature and are involved in thermo-sensing (for review: Tominaga 2007), whereas TRPM8 is activated by cold temperatures (McKemy et al. 2002; Peier et al. 2002a). Other TRP channels like TRPM5 are part of the signal transduction pathway engaged by taste receptors (Perez et al. 2002; Zhang et al. 2003). Of note, TRPM5 channel activity also depends on temperature (Talavera et al. 2005), a fact that explains why sweet or bitter food has a more intense taste in higher temperatures. Furthermore, TRP channels like TRPV4 react to mechanical stimuli in various cell types and organs (for review: Liedtke 2007; Plant and Strotmann 2007; Yin and Kuebler 2010) including the lung vasculature (Jian et al. 2008).

The most important function of TRP channels in the context of this review is their role as chemosensory molecules. In the respiratory system, especially TRPV1, TRPV4, TRPA1, and TRPM8 are involved in the detection of potentially harmful inhalants. However, these channels display a remarkable promiscuity with regard to their activators. For example, TRPA1 is activated by a large number of chemically unrelated substances (Table 1). Furthermore, TRP channels of the TRPC family are expressed in the lung and form part of redox-regulated signaling pathways. The activation of these TRP channels by TIHs results either from covalent modification of cystein residues of the ion channel or from phospholipase C (PLC)-regulated changes in lipid messengers (diacylglyercol, phosphatidyl-inositolphosphate) or intracellular calcium levels. In the first scenario, the TRP channel can be activated directly by reaction with an electrophilic TIH (Brone et al. 2008; Hinman et al. 2006; Macpherson et al. 2007a) or the reaction of the TIH with constituents of the plasma membrane leading to the generation of secondary TRP activators (Taylor-Clark et al. 2009; Trevisani et al. 2007). Regarding PLC-promoted signaling events leading to the activation of TRP channels, either stimulation of G_q/11-coupled protease-activated receptors (PARs) or purinergic receptors with subsequent activation of PLC-β or stimulation of redox-sensitive PLC isoforms is involved (see below).

Of note, some pulmonary TRP channels are involved in O₂ sensing by detecting hypoxic as well as hyperoxic states (for review: Takahashi et al. 2012). Hypoxia can lead to an activation of TRPA1- or TRPC6-promoted calcium responses (Fuchs et al. 2011; Takahashi et al. 2011; Weissmann et al. 2006). TRPA1 acting as an O₂ sensor is expressed in vagal neurons in the lung and its activation by hypoxia occurs via reduced activity of proline hydroxylases (PHDs). In normoxia PHDs oxidize a proline residue in TRPA1 (Pro394) thus inhibiting the channel (Takahashi et al. 2011). Hyperoxic states lead to the direct oxidation of cystein residues of TRPA1 (Cys633 and Cys856), a modification, which overrides the inhibition of TRPA1 by proline hydroxylation (Takahashi et al. 2011). Whereas TRPA1 is directly modified by the ambient O₂ concentration, TRPC6 is activated by hypoxic states via the enhanced generation of reactive oxygen species during hypoxia, which in turn lead to an accumulation of diacyl glycerol (DAG) (Weissmann et al. 2006). Thus, TIHs leading to obstruction of the airways via bronchoconstriction or increased mucus secretion can indirectly activate TRPA1 or TRC6. In contrast, oxidizing TIHs could potentially mimick hyperoxic conditions and by this means directly activate TRPA1.

To explain the role of TRP channels in pulmonary responses upon exposure to TIHs in more detail, this review will first focus on the involvement of TRP channels in irritative responses of the upper respiratory tract. Afterwards, we will discuss the special role of TRPA1 and TRPV1 in the cough reflex. Thereafter, the impact of TRP channels, especially TRPV4, in the regulation of mucociliary clearance will be addressed. Finally, the role of members of the canonical family of TRP channels, TRPCs, in redox-regulated pathways will be discussed.

As stated above, the mucosa of the eyes and the mucosa of the nasopharynx are first-line target of TIHs, especially those with high water solubility. Of note, all features of mucosal responses after exposure to TIHs, i.e. lacrimation, sternutation, nasal discharge, and irritative pain, are regulated by TRP channels.

In recent years evidence for an involvement of TRPC channels in lung function and toxicity became evident. Although TRPC channels are not the primary sensor molecules in different lung tissues, they are essential members of signal transduction cascades mediating toxicity in the targeted cells. TRPC channel activity is induced after ligand binding to a receptor (e.g. a G_q/11 protein-coupled receptor) and complex intracellular signaling cascades (Dietrich et al. 2005).

The seven TRPC family members can be grouped into subfamilies on the basis of their amino acid sequence similarity. While TRPC1 and TRPC2 are almost unique, TRPC4 and TRPC5 share ~65 % identity. TRPC3, 6 and 7 form a structural and functional subfamily with 70–80 % identity at the amino acid level and their ability to be activated by DAG (Dietrich et al. 2005; Hofmann et al. 1999; Okada et al. 1999), a product of phosphatidylinositol 4,5-bisphosphate cleavage by activated phospholipases C. In all eukaryotic cells, activation of phospholipase C (PLC)-coupled membrane receptors by hormones or neurotransmitters leads to an increase in the intracellular Ca²⁺ concentration [Ca²⁺]_i. Both activation of PLCβ and ε-isozymes by G protein coupled receptors and of PLCγ isoforms by receptor tyrosine kinases results in the hydrolysis of phosphatidyinositol 4,5-bisphosphate and the generation of inositol 1,4,5-trisphosphate (IP₃) and DAG. While it seems clear that TRPC3/6/7 channels are directly activated by DAG and are responsible for receptor-operated calcium entry (ROCE) (Hofmann et al. 1999; Okada et al. 1999), the regulation of other members and their involvement in store operated calcium entry (SOCE) is discussed controversially. SOCE occurs when inositol 1,4,5-trisphosphate (IP₃) or some other signal discharges Ca²⁺ from intracellular stores in the endoplasmic reticulum (ER). The subsequent fall in the ER Ca²⁺ concentration then signals to the plasma membrane and activates store-operated channels. A major breakthrough in understanding SOCE was the identification of the calcium sensor STIM (Liou et al. 2005; Roos et al. 2005; Zhang et al. 2005) and the Orai channels (Cahalan 2009; Feske et al. 2006; Vig et al. 2006) in 2005 and 2006, respectively. STIM 1 which is predominantly located in the ER, is a single transmembrane domain protein containing two N-terminal Ca²⁺-binding EF hands in the ER lumen and is therefore ideally situated to detect calcium levels in the ER. Upon Ca²⁺ release reduced calcium levels induce STIM redistribution to punctae and opening of store-operated channels in the plasma membrane. The so called Orai proteins with only four predicted transmembrane domains and intracellular C- and N-termini, were identified as the pore forming unit of store-operated channels by the analysis of T cells from patients with severe combined immunodeficiency (SCID) reviewed in Cahalan (2009). Although Orai 1 channels can multimerize with themselves to form pore forming units, it was also suggested that Orai molecules might interact with TRPC channels heterologously expressed in HEK293 cells to form store-operated channels (Liao et al. 2009). Moreover, evidence supporting the interaction of the calcium sensor STIM1 with TRPC proteins was presented for the same heterologous expression system (Yuan et al. 2009a, b).

TRPCs are also regulated by phosphorylation. TRPC3 channels can be inactivated by direct phosphorylation of amino acids threonine 11 (Thr 11) and serine 263 (Ser 263) by protein kinase G (PKG) (Kwan et al. 2004) and by protein kinase C (PKC)-mediated phosphorylation of Ser 712 (Trebak et al. 2005). The receptor tyrosine kinase Src phosphorylates TRPC3 at Tyrosine 226 (Tyr 226) and inhibits TRPC3 activity (Kawasaki et al. 2006), while Fyn another member of the Src family increases TRPC6 activity (Hisatsune et al. 2004). It has been shown that TRPC6 is negatively regulated after phosphorylation of Thr 69 by PKG (Takahashi et al. 2008b). Most interestingly, this phosphorylation is essential for anti-hypertrophic effects of phosphodiesterase 5 inhibitors (Kinoshita et al. 2010; Koitabashi et al. 2010; Nishida et al. 2010), which are also important therapeutics for pulmonary hypertension (Reichenberger et al. 2007).

TRPC channels can form functional homo- and heterotetramers within two defined subgroups (TRPC1/4/5 and TRPC3/6/7) excluding TRPC2 which is a non-functional pseudogene in humans (Goel et al. 2002; Hofmann et al. 2002). Novel combinations of TRPC1- with TRPC4 or 5 together with TRPC3, 6 or 7 in tetrameric complexes with three different TRPC family members have also been identified in HEK293 cells, as well as in embryonic brain, but not in adult rat tissues (Strubing et al. 2003). Evidence for the importance of the highly conserved pore region in the TRPC tetramer is derived from site-directed mutagenesis which results not only in the complete loss of channel activity upon heterologous expression, but also in a dominant-negative effect of a mutated channel monomer on functional homo- or heteromeric channel tetramers (Hofmann et al. 2002).

The TRPC family includes seven members (TRPC1-7), but only six are functional in humans, because TRPC2 is a pseudogene in the human genome. TRPC7 expression in lung is sparse and is predominantly identified in alveolar macrophages (Finney-Hayward et al. 2010). Expression of the other TRPC channels in pulmonary tissues and recent evidence concerning their potential function is summarized in the next paragraph.

Although TRPC1 was the first mammalian TRP channel to be cloned, its physiological functions are still elusive. It is not even clear, if TRPC1 is an ion channel or merely an adaptor protein regulating ion channel activity of other TRPC members (Storch et al. 2012). Homomeric TRPC1 complexes do not translocate to the plasma membrane (Hofmann et al. 2002), but TRPC1 as a member of heteromeric TRPC1/5 or TRPC1/4 complexes has a high impact on TRPC4/5 currents (Strubing et al. 2001) and decreases calcium permeability in such channel complexes (Storch et al. 2012). It has been reported that TRPC1 is important for the proliferation of bronchial smooth muscle cells, which may induce thickening of the airways facilitating airway hyperreactivity (summarized in Li et al. 2003). Along these lines, TRPC1 is up-regulated in proliferating airway smooth muscle cells while TRPC1 down-regulation inhibits cell proliferation (Sweeney et al. 2002a, b). However, it is not clear, if proliferation is influenced exclusively by TRPC1 channels or by TRPC1/4 channel complexes, because heteromeric TRPC1/4 channel complexes seem to be more important for the lung extra-alveolar endothelial barrier than for the capillary endothelial barrier (summarized in Cioffi et al. 2009). TRPC3 expression is up-regulated by tumour necrosis factor (TNF)-α in human airway smooth muscle cells (White et al. 2006). TNF-α is a pro-inflammatory cytokine which has been implicated in asthma and COPD because it increases airway hyperresponsiveness (AHR) by increasing airway smooth muscle Ca²⁺. Therefore, TNF-α blockage might represent a promising pharmacological target for the treatment of asthma and COPD (summarized in Amrani 2007). The function of TRPC4 in lung vascular endothelial cells was analysed using TRPC4−/− mice and wild-type controls. A defect in thrombin-induced Ca²⁺ influx is associated with reduced actin stress fiber formation and reduced endothelial cell retraction response. In isolated perfused lungs thrombin receptor activation increased the microvessel filtration coefficient (Kfc) by 2.8-fold in wild-type lungs but only 1.4 in TRPC4−/− lungs. This data provides evidence for a role of TRPC4 together with other ion channels in lung endothelial permeability (Tiruppathi et al. 2002). Recently TRPC5 was detected in the apical membrane of neuroepithelial bodies in the intrapulmonary airway epithelium by TRPC5 specific antibodies. However, it remains elusive if and how TRPC5 is involved in signal transduction cascades of these cells (Lembrechts et al. 2012).

Many studies were already published on the role of TRPC6 channels in the airways. Indeed, TRPC6 function in the lung might be particularly important, because it is the most prominently expressed TRPC channel in lungs (Hofmann et al. 2000). Therefore, we set out to analyze allergic responses in a TRP6-deficient mouse model expecting a reduced response in comparison to wild-type (WT) mice. However, we found an increased methacholine-induced AHR in TRPC6-deficient mice compared to WT mice. This surprising finding is most probably due to compensatory up-regulation of TRPC3 in airway smooth muscle cells (Sel et al. 2008). By experimental inflammation induced by intraperitoneal ovalbumin (OVA) sensitization followed by OVA aerosol challenges we were able to detect a decreased level of T-helper type 2 (Th2) cytokines (IL-5 and IL-13) as well as reduced IgE levels in the blood of TRPC6−/− mice compared to WT mice. Mucus production in goblets cells from challenged mice however was not altered in TRPC6-deficient mice (Sel et al. 2008). This data points to an important role of TRPC6 in the immune responsive rather than in airway smooth muscle tissues. Because neutrophil numbers are increased in the sputum of COPD and asthma patients, TRPC6 function in these cells might be also important for the progress of the diseases. Migration of TRPC6−/− neutrophils in response to macrophage inflammatory protein-2 (MIP2 also known as CXCL2) was reduced compared to WT neutrophils (Damann et al. 2009). In the same report an involvement of TRPC6 in cytoskeletal rearrangements during neutrophil migration was demonstrated suggesting an important role of TRPC6 in the migration of lung neutrophils. TRPC6 was also identified in other immune cells of the lung like alveolar macrophages (Finney-Hayward et al. 2010). Most interestingly, TRPC6 mRNA expression was significantly increased in macrophages obtained from COPD patients compared to healthy controls, while TRPC3 and TRPC7 levels remained unchanged (Finney-Hayward et al. 2010).