Our data provide what we believe to be the first in vitro and in vivo evidence that TRPA1, an ion channel of the TRP gene family, serves as a major neuronal sensor for oxidants in the airways. We showed that TRPA1 channels in primary sensory neurons and heterologous cells were activated by OCl– and H2O2, a reactive oxygen species. Oxidant activation of TRPA1 occurred within the range of established concentrations for irritancy in humans and rodents. OCl–- and H2O2-induced Ca2+ influx was eliminated in cultured neurons from Trpa1–/– mice. Cellular insensitivity to oxidants was reflected on the whole-animal level. Trpa1–/– mice showed profoundly diminished respiratory responses to low-level chlorine exposure and H2O2 exposure. Differences in respiratory sensitivity were specific for oxidants, because responses to a nonoxidizing activator of airway neurons, acetic acid, remained intact in Trpa1–/– mice. Diminished oxidant-induced pain behavior in Trpa1–/– mice indicated that the role of TRPA1 in oxidant sensing is not restricted to airway neurons, but extends to other peripheral tissues.
TRPA1 was initially identified as a potential sensor for cold temperature and a receptor for plant-derived noxious chemicals including mustard oil, the pungent ingredient in mustard (37
). Mustard oil is known as a potent activator of sensory nerve endings in airway mucosa (63
). There are several possible mechanisms by which oxidants might activate TRPA1. OCl–
may activate TRPA1 by oxidizing thiols to sulfinate and sulfonate groups or by modifying primary amines (64
). Indeed, we found that OCl–
failed to activate TRPA1 following mutation of cysteine and lysine residues previously identified as potential acceptor sites for electrophilic agonists (60
). Alternatively, oxidants may produce reactive intermediates such as 4-hydroxynonenal, a lipid peroxidation recently found to activate TRPA1 (65
). The capabilities of different oxidants to activate TRPA1 may vary widely depending on their reactivity and membrane permeability as well as other factors. This is illustrated by the higher potency and efficacy of OCl–
, a strong oxidant, compared with H2
for TRPA1 activation and induction of respiratory depression (66
). In addition to sensing exogenous oxidants, TRPA1 may also be involved in the detection of endogenous reactive species. Heightened oxidative stress accompanies many inflammatory and painful conditions, including ischemia, neurodegeneration, diabetes, arthritis, and other chronic inflammatory conditions (67
Our results support an essential role for sensory TRPA1 channels in the induction of upper airway irritant responses. Because of the low exposure concentrations used in our study, the observed oxidant-induced respiratory rate depression was likely caused by activation of nasal irritant receptors in trigeminal sensory nerve endings. Our data suggest that oxidants activate TRPA1 channels in nasal trigeminal nerve endings, resulting in neuronal depolarization and activation of neuronal reflexes. In addition, influx of Ca2+
through TRPA1 may promote the release of proinflammatory peptides from nasal nerve endings. Sensory neuropeptides such as calcitonin gene–related peptide or substance P are known to induce dilation of blood vessels in the nasal mucosa and promote glandular secretion, contributing to irritant-induced nasal obstruction (71
In addition to TRPA1, peripheral chemosensory C-fibers express the capsaicin receptor, TRPV1 (37
). Similar to oxidants, capsaicin is a potent inducer of respiratory reflex responses and airway obstruction (1
). The important role of TRPV1 in the regulation of respiratory reflexes was confirmed by recent pharmacological studies in guinea pigs in which a TRPV1 antagonist displayed antitussive activity (74
). TRPA1 is activated by a much broader range of chemical stimuli than TRPV1 and is coexpressed with TRPV1 in chemosensory C-fibers. Recent in vitro studies found that TRPA1 is activated by the α,β-unsaturated aldehyde acrolein, a toxicant in photochemical smog and smoke and a potent activator of respiratory reflexes (49
). TRPA1 is also required for the induction of pain responses to formaldehyde and acetaldehyde (77
). Our present results support the notion that TRPA1 may mediate respiratory irritant responses to these and many other reactive environmental toxicants in vivo. The existence of a shared neuronal receptor for oxidants and noxious electrophiles, including aldehydes, is also supported by the fact that inhalation of formaldehyde diminishes respiratory irritant responses to subsequent challenges with chlorine and acetaldehyde, and vice versa (12
). Cross-tolerance can be explained by the saturation or desensitization of TRPA1 by the different agonists.
Our results showed that sensory neurons derived from the nodose ganglia and DRG, innervating the lower airways, were as sensitive to chlorine as were trigeminal neurons that innervate the nasal passages. These responses were abolished in neurons cultured from Trpa1–/–
mice. Based on these data, we assume that TRPA1 activation may also contribute to the effects of chlorine and other TRPA1 agonists on chemosensory nerve endings in the lower airways. Because reactive irritants are efficiently cleared in the upper airways, sensory activation in the lower airways requires higher exposure levels. Extended or high-level exposure to oxidants, such as that observed in victims of chlorine gas exposures, induce severe pain, cough, mucus secretion, and bronchospasm (5
). At higher exposure levels, oxidants increase TRPA1 activity through direct activation as well as indirectly through phospholipase C–coupled signaling pathways (36
). Inhalation of oxidants induces the release of bradykinin, ATP, and lipid products with cognate receptors on airway sensory nerve endings (23
). In addition to TRPA1, these mediators may increase the activity of other sensory neuronal detection systems. Indeed, a recent study showed that H2
-induced changes in respiratory frequency can be inhibited, at least in part, by a purinergic receptor antagonist (23
). Involvement of additional neuronal pathways may explain why we observed residual respiratory depression and pain behavior in Trpa1–/–
mice in response to the high concentrations of H2
used in our experiments.
Individuals affected by allergic airway conditions such as rhinitis and asthma often display respiratory hypersensitivity responses to chemical irritants including chlorine, acrolein, and other TRPA1 agonists (4
). These exaggerated responses may be the result of sensitization of TRPA1 downstream of neuronal phospholipase C–coupled receptor systems activated during inflammation, including the receptors for bradykinin, histamine, proteases, and other inflammatory stimuli (36
). TRPA1 antagonists may be used to suppress sensory neuronal hyperexcitability in airway disease. We conclude that TRPA1 represents a promising new target for the development of drug candidates with potential antitussive, analgesic, and antiinflammatory properties.