Thomas E. Taylor-Clark

Prostaglandin-Induced Activation of Nociceptive Neurons via Direct Interaction with Transient Receptor Potential A1 (TRPA1)

Inflammation contributes to pain hypersensitivity through multiple mechanisms. Among the most well characterized of these is the sensitization of primary nociceptive neurons by arachidonic acid metabolites such as prostaglandins through G protein-coupled receptors. However, in light of the recent discovery that the nociceptor-specific ion channel transient receptor potential A1 (TRPA1) can be activated by exogenous electrophilic irritants through direct covalent modification, we reasoned that electrophilic carbon-containing A- and J-series prostaglandins, metabolites of prostaglandins (PG) E2 and D2, respectively, would excite nociceptive neurons through direct activation of TRPA1. Consistent with this prediction, the PGD2 metabolite 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) activated heterologously expressed human TRPA1 (hTRPA1-HEK), as well as a subset of chemosensitive mouse trigeminal neurons. The effects of 15dPGJ2 on neurons were blocked by both the nonselective TRP channel blocker ruthenium red and the TRPA1 inhibitor (HC-030031), but unaffected by the TRPV1 blocker iodo-resiniferatoxin. In whole-cell patch-clamp studies on hTRPA1-HEK cells, 15dPGJ2 evoked currents similar to equimolar allyl isothiocyanate (AITC) in the nominal absence of calcium, suggesting a direct mechanism of activation. Consistent with the hypothesis that TRPA1 activation required reactive electrophilic moieties, A- and J-series prostaglandins, and the isoprostane 8-iso-prostaglandin A2-evoked calcium influx in hTRPA1-HEK cells with similar potency and efficacy. It is noteworthy that this effect was not mimicked by their nonelectrophilic precursors, PGE2 and PGD2, or PGB2, which differs from PGA2 only in that its electrophilic carbon is rendered unreactive through steric hindrance. Taken together, these data suggest a novel mechanism through which reactive prostanoids may activate nociceptive neurons independent of prostaglandin receptors.

Expression and function of the ion channel TRPA1 in vagal afferent nerves innervating mouse lungs

Transient receptor potential (TRP) A1 and TRPM8 are ion channels that have been localized to afferent nociceptive nerves. These TRP channels may be of particular relevance to respiratory nociceptors in that they can be activated by various inhaled irritants and/or cold air. We addressed the hypothesis that mouse vagal sensory nerves projecting to the airways express TRPA1 and TRPM8 and that they can be activated via these receptors. Single cell RT‐PCR analysis revealed that TRPA1 mRNA, but not TRPM8, is uniformly expressed in lung‐labelled TRPV1‐expressing vagal sensory neurons. Neither TRPA1 nor TRPM8 mRNA was expressed in TRPV1‐negative neurons. Capsaicin‐sensitive, but not capsaicin‐insensitive, lung‐specific neurons responded to cinnamaldehyde, a TRPA1 agonist, with increases in intracellular calcium. Menthol, a TRPM8 agonist, was ineffective at increasing cellular calcium in lung‐specific vagal sensory neurons. Cinnamaldehyde also induced TRPA1‐like inward currents (as measured by means of whole cell patch clamp recordings) in capsaicin‐sensitive neurons. In an ex vivo vagal innervated mouse lung preparation, cinnamaldehyde evoked action potential discharge in mouse vagal C‐fibres with a peak frequency similar to that observed with capsaicin. Cinnamaldehyde inhalation in vivo mimicked capsaicin in eliciting strong central‐reflex changes in breathing pattern. Taken together, our results support the hypothesis that TRPA1, but not TRPM8, is expressed in vagal sensory nerves innervating the airways. TRPA1 activation provides a mechanism by which certain environmental stimuli may elicit action potential discharge in airway afferent C‐fibres and the consequent nocifensor reflexes.

Relative contributions of TRPA1 and TRPV1 channels in the activation of vagal bronchopulmonary C‐fibres by the endogenous autacoid 4‐oxononenal

Transient receptor potential (TRP) A1 channels are cation channels found preferentially on nociceptive sensory neurones, including capsaicin‐sensitive TRPV1‐expressing vagal bronchopulmonary C‐fibres, and are activated by electrophilic compounds such as mustard oil and cinnamaldehyde. Oxidative stress, a pathological feature of many respiratory diseases, causes the endogenous formation of a number of reactive electrophilic alkenals via lipid peroxidation. One such alkenal, 4‐hydroxynonenal (4HNE), activates TRPA1 in cultured sensory neurones. However, our data demonstrate that 100 μm 4HNE was unable to evoke significant action potential discharge or tachykinin release from bronchopulmonary C‐fibre terminals. Instead, another endogenously produced alkenal, 4‐oxononenal (4ONE, 10 μm), which is far more electrophilic than 4HNE, caused substantial action potential discharge and tachykinin release from bronchopulmonary C‐fibre terminals. The activation of mouse bronchopulmonary C‐fibre terminals by 4ONE (10–100 μm) was mediated entirely by TRPA1 channels, based on the absence of responses in C‐fibre terminals from TRPA1 knockout mice. Interestingly, although the robust increases in calcium caused by 4ONE (0.1–10 μm) in dissociated vagal neurones were essentially abolished in TRPA1 knockout mice, at 100 μm 4ONE caused a large TRPV1‐dependent response. Furthermore, 4ONE (100 μm) was shown to activate TRPV1 channel‐expressing HEK cells. In conclusion, the data support the hypothesis that 4‐ONE is a relevant endogenous activator of vagal C‐fibres via an interaction with TRPA1, and at less relevant concentrations, it may activate nerves via TRPV1.

Nitrooleic acid, an endogenous product of nitrative stress, activates nociceptive sensory nerves via the direct activation of TRPA1.

Transient Receptor Potential A1 (TRPA1) is a nonselective cation channel, preferentially expressed on a subset of nociceptive sensory neurons, that is activated by a variety of reactive irritants via the covalent modification of cysteine residues. Excessive nitric oxide during inflammation (nitrative stress), leads to the nitration of phospholipids, resulting in the formation of highly reactive cysteine modifying agents, such as nitrooleic acid (9-OA-NO2). Using calcium imaging and electrophysiology, we have shown that 9-OA-NO2 activates human TRPA1 channels (EC50, 1 μM), whereas oleic acid had no effect on TRPA1. 9-OA-NO2 failed to activate TRPA1 in which the cysteines at positions 619, 639, and 663 and the lysine at 708 had been mutated. TRPA1 activation by 9-OA-NO2 was not inhibited by the NO scavenger carboxy-PTIO. 9-OA-NO2 had no effect on another nociceptive-specific ion channel, TRPV1. 9-OA-NO2 activated a subset of mouse vagal and trigeminal sensory neurons, which also responded to the TRPA1 agonist allyl isothiocyanate and the TRPV1 agonist capsaicin. 9-OA-NO2 failed to activate neurons derived from TRPA1(-/-) mice. The action of 9-OA-NO2 at nociceptive nerve terminals was investigated using an ex vivo extracellular recording preparation of individual bronchopulmonary C fibers in the mouse. 9-OA-NO2 evoked robust action potential discharge from capsaicin-sensitive fibers with slow conduction velocities (0.4-0.7 m/s), which was inhibited by the TRPA1 antagonist AP-18. These data demonstrate that nitrooleic acid, a product of nitrative stress, can induce substantial nociceptive nerve activation through the selective and direct activation of TRPA1 channels.

Ozone activates airway nerves via the selective stimulation of TRPA1 ion channels

Inhalation of ozone is a major health risk in industrialized nations. Ozone can impair lung function and induce respiratory symptoms through sensory neural‐mediated pathways, yet the specific interaction of ozone with airway sensory nerves has yet to be elucidated. Here we demonstrate, using a vagally innervated ex vivo tracheal–lung mouse preparation, that ozone selectively and directly evokes action potential discharge in a subset of nociceptive bronchopulmonary nerves, namely slow conducting C‐fibres. Sensitivity to ozone correlated with the transient receptor potential (TRP) A1 agonist, cinnamaldehyde, with ozone having no effect on cinnamaldehyde‐insensitive fibres. C‐fibre responses to ozone were abolished by ruthenium red (TRP inhibitor). Ozone also stimulated a subset of nociceptive sensory neurones isolated from vagal ganglia of wild‐type mice, but failed to activate neurones isolated from transient receptor potential ankyrin 1 (TRPA1) knockout mice. Ozone activated HEK293 cells transfected with TRPA1, but failed to activate non‐transfected HEK293 or HEK293 transfected with the capsaicin‐sensitive transient receptor potential vanilloid 1 (TRPV1) channel. Thus, ozone is not an indiscriminate neuronal activator, but rather it potently and selectively activates a subset of airway C‐fibres by directly stimulating TRPA1.

Transient Receptor Potential Ankyrin 1 Mediates Toluene Diisocyanate–Evoked Respiratory Irritation

Toluene diisocyanate (TDI), a reactive, hazardous irritant, causes respiratory symptoms such as cough, rhinitis, dyspnea, and chest tightness in exposed workers. Although previous animal studies have shown that TDI causes respiratory reflexes that are abolished by desensitization of capsaicin-sensitive sensory nerves, the specific molecular identity of the transducer(s) responsible for sensing this noxious stimulus has, to date, remained elusive. Recent studies have demonstrated that transient receptor potential ankyrin 1 (TRPA1), an ion channel largely restricted to a subset of capsaicin-sensitive sensory nerves, functions as a transducer capable of initiating reflex responses to many reactive chemical stimuli. We therefore hypothesized that TRPA1 is the primary molecular transducer through which TDI causes sensory nerve activation and respiratory reflexes. Consistent with this hypothesis, TDI activated TRPA1, but not the capsaicin-sensitive transient receptor potential vanilloid 1 channel, in heterologous expression systems. TDI also activated a subset of dissociated trigeminal sensory neurons from wild-type but not TRPA1-deficient mice. In vivo, TDI mimicked known TRPA1 agonists by causing a pronounced decrease in breathing rate, indicative of respiratory sensory irritation, and this reflex was abolished in TRPA1-deficient mice. Together, our data suggest that TDI causes sensory nerve activation and airway sensory irritation via the activation of the ion channel, TRPA1.

Sensing pulmonary oxidative stress by lung vagal afferents

Oxidative stress in the bronchopulmonary airways can occur through a variety of inflammatory mechanisms and also following the inhalation of environmental pollutants. Oxidative stress causes cellular dysfunction and thus mammals (including humans) have developed mechanisms for detecting oxidative stress, such that defensive behavior and defensive biological mechanisms can be induced to lessen its potential damage. Vagal sensory nerves innervating the airways play a critical role in the detection of the microenvironment in the airways. Oxidative stress and associated compounds activate unmyelinated bronchopulmonary C-fibers, initiating action potentials in these nerves that conduct centrally to evoke unpleasant sensations (e.g. urge to cough, dyspnea, chest-tightness) and to stimulate/modulate reflexes (e.g. cough, bronchoconstriction, respiratory rate, inspiratory drive). This review will summarize the published evidence regarding the mechanisms by which oxidative stress, reactive oxygen species, environmental pollutants and lipid products of peroxidation activate bronchopulmonary C-fibers. Evidence suggests a key role for transient receptor potential ankyrin 1 (TRPA1), although transient receptor potential vanilloid 1 (TRPV1) and purinergic P2X channels may also play a role. Knowledge of these pathways greatly aids our understanding of the role of oxidative stress in health and disease and represents novel therapeutic targets for diseases of the airways.

Histamine receptors that influence blockage of the normal human nasal airway

1 The aim of this study was to investigate the mechanisms by which histamine causes nasal blockage. Histamine, 40–800 μg, intranasally into each nostril, induced significant blockage of the nasal airway in normal human subjects, as measured by acoustic rhinometry. 2 Oral pretreatment with cetirizine, 5–30 mg, the H1 antagonist, failed to reverse completely the nasal blockage induced by histamine, 400 μg. 3 Dimaprit, 50–200 μg, the H2 agonist, intranasally, caused nasal blockage, which was reversed by oral pretreatment with ranitidine, 75 mg, the H2 antagonist. 4 A combination of cetirizine, 20 mg, and ranitidine, 75 mg, caused greater inhibition of the nasal blockage caused by histamine, 400 μg, than cetirizine alone. In the presence of both antagonists, there was residual histamine‐induced nasal blockage. 5 R‐α‐methylhistamine (R‐α‐MeH), 100–600 μg, the H3 agonist, intranasally, caused nasal blockage, which was not inhibited by either cetirizine or ranitidine. 6 Thioperamide, 700 μg, the H3 antagonist, intranasally, reversed the R‐α‐MeH‐induced nasal blockage. Thioperamide alone had no significant action on the nasal blockage induced by histamine, 400 and 1000 μg, but, in the presence of cetirizine, 20 mg, thioperamide further reduced the histamine‐induced nasal blockage. 7 Corynanthine, 2 mg, the α1‐adrenoceptor antagonist, administered intranasally, caused nasal blockage. 8 Corynanthine produced a greater increase in nasal blockage when in combination with bradykinin compared to its combination with R‐α‐MeH. 9 There appears to be a contribution of H1, H2 and H3 receptors to histamine‐induced nasal blockage in normal human subjects. The sympathetic nervous system actively maintains nasal patency and we suggest that activation of nasal H3 receptors may downregulate sympathetic activity.

Leukotriene D4 increases the excitability of capsaicin‐sensitive nasal sensory nerves to electrical and chemical stimuli

Clinical studies have demonstrated significant reductions in allergen‐induced nasal symptoms of atopic rhinitis subjects by CysLT1 antagonists, including neuronally mediated symptoms such as sneeze, itch and reflex hypersecretion. Here, we test the hypothesis that cysteinyl leukotrienes activate and/or alter the activity of nasal nociceptive (capsaicin‐sensitive) sensory neurones.

TRPA1: a potential target for anti-tussive therapy.

Cough occurs as a result of the activation of specific airway sensory nerves. The mechanisms by which tussive stimuli activate these sensory nerves are starting to be understood and suggest that TRPA1 channels are heavily involved. TRPA1 channels are nociceptor-specific ion channels that are gated by a wide range of exogenous irritants and endogenously-produced inflammatory mediators, suggesting that the blockade of TRPA1 represents a novel therapy for the treatment of cough in humans.