Christina Nassenstein

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.

P2X2 receptors differentiate placodal vs. neural crest C-fiber phenotypes innervating guinea pig lungs and esophagus

The lungs and esophagus are innervated by sensory neurons with somata in the nodose, jugular, and dorsal root ganglion. These sensory ganglia are derived from embryonic placode (nodose) and neural crest tissues (jugular and dorsal root ganglia; DRG). We addressed the hypothesis that the neurons embryonic origin (e.g., placode vs. neural crest) plays a greater role in determining particular aspects of its phenotype than the environment in which it innervates (e.g., lungs vs. esophagus). This hypothesis was tested using a combination of extracellular and patch-clamp electrophysiology and single-cell RT-PCR from guinea pig neurons. Nodose, but not jugular C-fibers innervating the lungs and esophagus, responded to alpha,beta-methylene ATP with action potential discharge that was sensitive to the P2X3 (P2X2/3) selective receptor antagonist A-317491. The somata of lung- and esophagus-specific sensory fibers were identified using retrograde tracing with a fluorescent dye. Esophageal- and lung-traced neurons from placodal tissue (nodose neurons) responded similarly to alpha,beta-methylene ATP (30 microM) with a large sustained inward current, whereas in neurons derived from neural crest tissue (jugular and DRG neurons), the same dose of alpha,beta-methylene ATP resulted in only a transient rapidly inactivating current or no detectable current. It has been shown previously that only activation of P2X2/3 heteromeric receptors produce sustained currents, whereas homomeric P2X3 receptor activation produces a rapidly inactivating current. Consistent with this, single-cell RT-PCR analysis revealed that the nodose ganglion neurons innervating the lungs and esophagus expressed mRNA for P2X2 and P2X3 subunits, whereas the vast majority of jugular and dorsal root ganglia innervating these tissues expressed only P2X3 mRNA with little to no P2X2 mRNA expression. We conclude that the responsiveness of C-fibers innervating the lungs and esophagus to ATP and other purinergic agonists is determined more by their embryonic origin than by the environment of the tissue they ultimately innervate.

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.

Thrombin and trypsin directly activate vagal C‐fibres in mouse lung via protease‐activated receptor‐1

The nature of protease‐activated receptors (PARs) capable of activating respiratory vagal C‐fibres in the mouse was investigated. Infusing thrombin or trypsin via the trachea strongly activated vagal lung C‐fibres with action potential discharge, recorded with the extracellular electrode positioned in the vagal sensory ganglion. The intensity of activation was similar to that observed with the TRPV1 agonist, capsaicin. This was mimicked by the PAR1‐activating peptide TFLLR‐NH2, whereas the PAR2‐activating peptide SLIGRL‐NH2 was without effect. Patch clamp recording on cell bodies of capsaicin‐sensitive neurons retrogradely labelled from the lungs revealed that TFLLR‐NH2 consistently evokes a large inward current. RT‐PCR revealed all four PARs were expressed in the vagal ganglia. However, when RT‐PCR was carried out on individual neurons retrogradely labelled from the lungs it was noted that TRPV1‐positive neurons (presumed C‐fibre neurons) expressed PAR1 and PAR3, whereas PAR2 and PAR4 were rarely expressed. The C‐fibres in mouse lungs isolated from PAR1−/− animals responded normally to capsaicin, but failed to respond to trypsin, thrombin, or TFLLR‐NH2. These data show that the PAR most relevant for evoking action potential discharge in vagal C‐fibres in mouse lungs is PAR1, and that this is a direct neuronal effect.