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

Abstract

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.

Figure 1. TRPA1, but not TRPM8, mRNA is expressed in vagal airway neurons

Single cell RT-PCR of TRPV1⁺ (A) or TRPV1⁻ (B) jugular/nodose cells; 1–12 or 1–10, respectively, represent 12 or 10 individual jugular/nodose ganglion neurons retrograde lung-labelled from the lungs. Samples in which the reverse transcriptase was omitted (‘−’) served as negative control. cDNA obtained from a whole jugular/nodose ganglion served as positive control (‘+’); B = bath control (bath solution was used as a template)

Figure 2. Cinnamaldehyde increases intracellular [Ca²⁺]_free in retrogradely labelled vagal afferents

Effect of cinnamaldehyde (100 μm) and capsaicin (1 μm) on intracellular [Ca²⁺]_free (expressed as 352/380 ratio) in dissociated DiI-labelled bronchopulmonary jugular/nodose afferents; mean ± s.e.m., n = 15.

Figure 3. Cinnamaldehyde induced inward current response in gramicidin-perforated patch clamp recordings

All recorded jugular/nodose neurons (n = 7) were labelled from lungs and airways. Depicted is a typical trace of inward current induced by vehicle (0.1% EtOH) and cinnamaldehyde (100 μm) application.

Figure 4. Cinnamaldehyde stimulation of nerve terminals evokes action potential discharges

A, representative trace of extracellular recordings in response to cinnamaldehyde (CA) during perfusion with vehicle (Krebs-bicarbonate buffer solution) and after 15 min pretreatment with ruthenium red (30 μm). B, repeated stimulation with cinnamaldehyde evokes similar action potential discharges (mean ± s.e.m., n = 8). C, cinnamaldehyde-induced action potential discharge can be blocked by pretreatment with ruthenium red (mean ± s.e.m.; *P < 0.05; n = 5).

Figure 5. Cinnamaldehyde aerosol provocations elicit central reflexes

The time of braking (Tb) was measured in response to vehicle (20% EtOH in PBS) and increasing doses of cinnamaldehyde. A, breathing pattern of a mice while breathing room air (baseline), during inhalation of vehicle (20% EtOH in PBS), and during cinnamaldehyde aerosol provocation, respectively. Scale bar = 1 s. B, quantification of Tb during inhalation of increasing concentrations of cinnamaldehyde aerosol (mean ± s.e.m., n = 16); *P < 0.05 compared to vehicle; **P < 0.01 compared to vehicle; #P < 0.05 compared to baseline.