Comparison of TRPA1-versus TRPV1-mediated cough in guinea pigs

Abstract

TRPA1 receptor is activated by endogenous inflammatory mediators and exogenous pollutant molecules relevant to respiratory diseases. Previous studies have implicated TRPA1 as a drug target for antitussive therapy. Here we evaluated the relative efficacy of TRPA1 activation to evoke cough. In conscious guinea pigs the TRPA1 agonist allyl-isothiocyanate (AITC) evoked cough with a maximally effective concentration of 10mM that was abolished by the selective TRPA1 antagonist AP-18. AITC (10mM) was approximately 3-times less effective in inducing cough than capsaicin (50 μM). Ex vivo single fiber extracellular recordings revealed that, similarly to capsaicin, AITC evoked activation in airway jugular C-fibers, but not in airway nodose Aδ-fibers. Consistent with the cough studies, AITC was approximately 3-times less effective than capsaicin in evoking sustained activation of the jugular C-fibers. Another TRPA1 agonist, cinnamaldehyde, was approximately twofold more effective than AITC in inducing cough. However, the cinnamaldehyde (10mM)-induced cough was only partially inhibited by the TRPA1 antagonist AP-18, and was abolished by combination of AP-18 and the TRPV1 antagonist I-RTX. We conclude that in naïve guinea pigs, TRPA1 activation initiates cough that is relatively modest compared to the cough initiated by TRPV1, likely due to lower efficacy of TRPA1 stimulation to induce sustained activation of airway C-fibers.

Figure 1. The TRPA1 agonist allyl isothiocyanate (AITC) is less effective in inducing cough than the TRPV1 activators capsaicin and citric acid

(A) Time course of the cough induced by inhalation of allyl isothiocyanate (AITC), capsaicin and citric acid (paired study, n=9). Note that increasing the concentration of AITC from 3mM to 10mM did not further increase coughing indicating that the maximally effective concentration of AITC was attained. AITC (10mM) was less effective in inducing cough than capsaicin (50μM) and citric acid (0.4M). (Friedman test at 5 min time point, P<0.01, followed by Dunn's Test: *P<0.05 AITC vs. capsaicin, ^#P<0.01 AITC vs. citric acid). (B) The efficacy of AITC in inducing cough was not influenced by the type of vehicle used. The vehicles were PBS, DMSO (1%) in PBS, ethanol (2%)/Tween80 (2%) in PBS and in paired control experiments caused 0.2, 0.6 and 0.3 coughs/min, respectively. The cough to vehicle was subtracted from the cough to AITC. Kruskal-Wallis test (P<0.01) followed by Dunn's Test: *P<0.01 AITC vs. capsaicin, ^#P<0.01 AITC vs. citric acid.

Figure 2. Characterization of the TRPA1-mediated cough in the guinea pig

(A) Inhibition of AITC-induced cough by TRPA1 antagonists HC-030031 and AP-18 (paired studies). Note that AP-18 abolished the cough induced by AITC. The inhibition of AITC-induced cough by AP-18 was reversible. After 5 min of recovery following the AP-18 study, AITC evoked 2.3±1.0 coughs /5min (control subtracted). The magnitude of cough is expressed as the number of coughs/5min above the appropriate control. For AITC alone the control is DMSO (1% in PBS), for AITC with a TRPA1 antagonist, the control is the TRPA1 antagonist with DMSO (1% in PBS). See METHODS for details. HC-030031 and AP-18 were tested in different groups of guinea pigs. *P<0.05 Wilcoxon signed rank test. (B) Extending AITC inhalation to 10 min did not reveal a more robust response to AITC (tested with two different vehicles, DMSO (1%) in PBS and ethanol (2%)/Tween80 (2%) in PBS, see Fig. 1 legend for more details).

Figure 3. TRPA1 stimulation is less effective than TRPV1 stimulation in inducing sustained action potential discharge in the cough-triggering afferent nerves (jugular C-fibers) in the guinea pig trachea

Extracellular recordings were made from the jugular neurons projecting C-fibers into the trachea in the isolated vagally-innervated guinea pig airway preparation. (A) Two representative examples of the jugular C-fiber response to AITC and capsaicin. Histograms show the time course of the AITC- and capsaicin-induced action potential discharge. Note that individual jugular C-fibers responded to AITC in episodic brief (1-3s) high frequency action potential bursts. In contrast, capsaicin evoked robust more sustained action potential discharge. The delay in the onset of the response is attributable to the method of the drug delivery (see discussion). (B) Averaged responses of jugular C-fibers to AITC (n=7) and capsaicin (n=7). Only the C-fibers responsive to AITC (7/9) and capsaicin (7/7) were included in analysis. Error bars are omitted for clarity. (C) Capsaicin is more effective than AITC to evoke sustained activation of the jugular C-fibers (P<0.05, unpaired T-test).

Figure 4. In addition to TRPA1, TRPV1 contributes to cough induced by inhaled cinnamaldehyde in naïve conscious guinea pigs

(A) Cough evoked by cinnamaldehyde was only partially inhibited by the selective TRPA1 antagonist AP-18, but was abolished by the combination of AP-18 and the selective TRPV1 antagonist I-RTX. (P<0.01 Kruskal-Wallis test, *P<0.05 vs. vehicle, #P<0.05 vs. AP-18, Dunn's test). The inhibition of cinnamaldehyde-induced cough by AP-18 was partially reversible (after 5 min of recovery following the antagonist study cinnamaldehyde evoked 5.4+1.2 coughs/5min, control subtracted). Vehicle, DMSO (1%) in PBS. (B) In control experiments I-RTX had no effect on AITC-induced cough (unpaired study, P>0.5, Mann Whitney test). Vehicle, DMSO (1%) in PBS.