Nasal challenges with (+) and (−) menthol (TRPM8 agonists) have beneficial effects in suppression of subjectively evaluated airway irritation, increasing capsaicin cough threshold and in inhibition of the cough response. On the other hand expected enhancement of cough due to activation of TRPA1 by nasal isocyanate and cinnamaldehyde was not observed.
This study assessed the modulation of cough response induced by nasal stimulation of TRPA1 and TRPM8 channels. TRPA1 is the channel most likely stimulated by air born pollutants, oxidizing substances, and endogenous inflammatory products and is categorized as nociceptive. It is responsible for induction of burning, irritation and pain . TRPM8 channel is mainly thermoreceptor with activation threshold around 24°C, giving the feeling of innocuous cold and freshness. It could also be activated by menthol [13, 14] which is known to have contra irritating effects in the airways .
Nasal menthol challenges have an antitussive effect in awake and anaesthetized guinea pigs . The main targets for menthol action in the airways are the trigeminal afferents which are distributed widely in the nasal mucosa. More than 60% or nasal trigeminal afferents express TRPM8 channel, some of those afferents are TRPV1-/TRPM8+  and expression of TRPM8 in nodose and jugular neurons innervating lower airways was less frequent. We propose, that these terminals, which are not nociceptors (as are TRPV1-) may be involved in the down regulating cough observed by nasal menthol application in guinea pigs. Animal models for cough studies and the results obtained in guinea pig models cannot be conclusively applied to the clinical conditions. Although the guinea pig vagus nerve pharmacology and physiology is very similar to human vagus nerve, these results should always be considered and interpreted with caveats . In the present study in human volunteers, menthol was delivered only to the nose (using a micropipette). It is believed that this nasal challenge [3, 19] minimizes leaking of menthol deeper into the airways. Although the menthol is highly volatile substance, the possibility for further movement of menthol vapours could not be completely excluded. Further we would suggest that these minimal amounts of menthol (if present) in lower airways would be insufficient to modulate cough reflex.
Studies estimating the effect of menthol on cough reflex in humans are conflicting. Morice and colleages reported antitussive effect of menthol vapours after oral inhalation . However, recent studies document no effect of inhaled (−) menthol on the cough reflex in children  or patients prior bronchoscopy . We think that effect of menthol in our study and also in the study by Morice et al. is local, influencing airway afferents involved in modulation of cough reflex.
It is also possibile that systemic exposure of lung and airway afferents to the menthol reabsorbed from nasal musoca is responsible for the observed effect. It was identified that TRPM8 nerve fibres are abundant in nasal mucosa particularly around blood vessels, and may mediate neurovascular reflexes . Johnson et al. in 2009  reported that menthol is involved in regulation of vascular tone, and it may be responsible for vasodilatation. This mechanism together with the abundance of nasal mucosal vasculature may be responsible for systemic absorption of menthol with subsequent influence on lung and airway afferents. However we did not measure the serum concentration of menthol after administration of nasal drops. A study of transdermal absorption and bioavailability of menthol after dermal application of patch by Martin & Valdes, reported detectable but very low plasma concentrations of menthol. As menthol has an antitussive effect after oral administration in guinea pigs in a dose 100 mg/kg of body weight (Brendan Canning, unpublished data), vapors entering the nose after oral administration and the systemic effects need to be considered.
Our controlled study shows that nasal menthol challenge in humans suppresses cough reflex induced by inhalation of capsaicin. Moreover, menthol can stimulate not only trigeminal but also olfactory terminals and activate cortical voluntary cough related pathways. This complex action of menthol brings cooling and freshness sensations, and reduces dyspnoea and respiratory drive perception  possibly with typical mint smell. Menthol also decreases trigeminal sensitivity to irritants . Both molecules of menthol isomers (+) and (−) were effective equally, but subjects tolerated (+) menthol challenge better. Subjects were blinded to the substances (+ and – menthol) during the challenge, however, questioning them after the testing they considered (+) menthol more pleasant compared to (−) menthol, which mimicked over the counter drug smell.
Cough is modulated by the cortical influences  and many other factors including behavioural, attentional or cognitive may also modulate cough response . We speculate that subjectively pleasant sensations may inhibit the urge to cough by the cortical mechanism, whereas unpleasant sensations may enhance it.
We expected that nasal challenges with TRPA1 agonists would enhance cough response similarly as it does TRPV1 agonist capsaicin challenge in animals and humans [2, 3]. Obviously, our data show that cough modulation via nasal TRPA1 is less intensive than that after TRPV1 agonist challenge. Activation of TRPA1 induces symptoms (e.g. burning) to 1/3 of those induced by equal concentration of agonists for TRPV1 (Figure 1). Afferent nociceptive drive induced by nasal capsaicin challenge was 7/10 on the VAS and considerable burning lasted up to 15–20 min. In present subjects (with nasal TRPA1 agonists) burning or unpleasant sensations reached 5/10 and disappeared after 2–3 minutes. It is possible that higher concentrations of isocyanate or cinnamaldehyde, repeated challenges, or challenges in subjects with trigeminal sensorineural hyperresponsiveness might modulate cough parameters. It had been documented that TRPA1 agonists induce cough in guinea pigs and humans after they are inhaled into the lower airways . The C2 concentration was around 200 mM of cinammaldehyde. For nasal challenges in mice 1% toluene diisocyanate had been used by Taylor Clark et al. . All of these studies proved a strong irritation effect of TRPA1 activating substances. However, naturally present irritants, either indoor, or outdoor, are present in inhaled air in very low concentrations. Thus, we used the lowest concentrations capable to induce sensory activation to mimic situation of natural exposure to air born irritants. In pre tests we identified appropriate concentrations for given agonists at 10-3 M, which was quite well tolerated, but induced nasal symptoms.
In addition, nasal challenge with isocyanate in our experimental set up modulated the urge to cough. We assume that perception of airway irritation induced by nasal AITC challenge could interfere with the irritation induced by the inhalation of capsaicin itself during the cough test. Subjects already reached the concentration for urge to cough, so they were conscious of the airway irritation and unpleasant sensations possibly leading to cough long before they reached the C2. Early presence of urge to cough during the capsaicin test after nasal isocyanate documented the nociceptive capabilities of nasal TRPA1 agonists by possibly enhancing afferent drive induced by the capsaicin inhalation. In contrast, nasal TRPM8 agonists reduced the urge to cough. After menthol nasal challenge subjects tolerated the capsaicin test much better, the urge to cough that proceed the cough motor act was detected later, close to C2 concentration. We documented coughs that had not been preceded by the urge to cough at all after nasal menthol pre-treatment in approximately 40% out of tests. Such coughs might have very much simple reflex origin without cortically processed urge to cough induced phenomena.
We mentioned possible trigeminal - olfactory interaction in modulation of cough response in our interpretaion of the observed menthol effects. The nature and extent of interactions between trigeminal and olfactory stimulation are poorly understood however TRPM8 and TRPA1 were detected both on trigeminal and olfactory nerve terminals . Exposure of the nose to the malodorants (unpleasant smells) increases the responsiveness of trigeminal nerves, and this mechanism is mediated by paracrine signalling pathway between olfactory and trigeminal nerves . Patients with acquired olfactory loss exhibit reduced trigeminal sensitivity, possibly due to the lack of these interactions. Considering possible modulation of cough by cortical activity induced by odours is a phenomenon that could contribute to the antitussive efficiency of placebo . In addition, the sweet taste of cough syrups or honey may have modulatory roles through oral/oropharyngeal receptors.
However, the specific role of particular channels in any biological process can only be conclusively proven by the use of selective channel antagonists, which at the moment, are not available for human use in clinical conditions . The results we have obtained in our studies lead us to surmize that the TRPA1 agonist isocyanate significantly modulates urge to cough probably via additional airway irritation instead of capsaicin test itself. Both TRPA1 agonists failed to modulate cough threshold and total cough response at these concentrations, suggesting that higher concentration or repeated exposure to them are necessary to modulate cough response. Both menthol isomers after nasal administration significantly modulated urge to cough, cough threshold, and total cough response probably via the reduction of airway irritation induced during capsaicin challenge. In addition to trigeminal afferents expressing TRP channels, olfactory nerve endings, trigemino – olfactoric relationships, smell perception process and other supramedullar influences should be considered as having potential to modulate the cough response in humans .