Tussive challenge with ATP and AMP: does it reveal cough hypersensitivity?

Discussion

When comparing healthy volunteers with chronic cough patients, the patient group coughed significantly more, and at lower concentrations of ATP. However, the degree of hypersensitivity demonstrated by our patient group to ATP does not appear to be any more than previously seen in other cough inhalational challenges [9, 11, 12]. This suggests that chronic cough patients do not have an intrinsically heightened sensitivity to ATP and, thus, it is not the acute, peripheral response to ATP that underlies cough hypersensitivity in these patients.

The use of citric acid as a tussive challenge in humans was first described by Bickerman and Barach in 1954 [13]. Since then, the technique has been used in a number of different settings with different tussive agents. The most commonly used are citric acid, capsaicin and fog challenge. These challenges stimulate cough by acting on different peripheral nerve receptors in the airways. Capsaicin is known to stimulate TRPV1 receptors on sensory afferent C-fibres [14]; citric acid shows some cross-reactivity with a number of receptors on C-fibres and also a-delta fibres [15]; whereas the specific receptors stimulated by the low chlorine solution of a fog challenge are poorly defined [16]. Other receptors implicated in the cough response are more difficult to stimulate with an inhalational challenge given the nature of the ligands involved; however, cinnamaldehyde has been shown to cause cough in humans and supports the involvement of the TRPA1 receptor in the cough reflex [17].

One hypothesis for the cause of hypersensitivity in chronic cough patients is that there is an up-regulation in one or more of the peripheral receptors of the afferent limb of the cough reflex [18]. The blockade of receptors stimulated by cough challenges are therefore of interest as therapeutic targets. In animal studies, there has been some success in reducing cough by antagonising these receptors [19, 20]. However, this success has not been replicated in human trials of TRPA1 (personal communication) and TRPV1 receptors [21], suggesting that these afferent sensory receptors, which are undoubtedly important in the sensation of irritation stimulating cough, are not the root cause of cough hypersensitivity.

A number of different lines of evidence now point to the purinergic system as being the most likely mechanism for inducing afferent hypersensitivity. ATP, acting through a class of purinergic receptors, was first postulated as an extracellular signalling molecule by Burnstock in 1972 [22]. It took a number of years for this mechanism to become established, and the role of ATP as an extracellular signal was not widely accepted until the 1990s when the first purinergic receptors were cloned [23, 24].

A number of findings support the presence of ATP-responsive P2 receptors within mammalian lungs. P2X3 receptors are thought to be mainly responsible for the effects of ATP in the lung, which have been demonstrated using immunohistochemistry on sensory nerve endings [25]. These receptors are found as either homotrimeric P2X3 receptors or heterotrimeric P2X2/3 receptors [26]. Vagal C-fibres can be stimulated by ATP via heteromeric P2X2/3 receptors [27].

The responses of peripheral neurons to ATP vary within the same ganglia, between different types of ganglia and within species. The response appears to be consistently due to the effect of ATP on P2X2 and P2X3, but there is probably a difference in the proportion of homo/hetero types of receptor expressed [26]. Of relevance to cough hypersensitivity is the observation that activation of P2X2/3 heterodimers produces a prolonged current, whereas stimulation of P2X3 receptors produces a rapidly inactivating current [28]. A recent study has, however, suggested that prolonged activation of the P2X3 receptor can be achieved by TRPV4 activation of pannexin, causing the continuous stimulation of P2X3 and thus leading to prolonged hypersensitivity [29].

Animal studies considering the role of ATP and P2X receptors in cough have been limited to guinea pigs, in which inhaled ATP accentuated the subsequent citric acid challenge response. ATP alone failed to stimulate cough and antagonist studies implicated the P2X4 receptor in this species [30].

In humans, intravenous ATP administered to palliative care patients caused breathlessness as its most common side effect [31]. Inhaling ATP has previously been noted to cause cough, although this response has not been systematically characterised. Prolonged inhalation of ATP causes bronchoconstriction in both healthy and asthmatic volunteers, with a greater response in the asthmatic individuals [32, 33]. Inhalation challenges using ATP and AMP in patients with chronic obstructive pulmonary disease, in smokers and in healthy volunteers found that ATP appeared to cause increased breathlessness and cough compared to AMP [34].

The lack of a significant cough response to inhaled AMP in our healthy participants seems to be in contrast with previous studies. This could be due to methodological differences given the brief exposure of our participants, a consequence of the use of the single-breath inhalation method. While the majority of our healthy volunteers coughed with ATP, two did not. This is in keeping with experiences with other cough challenges, such as citric acid and capsaicin, in which a proportion of healthy volunteers do not cough within the range of the challenge. ATP challenge does not appear, therefore, to be exceptional in its sensitivity or persistence. Thus, while our findings support the importance of purinergic receptors in the normal cough reflex pathway, it is not possible to differentiate between P2X3 and P2X2/3 receptors as the main modulators of this tussive response to ATP.

AF-219 (a P2X3 receptor antagonist) (Afferent Pharmaceuticals, San Mateo, CA, USA) has been trialled in a phase 2 study and has been found to dramatically reduce 24 h cough counts after 2 weeks of administration in patients with chronic cough [10]. Our data would tend to support the hypothesis that, while P2X3 receptor activation could be the final common pathway producing hypersensitivity, acute activation of the receptor does not infer this state on afferent nerves, and that other mechanisms such as activation of TRPV4/pannexin are required.

Our study has several limitations. Our two study populations were gender matched because of the known influence of gender on cough reflex sensitivity. They were, however, not age matched and our patient group was older than our healthy volunteer group. Although chronic cough is more prevalent in older patients [35], this does not seem to influence the cough reflex but rather the increased prevalence of the underlying cough-provoking conditions in older patients. Previous studies have shown no influence of age on the cough reflex, as opposed to a comorbidity such as dementia [36].

Comorbidities were also more prevalent in the patient group. The majority of these were conditions that are often associated with cough, such as gastro-oesophageal reflux disease, asthma and post-nasal drip as well as irritable bowel syndrome and lymphoedema. These comorbidities were self-reported by patients. One participant in each group was taking ACE inhibitors, and while this could have influenced the cough threshold, these individuals did not appear as outliers.

Within the population of 40 participants, we only had one adverse effect to ATP. This urticarial rash appears to have been a hypersensitivity reaction.

Given that some of our participants commented on ongoing throat irritation after the challenge was completed, ATP could potentially be involved in cough hypersensitivity via mechanisms other than a direct pro-tussive effect.