Chronic Cough: Enigmatic Role of Purinergic ATP Receptors

Abstract

Chronic cough is defined as cough lasting more than 8 weeks [26]. This cough condition evades any diagnoses and no underlying causes are normally found. This makes the cough both refractory to conventional therapy and deleteriously affect the quality of life of patients with this condition. The prevalent notion is that persistent cough conditions that are not immediately or easily diagnosable in this way may have a distinct diagnosable entity, a form of idiopathy with an underlying pathophysiological mechanism [28]. In general, a hypersensitive cough reflex is observed, giving rise to the coinage of a new syndrome called “Cough Hypersensitivity Syndrome” [27]. The aim of this dissertation is to unveil current established findings about the nature of chronic cough as well as to critically examine the possible operational mechanisms of P2X receptor antagonists that have proven to be effective therapies for Chronic cough. The dissertation will also look at other therapies for Chronic Cough and will seek to determine any central underlying mechanism, and the possible varying entry points along the mechanism at which treatment can be delivered to mitigate and treat the condition.

Introduction

Cough is one of the commonest symptoms reported whenever patients seek medical attention. Most cough instances are easily traceable to some acute illness such as a viral upper respiratory tract infection, tuberculosis and GERD among others. But there are some cough conditions that are not easily diagnosable and fail to be traceable as a symptom of some other illness but rather exist seemingly as an actual disease condition itself. The prevalence of such a condition in the population of patients seeking medical attention has necessitated the categorization of a discrete pathological entity wherein chronic cough is by itself a disease and not just a symptom of another primary pathology. The main focus of this dissertation was to determine the possible pathological mechanism causing the chronic cough condition and how the recent use of ATP antagonism may be producing the promising results it has been showing in recent studies for mitigating the chronic cough condition. It first looks at the generally accepted physiology of cough reflex; looking at the agents that trigger cough as well as known receptors and innervating fibres. Increased sensitivity of the cough reflex is then alluded, following which the possible mechanism for the cough hypersensitivity is dissected. It was then established that in many of the cases; including chronic acid reflux, viral infection, COPD and other conditions that exhibited a sensitivity to tussive stimuli that sometimes persisted beyond any diagnosable pathology, that a common driving mechanism existed, that of chronic activation of purinergic ATP receptors. It was found that many of the instances of chronic cough coincided with high levels of extracellular ATP that would gate the purinergic receptors and activate otherwise normally silent fibres.

Body Of Dissertation

A tussive agent is a chemical or substance that causes coughing when it interacts with specific receptors in the respiratory system. Well known tussive agents include Capsaicin [2] and (citric) acid [1]. Capsaicin and acid have been shown to trigger cough by acting via TRPV1 receptors found on vagal C-fibre afferents [3]. Widdicombe [4] further established that several elements of the Vagus nerve - such as Rapidly Adapting Receptors (RARs), unmyelinated C-fibres and myelinated A-ɗ fibres express TRPV1 receptors, allowing them to mediate cough response to the inhalation of capsaicin or citric acid.

Fig 1: A drawing from Widdicombe (2003) showing the different afferent pathways involved in cough, with their stimuli and probable mechanism of action [4]. The vanilloid capsaicin is the principal agonist of TRPV1 ion-channels (also called VR1), which can be opened by either acid solutions or warmed-water solutions (37-50C). TRPV1 (VR1) is therefore a polymodal receptor on sensory vagal nerves. Here, citric acid was used as surrogate for refluxed gastric acid (HCl). Acid reflux is a potent stimulator of the coughing reflex in humans.

Source:[4]

The Capsaicin-cough challenge was introduced in early studies, such as one by Torrego et al [5], where it was established that patients with any form of chronic cough seemed to have a heightened sensitivity to otherwise innocuous stimulation of airway afferents with tussive agents [5], by a process likened to allodynia in cutaneous afferents [5]. This and other findings drove the idea that patients of chronic cough were suffering from a chronic “cough hypersensitivity syndrome” [8] in which the sensory neurons innervating the airways and lungs were sensitized to tussive stimuli. Subsequently, it was proposed that antagonists for different receptors of differing tussive agents could be applied as a therapeutic agent for chronic cough, such as the use of the TRPV1 antagonist; XEN-DO501, which was shown by Wortley et al [9], to be an effective blocker of TRPV1. However, subsequent experiments on the efficacy of the TRPV1 antagonists showed that Xen-D0501 did not reduce episodic coughing in refractory cough patients [10]. This strategy may have failed only because it was directed at a single tussive agent of heightened sensitivity to multiple tussive agents. A better outcome would probably be achieved by dissecting the mechanism that led to the heightened state of sensitivity of airway afferents in the first place.

Chuaychoo and colleagues [11] established that a particular set of afferent C-fibres called the nodose C-fibres (because their origin is from the epibranchial placode) are strongly responsive to autocoids; including adenosine, serotonin; and important to this dissertation, adenosine triphosphate (ATP). Thereafter, Weigand and colleagues [12] showed that not only does ATP cause the firing of C-fibres via interactions with its P2X receptors, but that the mechanisms of C-fibre activation by ATP may possibly be secondary to bronchoconstriction [12]. To come to this hypothesis, Weigand and colleagues [13] showed an increase in interstitial ATP following histamine/methacholine mediated constriction of bronchi smooth muscle. This finding corroborated an earlier paper by Hanna and Kaufman [13] where it was shown that interstitial ATP concentration increases during smooth muscle contraction. Y. Takahashi and colleagues [14] had previously demonstrated that ATP contributed to enhancement of the sensitivity of the cough reflex by demonstrating that guinea pigs pre-treated with nebulized ATP had a high frequency of coughing episodes in response to primary tussive agents, capsaicin and citric acid (Fig. 2)

​Fig. 2. Effect of different ATP concentrations in a nebuliser on the number of coughs evoked by citric acid inhalation, with saline as the control. ATP (6 and10 μM) significantly increased the number of coughing episodes (p< 0.05). However, ATP (3 mM) was not effective in potentiating the cough response to acid.

Source:[14]

The increase in cough responses to the tussive agent (citric acid) was clearly seen to be dependent on the concentration of ATP, with concentrations (higher than 3 μM) increasing the total number of cough responses to citric acid. ATP increased the cough response to the same tussive agent challenge in a directly proportional manner to the concentration of ATP applied. ATP receptor antagonists, such as TNP-ATP, PPADS and RB2, led to a reduced cough response to the primary tussive agent particularly TNP-ATP which blocks P2X receptor subtypes P2X1-4 [14] (fig. 3)

​Fig. 3 A graph showing the effects of P2X and P2Y receptor antagonists on ATP-induced enhancement of the number of citric acid-induced coughs. The guinea pigs were exposed to 10 μM concentration of ATP aerosol for 2 min during the 5 min before the inhalation of citric acid. The guinea pigs were exposed to 50 μM of PPADS, 50 μM of TNP-ATP or 30 μM of RB2 aerosol for 2 min during the 5 min preceding the inhalation of ATP aerosol. The number of coughs during the 10 min of exposure of citric acid was counted before (white column) and after exposure to ATP (crossed column). Each column represents the mean with S.E. of 6 animals. *P < 0.05 vs. the value before exposure to ATP

Source: [14]

Taken together these results indicated that stimulation of P2X receptors (more especially the P2X2/3 subtype) is required to enhance the cough reflex. This finding helped implicate nodose C-fibres as being part of the cough mechanism, since Chuaychoo and colleagues showed that not only did nodose C-fibres express purinergic P2X receptors for ATP, but that direct cell recordings from these fibres show an enhanced firing response to the application of ATP (as well as histamine) [11].

To prove that smooth muscle contraction was the source of the ATP, Weigand and colleagues established that whilst smooth muscles of the bronchi expressed M3-receptor for the well-known bronchoconstrictor Methacholine; the nodose C-fibres do not [12]. This would mean that if there was no influencing interaction between the smooth muscles and the C-fibres, then the C-fibres would not be expected to show any change in Action Potential discharge following application of Methacholine; simply because we do not expect the C-fibres to be able to transduce the Methacholine signal. But this was not the case when Weigand and colleagues carried out experiments. They found that Methacholine application caused action potential discharge in the nodose C-fibres (Fig. 4); with about 123±32 action potentials being recorded.

​Fig. 4. Single-unit recording of action potential discharge in a nodose C-fibre, in response to methacholine (arrow; 10μM). The background discharge rate was potentiated by the M3 receptor agonist methacholine, over a period in excess of 20 seconds. Source:[12]

Contraction of airway smooth muscle mediated by M3-receptor activation upon methacholine application may have led to an increase in interstitial ATP levels which then interacted with nodose C-fibres purinergic receptors to mediate the observed firing rates. To test this possibility, ATP-receptor antagonist where shown to reduce the number of action potential discharge by nodose C-fibres after the same injection of methacholine; dropping from 123±32 impulses to 21±5, as well lowering the frequency of impulse traffic (Fig. 5)

​Fig. 5. Total action potentials elicited from nodose C-fibres by exposure to histamine (HA; 30µM) and methacholine (MCh; 10µM) were significantly reduced (p< 0.05) by AF-353 (100 µM).

Source: [12]

These observations helped formulate the idea that ATP receptor antagonism may constitute a viable therapeutic strategy for treating chronic cough. The observations here also drove the conclusion that the mechanical effect of bronchoconstriction somehow releases ATP and led to the increase in the interstitial levels of ATP. These possibilities then suggested that bronchoconstriction is the primary mechanism driving C-fibre activation in this case.

Results obtained with the tussive agent histamine, where injection of histamine into either the trachea or pulmonary artery of the guinea pig led to similar increases in firing rates of nodose C-fibres, was inconclusive in establishing the mechanisms of C-fibre activation Undem and colleagues [16] had already established that C-nodose fibres have H1.Thus the increase in action potential firing rates in response to histamine could be just as easily explained in terms of histamine’s interaction with H1-receptors on the nodose C-fibres themselves as its actions as a bronchoconstrictor like Methacholine, causing contraction of smooth muscle and the subsequent increase in interstitial ATP. To answer these issues, Weigand and colleagues [12] antagonised the contraction of the smooth muscles using 1) Myosin Light Kinase Inhibitor (ML-7) and 2) Isoproterenol. Both these spasmolytics succeeded in antagonising smooth muscle contraction by significantly inhibiting tracheal perfusion pressures evoked by histamine application, reducing the tracheal perfusion pressures by up to 83±8%. Importantly, this corresponded in a sharp decrease in the histamine-induced action potential discharge of nodose C-fibres (dropping from 171±36 impulses to 55±23 impulses). (Fig. 6)

​Fig. 6. The averaged histamine-induced increase in tracheal pressure in guinea-pig, as a percentage of the maximum response to the tussive agent after perfusion with the 1) Myosin Light Chain Kinase Inhibitor ML-7 (30μM) or 2) Isoproterenol (100μM). Both spasmolytics significantly reduced (p<0.01) the evoked bronchoconstriction.

Source:[12]

These results strongly suggested that the nodose C-fibre activation observed after histamine application worked downstream of smooth muscle contraction and was not a result of direct interaction with H1-receptors. This hypothesised downstream mechanism of histamine was further consolidated by that peak activity of a single nodose C-fibre correlated with the dynamic phase of increase in tracheal perfusion pressure as shown in Figure 7.

​Fig. 7. The arrow labelled 1 shows time of histamine application, dotted line shows how the peak electrical activity of the nodose C-fibre coinciding with the dynamic phase of increase in tracheal perfusion pressure. Arrow head labelled 3 indicates the pressure artefact generated during infusion of the histamine.

Source:[12]

This suggested that the smooth muscle cells are the ones that mediated the signalling of Histamine first, causing the initial rise/increase in tracheal perfusion pressures; nodose C-fibre activation then followed later, suggesting that it came a little later in the signalling cascade. Thus this study by Weigand and colleagues [12] is again one of the several studies that increasingly point to ATP antagonism as a possible therapy for treating the chronic cough condition.

Sensitization of the cough reflex by extracellular ATP is also observed when there is an upregulation of TRPV1 expression in sub-epithelial nerves after a viral infection; where TRPV1 activation has been suggested to lead to the rise in extracellular ATP levels via the TRPV1/4 Pannexin-1 axis [22]. In a study by Zaccone and colleagues, hypersensitization of the cough reflex in guinea pigs to three tussive stimuli; capsaicin, bradykinin and citric acid, was observed in guinea pigs following 4 days of intranasal injection of Parainfluenza-3 [19]. Fig 8