Chronic cough: ATP activation of vagal afferent nerve supplying the airway

Abstract

Cough is the most common symptom seen by medical doctors in their general practices. While cough is a normal and necessary defense mechanism that protects the lower airway, it can become excessive or chronic, damaging the airway and deteriorating the patient’s quality of life. Cough is a reflex that involves an afferent pathway, a cough center, an efferent pathway and effector organs and muscles. In the terminals of the afferent pathway several types of receptors can be found. These receptors can be classified as mechanoreceptors and chemoreceptors. There are two main types of mechanosensors: rapidly adapting receptors and slowly adapting receptors. Chemoreceptors are located in two different nerve fibers: C-fibers and Aδ fibers. C-fibers and rapidly adapting receptors are the most commonly associated with the cough response. TRP receptors, present on the airway, have also been associated with cough. TRPV4 can induce cough in a mechanism that is dependent on the activation of the P2X3 receptor. However, cough can also be initiated by activating the P2X3 receptor in a TRPV4-independent manner. P2X3 is abundant on the airway and it is a member of the P2 receptor family. This family consists on ion channels gated by ATP. ATP is present on the airway, and its concentrations are increased in several respiratory pathologies, including chronic cough. The use of a P2X3 receptor antagonist showed improvement in the symptoms of chronic cough patients. All of these data supports the hypothesis that ATP and the P2X3 receptor have a role in chronic cough, and that P2X3 antagonists could have pharmacological uses in the treatment of this condition. This would clinically relevant, given that current treatments for chronic cough are mostly opiates, which not only have shown limited therapeutic efficiency, but are also linked to several negative side effects, including the development of physical dependency.

Declaration of Contribution

Introduction

Cough is a defensive reflex that protects the lower airway from particulate matter and foreign objects. Cough also protects the respiratory system form pathogens, mucus, inflammation mediators and other irritants than can be inhaled (Widdicombe, 1995).

There are three physiological phases in cough: inspiratory, compressive and expiratory. The inspiratory phase consists in an inhalation to gather sufficient air to expand the lungs. In the compressive phase the glottis briefly closes and the vocal cords contract. Besides, the chest wall closes and the diaphragm and abdominal muscles also contract. In the expiratory and final phase the glottis opens and the air is rapidly and noisily expelled (Chang, 2006). From the neurophysiological standpoint, cough consists on afferent fibers, a cough center that coordinates the received signals, and efferent fibers, which transmit the impulse to the effector organs and muscles (Widdicombe, 1995). The cough reflex begins with the stimulation of receptors placed on the terminal of the afferent nerves. These receptors can translate the signal induced by a stimulus into an electric impulse that travels through the efferent nerves to the effector organs and muscles. There are different types of receptors, including mechanoreceptors and chemoreceptors. The first kind is sensitive to mechanical stimuli, like touch and pressure; while chemoreceptors are sensitive to different chemical compounds. Mechanosensors are further divided into rapidly adapting receptors and slowly adapting receptors, while chemoreceptors can be found in C-fibers and Aδ fibers. Both mechanoreceptors and chemosensors have been associated with the initiation of a cough response, but rapidly adapting receptors and C-fibers are the most commonly implicated (Mazzone, 2005).

The airway expresses different types of receptors, including TRP channels and P2X receptors. TRP channels belong to a family of cationic channels that are able to sense temperature, touch, pain, osmolality, pheromones, taste and other stimuli (Clapham, 2003). TRPV1 channels, particularly, are expressed in the smooth muscle cells found on the airway. TRPV4, on the other hand, is present in airway cells and it induces the secretion of ATP into the airway. ATP is the ligand of P2 receptors, which are also present in the airway. The P2X3 receptor, particularly, is abundant on the lower airway. TRPV4 activation induces cough in a mechanism that is dependent on P2X3 receptor activation, suggesting a role for ATP and P2X3 receptor in the cough reflex response. ATP can also initiate a cough response independently from TRPV4 activation (Bonvini, 2015).

While cough is a normal physiological process, it has been associated with different pathologies and it is one of the more common reasons for patients to consult with a medical doctor (Cornford, 1998). Cough can become excessive and unhelpful, and when it lasts more than eight weeks, it is considered chronic. In these cases, patients find themselves negatively affected in their regular lives; chronic cough diminishes their quality of life (Chung, 2008). Regrettably, there are no efficient and safe treatments for chronic cough. Currently, one of the accepted treatment options for cough is the use of opiates, but their efficiency in the treatment of this condition is uncertain. Besides, opiates induce a wide range of negative physical side effects, one of the main ones being the induction of physical dependency (Morice, 2007). For these reasons, it is important to investigate new potential pharmaceutical options.

The P2X3 receptor and ATP are considered to be important mediators of cough (Abdulqawi, 2015). Therefore, the study of these compounds regarding their role in the cough reflex could result in valuable information about different treatment options. Antagonists of the P2X3 receptor, particularly, are a possible therapeutic option for cough patients. In fact, one study showed that patients with chronic cough showed improvements of their symptoms after being treated with a P2X3 antagonist, supporting the importance of ATP and the P2X3 receptor in the cough reflex (Abdulqawi, 2015).

Other mechanisms and signaling pathways are also involved in he cough reflex, and targeting them could be of benefit for chronic cough patients. Also, non-pharmaceutical therapies have been developed, including speech therapy, which has proven to have beneficial effects for chronic cough patients. Another potential pharmaceutical target is TRPV1, whose levels have been to be increased in the airway of chronic cough patients. Other treatment options include ion sodium and potassium blockers, which have shown promise in cough pharmacology. The rationale behind their use is that sodium and potassium channels need to be working properly in order for an action potential to be initiated (Reynolds, 2004). Also, it has been proposed that alterations in these channels are behind the hyper sensibility of chronic cough. GABA and NMDA receptors are other novel and attractive therapeutic targets. All of the above mentioned therapies and pathways pathways present novel treatment options for cough, and they could be used in synergy with drugs targeting the P2X3 receptor.

Considering the lack of safe and efficient treatment options for chronic cough patients, a better understanding of the molecular mechanisms behind this pathology and the development of new drugs could have a positive impact on these patients’ quality of life.

Main Body of Text

Cough is a modified respiratory act that consists on a rapid expulsion of air from the lungs (Lee, 2012). This rapid expulsion is accompanied by a characteristically sudden and sharp sound. Also known as tussis, cough is a defense mechanism that removes mucus and other foreign objects from the lower respiratory tract, thus clearing the airway from secretion particulates and protecting it from the aspiration of particulate matter (Widdicombe, 1995). Besides particulate objects, the cough reflex also protects the airway from pathogens, accumulated secretions, postnasal drip, and other irritants that are inhaled or formed wherever there is mucosal inflammation. Cough, therefore, is an important defensive reflex, necessary for maintaining the health of the lungs and of the overall respiratory system (Canning, 2014).

The importance of a normal cough response is highlighted in patients that lack an appropriate cough reflex. These patients have an increase risk of developing conditions such as atelectasis (complete or partial collapse of a lung), recurrent pneumonia (two or more pneumonia episode every year) and several other chronic airway diseases (Niimi, 2003).

While, as stated above, cough has normal defensive roles in healthy patients and in the combat of some respiratory diseases, this rapid air expulsion has also been linked to respiratory morbidity. For instance, in a vigorous cough process the intra-thoracic pressure can reach 300 mm Hg, while the velocity of the expiratory air flow can go up to 800 km/hr (Ford, 2007). Even though these elevated pressures and velocities are necessary for cough to accomplish its beneficial effects, they can also create complications for the patient, including exhaustion, insomnia, headaches, dizziness, musculoskeletal pain, hoarseness, excessive perspiration, urinary inconstancies, and in some cases, even rib fractures. Cough can also cause psychological issues in some patients, like embarrassment or self-consciousness (Irwin, 2006). Besides, under certain pathological conditions, cough can become excessive and unproductive, even becoming harmful to the airway mucosa (Lee, 2012). This is the case of chronic cough, defined as a cough that lasts eight weeks or longer in adults or four weeks in children, which can have a negative impact on patients’ quality of life (Chung, 2008).

Neurophysiology of cough

Physiologically, there are two types of cough: laryngeal and tracheobronchial. Laryngeal cough is a truly involuntary reflex that is triggered when laryngeal receptors are mechanically stimulated by foreign objects, like particulate material. Tracheobronchial cough, in contrast, can begin in a voluntarily manner and it is started distal to the larynx (Chang, 2006).

There are three mechanical distinct phases of cough: inspiratory, compressive and expiratory (Chang, 2006). The inspiratory phase consists on air inhalation, which lengthens the expiratory muscles and optimizes their length-tension ratio. This phase generates the necessary air volume for cough to occur. In the case of stimulation of the distal larynx (tracheobronchial cough), there is a more prominent inspiratory phase, possibly because a bigger airflow is needed in order to remove the distal stimulus. The cough reflex can also begin after a stimulus at the level of the vocal cords or a stimulus on the upper tracheal segments. In this second case there is no inspiratory phase. The second stage, known as the compressive phase, includes a brief closure of the glottis by laryngeal adductor muscles. The aim of this process is to maintain the lung volume even as intra-thoracic pressure increases. Together with this glottis closure the vocal cords contract, the chest wall closes, and the diaphragm and abdominal muscles also contract. The final stage, the expiratory phase, includes the re-opening of the glottis and the expulsion of a brief respiratory flow. In this final phase the airway is dynamically compressed after contraction of expiratory muscles, and debris are expelled together with air and, sometimes, mucus. In a chronological manner, the events of a cough reflex are the following: the diaphragm and external intercostal muscles contract, the glottis closes, the trachea becomes rigid thanks to the contraction of the trachealis muscle, and finally the glottis opens and through it passes a forced release of air from the lower respiratory tract. This release of air is accompanied by the characteristic sound of cough (Chang, 2006).

In order for a cough reflex to begin, a stimulus needs to be present. Cough can be triggered by a wide array of stimuli, including but not limited to smoke and smoky atmospheres, cold temperatures, eating or drinking, perfume smells, laughing, talking and shouting, contact with pets, pollen and post-nasal drip (Matsumoto, 2012). These triggers are sensed by receptors located at the nerve terminals, transforming the stimuli into an electric impulse that travels through nerve fibers (Widdicombe, 1995).

The cough reflex, therefore, also involves nerve fibers that transport the cough signal from its receptors to effector organs and muscles. These fibers include an afferent or sensory pathway that guides the signal to the bulbo-pontine neuronal aggregates, and an efferent pathway that connects and carries the signal to the effector organs and muscles. The afferent nerves include trigeminal, glossopharyngeal, superior laryngeal and vagus nerves. The vagus nerve is the most heavily implied in the cough reflex (Nishino, 1988). The signal that travels through the afferent pathway arrives to the cough center in the medulla; the cough center is under the control of a higher cortical center, located in the upper brain stem and pons, and it has the function of coordinating the cough response. The signal continues through an efferent pathway from the cough center, traveling through the vagus, phrenic and spinal motor nerves to the diaphragms, abdominal walls and muscles. This way the signal arrives to the expiratory muscles. In short, the afferent arm of the cough reflex connects the receptors to the respiratory or cough center, while the efferent arm unites the respiratory center to muscles of the larynx and respiratory muscles (Widdicombe, 1995).

Cough Afferent Fibers and Receptors

The afferent fibers of the intrapulmonary airway are divided into four subtypes according to their physiochemical sensitivity, adaptation to lung inflation, neurochemistry, whether they are myelinated, conduction velocity and site of termination in the airway. These neuronal subtypes correspond to rapidity adapting afferent nerves, slowly adapting afferent nerves, C-fibers, and Aδ fibers (Mazzone, 2005). In the terminations of the vagal afferent nerves several types of receptors can be found, which are located in the trachea, main carina, branching points of the large airways and small distant airway in the pharynx (Sant’Ambrogio, 1978).

One classification divides afferent nerves into mechanically sensitive and primarily chemically sensitive. Mechanically sensitive fibers are also known for expressing low threshold mechanosensors, which are activated by one or more mechanical stimuli, including lung inflation, bronchospasm and touch. While they do not normally respond to chemical triggers, a chemical stimulus can indirectly activate a mechanosensor by mechanically distorting the nerve terminal. Chemosensors, on the other hand, can be activated by several chemical compounds, including capsaicin, bradykinin, adenosine, prostaglandin E2, but are not sensitive to mechanical input (Mazzone, 2005). However, the distinction between mechanoreceptor and chemoreceptor is not absolute, as some mechanoreceptors can respond to chemical stimuli. For example, acid and ATP can sometimes activate mechanoreceptors (Mazzone, 2005).

Low threshold mechanosensors are further divided into two classic types: rapidly adapting receptors and slowly adapting receptors. The fibers expressing both of these types of receptors originate in the nodose ganglia and terminate in the intrapulmonary airway and lung parenquima (Canning, 2004).

The main characteristic of rapidly adapting receptors is their rapid adaptation to sustained lung inflation; their high sensitivity to lung collapse or deflation; and an elevated velocity of impulse conduction, ranging from 4 to 18 m/s, which is consistent with myelinated axons. These mechanoreceptors are active during the respiratory cycle and become more active when the lung inflates or increases in volume. The activation of this subtype of fiber induces a bronchospasm reflex and mucus secretion, and it has a role in coughing, possibly working in a synergetic manner with other nerve subtypes (Sant’Ambrogio, 2001).

Slowly adapting afferent nerves, on the other hand, are highly sensitive to mechanical inputs received by the lung while breathing. Their activity has been found to be increased during inspiration, just before the initiation of expiration. Slowly adapting receptors are different from rapidly adapting receptors in their conduction velocity and in their inability to adapt to sustained lung inflation. Their activation leads to decreased phrenic nerve activity and decreased smooth muscle tone (Sant’Ambrogio, 1983).

The afferent fibers from the cough reflex can also be chemosensitive. These types of fibers are present both in the airways and lungs and are normally quiescent, but are activated during inflammation or irritation. There are several types of chemosensitive fibers in the airway, including C-fibers and Aδ fibers. These fibers are not activated by mechanical stimulation like bronchoconstriction, lung inflation or touch, and are activated instead by chemical compounds like capsaicin, acid, heat, bradykinin, prostaglandin E2, adrenalin and adenosine, among others. Unlike rapidly adapting fibers and slowly adapting fibers, C-fibers are not myelinated. These fibers correspond to most of the nerves innervating the airways and the lung (Mazzone, 2005).

It has been proposed that several subtypes of nerves are responsible for inducing the cough response. Both mechanical and chemical stimuli can evoke reflex coughing, suggesting that both mechanosensor and chemosensor nerves are involved (Narula, 2014). Rapidly adapting receptors have been recognized as the primary nerve nerves of the cough reflex, since coughing can be initiated by activation of these rapidly adapting receptors (through constriction of the airway smooth muscle, mechanical irritation and airway constriction after capsaicin and bradykinin stimuli) (Undem, 2002). Nevertheless, some stimuli induce rapidly adapting receptor activation but do not induce cough; these stimuli include thromboxane, leukotriene C₄, histamine and methacholine. Chemosensitive receptors also seem to have a role in the cough response, since capsaicin, bradykinin and citric acid induce a cough response in conscious animals and humans. Therefore, usually C fibers and rapidly adapting fibers are the most often implicated in the cough reflex; however, there are several compounds that stimulate rapidly adapting fibers but do not provoke cough. Also, the activation of C-fibers alone is ineffective as tussive agent in anesthetized animals (Mazzone, 2005). In short, a complete understanding of the nerves that regulate the cough response and related receptors remains to be achieved.

TRP and P2X channels

The notion that ATP can act as an agonist for cell surface receptors was first described in the cardiovascular system, where it was discovered that ATP acts as an agonist for the heart and blood vessels. Afterwards, ATP was identified as a neurotransmitter in different kinds of nerves. ATP is now recognized as an extracellular messenger in several physiological processes, including exocrine and endocrine systems, secretory pathways, and in different types of cells, such as endothelial, musculoskeletal, immune and inflammatory cells (Edwards, 1992).

ATP as an agonist acts on purinireceptors, abbreviated as P2 receptors. There are two types of P2 receptors: P2X and P2Y, based mostly on pharmacological criteria. P2X receptors are ionotropic receptors, which consist on ionic channels that open after they interact with ATP. P2Y receptors, on the other hand, are metabotropic receptors, meaning that they are linked to G proteins (known as G protein coupled receptors) (Ralevic, 1998). While they have important roles on embryonic development, these receptors are also potential therapeutic targets, since several pathological conditions alter their signaling pathway. Among these conditions there are diseases of the airway; for instance, nucleotides increase the mucus secretion and ciliary beat frequency of the epithelial cells of the airway (Idzko, 2007).

ATP can be released into the extracellular space by a range of physiological mechanisms, and ATP is an important first messenger molecule. P2X receptors work as homodimers, (P2X1-7) or as heterodimers or multimers. Their structure changes depending on the tissue they are found, as does their ion permeability, sensitivity to agonists and antagonists, and de-sensibilization velocity. Besides, all P2X subunit can be glycosylated, which is important for their transport to the cell surface. After ATP interacts with a P2X receptor, there is an increase of intracellular calcium, which in turn stars a depolarization wave that travels through the afferent fiber. Besides, there is involvement of other ion channels, including potassium outflow and sodium inflow. The activation of P2X receptors is also linked to other signaling responses, besides the opening of ion channels, including the activation of phospholipase A2, activation of phospholipase D, activation of the MAPKs pathway and activation of the transcription factor NFkB (Ralevi, 1998).

P2Y receptors are different from P2X receptors: they have a different structure, sensitivity and mechanism of action. Like all G protein coupled receptors, their structure consists on a seven transmembrane domains next to intracellular loops. They are sensible to both purines and pyrimidine nucleotides. After interacting with ATP, these receptors activate several signaling transduction pathways and secondary messengers. The metabotropic mechanism is slower than the ionotropic mechanism of P2X receptors. P2Y receptors respond in one of two mechanisms, being classified as P2Y1-like or P2Y12-like. P2Y1 receptors activate the phospholipase C protein through interaction with the Gq protein, while P2Y12-like receptor act by inhibiting adenylate cyclase after the ligand interacts with a receptor that is linked to a Gi protein (Ralevic, 1998).

In the airway, P2X3 and P2X4 have been found in epithelial cells. It has been proposed that these receptors could be involved in the cough reflex (Burnstock, 2012).

Chemosensors can be defined by their ability to respond to capsaicin, therefore, by the presence of the capsaicin receptor: TRPV1. TRP receptors are a family of cationic channels and they are part of the sensory systems. These receptors are able to respond to temperature, touch, pain, osmolality, pheromones, taste and other stimuli (Clapham, 2003). TRP channels are abundant in the airways; TRPV1 channels, particularly, are expressed in the smooth muscle cells found on the airway. Both C-fibers and Aδ fibers express TRPV1, with a possible role in the regulation of the cough mechanism. TRPV1 integrates triggering stimuli in several protective reflexes, including cough. This role is further demonstrated by the fact that in patients with idiopathic chronic cough, TRPV1 expression is elevated. Also, capsaicin, the main agonist of TRPV1, is often used in research as a tussive (Adcock, 2009).

Another TRP channel that has been linked to cough is TRPV4 (Belvisi, 2013). This TRP channel can be activated by different stimuli, including mechanical stress, interaction with phorbol ester, arachidonic acid and other metabolites (Watanabe, 2003). The TRPV4 receptor is expressed in different areas of the respiratory tract, including the epithelium and the airway muscles (Belvisi, 2013). Interestingly, the TRPV4 signaling pathway has been linked to a different type of receptor: P2X receptors. When nerve fibers are stimulated with TRPV4 agonists, there is a sustained stimulation of all Aδ fibers and a similar effect on C-fibers. This activation is relatively slow compared to the normally observed activation with ligands such as capsaicin and citric acid, suggesting that the effect was is direct, but mediated by another pathway. The coughing effects induced by TRPV4 agonists were inhibited when there was also presence of a P2X3 antagonist, indicating that the effect of TRPV4 was mediated by this second receptor and suggesting a possible role for P2X3 agonist, ATP. ATP was found to be released from epithelial cells of the airway after stimulation with TRPV4 agonist. The mechanism of ATP release involves the opening of pannexin 1 channels, which are regulated in a Rho-dependent manner. This ATP release by TRPV4 has also been observed in the activation of vagal afferent nerves. ATP can also directly activate both C-fibers and Aδ fibers in a manner that is independent from TRPV4 activation (Bonvini, 2015).

In the airway, the activation of P2X receptors by endogenous ATP is an important mechanism. ATP can be released into the airway after being stimulated with several different compounds, like acid-pepsin (Tsai, 2009). An increase of oxygen-reactive species also promotes the release of ATP into the larynx, increase that can be inhibited by scavengers of oxygen-reactive species. ATP activates P2X receptors, and it has been suggested that these receptors, which are present in the terminals of afferent fibers, can sensitize these fibers to capsaicin. This theory proposes that laryngeal insult with acid-pepsin or oxygen-reactive species, including oxygen peroxide, induces inflammation and leads to the production of an excess of reactive oxygen species, which could, in turn, promote the release of ATP into the airway. ATP would then interact with P2X receptors, resulting in the sensitization to capsaicin in laryngeal afferent fibers (Lin, 2010).

Chronic cough and treatment options

As mentioned above, chronic cough is defined as a cough that last eight weeks or longer in adults. The most common causes are tobacco use, postnasal drip, asthma and acid reflux. Usually, addressing the underlying problem resolves the cough, however, some patients develop refractory cough, which remains despite guideline based treatment (Chung, 2008).

Historically, there have not been many treatments options for patients with chronic cough. Laudanum, a bitter fluid extracted from opium, was used a cough remedy in the 18^th century. Laudanum is an addictive substance that besides being used as a cough remedy was used as a recreational drug and against several other pathologies. Opiates in general have been used against cough and other diseases since the 1700s (Marketos, 1986).

Opioid compounds act on opioid receptors, which are located on neuronal cell membranes. There are three main types of opioid receptors: µ, δ, κ; all of them have seven transmembrane spaces. These three different kinds of receptor induce analgesia after binding to any opioid compound; however, unlike the other two, the κ receptor does not induce much of a physical dependency. Opioid receptors are coupled with G proteins, specifically with a Gi protein. On a neuron, the interaction of an opioid ligand with its receptor induces an increase of potassium release from the intracellular space to the extracellular medium, which generates a shortening on the repolarization time and a decrease in the duration of the action potential. Opioids produce these effects thanks to the direct coupling that exists between the G protein and potassium channels, as well as with voltage-sensitive calcium channels. There are other known mechanisms related to opioids and their receptor, including interaction with G alpha protein (Dickenson, 1991).

Laudanum and other opioids have been used against chronic cough given the negative effects that this condition has on patients’ quality of live. Because of the lack of alternative treatments, patients usually turn to these kinds of drugs. In fact, destromethorphan was the last approved new treatment for cough, more than 50 years ago (Banken, 2008). This drug is a synthetic opioid widely used as a cough suppressant; however, a study shown only a 12% reduction of cough frequency in human patients, which coupled with the dangers of opiates uses and its limited uses in children, put in question the effectiveness if this drug. Historically, codeine has also been used for diminishing chronic cough, however, the efficacy is not certain either (Matthys, 1083). While opiates are still being recommended for cough suppression, few studies support their efficacy, and their use has been linked to concerns regarding their safety, including their tendency to generate physical dependency (Morice, 2007).

The mechanism of action of opiates against cough would be through sedative action, but there is not much clinical data supporting their effectiveness. Indeed, some studies have found that opiates only have a placebo effect against cough (Morice, 2007). One study used morphine in chronic cough patients to evaluate their response, and they found a trend towards improvement. However, the use of morphine brings with it concerns regarding its side effects and dependence risk, specially considering that cough is not a life threatening condition (Morice, 2007).

Another promising option against a cough that is disrupting a patient’s life is associated with P2X receptors. P2X3 are mainly expressed in vagal C-fibers from primary afferent nerves in the cranial and dorsal ganglia (Abdulqawi, 2013). Patients with chronic cough experiment an increase in symptoms after being exposed to some environmental conditions, including changes in temperature, smoke or perfumes. Besides, these patients also experience a higher cough response to stimulus like capsaicin, citric acid, hypertonic saline solution and mannitol. It has been suggested that the P2X3 receptor increases responsiveness to several stimuli by priming the afferent nerve terminal and/or by modulating central synapses (Ford, 2013). One study demonstrated a role for P2X3 receptors in the hyper responsiveness to cough-inducing signals. In this study, 34 patients with chronic cough were assigned to treatment with a substance called AF-219, a P2X3 agonist. After treatment, cough frequency was reduced by 75%. The most marked improvement was found in patients with the highest cough frequency. There were no serious adverse events detected on this study. This research supports the idea that P2X3 receptors have an important role in the molecular mechanisms involved with refractory chronic cough (Abdulqawi, 2015). This is of special importance when considering that patients with chronic cough have few efficient treatment options if the cough does not improve after addressing the underlying disorder that was believed to be responsible for the condition.

ATP can be released from tissues after inflammation. Since most pulmonary diseases have an inflammation component, it is to be expected that inflammation has a role in chronic cough (Boulet, 1994). Indeed, increased ATP concentrations have been found in the airway of patients with different respiratory diseases, including pulmonary disease, asthma and idiopathic pulmonary fibrosis (Riteau, 2010). This is an indication that ATP and P2X3 could be related to chronic cough, as well as other respiratory diseases. It still remains unknown whether ATP can by itself induce cough or if ATP receptors expression changes in patients suffering from chronic cough. Since a P2X3 receptor agonist generated improvement in chronic cough patients by reducing cough frequency, there are basis to suggest that P2X3 agonist could be used as a new class of antitussive (Abdulqawi, 2015).

While, as mentioned above, opiates are still being used for chronic cough treatment, new treatments and potential targets have emerged in the last few years. One of these treatments has been developed based on the observation that more than half of cough patients also have a condition named paradoxical vocal cough movement, also known as vocal chord dysfunction. Speech pathologists and physiotherapists have shown that voice therapy, which treats laryngeal symptoms and breathing disorders, is an effective therapeutic alternative against chronic cough (Murry, 2010). Unlike other classic treatment options, this approach recognizes the importance of the upper airway in the cough reflex (Ryan, 2014).

Chronic cough has traditionally been considered a product of an underlying condition, like asthma or gastro esophageal reflux. However, in more recent years it has been proposed that chronic cough is a primary condition, caused by hypersensitivity in the afferent nerve fibers. Chronic cough patients experience abnormal laryngeal sensations and an excessive response to cough stimulants. It has been speculated that this hypersensitivity could be a consequence of the up regulation of certain receptors, like TRP receptors. Indeed, chronic cough patients have shown to express up to five time the normal amount of TRPV1 containing nerves (Groneberg, 2004). The development of new drugs targeting these receptors could potentially have clinical benefits in the management of chronic cough.

New antitussive approaches are also possible if the possibility of blocking of the pathways of neurophatic inflammation is considered. The hyper activation of the afferent nerves, in addition to overexpression of certain receptors, could also be due to the action of neuroactive molecules, such as the Nerve Growth Factor, whose levels have been found to be increased in some chronic cough patients. It is also possible that normal afferent signals are increased by interacting with different types of neurons in the brain stem, leading to an amplification of the signal. Another possible explanation for hyper sensibility is that exposure to virus or allergens induce the sensitization of the center in which afferent nerves terminate. Whatever the mechanism, it is possible to consider that chronic cough could have a neuropathic component, consistent with the evidence that in chronic patients the airway undergoes a remodeling process, including chronic inflammatory infiltration, airway wall remodeling with an increase in vascularization, fibrosis and hyperplasia of goblet cells. There have also been reports of elevated levels of histamine, prostaglandin D2 and E2, TNF α, IL-8, and TGF β. If chronic cough is considered a neuropathic condition, then several treatment options can be developed: NMDA receptors and AMPA receptors interact with glutamate, a major excitatory neurotransmitter in the brain. If these receptors are activated in excess, there is excess in the production of reactive oxygen species. These signals have been associated with cough, and microinjections of a glutamate receptor antagonist inhibited coughing induced by citric acid in guinea pigs (Canning, 2011). GABA receptors are another potential target for cough treatment; baclofen, a GABA agonist, is a compound that that inhibits capsaicin-induced cough in guinea pigs. A GABA antagonist, on the other hand, inhibited the anti cough effect of baclofen in cats and guinea pigs. In humans, baclofen was shown to have an antitussive effect and it had a positive effect on chroni cough patients by improving cough frequency and severity (Chung, 2015).

Another possibility is to act in the initiation of the action potential, which happens after the opening of voltage-gated sodium channels. There are nine subtypes of sodium channels, three of which are the most expressed on the airway. It is possible that these channels are responsible for the hypersensitivity of the afferent nerves, and therefore they would have a role in the etiology of chronic cough. A selective sodium channel antagonist has shown to be able to block nodose sensory nerve activity (Muroi, 2011)

Similarly to targeting sodium channels, potassium ion channels maintain the resting potential of the nerve membranes, and some compounds that are able to open potassium ion channels have been proposed to have a role in the modulation of cough responses. NS1619, a compound that opens potassium channels, can inhibit citric acid-induced cough in conscious guinea pigs. Other compounds that open potassium channels, pinacidil and cromakalin, can also inhibit the cough reflex in guinea pigs. However, there have been no clinical trials testing their efficiency and safety in human patients (Dicpinigaitis, 2014).

In summary, other mechanisms could be developed in order to address the lack of treatment options for cough patients. For instance, Speech therapy has helped patients with chronic cough. TRPV1 activity is another possible target, given its role in the cough reflex. Other treatment options include ion channel blockers, which have shown promise in cough pharmacology (Reynolds, 2004). Given the necessity of stimulating receptors in order to induce the cough response, and the possibility that at least in some chronic cough patients these receptors are hyper sensible, a possible strategy could be to desensitize mechanoreceptors. Also, it is possible that cough is associated to an overexpression of these receptors; research shows that TRPV1 are indeed overexpressed in chronic cough patients (Groneberg, 2004), which would explain a higher than normal response. Also, GABA and NMDA receptors are attractive therapeutic targets. These pathways present novel treatment options for cough, and they could be used together with drugs targeting the P2X3 receptor.

Considering the current research regarding cough mechanisms and chronic cough pathways, the hypothesis raised in this work is that ATP plays a role in the activation of vagal afferent nerves and cough, and therefore the P2X3 is a potential pharmaceutical target. The aim of this work is to determine the role of ATP in activating vagal afferent nerve and cough and the possibility of using the P2X3 receptor as a pharmaceutical target.

Discussion

Cough is the most common symptom for which patients consult with a medical doctor (Conford, 1998). This reflex is a normal defensive mechanism that protects the airway from foreign objects. In healthy individuals, cough prevents particulate objects to enter the lower airway, including food and other irritants. Cough is also present in individual that are fighting an immunological disease, protecting the airway from pathogen and microorganisms. Cough is also associated with respiratory diseases, including asthma, cystic fibrosis and pulmonary obstructive disease. While in all these cases cough is a normal and expected response, it can also be the cause of medical problems if it becomes excessive (Widdicombe, 1995). Chronic cough, for instance, can be associated with physical and psychological symptoms that lead to a deterioration of the patient’s quality of life (Chung, 2008).

One of the main concerns for patients suffering form chronic cough is the lack of efficient treatment options. Historically, chronic cough has been treated with opioid agonists, including the opium extract laudanum (Marketos, 1986). There are several issues with treating chronic cough with opiates. One of the main problems with the use of opiates drugs is that they are highly addictive, being prone to induce patients to develop physical dependency. Another concern is their side effects, which include nausea and vomiting, bloating, constipation, potential liver and brain damage, besides the development of physical tolerance (Chau, 2008). Besides, there is no robust evidence proving that opiates are a good option for chronic cough patients. Morphine has been tested as an option for the treatment of chronic cough, and while it showed to produce improvements, the risks associated with morphine use for a non-life threatening condition limit its use in a clinical setting (Morice, 2007). One of the drugs approved for chronic cough is dextromethorphan, a synthetic opioid agonist. However, a study puts its efficacy at the same level as the placebo (Eccles, 2002). Importantly, dextromethorphan is the last approved drug for chronic cough treatment. All of the above argues in favor of the need of producing novel treatment options for chronic cough patients and for individuals suffering from cough-related disorders, as well as the need to study treatment options that do not involve the use of opiates.

Equally to all reflex arcs, the cough reflex involves afferent nerves, a cough center, efferent fibers and effector organs and muscles. The vagal nerve is the main nerve innervating this whole arc (Chang, 2006). There are three main types of nerve fibers in this arc: rapidly adapting fibers, slowly adapting fibers and C-fibers. These fibers have, on their terminals, different subtypes of receptors that can sense a signal and then transform it into an electrical impulse that travels through the nerve to the effector organs, generating the cough response. These receptors can be mechanoreceptors or chemoreceptors, responding to different types of stimulus. Mechanosensors are activated by one or more mechanical stimuli, including lung inflation, bronchospasm and touch; while chemosensors can be activated by several chemical compounds, including capsaicin, bradykinin, adenosine and prostaglandin E2 (Mazzone, 2005).

The receptors found on the fibers that initiate the cough impulse are an attractive therapeutic option. There are different types of receptors, inducing different types of signal transduction. One type of receptor that could potentially be used as a therapeutic target are pyrimidine receptors (P2). There are two types of P2 receptors: P2X and P2Y. P2X receptors consist on ion channels that are gated by ATP, while P2Y receptors are coupled with G proteins (G protein coupled receptors) (Ralevic, 1998). In the airway there is abundance of P2X receptors, particular P2X3, making this receptor an attractive potential focus for new drug development (Abdulqawi, 2015).

Several evidences potentially link P2X3 receptor to cough and chronic cough. ATP is present in the airway, and its presence has been associated with inflammatory process. Inflammation occurs in several diseases that have cough as a symptom, suggesting that ATP has a role in the cough reflex (Boulet, 1994). Also, TRP receptors, which have also been linked to the cough reflex, have been associated to P2X receptors. After TRP receptors are stimulated, they release ATP into the airway. This ATP acts on P2X receptors, possibly inducing a cough reflex response. It is possible that TRP stimulation increases the sensibility of the nerves that afterward will induce a cough response (Bonvini, 2015).

There are antagonists available for the P2X3 receptor (Ford, 2012), which could be used in a pharmaceutical setting in order to alleviate the symptoms of cough patients. One study found that the use of a P2X3 receptor antagonist reduced the frequency of cough in chronic cough patients . Therefore, it is possible to hypothesize that ATP and P2X3 receptor have a role in the development of reparatory diseases in general and of chronic cough disease in particular, and that P2X3 receptor antagonists could be a potential pharmaceutical treatment option (Abdulqawi, 2015).

It is also important to consider other mechanism that could have a role in chronic cough, since targeting them could offer new and attractive treatment alternatives. Besides, the understanding of the mechanisms governing chronic cough remain to be completely elucidated; for instance, it remains unclear if P2X3 expression is altered in the airway of chronic cough patients.

Other options have been developed and are currently being investigated in order to offer better options to chronic cough patients. Speech therapy, for example, has been able to become effective therapeutic alternative against chronic cough (Murry, 2010).

While chronic cough has traditionally been considered a product of an underlying condition, the current paradigm is changing, and now chronic cough is starting to be viewed as a primary condition characterized by hypersensitivity in the afferent nerve fibers. This hypersensitivity to cough stimulants could be the result of several conditions, one of which is the overexpression of the receptors found on the afferent vagal nerves. TRPV1 receptors are, in fact, increased in the airway of chronic cough patients compared to the normal airway, and the development of TRPV1 antagonist as potential treatment for cough is currently being studied. (Groneberg, 2004). A new approach considers the possibility that chronic cough is related to neurophatic pathways. Neuroactive molecules, including the Nerve Growth Factor, have been implicated in cough development. This possibility offers a whole new array of options for chronic cough treatment, including the development of NMDA antagonists and GABA agonists. Studies with human patients have shown some positive results (Chung, 2015). Another important target could be the initiation of the action potential, necessary for the cough signal to travel to the effector organs and muscles. Sodium channels and potassium channels are necessary for the initiation of an action potential, and therefore blocking them could have a positive effect on chronic cough patients (Muroi, 2011).

An efficient and effective therapy could target more than one of the above-mentioned mechanisms. For instance, targeting the receptors responsible for the hyper sensibility of the afferent nerves belonging to the cough reflex could be coupled with the targeting of sodium and potassium channels responsible for the production of an action potential. Further studies need to be done in order to gain understanding of the molecular mechanisms that govern the cough reflex, which would in turn offer new and more attractive targets. P2X2 is an appealing therapeutic target, and it could offer benefits to chronic cough patients, especially in combination with other therapies. This is of special importance considering the lack of safe and efficient therapeutic options available today.

References

Abdulqawi, R., Dockry, R., Holt, K., Layton, G., McCarthy, B.G., Ford, A.P. and Smith, J.A., 2015. P2X3 receptor antagonist (AF-219) in refractory chronic cough: a randomised, double-blind, placebo-controlled phase 2 study.The Lancet, 385(9974), pp.1198-1205.

Abdulqawi, R., Dockry, R., Holt, K., Woodcock, A., Layton, G., McCarthy, B., Ford, A. and Jaclyn, S., 2013. Inhibition of ATP-gated P2X3 channels by AF-219: An effective anti-tussive mechanism in chronic cough. European Respiratory Journal, 42(Suppl 57), p.1965.

Adcock, J.J., 2009. TRPV1 receptors in sensitisation of cough and pain reflexes. Pulmonary pharmacology & therapeutics, 22(2), pp.65-70.

Belvisi, M.G., Bonvini, S.J., Grace, M.S., Ching, Y.M., Dubuis, E., Adcock, J.J. and Birrell, M.A., 2013. Activation of airway sensory nerves: a key role for the TRPV4 channel. In D19. WHAT’S GOING ON? MECHANISMS OF REMODELING AND HYPERRESPONSIVENESS (pp. A5265-A5265). American Thoracic Society.

Bonvini, S., Birrell, M., Grace, M., Maher, S., Adcock, J., Wortley, M., Dubuis, E., Ching, Y.M., Ford, A., Shala, F. and Miralpiex, M., 2015. TRPV4 and activation of airway sensory nerves: the role of ATP.

Boulet, L.P., Milot, J.O.A.N.N.E., Boutet, M.I.C.H.E.L., St Georges, F. and Laviolette, M.I.C.H.E.L., 1994. Airway inflammation in nonasthmatic subjects with chronic cough. American journal of respiratory and critical care medicine,149(2), pp.482-489.

Burnstock, G., Brouns, I., Adriaensen, D. and Timmermans, J.P., 2012. Purinergic signaling in the airways. Pharmacological reviews, 64(4), pp.834-868.

Canning BJ, Chang AB, Bolser DC, Smith JA, Mazzone SB, McGarvey L., 2014. Anatomy and Neurophysiology of Cough: CHEST Guideline and Expert Panel Report. Chest, 146(6), pp.1633–48.

Canning, B.J., Mazzone, S.B., Meeker, S.N., Mori, N., Reynolds, S.M. and Undem, B.J., 2004. Identification of the tracheal and laryngeal afferent neurones mediating cough in anaesthetized guinea pigs. The Journal of physiology, 557(2), pp.543-558.

Canning, B.J. and Mori, N., 2011. Encoding of the cough reflex in anesthetized guinea pigs. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 300(2), pp.R369-R377.

Chang, A.B., 2006. The physiology of cough. Paediatric Respiratory Reviews, 7(1), pp.2–8.

Chau, D.L., Walker, V., Pai, L. and Cho, L.M., 2008. Opiates and elderly: use and side effects. Clinical interventions in aging, 3(2), p.273.

Chung KF, Pavord ID. 2008. Prevalence, pathogenesis, and causes of chronic cough. The Lancet pp.1364–74.

Chung, K.F., 2015. NMDA and GABA receptors as potential targets in cough hypersensitivity syndrome. Current opinion in pharmacology, 22, pp.29-36.

Clapham, D.E., 2003. TRP channels as cellular sensors. Nature, 426(6966), pp.517-524.

Cornford, C.S., 1998. Why patients consult when they cough: a comparison of consulting and non-consulting patients. Br J Gen Pract, 48(436), pp.1751-1754.

Dickenson, A.H., 1991. Mechanisms of the analgesic actions of opiates and opioids. British medical bulletin, 47(3), pp.690-702.

Dicpinigaitis, P.V., Morice, A.H., Birring, S.S., McGarvey, L., Smith, J.A., Canning, B.J. and Page, C.P., 2014. Antitussive drugs—past, present, and future. Pharmacological reviews, 66(2), pp.468-512.Edwards, F.A., Gibb, A.J. and Colquhoun, D., 1992. ATP receptor-mediated synaptic currents in the central nervous system.

Eccles, R., 2002. The powerful placebo in cough studies?. Pulmonary pharmacology & therapeutics, 15(3), pp.303-308.

Ford, A.P., 2012. In pursuit of P2X3 antagonists: novel therapeutics for chronic pain and afferent sensitization. Purinergic signalling, 8(1), pp.3-26.

Ford, A.P. and Undem, B.J., 2013. The therapeutic promise of ATP antagonism at P2X3 receptors in respiratory and urological disorders.

Ford, P.A., Barnes, P.J. and Usmani, O.S., 2007. Chronic cough and Holmes-Adie syndrome. The Lancet, 369(9558), p.342.

Groneberg, D.A., Niimi, A., Dinh, Q.T., Cosio, B., Hew, M., Fischer, A. and Chung, K.F., 2004. Increased expression of transient receptor potential vanilloid-1 in airway nerves of chronic cough. American journal of respiratory and critical care medicine, 170(12), pp.1276-1280.

Idzko, M., Hammad, H., Van Nimwegen, M., Kool, M., Willart, M.A., Muskens, F., Hoogsteden, H.C., Luttmann, W., Ferrari, D., Di Virgilio, F. and Virchow, J.C., 2007. Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells. Nature medicine, 13(8), pp.913-919.

Irwin, R.S., 2006. Complications of Cough. CHEST Journal, 129(1_suppl), p.54S.

Lee KK, Birring SS. 2012. Cough. Medicine (Baltimore), 40(4), pp. 173–6.

Lin, Y.S., Hsu, C.C., Bien, M.Y., Hsu, H.C., Weng, H.T. and Kou, Y.R., 2010. Activations of TRPA1 and P2X receptors are important in ROS-mediated stimulation of capsaicin-sensitive lung vagal afferents by cigarette smoke in rats. Journal of Applied Physiology, 108(5), pp.1293-1303.

Marketos, S.G. and Eftychiades, A.C., 1986. Historical Perspectives: Bronchial Asthma According to Byzantine Medicine. Journal of Asthma,23(3), pp.149-155.

Matsumoto, H., Tabuena, R.P., Niimi, A., Inoue, H., Ito, I., Yamaguchi, M., Otsuka, K., Takeda, T., Oguma, T., Nakaji, H. and Tajiri, T., 2012. Cough triggers and their pathophysiology in patients with prolonged or chronic cough. Allergology International, 61(1), pp.123-132.

Matthys, H., Bleicher, B. and Bleicher, U., 1983. Dextromethorphan and codeine: objective assessment of antitussive activity in patients with chronic cough. Journal of International Medical Research, 11(2), pp.92-100.

Mazzone, S.B., 2005. An overview of the sensory receptors regulating cough.Cough, 1(1), p.2.

Morice, A.H., Menon, M.S., Mulrennan, S.A., Everett, C.F., Wright, C., Jackson, J. and Thompson, R., 2007. Opiate therapy in chronic cough.American journal of respiratory and critical care medicine, 175(4), pp.312-315.

Muroi, Y., Ru, F., Kollarik, M., Canning, B.J., Hughes, S.A., Walsh, S., Sigg, M., Carr, M.J. and Undem, B.J., 2011. Selective silencing of NaV1. 7 decreases excitability and conduction in vagal sensory neurons. The Journal of physiology, 589(23), pp.5663-5676.

Murry, T. and Sapienza, C., 2010. The role of voice therapy in the management of paradoxical vocal fold motion, chronic cough, and laryngospasm. Otolaryngologic Clinics of North America, 43(1), pp.73-83.

Narula, M., McGovern, A.E., Yang, S.K., Farrell, M.J. and Mazzone, S.B., 2014. Afferent neural pathways mediating cough in animals and humans. Journal of thoracic disease, 6(7), pp.S712-S719.

Niimi A, Matsumoto H, Ueda T, Takemura M, Suzuki K, Tanaka E, et al, 2003. Impaired cough reflex in patients with recurrent pneumonia. Thorax, 58, pp 152–3.

Nishino, T. et al., 1988. Respiratory reflex responses to stimulation of tracheal mucosa in enflurane-anesthetized humans. Journal of Applied Physiology, 65(3), pp.1069–1074.

Ralevic, V. and Burnstock, G., 1998. Receptors for purines and pyrimidines.Pharmacological reviews, 50(3), pp.413-492.

Reynolds, S.M., Mackenzie, A.J., Spina, D. and Page, C.P., 2004. The pharmacology of cough. Trends in pharmacological sciences, 25(11), pp.569-576.

Ryan, N.M. and Gibson, P.G., 2014. Recent additions in the treatment of cough. Journal of thoracic disease, 6(Suppl 7), p.S739.

Riteau, N., Gasse, P., Fauconnier, L., Gombault, A., Couegnat, M., Fick, L., Kanellopoulos, J., Quesniaux, V.F., Marchand-Adam, S., Crestani, B. and Ryffel, B., 2010. Extracellular ATP is a danger signal activating P2X7 receptor in lung inflammation and fibrosis. American journal of respiratory and critical care medicine, 182(6), pp.774-783.

Sant’Ambrogio, G. and Widdicombe, J., 2001. Reflexes from airway rapidly adapting receptors. Respiration physiology, 125(1), pp.33-45.

Sant’Ambrogio, G., Remmers, J.E., De Groot, W.J., Callas, G. and Mortola, J.P., 1978. Localization of rapidly adapting receptors in the trachea and main stem bronchus of the dog. Respiration physiology, 33(3), pp.359-366.

Sant’Ambrogio, G., Sant’Ambrogio, F.B. and Davies, A., 1983. Airway receptors in cough. Bulletin europeen de physiopathologie respiratoire, 20(1), pp.43-47.

Tsai, T.L., Chang, S.Y., Ho, C.Y. and Kou, Y.R., 2009. Role of ATP in the ROS-mediated laryngeal airway hyperreactivity induced by laryngeal acid-pepsin insult in anesthetized rats. Journal of Applied Physiology, 106(5), pp.1584-1592.

Undem, B.J., Carr, M.J. and Kollarik, M., 2002. Physiology and plasticity of putative cough fibres in the Guinea pig. Pulmonary pharmacology & therapeutics, 15(3), pp.193-198.

Watanabe, H., Vriens, J., Prenen, J., Droogmans, G., Voets, T. and Nilius, B., 2003. Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels. Nature, 424(6947), pp.434-438.

Widdicombe JG, 1995. Neurophysiology of the cough reflex. European Respiratory Journal p.1193–202.