BackgroundCough is the most frequent reason for consultation with a family doctor, or with a general or respiratory physician. Treatment options are limited and a recent meta-analysis concluded that over-the-counter remedies are ineffective and there is increasing concern about their use in children. Endogenous inflammatory mediators such as prostaglandin E2 (PGE2) and bradykinin (BK), which are often elevated in respiratory disease states, are also known to cause cough by stimulating airway sensory nerves. However, how this occurs is not understood.MethodsWe hypothesised that the transient receptor potential (TRP) channels, TRPA1 and TRPV1, may have a role as ‘common effectors’ of tussive responses to these agents. We have employed a range of in vitro imaging and isolated tissue assays in human, murine and guinea pig tissue and an in vivo cough model to support this hypothesis.ResultsUsing calcium imaging we demonstrated that PGE2 and BK activated isolated guinea pig sensory ganglia and evoked depolarisation (activation) of vagal sensory nerves, which was inhibited by TRPA1 and TRPV1 blockers (JNJ17203212 and HC-030031). These data were confirmed in vagal sensory nerves from TRPA1 and TRPV1 gene deleted mice. TRPV1 and TRPA1 blockers partially inhibited the tussive response to PGE2 and BK with a complete inhibition obtained in the presence of both antagonists together in a guinea pig conscious cough model.ConclusionThis study identifies TRPA1 and TRPV1 channels as key regulators of tussive responses elicited by endogenous and exogenous agents, making them the most promising targets currently identified in the development of anti-tussive drugs.

Over the last few decades, there has been an explosion of scientific publications reporting the many and varied roles of transient receptor potential (TRP) ion channels in physiological and pathological systems throughout the body. The aim of this review is to summarize the existing literature on the role of TRP channels in the lungs and discuss what is known about their function under normal and diseased conditions. The review will focus mainly on the pathogenesis and symptoms of asthma and chronic obstructive pulmonary disease and the role of four members of the TRP family: TRPA1, TRPV1, TRPV4 and TRPM8. We hope that the article will help the reader understand the role of TRP channels in the normal airway and how their function may be changed in the context of respiratory disease. LINKED ARTICLESThis article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-10 Abbreviation 4αPDD, 4α-phorbol-12,13-didecanoate; AHR, airway hyper-responsiveness; CGRP, calcitonin gene-related peptide; COPD, chronic obstructive pulmonary disease; DRG, dorsal root ganglia; HASM, human airway smooth muscle; LAR, late asthmatic response: OTC, over-the-counter; PAR, protease-activated receptors; SNP, single nucleotide polymorphism; TRP, transient receptor potential Introduction to transient receptor potential (TRP) ion channelsTRP channels were discovered in the eye of the Drosophila melanogaster fly, and named for their transient response to bright light (Montell and Rubin, 1989). Several homologues have since been identified that have a well conserved 'TRP domain' consisting of 23-25 amino acids. There are 28 mammalian TRP subunits, categorized in to six related protein subfamilies, based on sequence homology (Clapham, 2003). TRP ion channels are widely expressed throughout the body, and can respond to a remarkable diversity of intracellular and extracellular stimuli. This capacity to be activated by seemingly disparate mechanisms has led to the perception of TRP channels as multiple signal integrators. The TRP channel superfamily comprises a group of cation-selective proteins, which display a general preference for calcium ions. TRPs exhibit six transmembrane-spanning domains with the channel pore located between transmembrane domains 5 and 6, intracellular C and N termini, and varying degrees of ankyrin repeats (Caterina et al., 1997;Ramsey et al., 2006). Current evidence suggests that active TRP channels are formed by four subunits, and could assemble as homo-or hetero-tetramers (Latorre et al., 2009). For more in-depth information on TRP channels, we suggest consulting some of the excellent reviews including Clapham, 2003;Nilius et al. 2005;Bessac & Jordt, 2008;Preti et al. 2012. Aim of the reviewThe aim of this review is to describe the TRP channels, which have been explored in more depth regarding their role in physiological and pathological mechanisms in the airways (TRPA1, TRPV1, TRPV4 and TRPM8; receptor nomenclature ...

BackgroundSensory nerves innervating the airways play an important role in regulating various cardiopulmonary functions, maintaining homeostasis under healthy conditions and contributing to pathophysiology in disease states. Hypo-osmotic solutions elicit sensory reflexes, including cough, and are a potent stimulus for airway narrowing in asthmatic patients, but the mechanisms involved are not known. Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is widely expressed in the respiratory tract, but its role as a peripheral nociceptor has not been explored.ObjectiveWe hypothesized that TRPV4 is expressed on airway afferents and is a key osmosensor initiating reflex events in the lung.MethodsWe used guinea pig primary cells, tissue bioassay, in vivo electrophysiology, and a guinea pig conscious cough model to investigate a role for TRPV4 in mediating sensory nerve activation in vagal afferents and the possible downstream signaling mechanisms. Human vagus nerve was used to confirm key observations in animal tissues.ResultsHere we show TRPV4-induced activation of guinea pig airway–specific primary nodose ganglion cells. TRPV4 ligands and hypo-osmotic solutions caused depolarization of murine, guinea pig, and human vagus and firing of Aδ-fibers (not C-fibers), which was inhibited by TRPV4 and P2X3 receptor antagonists. Both antagonists blocked TRPV4-induced cough.ConclusionThis study identifies the TRPV4-ATP-P2X3 interaction as a key osmosensing pathway involved in airway sensory nerve reflexes. The absence of TRPV4-ATP–mediated effects on C-fibers indicates a distinct neurobiology for this ion channel and implicates TRPV4 as a novel therapeutic target for neuronal hyperresponsiveness in the airways and symptoms, such as cough.

Rationale: Most airway diseases, including chronic obstructive pulmonary disease (COPD), are associated with excessive coughing. The extent to which this may be a consequence of increased activation of vagal afferents by pathology in the airways (e.g., inflammatory mediators, excessive mucus) or an altered neuronal phenotype is unknown. Understanding whether respiratory diseases are associated with dysfunction of airway sensory nerves has the potential to identify novel therapeutic targets.Objectives: To assess the changes in cough responses to a range of inhaled irritants in COPD and model these in animals to investigate the underlying mechanisms.Methods: Cough responses to inhaled stimuli in patients with COPD, healthy smokers, refractory chronic cough, asthma, and healthy volunteers were assessed and compared with vagus/airway nerve and cough responses in a cigarette smoke (CS) exposure guinea pig model. Measurements and Main Results:Patients with COPD had heightened cough responses to capsaicin but reduced responses to prostaglandin E 2 compared with healthy volunteers. Furthermore, the different patient groups all exhibited different patterns of modulation of cough responses. Consistent with these findings, capsaicin caused a greater number of coughs in CS-exposed guinea pigs than in control animals; similar increased responses were observed in ex vivo vagus nerve and neuron cell bodies in the vagal ganglia. However, responses to prostaglandin E 2 were decreased by CS exposure.Conclusions: CS exposure is capable of inducing responses consistent with phenotypic switching in airway sensory nerves comparable with the cough responses observed in patients with COPD. Moreover, the differing profiles of cough responses support the concept of disease-specific neurophenotypes in airway disease.Clinical trial registered with www.clinicaltrials.gov (NCT 01297790).

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BackgroundChronic Obstructive Pulmonary Disease (COPD) is an inflammatory airway disease often associated with cigarette smoke (CS) exposure. The disease is increasing in global prevalence and there is no effective therapy. A major step forward would be to understand the disease pathogenesis. The ATP-P2X7 pathway plays a dominant role in murine models of CS induced airway inflammation, and markers of activation of this axis are upregulated in patients with COPD. This strongly suggests that the axis could be important in the pathogenesis of COPD. The aim of this study was to perform a detailed characterisation of the signalling pathway components involved in the CS-driven, P2X7 dependent airway inflammation.MethodsWe used a murine model system, bioassays and a range of genetically modified mice to better understand this complex signalling pathway.ResultsThe inflammasome-associated proteins NALP3 and ASC, but not IPAF and AIM2, are required for CS-induced IL-1β/IL-18 release, but not IL-1α. This was associated with a partial decrease in lung tissue caspase 1 activity and BALF neutrophilia. Mice missing caspase 1/11 or caspase 11 had markedly attenuated levels of all three cytokines and neutrophilia. Finally the mechanism by which these inflammatory proteins are involved in the CS-induced neutrophilia appeared to be via the induction of proteins involved in neutrophil transmigration e.g. E-Selectin.ConclusionThis data indicates a key role for the P2X7-NALP3/ASC-caspase1/11-IL-1β/IL-18 axis in CS induced airway inflammation, highlighting this pathway as a possible therapeutic target for the treatment of COPD.

BackgroundDiesel exhaust particles (DEPs) are a major component of particulate matter in Europe's largest cities, and epidemiologic evidence links exposure with respiratory symptoms and asthma exacerbations. Respiratory reflexes are responsible for symptoms and are regulated by vagal afferent nerves, which innervate the airway. It is not known how DEP exposure activates airway afferents to elicit symptoms, such as cough and bronchospasm.ObjectiveWe sought to identify the mechanisms involved in activation of airway sensory afferents by DEPs.MethodsIn this study we use in vitro and in vivo electrophysiologic techniques, including a unique model that assesses depolarization (a marker of sensory nerve activation) of human vagus.ResultsWe demonstrate a direct interaction between DEP and airway C-fiber afferents. In anesthetized guinea pigs intratracheal administration of DEPs activated airway C-fibers. The organic extract (DEP-OE) and not the cleaned particles evoked depolarization of guinea pig and human vagus, and this was inhibited by a transient receptor potential ankyrin-1 antagonist and the antioxidant N-acetyl cysteine. Polycyclic aromatic hydrocarbons, major constituents of DEPs, were implicated in this process through activation of the aryl hydrocarbon receptor and subsequent mitochondrial reactive oxygen species production, which is known to activate transient receptor potential ankyrin-1 on nociceptive C-fibers.ConclusionsThis study provides the first mechanistic insights into how exposure to urban air pollution leads to activation of guinea pig and human sensory nerves, which are responsible for respiratory symptoms. Mechanistic information will enable the development of appropriate therapeutic interventions and mitigation strategies for those susceptible subjects who are most at risk.

BackgroundRecent studies have suggested that the long-acting muscarinic receptor antagonist tiotropium, a drug widely prescribed for its bronchodilator activity in patients with chronic obstructive pulmonary disease and asthma, improves symptoms and attenuates cough in preclinical and clinical tussive agent challenge studies. The mechanism by which tiotropium modifies tussive responses is not clear, but an inhibition of vagal tone and a consequent reduction in mucus production from submucosal glands and bronchodilation have been proposed.ObjectiveThe aim of this study was to investigate whether tiotropium can directly modulate airway sensory nerve activity and thereby the cough reflex.MethodsWe used a conscious cough model in guinea pigs, isolated vagal sensory nerve and isolated airway neuron tissue– and cell-based assays, and in vivo single-fiber recording electrophysiologic techniques.ResultsInhaled tiotropium blocked cough and single C-fiber firing in the guinea pig to the transient receptor potential (TRP) V1 agonist capsaicin, a clinically relevant tussive stimulant. Tiotropium and ipratropium, a structurally similar muscarinic antagonist, inhibited capsaicin responses in isolated guinea pig vagal tissue, but glycopyrrolate and atropine did not. Tiotropium failed to modulate other TRP channel–mediated responses. Complementary data were generated in airway-specific primary ganglion neurons, demonstrating that tiotropium inhibited capsaicin-induced, but not TRPA1-induced, calcium movement and voltage changes.ConclusionFor the first time, we have shown that tiotropium inhibits neuronal TRPV1-mediated effects through a mechanism unrelated to its anticholinergic activity. We speculate that some of the clinical benefit associated with taking tiotropium (eg, in symptom control) could be explained through this proposed mechanism of action.