Novel airway smooth muscle-mast cell interactions and a role for the TRPV4-ATP axis in non-atopic asthma.

Mast cell-airway smooth muscle (ASM) interactions play a major role in the immunoglobulin (Ig)E- dependent bronchoconstriction seen in asthma but less is known about IgE-independent mechanisms of mast cell activation. Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) activation causes contraction of human ASM via the release of cysteinyl leukotrienes (cysLTs) but the mechanism is unknown. The objective of the present study was to investigate a role for IgE-independent, mast cell-ASM interaction in TRPV4-induced bronchospasm.Bronchoconstriction was measured in anaesthetised guinea pigs and contraction of human and guinea-pig airway tissue assessed using isometric tension measurements. Increases in intracellular [Ca2+] were imaged using the Ca2+-sensitive dye FURA2, and time-lapse ptychography was utilised as a surrogate for contraction of ASM cells.The TRPV4 agonist GSK1016790A caused contraction in vivo in the guinea pig, and in human and guinea-pig tracheal tissue, which was inhibited by the TRPV4 antagonist GSK2193874. GSK1016790A increased [Ca2+]i and released ATP in human ASM cells without causing contraction. TRPV4 and ATP evoked contraction in isolated tracheal tissue but co-culture experiments indicated a requirement for human lung mast cells. Expression profiling and pharmacological studies demonstrated that mast cell activation was dependent upon ATP activating the P2X4 receptor. Trypsin was shown to evoke contraction of tracheal tissue via activation of PAR-2-TRPV4-ATP-cysLT axis indicating the potential disease relevance of this signalling pathway.TRPV4 activation increases [Ca2+]i and releases ATP from ASM cells triggering P2X4-dependent release of cysLTs from mast cells resulting in ASM contraction. This study delineates a novel mast cell-ASM interaction and TRPV4 as a driver of IgE-independent mast cell-dependent bronchospasm.

Mechanistic link between diesel exhaust particles and respiratory reflexes.

BACKGROUND: Diesel 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. OBJECTIVE: We sought to identify the mechanisms involved in activation of airway sensory afferents by DEPs. METHODS: In 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. RESULTS: We 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. CONCLUSIONS: This 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.

Targeting fatty acid amide hydrolase as a therapeutic strategy for antitussive therapy.

Cough is the most common reason to visit a primary care physician, yet it remains an unmet medical need. Fatty acid amide hydrolase (FAAH) is an enzyme that breaks down endocannabinoids, and inhibition of FAAH produces analgesic and anti-inflammatory effects. Cannabinoids inhibit vagal sensory nerve activation and the cough reflex, so it was hypothesised that FAAH inhibition would produce antitussive activity via elevation of endocannabinoids.Primary vagal ganglia neurons, tissue bioassay, in vivo electrophysiology and a conscious guinea pig cough model were utilised to investigate a role for fatty acid amides in modulating sensory nerve activation in vagal afferents.FAAH inhibition produced antitussive activity in guinea pigs with concomitant plasma elevation of the fatty acid amides N-arachidonoylethanolamide (anandamide), palmitoylethanolamide, N-oleoylethanolamide and linoleoylethanolamide. Palmitoylethanolamide inhibited tussive stimulus-induced activation of guinea pig airway innervating vagal ganglia neurons, depolarisation of guinea pig and human vagus, and firing of C-fibre afferents. These effects were mediated via a cannabinoid CB2/Gi/o-coupled pathway and activation of protein phosphatase 2A, resulting in increased calcium sensitivity of calcium-activated potassium channels.These findings identify FAAH inhibition as a target for the development of novel, antitussive agents without the undesirable side-effects of direct cannabinoid receptor agonists.

CD4⁺ and CD8⁺ T cells play a central role in a HDM driven model of allergic asthma.

BACKGROUND: The incidence of asthma is increasing at an alarming rate and while the current available therapies are effective in the majority of patients they fail to adequately control symptoms at the more severe end of the disease spectrum. In the search to understand disease pathogenesis and find effective therapies animal models are often employed. As exposure to house dust mite (HDM) has a causative link, it is thought of as the allergen of choice for modelling asthma. The objective was to develop a HDM driven model of asthmatic sensitisation and characterise the role of key allergic effector cells/mediators. METHODS: Mice were sensitised with low doses of HDM and then subsequently challenged. Cellular inflammation, IgE and airway responsiveness (AHR) was assessed in wild type mice or CD4(+)/CD8(+) T cells, B cells or IgE knock out mice. RESULTS: Only those mice sensitised with HDM responded to subsequent low dose topical challenge. Similar to the classical ovalbumin model, there was no requirement for systemic alum sensitisation. Characterisation of the role of effector cells demonstrated that the allergic cellular inflammation and AHR was dependent on CD4(+) and CD8(+) T cells but not B cells or IgE. Finally, we show that this model, unlike the classic OVA model, appears to be resistant to developing tolerance. CONCLUSIONS: This CD4(+)/CD8(+) T cell dependent, HDM driven model of allergic asthma exhibits key features of asthma. Furthermore, we suggest that the ability to repeat challenge with HDM means this model is amenable to studies exploring the effect of therapeutic dosing in chronic, established disease.

Neurophenotypes in Airway Diseases. Insights from Translational Cough Studies.

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 E2 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 E2 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).

Transient receptor potential cation channel, subfamily V, member 4 and airway sensory afferent activation: Role of adenosine triphosphate.

BACKGROUND: Sensory 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. OBJECTIVE: We hypothesized that TRPV4 is expressed on airway afferents and is a key osmosensor initiating reflex events in the lung. METHODS: We 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. RESULTS: Here 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. CONCLUSION: This 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.

Hyperpolarized 83Kr magnetic resonance imaging of alveolar degradation in a rat model of emphysema.

Hyperpolarized (83)Kr surface quadrupolar relaxation (SQUARE) generates MRI contrast that was previously shown to correlate with surface-to-volume ratios in porous model surface systems. The underlying physics of SQUARE contrast is conceptually different from any other current MRI methodology as the method uses the nuclear electric properties of the spin I = 9/2 isotope (83)Kr. To explore the usage of this non-radioactive isotope for pulmonary pathophysiology, MRI SQUARE contrast was acquired in excised rat lungs obtained from an elastase-induced model of emphysema. A significant (83)Kr T1 relaxation time increase in the SQUARE contrast was found in the elastase-treated lungs compared with the baseline data from control lungs. The SQUARE contrast suggests a reduction in pulmonary surface-to-volume ratio in the emphysema model that was validated by histology. The finding supports usage of (83)Kr SQUARE as a new biomarker for surface-to-volume ratio changes in emphysema.

The role of adenylyl cyclase isoform 6 in ß-adrenoceptor signalling in murine airways.

BACKGROUND AND PURPOSE: Adenylyl cyclase (AC) is a key signalling enzyme for many GPCRs and catalyses the conversion of ATP to cAMP which, in turn, is a crucial determinant of many biological responses. ß-Adrenoceptor agonists are prescribed as bronchodilators for asthma and chronic obstructive pulmonary disease, and it is commonly assumed that they elicit their actions via AC-dependent production of cAMP. However, empirical evidence in support of this is lacking and the exact mechanism by which these drugs acts remains elusive. This is partly due to the existence of at least 10 different isoforms of AC and the absence of any truly selective pharmacological inhibitors. Here, we have used genetically modified mice and model systems to establish the role of AC isoforms in the airway responses to ß-adrenoceptor agonists. EXPERIMENTAL APPROACH: Receptors mediating responses to ß-adrenoceptor agonists in airway smooth muscle (ASM) and sensory nerve were identified in isolated tissue systems. Expression of mRNA for the AC isoforms in ASM and neurones was determined by qPCR. Functional responses were assessed in AC isoform KO mice and wild-type controls. KEY RESULTS: Airway and vagal tissue expressed mRNA for various isoforms of AC. AC6 was the most prominent isoform. Responses to ß-adrenoceptor agonists in tissues from AC6 KO mice were virtually abolished. CONCLUSIONS AND IMPLICATIONS: AC6 played a critical role in relaxation of ASM to ß1 -adrenoceptor agonists and in modulation of sensory nerves by ß1-3 -adrenoceptor agonists. These results further unravel the signalling pathway of this extensively prescribed class of medicine.

Prostaglandin D2 and the role of the DP1, DP2 and TP receptors in the control of airway reflex events.

Prostaglandin D2 (PGD2) causes cough and levels are increased in asthma suggesting that it may contribute to symptoms. Although the prostaglandin D2 receptor 2 (DP2) is a target for numerous drug discovery programmes little is known about the actions of PGD2 on sensory nerves and cough. We used human and guinea pig bioassays, in vivo electrophysiology and a guinea pig conscious cough model to assess the effect of prostaglandin D2 receptor (DP1), DP2 and thromboxane receptor antagonism on PGD2 responses. PGD2 caused cough in a conscious guinea pig model and an increase in calcium in airway jugular ganglia. Using pharmacology and receptor-deficient mice we showed that the DP1 receptor mediates sensory nerve activation in mouse, guinea pig and human vagal afferents. In vivo, PGD2 and a DP1 receptor agonist, but not a DP2 receptor agonist, activated single airway C-fibres. Interestingly, activation of DP2 inhibited sensory nerve firing to capsaicin in vitro and in vivo. The DP1 receptor could be a therapeutic target for symptoms associated with asthma. Where endogenous PGD2 levels are elevated, loss of DP2 receptor-mediated inhibition of sensory nerves may lead to an increase in vagally associated symptoms and the potential for such adverse effects should be investigated in clinical studies with DP2 antagonists.

Role of the inflammasome-caspase1/11-IL-1/18 axis in cigarette smoke driven airway inflammation: an insight into the pathogenesis of COPD.

BACKGROUND: Chronic 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. METHODS: We used a murine model system, bioassays and a range of genetically modified mice to better understand this complex signalling pathway. RESULTS: The inflammasome-associated proteins NALP3 and ASC, but not IPAF and AIM2, are required for CS-induced IL-1ß/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. CONCLUSION: This data indicates a key role for the P2X7-NALP3/ASC-caspase1/11-IL-1ß/IL-18 axis in CS induced airway inflammation, highlighting this pathway as a possible therapeutic target for the treatment of COPD.

Role of transient receptor potential and pannexin channels in cigarette smoke-triggered ATP release in the lung.

BACKGROUND: COPD is an inflammatory disease usually associated with cigarette smoking (CS) with an increasing global prevalence and no effective medication. Extracellular ATP is increased in the COPD affected lung and may play a key role in driving CS-induced airway inflammation, but the mechanism involved in ATP release has eluded researchers. Recently, the transient receptor potential (TRP) and pannexin-1 channels have been suggested to play a role in other experimental paradigms. Thus, the aim of this work is to investigate if these channels are involved in CS-induced ATP release in the lung. METHODS: Primary human cells were exposed to CS and extracellular ATP levels measured. Mice were exposed to mainstream CS and airway inflammation assessed. TRPV1/4 mRNA expression was assessed in human lung parenchyma. RESULTS: CS exposure caused a dose-related increase in ATP from primary airway bronchial epithelial cells. This was attenuated by blockers of TRPV1, TRPV4 and pannexin-1 channels. Parallel data was obtained using murine acute CS-driven model systems. Finally, TRPV1/4 mRNA expression was increased in lung tissue samples from patients with COPD. CONCLUSIONS: Extracellular ATP is increased in the COPD affected lung and may play a key role in driving disease pathophysiology. These experiments uncover a novel mechanism which may be responsible for CS-induced ATP release. These findings highlight novel targets that could lead to the development of medicine to treat this devastating disease.

Longstanding complex regional pain syndrome is associated with activating autoantibodies against alpha-1a adrenoceptors.

Complex regional pain syndrome (CRPS) is a limb-confined posttraumatic pain syndrome with sympathetic features. The cause is unknown, but the results of a randomized crossover trial on low-dose intravenous immunoglobulins (IVIG) treatment point to a possible autoimmune mechanism. We tested purified serum immunoglobulin G (IgG) from patients with longstanding CRPS for evidence of antibodies interacting with autonomic receptors on adult primary cardiomyocytes, comparing with control IgG from healthy and diseased controls, and related the results to the clinical response to treatment with low-dose IVIG. We simultaneously recorded both single-cell contractions and intracellular calcium handling in an electrical field. Ten of 18 CRPS preparations and only 1/57 control preparations (P<0.0001) increased the sensitivity of the myocytes to the electric field, and this effect was abrogated by preincubation with α-1a receptor blockers. By contrast, effects on baseline calcium were blocked by preincubation with atropine. Interestingly, serum-IgG preparations from all 4 CRPS patients who had responded to low-dose IVIG with meaningful pain relief were effective in these assays, although 4/8 of the nonresponders were also active. To see if there were antibodies to the α-1a receptor, CRPS-IgG was applied to α-1a receptor-transfected rat-1 fibroblast cells. The CRPS serum IgG induced calcium flux, and fluorescence-activated cell sorting showed that there was serum IgG binding to the cells. The results suggest that patients with longstanding CRPS have serum antibodies to α-1a receptors, and that measurement of these antibodies may be useful in the diagnosis and management of the patients.

TLR4 activation induces IL-1ß release via an IPAF dependent but caspase 1/11/8 independent pathway in the lung.

BACKGROUND: The IL-1 family of cytokines is known to play an important role in inflammation therefore understanding the mechanism by which they are produced is paramount. Despite the recent plethora of publications dedicated to the study of these cytokines, the mechanism by which they are produced in the airway following endotoxin, Lipopolysaccharide (LPS), exposure is currently unclear. The aim was to determine the mechanism by which the IL-1 cytokines are produced after LPS inhaled challenge. METHODS: Mice were challenged with aerosolised LPS, and lung tissue and bronchiolar lavage fluid (BALF) collected. Targets were measured at the mRNA and protein level; caspase activity was determined using specific assays. RESULTS: BALF IL-1b/IL-18, but not IL-1a, was dependent on Ice Protease-Activating Factor (IPAF), and to a lesser extent Apoptosis-associated Speck-like protein containing a CARD (ASC). Interestingly, although we measured an increase in mRNA expression for caspase 1 and 11, we could not detect an increase in lung enzyme activity or a role for them in IL-1a/b production. Further investigations showed that whilst we could detect an increase in caspase 8 activity at later points in the time course (during resolution of inflammation), it appeared to play no role in the production of IL-1 cytokines in this model system. CONCLUSIONS: TLR4 activation increases levels of BALF IL-1b/IL-18 via an IPAF dependent and caspase 1/11/8 independent pathway. Furthermore, it would appear that the presence of IL-1a in the BALF is independent of these pathways. This novel data sheds light on innate signalling pathways in the lung that control the production of these key inflammatory cytokines.

Tiotropium modulates transient receptor potential V1 (TRPV1) in airway sensory nerves: A beneficial off-target effect?

BACKGROUND: Recent 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. OBJECTIVE: The aim of this study was to investigate whether tiotropium can directly modulate airway sensory nerve activity and thereby the cough reflex. METHODS: We 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. RESULTS: Inhaled 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. CONCLUSION: For 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.

Theophylline inhibits the cough reflex through a novel mechanism of action.

BACKGROUND: Theophylline has been used in the treatment of asthma and chronic obstructive pulmonary disease for more than 80 years. In addition to bronchodilator and anti-inflammatory activity, clinical studies have suggested that theophylline acts as an antitussive agent. Cough is the most frequent reason for consultation with a family doctor, and treatment options are limited. Determining how theophylline inhibits cough might lead to the development of optimized compounds. OBJECTIVE: We sought to investigate the inhibitory activity of theophylline on vagal sensory nerve activity and the cough reflex. METHODS: Using a range of techniques, we investigated the effect of theophylline on human and guinea pig vagal sensory nerve activity in vitro and on the cough reflex in guinea pig challenge models. RESULTS: Theophylline was antitussive in a guinea pig model, inhibited activation of single C-fiber afferents in vivo and depolarization of human and guinea pig vagus in vitro, and inhibited calcium influx in airway-specific neurons in vitro. A sequence of pharmacological studies on the isolated vagus and patch clamp and single-channel inside-out experiments showed that the effect of theophylline was due to an increase in the open probability of calcium-activated potassium channels. Finally, we demonstrated the antitussive activity of theophylline in a cigarette smoke exposure model that exhibited enhanced tussive responses to capsaicin. CONCLUSION: Theophylline inhibits capsaicin-induced cough under both normal and "disease" conditions by decreasing the excitability of sensory nerves through activation of small- and intermediate-conductance calcium-activated potassium channels. These findings could lead to the development of optimized antitussive compounds with a reduced side effect potential.

Pre-clinical studies in cough research: role of Transient Receptor Potential (TRP) channels.

Cough is a protective reflex and defence mechanism in healthy individuals, which helps clear excessive secretions and foreign material from the lungs. Cough often presents as the first and most persistent symptom of many respiratory diseases and some non-respiratory disorders, but can also be idiopathic, and is a common respiratory complaint for which medical attention is sought. Chronic cough of various aetiologies is a regular presentation to specialist respiratory clinics, and is reported as a troublesome symptom by a significant proportion of the population. Despite this, the treatment options for cough are limited. The lack of effective anti-tussives likely stems from our incomplete understanding of how the tussive reflex is mediated. However, research over the last decade has begun to shed some light on the mechanisms which provoke cough, and may ultimately provide us with better anti-tussive therapies. This review will focus on the in vitro and in vivo models that are currently used to further our understanding of the sensory innervation of the respiratory tract, and how these nerves are involved in controlling the cough response. Central to this are the Transient Receptor Potential (TRP) ion channels, a family of polymodal receptors that can be activated by such diverse stimuli as chemicals, temperature, osmotic stress, and mechanical perturbation. These ion channels are thought to be molecular pain integrators and targets for novel analgesic agents for the treatment of various pain disorders but some are also being developed as anti-tussives.

Harvesting, isolation, and functional assessment of primary vagal ganglia cells.

Airway sensory nerves play an important defensive role in the lungs, being central in mediating protective responses like cough and bronchoconstriction. In some cases, these responses become excessive, hypersensitive, and deleterious. Understanding the normal function of airway nerves and phenotype changes associated with disease will help in developing new therapeutics for treating chronic obstructive pulmonary disease and chronic cough. Guinea pigs, and to a lesser extent ferrets, are commonly employed for studying the cough reflex because they have a cough response similar to humans. While rats and mice do not exhibit a cough response, they do possess sensory nerves that respond to the same range of tussive stimuli as guinea pigs and humans. Described in this unit are protocols for harvesting guinea pig, mouse, and rat sensory nerve cell bodies to assess molecular and functional changes associated with pulmonary disease, and to identify new targets for therapeutic intervention.

Transient receptor potential channels mediate the tussive response to prostaglandin E2 and bradykinin.

BACKGROUND: Cough 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. METHODS: We 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. RESULTS: Using 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. CONCLUSION: This 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.

TRP channel antagonists as potential antitussives.

Cough is a troublesome symptom associated with many respiratory diseases. In some instances cough can become prolonged and excessive, and chronic cough of various aetiologies is a common presentation to specialist respiratory clinics. However, current treatment options are limited. Despite its importance, our understanding of the mechanisms that provoke cough is poor. Recent investigation has focused on the interaction between G-protein-coupled receptors and ion channels expressed on airway sensory nerves that are responsible for driving the cough reflex. In particular, the Transient Receptor Potential class of ion channels appears to play a major role as a regulator of the afferent arm of the cough reflex and could be involved in the heightened cough response observed in disease states. Current research investigating the pathogenesis of cough supports the development of TRP channel inhibitors as novel and selective treatment modalities.

Transient receptor potential A1 channels: insights into cough and airway inflammatory disease.

Cough is a common symptom of diseases such as asthma and COPD and also presents as a disease in its own right. Treatment options are limited; a recent meta-analysis concluded that over-the-counter remedies are ineffective, and there is increasing concern about their use in children. Transient receptor potential cation channel, subfamily A, member 1 (TRPA1) channels are nonselective cation channels that are activated by a range of natural products (eg, allyl isothiocyanate), a multitude of environmental irritants (eg, acrolein, which is present in air pollution, vehicle exhaust, and cigarette smoke), and inflammatory mediators (eg, cyclopentenone prostaglandins). TRPA1 is primarily expressed in small-diameter, nociceptive neurons where its activation probably contributes to the perception of noxious stimuli. Inhalational exposure to irritating gases, fumes, dusts, vapors, chemicals, and endogenous mediators can lead to the development of cough. The respiratory tract is innervated by primary sensory afferent nerves, which are activated by mechanical and chemical stimuli. Recent data suggest that activation of TRPA1 on these vagal sensory afferents by these irritant substances could lead to central reflexes, including dyspnea, changes in breathing pattern, and cough, which contribute to the symptoms and pathophysiology of respiratory diseases.