John J. Adcock

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.

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.

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.

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. Prostaglandin D2 activates sensory nerves and evokes cough via DP1 receptors http://ow.ly/BR1kp

Mechanistic link between diesel exhaust particles and respiratory reflexes

Background: Diesel exhaust particles (DEPs) are a major component of particulate matter in Europes 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. Fatty acid amide hydrolase inhibition as a target for the development of novel, safe antitussive therapy http://ow.ly/l4ZE30dbbB1

T2 Oestrogen: an endogenous agonist for trpm3 triggered sensory nerve activation in the airway?

Introduction In chronic lung diseases, activation of airway sensory nerves initiates respiratory reflexes including cough for which there is currently no safe and effective treatment. Ion channels on sensory afferents can activate these reflexes and as such are attractive therapeutic targets. Using synthetic ligands we have shown that activation of TRPM3 can trigger human and guinea pig airway sensory nerves.1 As TRPM3 is thought as a “steroid receptor” and women are consistently over represented in chronic cough clinics,2 we hypothesised that oestrogen could be an endogenous agonist of TRPM3 mediated activation of airway sensory nerves. Methods Ex vivo tissue and neuron assay systems were employed, in conjunction with in vivo electrophysiology and a guinea pig cough model to investigate this hypothesis. Results In vivo, β-oestradiol caused firing of both C and Aδ fibres and also elicited a cough response in conscious unrestrained guinea pigs. Ex vivo, β-Oestradiol caused a concentration dependant depolarisation of isolated guinea pig vagal nerves which was inhibited by the non-selective ER receptor antagonist ICI182780 (92.7%±4.5%) and by the TRPM3 antagonist Isosakuranetin (86.5%±6.8%). The ER receptor antagonist had no effect on the TRPM3 aganist (CIM0216) mediated depolarisation. Translational responses were obtained in human vagal tissue. Single cell PCR indicated that the TRPM3 ion channel and two oestrogen receptors GPER and ERα were expressed in airway specific nodose and jugular ganglia, and were co-expressed with TRPM3. Conclusion These data show that the oestrogen can activate airway sensory nerves, and suggests that ER receptors may be activated upstream of TRPM3 activation. Further investigation is required, however this data may help to explain the higher number of females attending chronic cough clinics and suggests TRPM3 could be a novel therapeutic target for chronic cough. References Bonvini et al. AJRCCM 2017;195:A7047. Kelsall et al. Thorax 2009;64:393–8.