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.

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.

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.

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.

TRPV1 receptors in sensitisation of cough and pain reflexes.

Preclinical studies suggest that the vanilloid receptor (TRPV1) is an important component of several disease areas such as pain (inflammatory, visceral, cancer and neuropathic), airway disease (including chronic cough), inflammatory bowel disease (IBD), interstitial cystitis, urinary incontinence, pancreatitis and migraine. TRPV1 is a member of a distinct subgroup of the transient receptor potential (TRP) family of ion channels. The neuronally expressed TRPV1 is a non-selective, Ca(2+)-preferring, cation channel. In addition to capsaicin, this channel is activated by a number of different stimuli including heat, acid, certain arachidonic acid derivatives and direct phosphorylation via protein kinase C (PKC). Moreover, there is also evidence that various inflammatory mediators such as adenosine triphosphate (ATP), bradykinin, nerve growth factor (NGF) or prostaglandin E(2) (PGE(2)) may indirectly lead to activation of the TRPV1 channel via activation of their respective receptors. There is strong experimental evidence that the combination of direct and indirect mechanisms finely tune the TRPV1 activity. Each of the different known modes of direct TRPV1 activation (protons, heat and vanilloids) is capable of sensitising the channel to other agonists. Similarly, inflammatory mediators from the external milieu found in disease conditions can indirectly sensitise the receptor. It is this sensitisation of the TRPV1 receptor in inflammatory disease that could hold the key and contribute to the transduction of noxious signalling for normally innocuous stimuli, i.e. either hyperalgesia in the case of chronic pain or airway hyperresponsivness/hypertussive responses in patients with chronic cough. It seems reasonable to suggest that the various mechanisms for sensitisation provide a scenario for TRPV1 to be tonically active and this activity may contribute to the underlying pathology -- providing an important convergence point of multiple pain producing stimuli in the somatosensory system and multiple cough-evoking irritants in the airways. The complex mechanisms and pathways that contribute to the pathophysiology of chronic pain and chronic cough have made it difficult for clinicians to treat patients with current therapies. There is an increasing amount of evidence supporting the hypothesis that the expression, activation and modulation of TRPV1 in sensory neurones appears to be an integral component of pain and cough pathways, although the precise contribution of TRPV1 to human disease has yet to be determined. So the question remains open as to whether TRPV1 therapeutics will be efficacious and safe in man and represent a much needed novel pain and cough therapeutic.

Airway nerves: in vivo electrophysiology.

Information about the activity of airway sensory afferent nerves in vivo can be obtained electrophysiologically by extracellular recording of action potentials. Apart from data capture, the basic techniques used for recording sensory nerve activity have not advanced greatly in 50 years. However, clearly they continue to contribute vastly to our understanding of the role of these nerves in the control and functions of the airways in disease. This is particularly noticeable with the insight gained into exactly which physiological, biochemical and pharmacological events in the control of lung function rely upon the airway sensory afferent nerves and the subsequent airway vagal reflex arcs.