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.

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.

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.

The size of guinea-pig isolated tracheal contractions induced by histamine was substantially augmented by pretreatment with the cyclo-oxygenase inhibitor, indomethacin. However, compounds which inhibit both the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism not only did not augment histamine-induced contractions of the guinea-pig trachea, but also completely reversed the increased reactivity to histamine produced by indomethacin. The SRS-A (slow reacting substance of anaphylaxis) antagonist, FPL55712, and the anti-allergic drug, cromoglycate, had no effect on the augmentation of histamineinduced contractions by indomethacin.

SUMMARY1. Capsaicin, prostaglandin E2 (PGE2) and histamine are potent stimuli for reflex coughing and bronchoconstriction in many species including man. We have studied the effects of solutions of capsaicin, PGE2 and histamine on airway sensory receptors when administered as inhaled aerosols to the lower respiratory tract in anaesthetized, paralysed and artificially ventilated cats.2. Histamine, administered by aerosol (6 breaths of a 1 mg ml-' solution) and intravenously (10 jug kg-'), caused an increase in the rate of discharge from rapidly adapting stretch receptors (RARs) and caused bronchoconstriction.3. Six breaths of a capsaicin aerosol generated from solutions of 0.1 or 1 mg ml-' stimulated six out of nine RARs tested. Bronchoconstriction occurred with and without RAR stimulation. The diluent for the capsaicin aerosol had no significant effect on pulmonary mechanics or rate of RAR discharge.4. Administration of increasing concentrations (0 001-1 mg ml-') of PGE2 aerosol given in six breaths (at 6 min intervals) caused a dose-dependent increase in the rate of discharge of eight RARs tested and caused bronchoconstriction. The diluent for the PGE2 aerosol had no effect on pulmonary mechanics or rate of RAR discharge.5. Inhalation of aerosols of histamine (6 breaths of 1 mg ml-' solution) and capsaicin (3 breaths of 0-1 mg ml-' solution) stimulated all six lung C fibre endings studied (3 pulmonary and 3 bronchial). These aerosols of capsaicin and histamine also caused bronchoconstriction.

1 Antitussive, antinociceptive and respiratory depressant effects of codeine, morphine and H.Tyr.DArg.Gly.Phe(4-NO2) Pro.NH2 (compound BW443C) were investigated in unanaesthetized guineapigs. Antagonism of the antitussive and antinociceptive effects was investigated by the use of nalorphine and N-methylnalorphine. Naloxone was used to antagonize respiratory depression.2 Antitussive ED50s (with 95% confidence limits) for inhibition of cough induced by citric acid vapour were for codeine, morphine and BW443C respectively, 9.1(5.8-15), 1.3(0.7-2.4) and 1.2(0.6-2.6) mg kg-' s.c. and 8.7(4.2-12), 1.6(1.2-1.9) and 0.67(0.002-3.3) mg kg-', i.v. The antitussive effects of subcutaneous codeine (25mg kg-') morphine (8.1 mg kg-') and BW443C (2.5mg kg-') were significantly antagonized by subcutaneous nalorphine (3.0 mg kg-') and N-methylnalorphine (3.0mg kg-').3 In the multiple toe-pinch test, the antinociceptive ED5s (with 95% confidence limits) of codeine and morphine were 18(16-22) and 2.3(0.4-4.3) mg kg-', s.c., respectively. Compound BW443C was ineffective in doses of 2.5 and 10 mg kg-' s.c., a result consistent with its lacking penetration into the CNS. Subcutaneous nalorphine (3.0mg kg-') antagonized the antinociceptive action of codeine (25mg kg-') and morphine (8.1 mg kg-'). In contrast, N-methylnalorphine (3.0mg kg-') had no significant effect on the antinociceptive action of codeine and morphine, suggesting lack of penetration of the CNS by N-methylnalorphine.4 At doses near to the i.v. ED50 values for the antitussive activity, morphine (1.5 mg kg-', i.v.) and codeine (10mg kg-', i.v.) caused small but significant depressions of ventilation (7.0 ± 2.3% and 16.5 ± 8.4% respectively). Higher doses of morphine (10, 30 and 60mg kg-',i.v.) caused further doserelated depression of ventilation (9.6 ± 5.3%, 22.4 ± 6.2% and 36.2 ± 9.6% respectively) whereas codeine (30 and 60 mg kg-' i.v.) caused stimulation of ventilation which was marked (191.3 ± 43.9%) at 60 mg kg-'. 5 Compound BW443C in doses of 1 or 10mgkg-',i.v. (approximately equal to, and 10 times the EDo for antitussive activity) did not cause significant depression of ventilation. Only at higher doses of 30 and 60mg kg-', i.v. was there a significant decrease in minute volume (13.1 ± 6.8% and 15.9 ± 1.89% respectively). The depression of ventilation caused by either BW443C (60mg kg- ', i.v.) or morphine (60mg kg-', i.v.) was prevented by pretreatment with naloxone (3mg kg- ', i.v.) administered 15 min before morphine or BW443C. 6 These results in the guinea-pig support the hypothesis that the antitussive action of the opiates codeine and morphine and the opioid pentapeptide BW443C do not require penetration of these drugs into the CNS.

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.

1 The anti-tussive eects, of the local anaesthetic, lidocaine and carcainium chloride (RSD931) have been investigated in guinea-pigs and rabbits. 2 Pre-treatment of guinea-pigs with aerosols of lidocaine or RSD931 at 0.1, 1.0 and 10 mg ml 71 reduced the number of citric acid-induced coughs by 9.3, 32.6 and 40.9% (P40.05) for lidocaine and by 25.3% (P40.05), 40.4% (P40.05) and 97.6% (P50.01) for RSD931, respectively and increased the latency to onset of cough at 10.0 mg ml 71 only. In addition, RSD931 at 10 mg ml 71 reduced citric acid-evoked cough responses in rabbits (with prior exposure to ozone at 3 p.p.m. for 1 h) from 22.1+5.1 to 2.7+0.9 coughs (P50.01). ) signi®cantly (P50.05 ± 50.01) reduced the spontaneous and histamineevoked discharges in Ad-®bres originating from airway, rapidly adapting stretch receptors (RARs) without aecting histamine-evoked bronchoconstriction. Lidocaine at 10.0 mg ml 71 also signi®cantly (P50.05) inhibited the spontaneous and histamine-induced discharges of RARs without aecting histamine-evoked bronchoconstriction. 5 Aerosols of RSD931 (10.0 mg ml 71 ) caused a transient, but signi®cant (P50.05), activation of pulmonary C-®bre endings 2.5 min after administration started. RSD931 had no signi®cant (P40.05) eects on discharges in bronchial C-®bres originating from bronchial C-®bre endings, capsaicin-evoked discharges of either pulmonary or bronchial C-®bre endings or on capsaicinevoked bronchoconstriction. In contrast, lidocaine (10.0 mg ml 71 ) signi®cantly (P50.05) inhibited spontaneous and capsaicin-induced discharges in both pulmonary and bronchial C-®bres respectively. Lidocaine also signi®cantly (P50.05) reduced capsaicin-evoked bronchoconstriction. 6 These studies suggest that the anti-tussive actions of RSD931 are mediated via inhibition of discharges in Ad-®bres originating from airway RARs. The mechanism of action of RSD931 is distinct from that of the local anaesthetic lidocaine and RSD931 may represent a novel class of antitussive agent.