Polymodal vagal afferent nerves terminating in the mucosa of the large conducting airways play essential roles in regulating cough. These cough receptors project bilaterally from the nodose ganglia, are activated by protons and by mechanical stimulation, and terminate centrally in the medial (SolM) solitary tract nuclei (nTS). Although many vagal afferent nerves utilize non-NMDA type glutamate receptors exclusively for transmission centrally, cough receptor signaling requires NMDA receptor activation. We hypothesized that nTS nitric oxide synthase (NOS) activation and a cGMP transduction cascade would act as downstream messenger systems during the encoding of cough. We also hypothesized that NOS expression may be a defining characteristic of cough receptor relay neurons. NADPH-diaphorase staining identified NOS-expressing neurons throughout the brain stem (especially in the trigeminal and cuneate nuclei and in the dorsal motor nuclei of the vagus nerves). NOS expressing nTS neurons were rare but found in SolM. Bilateral SolM microinjections of NOS inhibitors or the NMDA receptor blocker SDZ 220581 markedly reduced coughing evoked by tracheal citric acid challenges. But citric acid evoked coughing was neither attenuated by inhibiting soluble guanylate cyclase in SolM nor potentiated by inhibiting the cGMP-selective phosphodiesterase-5. No changes in basal respiratory patterns were observed with NOS inhibitor or nitric oxide (NO) donor microinjections, but NMDA microinjection into SolM induced respiratory responses including cough that were at least partially NOS dependent. We conclude that NO is an essential downstream regulator of NMDA receptor mediated encoding of cough in nTS but does not act via soluble guanylate cyclase or cGMP. NEW & NOTEWORTHY Nitric oxide (NO) acts downstream from NMDA receptor activation to encode the cough reflex but without involvement of soluble guanylate cyclase. We propose that the enhancement of glutamatergic signaling by NO, perhaps through nitrosylation mechanisms, is an essential component of signal transduction at the synapses between cough receptor afferent nerves and their solitary tract nuclei (nTS) relay neurons.

Cough is essential to airway defence following aspiration. Using a relevant animal model, we set out to identify the mechanisms by which gastric fluid evokes coughing, the vagal afferents responsible for initiating this reflex and the ion channels directly activated by components of gastric fluid. We studied gastric fluid and citric acid evoked cough reflexes in guinea-pigs and, in parallel, their ability to activate airway vagal afferent nerves. Additionally, we utilized a single cell RT-PCR approach to determine the expression of acid sensitive ion channels by the vagal afferent neurones regulating cough. We observed that gastric fluid evoked coughing following direct application to the tracheal and laryngeal mucosa of anaesthetized guinea-pigs. An acidic pH of the gastric fluid was essential to its ability to evoke coughing, and the tussive actions of gastric fluid were mimicked by citric acid. The vagal afferent nerves regulating cough expressed mRNA for the acid-sensitive ion channels (ASICs) ASIC1, ASIC2 and ASIC3. The coughing evoked by gastric fluid and by acid and the vagal afferent nerve discharge evoked by protons were prevented by the ASIC inhibitors diminazene and diclofenac but not by transient receptor potential vanilloid 1 blockade. Based on these results, we conclude that airway mucosal afferent neuronal ASIC channels are essential to airway defence against aspiration of gastric fluid. We speculate that dysfunction of the reflex pathways initiated by vagal afferent neurone ASIC channel engagement may be a risk factor for aspiration pneumonia in susceptible patients. KEY POINTS: Aspiration of gastric contents can induce an acute lung injury that may progress to life-threatening aspiration pneumonia or acute respiratory distress syndrome. Cough is an essential defensive reflex that protects the airways from aspiration. Patients with an absent or ineffective cough reflex are at significantly greater risk for developing aspiration pneumonia. Using an animal model, we have determined the potential mechanisms driving cough in response to gastric fluid aspiration. We found that gastric fluid acidity is essential to the initiation of cough and we identified acid-sensing ion channels expressed by vagal sensory nerves terminating in the airway mucosa as key effectors of this reflex. We speculate that patients with dysfunctional acid-sensing mechanisms in their bronchopulmonary vagal afferent nerves may be at increased risk of aspiration pneumonia. We also summarize the evidence suggesting that this signalling pathway could explain the emergence of cough in patients with gastroesophageal reflux disease.

We have addressed the hypothesis that the opposing effects of bronchopulmonary C-fiber activation on cough are attributable to the activation of C-fiber subtypes. Coughing was evoked in anesthetized guinea pigs by citric acid (0.001-2 M) applied topically in 100-µl aliquots to the tracheal mucosa. In control preparations, citric acid evoked 10 ± 1 coughs cumulatively. Selective activation of the pulmonary C fibers arising from the nodose ganglia with either aerosols or continuous intravenous infusion of adenosine or the 5-HT3 receptor-selective agonist 2-methyl-5-HT nearly abolished coughing evoked subsequently by topical citric acid challenge. Delivering adenosine or 2-methyl-5-HT directly to the tracheal mucosa (where few if any nodose C fibers terminate) was without effect on citric acid-evoked cough. These actions of pulmonary administration of adenosine and 2-methyl-5-HT were accompanied by an increase in respiratory rate, but it is unlikely that the change in respiratory pattern caused the decrease in coughing, as the rapidly adapting receptor stimulant histamine also produced a marked tachypnea but was without effect on cough. In awake guinea pigs, adenosine failed to evoke coughing but reduced coughing induced by the nonselective C-fiber stimulant capsaicin. We conclude that bronchopulmonary C-fiber subtypes in guinea pigs have opposing effects on cough, with airway C fibers arising from the jugular ganglia initiating and/or sensitizing the cough reflex and the intrapulmonary C fibers arising from the nodose ganglia actively inhibiting cough upon activation.

Bradykinin has been implicated as a mediator of the acute pathophysiological and inflammatory consequences of respiratory tract infections and in exacerbations of chronic diseases such as asthma. Bradykinin may also be a trigger for the coughing associated with these and other conditions. We have thus set out to evaluate the pharmacology of bradykinin-evoked coughing in guinea pigs. When inhaled, bradykinin induced paroxysmal coughing that was abolished by the bradykinin B2 receptor antagonist HOE 140. These cough responses rapidly desensitized, consistent with reports of B2 receptor desensitization. Bradykinin-evoked cough was potentiated by inhibition of both neutral endopeptidase and angiotensin-converting enzyme (with thiorphan and captopril, respectively), but was largely unaffected by muscarinic or thromboxane receptor blockade (atropine and ICI 192605), cyclooxygenase, or nitric oxide synthase inhibition (meclofenamic acid and NG-nitro-L-arginine). Calcium influx studies in bronchopulmonary vagal afferent neurons dissociated from vagal sensory ganglia indicated that the tachykinin-containing C-fibers arising from the jugular ganglia mediate bradykinin-evoked coughing. Also implicating the jugular C-fibers was the observation that simultaneous blockade of neurokinin2 (NK2; SR48968) and NK3 (SR142801 or SB223412) receptors nearly abolished the bradykinin-evoked cough responses. The data suggest that bradykinin induces coughing in guinea pigs by activating B2 receptors on bronchopulmonary C-fibers. We speculate that therapeutics targeting the actions of bradykinin may prove useful in the treatment of cough.

We sought to locate and characterize the central terminations of cough receptor airway afferents in the nucleus tractus solitarii (nTS) of guinea pigs. Neuronal tracing indicated that cough receptors terminate in locations bordering the medial and intermediate nTS. Microinjection of NMDA receptor antagonists into these locations nearly abolished cough evoked by citric acid (0.001‐2M; 100μl aliquots) applied to the trachea of anaesthetised animals (vehicle: 10±3; AP‐5: 3±2; SDZ 220‐581: 0±0 coughs; n=3‐6).Microinjection of NMDA produced respiratory perturbations including tachypnoea (0.01nmol NMDA) and bradypnoea/apnoea (0.1nmol NMDA) that were eliminated by NMDA receptor antagonists or inhibitors of nitric oxide synthase (NOS): L‐NNA (non‐specific) or N‐propyl‐L‐arginine (NPLA) (neuronal NOS‐specific). NOS inhibitors also reduced citric acid‐evoked coughing (Vehicle: 11±2; L‐NNA: 4±1; NPLA 4±2 coughs; n=6‐8). Citric acid‐induced cough was not attenuated after inhibition of soluble guanylate cyclase (sGC) with ODQ (vehicle: 11±3 coughs; ODQ: 14±2 coughs; n=8) or potentiated after inhibition of phosphodiesterase‐5 (PDE5) with zaprinast (vehicle: 9±3 coughs; zaprinast 10±1 coughs; n=3). Our data indicates that reflex cough is dependent on NMDA receptor activation and/or NO in the nTS. NO signalling occurs independent of either sGC or PDE5, suggesting that alternative signalling mechanisms are at play.Work supported by NIH.

Citric acid (CA) evoked cough in anesthetized guinea pigs (GPs) depends upon the activation of nodose ganglia neurons that are acid‐sensitive but capsaicin‐insensitive and project to the laryngeal, tracheal and mainstem bronchial mucosa. Using single cell PCR and diminazene (DZ), a potent and selective inhibitor of ASICs, we have addressed the hypothesis that acid evoked cough in anesthetized guinea pigs is ASIC dependent. In vitro, DZ inhibited 1mM CA evoked action potential (AP) discharge in the capsaicin‐insensitive afferent neurons projecting to the trachea (CA evoked 44±7, 36±9, 15±4 and 1±1 APs in control preparations and following treatment with 1, 10 and 100μM DZ, respectively; n=3–5). In vivo, DZ inhibited cough evoked by CA (0.001–2M) applied topically to the tracheal mucosa (CA evoked 8±1, 6±1, 3±2 and 0±0 coughs in control and in GPs pretreated topically with 1, 10 and 100μM DZ, respectively; n=4–8). By contrast, the TRPV1 antagonist SB366791 (10μM) nearly abolished 0.1μM capsaicin‐evoked contractions of the GP trachea in vitro but was without effect on CA evoked cough (8±1 coughs; n=5). Single cell PCR revealed that nodose ganglia neurons retrogradely labeled from the trachea do not express TRPV1 mRNA (0/9) but do (8/9) express either ASIC1 (6/9) and/or ASIC3 (5/9) mRNA. Together, these data implicate ASICs in the acid evoked coughing evoked from the airway mucosa.

Miniature inverted-repeat transposable elements (MITEs) are widespread in both prokaryotic and eukaryotic genomes, where their copy numbers can attain several thousands. Little is known, however, about the genetic factor(s) affecting their transpositions. Here, we show that disruption of a gene encoding ubiquitin-like protein markedly enhances the transposition activity of a MITE mPing in intact rice plants without any exogenous stresses. We found that the transposition activity of mPing is far higher in the lines harboring a non-functional allele at the Rurm1 (Rice ubiquitin-related modifier-1) locus than in the wild-type line. Although the alteration of cytosine methylation pattern triggers the activation of transposable elements under exogenous stress conditions, the methylation degrees in the whole genome, the mPing-body region, and the mPing-flanking regions of the non-functional Rurm1 line were unchanged. This study provides experimental evidence for one of the models of genome shock theory that genetic accidents within cells enhance the transposition activities of transposable elements.

Gastroesophageal reflux disease (GERD) is a common cause of chronic cough. Both acid and nonacid reflux is thought to play a role in the initiation of coughing and cough hypersensitivity. The GABAB receptor agonist lesogaberan was developed as a peripherally restricted anti-reflux therapy that reduces the frequency of transient lower esophageal sphincter relaxations (TLESR; the major cause of reflux) in animals and in patients with GERD. GABAB receptor agonists have also been shown to possess antitussive effects in patients and in animals independent of their effects on TLESR, suggesting that lesogaberan may be a promising treatment for chronic cough.

Coughing protects and clears the airways and lungs of inhaled irritants, particulates, pathogens, and accumulated secretions. An initial urge to cough, and an almost binary output suggests gating mechanisms that encode and modulate this defensive reflex. Whether this “gate” has a physical location for the physiological barrier it poses to cough is unknown. Here we describe a critical component to cough gating, the central terminations of the cough receptors. A novel microinjection strategy defined coordinates for microinjection of glutamate receptor antagonists that nearly abolished cough evoked from the trachea and larynx in anesthetized guinea pigs while having no effect on basal respiratory rate and little or no effect on reflexes attributed to activating other afferent nerve subtypes. Comparable microinjections in adjacent brainstem locations (0.5–2 mm distal) were without effect on coughing. Subsequent transganglionic and dual tracing studies confirmed that the central terminations of the cough receptors and their primary relay neurons are found bilaterally within nucleus tractus solitarius (nTS), lateral to the commissural subnucleus and perhaps in the medial subnuclei. These synapses possess the physiological characteristics of a cough gate. Their localization should facilitate more mechanistic studies of the encoding and gating of cough.—Canning, B. J., Nanako Mori. An essential component to brainstem cough gating identified in anesthetized guinea pigs. FASEB J. 24, 3916–3926 (2010). www.fasebj.org

The airways contain a dense subepithelial microvascular plexus that is involved in the supply and clearance of substances to and from the airway wall. We set out to test the hypothesis that airway smooth muscle reactivity to bronchoconstricting agents may be dependent on airway mucosal blood flow. Immunohistochemical staining identified vasoconstrictor and vasodilator nerve fibers associated with subepithelial blood vessels in the guinea pig airways. Intravital microscopy of the tracheal mucosal microvasculature in anesthetized guinea pigs revealed that blockade of α-adrenergic receptors increased baseline arteriole diameter by ~40%, whereas the α-adrenergic receptor agonist phenylephrine produced a modest (5%) vasoconstriction in excess of the baseline tone. In subsequent in vivo experiments, tracheal contractions evoked by topically applied histamine were significantly reduced (P < 0.05) and enhanced by α-adrenergic receptor blockade and activation, respectively. α-Adrenergic ligands produced similar significant (P < 0.05) effects on airway smooth muscle contractions evoked by topically administered capsaicin, intravenously administered neurokinin A, inhaled histamine, and topically administered antigen in sensitized animals. These responses were independent of any direct effect of α-adrenergic ligands on the airway smooth muscle tone. The data suggest that changes in blood flow in the vessels supplying the airways regulate the reactivity of the underlying airway smooth muscle to locally released and exogenously administered agents by regulating their clearance. We speculate that changes in mucosal vascular function or changes in neuronal regulation of the airway vasculature may contribute to airways responsiveness in disease.

We have previously described the physiological and morphological properties of the cough receptors and their sites of termination in the airways and centrally in the nucleus tractus solitarius (nTS). In the present study, we have addressed the hypothesis that the primary central synapses of the cough receptors subserve an essential role in the encoding of cough. We found that cough requires sustained, high-frequency (≥8-Hz) afferent nerve activation. We also found evidence for processes that both facilitate (summation, sensitization) and inhibit the initiation of cough. Sensitization of cough occurs with repetitive subthreshold activation of the cough receptors or by coincident activation of C-fibers and/or nTS neurokinin receptor activation. Desensitization of cough evoked by repetitive and/or continuous afferent nerve activation has a rapid onset (<60 s) and does not differentiate between tussive stimuli, suggesting a central nervous system-dependent process. The cough reflex can also be actively inhibited upon activation of other airway afferent nerve subtypes, including slowly adapting receptors and pulmonary C-fibers. The sensitization and desensitization of cough are likely attributable to the prominent, primary, and unique role of N-methyl-d-aspartate receptor-dependent signaling at the central synapses of the cough receptors. These attributes may have direct relevance to the presentation of cough in disease and for the effectiveness of antitussive therapies.