Synergistic interactions between airway afferent nerve subtypes regulating the cough reflex in guinea-pigs

Abstract

Cough initiated from the trachea and larynx in anaesthetized guinea-pigs is mediated by capsaicin-insensitive, mechanically sensitive vagal afferent neurones. Tachykinin-containing, capsaicin-sensitive C-fibres also innervate the airways and have been implicated in the cough reflex. Capsaicin-sensitive nerves act centrally and synergistically to modify reflex bronchospasm initiated by airway mechanoreceptor stimulation. The hypothesis that polymodal mechanoreceptors and capsaicin-sensitive afferent nerves similarly interact centrally to regulate coughing was addressed in this study. Cough was evoked from the tracheal mucosa either electrically (16 Hz, 10 s trains, 1-10 V) or by citric acid (0.001-2 m). Neither capsaicin nor bradykinin evoked a cough when applied to the trachea of anaesthetized guinea-pigs, but they substantially reduced the electrical threshold for initiating the cough reflex. The TRPV1 receptor antagonist capsazepine prevented the increased cough sensitivity induced by capsaicin. These effects of topically applied capsaicin and bradykinin were not due to interactions between afferent nerve subtypes within the tracheal wall or a direct effect on the cough receptors, as they were mimicked by nebulizing 1 mg ml(-1) bradykinin into the lower airways and by microinjecting 0.5 nmol capsaicin into nucleus of the solitary tract (nTS). Citric acid-induced coughing was also potentiated by inhalation of bradykinin. The effects of tracheal capsaicin challenge on cough were mimicked by microinjecting substance P (0.5-5 nmol) into the nTS and prevented by intracerebroventricular administration (20 nmol h(-1)) of the neurokinin receptor antagonists CP99994 or SB223412. Tracheal application of these antagonists was without effect. C-fibre activation may thus sensitize the cough reflex via central mechanisms.

Figure 1. Capsaicin and bradykinin-induced sensitization of the cough reflex initiated by electrical stimulation of the tracheal mucosa of anaesthetized guinea-pigs

The voltage threshold for evoking cough was estimated by electrically stimulating the tracheal mucosa at varying stimulation voltages (16 Hz, 10 s train, 1 ms pulse duration). Capsaicin (A) bradykinin (B) or vehicle were added to the tracheal perfusate prior to constructing the voltage–response curves. Capsazepine or its vehicle was added to the tracheal perfusate 10 min prior to the addition of capsaicin. The results are presented as the percentage of animals coughing in response to a given stimulation voltage. Challenge with either capsaicin or bradykinin increased the likelihood of coughing at lower stimulation intensities (2–4 V) relative to control (P < 0.001).

Figure 2. Inhalation of bradykinin sensitizes the cough reflex initiated by electrical stimulation of the tracheal mucosa of anaesthetized guinea-pigs

Bradykinin (1 mg ml⁻¹) or vehicle (saline) was nebulized into the air inspired by the animals 30 s prior to and during the electrical stimulation used to initiate coughing. No animals coughed in response to inhalation of either bradykinin or vehicle, but bradykinin challenge increased respiratory rate (see Fig. 3). The results are presented as the percentage of animals coughing in response to a given stimulation voltage. Inhalation of bradykinin increased the likelihood of coughing at lower stimulation intensities (2–4 V) relative to control (P < 0.001).

Figure 3. Inhalation of bradykinin sensitizes the cough reflex initiated by citric acid applied topically to the tracheal mucosa of anaesthetized guinea-pigs

Bradykinin (1 mg ml⁻¹) or vehicle (saline) was nebulized into the air inspired by the guinea-pigs 60 s prior to and throughout the entire citric acid concentration–response curve. A, representative traces (∼5 min) of citric acid-evoked cough during inhalation of saline (top) and bradykinin (bottom). Bradykinin and saline inhalation evoked augmented breaths but no coughing. Bradykinin evoked more augmented breaths than saline. Citric acid, delivered in 100 μl aliquots in increasing concentrations from 0.001 to 0.1 m (the time of first dose administration is marked by an arrow on each trace) were administered at 1 min intervals and evoked augmented breaths or coughing, usually followed by a brief slowing of respiration, particularly upon challenge with higher concentrations of citric acid (≥ 0.1 m). Vertical and horizontal bars denote 5 cmH₂O and 1 min, respectively. B, effects of saline and 1 mg ml⁻¹ bradykinin inhalation (beginning at time 0) on respiratory rate. Respiratory rate increased more during bradykinin inhalation (18 ± 3% peak increase, n = 14) than during inhalation of saline (6 ± 1% peak increase, n = 13; P < 0.01). C, percentage of animals coughing in response to increasing concentrations of citric acid following inhalation of saline (filled bars) or 1 mg ml⁻¹ bradykinin (open bars). Bradykinin significantly increased the probability of 0.01 m citric acid challenge to evoke cough in the anaesthetized guinea-pigs (P < 0.05). D, mean ± s.e.m. cumulative coughs evoked by increasing concentrations of citric acid during inhalation of saline (open circles) and 1 mg ml⁻¹ bradykinin (filled circles). Inhalation of bradykinin significantly increased the total number of coughs evoked cumulatively by 0.001–0.1 m citric acid and increased the potency of citric acid for inducing cough (see Table 2).

Figure 4. Effect of microinjecting capsaicin and substance P into the cnTS on respiratory rate in anaesthetized guinea-pigs

Capsaicin (0.5 nmol), substance P (0.5–5 nmol) or vehicle (20% ethanol in saline) was microinjected (0.5 μl) into the cnTS. A, representative injection site, indicated by arrow (∼0.5 mm caudal to obex). CC, central canal. B, representative traces showing respiration before and 10 min after microinjection of capsaicin. C, mean ± s.e.m. effects of cnTS microinjection of capsaicin, substance P and vehicle (0.5 μl over 3 min beginning at time 0) on respiratory rate. Microinjecting 0.5 nmol capsaicin into brainstem subnuclei rostral and lateral to cnTS, including the interpolar trigeminal nucleus (n = 4) and the intermediate and ventrolateral nTS (n = 3) was without effect on respiration (not shown).

Figure 5. Microinjection of capsaicin or substance P into the cnTS sensitizes the cough reflex in anaesthetized guinea pigs

Cough was evoked electrically from the trachea (16 Hz, 10 s train, 1–10 V). The effects of capsaicin (A) and substance P (B) microinjection on the voltage–coughing-response curve are depicted. The results are presented as the percentage of animals coughing in response to a given stimulation voltage. Microinjection of 0.5 nmol capsaicin or 5 nmol substance P into the cnTS increased the likelihood of coughing at lower stimulation intensities (2–6 V) relative to control (P < 0.001).

Figure 6. Neurokinin receptor antagonists have no effect on cough evoked electrically or by citric acid in control animals

Cough was evoked from the tracheal mucosa electrically (16 Hz, 10 s train, 1 ms pulse duration, 1–10 V) (A) and subsequently by citric acid (0.001–2 m) applied topically to the tracheal mucosa (B). Animals were first pretreated intraperitoneally either with vehicle (dimethyl sulfoxide; n = 6) or with a combination of three neurokinin receptor antagonists CP99994, SR48968 and SB223412 (1 mg kg⁻¹ each, n = 5). The antagonists (administered at doses known to abolish C-fibre-mediated reflexes in guinea pigs; Bolser et al. 1997; Canning et al. 2001; Mazzone & Canning, 2002a) had no effect on the percentage of animals coughing in response to any stimulation voltage or citric acid challenge and had no effect on the tussigenic potency or efficacy of citric acid (P > 0.1). Moreover, in these animals, which were not challenged with capsaicin prior to evoking cough (see Figs 7 and 8), the antagonists had no effect on the peak expiratory pressures during cough evoked either electrically or by citric acid (the peak expiratory pressures of coughs evoked following antagonist administration averaged 106 ± 13% of that evoked in stimulation (voltage or citric acid concentration) matched controls; P > 0.1).

Figure 7. Intracerebroventricular administration of CP99994 or SB223412 prevents tracheal capsaicin-induced sensitization of cough in anaesthetized guinea-pigs

Cough was evoked electrically from the trachea (16 Hz, 10 s train, 1–10 V). Antagonists (1 nmol μl⁻¹) or vehicle (dimethyl sulfoxide) were administered into the lateral ventricles by continuous infusion (20 μl h⁻¹) beginning 10 min prior to adding 3 μm capsaicin to the tracheal perfusate. The results are depicted as the percentage of animals coughing in response to a given stimulation voltage. Tracheal capsaicin increased the likelihood of coughing at lower stimulation intensities (2–4 V) relative to control (P < 0.001), an effect that was prevented by CP99994 and by SB223412.

Figure 8. CP99994 and SB223412 reduce peak expiratory pressures during cough initiated from the tracheal or laryngeal mucosa

Data are presented as the mean ± s.e.m. expiratory pressure during cough relative to peak tidal expiratory pressures. All animals were challenged with 3 μm capsaicin administered to the tracheal perfusate. At least one cough evoked electrically (A) or mechanically (B) in each of 5 animals in each treatment group was analysed.