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J Appl Physiol 101: 635-643, 2006. First published April 20, 2006; doi:10.1152/japplphysiol.00106.2006
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HIGHLIGHTED TOPIC
Reflexes from the Lungs and Airways

Na+-K+-2Cl– cotransporters and Cl– channels regulate citric acid cough in guinea pigs

Stuart B. Mazzone and Alice E. McGovern

Howard Florey Institute, University of Melbourne, Parkville, Victoria, Australia

Submitted 20 January 2006 ; accepted in final form 6 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Loop diuretics have been shown to inhibit cough and other airway defensive reflexes via poorly defined mechanisms. We test the hypothesis that the furosemide-sensitive Na+-K+-2Cl– cotransporter (NKCC1) is expressed by sensory nerve fibers innervating the airways where it plays an important role in regulating sensory neural activity. NKCC1 immunoreactivity was present on the cell membranes of most nodose and jugular ganglia neurons projecting to the trachea, and it was present on the peripheral terminals of putative mechanosensory nerve fibers in the airways. In urethane-anesthetized, spontaneously breathing guinea pigs, bolus application of citric acid (1 mM to 2 M) to an isolated and perfused segment of the tracheal mucosa evoked coughing and respiratory slowing. Removal of Cl– from the tracheal perfusate evoked spontaneous coughing and significantly potentiated cough and respiratory slowing reflexes evoked by citric acid. The NKCC1 inhibitor furosemide (10–100 µM) significantly reduced both the number of coughs evoked by citric acid and the degree of acid-evoked respiratory slowing (P < 0.05). Localized tracheal pretreatment with the Cl– channel inhibitors DIDS or niflumic acid (100 µM) also significantly reduced cough, whereas the GABAA receptor agonist muscimol potentiated acid-evoked responses. These data suggest that vagal sensory neurons may accumulate Cl– due to the expression of the furosemide-sensitive Cl– transporter, NKCC1. Efflux of intracellular Cl–, in part through calcium-activated Cl– channels, may play an important role in regulating airway afferent neuron activity.

airway; cough receptor; furosemide; sensory nerves; apnea

FUROSEMIDE AND OTHER LOOP diuretics relieve a variety of asthmatic symptoms, including airway obstruction evoked by allergen, bradykinin, distilled water, exercise, cold air, and hypernea in humans (12, 24, 32, 33, 36, 38, 47). Loop diuretics also reduce cough evoked by citric acid, low-Cl– solutions, and angiotensin-converting enzyme inhibitors in conscious guinea pigs and humans, and they alleviate the sensations of dyspnea in humans (13, 15, 20, 31, 34, 49, 54). Although little is known about the specific mechanisms underlying these beneficial effects of loop diuretics, inhibition of a neuronally expressed Cl– transporter in the airways may be involved. For example, furosemide prevents the activation of laryngeal and tracheobronchial sensory nerves by solutions deficient in Cl– (14, 16, 45) and selectively reduces the respiratory-related activity of rapidly adapting airway mechanosensors (50). Furthermore, furosemide inhibits neurally mediated contractions of guinea pig and human airways via prejunctional mechanisms (11, 55).

Studies of the somatic nervous system also suggest that loop diuretics inhibit sensory nerves. Many neurons in the dorsal root ganglia (DRG) express the electroneutral, furosemide-sensitive Na+-K+-2Cl– cotransporter (NKCC1) (1, 51). NKCC1 functions to accumulate intracellular Cl– above the electrochemical equilibrium in DRG neurons (51). Under these circumstances, the opening of membrane Cl– channels evokes an inward (depolarizing) current, distinct from the typical hyperpolarizing current mediated by Cl– channels in most other mature neuron subtypes. Indeed, GABA-evoked primary afferent depolarization is a major mechanism inhibiting neurotransmitter release from the central projections of DRG neurons in the spinal cord (reviewed in Ref. 44). In the presence of furosemide or in NKCC1 knockout mice, DRG neurons are unable to maintain their high intracellular Cl– concentration and as a consequence normal sensory driven processes are diminished (17, 22, 51). Similar observations have also been made for olfactory sensory neurons (19, 40, 41).

Less is known about the regulation of Cl– by Cl– transporters in vagal sensory nerves. Muscimol and GABA depolarize neurons in guinea pig and rabbit nodose and jugular ganglia, consistent with a high intracellular Cl– concentration in these cells (23, 56). In guinea pigs, bradykinin depolarizes subsets of vagal sensory neurons, a response that is reduced by a variety of Cl– channel blockers (23, 37). This suggests that an inward Cl–-mediated current is involved in bradykinin-evoked nerve activation. In the present study, we test the hypothesis that NKCC1 is expressed by vagal sensory neurons innervating the airways. We also assess whether Cl– channels likely expressed by airway sensory nerve terminals play an important role in regulating defensive respiratory reflexes. Some of the results of these studies have been previously reported in the form of an abstract (28).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
The Howard Florey Institute Animal Ethics Committee approved all experiments conducted in this study. Experiments were performed on male albino Hartley guinea pigs [200–300 g (mean = 287 ± 13 g); n = 69], IVMS, South Australia, Australia).

Retrograde Tracing and Immunohistochemical Staining of Vagal Ganglia and Trachea

Guinea pigs (n = 3) were anesthetized with ketamine (50 mg/kg im) and xylazine (2.5 mg/kg im), and 5 µl of a 1% solution of cholera toxin B subunit conjugated to Alexa Fluor 594 (Molecular Probes, Eugene, OR) were injected into the lumen of the extrathoracic trachea via a small incision in the neck (26). Animals were allowed to recover for 8 days, at which time they were deeply anesthetized with sodium pentobarbital (100 mg/kg) and transcardially perfused with 10 mM PBS followed by 4% paraformaldehyde. In some experiments (n = 5), guinea pigs were anesthetized and perfused without injection of retrograde tracer. Nodose and jugular ganglia were removed, rapidly frozen, and cut (16 µm) using a cryostat. Slide-mounted sections were incubated for 1 h in blocking solution (PBS and 10% horse serum) and then overnight (at room temperature) with antisera raised against the two known isoforms of NKCC: rabbit anti-NKCC1 (1:1,000, gift from R. J. Turner, National Institute of Dental and Craniofacial Research, Bethesda, MD) (21) or rabbit anti-NKCC2 (1:1,000, gift from M. Knepper, National Heart, Lung, and Blood Institute, Bethesda, MD) (10). Staining was detected with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Molecular Probes, Eugene, OR) and visualized using an Olympus BX51 microscope equipped with an Optronics digital camera. Negative-control experiments, in which the primary antibody was replaced with nonimmune serum, were conducted where appropriate.

For staining of nerve fibers in tracheal whole mounts, guinea pigs (n = 4) were deeply anesthetized with pentobarbital sodium (100 mg/kg ip) and transcardially perfused with 10 mM PBS. The entire trachea was removed, cleaned of excess connective tissue, and opened longitudinally via a ventral incision. The trachea was then pinned flat (mucosal side up) to a piece of cork board and fixed (2–3 h, 4°C) in 4% paraformaldehyde. The tissue was then incubated (en bloc) overnight (37°C) with rabbit anti-NKCC1 (1:1,000), and either rat anti-substance P (1:200, Chemicon, Boronia, Victoria, Australia) to visualize capsaicin-sensitive nociceptor nerve terminal (3, 53) or mouse anti-{alpha}3-Na+-K+-ATPase (1:400, Biomol, Plymouth Meeting, PA) to visualize low-threshold mechanosensors (5, 6, 58). Tissues were then washed and incubated (1 h at room temperature) with Alexa Fluor 594-conjugated goat anti-rabbit IgG and either Alexa Fluor 488-conjugated goat anti-rat or goat anti-mouse IgG (1:200; Molecular Probes), and visualized as described above.

Cough and Respiration in Anesthetized Guinea Pigs

The methods for studying cough in urethane (1.2–1.5 g/kg ip)-anesthetized spontaneously breathing guinea pigs have been described elsewhere (7). The caudal extrathoracic trachea was cannulated, and the tracheal and laryngeal mucosa (rostral to the cannula) were superfused (4 ml/min) with 37°C, oxygenated Krebs bicarbonate buffer [composition (in mM): 118 NaCl, 5.4 KCl, 1 NaHPO4, 1.2 MgSO4, 1.9 CaCl2, 25 NaHCO3, and 11.1 dextrose, containing 3 µM indomethacin] (25). The pH of the buffer was kept consistent at 7.35–7.45 in all experiments. The buffer was collected by gentle suction at the rostral end of the larynx. Respiratory pressures were amplified (Neurolog System, Digitimer, Hertfordshire, UK), digitized (Micro1401 A-D converter, CED, Cambridge, UK) and recorded using Spike II software (CED). At the end of each experiment, animals were killed by an intracardiac injection of anesthetic.

Respiratory reflexes (cough and respiratory slowing) were evoked by applying 100-µl aliquots of increasing concentrations of citric acid (1 mM to 2 M) to the bypassed segment of trachea at 2-min intervals (7). We first assessed the effect of reducing the Cl– content of the tracheal perfusion buffer on evoked reflexes. Low (3.8 mM)- or normal (127 mM)-Cl– buffer (n = 5 each) was superfused over the tracheal mucosa for 5 min before initiation of acid challenges. Low-Cl– buffer was prepared by replacing 118 mM sodium chloride with an isoosmotic concentration of sodium gluconate. In subsequent experiments, we treated animals with the NKCC1 inhibitor furosemide (10–100 µM, n = 4–6), the Cl– channel inhibitors DIDS or niflumic acid (100 µM, n = 5 each) or the GABAA receptor agonist muscimol (10 µM, n = 5). Agents were added to the tracheal perfusate 10 min before acid challenges. Drug concentrations employed were based on preliminary experiments and other published studies (8, 23). Vehicle control animals were studied in parallel (n = 7–9).

After citric acid challenges in all animals, we attempted to evoke cough via mechanical stimulation of the trachea and larynx. Mechanical stimulation was conducted over 5 s using 10 individual punctate stimuli along the rostrocaudal extent of the extrathoracic trachea and larynx with a blunt probe that provides a supramaximal mechanical force for activating all airway afferents (7).

Drugs and Reagents

Indomethacin, furosemide, DIDS, niflumic acid, and muscimol, were all purchased from Sigma (St. Louis, MO). Concentrated stock solutions were made (10–100 mM) of all drugs added to the tracheal perfusate: indomethacin (30 mM) and niflumic acid (100 mM) were dissolved in ethanol; DIDS (100 mM) was dissolved in DMSO.

Data Analysis

Cough was defined as a ≥500% increase in peak expiratory pressure preceded by an enhanced inspiratory effort and was readily differentiated from augment breaths by the rate and magnitude of the effort (7). Data are presented as the cumulative number of coughs evoked over an entire citric acid dose-response curve and the threshold concentration of citric acid required to evoke cough. Peak expiratory pressures during cough (a measure of cough quality) were expressed as a percentage of tidal expiratory pressures as described previously (29). Baseline respiratory rates and inspiratory (TI), expiratory (TE), and postexpiratory pause times (TP) were calculated from 10-s bins 1 min before and/or after a given challenge. Apnea was defined as a cessation of breathing lasting longer than 1 s immediately after a cough effort. Data are presented as means ± SE, and statistical differences between groups are compared using analysis of variance (Systat Software, Richmond, CA). Tukey tests were performed when multiple comparisons were appropriate. {chi}2 Analysis was used to compare the percentage of animals responding. P < 0.05 was considered statistically significant. Occasionally (<10% of experiments), the baseline respiratory rate was below 40 breaths/min, and animals failed to cough in response to any stimuli. In such cases, animals were excluded from subsequent analyses.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Immunohistochemical Localization of NKCC1 and NKCC2 in Guinea Pig Airway Sensory Neurons

Retrograde tracing from the trachea with cholera toxin B revealed tracheal-projecting sensory neurons in the jugular and nodose ganglia of all animals studied (Fig. 1). Immunostaining for NKCC1 revealed that 83.9 ± 0.8% of jugular ganglia neurons projecting to the trachea and that 92.9 ± 0.9% of nodose ganglia neurons projecting to the trachea express detectable levels of NKCC1 immunoreactivity (Fig. 1). Close inspection of the staining revealed that NKCC1 immunoreactivity was typically concentrated to the plasma membrane of ganglia neurons (Fig. 1, insets). In tracheal whole mounts, NKCC1 immunoreactivity was readily observed on subsets of mucosal nerve fibers (Fig. 2). These fibers coexpressed the {alpha}3-subunit of Na+-K+-ATPase but not the neuropeptide substance P (Fig. 2). Interestingly, NKCC1 staining was not uniform along the entire nerve terminal, but rather it was concentrated on the axon and primary arborizations of {alpha}3-Na+-K+-ATPase-positive fibers. Exclusion of the NKCC1 primary antisera prevented staining in all preparations tested (not shown). NKCC2 immunoreactivity was undetectable in vagal ganglia (Fig. 1, C and D) and tracheal whole mounts (not shown).


Figure 1
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  		Fig. 1. Photomicrographs showing the expression of Na+-K+-2Cl– cotransporters (NKCC1 and NKCC2) in the nodose and jugular ganglia. Nodose (A) and jugular (B) neurons projecting to the trachea were visualized by retrograde tracing with cholera toxin B subunit (CTb; arrows). NKCC1 expression (A’ and B’) was present in the majority of ganglia neurons. Insets, high-power magnification of NKCC1 expression on the cell membrane of retrogradely labeled neurons. NKCC2 immunoreactivity was not present in the nodose (C) or jugular (D) ganglia. Scale bars = 50 µm (A, A’, B, and B’) and 200 µm (C, D).

 

Figure 2
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  		Fig. 2. Photomicrographs showing NKCC1 expression by sensory nerve fibers in whole-mount preparations of the guinea pig trachea. Nodose ganglia-derived low-threshold mechanoosensitive nerve fibers were visualized using immunoreactivity for the {alpha}3-subunit of Na+-K+-ATPase (A and B). Jugular ganglia-derived nociceptors were visualized using immunoreactivity for the neuropeptide substance P (C). NKCC1 is expressed in the primary arborizations of {alpha}3-Na+-K+-ATPase-immunoreactive nerve fibers (A’, A’’, B’, and B’’) but not in fibers immunoreactive for substance P (C’ and C’’). In C’, the arrow shows an NKCC1-positive axon that clearly is devoid of substance P immunoreactivity (C and C’’). Scale bar = 100 µm (A and B) and 50 µm (C). See text for further details.

 
Effect of Low Cl– on Citric Acid-Evoked Cough and Respiratory Slowing in Anesthetized Guinea Pigs

Baseline (tidal) respiration in anesthetized guinea pigs was spontaneous and rhythmical and was characterized by an inspiratory phase, expiratory phase, and a postexpiratory pause (Fig. 3). In all animals used in this study (n = 57), baseline breathing parameters were as follows; expiratory pressure (1.49 ± 0.18 cmH2O), respiratory rate (55.9 ± 0.7 breaths/min), TI (0.21 ± 0.01 s), TE (0.36 ± 0.01 s), and TP (0.68 ± 0.04 s) (n = 57). There were no detectable differences in baseline respiration between any of the groups studied (not shown).


Figure 3
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  		Fig. 3. Representative trace (original record) showing the effect of bolus application of 1 M citric acid to the tracheal mucosa in urethane-anesthetized, spontaneously breathing guinea pigs. At this concentration, citric acid evokes 1 or more coughs followed by a period of respiratory cessation (apnea). At lower concentrations of citric acid a transient, dose-dependent reduction in respiratory rate, rather than apnea, follows coughing (not shown).

 
Superfusion of the extrathoracic tracheal mucosa with Krebs buffer containing the normal-Cl– content produced no detectable changes in respiratory pattern (not shown). As previously reported (7), bolus application of increasing concentrations of citric acid evoked coughing in all control animals studied (Figs. 3 and 4, Table 1). Animals typically responded with one or more coughs to each citric acid challenge, once a given threshold was reached (Fig. 4). Cough was immediately followed by a transient dose-dependent reduction in respiratory rate (and eventually apnea at high concentrations of citric acid) that recovered completely prior to subsequent acid challenges (Fig. 3, Table 2). Respiratory slowing in response to acid challenges was primarily due to an elongation of TP (e.g., TI = 0.22 ± 0.02 s; TE = 0.42 ± 0.02 s, and TP = 1.12 ± 0.11 s after 100 mM citric acid, compared with baseline parameters described above; n = 21). In control animals, the concentrations of citric acid that were threshold for the appearance of cough and the respiratory slowing reflex were similar (16.5 ± 2.6 and 22.0 ± 2.5 mM, respectively).


Figure 4
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  		Fig. 4. Representative traces showing citric acid and mechanically evoked cough in control (top), low Cl–-treated (middle) and 100 µM furosemide-treated (bottom) guinea pigs. In these experiments, normal Krebs-bicarbonate buffer or low-Cl– buffer was superfused continuously over an isolated segment of the extrathoracic trachea. Cough was evoked by bolus application of 100 µl of increasing concentrations of citric acid (1–2,000 mM) to the tracheal mucosa. Furosemide and other pharmacological treatments were added directly to the tracheal perfusate 10 min before the initiation of citric acid challenges. Low-Cl– Krebs buffer was prepared by replacing 118 mM sodium chloride with an isoosmotic concentration of sodium gluconate. Note the spontaneous coughs (arrows) in the presence of low-Cl– buffer and the reduction in coughing in the presence of furosemide. At the end of each citric acid dose-response curve, punctate mechanical stimulation of the tracheal mucosa was used to assess mechanically evoked coughing (Mech). Traces are representative of 5–9 similar experiments and are ~40 min in duration. Mean data are presented in Tables 1–3.

 

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  		Table 1. Pharmacological modulation of citric acid-evoked cough in anesthetized guinea pigs

 

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  		Table 2. Duration of citric acid-evoked apnea

 
Superfusion of the trachea with low-Cl– buffer evoked a modest change in baseline breathing pattern (not shown). Whereas no animals coughed in response to normal-Krebs buffer perfusion alone (n = 57), low-Cl– buffer evoked spontaneous coughing within 1 min of the challenge in three of the five animals tested (Fig. 4). In addition, superfusion of the trachea with low-Cl– buffer significantly (P < 0.05) lowered the threshold for citric acid-evoked respiratory reflexes (Fig. 4, Tables 1 and 2). For example, whereas no control animals (n = 5) coughed after challenge with 1 or 2 mM citric acid and only one animal coughed in response to 5 mM, in the low-Cl–-challenged group (n = 5) three animals coughed at 1 mM, four animals coughed at 2 mM, and all animals coughed at 5 mM citric acid. The threshold concentration for citric acid-evoked cough was potentiated at least eightfold lower in the animals treated with low-Cl– buffer (P < 0.05, Table 1). Low-Cl– buffer also reduced the threshold for the onset of citric acid-evoked respiratory slowing. Whereas control animals showed no signs of respiratory slowing in response to 1 mM citric acid, this concentration of citric acid evoked a 5–10% reduction in respiratory rate in low-Cl–-treated animals (Fig. 5A). Furthermore, in the presence of low Cl–, 10 mM citric acid evoked an apnea lasting 2.2 ± 0.4 s, compared with 0 ± 0 s in the control animals (Table 2).


Figure 5
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  		Fig. 5. Effect of low-Cl– buffer (A), 100 µM furosemide (B), 100 µM DIDS (C), or 100 µM niflumic acid (D) and 10 µM muscimol on citric acid-evoked respiratory (Resp) slowing in anesthetized guinea pigs. Values are means ± SE of 5–9 experiments. *Significantly different from vehicle control, P < 0.05.

 
The potentiation of citric acid-evoked cough and respiratory slowing responses was not sustained in the continued presence of low-Cl– buffer. For example, the higher doses of citric acid (0.5–2 M, given ~24–30 min after the onset of the experiment) tended to readily evoke cough in control animals but often failed to evoke cough in the presence of low Cl–. Similarly, respiratory slowing and apnea evoked by citric acid were significantly potentiated in the early stages of the experiments but significantly reduced in the latter stages (Figs. 4 and 5A, Table 2).

Effect of Furosemide on Citric Acid-Evoked Cough and Respiratory Slowing in Anesthetized Guinea Pigs

Tracheal pretreatment with furosemide (10–100 µM) produced no detectable changes in baseline respiration. Furosemide, however, evoked a dose-dependent reduction in the number of coughs evoked by citric acid (Fig. 4, Table 1). For example, ~90% of control animals but only 25% of animals pretreated with 100 µM furosemide coughed in response to 20 mM citric acid. Pretreatment with 100 µM furosemide also reduced the magnitude of the respiratory slowing response and the duration of apnea evoked by citric acid (Fig. 5B, Table 2).

Effect of DIDS, Niflumic Acid, and Muscimol on Citric Acid-Evoked Cough and Respiratory Slowing in Anesthetized Guinea Pigs

The Cl– channel blockers DIDS and niflumic acid (both tested at 100 µM) reduced the number of coughs evoked by citric acid (Table 1). DIDS appeared to be the more effective inhibitor at this concentration, reducing cough by >50% (P < 0.05, significantly different from control). A similar inhibitory effect was also observed with respect to the acid-evoked respiratory slowing and apnea, although DIDS was less effective then niflumic acid at inhibiting the duration of apnea evoked by 1 M citric acid (Fig. 5C, Table 2).

By contrast, the GABAA receptor agonist muscimol (10 µM) significantly potentiated acid-evoked reflexes (Fig. 5D, Tables 1 and 2). For example, only 15 and 40% of control animals coughed in response to 2 and 5 mM citric acid, compared with 75 and 100% of muscimol-treated animals. Muscimol also significantly potentiated the respiratory slowing and apnea responses, although this was only evident at the lower concentrations of citric acid employed (Fig. 5D, Table 2). Unlike in the presence of low Cl–, muscimol-treated animals did not cough spontaneously in the absence of acid challenges.

Effect of Low Cl–, Furosemide, DIDS, and Niflumic Acid on Cough Intensity in Anesthetized Guinea Pigs

In addition to assessing the effect of the various pretreatments on the quantity of coughs evoked by citric acid, we also compared individual cough magnitudes after citric acid and mechanical challenges as an index of cough quality. Bolus application of citric acid to the tracheal mucosa evoked coughs with maximum expiratory intensity that was ~1,100% of tidal expirations (Table 3). Furosemide treatment produced a modest but significant (P < 0.05) reduction in citric acid cough intensity. Citric acid-evoked cough intensity was not significantly altered in the presence of any other treatments (Table 3). Supramaximal mechanical stimulation of the trachea with a blunt probe (after the completion of citric acid dose-response curves) also evoked coughs (Fig. 4), albeit with slightly lower maximum intensity compared with citric acid (Table 3). Mechanically evoked cough intensity was not altered by furosemide, DIDS, niflumic acid, or muscimol. Although most animals responded with a coughlike respiratory response to mechanical stimulation in the presence of low-Cl– buffer, the magnitude of the expiratory effort was on average below our predetermined definition of a cough (Table 3).


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  		Table 3. Peak expiratory pressures associated with citric acid- and mechanically evoked coughing in anesthetized guinea pigs

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGMENTS
 REFERENCES
 
In the present study, we show that sensory neurons originating in the nodose and jugular, and projecting to the guinea pig trachea, express the furosemide-sensitive Cl– cotransporter NKCC1. In the tracheal submucosa, NKCC1 was observed on a subset of putative mechanosensitive nerve fibers. Our functional studies indicate that a reduction in extracellular Cl– concentration or the opening of membrane Cl– channels increases the excitability of airway afferent nerve fibers, whereas inhibition of NKCC1 or blockade of Cl– channels significantly inhibits airway sensory neuron-mediated functions in anesthetized guinea pigs. These effects generally manifested as an alteration in cough number and/or magnitude of respiratory slowing evoked by citric acid, rather than an effect on the quality (size) of individual cough efforts. Accumulation of intracellular Cl– in peripheral sensory nerve terminals via the actions of NKCC1, and the subsequent efflux of intracellular Cl– through membrane Cl– channels, may be involved in the activation of airway sensory nerve terminals by acidic stimuli in vivo. These data may explain in part the inhibitory effects that loop diuretics have on cough and other symptoms associated with airway diseases.

Sensory Pathways Involved in Citric Acid-Evoked Respiratory Reflexes in Guinea Pigs

Previous reports have described in detail the sensory pathways involved in acid-evoked cough in guinea pigs (7, 27, 29). In conscious and anesthetized guinea pigs, transient bolus application of acid to the laryngeal or tracheal mucosa evokes cough via capsaicin-insensitive (mechanically sensitive) vagal afferent neurons (7, 52). These putative cough receptors are readily differentiated from other types of airway and intrapulmonary mechanosensitive afferent fiber types (for review see Ref. 27). They are highly sensitive to light punctate mechanical stimuli and transient airway acidification, originate in the nodose ganglia, and innervate only the extrapulmonary airways (7). Cough receptors are not activated by airway stretch, bronchoconstriction, or other stimuli that activate intrapulmonary rapidly and slowly adapting stretch receptors (7). The sensory pathways that mediate respiratory slowing and transient respiratory arrest with increasing concentrations of citric acid are not well defined, but they may occur secondary to activation of capsaicin-sensitive C fibers that innervate the guinea pig trachea. Consistent with this application of capsaicin to the trachea or larynx in anaesthetized guinea pigs similarly evokes respiratory slowing and apnea (7, 29). Unlike cough receptors, tracheal capsaicin-sensitive afferent nerves (nociceptors) originate in the jugular ganglia and display a very low sensitivity to mechanical stimuli (7, 29, 42).

NKCC1 and Airway Sensory Neuron Excitability

Two NKCC isoforms exist (NKCC1 and NKCC2), differing in their tissue and cellular distribution. NKCC2 has not yet been identified outside of the kidney. NKCC1, on the other hand, is present in the kidney but in addition is expressed by several other tissues and cell types, including neurons. In the DRG, most cells express detectable levels of NKCC1, and the reversal potential for Cl– is consistent with intracellular accumulation in these neurons (1, 51). GABA depolarizes DRG neurons via Cl– efflux through GABAA receptors, a response that is absent in NKCC1 knockout mice or in the presence of furosemide (17, 22, 51). Similar observations have been made in olfactory sensory neurons (19, 40, 41). Our data showing NKCC1 (but not NKCC2) on the plasma membrane of neurons with their soma in the vagal sensory ganglia suggest that NKCC1 expression is perhaps a common feature of all mammalian somatosensory and viscerosensory neurons, regardless of their sensory modality.

Although the role that NKCC1 plays in central presynaptic inhibition and nociception is well described (48), less is known about the possible regulatory role that NKCC1 plays on the excitability of the peripheral projections of sensory nerves fibers. Circumstantial evidence suggests that peripheral sensory terminals express NKCC1. GABAA receptors are expressed on nerve terminals in the cat paw, and exogenous administration of GABAA agonists excites primary afferent fibers in this tissue (8). In rats, local peripheral administration of loop diuretics reduces pain-related behavioral responses (17), and immunohistochemical experiments have detected NKCC1 in peripheral myelinated axons of rat and cat somatosensory nerves and on the soma of mouse olfactory receptor neurons (1, 41). With respect to the airways, furosemide has been shown to inhibit sensory nerve activation by a variety of stimuli (11, 14, 16, 45, 50). Our data indicate that muscimol and low extracellular Cl– concentrations increase the excitability of airway afferent nerves, thereby potentiating acid-evoked defensive reflexes. Conversely, furosemide applied topically to the airway mucosa dose dependently reduced afferent responses. This is strongly suggestive that peripheral afferent nerve terminals in the airways accumulate Cl– via a furosemide-sensitive mechanism.

We directly observed a subset of subepithelial nerve fibers in the airways that express NKCC1 immunoreactivity. These fibers were presumably associated with tracheal mechanosensors (cough receptors) because they coexpressed the {alpha}3-subunit of Na+-K+-ATPase (5, 6, 58). It is intriguing that NKCC1 immunoreactivity was not uniformly expressed over the entire mechanosensor nerve terminal, but rather it was limited to the axon and primary arborizations. This may suggest that pump activity in the major nerve branches and subsequent passive intracellular equilibration are responsible for elevating Cl– concentration in the terminal boutons. It is also interesting that substance P-containing nociceptors innervating the trachea do not express detectable levels of NKCC1 on their peripheral terminals, despite the majority of their soma in the jugular ganglia displaying NKCC1 immunoreactivity. NKCC1 protein may not be transported peripherally in these fibers. Nevertheless, the present study showing that furosemide and Cl– channel inhibitors prevent citric acid-evoked respiratory slowing and previous data suggesting that Cl– channel inhibitors reduce bradykinin-evoked action potential formation in capsaicin-sensitive tracheal nerve fibers (23) suggest that Cl– are also elevated in jugular ganglia-derived nociceptor terminals. Whether intracellular diffusion of Cl– from NKCC1 activity at a more proximal site on the axon, or an alternative (furosemide sensitive) mechanism not involving NKCC1 is involved in Cl– accumulation in these fibers, is unknown.

The effects of low-Cl– buffer observed in the present study deserve further discussion. Removal of Cl– initially potentiated acid-evoked respiratory reflexes. However, in the continued presence of low- Cl–, respiratory responses were dramatically reduced. The initial potentiation of cough and respiratory slowing supports the hypothesis that airway afferent nerves accumulate Cl–. Rapid removal of extracellular Cl– would be expected to depolarize nerve terminals and increase excitability (41, 51). The mechanism underlying the reduction in reflex responsiveness that was observed in the continued presence of low-Cl– buffer is unknown. Low-Cl– buffer may eventually evoke a generalized desensitization of airway afferent terminals, perhaps due to the depletion of intracellular Cl–, or secondary effects on other ion concentrations. In this regard, it is interesting that the loss of airway afferent nerve responsiveness was not limited to acid-evoked reflexes, because we also observed a reduction in the magnitude of mechanically evoked coughs after prolonged removal of extracellular Cl–. Also of interest is the observation that muscimol was less effective at potentiating respiratory depression than cough. Although the mechanism underlying this observation is unknown, the differential effect of muscimol supports the hypothesis that distinct afferent pathways mediate the two reflexes evoked by citric acid.

Role of Cl– Channels in Acid-Evoked Responses

The expression of NKCC1 by airway afferent nerves suggests that afferent excitability may involve an efflux of intracellular Cl– through membrane Cl– channels (9). Consistent with this, acid-evoked respiratory responses were significantly inhibited by the nonselective anion channel blocker DIDS and by the selective calcium-activated Cl– channel blocker niflumic acid. Calcium-activated Cl– channels have previously been shown to play a role in citric acid-evoked activation of taste cells, as well as the activation of DRG and olfactory neurons by various stimuli (2, 30, 40). In addition, niflumic acid and the related Cl– channel blocker 2-(3-phenylpropylamino)benzoic acid inhibit bradykinin-evoked activation of airway vagal afferents (23, 37). These data would argue that calcium-activated Cl– channels likely play a key role in vagal (and other) sensory neuron excitability. Nevertheless, a role for alternative types of Cl– channels cannot be excluded. Extracellular acidification of HEK-293 has been shown to open volume-activated Cl– channels, also sensitive to hypotonic stimuli (35). However, acid was shown to render the selective volume-activated Cl– channel blocker tamoxifen inactive, making it difficult to study pharmacologically the involvement of this channel in acid-evoked responses (35). It should also be noted that acid-evoked activation of airway afferent nerves is unlikely solely mediated by a Cl– current, because Cl– channel blockers never abolished reflexes. Furthermore, mechanically evoked cough appeared to be unaffected by Cl– channel blockers (or furosemide), suggesting that not all stimuli depend on a Cl– current to activate peripheral sensory nerve terminals in the airways.

Significance

The mechanisms that underlie altered neural activity in abnormal pain states and some symptoms in airways disease are similar (4, 18, 26, 42, 43, 46, 57). Inflammation has been shown to evoke upregulation of NKCC1 expression in somatosensory neurons, leading to an overactivity of the neural pathways involved in signaling pain-related information (39). Inhibition of NKCC1 may therefore represent a novel approach for the treatment of hyperalgesia. Although it is not known whether neuronal NKCC1 is upregulated during airways inflammation, the present and published data showing that loop diuretics effectively reduce many asthmalike symptoms supports this hypothesis (12, 13, 20, 31–34). Furthermore, our data suggest that neuronal Cl– channels may represent an alternative novel target for relieving symptoms of airways diseases.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
REFERENCES
 
This research was funded by National Health and Medical Research Council of Australia Grants 350333 and 007188 (to S. B. Mazzone).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. B. Mazzone, Howard Florey Institute, University of Melbourne, Parkville 3010, Victoria, Australia (e-mail: s.mazzone{at}hfi.unimelb.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
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