jpet #230383 1 title page pharmacology of bradykinin

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TITLE PAGE

PHARMACOLOGY OF BRADYKININ EVOKED COUGHING IN GUINEA PIGS.

Matthew M. Hewitt, Gregory Adams, Jr. , Stuart B. Mazzone, Nanako Mori, Li Yu and Brendan

J. Canning

The Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland (GA, NM, BJC)

University of Pennsylvania, Philadelphia, PA (MMH)

University of Queensland, Australia (SBM)

Department of Respiratory Medicine, Tongji Hospital, Tongji University School of Medicine,

Shanghai, China (LY)

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on March 21, 2016 as DOI: 10.1124/jpet.115.230383

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Running Title Page

Running title: Bradykinin evoked coughing

Send correspondence to:

Brendan J. Canning, Ph.D., Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, Phone: 410-550-2156, Fax: 410-550-2130, e-mail: [email protected]

Text pages: 27 Tables: 1 Figures: 6 References: 88 Words in Abstract: 219 Words in Introduction: 552 Words in Discussion: 1558

List of nonstandard abbreviations: 15-HETE: 15-Hydroxyeicosatetraenoic acid, ACE:

angiotensin converting enzyme, DiI (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine

Perchlorate), L-NNA: NG-nitro-L-Arginine, NK: neurokinin, NMDA: n-methyl-d-aspartic acid,

NO: nitric oxide, NOS: nitric oxide synthase, PGE2: prostaglandin E2, PIP: pulmonary inflation

pressure, RAR: rapidly adapting receptor


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ABSTRACT

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 L-NNA). 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 that target the actions

of bradykinin may prove useful in the treatment of cough.


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INTRODUCTION

Bradykinin is a peptide autacoid formed from precursor kininogens by tissue and plasma

peptidases. Multiple chemical insults and pathological conditions result in bradykinin

generation in tissues and on mucosal surfaces. The ability of bradykinin to initiate

vasodilatation, plasma exudation, leukocyte activation, and reflexes and sensations attributed to

the stimulation of visceral and somatic nociceptors established bradykinin as a mediator of many

acute and chronic inflammatory conditions (Joseph and Kaplan, 2005; Leeb-Lundberg et al.,

2005; Kaplan and Joseph, 2014).

Kinins have been implicated in inflammatory responses of the airways and lungs initiated

by allergen, airway acidification, cold, dry air inhalation, viral infections, gram negative bacterial

infections and in other inflammatory conditions promoting recruitment of neutrophils and/ or

eosinophils to the airways (Proud et al., 1983; Proud et al., 1988; Bertrand et al., 1993;

Ricciardolo et al., 1994; Coyle et al., 1995; Featherstone et al., 1996; Yoshihara et al., 1996;

Grünberg et al., 1997; Ricciardolo et al., 1999; Folkerts et al., 2000; Scuri et al., 2000; Turner et

al., 2001; Abraham et al., 2006; Arndt et al., 2006; Hewitt and Canning, 2010; Broadley et al.,

2010; Taylor et al., 2013; Sahoo et al., 2014). The bronchospasm, mucus secretion, airway

microvascular dilatation and plasma exudation evoked by exogenously administered bradykinin

are primarily mediated by bradykinin B2 receptor activation (Nakajima et al., 1994; Abraham et

al., 2006; Broadley et al., 2010). B1 receptor dependent effects have also been implicated in

respiratory diseases, but bradykinin has little or no affinity for B1 receptors. The actions of

bradykinin are limited by metabolizing peptidases including angiotensin converting enzyme

(ACE) and neutral endopeptidase, and by rapid B2 receptor desensitization (Wolsing and

Rosenbaum, 1993; Leeb-Lundberg et al., 2005; Broadley et al., 2010; Zimmerman et al., 2011).


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When inhaled, bradykinin causes coughing in humans and in guinea pigs (Choudry et al.,

1989; Katsumata et al. 1991; Canning et al., 2004; Grace et al., 2012; Smith et al., 2012). This

peptide autacoid may also cause the coughing associated with ACE inhibitor therapy (Fox et al.,

1996; Morice et al., 1997; Hirata et al., 2003; Dicpinigaitis, 2006; Cialdai et al., 2010; Mutolo et

al., 2010; Mahmoudpour et al., 2013). Bradykinin-induced cough likely results from its direct

effects on bronchopulmonary C-fibers (Kaufman et al.; 1980; Bergren, 1997; Kajekar et al.,

1999). But the indirect effects of B2 receptor activation might also contribute to its capacity to

initiate cough (Grace et al., 2012). For example, bradykinin induces eicosanoid formation in the

airways, including PGE2, thromboxane and 15-HETE (Salari and Chan-Yeung, 1989; Arakawa

et al., 1992). Prostanoids can both enhance and directly initiate coughing (Shinagawa et al.,

2000; Liu et al., 2001; Xiang et al., 2002; Gatti et al., 2006; Maher et al., 2009; Ishiura et al.,

2014). These effects of the bronchoconstrictor prostanoids could result from the activation of

mechanically sensitive vagal afferent nerves innervating the airways and lungs (Bergren, 1997;

Canning et al., 2001).

We have addressed the hypothesis that bradykinin evokes coughing by activating

bronchopulmonary C-fibers as well as the mechanically sensitive vagal afferents innervating the

airways and lungs. We further hypothesized that these vagal afferent nerve subtypes act

synergistically to promote coughing in response to bradykinin. On the contrary, the results

suggest dissociation of the prostanoid dependent bronchospasm evoked by bradykinin and the

prostanoid independent effect of this peptide on the cough reflex.


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METHODS

Our institutional Animal Care and Use Committees approved all of the experiments

described in this study. Male Hartley strain guinea pigs (200-400 grams, Charles River) were

purchased pathogen free and housed in accredited housing facilities with food and water

provided ad-libidum.

Bradykinin-induced coughing was studied in awake guinea pigs placed in a recording

chamber continuously filled with fresh, room temperature air. Bradykinin was delivered by

aerosol to the chambers using an ultrasonic nebulizer (particle size: <5 µm). Breathing patterns,

respiratory rate and cough were monitored visually and by measuring pressure changes within

the chambers, which were recorded digitally (Biopac Systems). Bradykinin was delivered as a

single dose (0.1-10 mg.ml-1) for 10 minutes, or with cumulatively increasing doses (1-10 mg.ml-

1), delivered for 5 minutes, with 5 minutes in between each dose. In some animals, citric acid

(0.01-0.1M) was also used to evoke cough. Results are presented as the mean±sem cumulative

coughs evoked. Differences amongst treatment groups were assessed by analysis of variance,

with differences amongst treatment groups evaluated by Scheffe’s f-test for unplanned

comparisons. Statistical significance was set at p<0.05.

We attempted to modify bradykinin-evoked coughing by drug pretreatments given

intraperitoneally or by aerosol. These interventions were chosen for their known ability to

modify bradykinin-evoked responses in the airways and lungs and were administered at doses

that were selected based on the results of previous studies or following validation studies

performed prior to the cough experiments. The neutral endopeptidase inhibitor thiorphan and the

angiotensin converting enzyme inhibitor captopril were delivered as aerosols prior to bradykinin

challenge to evaluate the role of metabolism in regulating bradykinin-evoked coughing. These


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peptidase inhibitors were dissolved in saline and administered at doses of 1 mg.ml-1, with vehicle

control experiments carried out in parallel. The bradykinin B2 receptor antagonist HOE 140 (1

mg.ml-1) was also administered by aerosol. The thromboxane/ TP receptor antagonist ICI192605

(1 mg.kg-1ip or 10 µM delivered as an aerosol for 10 minutes) and the cyclooxygenase inhibitor

meclofenamic acid (1mg.kg-1 ip), were used to measure the contribution of prostanoids in the

response to bradykinin. The nitric oxide synthase (NOS) inhibitor L-NNA (0.1mM) and the

muscarinic receptor antagonist atropine (1 mg.ml-1) were administered to modify bronchospasm

during bradykinin evoked cough. Both were administered by aerosol for 10 minutes prior to

bradykinin challenge. Atropine was dissolved in saline, while L-NNA was first dissolved in

0.1N HCl at a concentration of 0.1M, and then diluted 1000-fold in saline to the concentration

used for aerosol delivery. The role of neurokinin receptors in bradykinin evoked coughing was

determined by pretreating the animals with various combinations of neurokinin1 (NK1; SR14033

and CP99994), NK2 (SR48968) and NK3 (SR142801 and SB223412) receptor antagonists,

administered at 3 mg.kg-1 each by ip injection or by aerosol (1 mg.ml-1 each). All studies were

designed as parallel group, unpaired experiments. Vehicle control experiments for each

intervention were carried out in parallel. Drugs used in an attempt to modify bradykinin evoked

coughing were administered 10-30 minutes prior to bradykinin challenge.

We also studied bradykinin-induced bronchospasm and coughing evoked by mechanical

stimulation of the airway mucosa in anesthetized guinea pigs. Guinea pigs were anesthetized

using urethane (1.5 g.kg-1, ip), which produces a stable anesthesia lasting well beyond the

duration of these experiments. The absence of withdrawal responses or cardiovascular responses

to a sharp pinch of a hindlimb was used to determine the adequacy of the anesthesia. Although

no animals required additional anesthesia in these experiments, supplemental urethane would


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have been given had arousal been noted. Once the animals were anesthetized and placed supine

on a warming pad, a midline incision exposed the trachea, which was cannulated at its caudal

most end. To study mechanically-induced cough, we either probed the laryngeal mucosa with a

von Frey filament (producing >1mN of force), or mechanically stimulated the intrathoracic

trachea and carina by threading a length of 6-0 suture through the tracheal cannula and towards

the carina (Canning et al. 2004). These stimuli typically evoke a single cough and no more than

2 and so results are presented as a percentage of the animals coughing.

Bradykinin-induced bronchospasm was studied as described previously (Canning et al.,

2001). Once the trachea had been cannulated, the animals were paralyzed with succinylcholine

(2.5 mg.kg-1, s.c.). Guinea pigs were then mechanically-ventilated (60 breaths min, 6 ml.kg-1

body weight tidal volume, 3-5 cmH2O of positive end-expiratory pressure (to limit the prominent

gas trapping that occurs during bronchospasm in guinea pigs; Stengel et al., 1995)). These

ventilation parameters created a baseline peak pulmonary inflation pressure (PIP) of 8-12

cmH2O. The abdominal aorta and vena cava were exposed by abdominal incision and

cannulated. Blood pressure and heart rate were monitored using a pressure transducer connected

to the cannula in the aorta. To assess adequacy of anesthesia following paralysis, we monitored

changes in heart rate and blood pressure in response to a sharp pinch of a forelimb. Additional

anesthetic would have been provided if responses to the pinches were noted (no animals required

additional anesthetic). Bradykinin (0.1-2 nmol.kg-1) was administered intravenously to evoke

bronchospasm, which was monitored by recording PIP with a pressure transducer connected to a

side-port of the tracheal cannula. We use PIP as a surrogate measure of bronchospasm and

interpret changes in PIP as the net effects of interventions on airways resistance and lung

compliance (Arakawa et al., 1992; Broadley et al., 2010; Keir et al., 2015). Heart rate, blood


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pressure and pulmonary inflation pressures were recorded digitally (Biopac). Doses were

administered at 5 minute intervals, with volume histories (1-2 tidal breath holds given by

preventing consecutive lung deflations) used to reverse any residual airways obstruction 1

minute prior to the administration of each dose. At the end of these experiments, guinea pigs

were asphyxiated by carbon dioxide, followed by exsanguination.

The effects of bradykinin on [Ca++] measured intracellularly were recorded in vitro in

retrogradely labeled vagal afferent neurons acutely dissociated from the nodose and jugular

ganglia. Guinea pigs (150-200g) were anesthetized with ketamine and xylazine (60 and 6mg.kg-

1, s.c.). Once anesthetized and placed supine on a warming pad, the neck was shaved and a small

(5-10 mm) incision was made in the neck to expose the trachea. The neuronal tracer DiI (1,1'-

Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate) was injected (3-4 µL) into 3

locations in the extrapulmonary airways (larynx, extrathoracic trachea, carina) using a Hamilton

syringe with 30 gauge needle. The incisions were sutured shut, coated with betadine and the

animals allowed to recover under close observation. After 2-3 weeks, the animals were

euthanized and the nodose and jugular ganglia removed. The ganglia neurons were dissociated

and adhered to coverslips in culture media overnight. Neurons retrogradely labeled from the

airways were visualized by fluorescent microscopy. Ca++ influx was recorded in these

retrogradely labeled neurons using a Fura-based assay as described elsewhere (Lee et al., 2005).

Responses were normalized to the effects of ionomycin.

Reagents

Atropine, bradykinin, captopril, citric acid, ionomycin, L-NNA, meclofenamic acid,

succinylcholine, thiorphan, urethane and xylazine were purchased from Sigma (St. Louis, MO).

ICI 192, 605 was purchased from Tocris. U46619 was purchased from Cayman (Ann Arbor,


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MI). Glaxosmithkline and Sanofi-Aventis kindly provided CP99994, SB223412, SR48968,

SR142801 and SR140333. HOE 140 was a generous gift from Hoechst. Drugs were dissolved

in 0.9% saline solutions except the neurokinin receptor antagonists, which were dissolved

initially in DMSO (30 mg.ml-1) and then further diluted into saline. Drugs were delivered by

aerosol (thiorphan, captopril, L-NNA, atropine, HOE140) or were administered by ip injection,

with injection volumes <500 µL.


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RESULTS

Bradykinin evokes paroxysmal coughing: Role of B2 Receptors and modulation by peptidases.

When delivered as an aerosol to awake guinea pigs, bradykinin (1-10 mg.ml-1) evoked

coughing in a dose-dependent manner. With no pretreatments (e.g. peptidase inhibitors) at the

outset of experimentation, 10 minute aerosol challenges with single doses of 0.1, 1 and 3 mg.ml-1

bradykinin were in most animals subthreshold for initiating cough. Only aerosol doses of 5 (9±4

coughs; n=7) and 10 (14±3 coughs; n=20) mg.ml-1 bradykinin reliably evoked cough in control

animals. The bradykinin B2 receptor antagonist HOE 140 (1 mg.ml-1 delivered as an aerosol)

completely abolished the cough responses evoked by bradykinin (figure 1).

As shown previously (Canning et al., 2004; Smith et al., 2012), bradykinin-induced

coughing occurred in repetitive, paroxysmal patterns, often with few or no tidal breaths

separating each cough effort. This occasionally produced expiration reflexes in lieu of coughing,

occurring when the animals had initiated a cough before much if any inspiratory efforts had been

completed at the end of the preceding cough. Initial, subthreshold challenges with bradykinin

appeared to sensitize the airways to higher concentrations of the kinin. Thus, as illustrated in

figure 2, 3 mg.ml-1 bradykinin, delivered as a single challenge without preceding or subsequent

challenges, was typically subthreshold for initiating cough, but in animals first challenged with 1

mg.ml-1 bradykinin, 3 mg.ml-1 was the optimal dose for cough challenge (figure 2).

Cough responses to bradykinin quickly desensitized. In animals challenged with

cumulatively increasing doses, 3 mg.ml-1 bradykinin evoked 9.0±2.5 coughs, while subsequent

(just 5 minutes later) challenge with 10 mg.ml-1 bradykinin resulted in 1 cough in just 1 of 8

animals studied (0.1±0.1 coughs overall; n=8). Desensitization could also be seen by quantifying

cough responses after the initial paroxysm of cough evoked by the peptide. In the 9 (out of 20)


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vehicle-treated animals challenged with 10 mg.ml-1 bradykinin that had paroxysmal bouts of ≥10

coughs in any 30 second interval of the 10 minute challenge (16.8±2.6 coughs in ≤30 seconds;

n=9), just 1.4±0.6 coughs occurred over the ensuing 5.1±0.7 minutes of continuous bradykinin

challenge. Similar results were seen in animals in all other treatment groups challenged with 10

mg.ml-1 bradykinin (15.0±0.5 coughs in ≤30 seconds of paroxysmal coughing, 1.8±0.5 coughs

over the ensuing 5.1±0.6 minutes of challenge; n=21) and in animals provoked with 5 mg.ml-1

bradykinin (15.6±1.7 coughs in ≤30 seconds of paroxysmal coughing, 1.6±0.5 coughs over the

ensuing 4.9±0.8 minutes of challenge; n=7)). Overall, only 12 out of 107 animals studied had

multiple bouts of 10 or more coughs in any 30 second intervals during bradykinin challenge,

with 8 of the 12 additional paroxysms occurring less than 2 minutes after the preceding bout,

suggestive of a continuation/ 2nd phase of an ongoing response.

Pretreatment with thiorphan or captopril alone (1 mg.ml-1 each delivered as aerosols) did

not evoke cough or alter cough responsiveness to 1 mg.ml-1 bradykinin challenges. When

administered in combination, however, captopril and thiorphan markedly potentiated the cough

evoked by bradykinin (figure 3). This combination of peptidase inhibitors decreased the time to

first cough (1.07±0.01 minutes after initiating a 1 mg.ml-1 bradykinin challenge with peptidase

pretreatment vs. 3.9±0.8 minutes after initiating a 10 mg.ml-1 bradykinin challenge in the absence

of peptidase inhibitors; n≥15; p<0.05). The paroxysmal pattern of coughing was unchanged,

however, and desensitization was still apparent, with no coughing observed over an average of

the last 4.3±0.7 minutes of the 10 minute 1 mg.ml-1 bradykinin challenge.

Bradykinin-evoked bronchospasm does not initiate coughing.

Bradykinin-induced bronchospasm occurs indirectly and is thought to result from the net

effects of dilating nitric oxide (NO) and constricting thromboxanes (Arakawa et al., 1992;


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Ricciardolo et al., 1994; Figini et al., 1996; Ricciardolo et al., 1996; Ricciardolo et al., 1999;

Canning et al., 2001; Keir et al., 2015). We have thus evaluated the effects of a thromboxane

(TP) receptor antagonist (ICI 192605) and an NO synthase inhibitor (L-NNA) on the cough

responses evoked by bradykinin. Using a pretreating dose and delivery scheme (0.1mM aerosol

for 10 minutes) identical to that used previously to enhance bradykinin-induced bronchospasm

(Ricciardolo et al., 1994), we found that L-NNA did not significantly modify bradykinin induced

coughing (figure 4). We then confirmed the adequacy of dosing of ICI 192605 (10µM delivered

as an aerosol) by showing that this TP receptor antagonist prevented the enhancement of citric

acid induced coughing evoked by the TP receptor agonist U46619 (1 µM delivered as an aerosol

30 minutes prior to cough challenge; Xiang et al., 2002), with citric acid (0.01-0.1M) evoking

2±1, 25±6 and 6±3 cumulative coughs in animals pretreated with vehicle, U46619, or U46619

with ICI192605 pretreatment, respectively (n=8-13; p<0.05 for control vs. U46619). But

aerosolized ICI192605 failed to significantly inhibit bradykinin evoked cough (15±6 and 11±5

coughs in control and ICI192605 pretreated animals, respectively; n=5-6/ treatment group;

p>0.1). Moreover, at twice the dose (0.5 mg.kg-1) that completely abolished bradykinin (0.1-2

nmol.kg-1 iv) induced increases in pulmonary inflation pressure (PIP) in anesthetized,

mechanically-ventilated guinea pigs, intraperitoneally administered ICI 192605 (1 mg.kg-1) failed

to significantly inhibit bradykinin evoked cough (figure 4). Incidentally, the TP receptor agonist

U46619 failed to directly evoke coughing upon aerosol administration but did induce labored

breathing, suggestive of bronchoconstriction, which was prevented by aerosolized ICI192605.

Prostanoids in addition to thromboxane may regulate the respiratory reflexes evoked by

bradykinin (Canning et al., 2001; Chou et al., 2008). Parasympathetic-cholinergic nerves and

neurokinins released in the airways through axonal reflexes also play a role in responses to


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bradykinin (Fuller et al., 1987; Nakajima et al., 1994; Canning et al., 2001). Their roles in the

cough responses evoked by bradykinin were evaluated in the present study. Neither

meclofenamic acid nor atropine reduced the number of bradykinin-evoked coughs. Atropine did,

however, change the time course of the coughing evoked. Thus, in control animals, the time

elapsed until ≥50% of the total number of coughs occurred averaged 7.0±0.5 minutes

(median=7.25 minutes; range: 2.5-10 minutes; n=16). This was significantly reduced by atropine

(3.4±0.7 minutes; n=5; p<0.05) but not by meclofenamic acid (5.6±1.2 minutes; n=5). The

combination of meclofenamic acid and atropine still failed to produce a statistically significant

inhibition of cough evoked by 10 mg.ml-1 bradykinin (8±4 coughs; n=10; p>0.05; compare to the

results in figure 5) but like atropine alone, reduced the time elapsed until ≥50% of the total

number of coughs occurred (3.5±0.6 minutes; n=4; p<0.05).

In contrast to the effects of atropine and meclofenamic acid, a combination of neurokinin1

(NK1), NK2 and NK3 receptor antagonists (SR140333, SR48968 and SB223412, respectively; 3

mg.kg-1 each, given ip) markedly inhibited bradykinin evoked cough (figure 5). When

administered directly to the airways by aerosol (1 mg.ml-1 each), however, a combination of NK1

(SR140333 (n=3) or CP99994 (n=2)) and NK2 (SR48968) receptor antagonists was without

effect on bradykinin evoked cough (13±5 coughs; n=5; compare to the results in figure 5).

Evidence that Bronchopulmonary C-fibers Regulate Bradykinin Evoked Coughing.

Bradykinin is thought to be a relatively selective stimulant of C-fibers but bradykinin can

activate other airway afferent nerves in guinea pigs, including rapidly adapting receptors (RARs)

and a poorly defined subset of capsaicin-sensitive myelinated afferents arising from the jugular

ganglia (Bergren, 1997; Kajekar et al., 1999). We have quantified the effects of bradykinin on

airway vagal afferent nerve subtypes using a Ca++ influx assay in neurons retrogradely labeled


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with DiI injected into the larynx, trachea and mainstem bronchi. Amongst the labeled afferent

neurons recovered from the vagal sensory ganglia, only those taken from the jugular ganglia

were activated by bradykinin (figure 6).

The sensitivity of bradykinin evoked coughing to blockade of all 3 neurokinin receptors

with combinations of either SR140333, SR48968 and SB223412 or CP99994, SR48968 and

SB223412 (figure 5) is consistent with a role for jugular C-fibers in cough (Ricco et al., 1996).

We have further characterized the specific neurokinin receptors involved in these responses. The

combinations of only NK1 and NK2 (CP99994 and SR48968) or NK1 and NK3 (CP99994 and

SB223412) receptor antagonists were without significant effect on bradykinin evoked coughing

(table 1). But in contrast to the combinations of NK1 and NK2 and NK1 and NK3 receptor

antagonists, which failed to modify responses to bradykinin, a combination of NK2 (SR48968)

and NK3 (SR142801) receptor antagonists markedly inhibited bradykinin evoked coughing,

approximating the effects of blocking all 3 neurokinin receptors. The NK3 receptor antagonist

SR142801 administered alone did not significantly inhibit bradykinin evoked coughing

(15.2±7.3 and 13.0±3.5 coughs in matched control and SR142801 (3mg/ kg) treated animals,

respectively; n=4-5; p>0.1). Respiratory rate was unchanged by these compounds (Table 1).

The inhibitory effects of the neurokinin receptor antagonists studied here were selective

for bradykinin evoked cough. Single, explosive coughing events evoked by mechanical

stimulation of the airway mucosa in anesthetized guinea pigs, reflexes we attribute to activation

of the bradykinin-insensitive nodose Aδ fibers innervating the trachea, larynx and mainstem

bronchi (Mazzone et al., 2009; Muroi et al., 2013), were still present in all animals treated with a

combination of the NK1, NK2 and NK3 receptor antagonists (SR140333, SR48968 and

SB223412, respectively; 3 mg.kg-1 each, ip; n=3) or the vehicle for these antagonists (n=3).


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DISCUSSION

We have described the physiological and pharmacological basis of bradykinin induced

cough in guinea pigs. Like many other effects of bradykinin in the lung, we anticipated that the

indirect effects of this inflammatory peptide on the structural cells of the airways would

contribute to the coughing we observed upon aerosol challenge. For example, the bronchospasm

induced by bradykinin occurs via indirect mechanisms, either secondary to thromboxane

formation (perhaps from platelets) or by reflex activation of airway parasympathetic-cholinergic

nerves (Fuller et al., 1987; Arakawa et al., 1992; Hulsmann et al., 1994; Arvidsson et al., 2001;

Canning et al., 2001; Keir et al., 2015). Neurokinin receptor dependent axonal reflexes may also

contribute to the airway responses evoked by bradykinin in guinea pigs (Nakajima et al., 1994;

Joad et al., 1997). But we observed that drugs that prevent bradykinin-induced bronchospasm

did not prevent bradykinin-induced coughing, while drugs that would be expected to enhance

bradykinin-induced bronchospasm (e.g. captopril, L-NNA and thiorphan; Ichinose and Barnes,

1990; Ricciardolo et al., 1994) did not, on their own, enhance bradykinin evoked coughing.

Cough resulting from bradykinin challenge likely depends upon the direct effects of the

inflammatory peptide on bronchopulmonary C-fibers (Kaufman et al.; 1980; Bergren, 1997;

Kajekar et al., 1999). Bradykinin can also activate RARs, perhaps due to its direct physiologic

effects (e.g. bronchospasm, vascular engorgement, vascular leakage, mucus secretion), and there

is also a bradykinin-sensitive, capsaicin-sensitive, myelinated afferent nerve subtype innervating

the extrapulmonary airways of guinea pigs (Kajekar et al., 1999; Kaufman et al.; 1980; Bergren,

1997). But RAR activation by bradykinin is largely abolished by cyclooxygenase inhibition or

by isoproterenol (Bergren, 1997; Canning et al., 2001), and neither meclofenamic acid nor the

TP receptor antagonist ICI 192605 (both of which abolish bradykinin evoked bronchospasm)

inhibited bradykinin-induced cough. Also arguing against a role for RARs in bradykinin evoked


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cough is the observation that thromboxane inhalation fails to evoke coughing in guinea pigs

(present study; Shinagawa et al., 2000; Xiang et al., 2002), although PGE2, via EP3 receptor

activation and PGD2 via DP1 receptor activation, can induce coughing in guinea pigs and

humans (Choudry et al., 1989; Maher et al., 2009; Maher et al., 2014).

The effects of the neurokinin receptor selective antagonists on bradykinin evoked cough

and the results of previous studies argue in favor of jugular C-fiber involvement and for a central

site of action for these agents in cough suppression (Bolser et al., 1997; Canning et al., 2001;

Mazzone and Canning, 2002; Mazzone et al., 2005). The primary local effects of bradykinin

occur through parasympathetic-cholinergic reflex bronchospasm and mucus secretion, prostanoid

formation, and perhaps an NK1 receptor dependent vasodilation and plasma exudation initiated

by axonal reflexes (Arakawa et al., 1992; Bertrand et al., 1993; Nakajima et al., 1994; Joad et al.,

1997; Canning et al., 2001). But neither cyclooxygenase inhibition nor atropine prevented

bradykinin-evoked cough. NK1 receptor antagonism also failed to modulate bradykinin evoked

cough when given in combination with either NK2 or NK3 receptor antagonists. Only when both

NK2 and NK3 receptors were blocked was bradykinin-evoked cough inhibited. Indeed, in 4

separate sets of experiments where both NK2 and NK3 receptor antagonists were administered,

with 2 structurally unrelated NK3 receptor antagonists used, bradykinin-evoked coughing was

markedly reduced relative to that observed in matched control animals. All neurokinin receptor

subtypes have been localized to mammalian brainstem (Geraghty and Mazzone, 2003). There is

precedent for NK2 and NK3 receptor-dependent cough in guinea pigs, as well as precedent for a

lack of effect of NK1 receptor antagonists in some (but not all) studies of cough in guinea pigs

(Advenier et al., 1993; Bolser et al., 1997; Emonds-Alt et al., 1997; Daoui et al., 1998; Emonds-

Alt et al., 2002; Hay et al., 2002; El-Hashim et al., 2004). The involvement of neurokinins also


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supports our previous assertions of jugular C-fiber involvement and against a role for either

jugular Aδ-fibers or nodose C-fibers (Ricco et al., 1996; Undem et al., 2004; Muroi et al., 2013).

Despite the profound antitussive effects of the neurokinin receptor antagonists reported

here and elsewhere, their promise as cough suppressants in patients remain unclear. In favor of

their utility in cough and in controlling other aberrant respiratory reflexes and sensations, we and

others have shown previously that, in guinea pigs, neurokinin receptor antagonists inhibit

capsaicin and citric acid evoked coughing, reflex bronchospasm evoked by tracheal/ laryngeal C-

fiber activation and the reflex bronchospasm evoked by bradykinin following cyclooxygenase

inhibition (Girard et al., 1995; Bolser et al., 1997; Canning et al., 2001; Mazzone and Canning,

2002; El-Hashim et al., 2004). Neurokinin receptor antagonists also inhibit coughing evoked in

rabbits, cats and dogs (reviewed in Canning, 2009), and a recent preliminary report suggests

modest cough suppression in lung cancer patients by the NK1 receptor antagonist aprepitant

(Harle et al, 2015). But we have also described how neurokinin receptor antagonists are without

effect on coughing evoked by acid or mechanical stimulation of the airways of anesthetized

guinea pigs, or the reflex bronchospasm evoked by histamine in anesthetized guinea pigs

(present study; Canning et al. 2001; Mazzone et al., 2005), each of which being reflexes that are

unlikely to depend upon C-fiber activation. The utility of these agents in human cough will thus

depend upon the predictive value of studies performed in animals but also on the relative

contribution of afferent nerve subtypes to cough in human disease.

We observed both sensitization and desensitization of the cough responses to bradykinin.

The desensitization to bradykinin-induced cough (and bradykinin-induced bronchospasm; see

figure 4A) is consistent with previous studies and likely reflects the rapid and pronounced

desensitization of B2 receptors with sustained receptor occupancy (Wolsing and Rosenbaum,


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1993; Leeb-Lundberg et al., 2005; Zimmerman et al., 2011). But we cannot rule out other

possible inhibitory mechanisms, including local inhibitory effects induced by autacoids formed

in response to bradykinin, adaptation at the central synapses for C-fibers, or perhaps negative

feedback regulation of the cough reflex. The sensitization of cough apparent when comparing

the responses to bradykinin evoked with or without preceding subthreshold challenge doses has

not been previously described. But a sensitizing effect of subthreshold doses of bradykinin on

subsequently evoked cough responses has been noted (Fox et al., 1996; El-Hashim et al., 2005;

Mazzone et al., 2005). These sensitizing effects may be both CNS and NK1 receptor dependent

(Joad et al., 2004; Mazzone et al., 2005; Canning and Mori, 2011; Cinelli et al., 2015) or could

depend upon local actions of eicosanoids released from structural cells following bradykinin B2

receptor activation (Salari and Chan-Yeung, 1989; Arakawa et al., 1992; Gatti et al., 2006; Petho

and Reeh, 2012).

The paroxysmal pattern of coughing evoked by bradykinin is unique to this peptide and

characteristic of certain pathologies, leading to the speculation that conditions that enhance

bradykinin actions or slow the rate of bradykinin receptor desensitization may result in

bradykinin dependent cough (Fox et al., 1996; Morice et al., 1997; Dicpinigaitis, 2006; Hewitt

and Canning, 2010; Mutolo et al., 2010). It is also interesting that captopril alone was unable to

enhance bradykinin-evoked coughing in this study. Only when angiotensin converting enzyme

and neutral endopeptidase were both inhibited was the cough response enhanced. This result

suggests a redundant effect of these enzymes in limiting the actions of bradykinin and may also

suggest that the small subset of patients that cough when on ACE inhibitor therapy have an

additional decrease/ defect in neutral endopeptidase activity that predisposes them to coughing

evoked by bradykinin (Dicpinigaitis, 2006; Mahmoudpour et al., 2013).


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Implications for Cough in Disease.

The results of our study suggest that neither bradykinin evoked bronchospasm, nor

eicosanoid formation within the airways, nor even CNS activation of NK1 receptors are

necessary for the initiation of cough evoked by this peptide. But this does not rule out

modulatory roles for these secondary effects of bradykinin in cough (Malini et al., 1997;

Shinagawa et al., 2000; Liu et al., 2001; Xiang et al., 2002; Mazzone et al., 2005; Gatti et al.,

2006; Canning and Mori, 2011). Thus, we saw atropine altered the kinetics of bradykinin-

evoked cough and that TP receptor activation enhances cough responsiveness, and bradykinin

certainly induces thromboxane release and TP receptor activation in the airways. We speculate

that the effects of atropine may highlight the importance of mucosal barrier function and

clearance of inhaled irritants on cough responses, while autacoids such as thromboxane may act

directly on the afferent nerve terminals to enhance their excitability. Precisely how the many

secondary effects of bradykinin might modulate cough awaits further study. The primary focus

of the present study was defining how bradykinin directly evokes this airway defensive reflex.

The most logical approach to preventing the actions of bradykinin in the lungs is B2

receptor antagonism. It may also be possible to inhibit bradykinin evoked cough by preventing

its effects on the ion channels TRPA1 and TRPV1 (Shin et al., 2002; Carr et al., 2003; Bandell et

al., 2004; Kollarik and Undem, 2004; Lee et al., 2005; Grace et al., 2012). Other approaches

targeting the central and peripheral terminals of bradykinin-sensitive C-fibers, preventing

bradykinin formation (e.g. kallikrein inhibitors), hastening its degradation (e.g. soluble neutral

endopeptidase) or promoting bradykinin B2 receptor desensitization or downregulation may also

prevent bradykinin-evoked coughing (Joseph and Kaplan, 2005; Leeb-Lundberg et al., 2005;

Smith et al., 2012; Zimmerman et al. 2011). No matter the approach, we speculate that targeting

bradykinin may prevent the coughing associated with several respiratory diseases.


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Authorship contributions

Matthew Hewitt, Gregory Adams, Nanako Mori, Stuart Mazzone and Li Yu carried out the experiments, performed data analysis on the results and wrote selected parts of the narrative. Brendan Canning designed the experiments, generated the final and revised draft of the manuscript and performed data analysis.


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FOOTNOTES

The work summarized in this manuscript was supported by a grant from the National Institutes

of Health (HL083192). SBM is funded by a National Health and Medical Research Council of

Australia fellowship grant (APP1025589). Gregory Adams, Jr. performed this work while at

Johns Hopkins. He is currently a postdoctoral fellow at the National Heart, Lung and Blood

Institute (NHLBI) of the National Institutes of Health (NIH, Bethesda, MD).

Conflict of interest: The authors declare no conflicts of interest relating to the conduct or

summary of these studies.


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Figure Legends.

Figure 1. Bradykinin evokes paroxysmal coughing in conscious guinea pigs via bradykinin B2

receptor activation. A) The representative trace of bradykinin (10 mg.ml-1) evoked coughing

illustrates the typical pattern of cough evoked by the inflammatory peptide. Within minutes of

initiating the aerosol challenge, multiple coughs in rapid succession occur, often ending abruptly

despite continued bradykinin challenge. Expiratory efforts manifest as upward deflections in

chamber pressure (see Methods for further details). B) The bradykinin B2 receptor antagonist

HOE140 (1 mg.ml-1, delivered as an aerosol 10 minutes prior to bradykinin challenge) prevented

bradykinin (0.1-10 mg.ml-1, delivered cumulatively as aerosols) evoked coughing (*p<0.05).

The results are presented as the mean±sem cumulative coughs evoked in 4-9 experiments.

Figure 2. Bradykinin both sensitizes and desensitizes its ability to evoke coughing in awake

guinea pigs. The peptide was delivered as an aerosol either as single doses to individual animals

or in cumulatively increasing concentrations, with 5 minutes in between each challenge dose.

When administered as a single dose, only concentrations of 5 (7±3 coughs; n=14; not shown) or

10 mg.ml-1 reliably evoked coughing. When administered in cumulatively increasing

concentrations however, 3 mg.ml-1 bradykinin was optimal for evoking cough. Increasing the

concentration of bradykinin from 3 to 10 mg.ml-1 evoked only 1 cough in 1 of 8 animals studied.

An asterisk (*) indicates that the number of coughs evoked by bradykinin was significantly less

than the number of coughs evoked by 3 mg.ml-1 bradykinin in the cumulative response curves

(p<0.05). These results are the mean±sem of 4-20 experiments. The data for coughing evoked

by cumulatively administered bradykinin were regraphed from figure 1B, showing only the

cough responses evoked by each dose studied and not the cumulative responses.


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Figure 3. Peptidases regulate bradykinin evoked cough in awake guinea pigs. Bradykinin (1

mg.ml-1) was administered as an aerosol after aerosol pretreatment with vehicle (saline), the

neutral endopeptidase inhibitor thiorphan (1 mg.ml-1), the angiotensin converting enzyme

inhibitor captopril (1 mg.ml-1) or the combination of thiorphan and captopril (1 mg.ml-1 each).

The drugs administered alone had little effect on 1 mg.ml-1 bradykinin evoked coughing, but

when administered in combination, the cough responses were markedly potentiated. Even

following peptidase inhibition, however, a lower dose of bradykinin (0.1 mg.ml-1) was still

largely ineffective at evoking cough (median = 1; n=14). The data are presented as a mean±sem

of 12-21 experiments. An asterisk (*) indicates a statistically significant potentiation of

bradykinin-evoked cough relative to vehicle treated animals (p<0.05).

Figure 4. Bradykinin-induced bronchospasm does not influence the number of coughs evoked

by bradykinin. A) The TP receptor antagonist ICI192605 (0.5 mg.ml-1 iv; n=3) abolishes

bradykinin evoked bronchoconstriction (measured as an increase in pulmonary inflation

pressure; PIP) in anesthetized guinea pigs (*: p<0.05). B) In contrast to bradykinin evoked

bronchospasm, 1 mg.ml-1 bradykinin evoked coughing in awake guinea pigs was not inhibited by

prior pretreatment with ICI 192, 605 (1 mg.ml-1 ip; n=8). Similarly, administered in a way shown

previously to enhance bradykinin-evoked bronchospasm (0.1mM aerosol for 10 minutes; see

Ricciardolo et al., 1994), the NO synthase inhibitor L-NNA (n=8) also failed to alter the number

of coughs evoked by bradykinin. The results are presented as a mean±sem of 3-15 experiments.

Bradykinin challenges were delivered 5 minutes after aerosol pretreatments with the peptidase

inhibitors captopril and thiorphan (1 mg.ml-1 each, 10 minute aerosols). Control animals

received either the vehicle for ICI 192605 or the vehicle for L-NNA. The cough responses in

these 2 control groups were similar and the data were therefore pooled.


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Figure 5. Neurokinin receptor antagonists but neither atropine nor meclofenamic acid reduced

the number of coughs evoked by aerosolized bradykinin (10 mg.ml-1) in awake guinea pigs.

Thirty minutes after administration of vehicle (ip; n=20), atropine (1 mg.ml-1 aerosol; n=7),

meclofenamic acid (1 mg.kg-1 ip; n=7) or the combination of SR140333, SR48968 and

SB223412 (3 mg.kg-1 each ip; n=5), bradykinin was delivered as an aerosol for 10 minutes and

the total number of coughs evoked was counted. Like the neurokinin receptor antagonists

administered alone, coadministering neurokinin receptor antagonists (CP99994, SR48968 and

SB223412; 3 mg.kg-1 each ip) along with either meclofenamic acid (1 mg.kg-1 ip; n=8) or ICI

192, 605 (1 mg.kg-1 ip; n=5) also markedly inhibited the bradykinin evoked coughing (4±2 and

4±2 coughs, respectively; p<0.05). The vehicle for atropine aerosol delivery (saline) was

without effect on bradykinin evoked coughing (14±4 coughs; data not shown; n=7). Some

animals pretreated with the ip vehicle (4/20), the vehicle for atropine aerosol (2/7),

meclofenamic acid (2/ 7), atropine (2/7), or the neurokinin receptor antagonists (4/5) coughed

once or not at all to the bradykinin challenges. These animals were still included in the mean

data, which are presented as a mean±sem of 5-20 experiments. An asterisk (*) indicates that the

neurokinin receptor antagonists significantly inhibited bradykinin evoked coughing relative to

vehicle control (p<0.05).


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Figure 6. Bradykinin fails to evoke intracellular Ca++ increases in laryngeal/ tracheal/ bronchial

vagal sensory neurons arising from the nodose ganglia (a-c) but does activate airway afferent

neurons arising from the jugular ganglia (d-f). Neurons were visualized in brightfield

microscopy (a, d) and by fluorescent microscopy at baseline (b, e) and at the peak of their

responses to 1 µM bradykinin (c, f). Images in the upper and lower panels are from the same

neurons visualized under differing conditions. Red and yellow coloring in these neurons

indicates higher concentrations of Ca++ in comparison to the green fluorescence at baseline.

Retrograde neuronal tracing with DiI was used to identify vagal sensory neurons projecting to

the larynx, trachea and mainstem bronchi. Neurons were dissociated from the ganglia, cultured

on coverslips and loaded with Fura for measurements of Ca++ influx. All 24 retrogradely

labeled jugular ganglia neurons studied responded to 1µM bradykinin challenge (Ca++ influx

≥10% of the response to ionomycin) while none of the 6 labeled nodose ganglia neurons

responded (even though many of the 63 nonlabeled nodose ganglia neurons studied were

activated by bradykinin). The micrographs shown are representative of experiments performed

on neurons recovered from 8 guinea pigs.


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Table 1. Effect of neurokinin receptor antagonists on bradykinin-evoked coughing in awake

guinea pigs.

Treatment Total Number of Coughs Basal Respiratory Rate

Vehicle Control 21±3 113±4

CP99994 and SR48968 17±3 113±4

CP99994 and SB223412 22±6 108±8

SR48968 and SR142801 6±3* 115±8

Neurokinin receptor antagonists were administered simultaneously at a dose of 3 mg.kg-1 by

intraperitoneal injection. Coughing was evoked by aerosol challenges with 1 mg.ml-1 bradykinin

30 minutes subsequent to drug pretreatments. Basal respiratory rates were measured just prior to

the bradykinin challenges. Bradykinin challenges were delivered 5 minutes after aerosol

pretreatments with the peptidase inhibitors captopril and thiorphan (1 mg.ml-1 each, 10 minute

aerosols). The results are presented as the mean±sem of 8-15 nonpaired experiments. An

asterisk (*) indicates that the combination of SR48968 and SR142801 inhibited bradykinin

evoked coughing (p<0.05).


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