J Appl Physiol 106: 1584–1592, 2009.
First published February 26, 2009; doi:10.1152/japplphysiol.91517.2008.
Role of ATP in the ROS-mediated laryngeal airway hyperreactivity induced
by laryngeal acid-pepsin insult in anesthetized rats
Tung-Lung Tsai,1,4,5 Shyue-Yih Chang,4,5 Ching-Yin Ho,4,5 and Yu Ru Kou2,3
Institutes of 1Clinical Medicine, 2Physiology, and 3Emergency and Critical Care Medicine and 4Department
of Otorhinolaryngology, School of Medicine, National Yang-Ming University, Taipei; and 5Department of Otolaryngology,
Taipei Veteran General Hospital, Taipei, Taiwan
Submitted 20 November 2008; accepted in final form 18 February 2009
extraesophageal reflux; laryngeal capsaicin-sensitive afferent fibers;
adenosine 5⬘-triphosphate; reactive oxygen species
(GERD) is a common disorder of the gastrointestinal tract caused by the backflow of
gastric contents into the upper digestive tract (32, 39). When
the effects of refluxed gastric contents extend beyond the
esophagus, it is referred to as extraesophageal reflux (EER)
(32, 39). EER is associated with several respiratory manifestations including laryngeal airway hyperreactivity (LAH),
which is characterized by increased sensitivity of laryngeal
vagally mediated reflexes such as cough, glottic stop, or laryngeal adductor responses (5, 13, 34, 40, 47). LAH is thus
defined as a sensory hyperresponsiveness of the laryngeal
mucosa leading to exaggerated reflex responses to stimuli.
LAH may contribute to chronic cough and paroxysmal laryn-
GASTROESOPHAGEAL REFLUX DISEASE
Address for reprint requests and other correspondence: Y. R. Kou, Institute
of Physiology, School of Medicine, National Yang-Ming Univ., Taipei 11221,
Taiwan (e-mail: [email protected]).
1584
gospasm seen in EER patients (5, 13, 29, 32, 34, 39, 41). Since
EER patients who display LAH have a high incidence of
laryngeal inflammation (5, 40, 41) and because inflammatory
mediators can increase the excitability of airway afferent fibers
(8, 18), it has been postulated that sensitization of laryngeal
sensory receptors by inflammatory mediators may be the cause
of LAH. However, the hypothesis remains unproven, and the
mediator mechanisms are still unclear.
We recently (49) established a rat model of EER with both
LAH and laryngeal inflammation induced by laryngeal insult
with acid (pH 5) and pepsin, two major components of refluxed
gastric contents that cause laryngeal injury (2, 25). This LAH
is mediated through sensitization of the capsaicin-sensitive
laryngeal afferent fibers (49), a type of nociceptive-like free
nerve endings (7, 8). This LAH is prevented by scavengers of
hydroxyl radical (•OH), suggesting a critical role of reactive
oxygen species (ROS) (49), an important category of inflammatory mediators in GERD (19, 33).
Although the role of ROS seems to be clear, they may not be
the sole mediator involved in the LAH. For example, the
ROS-induced activation of capsaicin-sensitive lung vagal afferent fibers is partly mediated through the action of the
released adenosine 5⬘-triphosphate (ATP) on P2X purinoceptors located on fiber terminals (42, 43). In addition, ROS may
damage cells, causing a subsequent release of cytosolic ATP
into their surroundings, which activates the P2X receptors
located on nearby nociceptors (10, 12, 35). Furthermore, ATP
can sensitize capsaicin-sensitive ion channels in the primary
sensory neurons, leading to hyperalgesia (26, 30, 48). These
findings support our hypothesis that laryngeal acid-pepsin
insult may sequentially induce increases in ROS and then in
ATP; the latter mediator may activate P2X receptors to increase the sensitivity of capsaicin-sensitive laryngeal afferents,
and this results in LAH.
To test our hypothesis, we undertook the present study in
anesthetized rats to investigate, first, whether laryngeal insults
with acid-pepsin and hydrogen peroxide (H2O2, a major type of
ROS) produce LAH with similar characteristics; second,
whether activation of P2X receptors by ATP is important to the
LAH induced by laryngeal insult with acid-pepsin or H2O2;
third, whether laryngeal application of an ATP analog also
induces an LAH that is mediated through the capsaicin-sensitive laryngeal afferent fibers; and finally, whether both laryngeal insults with acid-pepsin and H2O2 promote increases in
levels of ATP, lipid peroxidation (an index of oxidative stress),
and inflammation of the larynx. To accomplish these objectives, we measured the reflex apneic response to laryngeal
ammonia provocation to reflect laryngeal reflex reactivity. The
8750-7587/09 $8.00 Copyright © 2009 the American Physiological Society
http://www. jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.3 on June 16, 2017
Tsai TL, Chang SY, Ho CY, Kou YR. Role of ATP in the
ROS-mediated laryngeal airway hyperreactivity induced by laryngeal acid-pepsin insult in anesthetized rats. J Appl Physiol
106: 1584 –1592, 2009. First published February 26, 2009;
doi:10.1152/japplphysiol.91517.2008.—The pathogenetic mechanisms of laryngeal airway hyperreactivity (LAH) in patients with
extraesophageal reflux are unclear. We recently reported that a laryngeal acid-pepsin insult produces LAH that is mediated through sensitization of the capsaicin-sensitive laryngeal afferent fibers by reactive oxygen species (ROS) in rats. Since ROS may promote the
release of ATP from cells, we hypothesized that activation of P2X
purinoceptors by ATP subsequent to an increase in ROS induces LAH
in an inflamed larynx that has been insulted by acid-pepsin or H2O2 (a
major type of ROS). The larynxes of 208 anesthetized rats were
functionally isolated while the animals breathed spontaneously. Ammonia vapor was delivered into the larynx to measure laryngeal reflex
reactivity. Laryngeal insult with acid-pepsin or H2O2 produced LAH
with similar characteristics. The H2O2-induced LAH was prevented
by laryngeal pretreatment with dimethylthiourea (a hydroxyl radical
scavenger), suggesting a critical role for ROS. The LAH induced by both
insults were completely prevented by ATP scavengers (a combination of
apyrase and adenosine deaminase) or a P2X receptor antagonist (isopyridoxalphosphate-6-azophenyl-2⬘,5⬘-disulfonate). Laryngeal application of a P2X receptor agonist (␣,␤-methylene-ATP) also produced LAH.
An insult with either acid-pepsin or H2O2 similarly promoted an increase
in the levels of ATP, lipid peroxidation, and inflammation in the larynx.
Our findings suggest that laryngeal insult with acid-pepsin or H2O2
induces inflammation and produces excess ROS in the rat’s larynx.
The latter may in turn promote the release of ATP to activate P2X
receptors, resulting in sensitization of capsaicin-sensitive laryngeal
afferent fibers and LAH.
ROS AND ATP CAUSE HYPERSENSITIVITY OF LARYNGEAL REFLEX
effects of laryngeal acid-pepsin or H2O2 insult on this apneic
response were determined to evaluate the LAH.
MATERIALS AND METHODS
dimethylthiourea (DMTU, a •OH scavenger; 4.8 M; Sigma) (15) were
prepared by dissolving the chemicals in saline. The solution of
apyrase (an enzyme that catalyzes breakdown of ATP to AMP; 800
U/ml; Sigma) was prepared by dissolving the chemical in PBS. The
solution of adenosine deaminase (ADA, an enzyme that catalyzes
breakdown of adenosine; 417 U/ml; Sigma) was prepared by dissolving the chemical in 50% glycerol and 50 mM potassium phosphate
and was further diluted to a concentration of 400 U/ml with PBS. A
combination of apyrase and ADA (apyrase ⫹ ADA) has previously
been used as an ATP scavenger (1).
Laryngeal provocation with ammonia or ␣,␤-meATP. The methods
used for laryngeal ammonia provocation have been described previously (46, 49). In brief, 5 ml of ammonia vapor (0.2% in air) was
continuously delivered into the isolated larynx and flowed out via the
oral tube. Laryngeal provocation with ␣,␤-meATP (5 mM, 30 ␮l)
were carried out by spraying the solution into the larynx via a spinal
needle.
Laryngeal insult with acid-pepsin or H2O2. For acid-pepsin insult,
a small piece of filter paper (⬃1 ⫻ 10 mm) was presoaked with the
acid-pepsin solution, carefully inserted into the larynx via the oral
tube, and then removed 40 s after insertion. For H2O2 insult, 30 ␮l of
H2O2 solution (1%) was sprayed into the larynx via a spinal needle.
After these insults, an elapsed time of at least 60 min was allowed
before ammonia provocation.
Laryngeal application of pharmacological agents. Solutions containing apyrase ⫹ ADA (apyrase, 800 U/ml, 2 ␮l; ADA, 400 U/ml, 2
␮l), iso-PPADS (2 ␮M, 2 ␮l), ␣,␤-meATP (5 mM, 30 ␮l), DMTU
(4.8 M, 2 ␮l), or their vehicles were sprayed into the laryngeal
segment via a spinal needle; these applications were made 30 min
before the laryngeal insult. In addition, applications of iso-PPADS or
its vehicle was made 30 min before each ammonia provocation. The
doses and treatment times of these agents were determined based on
our preliminary investigations, which indicated that the drug effects of
Fig. 1. Schematic illustrations showing the experimental protocols of this study. Timelines in A–F depict protocols for studies 1– 6 described in MATERIALS AND
METHODS. Acid-pepsin, H2O2, or ␣,␤-methylene-ATP (␣,␤-meATP) represent laryngeal treatment for induction of laryngeal airway hyperreactivity. NH3
represents laryngeal ammonia provocation. Apyrase and adenosine deaminase (apyrase ⫹ ADA), dimethylthiourea (DMTU), iso-pyridoxalphosphate-6azophenyl-2⬘,5⬘-disulfonate (iso-PPADS), and their vehicles were given as pretreatments 30 min before laryngeal insult with acid-pepsin or H2O2. Two additional
pretreatments with iso-PPADS and its vehicle were made 30 min before laryngeal ammonia provocations due to the short effective time of iso-PPADS. SLN
cut represents denervation of superior laryngeal nerves. See text for further explanations.
J Appl Physiol • VOL
106 • MAY 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.3 on June 16, 2017
Animal preparation. All protocols were approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats
were initially anesthetized with an intraperitoneal injection of ␣-chloralose (100 mg/kg; Sigma Chemical, St. Louis, MO) and urethane
(500 mg/kg; Sigma) dissolved in a borax solution (2%). The depth of
anesthesia was maintained by supplemental doses of chloralose (20
mg 䡠 kg⫺1 䡠 h⫺1) and urethane (100 mg 䡠 kg⫺1 䡠 h⫺1). The right femoral
artery and jugular vein were cannulated to record arterial blood
pressure and for administration of pharmacological agents, respectively. The animal’s neck was opened, and the superior laryngeal
nerves (SLNs) were isolated. Body temperature was maintained at
about 37°C.
Functionally isolated laryngeal preparation. The methods used for
the preparation of a functionally isolated larynx have been reported
previously (28, 49). In brief, a lower tracheal catheter (PE-260) was
inserted caudally just above the thoracic inlet, whereas an upper
tracheal catheter (PE-200) was inserted cranially with its tip placed
slightly below the cricoid cartilage. An oral tube was introduced
through the mouth with its tip placed at the vallecula. During the
experiment, rats breathed spontaneously via the lower tracheal catheter. Respiratory flow and tidal volume were measured.
Preparation of the pharmacological agents. The acid-pepsin solution was prepared by adding pepsin to normal saline (0.9% NaCl)
(0.0025–2.5 mg/ml) and adjusted to pH 5 with 1 N NaOH. The H2O2
solution (35%; Shimakyu, Osaka, Japan) was diluted with saline to a
concentration of 1% (pH 7.4). The solutions of ␣,␤-methylene-ATP
(␣,␤-meATP, a P2X receptor agonist; 5 mM; Sigma), iso-pyridoxalphosphate-6-azophenyl-2⬘,5⬘-disulfonate (iso-PPADS, a P2X receptor
antagonist; 2 ␮M; Tocris Cookson, Ellisville, MO) (17, 37) and
1585
1586
ROS AND ATP CAUSE HYPERSENSITIVITY OF LARYNGEAL REFLEX
depicts the experimental protocols. Study 1 (Fig. 1A) was performed
to obtain the control response. The reflex apneic responses to four
laryngeal ammonia provocations were studied. The first provocation
was made 40 min before laryngeal insult with 0.0025–2.5 mg/ml
pepsin (groups 1– 4) in pH 5 solution. The other provocations were
made at 1, 2, and 3 h after insult. Study 2 (Fig. 1B) was performed to
test the possibility that ATP might be involved in the acid-pepsininduced LAH. The reflex apneic responses to four laryngeal ammonia
provocations were studied. The first provocation was made 40 min
before laryngeal application of apyrase ⫹ ADA (group 5), vehicle of
apyrase ⫹ ADA (group 6), iso-PPADS (group 7), or vehicle of
iso-PPADS (group 8). Subsequently, 30 min elapsed before laryngeal
acid-pepsin insult. The other provocations were made at 1, 2, and 3 h
after insult. Study 3 (Fig. 1C) was performed to investigate the
H2O2-induced LAH. The reflex apneic responses to five laryngeal
ammonia provocations were studied. The first provocation was made
at 40 min before laryngeal insult with H2O2 (group 9) or its vehicle
(group 10). The other provocations were made at 1, 2, 3, and 4 h after
insult. SLN denervation was performed at 30 min before the last
provocation. The effect of SLN denervation on the acid-pepsininduced LAH was investigated previously (49) and thus was not
evaluated in this study. Study 4 (Fig. 1D) was performed to test the
possibility that ROS and ATP might be involved in the H2O2-induced
LAH. The reflex apneic responses to four laryngeal ammonia provocations were studied. The first provocation was made 40 min before
laryngeal application of DMTU (group 11), vehicle of DMTU (group
12), apyrase ⫹ ADA (group 13), vehicle of apyrase ⫹ ADA (group
14), iso-PPADS (group 15), or vehicle of iso-PPADS (group 16).
Subsequently, 30 min elapsed before laryngeal H2O2 insult. The other
provocations were made at 1, 2, and 3 after insult. Study 5 (Fig. 1E)
Fig. 2. Immediate responses to laryngeal ammonia provocations before and after laryngeal acid-pepsin insult in 2 anesthetized rats pretreated with laryngeal
application of ATP scavengers or vehicle. The laryngeal application of ATP scavengers (apyrase ⫹ ADA) was performed 30 min before acid-pepsin insult.
Responses were obtained 40 min before, 2 h after, and 3 h after laryngeal acid-pepsin insult. V̇R, respiratory flow; VT, tidal volume; ABP, arterial blood pressure.
Arrows indicate onset of ammonia provocation.
J Appl Physiol • VOL
106 • MAY 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.3 on June 16, 2017
iso-PPADS, apyrase ⫹ ADA, DMTU, and ␣,␤-meATP were able to
last ⬃40, 140, 180, and ⬎360 min, respectively.
Measurements of lipid peroxidation in laryngeal tissues and ATP
concentrations in laryngeal fluid. Levels of lipid peroxidation in the
laryngeal tissue (20 mg) were determined by measuring malondialdehyde and its dihydropyridine polymers at 356-nm excitation and
426-nm emission by using a fluorescence spectrophotometer (F-4500;
Hitachi, Tokyo, Japan) (14). ATP concentrations in laryngeal fluids
were determined immediately (⬍30 s) after sample collection by
using an ATP assay kit (ATPLite; PerkinElmer, Waltham, MA) and a
luminescence assay system (Multilabel Readers; PerkinElmer) with
the firefly luciferin-luciferase reaction (20).
Histological preparation and examination. After the animals were
killed by intravenous injection of an overdose of the anesthetics, their
larynxes were excised and fixed by immersion in a buffered neutral
formalin solution for 48 h. Tissue specimens were embedded in
paraffin and were cut transversely into 5-␮m-thick sections, which
were subsequently stained with hematoxylin and eosin. These sections
were examined by a qualified pathologist in a blind fashion. The
histological assessment was quantified using a scoring system (49)
that assigned a value from 0 (none or normal) to 3 (severe or diffuse)
for epithelial damage and infiltration of inflammatory cells of the
laryngeal tissues. Total injury scores were obtained by adding the
values assessed from these two injury variables. For each rat, three
histological sections were examined. For each section, structures at
three different areas were randomly selected, and their injury scores
were averaged.
Experimental design and protocol. In this study, 208 rats (weight
377 ⫾ 23 g) were randomly divided into 25 groups (groups 1–18 and
23–25, each n ⫽ 8; groups 19 –22, each n ⫽ 10) of animals. Figure 1
ROS AND ATP CAUSE HYPERSENSITIVITY OF LARYNGEAL REFLEX
RESULTS
Laryngeal acid-pepsin insult produces LAH. In the control
animals, an apneic response was elicited within 1 s after
laryngeal ammonia provocation (Fig. 2). After acid (pH 5)pepsin (2.5 mg/ml) insult, the same ammonia provocation
elicited a similar magnitude of apneic response after the first
hour but evoked a significantly augmented apneic response
after the second and third hour compared with the control
response (Figs. 2A and 3). When the concentration of pepsin
was decreased from 2.5 mg/ml to 2.5 ␮g/ml, the time of
occurrence of LAH was delayed and the number of animals
displaying LAH was reduced (Table 1). As a group, the mean
apneic responses in animals treated with pH 5-pepsin at concentrations of 2.5 and 25 ␮g/ml were not significantly different
from control values at any time point (Table 1). Insult with pH
5-pepsin (2.5 mg/ml) was then chosen as the standard insult for
the subsequent studies.
J Appl Physiol • VOL
Fig. 3. Mean apneic responses to laryngeal ammonia provocations before and
after laryngeal insult with acid-pepsin in 4 study groups. A: animals pretreated
with apyrase ⫹ ADA (ATP scavengers) or vehicle. B: animals pretreated with
iso-PPADS (P2X receptor antagonist) or vehicle. Recovery of the response
was obtained after the drug effect had worn off. Baseline expiratory duration
(TE) was calculated as the mean over 10 breaths immediately before provocation. *P ⬍ 0.05, significantly different from the response before acid-pepsin
insult in the same group. #P ⬍ 0.05, significantly different from the response
of the vehicle group at the same time point. Data in each group are means ⫾
SE from 8 rats.
Activation of P2X receptors by ATP is important in acidpepsin-induced LAH. In the groups with laryngeal pretreatment
with apyrase ⫹ ADA (ATP scavengers) or iso-PPADS (a P2X
receptor antagonist), ammonia provocation evoked an apneic
response that did not significantly differ from the control
response at the second hour after insult with acid-pepsin (Figs.
2B and 3), a time during which LAH was supposed to occur.
The blocking effect of iso-PPADS on the apneic response to
laryngeal ␣,␤-meATP provocation was proven to vanish at 90
min after its pretreatment (Table 2). Hence, after the effects of
apyrase ⫹ ADA and iso-PPADS had worn off, the same
ammonia provocation elicited a significantly greater apneic
response at the third hour after insult with acid-pepsin (Figs.
2B and 3) compared with the responses under baseline condition. In contrast, in the groups pretreated with vehicles of
apyrase ⫹ ADA or iso-PPADS, the LAH induced by acidpepsin insult was unaffected (Figs. 2A and 3).
106 • MAY 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.3 on June 16, 2017
was performed to test the possibility that laryngeal ATP might induce
LAH. The reflex apneic responses to three laryngeal ammonia provocations were studied. The first provocation was made 40 min before
laryngeal application with ␣,␤-meATP (group 17) or its vehicle
(group 18). The other provocations were made at 1 and 2 h after
␣,␤-meATP application. Study 6 (Fig. 1F) was performed to investigate whether laryngeal insult with acid-pepsin or H2O2 might induce
an increase in levels of inflammation, lipid peroxidation, and ATP in
the larynx. Laryngeal insult with acid-pepsin (group 19), H2O2 (group
20), or their respective vehicles (groups 21 and 22) were performed.
Two hours later, the laryngeal fluids of five animals in each group
were collected, and their laryngeal tissues were sampled for measurements of ATP and lipid peroxidation, whereas the larynxes of the
other five animals were excised for the pathohistological study. To
collect laryngeal fluids, a suction catheter (PE-50) was inserted into
the upper tracheal catheter with its tip close to the laryngeal segment.
The laryngeal fluids were sucked by a syringe via this suction catheter.
Study 7 was performed to investigate the potency and duration of
blocking effect of iso-PPADS. The reflex apneic responses to four
laryngeal provocations with ␣,␤-meATP were investigated. One hour
was allowed to elapse between any two ␣,␤-meATP provocations.
Laryngeal pretreatments with iso-PPADS (group 23) or its vehicle
(group 24) were made 30 min before the second and third ␣,␤-meATP
provocations. Study 8 was performed to investigate the possible type
of laryngeal afferents that responded to ␣,␤-meATP. The reflex
apneic responses to laryngeal ␣,␤-meATP provocations and mechanical provocations were investigated before and during perineural
capsaicin treatment (PCT) of superior laryngeal nerves (group 25)
with a procedure described previously (49). Laryngeal mechanical
provocations were carried out by probing the laryngeal segment with
a nylon thread.
Data analysis and statistics. Respiratory flow, tidal volume, and
expiratory duration (TE) were analyzed on a breath-by-breath basis.
Mean arterial blood pressure and heart rate were measured at 1-s
intervals. These physiological parameters were analyzed using a
computer system (BioCybernatics, 1.0; Taipei, Taiwan). The longest
TE occurring during the first 5 s after laryngeal provocation was
divided by the baseline TE, and the value was then multiplied by 100
to give a percentage apneic index. The data for cardiopulmonary
parameters were compared using one-way repeated-measures
ANOVA followed by Fisher’s least significant difference procedure
where appropriate. The injury scores were compared using the MannWhitney U-test. The data for lipid peroxidation and ATP concentrations were compared using Student’s t-test. A value of P ⬍ 0.05 was
considered significant. All data are means ⫾ SE.
1587
1588
ROS AND ATP CAUSE HYPERSENSITIVITY OF LARYNGEAL REFLEX
Table 1. Apneic response to laryngeal ammonia provocation
before and after laryngeal insults with 4 concentrations of
pepsin in pH 5 solution in 4 study groups
Pepsin, mg/ml
Study Group
Control
Apneic index, %
Animals with LAH,
1 h after insult
Apneic index, %
Animals with LAH,
2 h after insult
Apneic index, %
Animals with LAH,
3 h after insult
Apneic index, %
Animals with LAH,
2.5
0.25
0.025
0.0025
%
504⫾30
N/A
517⫾30
N/A
510⫾10
N/A
504⫾15
N/A
%
537⫾34
0
534⫾32
0
503⫾27
0
496⫾27
0
%
980⫾115*
100
609⫾125
25
533⫾47
12.5
503⫾28
0
808⫾98*
50
607⫾75
25
536⫾48
12.5
%
1038⫾98*
100
Laryngeal H2O2 insult also produces LAH. To examine
whether a direct increase in ROS level might produce LAH, the
larynx was insulted with H2O2. Laryngeal H2O2 insult produced an LAH with characteristics similar to those of acidpepsin-induced LAH (Fig. 4). A subsequent denervation of the
SLNs abolished the apneic response to ammonia provocation
(Fig. 4A), indicating that the H2O2-induced LAH was mediated
through the SLNs. This neural mechanism is similar to that of
acid-pepsin-induced LAH reported previously (49). In contrast,
in animals with laryngeal insult with vehicle of H2O2, this
LAH was not observed over the same time period (Fig. 4A). In
addition, the H2O2-induced LAH was prevented by laryngeal
pretreatment with DMTU but not with its vehicle, suggesting a
critical role for •OH (Fig. 4B).
Activation of P2X receptors by ATP is important in H2O2induced LAH. The effects of laryngeal pretreatment with
apyrase ⫹ ADA or iso-PPADS on the LAH induced by
laryngeal H2O2 insult (Fig. 5) were similar to those on the LAH
induced by laryngeal acid-pepsin insult. The H2O2-induced
LAH was prevented by laryngeal pretreatment with these drugs
but was unaffected by pretreatment with their vehicles (Fig. 5).
Table 2. Effect of pretreatment with iso-PPADS or its
vehicle on the apneic response to 4 laryngeal ␣,␤-meATP
provocations in 2 study groups
Study Group
Control Apneic
Index, %
Response to 2nd
Provocation, %
Response to 3rd
Provocation, %
Response to 4th
Provocation, %
iso-PPADS
Vehicle
397⫾78
487⫾86
102⫾1*
454⫾96
101⫾1*
617⫾141
783⫾210
754⫾215
Data in each group are means ⫾ SE from 8 rats. One hour was allowed to
elapse between any two ␤-methylene-ATP (␣,␤-meATP) provocations. Layngeal pretreatment with iso-pyridoxalphosphate-6-azophenyl-2⬘,5⬘-disulfonate (iso-PPADS) or its vehicle were made 30 min before the 2nd and 3rd
␣,␤-meATP provocations. Apneic response is expressed as apneic index
(see Table 1 legend for definition). *P ⬍ 0.05, significantly different from
vehicle.
J Appl Physiol • VOL
Fig. 4. Mean apneic responses to laryngeal ammonia provocations before and
after insult with H2O2 or its vehicle in 4 study groups. A: responses before and
after laryngeal insult with H2O2 or its vehicle. Denervation of SLNs was
performed at the 4th hour after insult. B: responses before and after laryngeal
insult with H2O2 in animals pretreated with laryngeal application of DMTU or
vehicle. Recovery of the response was obtained after the drug effect had worn
off. Baseline TE was calculated as the mean over 10 breaths immediately
before provocation. *P ⬍ 0.05, significantly different from the response before
H2O2 or vehicle insult in the same group. #P ⬍ 0.05, significantly different
from the response of the vehicle group at the same time point. Data in each
group are means ⫾ SE from 8 rats.
Activation of P2X receptors by ATP analog alone also
produces LAH. In animals with laryngeal application of ␣,␤meATP, ammonia provocation evoked a significantly augmented apneic response during the first and second hours after
application compared with the control response (Fig. 6). In
contrast, in animals with laryngeal application of the vehicle
for ␣,␤-meATP, the LAH was not observed over the same time
period (Fig. 6). To identify the type of laryngeal afferents that
responded to ␣,␤-meATP, we investigated the reflex apneic
responses to laryngeal provocation by this agonist before and
during PCT of SLNs, a procedure that selectively blocks the
neural conduction of capsaicin-sensitive afferent fibers (49).
It was found that PCT nearly abolished the apneic response
to ␣,␤-meATP provocation (apneic index before vs. during
PCT, 565 ⫾ 171 vs. 109 ⫾ 3%), whereas it did not
significantly affect the apneic response to mechanical provocation in the same animals (apneic index before vs. during
106 • MAY 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.3 on June 16, 2017
Data in each group are means ⫾ SE from 8 rats.The longest expiratory
duration (TE) occurring during the first 5 s after laryngeal provocation was
divided by the baseline TE, and the value was then multiplied by 100 to give
a percentage apneic index. When the ammonia-evoked apneic index exceeded
the baseline by at least 20%, the animal was defined as displaying laryngeal
airway hyperreactivity (LAH). *P ⬍ 0.05, significantly different from baseline
in the same group.
ROS AND ATP CAUSE HYPERSENSITIVITY OF LARYNGEAL REFLEX
1589
Hemodynamics. In all animals, the arterial blood pressure
and heart rate were stable until the end of each experiment.
Before and at the end of experiment, the mean values for
arterial blood pressure were 101.2 ⫾ 14.1 and 99.9 ⫾ 11.3
mmHg, respectively, and the mean values for heart rate were
367.2 ⫾ 40.0 and 360.3 ⫾ 34.3 beats/min, respectively.
DISCUSSION
PCT, 630 ⫾ 103 vs. 559 ⫾ 77%), indicating a selective
blocking effect of PCT.
Laryngeal insults with acid-pepsin or H2O2 promote increases in lipid peroxidation, ATP, and inflammation in the
larynx. The levels of lipid peroxidation in laryngeal tissues and
ATP in the laryngeal fluid sampled at 2 h after laryngeal insults
with acid-pepsin (Fig. 7, A and B) and H2O2 (Fig. 7, C and D)
were both significantly higher than those sampled at 2 h after
vehicle insult. Histological examination revealed that laryngeal
insults with acid-pepsin (Fig. 8B) or H2O2 (Fig. 8D) produced
a mild epithelial damage, as evidenced by epithelial shedding
and/or observable infiltration of inflammatory cells, compared
with insults with their vehicles (Fig. 8, A and C). Overall, the
total injury score in the acid-pepsin group (2.6 ⫾ 0.2) was not
significantly different from that in the H2O2 group (2.2 ⫾ 0.2),
and both scores were significantly greater than scores for their
corresponding controls (vehicle of acid-pepsin, 1.1 ⫾ 0.1;
vehicle of H2O2, 1.4 ⫾ 0.2).
J Appl Physiol • VOL
Fig. 6. Mean apneic responses to laryngeal ammonia provocations before and
after laryngeal application with ␣,␤-meATP vehicle in 2 study groups.
Baseline TE was calculated as the mean over 10 breaths immediately before
provocation. *P ⬍ 0.05, significantly different from the response before
␣,␤-meATP in the same group. #P ⬍ 0.05, significantly different from the
response of the vehicle group at the same time point. Data in each group
are means ⫾ SE from 8 rats.
106 • MAY 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.3 on June 16, 2017
Fig. 5. Mean apneic responses to laryngeal ammonia provocations before
and after laryngeal insult with H2O2 in 4 study groups. A: animals
pretreated with apyrase ⫹ ADA (ATP scavengers) or vehicle. B: animals
pretreated with iso-PPADS (P2X receptor antagonist) or vehicle. Recovery
of the response was obtained after the drug effect had worn off. Baseline TE
was calculated as the mean over 10 breaths immediately before provocation. *P ⬍ 0.05, significantly different from the response before H2O2
insult in the same group. #P ⬍ 0.05, significantly different from the
response of the vehicle group at the same time point. Data in each group are
means ⫾ SE from 8 rats.
We (49) recently reported that a laryngeal acid-pepsin insult
produces an LAH that is mediated through sensitization of the
capsaicin-sensitive laryngeal afferent fibers by ROS. In the
current study, we further demonstrated that this LAH was
prevented by laryngeal pretreatment with either ATP scavengers or a P2X receptor antagonist, indicating the importance of
activation of P2X receptors by endogenous ATP. A plausible
hypothesis is that ATP release following acid-pepsin insult
activates P2X receptors located on the nerve endings and that
this induces sensitization of these afferents fibers, resulting in
LAH. This notion is strongly supported by the observations
that direct application of ␣,␤-meATP alone could also produce
LAH and that the acid-pepsin insult indeed promoted the
release of endogenous ATP as shown by the elevated level in
the laryngeal fluid. The ␣,␤-meATP-induced LAH also appears to be mediated through capsaicin-sensitive afferent fibers, because the laryngeal reflex response to this agonist
can be eliminated by a procedure that selectively blocks the
neural conduction of these afferent fibers. The onset of LAH
induced by laryngeal ␣,␤-meATP was sooner than that
induced by acid-pepsin insult. This may possibly be due to
the fact that exogenous ␣,␤-meATP has a direct action,
whereas endogenous ATP requires a certain period of time
to be released following acid-pepsin insult. Taken together,
in addition to ROS, the present findings suggest the crucial
role of activation of P2X receptors by ATP in the acidpepsin-induced LAH.
1590
ROS AND ATP CAUSE HYPERSENSITIVITY OF LARYNGEAL REFLEX
Fig. 7. Increases in levels of lipid peroxidation in laryngeal
tissues (A and C) and ATP in the laryngeal fluid (B and D)
sampled at 2 h after laryngeal insult with acid-pepsin (A and B)
or H2O2 (C and D). RFU, relative fluorescent unit. *P ⬍ 0.05,
significantly different from the vehicle. Data in each group are
means ⫾ SE from 5 rats.
the levels ATP, lipid peroxidation, and inflammation in the
larynx. These findings suggest that an increase in laryngeal
ROS promotes the release of ATP in the inflamed larynx,
which may subsequently activate P2X receptors, serving as a
downstream mechanism responsible for the development of the
observed LAH. Since these H2O2-induced pathophysiological
Fig. 8. Light micrographs of laryngeal tissues from 4 anesthetized rats excised at 2 h after laryngeal insult with acid-pepsin
(B), H2O2 (D) or their respective vehicles (A and C). All
magnifications, ⫻100. e, Epiglottis; vc, vocal cord. Arrows
indicate epithelium damage; arrowheads indicate subepithelial
inflammatory cell infiltration.
J Appl Physiol • VOL
106 • MAY 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.3 on June 16, 2017
Since scavenging either •OH or ATP completely abrogated
the acid-pepsin-induced LAH, their functional contributions
ought to be interrelated in series. In this study, we found that
laryngeal H2O2 insult induced an LAH that was abrogated by
a •OH scavenger, ATP scavengers, or a P2X receptor antagonist. In addition, laryngeal H2O2 insult caused an increase in
ROS AND ATP CAUSE HYPERSENSITIVITY OF LARYNGEAL REFLEX
J Appl Physiol • VOL
are consistent with the report that EER patients show a longlasting increase in cough reflex sensitivity to inhaled capsaicin
aerosol (5, 13, 34, 40). Furthermore, our observations are in
agreement with the emerging concept that a new category of
weakly acidic reflux (pH 4-7) should be defined and that
GERD patients in this category are associated with chronic
cough (6, 44, 45). Second, capsaicin-sensitive laryngeal afferent fibers are polymodal fibers that also can be stimulated by
various mediators such as acid, ATP, ROS, histamine, and
arachidonate metabolites or by many inhaled irritants (7, 8, 27,
42, 43). The accessibility of laryngeal stimuli to these afferent
fibers may be increased in inflamed larynx when laryngeal
mucosal permeability is elevated. Thus the unexplained
chronic cough found in patients with GERD or EER could be
due to the fact that the cough is more vulnerable to being
triggered when sensitized capsaicin-sensitive laryngeal afferent fibers are stimulated by acidic gastric juices during
reflux episodes, or by inflammatory mediators and inhaled
irritants when there is no reflux (4, 5, 6, 32, 41). These two
scenarios probably can explain the clinical observations that
the temporal pattern of reflux may or may not correlate well
with that of the cough (3, 4, 11) and that proton pump
inhibitor treatment may or may not relieve chronic cough in
these patients (5, 9, 21, 38).
In conclusion, our findings suggest that laryngeal insult with
acid-pepsin or H2O2 induces inflammation and produces excess ROS in the rat’s larynx. The latter may in turn promote the
release of ATP to activate P2X receptors, resulting in sensitization of capsaicin-sensitive laryngeal afferent fibers and LAH.
Although the optimal therapy to treat chronic cough in these
patients is still evolving, the functional significance of ROS,
ATP, and P2X receptors in the development of LAH induced
by laryngeal acid-pepsin or H2O2 insult may provide a basis for
possible target choices when investigating potential therapeutic
regimes.
ACKNOWLEDGMENTS
We are grateful to Dr. Tien Huan Hsu for statistical analysis of data and Dr.
Ralph Kirby for help in language editing.
GRANTS
This study was supported by a grant (95-2320-B-010-025-MY3) from the
National Science Council of the Republic of China, grants (VGH94-061 and
V95A-037, V96B1-009) from Taipei Veterans General Hospitals, a grant
(VGHUST95-P7-28) from the joint program of the Veterans General Hospitals
and the University System of Taiwan, and a grant (96A-D-P208) from the Aim
for the Top University Plan supported by the Ministry of Education of the
Republic of China.
REFERENCES
1. Ahmad S, Ahmad A, Ghosh M, Leslie CC, White CW. Extracellular
ATP-mediated signaling for survival in hyperoxia-induced oxidative
stress. J Biol Chem 279: 16317–16325, 2004.
2. Adhami T, Goldblum JR, Richter JE, Vaezi MF. The role of gastric and
duodenal agents in laryngeal injury: an experimental canine model. Am J
Gastroenterol 99: 2098 –2106, 2004.
3. Avidan B, Sonnenberg A, Schnell TG, Sontag SJ. Temporal associations between coughing or wheezing and acid reflux in asthmatics. Gut 49:
767–772, 2001.
4. Benini L, Ferrari M, Talamini G, Vantini I. Reflux associated cough is
usually not associated with reflux: role of reduced cough threshold. Gut
55: 583–584, 2006.
5. Benini L, Ferrari M, Sembenini C, Olivieri M, Micciolo R, Zuccali V,
Bulighin GM, Fiorino F, Ederle A, Cascio VL, Vantini I. Cough
106 • MAY 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.3 on June 16, 2017
consequences were similar to those induced by acid-pepsin
insult, it is possible that a comparable mechanism with this
hierarchy of ROS and ATP is involved in the development of
the acid-pepsin-induced LAH. Alternatively, H2O2 may act
nonspecifically as an irritant leading to LAH in the same
manner as acid-pepsin insult.
Our results fully support the hypothesis that inflammatory
mediators can sensitize laryngeal afferent fibers, resulting in
LAH in EER patients, as postulated by other investigators (5,
40, 41). The hierarchy of ROS and ATP as the sensitizing
mediators is not surprising because, in other organs, inflamed tissues usually generate excess ROS that may damage or stimulate cells, resulting in release of their cytosolic
ATP to act on other cells in the vicinity (10, 12, 35). The
source of ATP could not be determined in this study.
However, because increased levels of ATP could be detected in the laryngeal fluid, airway epithelial cells are a
plausible candidate (24).
Capsaicin-sensitive afferent fibers are present in the larynx
(23, 31). Functional studies in the stimulation of capsaicinsensitive airway afferent fibers by receptor agonist suggest that
P2X receptors may be located at the terminals of these afferent
fibers (37, 42). The possibility that activation of these P2X
receptors by ATP may sensitize capsaicin-sensitive laryngeal
afferent fibers, resulting in LAH, however, has not been
previously reported. In another visceral organ, activation of
P2X receptor by ␣,␤-meATP induces sensitization of vagal
mechanoreceptors during esophageal inflammation in ferrets
(36). In addition, in vivo studies have demonstrated that
activation of P2X receptor by ␣,␤-meATP produces hyperalgesia in mice or rats, a response that is mediated through
capsaicin-sensitive nociceptive afferent fibers (50, 51, 52).
In vitro studies have shown that activation of P2X receptors
by ␣,␤-meATP sensitizes rat cutaneous nerve terminals of
the nociceptive C- and A-␦ fibers to mechanical or heat
stimulation (26, 52, 53). Thus it appears that capsaicinsensitive afferent fibers in both somatic and visceral organs
are vulnerable to sensitization following activation of P2X
receptors by ATP.
The pepsin concentrations in the sputum samples collected
from EER patients range from 0.003 to 22 ␮g/ml (22). Thus
one potential limitation of our experimental model is that the
effective concentration of pepsin (2.5 mg/ml) for induction of
LAH is higher than physiological concentrations measured in
patients. However, the laryngeal insults by gastric contents in
EER patients usually are repeated and recurrent during reflux
episodes. This scenario is obviously different from that with
only one laryngeal insult by acid-pepsin or H2O2 in our
experimental model. The other limitation of this study is that
our findings were based on the measurement of reflex apneic
responses elicited by laryngeal irritation. We did not measure
the responses mediated by other efferent arms, such as laryngeal adduction or cough. We assumed that our findings could
be extrapolated to these two responses, which are commonly
observed in EER patients.
Our results may have several clinical implications. First, we
found that a single laryngeal exposure to pepsin at a weakly
acidic pH (pH 5) for only 40 s can sensitize capsaicin-sensitive
laryngeal afferent fibers, resulting in an LAH that persists for
at least 1 h. Since stimulation of these afferent fibers can
trigger cough reflex in human subjects (11, 16), our findings
1591
1592
6.
7.
8.
9.
10.
11.
12.
13.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
threshold in reflux oesophagitis: influence of acid and of laryngeal and
oesophageal damage. Gut 46: 762–767, 2000.
Blondeau K, Dupont LJ, Mertens V, Tack J, Sifrim D. Improved
diagnosis of gastro-oesophageal reflux in patients with unexplained
chronic cough. Aliment Pharmacol Ther 25: 723–32, 2007.
Carr MJ, Undem BJ. Bronchopulmonary afferent nerves. Respirology 8:
291–301, 2003.
Carr MJ, Undem BJ. Inflammation-induced plasticity of the afferent
innervation of the airways. Environ Health Perspect 109: 567–571, 2001.
Chang AB, Lasserson TJ, Kiljander TO, Connor FL, Gaffney JT,
Garske LA. Systematic review and meta-analysis of randomised controlled trials of gastro-oesophageal reflux interventions for chronic cough
associated with gastro-oesophageal reflux. BMJ 332: 11–17, 2006.
Chizh BA, Illes P. P2X receptors and nociception. Pharmacol Rev 53:
553–568, 2001.
Collier JG, Fuller RW. Capsaicin inhalation in man and the effects of
sodium cromoglycate. Br J Pharmacol 81: 113–117, 1984.
Cook SP, McCleskey EW. Cell damage excites nociceptors through
release of cytosolic ATP. Pain 95: 41– 47, 2002.
Ferrari M, Olivieri M, Sembenini C, Benini L, Zuccali V, Bardelli E,
Bovo P, Cavallini G, Vantini I, Lo Cascio V. Tussive effect of capsaicin
in patients with gastroesophageal reflux without cough. Am J Respir Crit
Care Med 151: 557–561, 1995.
Fletcher BL, Dillard CJ, Tappel AL. Measurement of fluorescent lipid
peroxidation products in biological systems and tissues. Anal Biochem 52:
1–9, 1973.
Fox RB. Prevention of granulocyte-mediated oxidant lung injury in rats by
a hydroxyl radical scavenger, dimethylthiourea. J Clin Invest 74: 1456 –
1464, 1984.
Hansson L, Wollmer P, Dahlback M, Karlsson JA. Regional sensitivity
of human airways to capsaicin-induced cough. Am Rev Respir Dis 145:
1191–1195, 1992.
Irnich D, Burgstahler R, Bostock H, Grafe P. ATP affects both axons
and Schwann cells of unmyelinated C fibres. Pain 92: 343–350, 2001.
Ishikawa T, Sekizawa SI, Sant’Ambrogio FB, Sant’Ambrogio G.
Larynx vs. esophagus as reflexogenic sites for acid-induced bronchoconstriction in dogs. J Appl Physiol 86: 1226 –1230, 1999.
Jimenez P, Piazuelo E, Sanchez MT, Ortego J, Soteras F, Lanas A.
Free radicals and antioxidant systems in reflux esophagitis and Barrett’s
esophagus. World J Gastroenterol 11: 2697–2703, 2005.
Kangas L, Gronroos M, Nieminen AL. Bioluminescence of cellular
ATP: a new method for evaluating cytotoxic agents in vitro. Med Biol 62:
338 –343, 1984.
Kiljander TO. The role of proton pump inhibitors in the management of
gastroesophageal reflux disease-related asthma and chronic cough. Am J
Med 115: 65S-71S, 2003.
Knight J, Lively MO, Johnston N, Dettmar PW, Koufman JA. Sensitive pepsin immunoassay for detection of laryngopharyngeal reflux.
Laryngoscope 115:1473–1478, 2005.
Koike S, Uno T, Bamba H, Shibata T, Okano H, Hisa Y. Distribution
of vanilloid receptors in the rat laryngeal innervation. Acta Otolaryngol
124: 515–519, 2004.
Novak I. ATP as a signaling molecule: the exocrine focus. News Physiol
Sci 18: 12–17, 2003.
Koufman JA. The otolaryngologic manifestations of gastroesophageal
reflux disease (GERD): a clinical investigation of 225 patients using
ambulatory 24-h pH monitoring and an experimental investigation of the
role of acid and pepsin in the development of laryngeal injury. Laryngoscope 101: 1–78, 1991.
Kress M, Guenther S. Role of [Ca2⫹]i in the ATP-induced heat sensitization process of rat nociceptive neurons. J Neurophysiol 81: 2612–2619,
1999.
Lee LY, Burki NK, Gerhardstein DC, Gu Q, Kou YR, Xu J. Airway
irritation and cough evoked by inhaled cigarette smoke: role of neuronal
nicotinic acetylcholine receptors. Pulm Pharmacol Ther 20: 355–364,
2007.
Lin YS, Kou YR. Reflex apneic response evoked by laryngeal exposure
to wood smoke in rats: neural and chemical mechanisms. J Appl Physiol
83: 723–730, 1997.
Maceri DR, Zim S. Laryngospasm: an atypical manifestation of severe
gastroesophageal reflux disease (GERD). Laryngoscope 111: 1976 –1979,
2001.
J Appl Physiol • VOL
30. Moriyama T, Iida T, Kobayashi K, Higashi T, Fukuoka T, Tsumura
H, Leon C, Suzuki N, Inoue K, Gachet C, Noguchi K, Tominaga M.
Possible involvement of P2Y2 metabotropic receptors in ATP-induced
transient receptor potential vanilloid receptor 1-mediated thermal hypersensitivity. J Neurosci 23: 6058 – 6062, 2003.
31. Mutoh T, Tsubone H. Hypersensitivity of laryngeal C-fibers induced by
volatile anesthetics in young guinea pigs. Am J Respir Crit Care Med 167:
557–562, 2003.
32. Napierkowski J, Wong RK. Extraesophageal manifestations of GERD.
Am J Med Sci 326: 285–299, 2003.
33. Naya MJ, Pereboom D, Ortego J, Alda JO, Lanas A. Superoxide anions
produced by inflammatory cells play an important part in the pathogenesis
of acid and pepsin induced oesophagitis in rabbits. Gut 40: 175–181, 1997.
34. Nieto L, de Diego A, Perpina M, Compte L, Garrigues V, Martinez E,
Ponce J. Cough reflex testing with inhaled capsaicin in the study of
chronic cough. Respir Med 97: 393– 400, 2003.
35. North RA. P2X3 receptors and peripheral pain mechanisms. J Physiol
554: 301–308, 2004.
36. Page AJ, O’Donnell TA, Blackshaw LA. P2X purinoceptor-induced
sensitization of ferret vagal mechanoreceptors in oesophageal inflammation. J Physiol 523: 403– 411, 2000.
37. Pelleg A, Hurt CM. Mechanism of action of ATP on canine pulmonary
vagal C fibre nerve terminals. J Physiol 490: 265–275, 1996.
38. Poe RH, Kallay MC. Chronic cough and gastroesophageal reflux disease:
experience with specific therapy for diagnosis and treatment. Chest 123:
679 – 684, 2003.
39. Poelmans J, Tack J. Extraoesophageal manifestations of gastro-oesophageal reflux. Gut 54: 1492–1499, 2005.
40. Prudon B, Birring SS, Vara DD, Hall AP, Thompson JP, Pavord ID.
Cough and glottic-stop reflex sensitivity in health and disease. Chest 127:
550 –557, 2005.
41. Rolla G, Colagrande P, Magnano M, Debernardi V, Dutto L, Delpiano
L, Cassolino P, Bucca C. Extrathoracic airway dysfunction in cough
associated with gastroesophageal reflux. J Allergy Clin Immunol 102:
204 –209, 1998.
42. Ruan T, Lin YS, Lin KS, Kou YR. Sensory transduction of pulmonary
reactive oxygen species by capsaicin-sensitive vagal lung afferent fibres
in rats. J Physiol 565: 563–578, 2005.
43. Ruan T, Ho CY, Kou YR. Afferent vagal pathways mediating respiratory
reflexes evoked by ROS in the lungs of anesthetized rats. J Appl Physiol
94: 1987–1998, 2003.
44. Sifrim D. Acid, weakly acidic and non-acid gastro-oesophageal reflux:
differences, prevalence and clinical relevance. Eur J Gastroenterol Hepatol 16: 823– 830, 2004.
45. Sifrim D, Dupont L, Blondeau K, Zhang X, Tack J, Janssens J.
Weakly acidic reflux in patients with chronic unexplained cough during 24
hour pressure, pH, and impedance monitoring. Gut 54: 449 – 454, 2005.
46. Szekely SM, Vickers MD. A comparison of the effects of codeine and
tramadol on laryngeal reactivity. Eur J Anaesthesiol 9: 111–120, 1992.
47. Thompson DM. Laryngopharyngeal sensory testing and assessment of
airway protection in pediatric patients. Am J Med 115: 166S–168S, 2003.
48. Tominaga M, Wada M, Masu M. Potentiation of capsaicin receptor
activity by metabotropic ATP receptors as a possible mechanism for
ATP-evoked pain and hyperalgesia. Proc Natl Acad Sci USA 98: 6951–
6956, 2001.
49. Tsai TL, Chang SY, Ho CY, Kou YR. Neural and hydroxyl radical
mechanisms underlying laryngeal airway hyperreactivity induced by laryngeal acid-pepsin insult in anesthetized rats. J Appl Physiol 101: 328 –
338, 2006.
50. Tsuda M, Ueno S, Inoue K. Evidence for the involvement of spinal
endogenous ATP and P2X receptors in nociceptive responses caused by
formalin and capsaicin in mice. Br J Pharmacol 128: 1497–504, 1999.
51. Tsuda M, Ueno S, Inoue K. In vivo pathway of thermal hyperalgesia by
intrathecal administration of alpha,beta-methylene ATP in mouse spinal
cord: involvement of the glutamate-NMDA receptor system. Br J Pharmacol 127: 449 – 456, 1999.
52. Wu G, Whiteside GT, Lee G, Nolan S, Niosi M, Pearson MS, Ilyin VI.
A-317491, a selective P2X3/P2X2/3 receptor antagonist, reverses inflammatory mechanical hyperalgesia through action at peripheral receptors in
rats. Eur J Pharmacol 504: 45–53, 2004.
53. Zhang Q, Zhao Y, Guo Y, Cao DY, Tang XD, Tian YL, Yao FR, Wang
HS. Activation and sensitization of C and Adelta afferent fibers mediated
by P2X receptors in rat dorsal skin. Brain Res 1102: 78 – 85, 2006.
106 • MAY 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.3 on June 16, 2017
14.
ROS AND ATP CAUSE HYPERSENSITIVITY OF LARYNGEAL REFLEX