Hyperpnea-induced bronchoconstriction is dependent
on tachykinin-induced cysteinyl leukotriene synthesis
X. X. YANG, W. S. POWELL, M. HOJO, AND J. G. MARTIN
Meakins-Christie Laboratories, McGill University Montreal, Quebec, Canada H2X 2P2
leukotriene D4; exercise-induced asthma; neurokinin antagonists
of exercise-induced asthma (EIA) has
been investigated extensively (11, 38). However, the
mechanism of this phenomenon is still uncertain. The
similarity between EIA and isocapnic dry air hyperpneainduced bronchoconstriction (HIB) has led to the speculation that they are mediated by similar mechanisms
(9, 22, 41). Heat and water loss have proved to play a
crucial role in both EIA and HIB (10, 20, 40). However,
hyperpnea-induced changes in airway caliber or blood
flow have been demonstrated in several species (1, 7),
suggesting that hyperpnea may induce changes in
airway function even in normal animals and raising the
possibility that exercise-induced bronchoconstriction
may not reflect a phenomenon unique to asthmatic
subjects.
THE PHENOMENON
538
The guinea pig is one of the most frequently studied
models of hyperpnea-induced airway responses (37). In
this species, the release of tachykinins, low-molecularweight neuropeptides, from sensory C fiber nerve endings found in airways appears to contribute to the
airway narrowing of HIB (36). Inhibition of neutral
endopeptidase, an enzyme that degrades tachykinin in
the airways and augments HIB and capsaicin, a neurotoxin that destroys tachykinin-containing nerve fibers,
diminishes HIB (15). Furthermore, selective neurokinin (NK) antagonists abolish HIB (3). It is possible that
tachykinins act through the production of other bronchoactive mediators such as cysteinyl leukotrienes (LTs). It
has been shown both in vivo and in vitro that airway
contractile responses to sensory nerve stimulation can
be inhibited by cysteinyl LT antagonists and that
afferent neural responses in vitro can be potentiated by
the addition of exogenous LTD4 (13). These findings
suggest the possibility of LT involvement in HIB in
guinea pigs. Various cyclooxygenase and lipoxygenase
inhibitors and a LTD4 antagonist have been shown to
reduce the magnitude of HIB in the guinea pig (15).
However, it is not known whether cysteinyl LTs act in
parallel or in series with tachykinins to induce airway
narrowing. It is possible that cysteinyl LTs and tachykinins may be involved in a cascade of reactions. Indeed,
there is evidence that cysteinyl LTs can release tachykinins in airway tissue (5, 30).
We hypothesized that cysteinyl LTs mediate HIB and
that their release is stimulated by the action of tachykinins on effector cells in the airways. To investigate the
role of cysteinyl LTs in HIB, we took advantage of the
fact that the bile is the major route for their excretion in
the guinea pig (17, 26). Biliary cysteinyl LT levels were
quantitated before and after hyperpnea challenge, and
the effects of selective NK antagonists on cysteinyl LT
levels were determined. We reasoned that cysteinyl LT
levels after hyperpnea challenge would be unaffected
by pretreatment of animals with NK antagonists if LT
synthesis were independent of the endogenously released tachykinins. However, if our hypotheses were
correct, the levels of cysteinyl LTs in bile should be
reduced by pretreatment with NK antagonists. Furthermore, to evaluate the possible contribution of mast cells
to the production of bronchoconstrictive mediators after hyperpnea, we measured histamine levels in bronchoalveolar lavage (BAL) fluid and tested the effects of
an antihistamine on the degree of bronchoconstriction
induced by dry gas challenge.
MATERIALS AND METHODS
Animal preparation. Forty-one male Hartley guinea pigs
(400–550 g) were purchased from Charles River (St. Constant, Quebec, Canada). Guinea pigs were anesthetized with
0161-7567/97 $5.00 Copyright r 1997 the American Physiological Society
Downloaded from http://jap.physiology.org/ by 10.220.33.1 on June 15, 2017
Yang, X. X., W. S. Powell, M. Hojo, and J. G. Martin.
Hyperpnea-induced bronchoconstriction is dependent on
tachykinin-induced cysteinyl leukotriene synthesis. J. Appl.
Physiol. 82(2): 538–544, 1997.—The purpose of the study was
to test the hypothesis that tachykinins mediate hyperpneainduced bronchoconstriction indirectly by triggering cysteinyl
leukotriene (LT) synthesis in the airways. Guinea pigs (350–
600 g) were anesthetized with xylazine and pentobarbital
sodium and received hyperpnea challenge (tidal volume
3.5–4.0 ml, frequency 150 breaths/min) with either humidified isocapnic gas (n 5 6) or dry gas (n 5 7). Dry gas challenge
was performed on animals that received MK-571 (LTD4
antagonist; 2 mg/kg iv; n 5 5), capsaicin (n 5 4), neurokinin
(NK) antagonists [NK1 (CP-99994) 1 NK2 (SR-48968) (1
mg/kg iv); n 5 6], or the H1 antihistamine pyrilamine (2
mg/kg iv; n 5 5). We measured the tracheal pressure and
collected bile for 1 h before and 2 h after hyperpnea challenge.
We examined the biliary excretion of cysteinyl LTs; the
recovery of radioactivity in bile after instillation of 1 µCi
[3H]LTC4 intratracheally averaged 24% within 4 h (n 5 2).
The major cysteinyl LT identified was LTD4 (32% recovery of
radioactivity). Cysteinyl LTs were purified from bile of animals undergoing hyperpnea challenge by using reversephase high-pressure liquid chromatography and quantified
by radioimmunoassay. There was a significant increase in the
peak value of tracheal pressure after challenge, indicating
bronchoconstriction in dry gas-challenged animals but not
after humidified gas challenge. MK-571, capsaicin, and NK
antagonists prevented the bronchoconstriction; pyrilamine
did not. Cysteinyl LT levels in the bile after challenge were
significantly increased from baseline in dry gas-challenged
animals (P , 0.05) and were higher than in the animals
challenged with humidified gas or dry gas-challenged animals treated with capsaicin or NK antagonists (P , 0.01).
The results indicate that isocapnic dry gas hyperpneainduced bronchoconstriction is LT mediated and the role of
tachykinins in the response is indirect through release of LTs.
Endogenous histamine does not contribute to the response.
TACHYKININS TRIGGER CYSTEINYL LEUKOTRIENE SYNTHESIS
capsaicin; day 2: 0.5 ml of 1.0 mg/ml capsaicin; day 3: 0.5 ml of
10 mg/ml capsaicin; day 4: 0.2 ml and subsequently 0.3 ml of
50 mg/ml capsaicin; and day 5: 0.5 ml and subsequently 0.7
ml of 50 mg/ml capsaicin. To counteract respiratory impairment caused by capsaicin, animals were treated with 2.5
mg/ml of aerosolized salbutamol for 5 min before each capsaicin treatment. All animals were anesthetized with 30 mg/kg
ip pentobarbital sodium and were administered supplemental O2. The dry gas hyperpnea challenge was performed 1 day
after the final capsaicin pretreatment.
The role of tachykinins in HIB was also evaluated by using
specific NK antagonists. The specific NK1-receptor antagonist
CP-99994 and the NK2-receptor antagonist SR-48968 were
dissolved in equal volumes of ethylene glycol and saline. All
animals (NK antagonists; n 5 6) were administered a combination of CP-99994 (1 mg/kg) and SR-48968 (1 mg/kg) into the
jugular vein. Dry gas hyperpnea challenge was performed 10
min after the administration of the NK antagonists.
The role of histamine in HIB in guinea pigs (pyrilamine;
n 5 5) was assessed by using a specific histamine type
1-receptor antagonist. The histamine type 1-receptor antagonist pyrilamine was dissolved in saline (2 mg/ml in 1 ml) and
administered intravenously through the jugular vein 10 min
before dry gas hyperpnea challenge was performed. BAL was
performed in these animals 2 h after hyperpnea challenge by
using 5 ml of saline and compared with the results of lavage
fluid analysis from unchallenged control animals (n 5 6).
Recovery of radioactive LT metabolites after tracheal instillation of [3H]LTC4. Two guinea pigs were examined for the
recovery of radioactive products in bile after instillation of
1 µCi [14,15-3H]LTC4 in 100 µl of saline into the trachea. The
bile was collected continuously for 4 h in 15-min fractions.
The radioactivity in 30% of each bile sample was measured by
liquid scintillation counting. The remaining volumes of each
of the 15-min samples were pooled so as to obtain fractions
corresponding to the following time periods: 0–1 h, 1–2 h,
2–3 h, and 3–4 h. Each fraction was analyzed by RP-HPLC as
described below.
Separation of LTs by precolumn extraction/RP-HPLC.
Methanol (1.6 ml) was added to an aliquot (0.4 ml) of each bile
sample to yield a final concentration of 80% methanol.
Samples were allowed to stand at room temperature for 30
min and were then centrifuged at 3,500 g for 10 min to remove
protein. Distilled water (3.3 ml) was added to the supernatant
to give a concentration of 30% methanol, and the pH was
adjusted to 3.0 by using 1 M phosphoric acid. The sample was
then filtered by using a 0.45-µm filter (Millex-HV, MilliporeWaters, Bedford, MA). With a Milton-Roy minipump, the
sample (5.3 ml total volume) was loaded via a six-port
switching valve onto a precolumn cartridge (µBondapak C18
GuardPak, Millipore-Waters), which had been preequilibrated
with 11 ml of 30% methanol adjusted to pH 3 by the addition
of 1 M phosphoric acid. The precolumn was washed with 10
ml of 20% methanol containing 1 mM EDTA and 0.1% acetic
acid and adjusted to a pH of 5.4 by the addition of ammonium
hydroxide. The six-port valve was then switched to the
‘‘inject’’ position, placing the precolumn in line with an HPLC
pump (model 510, Millipore-Waters) and an octadecylsilylsilica HPLC column (Adsorbosphere HS C18, 3-µm particle
size, 4.6 3 150 mm, Alltech). The mobile phase consisted of
methanol-water-acetic acid (64:36:0.1) containing 1 mM EDTA
and adjusted to a pH of 5.4 by the addition of ammonium
hydroxide. The flow rate was 0.7 ml/min. Ultraviolet absorbance was monitored by a variable wavelength ultraviolet
detector (model 481, Millipore-Waters). Fractions collected
every 1 min were monitored either for radioactivity by using a
liquid scintillation counter (model LS 6000IC, Beckman
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xylazine (7 mg/kg) and pentobarbital sodium (65 mg/kg)
intraperitoneally. A high cervical tracheostomy was performed, and a short piece of polyethylene tubing (PE-240;
0.165 cm ID, 2.5 cm long) was inserted into the trachea below
the second cartilaginous ring and connected to a smallanimal respirator (model 360 rodent ventilator, Harvard,
South Natick, MA). Silastic medical-grade tubing (Dow Corning Medical Products, Midland, MI) was used to cannulate
the internal jugular vein for fluid replacement and administration of drugs. The inspiratory and expiratory tubes of the
ventilator were attached to the tracheal cannula through a Y
connector with a 3-cm common segment (total dead space 0.24
ml) to minimize conditioning of the inspired gas. The inspiratory port of the ventilator was connected to a warm (35–37°C)
humidifier through which room air was passed. The tracheal
pressure (Ptr) was measured at the tracheal cannula by a
piezoresistive differential pressure transducer (model
SCX01DN; SenSym, Sunnyvale, CA). The animals were
mechanically ventilated with a tidal volume of 6 ml/kg and 60
breaths/min. Ptr signals were amplified, passed through
eight-pole Bessel filters (model 902 lpf, Frequency Devices,
Haverhill, MA), and passed through an analog-to-digital
converter (model D7–2801-a, Data Translation, Marlborough, MA) for final storage in an IBM compatible personal
computer. A commercial software package (RHT Infodat,
Montreal, Quebec, Canada) was used to analyze the changes
in Ptr. Increases in Ptr, which reflect increases in respiratory
system impedance, were interpreted to indicate airway narrowing and, for convenience, are referred to throughout the
text as bronchoconstriction even though the possible contributions of microvascular leak and other mechanical changes
were not quantitated.
Collection of bile. The bile duct was cannulated with
polyethylene tubing (PE-60) through a small incision in the
abdominal wall. After surgery, the animals were allowed to
stabilize for a period of 90 min before challenge. For 1 h before
and for 2 h after hyperpnea challenge, bile was collected
under a stream of argon into Eppendorf tubes that were kept
on ice. Bile was stored at 280°C before analysis by reversephase high-pressure liquid chromatography (RP-HPLC) and
radioimmunoassay.
Experimental protocol. Dry gas hyperpnea (test; n 5 7) was
performed by introducing a dry mixture of 5% CO2-95% O2
from a balloon into the inspiratory port of the mechanical
ventilator. The ventilatory rate was set at 150 breaths/min,
and the tidal volume was increased to 4 ml; hyperpnea was
continued for 5 min. Humidified gas hyperpnea challenge
(control; n 5 6) was performed in an identical fashion, except
that the 5% CO2-95% O2 was bubbled through a water bath at
37°C before it passed through the ventilator. Baseline ventilator settings and gas mixtures were resumed after hyperpnea
challenge for a period that lasted until baseline Ptr was
reestablished (usually 10–40 min). Ptr measurements were
obtained at 1-min intervals for the first 10 min into the
recovery period, at 5-min intervals for the next 20 min, and at
15-min intervals thereafter for up to 2 h.
To evaluate the role of LTD4 in HIB, a specific LTD4
antagonist, MK-571 (2 mg/kg dissolved in 1 ml of 0.9% saline),
was administered to a group of guinea pigs (MK-571; n 5 5)
through the jugular vein catheter 10 min before the dry gas
hyperpnea challenge.
To deplete airway sensory nerves of neuropeptides, animals were pretreated with capsaicin (n 5 4; total dose 5 91
mg injected subcutaneously) in eight increasing doses over 5
days. Capsaicin was dissolved in 10% Tween 20 (Sigma
Chemical, St. Louis, MO), 10% ethanol, and 80% saline. Each
dosage was given as follows: day 1: 0.1 ml of 1.0 mg/ml
539
540
TACHYKININS TRIGGER CYSTEINYL LEUKOTRIENE SYNTHESIS
RESULTS
Recovery of radioactive products in bile after tracheal
instillation of [3H]LTC4. The time course of the appearance of radioactivity in the bile of the two guinea pigs
that received [14,15-3H]LTC4 by tracheal instillation is
shown in Fig. 1. The radioactivity in the bile samples
Fig. 1. Total radioactivity and radioactivity corresponding to each
product recovered after tracheal instillation of [3H]leukotriene (LT)
C4 plotted as function of time after administration of [3H]LTC4. j,
Total radioactivity recovered (inset); l, polar metabolites; s, LTE4;
5, LTC4; n, LTD4; 6, N-acetyl LTE4. cpm, counts/min. Each species is
expressed as percentage of total counts instilled into trachea.
increased almost linearly with time for the first 3 h and
appeared to reach a plateau by 4 h. About 23% of the
instilled radioactivity was recovered within 4 h (21 and
24% in each animal). The LT species contained in the
radioactive material appearing in the bile samples
were determined by RP-HPLC analysis. For this purpose, the bile samples were pooled to give four fractions, each corresponding to a collection period of 1 h,
after instillation of [3H]LTC4. The major species identified was LTD4 (38.4% of the total recovered radioactivity in the bile), whereas smaller amounts of LTC4
(10.7%), LTE4 (19.8%), and N-acetyl LTE4 (8.7%) were
detected. The balance of the radioactivity was associated with unidentified polar species. There was no
obvious change in the proportions of the different
species over time.
Airway response to hyperpnea challenge. The baseline values of inspiratory Ptr were not significantly
different among the various treatment groups. The
time course of the change in the values of Ptr after
isocapnic dry gas hyperpnea challenge was consistent
with the development of bronchoconstriction (Fig. 2).
The inspiratory Ptr showed an increase as early as 1
min after dry gas challenge compared with control
animals challenged with humidified gas. The greatest
change was observed by 10 min, so we chose this time
point to test the statistical significance of the changes
among groups (ANOVA and Fisher’s least significant
differences test; P , 0.05), and there was a gradual
recovery over the subsequent 40–50 min. In contrast,
no changes in Ptr were observed in control animals that
received humidified gas. The constrictive response to
dry gas was virtually abolished by pretreatment with
either the LTD4-receptor antagonist MK-571 or a combination of the NK1-receptor antagonist CP-99994 and
the NK2-receptor antagonist SR-48968 (Fig. 2). The
response was also abolished by depletion of NKs from
sensory nerves with capsaicin (Fig. 2). The peak value
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Instruments, Irvine, CA) or for immunoreactive cysteinyl LTs
by radioimmunoassay. The retention times of standards
using these conditions were 10 min for LTC4, 15 min for
N-acetyl LTE4, 20 min for LTD4, and 24 min for LTE4. After
each chromatography, the precolumn was washed successively with water (11 ml) and 100% acetonitrile (11 ml).
Radioimmunoassay. Column fractions were evaporated to
dryness in a vacuum centrifuge, and the residues were
dissolved in phosphate-buffered saline (0.1 ml at pH 8).
Cysteinyl LTs were measured in each fraction by using a
monoclonal antibody directed against LTC4, which crossreacted with LTD4 (63.8 6 10.9%), LTE4 (49.7 6 3.7%), and
N-acetyl LTE4 (59.5 6 7.4). [14,15-3H]LTC4 was used as the
radioactive ligand. All values were corrected for the degree of
cross-reactivity of the LTs with the antibody, but they were
not corrected for the recovery of cysteinyl LTs, which was
,60% in the case of LTC4. The background values for
materials displaying LTC4-like immunoreactivity were quite
low in the HPLC fractions and were not subtracted from the
reported values for LTs. The total amounts of cysteinyl LTs in
bile were calculated by adding the amounts (in pmol/time
period) for LTC4, LTD4, LTE4, and N-acetyl LTE4.
Histamine assay. BAL was performed immediately after
the peak increase in Ptr by instilling 5 ml of normal saline via
the endotracheal tube. The saline was immediately reaspirated and kept at 240°C until assayed. The histamine
content in BAL fluid was measured by using a colorimetric
assay based on the imidazole group of histamine. In brief, 0.5
ml of sample mixed with both 0.1 ml of 1% sulfanilic acid and
0.1 ml of 5% aqueous sodium nitrite solution was incubated
for 10 min. Then 1.3 ml of aqueous 5% sodium carbonate
solution were added. Two minutes later, 1 ml of 75% of
ethanol was added. The absorbance of the orange-red complex
in 200 µl of reaction mixture was measured at 530 nm within
20 min using a microplate reader (400 ATC, SLT Labinstruments, Unterbergstrassel. A5082 Grödig/Salzburg, Austria).
A standard curve was performed by using histamine dihydrochloride in concentrations ranging from 0 to 40 µg/ml.
Statistical analysis. All results are expressed as means 6
SE. Comparison of two means was performed by using paired
or unpaired t-tests or Wilcoxon’s signed ranks test as appropriate. For comparison of several means, an analysis of variance
(ANOVA) followed by the Fisher’s least significant differences
test was used. Differences were considered to be statistically
significant at P , 0.05.
Drugs and chemicals. LTC4, LTD4, LTE4, N-acetyl LTE4,
monoclonal antibody to LTC4, and the specific LTD4-receptor
antagonist MK-571 were kindly provided by Dr. A. W. FordHutchinson (Merck-Frosst, Pointe Claire, Quebec, Canada).
Capsaicin, histamine hydrochloride, and sulfanilic acid were
purchased from Sigma Chemical. The NK1-receptor antagonist CP-99994 and NK2-receptor antagonist SR-48968 were
kindly provided by Dr. Ian Rodger (Merck-Frosst). Salbutamol was purchased from Glaxo Canada (Toronto, Ontario).
Pentobarbital sodium was supplied by MTC Pharmaceuticals
(Cambridge, Ontario, Canada). Pyrilamine was purchased
from Research Biochemicals International (Natick, MA). Sodium nitrite and sodium carbonate were purchased from
Fisher Scientific (Fair Lawn, NJ).
541
TACHYKININS TRIGGER CYSTEINYL LEUKOTRIENE SYNTHESIS
of Ptr after challenge in the test group was significantly
higher than the baseline value (paired t-test; P , 0.05)
and the peak values of Ptr after challenge in the other
groups (ANOVA and Fisher’s least significant differences test; P , 0.05), with the exception of the pyrilamine-treated animals, which were not significantly
different in their responses to the test group (Fig. 3).
Fig. 4. Level of total biliary cysteinyl (cys) LTs before hyperpnea
challenge (open bars) was no different among groups (ANOVA; P 5
0.12). Cysteinyl LT levels in bile after hyperpnea challenge (hatched
bars) were significantly higher in test group animals than in other
groups (ANOVA; *P , 0.01), and Fisher’s least significant differences
test confirmed significant differences between test and control and
capsaicin and NK antagonist-treated groups.
Biliary cysteinyl LTs and hyperpnea challenge. To
minimize the contribution of the surgical procedure to
biliary LTs (12), we waited for 60 min before collecting
bile for baseline measurements of cysteinyl LTs. The
total cysteinyl LTs (expressed as the sum of LTC4,
LTD4, LTE4, and N-acetyl LTE4 ) in bile before and after
hyperpnea challenge are shown for the different treatment groups in Fig. 4. The biliary cysteinyl LTs were
not analyzed for the MK-571-treated animals as it was
not anticipated that the results in this group would be
different from those for the dry gas-challenged test
group. The total biliary levels of cysteinyl LTs before
hyperpnea challenge were not significantly different
among the groups (Fig. 4). There were few differences
in the baseline levels of the various cysteinyl LTs
among treatment groups (Table 1). However, LTD4 was
significantly higher in the NK group compared with
control and capsaicin-treated animals. After hyperpnea
challenge, the total cysteinyl LT levels rose significantly (Wilcoxon’s signed ranks test; P , 0.05) in the
Table 1. Baseline prechallenge level of biliary
cysteinyl leukotrienes
Biliary Leukotrienes,
pmol/h
Leukotriene C4
Fig. 3. Peak inspiratory value of Ptr after hyperpnea challenge
(hatched bars). There were significant difference among groups
(ANOVA; P , 0.005), and post hoc testing using Fisher’s least
significant differences test confirmed significant differences between
test and control and MK-571, capsaicin, and NK antagonists but not
pyrilamine-pretreated animals. Peak value of Ptr after challenge was
significantly higher in test group and pyrilamine-pretreated animals
compared with value of Ptr before challenge (open bars).
Leukotriene D4
Leukotriene E4
N-acetyl leukotriene E4
Control
(n 5 6)
Test
(n 5 7)
Capsaicin
(n 5 4)
Neurokinin
Antagonists
(n 5 6)
0.6 6 0.1
(11.9)
1.4 6 0.7
(29.9)
2.0 6 0.6
(40.4)
0.9 6 0.3
(17.7)
0.9 6 0.5
(6.8)
4.6 6 1.6
(36.2)
4.6 6 2.5
(36.1)
2.6 6 1.4
(20.8)
0.2 6 0.04
(10.9)
0.5 6 0.1
(30.0)
0.7 6 0.04
(42.1)
0.3 6 0.06
(18.0)
0.6 6 0.3
(5.6)
5.1 6 0.9*
(77.2)
1.1 6 0.6
(9.1)
1.0 6 0.8
(8.1)
Values are means 6 SE; n, no. of animals. Percentage of total
biliary cysteinyl leukotrienes shown in parentheses.
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Fig. 2. Value of inspiratory tracheal pressure (Ptr) measured at 1, 3,
10, 30, and 60 min after challenge in test [dry gas hyperpnea
challenge (s)], control [humidified gas (r)], and MK-571 (k)-,
capsaicin (j)-, neurokinin (NK) antagonist (n)-, and pyrilamine
(l)-pretreated groups. Hyperpnea with dry gas caused bronchoconstriction in test and pyrilamine-treated animals. There were significant differences among groups [analysis of variance (ANOVA); P ,
0.005]. Post hoc comparisons using Fisher’s least significant differences test confirmed significant differences among test and control
and MK-571, capsaicin, and NK antagonists but not pyrilaminepretreated animals.
542
TACHYKININS TRIGGER CYSTEINYL LEUKOTRIENE SYNTHESIS
test group but not in the control group in which the
challenge was performed with humidified air (Fig. 4).
Nor did the levels of cysteinyl LTs increase after
hyperpnea challenge in the groups of animals that were
pretreated with either capsaicin or a combination of the
NK1- and NK2-receptor antagonists. The bile was not
analyzed from pyrilamine-treated animals. The change
in cysteinyl LTs in the test group is attributable to
LTD4, which rose from 4.6 6 1.6 to 12.5 6 5.0 pmol/h
(ANOVA and Fisher’s least significant differences test;
P , 0.05). LTE4 also tended to be higher in the test
group, but the difference did not quite reach statistical
significance (Fig. 5).
Histamine levels in BAL fluid. The concentration of
histamine in the BAL fluid of unchallenged guinea pigs
was 5.09 6 0.6 µg/ml. There was no significant difference in the concentration of histamine in the lavage
fluid of dry gas-challenged animals (6.43 6 2.05 µg/ml;
P 5 0.24).
The principal aim of this study was to determine the
relationship between the airway response to isocapnic
dry gas hyperpnea challenge and the synthesis of
cysteinyl LTs. The blockade of HIB by selective antagonists of both LTD4 and NK receptors indicates that
tachykinins and cysteinyl LTs are both important mediators of the bronchoconstrictive response to hyperpnea. The completeness of the blockade caused by the
antagonists of either pathway suggests an important
interaction between these two classes of mediators. The
finding that a selective LTD4 antagonist eliminated the
airway response to hyperpnea suggests that tachykinins may evoke airway narrowing indirectly by the
release of cysteinyl LTs from airway effector cells. The
abolition of an increase in cysteinyl LT synthesis after
hyperpnea challenge by pretreatment with NK antago-
Fig. 5. Individual biliary cys LTs after hyperpnea challenge (expressed as pmol/h). N-Ac LTE4, N-acetyl LTE4. Only LTD4 was
significantly different among groups (ANOVA; *P , 0.05) and was
elevated in test group compared with control, capsaicin, and NK
antagonist-pretreated animals (Fisher’s least significant differences
test). LTE4 also tended to be elevated after challenge, but difference
was not quite significant.
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DISCUSSION
nists confirms that airway tachykinins evoke cysteinyl
LT release.
A limitation of our study is that we quantified the
airway responses to hyperpnea challenge from changes
in Ptr that reflect alterations in respiratory system
impedance. The pulmonary response to hyperpnea
challenge is clearly complex and involves changes in
both airway and tissue resistance (31) as well as
microvascular leak (15). However, we have assumed
that most of the measured response is a consequence of
airway narrowing resulting from airway smooth muscle
contraction, which has been confirmed by morphometric studies (31).
Measurement of biliary cysteinyl LTs is a convenient
method to evaluate the synthesis of these substances in
vivo (17). The biliary recovery of radioactivity after
intratracheal instillation of [3H]LTC4 was ,23% in 4 h.
Although this value is substantially less than the
recovery of intravenously injected [3H]LTC4, which has
been reported to be 60% over 6 h, it is similar to the recovery of the intratracheally instilled label in rats (29).
The conversion of LTC4 to LTD4 was almost complete,
with only relatively small amounts of LTC4 excreted
unchanged. LTD4 was the major cysteinyl LT identified
in bile in most circumstances, in contrast to the rat, in
which N-acetyl LTE4 is the major identifiable species
but in agreement with other studies on the metabolism
of LTC4 in the guinea pig. LTE4 is the major cysteinyl
LT metabolite in humans and other primates (19).
Our study and previous observations have demonstrated that HIB in guinea pigs can be entirely attributed to tachykinins (15). Sensory neuropeptides [substance P (SP); NKA, and calcitonin gene-related peptide
(CGRP)] have been shown to be capable of producing
constriction of airway smooth muscle, edema and
plasma extravasation, and mucus hypersecretion both
in animals and humans (2, 8, 27, 34). Tachykinins
released by exogenous agonists or exogenously administered can cause bronchoconstriction (6, 16). Garland et
al. (15) have shown that capsaicin pretreatment blunts
the bronchoconstriction evoked by isocapnic dry gas
hyperpnea. As expected, phosphoramidon, a neutral
endopeptidase inhibitor, potentiates the effects of HIB,
which is further, albeit indirect, evidence of participation of neuropeptides in the airway response (25). More
recent data from studies that used the selective NK1
(SP)-receptor antagonist CP-96345 and the NK2 (NKA)receptor antagonist SR-48968 have demonstrated attenuation of the airway response to HIB (39), providing
more support for the role of tachykinins in this phenomenon. Our experiments have provided similar results:
HIB is abolished after depletion of tachykinins by
capsaicin and significantly blunted by pretreatment
with a combination of the NK1-receptor antagonist
CP-99994 and the NK2-receptor antagonist SR-48968.
The biliary LTD4 level significantly increases after
isocapnic dry gas hyperpnea challenge, indicating increased synthesis of cysteinyl LTs. The absence of such
change after a similar challenge with humidified gas
indicates the specificity of the response to the airway
challenge. An important role for cysteinyl LTs in the
TACHYKININS TRIGGER CYSTEINYL LEUKOTRIENE SYNTHESIS
useful model of EIA. The results of our study demonstrate a further similarity to EIA in that cysteinyl LTs
have been convincingly implicated in the airway narrowing triggered in this way. Pliss et al. (35) have showed
that the concentrations of cysteinyl LTs in BAL fluid
are significantly increased after isocapnic hyperpnea in
asthmatic subjects. Israel et al. (24) have found that
A-64077, a 5-lipoxygenase inhibitor, increased the tolerance of asthmatic subjects to hyperventilation with cold
dry air and decreased the level of plasma LTB4. Furthermore, the LTD4-receptor antagonist MK-571 attenuates EIA in asthmatic subjects (28).
In conclusion, HIB in guinea pigs is mediated by
cysteinyl LTs. The release of these mediators appears to
be triggered by tachykinins that are presumably released from sensory nerve endings stimulated by altered osmolarity or thermal fluxes related to inhalation
of dry air. Histamine was not involved in dry gas HIB in
guinea pigs. The airway cells responsible for cysteinyl
LT synthesis remain to be identified.
The authors thank Liz Milne for help in the preparation of the
manuscript.
This study was supported by the Medical Research Council of
Canada Grant 7852 and the Respiratory Health Network of Centres
of Excellence of Canada.
Address for reprint requests: J. G. Martin, Meakins-Christie
Laboratories, McGill Univ., 3626 St. Urbain St., Montreal, Quebec,
Canada, H2X 2P2.
Received 7 November 1995; accepted in final form 16 October 1996.
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